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U N I V E R S I T Y O F C O P E N H A G E NU N I V E R S I T Y O F C O P E N H A G E N
PhD thesis
Chad Alton Keyser
Protecting plants against pests and pathogens with
entomopathogenic fungi:
The biocontrol agent Metarhizium, its distribution, application, and interaction with other
beneficial fungi
Academic Advisor: Nicolai Vitt Meyling
Submitted: 30 January, 2015
This thesis has been submitted to the PhD School of The Faculty of Science, University of Copenhagen
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Institution: University of Copenhagen, Faculty of Science
Department: Department of Plant and Environmental Sciences (PLEN)
Author: Chad Alton Keyser
Title: Protecting plants against pests and pathogens with entomopathogenic fungi:
the biocontrol agent Metarhizium, its distribution, application, and interaction
with other beneficial fungi.
Academic advisor: Nicolai Vitt Meyling
Co-advisor: Kristian Thorup-Kristensen
Submitted: 30 January 2015
3
Acknowledgments
“It is only with the heart that one can see clearly, for the most essential things are invisible to the eye."
― Hans Christian Andersen, The Ugly Duckling
Through this acknowledgment I would like to express my profound appreciation to the many
individuals that have supported and encouraged me during the course of my PhD program. Throughout the
last three years I have grown significantly as a scientist and as a person – this growth is due mainly to the
collaborations, friendships and nurturing influence of the many people I have had the opportunity to interact
with.
I first met Nicolai Meyling in 2009 at a scientific conference in Park City, Utah, as we stood in a
crowded hall waiting for seating to begin. During our short conversation I was impressed by his attentive
interest as I explained what I was working on as a Masters student. As my supervisor, Nicolai has continued
to support and facilitate my efforts and growth. He has patiently steered my ideas in scientifically-relevant
directions and ensured that my project was progressing. I am very grateful to Nicolai for his relaxed manner,
thoughtful comments and suggestions, and the diligence and effort he has put into mentoring me.
The Section for Organismal Biology (SOBI) has assembled an exemplary team of insect
pathologists. It has been a pleasure to work alongside such a cohesive group, to share ideas, experience and
encouragement – my experience here will always serve as a paragon for future collaborations. As the head
of this research group, I am grateful to Jørgen Eilenberg for his infectious enthusiasm, his example of
efficiency and his effort to make me feel part of the team. Also, I am very grateful to Bernhardt Steinwender
for both his friendship and constant willingness to listen and discuss even the most ridiculous of ideas. I am
also thankful to Henrik de Fine Licht for his patients in instructing me in art of AFLP analysis. The Team
consists of many more PhD fellows, post-docs and professors who have each individually encouraged,
inspired, taught and helped me in many ways and for which I am truly appreciative.
It has been a great pleasure to work alongside many skilled technicians and assistants. I am
especially grateful to Louise Munk Larsen for her ample technical skills and continued willingness to drop
what she is doing to help. I thank Sylvia Mathiasen and Vinnie Deichmann for their assistance with my
molecular work. I am also grateful to Line Lykke, Lærke Thordsen, Martina Falagiarda, Jesper Anderson,
Azmi Mahmood, Darren Thomsen and the many other student helpers that have assisted me in many aspects
of my experiments – I would not have been able to accomplish what I have needed to do without their
diligent effort and skills.
I have also been fortunate to have Kristian Thorup-Kristensen as a co-advisor. I am thankful to him
for sharing his expertise in working with plants and in experimental design, as well as including me as part
of his team. I also thank Birgit Jensen for her assistance in working with Fusarium and Clonostachys and her
interest and willingness to train and assist me throughout the study for the second Manuscript.
Living in Denmark and attending the University of Copenhagen has been the experience of a
lifetime. I am thankful to Plant Biosystems Elite Environment at the University of Copenhagen for funding
my PhD research. I am also grateful to the Department of Plant and Environmental Sciences and the Section
for Organismal Biology for hosting and providing necessary facilities for my studies.
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I am also supremely grateful for the support and encouragement I have received from my families. I
recognize that living overseas has been difficult for both my family and my wife’s family; I sincerely
appreciate their willingness to accept my decision to pursue this degree and the support and love they have
shown which has been an enabling power to finish. I also appreciate the family and friends that have found
the opportunity to visit us and share in this experience – their refreshing visits have made the distance more
bearable.
Most importantly, I would like to express sincere gratitude to my beautiful wife Shannon and three
energetic children: Myra, Alexys and Noah. They willingly left behind their friends, families, job and
comforts to follow me on an unknown path. My children have worked hard, learned the difficult language
and adjusted quickly to the Danish lifestyle – their adaptability and bravery has given me strength. Shannon
has also thrived and grown to love European living – her infectious eagerness to explore and discover the
world around us has lifted and strengthened our whole family. I could not have succeeded in completing this
program if not for her. Having my best friend by my side and knowing that she supports my ambitions has
made this experience truly enjoyable. It is to my wife and children I dedicate this work and my life.
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TABLE OF CONTENTS
ACKNOWLEDGMENTS .......................................................................................................................................3
I. LIST OF INCLUDED MANUSCRIPTS ...........................................................................................................6
II. SUMMARY ..................................................................................................................................................7
III. DANSK RESUMÈ .........................................................................................................................................9
IV. THESIS OBJECTIVES................................................................................................................................11
1. INTRODUCTION ...........................................................................................................................................12
2. THE ENTOMOPATHOGENIC FUNGAL GENUS METARHIZIUM ...................................................................13
2.1 Phylogeny and Taxonomy..................................................................................................................... 15
2.2 Ecology.................................................................................................................................................. 18
2.2.1 Abundance and distribution............................................................................................................ 18
2.2.2. Abiotic factors that affect survival and growth ............................................................................. 21
2.2.3 Environmental dissemination pathways ......................................................................................... 22
3. TROPHIC INTERACTION ..............................................................................................................................23
3.1 Metarhizium ↔ Insects.......................................................................................................................... 24
3.2 Metarhizium ↔ Plants........................................................................................................................... 26
3.3 Metarhizium ↔ Other microorganisms................................................................................................. 29
3.4 Multi-trophic interactions with Metarhizium ........................................................................................ 31
3.4.1 Metarhizium ↔ Other microorganisms ↔ Insects......................................................................... 31
3.4.2 Metarhizium ↔ Other microorganisms ↔ Plants .......................................................................... 32
3.4.3 Metarhizium ↔ Plants ↔ Insects................................................................................................... 32
3.4.4 Metarhizium ↔ Other microorganisms ↔ Plants ↔ Insects ......................................................... 33
4. METHODOLOGY.........................................................................................................................................34
4.1 Selective media...................................................................................................................................... 34
4.2 Bioassay statistics.................................................................................................................................. 37
4.2.1 Experimental design ....................................................................................................................... 37
4.2.2 Types of Statistical Analyses.......................................................................................................... 38
V. CONCLUSION AND FUTURE PERSPECTIVES ..............................................................................................42
VI. REFERENCES ............................................................................................................................................44
VII. APPENDIX ................................................................................................................................................52
Manuscript 1................................................................................................................................................ 52
Manuscript 2................................................................................................................................................ 63
Manuscript 3................................................................................................................................................ 90
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i. List of included Manuscripts
Manuscript 1
METARHIZIUM SEED TREATMENT MEDIATES FUNGAL DISPERSAL VIA ROOTS AND INDUCES
INFECTIONS IN INSECTS
Chad A. Keyser, Kristian Thorup-Kristensen, & Nicolai V. Meyling
Status: Published in Fungal Ecology, October 2014, Vol. 11, pg. 122-131
License Number: 3531750730534
Manuscript 2
BEST OF BOTH WORLDS: DUAL EFFECTS OF METARHIZIUM SPP. AND CLONOSTACHYS ROSEA
AGAINST AN INSECT AND A SEED-BORNE PATHOGEN IN WHEAT
Chad A. Keyser, Birgit Jensen & Nicolai V. Meyling
Status: Under Review - Pest Management Science, submitted 22 Dec, 2014
Manuscript 3
DIVERSITY OF METARHIZIUM FLAVOVIRIDE POPULATIONS ASSOCIATED WITH ROOTS OF CROPS IN
DENMARK
Chad A. Keyser, Henrik H. de Fine Licht, Bernhardt M. Steinwender & Nicolai V. Meyling
Status: Manuscript
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ii. Summary
Background: Insect-pest management is an increasingly important area of research. Efforts
to maximize agricultural output are significantly dependent on reliable means for pest suppression.
Biological control, or the use of living organisms to suppress a pest population, is among one of the
leading alternatives to traditional chemical-based pesticides for crop protection. For the past 130
years several isolates of the fungal genus Metarhizium has been lead candidates among potential
fungal-based biological control agents (BCAs) for insect pest control in agriculture. However, the
majority of Metarhizium research has emphasized product development and application, largely
neglecting the ecological and fundamental aspects. Inconsistent field reliability and economic
viability have limited wider implementation of many BCAs, including Metarhizium-based products;
an increased understanding of the fundamental ecology and environmental interactions has
substantial potential to improve biological control efforts.
The overall aim of this thesis was to improve the current understanding of how members of
the fungal genus Metarhizium naturally occur in association with roots of crops in Denmark and
interact with other organisms in relation to plant roots when applied as BCAs. Several scientific
studies were conducted to answer important ecological questions regarding Metarhizium spp.
interactions and their use as BCAs. The results of these studies are presented in three manuscripts.
Manuscript 1: Recent research has revealed that many Metarhizium spp. interact with plants
in the rhizosphere and have been shown to increase nutrient uptake and promote plant growth. In
Manuscript 1 we investigate how might the fungus benefit from a plant association; namely,
whether the plant provides a means of dispersal for the otherwise immobile fungus; as well as if the
fungus maintains pathogenicity to insects while interacting with the plant. We found that when
Metarhizium spp. were inoculated as conidia on wheat seeds they were able to disperse through the
soil with the growing root and be re-isolated from lower portions of the root. Furthermore we
observed that when washed roots were placed with Tenebrio molitor larvae, the larvae would
succumb to Metarhizium spp. infection.
Manuscript 2: Agricultural yields are threatened by multiple pests including insects and
plant pathogens. Often the control of these pests requires the application of multiple biological
control agents. In Manuscript 2 we investigate whether the mycoparasite Clonostachys rosea,
commonly used to control plant-fungal pathogens, can be applied jointly with Metarhizium spp. to
control both a plant pathogen and an insect pest. In this study we observed that C. rosea was highly
efficacious at controlling Fusarium culmorum alone and in combination with Metarhizium – when
8
applied as a conidial seed treatment to wheat seeds. Additionally, we observed that while a
significant level of T. molitor were infected with Metarhizium spp. after a combined treatment,
there was a slight reduction in virulence when either C. rosea or F. culmorum were also present
when compared to Metarhizium spp. only seed treatments. Based on the result of the direct
inoculation bioassay of T. molitor larvae in which we did not observe a reduction in virulence when
comparing combination treatments to individual treatments, we suspect that the virulence reduction
is the result of resource competition on the growing root and not direct mycoparasitism.
Manuscript 3: An awareness of the composition and distribution of naturally occurring
Metarhizium spp. communities is important to understanding their role to insect host regulation.
However there is an acute lack of ecological studies that assess the occurrence and community
structure of entomopathogenic fungi. The objective of Manuscript 3 was to evaluate the
occurrence, diversity and community structure of Metarhizium spp. isolates obtained from different
crops at geographically separated agricultural fields in Denmark. Root and root-associated soil was
sampled from wheat, oilseed rape, and bordering uncultivated grass fields at three different
locations; 132 new Metarhizium isolates were obtained. Morphological data and sequencing of the
rDNA intergenic spacer region (IGS) revealed that 118 of the isolates belonged to Metarhizium
flavoviride, 13 M. brunneum and one M. majus. We then further characterized the intraspecific
variability within M. flavoviride by unspecific markers (i.e., AFLP identification) to evaluate
diversity and potential crop and/or area associations. We found there was a high level of diversity
among the M. flavoviride isolates indicating that the isolates were not of the same clonal origin,
however due to insufficient loci in the AFLP analysis we were not able to determine haplotype
groupings or confirm any habitat associations. We suggest that the development of more specific
markers would greatly improve our ability to evaluate M. flavoviride diversity. This represents the
first time that an in-depth analysis of the molecular diversity within a large isolate collection of the
species M. flavoviride has been reported.
Overall the scientific studies presented in this thesis are both important and novel to the field
of Metarhizium research; these studies advance the current knowledge of the ecological significance
of Metarhizium spp. as a naturally occurring microorganism and increase our understanding of their
interactions as biological control agents with other organisms. Furthermore, this thesis presents the
background literature and motivation for the research and their implication to the field of insect
pathology.
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iii. Dansk resumè
Baggrund: Bekæmpelse af skadedyr er et vigtigt forskningsområde. Mulighederne for at øge
landbrugsudbyttet er afhængigt af pålidelige bekæmpelsesmetoder. Biologisk bekæmpelse, brugen
af levende organismer til at begrænse skadedyrspopulationer, er en af de væsentligste alternativer til
kemisk baseret plantebeskyttelse. I løbet af de seneste 130 år har flere isolater af den insektpatogene
svampeslægt Metarhizium blevet førende kandidater til svampebaseret biologisk bekæmpelse af
skadedyr. Dog har hovedparten af forskning i Metarhizium fokuseret på produktudvikling og
udbringning, hvor forståelse af grundlæggende økologiske aspekter har blevet negligeret.
Varierende pålidelighed har begrænset implementeringen af mange biologiske
bekæmpelsesprodukter, inklusiv flere baseret på Metarhizium. En øget forståelse af de
fundamentale økologiske og miljømæssige interaktioner, hvori Metarhizium indgår, har stort
potentiale til at forbedre brugen af biologisk bekæmpelse.
Det overordnede formål med denne afhandling var at øge forståelsen af hvordan medlemmer
af svampslægten Metarhizium indgår i naturlig associering med rødder af afgrøder i Danmark og
hvordan de interagerer med andre organismer i relation til planterødder når de tilføres ved biologisk
bekæmpelse. Flere videnskabelige studier blev gennemført for at svare på dette, og resultaterne er
præsenteret i tre manuskripter.
Manuskript 1: Det er for nyligt blevet videnskabeligt vist at flere arter af Metarhizium
interagerer med planter i rhizosfæren og at dette kan øge næringsstofoptag og vækst hos planten. I
Manuskript 1 undersøges det om svampen kan have fordel af denne interaktion ved at bruge
plantens rødder til transport i jorden, og om svampen bevarer sin evne til at inficere insekter ved at
interagere med planten. Det blev fundet, at når Metarhizium spp. blev inokuleret som konidier på
frø af hvede, kunne svampen sprede sig gennem jord med de voksende rødder og blive genisoleret
fra den nedre del af rødderne. Desuden var vaskede rødder infektive mod larver af melorme
Tenebrio molitor, som efter inkubering sammen med rødder døde af Metarhizium infektion.
Manuskript 2: Afgrøder bliver ofte angrebet af flere organismer, primært insekter og
plantepatogener, hvilket kræver samtidig brug af flere biologiske bekæmpelsesmidler. I Manuskript
2 blev det undersøgt, om nyttesvampen Clonostachys rosea, som normalt bruges til at bekæmpe
plantepatogener, kan blive anvendt sammen med Metarhizium spp. til at bekæmpe både insekter og
plantesygdomme på samme tid. Det blev observeret at C. rosea var meget effektiv til at bekæmpe
Fusarium culmorum alene og sammen med Metarhizium spp., når de to typer af svampe blev tilført
som konidier på frø af hvede. Desuden blev larver af melorme inficeret af Metarhizium spp. både
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når denne svamp blev tilført alene og sammen med C. rosea eller F. culmorum, men infektionen var
en smule nedsat i kombinationerne. Ved direkte inokulering af larverne med sammen
svampekombinationer sås ikke denne reduktion. Reduktionen observeret ved planteinokuleringen
kunne derfor skyldes en indirekte interaktion på planteroden og ikke direkte interaktion som
mykoparasitisme mellem svampene.
Manuskript 3: Det er afgørende af kende til den naturlige forekomst og udbredelse af
Metarhizium spp. for at forstå deres bidrag til regulering af insektpopulationer. Der findes dog
fortsat kun få studier af den naturlige forekomst af insektpatogene svampe. Formålet med studiet
præsenteret i det tredje manuskript var at beskrive forekomst, udbredelse og diversitet af
Metarhizium spp. i forbindelse med rødder af forskellige afgrøder i Danmark på tre geografisk
afgrænsede lokaliteter. Rødder og rod-associeret jord blev indsamlet på hver lokalitet fra
vinterhvede, raps, og fra et uforstyrret græsningsareal. 132 Metarhizium isolater blev indsamlet.
Morfologisk og DNA sekventering viste, at 118 af isolaterne tilhørte arten Metarhizium flavoviride,
13 var M. brunneum, og et var M. majus. Ved yderligere karakterisering af intraspecifik variation i
M. flavoviride med uspecifikke markører (AFLP) blev diversitet og potentiel lokalitet- eller
afgrødespecificitet undersøgt. En høj grad af diversitet blev fundet med de anvendte markører,
hvilket indikerer at de ikke alle har samme klonale ophav, men metoden var ikke tilstrækkelig til at
identificere lokalitet- eller afgrødespecificitet. Specifikke markører udviklet til M. flavoviride vil
være nødvendige for at undersøge dette. Dette studie er det første hvor arten M. flavoviride blev
undersøgt mht. molekylær diversitet ved brug af en større samling af isolater.
De videnskabelige studier præsenteret i denne afhandling er både vigtige og nye indenfor
forskningen i Metarhizium. Studierne øger den nuværende viden om økologisk betydning af
Metarhizium spp. som naturlig forekommende mikroorganisme samt ved interaktioner med planter
og med andre mikroorganismer. Desuden giver denne afhandling en gennemgang af litteraturen om
emnet og betydningen af den præsenterede forskning for insektpatologi.
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iv. Thesis Objectives
The aim of this thesis is to advance our understanding of how members of the entomopathogenic
fungal genus Metarhizium interact with other organisms, with an emphasis placed on its role as a
biological control agent. To accomplish this goal the following research questions were
investigated:
■ Can plant roots act as a vehicle to disseminate Metarhizium in soil? Additionally, while
associating with plants, do Metarhizium spp. maintain their pathogenicity to insects?
■ In a seed-treatment biocontrol context, what interactions occur between other organisms,
namely: the mycoparasitic fungus Clonostachys rosea, and the plant pathogenic fungus
Fusarium culmorum?
■ What is the prominence and species composition of naturally occurring Metarhizium in roots
and soil within different agro-ecosystems in Sjælland, Denmark?
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1. Introduction
Agricultural production is incalculably important to human existence both from an
economical as well as a nutrient-resources perspective. Worldwide estimates suggest that currently
38% of the Earth’s terrestrial surface is dedicated to agricultural endeavors (Foley et al., 2011).
Furthermore, it has been projected that from 2005 to 2050 global crop production will need to
increase as much as 110% to meet the caloric and protein demands of the world population (Tilman
et al., 2011). We cannot expect to meet the growing agricultural needs by merely dedicating more
land to food production; increasing the productivity of the current agricultural systems is vital to our
future (Tilman et al., 2011). A significant limiting factor to agriculture yields are pests – this
includes herbivores (e.g., arthropods), weeds and plant pathogens. Over the last century advances
in chemical pesticide development have greatly mitigated pest related crop losses. However, due to
pesticide resistance and an increased awareness of deleterious effects on non-target organisms,
including humans, reliable alternative methods for controlling pests are desperately needed.
Biological control is an important alternative which has potential to effectively control pest
populations with limited risk (Hajek, 2004).
Biological control can be defined as “the use of living organisms to suppress the population
of a specific pest organism, making it less abundant or less damaging then it would otherwise be”
(Eilenberg et al., 2001). There are several types of organisms that have been identified as biological
Glossary of Terms
Agroecosystem Agricultural ecosystem - Specialized ecosystem which has been manipulated by human activities
with the aim to produce high levels of organic output. Includes living and non-living components
and their interactions.
Anamorph The asexual (conidial or imperfect) stage in the life history of a fungus.
BCA Biological control agent – the organism used for Biological control
Bioassay The measurement of the potency of any BCA, by means of the response which it produces in a
living host.
Biological control The use of living organisms to control (usually meaning to suppress) undesirable animals and
plants.
Entomopathogen A microbe affecting insects (or in a more general sense, other terrestrial arthropods including
arachnids), usually causing mortality in the host (as opposed to a more benign relationship).
Fungal endophyte An asymptomatic plant-colonizing fungus that lives a portion of its life cycle inside the plant.
Pathogenicity The quality or state of being pathogenic. The potential ability to produce disease. Pathogenicity
is qualitative, an all-or-none concept
Rhizosphere The narrow region around the plant root that is directly influenced by root secretions and
associated soil microorganisms.
Teleomorph The sexual stage in the life history of a fungus.
Virulence The disease-producing power of a microorganism. Virulence can be quantified.
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control agents (BCAs), including: predators, parasitoids, parasitic nematodes, bacteria, viruses,
fungi and microsporidia (Hajek, 2004), the latter group is now recognized and a basal fungal group
(James et al., 2006). Each BCA has different characteristics which determine their effectiveness in
a particular circumstance. Understanding the environmental factors that contribute to effectiveness
will largely determine the success of a BCA. For many years research regarding the fundamental
ecology of these organisms has had far less priority than product development; this has likely led to
inconsistent results in the field (Vega, 2008). The present thesis contributes in part, to increasing
our understanding of the complex interactions involved between a particular fungal BCA and other
important organisms in its environment, within a biological control context.
2. The entomopathogenic fungal genus Metarhizium
Entomopathogenic fungi are fungal organisms that have the ability to infect and cause
disease in an arthropod. The kingdom Fungi is estimated to contain more than 5.1 million species
(O'Brien et al., 2005), of those, 750-1000 species are pathogenic to insects (Vega et al., 2012).
Fungi of the Ascomycota genus Metarhizium (Hypocreales: Clavicipitaceae) are among one
of the most studied groups of entomopathogenic fungi, in fact in the last decade the number of peer-
reviewed publications has increased substantially (Figure 1). Metarhizium spp. are ubiquitously
found in terrestrial ecosystems worldwide, having been isolated from every continent except
Antarctica (Roberts and St. Leger, 2004). Many species of Metarhizium have a wide host range,
like M. brunneum and M. robertsii, infecting at least seven insect orders (Veen, 1968;
Zimmermann, 1993); however a few species are known to be host specific like M. acridum which
only infects some taxa in the order Orthoptera (Driver et al., 2000; Wang et al., 2011). In addition
to their host range there are several other key attributes that make Metarhizium spp. ideal candidates
for BCAs, including: the infection process is topical (does not need to be ingested by host),
infectious conidia can be mass produced on artificial media, remain viable and can be easily
0
300
600
900
1200
1500
1975-1979 1980-1984 1985-1989 1990-1994 1995-1999 2000-2004 2005-2009 2010-2014
Numberof
Publications
Figure 1. The number of peer-reviewed publications published of 5 year periods for the last 40
years according to a search on web of science with the key word “Metarhizium”.
14
disseminated, and many species of Metarhizium produce secondary metabolites known as
destruxins which are toxic to invertebrates and help the pathogen overcome the host immunity
quickly (Dorta et al., 1996; Zimmermann, 2007).
The infection process of Metarhizium spp. to a suitable host was reviewed by Zimmermann
(2007) and is summarized in Figure 2. He describes the process in 6 steps including: 1. Attachment,
in which the conidia adheres to the cuticle using a combination of hydrophobic interaction and
specialized adhesion proteins; 2. Germination and appressoria formation; 3. Penetration through the
cuticle, which is mechanical but aided by the production of enzymes including proteases, chitinases
and lipases; 4. Overcoming host defenses, often by the production of novel destruxins; 5.
Proliferation within the host, generally via the production of blastospores or hyphae; and lastly, 6.
Outgrowth and production of new infective conidia.
Figure 2: The infection process, an illustration showing the 6 stages of fungal infection in an insect:
1. Attachment; 2. Germination and appressoria formation; 3. Penetration through the cuticle; 4.
Overcoming host defenses; 5. Proliferation within the host; 6. Outgrowth and production of new
infective conidia. Illustration by C. A. Keyser.
15
2.1 Phylogeny and Taxonomy
The taxonomy of the Genus Metarhizium (Family: Clavicipitaceae, Order: Hypocreales,
Class: Sordariomycetes Phylum: Ascomycota, Kingdom: Fungi) has been revised many times over
the years. In 1883 Russian mycologist Sorokin first introduced the name Metarhizium as the genus
name for fungi that are the causal agent of insect disease “green muscardine”, which had originally
been called Entomophthora anisopliae by Elia Metchnikoff in 1879 (Zimmermann et al., 1995).
For the last 130 years the genus name Metarhizium has persisted, however many of the species
names belonging to this genus have changed.
Many fungi are pleomorphic, i.e., they have different life stages which are morphologically
distinct – often the different stages have been identified as different organisms. To help standardize
the descriptions the term teleomorph is used to describe the sexual stage, anamorph to describe the
asexual stage, and holomorph when both are present (Hennebert and Weresub, 1977). Species of
the genus Metarhizium have historically been considered anamorphic fungi (i.e., only exhibiting the
asexual stage and reproduce clonally by mitosporic conidia production) and placed in the former-
division Deuteromycota; however the discovery of a sexual and asexual stage (Liang et al., 1991),
as well as molecular analysis have facilitated their placement in the phylum Ascomycota (Kepler et
al., 2014). In this thesis only the anamorphic stage of Metarhizium is discussed.
Another challenge for Metarhizium taxonomy is that many species of Metarhizium are
morphologically similar, so identification based on morphological attributes is difficult. Tulloch
(1976) made a major revision of the genus Metarhizium based on drawings and the morphological
descriptions available, she reduced all known taxa to two speices, M. flavoviride and M. anisopliae.
The next major revision of the genus was performed by Driver et al. (2000) using ITS sequence data
for phylogenetic analyses; they observed a high level of genetic diversity and were able to identify
ten distinct clades, however they restricted their descriptions to varieties rather than species due to
limited resolution and support in the sequence analysis. Bischoff et al. (2009) used a multigene
phylogenetic approach to resolve the M. anisopliae group which at the time consisted of 4 varieties
as defined by Driver et al. (2000); i.e., M. anisopliae var. acridum, M. anisopliae var. anisopliae,
M. anisopliae var. lepidiotae, and M. anisopliae var. majus. They describe ten species within the
M. anisopliae complex (viz., M. anisopliae, M. acridum, M. brunneum, M. globosum, M.
guizhouense, M. lepidiotae, M. majus, M. pingshaense, and M. robertsii) (Bischoff et al., 2009).
The taxonomic clarification of the Metarhizium genus was continued by Kepler et al. (2014) and the
M. flavoviride complex was resolved into four species (viz., M. flavoviride, M. koreanum, M.
16
minus, and M. pemphigi). Additionally, in this revision an effort was made to reevaluate fungal
nomenclature with the intention to give one name to one fungus, regardless of life stage (Taylor,
2011). In doing this Kepler et al. (2014) transferred many taxa from Chamaeleomyces,
Metacordyceps and Nomuraea to Metarhizium. The genus Metarhizium now includes 36 species
(Table 1). Isolates described as “M. anisopliae” or “M. flavoviride” in studies published prior to
(and sometimes after) the 2009 and 2014 taxonomic revisions are now designated as “M. anisopliae
sensu lato” (s.l.) or “M. flavoviride s.l.” if the new taxonomic identity according to Bischoff et al.
(2009) or Kepler et al. (2014) is unknown or not specified, M. anisopliae and M. flavoviride
identified using the current taxonomic criteria are labeled using the sensu stricto (s.s.) designation.
As noted by Steinwender (2013) “taxonomy is not set in stone but rather a snapshot of a given
moment”, it is likely the genus Metarhizium will continue to develop and change as our
understanding of the complexity of life improves.
17
Table 1: Current Species of the Genus Metarhizium, the authorities and year of description –
arranged alphabetically.
Species name Authorities, year of description
M. acridum (Driver & Milner) J.F. Bisch., S.A. Rehner & Humber, 2009
M. album Petch, 1931
M. anisopliae (Metsch.) Sorokin, 1883
M. atrovirens (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber, 2014
M. brasiliense Kepler, S.A. Rehner & Humber, 2014
M. brittlebankisoides (Zuo Y. Liu, Z.Q. Liang, Whalley, Y.J. Yao & A.Y. Liu) Kepler, S.A. Rehner & Humber, 2014
M. brunneum Petch, 1935
M. campsosterni (W.M. Zhang & T.H. Li) Kepler, S.A. Rehner & Humber, 2014
M. carneum (Duché & R. Heim) Kepler, S.A. Rehner & Humber, 2014
M. flavoviride W. Gams & Rozypal, 1973
M. frigidum (Driver & Milner) J.F. Bisch. & S.A. Rehner, 2006
M. globosum J.F. Bisch., S.A. Rehner & Humber, 2009
M. granulomatis (Sigler) Kepler, S.A. Rehner & Humber, 2014
M. guizhouense Q.T. Chen & H.L. Guo, 1986
M. guniujiangense (C.R. Li, B. Huang, M.Z. Fan & Z.Z. Li) Kepler, S.A. Rehner & Humber, 2014
M. indigoticum (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber, 2014
M. khaoyaiense (Hywel-Jones) Kepler, S.A. Rehner & Humber, 2014
M. koreanum Kepler, S.A. Rehner & Humber, 2014
M. kusanagiense (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber, 2014
M. lepidiotae (Driver & Milner) J.F. Bisch., S.A. Rehner & Humber, 2009
M. majus (J.R. Johnst.) J.F. Bisch., S.A. Rehner & Humber, 2009
M. marquandii (Massee) Kepler, S.A. Rehner & Humber, 2014
M. martiale (Speg.) Kepler, S.A. Rehner & Humber, 2014
M. minus (Rombach, Humber & D.W. Roberts) Kepler, S.A. Rehner & Humber, 2014
M. novozealandicum Kepler, S.A. Rehner & Humber, 2014
M. owariense (Kobayasi) Kepler, S.A. Rehner & Humber, 2014
M. owariense f. viridescens (Uchiy. & Udagawa) Kepler, S.A. Rehner & Humber, 2014
M. pemphigi (Driver & R.J. Milner) Kepler, S.A. Rehner & Humber, 2014
M. pingshaense Q.T. Chen & H.L. Guo, 1986
M. pseudoatrovirens (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber, 2014
M. taii Z.Q. Liang & A.Y. Liu, 1991
M. rileyi (Farl.) Kepler, S.A. Rehner & Humber, 2014
M. robertsii J.F. Bisch., S.A. Rehner & Humber, 2009
M. viride
(Segretain, Fromentin, Destombes, Brygoo & Dodin ex Samson) Kepler, S.A. Rehner &
Humber, 2014
M. viridulum (Tzean, L.S. Hsieh, J.L. Chen & W.J. Wu) B. Huang & Z.Z. Li, 2004
M. yongmunense (G.H. Sung, J.M. Sung & Spatafora) Kepler, S.A. Rehner & Humber, 2014
18
2.2 Ecology
Despite having more than 100 years’ worth of research interest, we are only beginning to
understand the ecology of Metarhizium spp. and the important role they play in the ecosystem.
There are two main reasons why the natural ecology of Metarhizium is important to biological
control: first, as a ubiquitous organism infectious to insects, understanding the natural occurrence
and distribution, and the contributions of Metarhizium in regulating insect populations is highly
relevant; and second, understanding how Metarhizium interacts with other organisms and is affected
by abiotic factors will help optimize how to most effectively use them as BCAs.
Bruck (2010) pointed out that in plant pathology a concept known as the “disease triangle”
is often used to describe the interaction between a host, a pathogen, and the environment; he
suggested that this same concept should also be applied to biological control. By emphasizing a
total ecological approach to Metarhizium spp. research, which focuses on both the direct and
indirect effects of biotic and abiotic factors in the environment, we gain greater clarity of the role
Metarhizium spp. play in the ecosystem. In this section I will discuss with regard to Metarhizium:
2.2.1. Abundance and distribution; 2.2.2. Abiotic factors that affect survival and growth; and 2.2.3.
Environmental dissemination pathways. In section 3 (below) I will discuss biotic interaction that
occur between Metarhizium and other organisms.
2.2.1 Abundance and distribution
The United States Department of Agriculture’s (USDA) Agricultural Research Service
Collection of Entomopathogenic Fungal Cultures (ARSEF), Ithaca, NY, hosts one of the largest
libraries of entomopathogenic fungal isolates collected from all over the world. This collection has
over 1500 different isolates of Metarhizium, however most of these isolates, as well as the many
others that have been collected over the years, were collected not with the intention to understand
abundance and distribution but rather to find new potential products for commercialization:
examples include the LUBILOSA project (Roberts and St. Leger, 2004), in which researchers
scoured the African and Australian continents searching for entomopathogenic fungi with the intent
to develop a BCA for locust control. More recently, an 8 year USDA-APHIS project which
surveyed 30,000 soil samples in the western US and collected more than 2,000 new isolates of
Metarhizium with a goal to find M. acridum in USA soil (C. A. Keyser, unpublished data). While
these “goal-orientated” types of surveys often produce useful information about the ecology and
distribution of Metarhizium in nature; they are not designed as ecological studies and so many of
the conclusions they can provide are incomplete.
19
There have, however, been several studies intended to investigate Metarhizium occurrence
in both natural habitats and agroecosystems (Table 2); it is noteworthy that there are a lack of
studies from latitudes below 40º (e.g., tropical regions). Many aspects differ between these studies
(e.g., sampling and isolation method) which makes direct comparison difficult; however, when
viewed as a group we are able to make some generalizations about the abundance and distribution
of Metarhizium spp. in different environments, several of which I would like to briefly outline:
First, Metarhizium spp. tend to be less abundant in colder regions than in temperate regions (Inglis
et al., 2008; Klingen et al., 2002; Vanninen, 1995), this observation is also supported by laboratory
work which has demonstrated that most species of Metarhizium , with the exception of M. frigidum,
do not grow at cold temperatures (Fernandes et al., 2010a; Fernandes et al., 2008). Second,
Metarhizium is more abundant than other entomopathogenic fungi in cultivated fields and open
meadows (Bidochka et al., 1998; Quesada-Moraga et al., 2007; Sun et al., 2008; Vanninen, 1995).
Third, Metarhizium is primarily found in the soil environment and not on above ground substrates
(Meyling et al., 2011; Vega et al., 2012). Lastly, Metarhizium spp. distribution tends to associate
with habitats and not with host insects (Bidochka et al., 2001; Fisher et al., 2011; Wyrebek et al.,
2011). Traditionally, insect association was thought to be the key factor in determining population
structure (Bridge et al., 1997; St. Leger et al., 1992), this shift away from the traditional paradigm
has led to many questions about what role these fungi truly play in the environment (Bidochka et
al., 2001; Vega, 2008).
Another interesting observation we can glean from viewing these studies together is that
Metarhizium spp. composition and dominance appears to be location specific. For example, a study
in the mid-east part of Canada found M. robertsii to be the most common species of Metarhizium
(Wyrebek et al., 2011), while a study in the western part of Canada found M. anisopliae s.l. to be
most common (Inglis et al., 2008). Even in Denmark survey studies have yielded inconsistent
species compositions. In 1995 Steenberg reported that M. anisopliae s.l. dominated cultivated soils
(Vega et al., 2012), also Steinwender et al. (2014) primarily found M. brunneum (a species within
the M. anisopliae complex) to be most often isolated from agricultural soil in Denmark. However,
Meyling and Eilenberg (2006) found that M. anisopliae s.l. was almost absent in a single organic
agroecosystem sampled. Also, as reported in Manuscript 3, we found that in three separate
agricultural areas, M. flavoviride was the most frequently isolated species. While further sampling
is needed to confirm these observations, it is clear that Metarhizium spp. occurrence is neither
random nor ubiquitous.
20
Table 2: Metarhizium abundance and distribution studies.
Country
(latitude) Reference
Isolation
method
Samples
taken
Habitat
types
Metarhizium
isolates
obtained General Metarhizium results
Finland
(62º N)
(Vanninen,
1995)
Insect soil
baiting
590 soil
samples
from 347
sites
Forests,
agricultural
fields
92 isolates Found in lower latitudes, not affected by
cultivation
Canada
(45 ºN)
(Bidochka et
al., 2001,
Bidochka et al.,
1998)
Insect soil
baiting
266 soil
samples
from 133
sites
Natural
(forests) and
agricultural
357 isolates Most abundant entomopathogen isolated,
most frequently recovered from soil baiting at
25ºC, more often in Agricultural soil.
Genotype association with habitat, no
association with insect host.
Norway
(66ºN)
(Klingen et al.,
2002)
Insect soil
baiting
200 samples Conventional
and organic
farms
9 isolates Not commonly found. No difference between
fields and field margins.
Denmark
(55 ºN)
(Meyling and
Eilenberg,
2006)
Insect Soil
baiting
544 soil
samples over
two years
experimental
research farm
and hedgerow
134 isolates Surprisingly low occurrence of M. anisopliae
s.l.
Spain
(40ºN)
(Quesada-
Moraga et al.,
2007)
Insect soil
baiting
244 samples Natural and
cultivated areas
71 isolates Most common in cultivated areas. Preferred
soil with low clay content.
Canada
(45 ºN)
(Inglis et al.,
2008)
Insect soil
baiting and
selective media
250 soil
samples
Urban,
agricultural and
forest
250 isolates Metarhizium very widespread and diverse
however one genotype dominated.
China
(40ºN)
(Sun et al.,
2008)
Insect soil
Baiting
>2300 soil
samples
cultivated fields
and orchards
60% of soil
samples
More frequent in cultivated fields than
orchards.
South
Africa
(33ºS)
(Goble et al.,
2010)
Insect soil
baiting
288 soil
samples
Conventional
and organic
citrus farms
16 isolates No difference between organic and
conventional.
USA
(44 ºN)
(Fisher et al.,
2011)
Insect baiting
with roots
339 root
samples
Strawberries,
blueberry,
grapes, and
Christmas trees
(roots)
94 isolates Species/ plant-root association; M. brunneum
= strawberries and blueberries, M.
guizhouense = Christmas trees, and M.
robertsii = Christmas trees
Canada
(45 ºN)
(Wyrebek et al.,
2011)
Washed root
homogenate on
selective media
200 root
samples
Natural
meadows, and
forests (roots)
102 isolates Species/ plant-root association; M. brunneum
= shrubs and trees, M. guizhouense = tree
roots, and M. robertsii = grasses and
wildflowers
Denmark
(55 ºN)
(Steinwender et
al., 2014)
Insect soil
baiting
53 soil
samples
Agricultural
field and
boarding
hedgerow
123+ isolates Five genotypes of M. brunneum , 6
genotypes of M. robertsii. One M. brunneum
genotype most dominant.
Denmark
(55 ºN)
Keyser. et al.
(2015) –
Manuscript 3
Insect soil
baiting and
selective media
450 soil and
root samples
Winter wheat,
Oilseed rape
and natural
meadow
132 isolates 118 of the isolates were M. flavoviride, 13 M.
brunneum and 1 M. majus. AFLP analysis
revealed high level of diversity with the M.
flavoviride species
21
2.2.2. Abiotic factors that affect survival and growth
Three of the most important abiotic factors that affect entomopathogenic fungal
performance are: temperature, humidity, and UV-radiation. Temperature has been shown to affect
Metarhizium spp. germination, hyphal growth and infection rates (Keyser, 2010; Keyser et al.,
2014a). Growth at high and low temperatures has even been used as phenotypic traits to distinguish
certain species of Metarhizium (Fernandes et al., 2010a). Fluctuating ambient temperatures have
also been indicted as one of the primary limiting factors in field success (Foster et al., 2010; Foster
et al., 2011). Likewise, relative humidity is an important factor in determining growth, infection,
sporulation and conidial longevity (Arthurs and Thomas, 2001; Daoust and Roberts, 1983; Milner et
al., 1997; Vega et al., 2012). UV radiation, especially UV-B radiation can be highly detrimental to
conidia survival (Braga et al., 2001; Rangel et al., 2005) and even sub-lethal UV-B radiation
exposure was observed to delay conidial germination (C.A. Keyser, unpublished data). These
factors are also highly relevant in understanding the natural distribution and abundance in the field.
Some species of Metarhizium have been shown to be less cold tolerant then Beauveria spp.
(Fernandes et al., 2008), which may explain why Vanninen (1995) found Metarhizium primarily in
the southern Finland areas while Beauveria was isolated all over Finland. In one area of southern
Alberta, Canada, Inglis et al. (2008) did not find any Metarhizium isolates, they suggested that a
possible explanation for the lack of isolates is that the area surveyed was semi-arid, had very cold
winters and short summers. Bidochka et al. (2001) also observed that isolates found in forested
areas were more likely to grow at low temperatures while those found in open fields showed a
propensity for growth at higher temperatures and UV-B tolerance. Abiotic factors have strong
influence on Metarhizium population structure and biological success.
One of the challenges that has slowed BCA implementation is that they are sometimes
viewed (especially by the end user) to be one-to-one substitutions for chemical pesticides (Cook,
1993). This leads the expectation that they will have a similar shelf life, can be applied in similar
conditions, and will have a similar mode of action and time to kill. Distinguishing the differences
between applying a living organism and a chemical is vital to successfully integrate BCAs as a
viable treatment option. One of the limitations is BCA susceptibility to abiotic environmental
factors (Jaronski, 2010; Starnes et al., 1993). Future research that uncovers the mechanisms and
limitation regarding responses to abiotic factors will greatly aid in improving BCA effectiveness.
22
2.2.3 Environmental dissemination pathways
Fungi are non-motile organisms, in nature the infection propagules (e.g., conidia) of
Metarhizium are dispersed passively. Generally dispersal is thought to occur by abiotic factors such
as wind and rain (Hajek, 1997; Inglis et al., 2001; Meyling and Eilenberg, 2007). Additionally, it is
possible that insects as well as other animals may act as vectors for conidia (Meyling et al., 2006).
In Manuscript 1 we show that plants also act as a vector in aiding conidia dispersal through the soil
environment (Keyser et al., 2014b), and we speculate that this may explain why entomopathogenic
fungi have evolved to associate with plants (see also section 3). Historically, biological control
programs utilizing Metarhizium as a bio-pesticide have tried to adapt chemical-pesticide application
methods for conidial dispersal (Bateman, 1997) (Figure 3). Often formulating conidia for
application in liquid substrate or as a powder, this is then applied topically to vegetation (Booth et
al., 2000; Caudwell and Gatehouse, 1996; Griffiths and Bateman, 1997).
The effectiveness of biological control programs using Metarhizium have been inconsistent,
one factor that may be contributing to the unpredictability of Metarhizium as a BCA is the
application method or delivery system. Several studies have shown that application method does
greatly influence infection rates and environmental persistence (Farenhorst et al., 2008; Fargues et
al., 1997; Jenkins and Thomas, 1996; Kanga et al., 2003); however nearly all of these studies are
focused solely on the BCA implementation and thus neglect further investigations to understand the
underlying ecological principles contributing to increased infectivity. Understandably, designing
Figure 3. Methods of Metarhizium application. (A) In China mortar shells filled with powdered
conidia were launched above a forested area using a grenade launcher (photo by Richard Soper),
arrow indicates conidial cloud. (B) Conidia formulated in oil is applied to grasshopper infected
rangeland in Utah, USA, using a rear-mounted sprayer (photo by C. A. Keyser).
23
BCAs to fit seamlessly into insecticide application methods greatly enhances their usability and
facilitates an easy transition away from chemical pesticides. Unfortunately, without an
understanding of the natural dispersal mechanisms which contribute to efficacy, consistent
biological control results will be more a matter of luck than design. Only by investigating the
ecological principles can we hope to improve reliability. For example, in a study to evaluate the M.
brunneum biocontrol product Met52 for control of Black vine weevil, Otiorhychus sulcatus (Col.
Curculionidae) in field grown strawberries, Ansari and Butt (2013) tested several application
methods using the granular formulation of the commercial product. The methods of application
included: 1. Premixed in soil; 2. Drench application to the base of each plant; and 3. Plant roots
being dipped in an aqueous suspension of the conidial product and then planted. They found that
Black vine weevil control was more efficient when the drench application method was used.
Further studies revealed that plant roots were colonized by significantly more conidia than when the
drench method was used (Ansari and Butt, 2013). This additional information allowed them to
conclude that the conidia concentration in the rhizosphere was a key component to improving
infectivity. It was recently shown, however that M. brunneum population dynamics in the
rhizosphere may depend on adaptations to the local environmental conditions; a Norwegian M.
brunneum isolate proliferated more in strawberry rhizopheres at ambient temperatures in Norway
than an isolate similar to Met52 which originated from Austria (Klingen et al., 2015). Clearly,
understanding how infectious propagules are dispersed naturally can improve BCA performance in
the field.
3. Trophic interaction
One of the greatest challenges to fully understanding the ecology of the world we live in is
accounting for all the trophic interactions that influence an organism; however for BCAs it is
crucial that we account for these interactions as they will greatly affect efficiency. If we truly hope
to exploit biological control to suppress pests, we must view insect pathology not as one organism
acting on another, but rather as a node in a complex web of intertwined organisms and response
variables which have co-evolved and adapted to each other. This can only be achieved by studying
the organisms in combination and not individually and valuing both direct and indirect interactions.
The following sections will highlight several key interactions that occur between
Metarhizium and other important organisms (Figure 4). Some of these interactions have been well
studied (e.g., Metarhizium ↔ Insects), while for others very little is known (e.g., Metarhizium ↔
Other microorganisms ↔ Plants). The overall message that I hope to convey is the importance of
24
not only understanding the various responses associated with individual bi-trophic interactions, but
also a broad perspective of how they affect each other.
Figure 4: Illustration depicting several trophic interactions that may occur between Metarhizium
spp. and other organisms. Blue arrows represent direct interactions and orange arrows represent
indirect interactions. Diagram by C. A. Keyser
3.1 Metarhizium ↔ Insects
Among the millions of fungal species in the world, Metarhizium spp. have garnered
significant scientific and economic interest primarily because of how they interact with arthropods.
A large portion of the research involving Metarhizium centers on its use as a BCA and consequently
25
deals with its relationship to various insects. As a generalist, many Metarhizium spp. are able to
infect a wide range of hosts (Veen, 1968; Zimmermann, 1993), however the virulence can vary
considerably between different host species (Butt et al., 1992), making it necessary to test virulence
towards each insect species of interest. Furthermore, virulence varies between isolates even within
the same fungal species (Keyser, 2010), which further complicates the selection of appropriate
isolates for BCA use. The variation in virulence should not necessarily be looked at as a negative
aspect for a potential BCA; it is important to remember that a very small percentage of all insect
species are important pests – many herbivores do not cause any economic damage and some
predators, parasitoids and pollinators are important beneficial insects. The variation in host
virulence allows selection for a BCA that is highly virulent to a particular pest insect while being
less virulent to non-target insects. For example, Falagiarda (2014) observed that a commercial
isolate of M. brunneum, the same isolate used in Manuscript 1 and 2, to be highly virulent to the
grain-pest insect T. molitor but having very low virulence to the beneficial coleopteran, Atheta
coriaria (Figure 5). Her results suggest that this isolate of M. brunneum could therefore be
considered a “low risk” substance under the proposed EU legislation. Further testing might
investigate whether there is an additive or synergistic effect when M. brunneum and A. coriaria are
used in combination as BCAs.
Many factors from both the host and the pathogen contribute to virulence; it is a give and
take relationship with each attempting to maximize their own fitness. Each step in the infection
process (see Figure 2) is controlled by specific cues and responses which determine the success of
the infection. For example, ambient pH levels on and around the insect cuticle regulate the
secretion of proteolytic and chitinolytic enzymes which aid in the penetration process (St. Leger et
al., 1998). In response to the infections, insects have evolved different immune responses, both
physiological and behavioral, to mitigate the pathogenic effects. For example, after exposure to
Metarhizium there are changes in insect biochemistry, including melanization of the cuticle and
antimicrobial defenses of the haemolymph (Dubovskiy et al., 2013; Gillespie et al., 2000). Also,
behavioral adaptations have been noted, including: increased grooming among infected ants and
termites (Hughes et al., 2002; Qiu et al., 2014; Yanagawa et al., 2008), or basking in the sun in
order to elicit a “behavioral fever” which restricts or kills the infecting pathogen (Blanford and
Thomas, 2001; Kemp, 1986; Ouedraogo et al., 2004). These immune responses can severely limit
the effects of a pathogen; however some species of Metarhizium have developed traits to more
efficiently overcome insect immunity. For example, to increase virulence many of the generalist
26
species of Metarhizium produce cyclic peptides known as destruxins (Roberts and St. Leger, 2004);
these insecticidal toxins cause muscle paralysis and suppress immunoresponses in the host (Pedras
et al., 2002; Roberts, 1981).
These examples represent only a small fraction of the many interactions that take place
between insects and Metarhizium; however, they begin to illustrate the complex dance that occurs
between host and pathogen. The level of complexity can be even greater when multiple trophic
levels of insects are considered. For example, Rännbäck et al. (2015) observed a reduction in
parasitoid egg laying when hosts were infected with Metarhizium. While the complexity of these
interactions can seem overwhelming, it is highly relevant that we understand what is occurring if we
hope to successfully implement BCA to control pest insects.
3.2 Metarhizium ↔ Plants
Most land plants form symbiotic relationships with soil fungi (van der Heijden et al., 1998).
It is therefore not surprising that several species of Metarhizium have been observed to interact with
some plants. A fungal endophyte is defined as an asymptomatic plant-colonizing fungus that lives a
portion of its life cycle inside the plant (Behie and Bidochka, 2014a). Metarhizium spp. have a dual
life cycle, persisting in the environment both as an insect pathogen and as a facultative saprophyte
(Wang et al., 2005). While soil has long been considered a reservoir for naturally occurring
Metarhizium, it has not been clear whether the recovered isolates were from dormant conidia in the
soil or hyphae actively growing on plant material (St Leger, 2008). Metarhizium has been viewed
for many years as only an insect pathogen – the realization that it also actively interacts with plants
a b
Figure 5. Bioassay survival curves after exposure to Metarhizium brunneum or Beauveria bassiana of
(a) Tenebrio molitor larvae and (b) Atheta coriaria after exposure to M. brunneum (Falagiarda, 2014).
27
is both novel and exciting for the field of Metarhizium research. For this reason and because it is of
special interest to the research contained in this thesis, I will give a more comprehensive outline of
the current status on Metarhizium ↔ Plants interactions.
As mentioned in section 2.2.1, specific species of Metarhizium have been shown to associate
closely with different plants. In a Canadian-field survey Wyrebek et al. (2011) observed that M.
robertsii was the only species associating with grass roots, while M. guizhouense tended to
associate with roots of trees and M. brunneum was found in the roots of shrubs and trees. In a
similar study Fisher et al. (2011) found that in Oregon, M. brunneum associated strongly with roots
of Strawberry and Blueberry plants while M. robertsii and M. guizhouense were isolated
predominantly from Christmas tree roots. The plant specificity observed in these two studies could
indicate a history of co-evolution between Metarhizium and certain plants.
The rhizosphere is a region of soil where plant-root exudate influences soil microorganisms.
M. anisopliae s.s was first discovered to be a rhizosphere competent isolate by Hu and St Leger
(2002) in a field study designed to evaluate the fate of the BCA after application. In a cabbage field
soil samples were collected 4-5 cm from the plant as well as directly next to the taproot. The
researchers observed that even after a year the population of M. anisopliae s.s. remained high in the
rhizosphere area while it declined over time in the bulk soil (Hu and St Leger, 2002). M. brunneum
was also observed to be rhizosphere competent in soilless potting media (Bruck, 2005) and several
Metarhizium spp. isolates have been shown to germinate and grow in root exudates (Pava-Ripoll et
al., 2011; Wang et al., 2005). Additionally, specialized genes which become active when
Metarhizium interacts with plants or plant compounds have been identified; like, the Mad2 gene
which is involved in adhesion to plants during colonization (Barelli et al., 2011; Wang and St
Leger, 2007), or the Mrt and Mlrnv genes which transport oligosaccharides found in root exudate
(Fang and St Leger, 2010; Liao et al., 2013). These studies clearly show that Metarhizium is more
than just an entomopathogen but that it has evolved to propagate while interacting with plants.
In an effort to determine if Metarhizium had a localized plant-tissue preference when
associating with plants in the field Behie et al. (2015) performed a field survey of grasses and forbs
in Canada. They found that endophytic Metarhizium spp. (95% of which were M. robertsii)
associations were exclusively with plant roots and not with hypocotyl or the stem and leafs of a
plant. In vitro laboratory studies with Haricot beans also showed endophytic root preference of
Metarhizium spp. (Behie et al., 2015). However, Batta (2013) was able to re-isolate M. anisopliae
s.s from untreated leaves, petioles and stems of the oilseed rape plants when other leaves were
28
sprayed with a high dose of M. anisopliae. Furthermore, Golo et al. (2014) observed that cowpea
and cucumber plants resulting from M. robertsii or M. acridum inoculated seeds had endophytic
association in both the roots and leaves of the plants after 12 days. They further observed the
production of destruxins by M. robertsii in the cowpea plants.
Akello and Sikora (2012) showed that M. anisopliae s.s. could live for at least a month
inside V. faba roots as an endophyte after seeds were soaked for 4 hours in a conidial suspension.
Interestingly, in a similar study Akutse et al. (2013) did not observe M. anisopliae s.s. colonizing
any part of V. faba plant when seeds were inoculated with a conidial suspension even though the
same fungal isolate and plant were used as well as similar methods as Akello and Sikora (2012);
although in addition to treating the seeds with conidia (as was done in Manuscript 1), Akello and
Sikora (2012) also drenched soil with a conidial suspension, the additional load of conidia in the
soil might explain why an endophytic interaction was observed in one and not the other.
Further implications regarding the importance of Metarhizium ↔ Plants interactions were
suggested when, in a proof of concept study, Behie et al. (2012) showed that nitrogen from a M.
robertsii-infected insect can be transferred to a plant via an endophytic hyphal connection. In a
follow-up, in-depth study Behie and Bidochka (2014b) tested the insect-derived nitrogen-
transferring abilities of five species of Metarhizium [i.e., M. acridum, M. brunneum, M. pemphigi
(=flavoviride var. pemphigi), M. guizhouense, and M. robertsii] to four types of plants (haricot
bean, soybean, switchgrass and wheat). They showed that all five Metarhizium species had the
capacity to transfer nitrogen to plants, although in varying degrees. In addition, they showed that
nitrogen was also transferred in the field, despite other competing microorganisms (Behie and
Bidochka, 2014b).
Metarhizium-plant associations have also been observed to promote plant growth. Sasan
and Bidochka (2012) observed that after inoculation with M. robertsii, switchgrass and haricot bean
plants both had increased root hair growth. Also, increased plant growth has been observed in
several agricultural crops, including soy bean (Khan et al., 2012), tomato (Elena et al., 2011), and
corn (Liao et al., 2014). In soy bean, Khan et al. (2012) observed endophytic interaction with M.
anisopliae s.l. increased biomass, chlorophyll contents, transpiration rate, photosynthetic rates and
leaf area compared to untreated control plants. While wild type M. robertsii, M. brunneum and M.
anisopliae s.s. increased many aspects of corn growth, Liao et al. (2014) observed that when
Metarhizium genes that are associated with either adhesion to the plant or the utilization of plant
exudates were knocked out, no plant growth promotion was observed.
29
The ecological significances of delving in to the complex systems of the Metarhizium ↔
Plants interactions are clear. Furthermore, these fundamental research studies also have important
implications for biological control. Nearly all the studies so far have described either beneficial or
neutral effects on the plant resulting from Metarhizium associations. Based on the results of these
studies, Metarhizium based BCA may do more than just protect crops from insect pests, they may
also aid in nutrient acquisitions and plant-growth promotion. However, more research is necessary
to illuminate the mechanisms involved in these processes and whether the benefits observed in the
laboratory are also seen in the field.
Thus far, the majority of the published studies involving Metarhizium ↔ Plants interaction
have focused on responses exhibited by the plant. This is most likely because traditionally
underlying justification for Metarhizium research has been in the interest of plant protection,
however, research into beneficial or deleterious effects to the fungal organism should also be of
interest. In Manuscript 1, after demonstrating that species of Metarhizium will disperse along a
growing root though soil, I suggest that a possible benefit that Metarhizium derives from associating
with plants is mobility and proximity to potential hosts. It is likely that the fungus is also affected
in other ways while interacting with plants so it is important that questions like, “Why would a
Metarhizium organism give up a limited resource such as nitrogen and what does it receive in
return”, continue to be investigated. Metarhizium ↔ Plants interactions are an important part of the
future of biological control research.
3.3 Metarhizium ↔ Other microorganisms
Very few studies have been conducted to investigate the interaction between Metarhizium
and other microorganisms. This is an important aspect of Metarhizium ecology that should be
addressed more thoroughly. Metarhizium spp. are abundantly found in the soil environment
(Tkaczuk et al., 2014). It is likely that an actively growing saprophyte will have developed
antimicrobial strategies to survive and compete in an environment teaming with natural
microorganisms. I observed evidence of this while performing isolation on nutrient agar.
Inadvertently, a petri plate which had been inoculated with a new isolate of M. flavoviride also
became contaminated with a fast growing unknown fungus, however a very clear inhibition zone
was present between the M. flavoviride and the contaminate (Figure 6a). Additionally, while
developing the methodology for Manuscript 2, media plates with M. brunneum, M. robertsii and M.
flavoviride were also inoculated with plugs of Fusarium culmorum. A small zone of inhibition was
observed on the plates with M. brunneum and M. robertsii and a much larger zone was present on
30
Figure 6: Inhibition zones observed between Metarhizium spp. isolates and other fungi on PDAY
media. (A) M. flavoviride isolate from bait insect on petri plate with un-known fungal contaminant.
(B) M. brunneum, M. robertsii, and M. flavoviride on petri plate with Fusarium culmorum.
those with M. flavoviride (Figure 6b). Sasan and Bidochka (2013) also observed a zone of
inhibition when M. robertsii and Fusarium solani were cultured on the same petri plate and they
noted a significant reduction of the colony size of the F. solani. Furthermore, Sasan and Bidochka
(2013) showed that liquid media, which had had M. robertsii growing in it but was then passed
through a filter to remove all fungal material suppressed F. solani germination. They suggested that
this indicates that M. robertsii secretes an anti-fungal compound that inhibits F. solani growth.
Krauss et al. (2004) tested the interaction between several entomopathogenic fungi,
including two strains of M. anisopliae s.l., and mycoparasitic fungi by completely colonizing a petri
plate with the entomopathogen and then placing an agar plug of the mycoparasite on top of the
entomopathogen colony. They claimed that because the mycoparasite was not in contact with the
media any growth indicated it was receiving nutrients from the fungus and not the media. They
found that of the isolates they tested M. anisopliae s.l. was the most susceptible to mycroparasitism
and allowed growth of all the mycoparasites tested including Clonostachys byssicola, C. rosea and
Lecanicillium lecanii (Krauss et al., 2004). These two studies demonstrate that Metarhizium spp.
are both affected by and effectors of other microorganisms. Further studies with regard to the
mechanisms involved in these interactions would greatly expand our understanding of the ecology
of Metarhizium spp. in the field and identify potential benefits and challenges to their use as BCAs.
31
3.4 Multi-trophic interactions with Metarhizium
Each additional trophic level included in a study greatly increases the size and complexity of
the experiment necessary to evaluate all the variable permutations. It is therefore not surprising that
most studies focus on bi-trophic interactions. Nevertheless, multi-trophic interactions which
include three or more levels are necessary to both better grasp what occurs in nature and provide
greater predictive power to BCA employment. Generally bi-trophic interactions are concerned with
direct effects while multi-trophic interactions must account for both direct and indirect effects. In
the following section I will review several Metarhizium studies which evaluate multi-trophic
interactions. The trophic levels that I focus on are those of different types of organisms (i.e., other
microorganisms, insect and plants); however multi-trophic interactions that involve multiple species
of the same type of organism (e.g., insect ↔ insect ↔ microorganism) – as was seen above in the
study by Rännbäck et al. (2015) in which interaction between a parasitoid insect as pest insect and
M. brunneum were evaluated – should not be neglected.
3.4.1 Metarhizium ↔ Other microorganisms ↔ Insects
There are a few examples of tri-trophic interaction involving Metarhizium, other microbes
and insects; generally they are geared towards evaluating how the biological control capacity of
Metarhizium is affected when other microorganisms are present. Hughes and Boomsma (2004)
found that the normally avirulent, opportunistic fungal pathogen Aspergillus flavus would out-
compete a highly virulent isolate of M. anisopliae s.l. when applied as a mixture on a leaf-cutting
ant host. They explained that alone A. flavus is generally unable to overcome the host immune
defenses, however, when coupled with a second pathogen like Metarhizium, which is more adept at
suppressing the immune responses, the avirulent fungus is then able to grow much faster and better
utilize the pilfered resources. Geetha et al. (2012) demonstrated that co-application of multiple
fungi on the same insect host negatively influence the virulence and sporulation of M. anisopliae s.l.
Also, Krauss et al. (2004) found that when applied in combination with mycoparasitic fungi on
three different species of insects, the virulence of M. anisopliae s.l. was not significantly affected,
despite the authors having found that M. anisopliae s.l. was highly susceptible to mycroparasitism.
In Manuscript 2, an insect bioassay was conducted in which Tenebrio molitor larvae were
inoculated with one of three species of Metarhizium (M. brunneum, M. flavoviride s.s., or M.
robertsii) combined with either the mycoparasite Clonostachys rosea or the plant pathogen
Fusarium culmorum or both. We observed that, when compared to the individual treatments, for
some of the combined treatments there was a slight reduction in virulence; however, in general,
32
virulence remained similar to the individual Metarhizium treatments. Based on this study and the
others mentioned it is obvious that interaction which affect the BCA performance of Metarhizium
occur when other microorganisms are present. Since microbes are always present in the field these
interactions are highly relevant – especially those with other BCA like C. rosea which are also often
applied for crop protection.
3.4.2 Metarhizium ↔ Other microorganisms ↔ Plants
In light of the recent emphasis placed on Metarhizium ↔ plant interactions, the tri-trophic
interaction between Metarhizium, other microorganisms and plants are highly relevant. There have
been very few studies which have looked at these interactions. Sasan and Bidochka (2013) found
Haricot bean seeds had higher germination when in the presence of M. robertsii and F. solani than
when only F. solani was present. The resulting plants also showed significantly less F. solani
infection symptoms when M. robertsii was also present. In Manuscript 2 we also evaluate the
interactions between Metarhizium (M. brunneum or M. flavoviride), C. rosea, F. culmorum and
Winter wheat. We observed that the presence of either of the Metarhizium species did not reduce
the F. culmorum infection or hinder the biocontrol efficacy of C. rosea. The two beneficial BCAs
may therefore potentially be used in concert against both insect pests and plant pathogens without
obstructing each other’s effects. But no additive nor synergistic effects should be expected
(Manuscript 2). Several important differences exist between Manuscript 2 and the study by Sasan
and Bidochka (2013) which might account for the different observations. For example, the species
of Metarhizium and Fusarium that were used are different, as was the plant species and inoculation
method; also Sasan and Bidochka (2013) allowed an establishment period for M. robertsii and F.
solani in the soil before introducing the bean seeds, where as in Manuscript 2, un-germinated seeds
were exposed to F. culmorum first and then Metarhizium spp. before germination.
3.4.3 Metarhizium ↔ Plants ↔ Insects
The indirect effects to plants are nearly always implied in Metarhizium ↔ insect studies
which have a biological control aspect to them. Nevertheless these indirect effects (i.e. increased
plant productivity caused by reduced effects of pest insects after BCA application) are often not
measured. One example is a study by Kabaluk and Ericsson (2007) in which they found that corn
seeds treated with the M. brunneum BCA F52 (called M. anisopliae by the authors) resulted in an
increased yield in comparison to untreated control plants when planted in fields with wireworm. On
the other hand, indirect effects may also be observed to occur to the insect when the plant interacts
with Metarhizium. For example, Akello and Sikora (2012) showed that aphids feeding on V. faba
33
plants which had been treated as seeds with different fungi including Metarhizium spp. had reduced
survival, offspring fitness, development and fecundity compared to untreated control. They
demonstrated that Metarhizium spp. isolates along with other endophytic isolates indirectly
protected the plant from aphid damage. However, the mechanisms behind this remain unknown.
3.4.4 Metarhizium ↔ Other microorganisms ↔ Plants ↔ Insects
The last type of interaction that I would like to mention is that which involves four or more
trophic levels. Very few studies have attempted to incorporate so many different trophic levels; in
fact I was unable to identify any other studies, involving Metarhizium, which evaluated response
variables for four trophic levels. One example, which uses another entomopathogenic fungus (B.
bassiana) tested whether insect pollinators could be utilized as delivery vehicles to vector two
BCAs to plants in order to control both an insect pest and a plant pathogen (Kapongo et al., 2008).
They found in laboratory greenhouse trials that the bees successfully vectored the BCAs to the
flowering plants and they observed a reduction in both plant pathogen and insect pest occurrence.
Manuscript 2 also describes a system with up to five trophic levels are evaluated. F.
culmorum infected seeds were treated with C. rosea, a mycoparasitic fungus which can be used as a
BCA against plant disease, and two Metarhizium spp. isolates. After the plants had grown for 14
days the roots were feed to T. molitor larvae which were then monitored for mortality and fungal
disease. There were two main response variables assessed in this study, plant disease incidence and
insect mortality. Combination effects could be assessed by comparing the data of the combination
treatments with those of the non-combined treatments.
Prior to this experiment we had several hypotheses on what combination effects we might
expect. Metarhizium has been shown to have antifungal properties (Sasan and Bidochka, 2013) and
therefore might aid in Fusarium disease suppression. As a potential mycoparasite of Metarhizium
spp. (Krauss et al., 2004), C. rosea may severely reduce the ability of Metarhzium to disperse along
the root and subsequently infect the larvae. On the other hand, isolates of Fusarium spp. and C.
rosea have been observed to be pathogenic to insects (Teetor-Barsch and Roberts, 1983; Vega et al.,
2008), and may therefore have an additive effect on insect virulence when in combination with
Metarhizium spp. As reported in Manuscript 2, no effect on plant-disease reduction by C. rosea
was observed when Metarhizium was added, however when C. rosea was present, alone or
combined with Metarhizium spp. there was little to no disease symptoms in the plants; the high
efficiency of the C. rosea treatment may however have masked any positive effects of Metarhizium.
In most of the treatments there was a significant reduction in virulence to the insect when the seeds
34
had been treated with Metarhizium spp. combined with another fungus. This indicates that there is
an indirect effect on insect virulence when other fungi are present. Future studies should evaluate if
this effect is due to direct interaction between the combined fungi or due to an indirect effect like
resource competition. It is clear however, that multi-trophic interactions are important and may
yield unexpected results.
4. Methodology
In fulfilling the research requirements of this thesis it was necessary that I develop and test
various experimental methodologies and expand my analytical skills. The following section will
briefly summarize and describe a few important methodological techniques I worked with.
4.1 Selective media
Selective media is often used to assist in the isolation of entomopathogenic fungi. The basic
principle of selective media is to provide a substrate that allows the growth of a desired organism
while discouraging the growth of others. This can be accomplished in several ways, for example,
many bacteria are inhibited by low pH levels while fungi are not. Also the nutrient composition of
many growth media favor particular organisms, e.g., Potato dextrose media and Sabouraud dextrose
media are better suited for fungal growth than bacterial. While usually not included as a selective
medium, insects used for the soil baiting method (Zimmermann, 1986) are essentially a “ready-
made” selective medium that screens out any non-pathogenic organisms and selects for those that
can infect the host.
Media are often amended with more specific growth inhibiting components to further select
for desired organisms. Antibiotics, like streptomycin or chloramphenicol, can be added to help to
reduce contaminating bacteria. Contaminating fungi are more problematic. For many species of
Beauveria and Metarhizium some fungicides, like dodine or cyclohexamide, in low concentration
help to eliminate the fast growing contaminants like Rhizopus or Trichoderma while allowing
Metarhizium and Beauveria growth (Fernandes et al., 2010b; Rangel et al., 2010) (Figure 7). Many
different media have been developed for the isolating of Metarhizium spp.; Table 3 lists some of the
major component combinations that have been found effective as well as the studies in which they
were presented.
35
Figure 7: Selective media test with several types of media (columns) to evaluate effectivness at
supressing naturally occuring microbes while allowing the growth of entomopathogenic fungi.
Both natural and Metarhizium “spiked soil” was used (rows). Soil was diluted in ddH2O and then
200µl was pipetted onto each plate. Plate incubated for 21 days at ~21ºC.
36
Table 3: Selective media ingredients that have been observed to be effective for the isolation of
Metarhizium spp isolates from soil. All concentratoins represent grams/liter unless otherwise
specified.
Media Ingredients
Veen&Ferron,(1966)
Beilharzetal.,(1982)
Chaseetal.,(1986)
Liuetal.,(1993)
Shimazu&Sato,(1996)
Strasseretal.,(1997)
Hughesetal.,(2004)
Shinetal.,(2010)
Rengaletal.,(2010)
Fernandesetal.,(2010b)
Wyrebeketal.,(2011)
Rocha&Luz,(2012)
Steinwender,(2013)
Keyseretal.,(2014b)
SelectiveIngredients
Dodine 0.065 0.46* 0.01* 1ml* 0.1 0.05 0.02 0.325 0.300 0.29 0.2
Cycloheximide 0.25 0.25 0.05 0.25 0.5 0.06 0.25
Chloramphenicol 0.5 0.5 0.05 0.5 0.2 0.014 0.5
Thiabendazole 0.001
Chlortetracycline 0.005 0.005
Copper chloride
(CuCl2) 0.02
Gentamicin
Streptomycin 0.6 0.1 1
Penicillin 0.4
Tetracycline 0.05
Crystal violet 0.01 0.01 0.01 0.01
NutrientIngredients
PDA X X X X
SDA 30% X X X
Yeast extract 1 1 X
Glucose 10 10 20
Peptone 10 10 10
Oxgall 15 15
Oatmeal infusion 20 20 20
Agar 35 20 20 35 18 20
pH 10+ 6.9 6.9
37
4.2 Bioassay statistics
A bioassay is a scientific study with the aim to determine the biological activity or potency
of a substance by testing its effect on the growth of an organism. In insect pathology an infection
bioassay is a study which evaluates the infectivity of a particular pathogen on a host insect. The
ability to cause infection in insects is one of the most basic and interesting traits of
entomopathogenic fungi like Metarhizium spp. It is for this reason that many studies of these fungi,
among other things include infection bioassays. Infection bioassays are often designed to evaluate
pathogenicity, the ability to cause infection, and virulence, the killing power of a pathogen.
The most common type of studies that include infection bioassays are those aimed at
determining the biological control potential of a pathogen for a particular host. However, infection
bioassays are regularly included in other types of studies as a phenotypic trait that can be used to
compare different genotypes to each other or measure changes within the same isolate after a
mutation or stress. For example Jin et al. (2012) used infection bioassays to show the effects of the
Hog1-type mitogen-activated protein kinase gene in M. acridum, or Lopes et al. (2013) who while
performing a diversity study of Beauveria spp. and Metarhizium spp. in banana fields included a
bioassay and interpreted low virulence to explain the low occurrence of epizootics. For any insect
pathologist an understanding of how to appropriately design and execute an infection bioassay is a
vital skill.
4.2.1 Experimental design
There are several factors that influence the outcome of an infection bioassay; however, the
two most important factors are time and dose. Therefore, experiments are usually designed around
these factors either as dose-mortality or time-mortality experiments. In a dose-mortality experiment
the pathogen dose or number of conidia administered to each host is varied while the time at which
the response is evaluated is kept constant. Often in a dose-mortality experiment the response is
reported as a value of LD50 or LD90 (dose at which 50% or 90% of individuals exhibit the desired
response, i.e., mortality) for a particular treatment. The most important factors are sample size and
dose range. It has been recommended that sample size of 120 to 240 insects is necessary to obtain a
reliable response (Robertson et al., 1984). Ideally the dose range will include three to eight doses
that result in 25 to 75% responses at the time of observations (Robertson et al., 1984), however, the
experiment can be checked at several time periods and then the most appropriate observation time
for the analysis can be selected afterwards.
38
In a time-mortality experiment, dose is kept constant (or multiple doses are considered
separately) and the response is measured over a period of time, often results are reported as LT50 or
LT90. To avoid data being correlated in a time-mortality experiment, either different groups of
insects must be used for each time measurement so that the data are not correlated or the method of
analysis must allow for correlated data. The bioassay performed in Manuscript 1 and 2 were
designed as time-mortality experiments with the data for each dose being analyzed separately.
I conducted a survey of 30 randomly selected scientific publications selected from a Google
Scholar search with the key words “Metarhizium” + “Bioassay” and limited to papers published
between Jan 2010 and April 2014. Of these 30 papers, 15 included time-mortality bioassay designs,
7 dose-mortality experiments and 8 with both (Table 4).
4.2.2 Types of Statistical Analyses
4.2.2.1 Abbot correction: Random mortality not related to a treatment but rather
experimental conditions being tested in an infection bioassay can be adjusted for in the analysis
using the control group. The most common method employed in insect pathology is the Abbot’s
formula (Abbot, 1925). The following formula is used to estimate the percentage of insects killed
by a particular treatment: 𝑃 = (
𝐶−𝑇
𝐶
) ∗ 100. Where P is the estimated percentage of insect mortality
due to treatment, C is the percentage dead in the control and T is the percentage of dead in
treatment. A few considerations before implementing an Abbot adjustment include: First, the
Abbot formula assumes pathogenicity of a treatment, and therefore should not be used if the
researcher wishes to determine if a particular treatment differs from the control. Second, if a
control has greater mortality than a treatment an Abbot correction will result in negative percent
mortality and is not appropriate. Lastly, an Abbot correction cannot be used when performing a
survival analysis because the binomial data used for the analyzed is of the individual and not the
group.
4.2.2.2 Probit analysis: Infection bioassays are typically analyzed in one of two ways, the
first is using a probit analysis model. A probit analysis is most appropriate for dose-response
designs but can be used in a time-response design if different groups of insects are used for each
time measurement otherwise the data are correlated and the model is not valid; however, examples
have been shown where correlated data can be analyzed using a probit transformation of proportion
of insects killed (Goettel and Inglis, 1997).
Probit analysis is a type of regression used to analyze binomial response variables. It
transforms the sigmoid dose-response curve to a straight line that can then be analyzed by linear
39
regression analysis. Probit analysis was first published in 1934 by Chester Bliss, an entomologist
working with pesticides and their effectiveness for pest control (Bliss, 1934). Later in 1952 David
Finney, a professor of statistics, published a book called Probit Analysis (Finney, 1952). Probit
analysis continues to be one of the preferred statistical methods in understanding dose-response
relationships.
4.2.2.3 Survival analysis: The survival analysis is historically one of the oldest fields of
statistics dating as far back as the 17th
century (Aalen et al., 2009). As suggested in the name, a
survival analysis traditionally is used to analyze survival or death rates, however today it is often
used in engineering to predict failure times of machines. A survival analysis is used to analyze data
in which time duration until an event is of interest occurs. The response is often referred to as a
failure time, survival time, or event time. One of the primary advantages of using a survival model
is that it has the ability to account for censored data, most statistical models don’t. Censored data is
data in which the response is unknown in the window of observation, for example, if an experiment
ends before all the individuals are dead those individuals are considered censored. An ordinary
linear regression model cannot handle censored data. Despite its early beginnings, it was not until
the late 1950s that the field of survival analysis was significantly advanced. A publication in 1958
by Kaplan and Meier which allowed a survival curve estimation was presented and then later in
1972 Cox published a method of comparing survival curves (Aalen et al., 2009). Both of these
modeling approaches advanced the usefulness and the applicability of survival analysis to the
current state. In Manuscripts 1 and 2 bioassays were performed. The experimental design of these
bioassays in each study was based on a survival analyses. A Cox proportional hazard model was
used to compare the survival curves between the various treatments. This model was most
appropriate for the type of data that I collected because it reduced the number of dose-treatments
needed, allowed for the same batch of insects to be checked daily and accounted for censored data
(insects surviving beyond the time frame of the experiment).
Of the 30 studies surveyed included in table 4, 13 used a probit analysis, 8 a survival
analysis, 3 used both, and 6 used either another type of analysis or did not specify.
40
Table 4: A survey of experimental design and statistical methods used in 30 studies; selected from
a Google Scholer search with key words “Metarhizium + Bioassay”, limited to 2010-2014.
Time –
Response
Method of
analysis
Host Treatment method
Gao et al.
(2013)
Survival analysis Bombyx mori Larvae immersed in 5 ×10
6
suspension for 1 min or injected
with 10µl of 10
6
conidia/ml. 15 insect (×3) checked every
12h.
Garza-
Hernandez et
al. (2013)
Survival analysis Aedes aegypti Infection of mosquitos co-infected with virus. 25 adults
exposed to filter paper impregnated with 1 × 10
8
conidia/ml.
Checked daily until all dead.
Goble et al.
(2014)
Survival analysis Anoplophora
glabripennis
For 36 individuals the ventral surfaces were pressed on to
two agar plugs with conidia for 5 sec. Also, for 24 beetles
were submerged for 10 sec in a 15ml suspension of 1 × 10
7
conidia/ml. Checked daily for 50 days.
Howard et al.
(2010)
Survival analysis Anopheles
gambiae
25 adult mosquitos were placed in a tube with netting that
had been treated in a fungus. Checked daily until all fungal
treated individuals died.
Jin et al. (2012) Method not
specified
Locusta
migratoria
Adults were either: dipped up to the head-thorax junction in a
soybean-oil suspension containing 1×10
7
conidia/ml; or,
injected with 5 µl of a 1 × 10
6
conidia/ml in the haemocoel
cavity through. Twenty insect per rep, checked every 12h.
Lopes et al.
(2013)
Survival analysis Cosmopolites
sordidus
Adults were submerged in conidial treatment of 1 × 10
8
conidia/ml for 90 sec. Sixteen-eighteen insects per
treatments, mortality assessed every other day for 14 or 16
days.
Orduno-Cruz et
al. (2011)
Logistic
regression
Metamasius
spinolae
Twenty adults were immersed in 500ml suspension with 1
×10
8
conidia/ml for 10 second. Checked for 41 days.
Peng et al.
(2011)
Survival analysis Anoplophora
glabripennis
Females exposed to fiber bands impregnated with fungi.
Males held on conidia-covered surface for 30 seconds.
Checked daily for 50 days.
Quinelato et al.
(2012)
Probit analysis &
non-parametric
Kruskal–Wallis
test
Rhipicephalus
microplus
Larvae (~100/tube) were immersed in 1.2 × 10
8
, 10
7
, 10
6
and
10
5
conidia/ml suspensions for 3 min. The suspension was
absorbed out of test tube, mortality checked every 5 days for
30 days. Mortality was estimated as a percentage.
Quintela et al.
(2013)
Probit analysis
and Survival
analysis
Tibraca
limbativentris
Adults were inoculated with 10µl of 5 × 10
7
conidia/mL
suspension on the dorsal region. Checked daily for 12 days.
Reyes-
Villanueva et al.
(2011)
Survival analysis Aedes aegypti Females exposed to filter paper impregnated with 6 × 10
8
conidia/ml for 48 hours and checked for survival. Also,
females exposed to exposed males that had been exposed
to impregnated filter paper for 48h and watched for survival.
San Andrés et
al. (2014)
Probit analysis Ceratitis
capitata
Adults exposed for 30s to an “infective dish”, a dish with
3.1 × 10
8
conidia spread uniformly over the dish. 10 host per
treatment checked daily for 10 days.
Tavassoli et al.
(2012)
General linear
model and
repeated measure
analysis
Ornithodoros
lahorensis
Eggs, larvae, and adult ticks were immersed in either 1 × 10
5
or 1 × 10
7
conidia/ml for 5s. Survival was checked every 3
days for 21days.
Wang et al.
(2012)
Survival analysis Bombyx mori &
Locusta
migratoria
Silkworms injected with 10 μL of suspension of 5 × 10
5
conidia/ml, locust injected with 10 µl of 1 × 10
7
. Ten insects
in each repetition. Mortality checked every 12 hours.
Wang et al.
(2014)
Probit analysis Galleria
mellonella &
Tenebrio
molitor
Each individual larva (30 per repetition) was sprayed with 1
ml of suspension of 10
7
conidia/ml. Monitored daily for
mortality.
41
Dose –
Response
Ansari et al.
(2010)
Probit analysis Culicoides
nubeculosus
Eggs suspended in 10
8
conidia/ml until hatch. Larvae
suspended in 10
4
, 10
5
, 10
6
, 10
7
, or 10
8
conidia/ml – checked
for 3 days.
Contreras et al.
(2014)
Probit analysis Tuta absoluta Eight pupae suspended in one of six concentrations (0.34-
11.15 × 10
9
viable conidia per liter), checked every 3 days
for up to 3 weeks.
Leemon and
Jonsson (2012)
Probit analysis Rhipicephalus
microplus &
Lucilia cuprina
Six fungal concentrations ranging from 1 × 10
9
to
1 × 10
4
conidia/ml. Ticks were either topically inoculated with
2µl or submerged in inoculum. Blowflies were either topically
treated with 2µl suspension or fungi was mixed with food
source. 20 insects per treatment, checked for 10 days.
Luz et al.
(2011)
Probit analysis Anopheles
gambiae and A.
arabiensis
Eggs were topically treated with 5 × 10
6
conidia/cm
2
. Also,
eggs in soil treated with oil formulation of 10
5
, 3.3 × 10
5
, 10
6
,
3.3 × 10
6
, and 10
7
conidia/cm
2
. Checked every 5 d for 30 d.
Nussenbaum
and Lecuona
(2012)
Probit analysis Anthonomus
grandis
Lots of isolates tested by submerging adults for 15 sec in a 5
× 10
8
conidia/ml, checked daily for 20 d. Also, 40 individuals
immersed treatments of: 1 × 10
6
, 5 × 10
6
, 1 × 10
7
, 5 × 10
7
,
1 × 10
8
and 5 × 10
8
conidia/ml; checked daily for 15 d.
Zayed et al.
(2013)
Probit analysis Phlebotomus
papatasi
Suspension of 1 × 10
6
, 5 × 10
6
, 1 × 10
7
, 5 × 10
7
, 1 × 10
8
, and
5 × 108 conidia/ml prepared. 0.5g of each was mixed with
0.15g finely ground larval diet. 10 larvae added to each vile.
9 replicates. Mortality based on failure for adult to emerge.
Behle and
Jackson (2014)
Method not
specified
Alphitobius
diaperinus
Larvae were exposed to treated soil. Conidial treatment
ranged from 1.69 × 10
7
to 2.08 × 10
5
conidia/ml. 30 larvae
per dosage was used. Mortality was monitored for 14 days.
Both Dose and Time Response
Jin et al. (2011) Method not
specified
Nilaparvata
lugens
Thirty-forty nymphs were sprayed at 5 different rates on with
one of three fungal concentrations (2 × 10
8
, 2 × 10
7
and 2 ×
10
6
conidia/mL). Checked daily.
Kirubakaran et
al. (2013)
Probit analysis
and ANOVA
Cnaphalocrocis
medinalis
Leaves sprayed with 5ml of 1 × 10
3
, 1 × 10
4
, 1 × 10
5
, 1 × 10
6
,
1 × 10
7
and 1 × 10
8
conidia/ml in water or oil formulation, 20
larvae exposed to treatments, checked daily for 8 days.
Maldonado-
Blanco et al.
(2013)
Probit analysis Aedes aegypti Submersion of 25 larvae in fungal concentrations of 1 × 10
5
,
3 × 10
5
, 5 × 10
5
, 8 × 10
5
or 1 × 10
6
submerged spores/ml.
Insects checked daily for 5 days.
Mishra et al.
(2011)
Probit analysis Musca
domestica
One ml fungal suspension of 10
3
, 10
5
, 10
6
, 10
7
, and
10
9
conidia/ml were applied to fly diet. Checked daily for 5
days. Also done in an arena setup.
Ortiz-Urquiza et
al. (2013)
Probit analysis
and Survival
analysis
Ceratitis
capitata & G.
mellenella
Adult flies were treated with 1 of 6 spore concentrations from
10
3
- 10
8
conidia/ml by spraying. Galleria larvae treated by
injecting 8µl of suspension. 10 insect per treatment.
Shan and Feng
(2010).
Method not
specified
Myzus persicae A leaf with 40 adult aphids was sprayed with 1 ml
suspension, 1 × 10
6
, 1 × 10
7
and 1 × 10
8
conidia/ml),
mortality was checked daily for 8 days.
Vázquez-
Martínez et al.
(2013)
Probit analysis Anopheles
albimanus
Larvae were placed in cups with 200 ml of 2.6×10
7
conidia/mL. Checked daily until dead or adult emergence.
Adults were immobilized with cold and inoculated with 5µl
2.7×10
8
conidia/mL suspension. Checked daily for 7 days.
Yousef et al.
(2013)
Probit analysis
and Survival
analysis
Bactrocera
oleae
Adults were treated with 1 ml of a 1.0 × 10
8
conidia/ml by
spraying. Checked daily for 12 d. Two assays for puparia. 1.
1 ml of a10
8
conidia/ml added to 30g soil with 63 puparia; 2.
Puparia immersed in fungal suspension for 10 seconds.
42
v. Conclusion and future perspectives
The use of living organisms to control pest insects is an important part of current and future
crop protection. Understanding the fundamental ecology of these organisms is vital to their success
as BCAs. For example, research regarding their natural occurrence and effect on host populations
greatly enhances their potential for more efficient utilization in pest regulation; e.g., conservation
biological control. Additionally, research which investigates how these organisms interact with
other organisms, viz. plants, plant pathogens and other BCAs, is crucial to predicting their
effectiveness and maintaining consistency as BCAs. This thesis advances the current scientific
knowledge regarding the ecology and biological control use of Metarhizium spp. fungi in several
areas, namely:
 M. flavoviride is the dominant species of the Metarhizium community found in some
agroecosystems in Denmark.
 Significant intra-species diversity exists within the understudied species M. flavoviride.
 M. brunneum, M. flavoviride and M. robertsii, applied as a seed treatment of conidia, will
disperse through soil with a growing plant root. Furthermore, these species of Metarhizium
will maintain pathogenicity to insects while interacting with plant roots.
 Dual BCA seed treatments of C. rosea and M. brunneum or M. flavoviride will control F.
culmorum infection in wheat seedlings.
 A reduction in virulence occurs when M. brunneum or M. flavoviride and C. rosea are
applied as a seed treatment on F. culmorum infected plants. However, virulence to T.
molitor was still observed at a significant level when compared to untreated controls.
Perhaps one of the most valuable components of any research is not the conclusion provided
but rather the new questions that remain unanswered. Based on the observations of this thesis there
are several research questions that I think should be addressed, including:
 Worldwide survey studies that endeavor to elucidate the distribution and occurrence of
Metarhizium spp. in agriculture. These studies should emphasize habitat associations as
well seeking to understand what characteristics promote abundance in particular areas.
Highly important to these studies will be the continued development of molecularly based
ecological tools, like isolating SSR markers (microsatellites) that can explicitly discriminate
genotypic diversity within the M. flavoviride species.
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PhD Thesis_Chad A. Keyser

  • 1. U N I V E R S I T Y O F C O P E N H A G E NU N I V E R S I T Y O F C O P E N H A G E N PhD thesis Chad Alton Keyser Protecting plants against pests and pathogens with entomopathogenic fungi: The biocontrol agent Metarhizium, its distribution, application, and interaction with other beneficial fungi Academic Advisor: Nicolai Vitt Meyling Submitted: 30 January, 2015 This thesis has been submitted to the PhD School of The Faculty of Science, University of Copenhagen
  • 2. 2 Institution: University of Copenhagen, Faculty of Science Department: Department of Plant and Environmental Sciences (PLEN) Author: Chad Alton Keyser Title: Protecting plants against pests and pathogens with entomopathogenic fungi: the biocontrol agent Metarhizium, its distribution, application, and interaction with other beneficial fungi. Academic advisor: Nicolai Vitt Meyling Co-advisor: Kristian Thorup-Kristensen Submitted: 30 January 2015
  • 3. 3 Acknowledgments “It is only with the heart that one can see clearly, for the most essential things are invisible to the eye." ― Hans Christian Andersen, The Ugly Duckling Through this acknowledgment I would like to express my profound appreciation to the many individuals that have supported and encouraged me during the course of my PhD program. Throughout the last three years I have grown significantly as a scientist and as a person – this growth is due mainly to the collaborations, friendships and nurturing influence of the many people I have had the opportunity to interact with. I first met Nicolai Meyling in 2009 at a scientific conference in Park City, Utah, as we stood in a crowded hall waiting for seating to begin. During our short conversation I was impressed by his attentive interest as I explained what I was working on as a Masters student. As my supervisor, Nicolai has continued to support and facilitate my efforts and growth. He has patiently steered my ideas in scientifically-relevant directions and ensured that my project was progressing. I am very grateful to Nicolai for his relaxed manner, thoughtful comments and suggestions, and the diligence and effort he has put into mentoring me. The Section for Organismal Biology (SOBI) has assembled an exemplary team of insect pathologists. It has been a pleasure to work alongside such a cohesive group, to share ideas, experience and encouragement – my experience here will always serve as a paragon for future collaborations. As the head of this research group, I am grateful to Jørgen Eilenberg for his infectious enthusiasm, his example of efficiency and his effort to make me feel part of the team. Also, I am very grateful to Bernhardt Steinwender for both his friendship and constant willingness to listen and discuss even the most ridiculous of ideas. I am also thankful to Henrik de Fine Licht for his patients in instructing me in art of AFLP analysis. The Team consists of many more PhD fellows, post-docs and professors who have each individually encouraged, inspired, taught and helped me in many ways and for which I am truly appreciative. It has been a great pleasure to work alongside many skilled technicians and assistants. I am especially grateful to Louise Munk Larsen for her ample technical skills and continued willingness to drop what she is doing to help. I thank Sylvia Mathiasen and Vinnie Deichmann for their assistance with my molecular work. I am also grateful to Line Lykke, Lærke Thordsen, Martina Falagiarda, Jesper Anderson, Azmi Mahmood, Darren Thomsen and the many other student helpers that have assisted me in many aspects of my experiments – I would not have been able to accomplish what I have needed to do without their diligent effort and skills. I have also been fortunate to have Kristian Thorup-Kristensen as a co-advisor. I am thankful to him for sharing his expertise in working with plants and in experimental design, as well as including me as part of his team. I also thank Birgit Jensen for her assistance in working with Fusarium and Clonostachys and her interest and willingness to train and assist me throughout the study for the second Manuscript. Living in Denmark and attending the University of Copenhagen has been the experience of a lifetime. I am thankful to Plant Biosystems Elite Environment at the University of Copenhagen for funding my PhD research. I am also grateful to the Department of Plant and Environmental Sciences and the Section for Organismal Biology for hosting and providing necessary facilities for my studies.
  • 4. 4 I am also supremely grateful for the support and encouragement I have received from my families. I recognize that living overseas has been difficult for both my family and my wife’s family; I sincerely appreciate their willingness to accept my decision to pursue this degree and the support and love they have shown which has been an enabling power to finish. I also appreciate the family and friends that have found the opportunity to visit us and share in this experience – their refreshing visits have made the distance more bearable. Most importantly, I would like to express sincere gratitude to my beautiful wife Shannon and three energetic children: Myra, Alexys and Noah. They willingly left behind their friends, families, job and comforts to follow me on an unknown path. My children have worked hard, learned the difficult language and adjusted quickly to the Danish lifestyle – their adaptability and bravery has given me strength. Shannon has also thrived and grown to love European living – her infectious eagerness to explore and discover the world around us has lifted and strengthened our whole family. I could not have succeeded in completing this program if not for her. Having my best friend by my side and knowing that she supports my ambitions has made this experience truly enjoyable. It is to my wife and children I dedicate this work and my life.
  • 5. 5 TABLE OF CONTENTS ACKNOWLEDGMENTS .......................................................................................................................................3 I. LIST OF INCLUDED MANUSCRIPTS ...........................................................................................................6 II. SUMMARY ..................................................................................................................................................7 III. DANSK RESUMÈ .........................................................................................................................................9 IV. THESIS OBJECTIVES................................................................................................................................11 1. INTRODUCTION ...........................................................................................................................................12 2. THE ENTOMOPATHOGENIC FUNGAL GENUS METARHIZIUM ...................................................................13 2.1 Phylogeny and Taxonomy..................................................................................................................... 15 2.2 Ecology.................................................................................................................................................. 18 2.2.1 Abundance and distribution............................................................................................................ 18 2.2.2. Abiotic factors that affect survival and growth ............................................................................. 21 2.2.3 Environmental dissemination pathways ......................................................................................... 22 3. TROPHIC INTERACTION ..............................................................................................................................23 3.1 Metarhizium ↔ Insects.......................................................................................................................... 24 3.2 Metarhizium ↔ Plants........................................................................................................................... 26 3.3 Metarhizium ↔ Other microorganisms................................................................................................. 29 3.4 Multi-trophic interactions with Metarhizium ........................................................................................ 31 3.4.1 Metarhizium ↔ Other microorganisms ↔ Insects......................................................................... 31 3.4.2 Metarhizium ↔ Other microorganisms ↔ Plants .......................................................................... 32 3.4.3 Metarhizium ↔ Plants ↔ Insects................................................................................................... 32 3.4.4 Metarhizium ↔ Other microorganisms ↔ Plants ↔ Insects ......................................................... 33 4. METHODOLOGY.........................................................................................................................................34 4.1 Selective media...................................................................................................................................... 34 4.2 Bioassay statistics.................................................................................................................................. 37 4.2.1 Experimental design ....................................................................................................................... 37 4.2.2 Types of Statistical Analyses.......................................................................................................... 38 V. CONCLUSION AND FUTURE PERSPECTIVES ..............................................................................................42 VI. REFERENCES ............................................................................................................................................44 VII. APPENDIX ................................................................................................................................................52 Manuscript 1................................................................................................................................................ 52 Manuscript 2................................................................................................................................................ 63 Manuscript 3................................................................................................................................................ 90
  • 6. 6 i. List of included Manuscripts Manuscript 1 METARHIZIUM SEED TREATMENT MEDIATES FUNGAL DISPERSAL VIA ROOTS AND INDUCES INFECTIONS IN INSECTS Chad A. Keyser, Kristian Thorup-Kristensen, & Nicolai V. Meyling Status: Published in Fungal Ecology, October 2014, Vol. 11, pg. 122-131 License Number: 3531750730534 Manuscript 2 BEST OF BOTH WORLDS: DUAL EFFECTS OF METARHIZIUM SPP. AND CLONOSTACHYS ROSEA AGAINST AN INSECT AND A SEED-BORNE PATHOGEN IN WHEAT Chad A. Keyser, Birgit Jensen & Nicolai V. Meyling Status: Under Review - Pest Management Science, submitted 22 Dec, 2014 Manuscript 3 DIVERSITY OF METARHIZIUM FLAVOVIRIDE POPULATIONS ASSOCIATED WITH ROOTS OF CROPS IN DENMARK Chad A. Keyser, Henrik H. de Fine Licht, Bernhardt M. Steinwender & Nicolai V. Meyling Status: Manuscript
  • 7. 7 ii. Summary Background: Insect-pest management is an increasingly important area of research. Efforts to maximize agricultural output are significantly dependent on reliable means for pest suppression. Biological control, or the use of living organisms to suppress a pest population, is among one of the leading alternatives to traditional chemical-based pesticides for crop protection. For the past 130 years several isolates of the fungal genus Metarhizium has been lead candidates among potential fungal-based biological control agents (BCAs) for insect pest control in agriculture. However, the majority of Metarhizium research has emphasized product development and application, largely neglecting the ecological and fundamental aspects. Inconsistent field reliability and economic viability have limited wider implementation of many BCAs, including Metarhizium-based products; an increased understanding of the fundamental ecology and environmental interactions has substantial potential to improve biological control efforts. The overall aim of this thesis was to improve the current understanding of how members of the fungal genus Metarhizium naturally occur in association with roots of crops in Denmark and interact with other organisms in relation to plant roots when applied as BCAs. Several scientific studies were conducted to answer important ecological questions regarding Metarhizium spp. interactions and their use as BCAs. The results of these studies are presented in three manuscripts. Manuscript 1: Recent research has revealed that many Metarhizium spp. interact with plants in the rhizosphere and have been shown to increase nutrient uptake and promote plant growth. In Manuscript 1 we investigate how might the fungus benefit from a plant association; namely, whether the plant provides a means of dispersal for the otherwise immobile fungus; as well as if the fungus maintains pathogenicity to insects while interacting with the plant. We found that when Metarhizium spp. were inoculated as conidia on wheat seeds they were able to disperse through the soil with the growing root and be re-isolated from lower portions of the root. Furthermore we observed that when washed roots were placed with Tenebrio molitor larvae, the larvae would succumb to Metarhizium spp. infection. Manuscript 2: Agricultural yields are threatened by multiple pests including insects and plant pathogens. Often the control of these pests requires the application of multiple biological control agents. In Manuscript 2 we investigate whether the mycoparasite Clonostachys rosea, commonly used to control plant-fungal pathogens, can be applied jointly with Metarhizium spp. to control both a plant pathogen and an insect pest. In this study we observed that C. rosea was highly efficacious at controlling Fusarium culmorum alone and in combination with Metarhizium – when
  • 8. 8 applied as a conidial seed treatment to wheat seeds. Additionally, we observed that while a significant level of T. molitor were infected with Metarhizium spp. after a combined treatment, there was a slight reduction in virulence when either C. rosea or F. culmorum were also present when compared to Metarhizium spp. only seed treatments. Based on the result of the direct inoculation bioassay of T. molitor larvae in which we did not observe a reduction in virulence when comparing combination treatments to individual treatments, we suspect that the virulence reduction is the result of resource competition on the growing root and not direct mycoparasitism. Manuscript 3: An awareness of the composition and distribution of naturally occurring Metarhizium spp. communities is important to understanding their role to insect host regulation. However there is an acute lack of ecological studies that assess the occurrence and community structure of entomopathogenic fungi. The objective of Manuscript 3 was to evaluate the occurrence, diversity and community structure of Metarhizium spp. isolates obtained from different crops at geographically separated agricultural fields in Denmark. Root and root-associated soil was sampled from wheat, oilseed rape, and bordering uncultivated grass fields at three different locations; 132 new Metarhizium isolates were obtained. Morphological data and sequencing of the rDNA intergenic spacer region (IGS) revealed that 118 of the isolates belonged to Metarhizium flavoviride, 13 M. brunneum and one M. majus. We then further characterized the intraspecific variability within M. flavoviride by unspecific markers (i.e., AFLP identification) to evaluate diversity and potential crop and/or area associations. We found there was a high level of diversity among the M. flavoviride isolates indicating that the isolates were not of the same clonal origin, however due to insufficient loci in the AFLP analysis we were not able to determine haplotype groupings or confirm any habitat associations. We suggest that the development of more specific markers would greatly improve our ability to evaluate M. flavoviride diversity. This represents the first time that an in-depth analysis of the molecular diversity within a large isolate collection of the species M. flavoviride has been reported. Overall the scientific studies presented in this thesis are both important and novel to the field of Metarhizium research; these studies advance the current knowledge of the ecological significance of Metarhizium spp. as a naturally occurring microorganism and increase our understanding of their interactions as biological control agents with other organisms. Furthermore, this thesis presents the background literature and motivation for the research and their implication to the field of insect pathology.
  • 9. 9 iii. Dansk resumè Baggrund: Bekæmpelse af skadedyr er et vigtigt forskningsområde. Mulighederne for at øge landbrugsudbyttet er afhængigt af pålidelige bekæmpelsesmetoder. Biologisk bekæmpelse, brugen af levende organismer til at begrænse skadedyrspopulationer, er en af de væsentligste alternativer til kemisk baseret plantebeskyttelse. I løbet af de seneste 130 år har flere isolater af den insektpatogene svampeslægt Metarhizium blevet førende kandidater til svampebaseret biologisk bekæmpelse af skadedyr. Dog har hovedparten af forskning i Metarhizium fokuseret på produktudvikling og udbringning, hvor forståelse af grundlæggende økologiske aspekter har blevet negligeret. Varierende pålidelighed har begrænset implementeringen af mange biologiske bekæmpelsesprodukter, inklusiv flere baseret på Metarhizium. En øget forståelse af de fundamentale økologiske og miljømæssige interaktioner, hvori Metarhizium indgår, har stort potentiale til at forbedre brugen af biologisk bekæmpelse. Det overordnede formål med denne afhandling var at øge forståelsen af hvordan medlemmer af svampslægten Metarhizium indgår i naturlig associering med rødder af afgrøder i Danmark og hvordan de interagerer med andre organismer i relation til planterødder når de tilføres ved biologisk bekæmpelse. Flere videnskabelige studier blev gennemført for at svare på dette, og resultaterne er præsenteret i tre manuskripter. Manuskript 1: Det er for nyligt blevet videnskabeligt vist at flere arter af Metarhizium interagerer med planter i rhizosfæren og at dette kan øge næringsstofoptag og vækst hos planten. I Manuskript 1 undersøges det om svampen kan have fordel af denne interaktion ved at bruge plantens rødder til transport i jorden, og om svampen bevarer sin evne til at inficere insekter ved at interagere med planten. Det blev fundet, at når Metarhizium spp. blev inokuleret som konidier på frø af hvede, kunne svampen sprede sig gennem jord med de voksende rødder og blive genisoleret fra den nedre del af rødderne. Desuden var vaskede rødder infektive mod larver af melorme Tenebrio molitor, som efter inkubering sammen med rødder døde af Metarhizium infektion. Manuskript 2: Afgrøder bliver ofte angrebet af flere organismer, primært insekter og plantepatogener, hvilket kræver samtidig brug af flere biologiske bekæmpelsesmidler. I Manuskript 2 blev det undersøgt, om nyttesvampen Clonostachys rosea, som normalt bruges til at bekæmpe plantepatogener, kan blive anvendt sammen med Metarhizium spp. til at bekæmpe både insekter og plantesygdomme på samme tid. Det blev observeret at C. rosea var meget effektiv til at bekæmpe Fusarium culmorum alene og sammen med Metarhizium spp., når de to typer af svampe blev tilført som konidier på frø af hvede. Desuden blev larver af melorme inficeret af Metarhizium spp. både
  • 10. 10 når denne svamp blev tilført alene og sammen med C. rosea eller F. culmorum, men infektionen var en smule nedsat i kombinationerne. Ved direkte inokulering af larverne med sammen svampekombinationer sås ikke denne reduktion. Reduktionen observeret ved planteinokuleringen kunne derfor skyldes en indirekte interaktion på planteroden og ikke direkte interaktion som mykoparasitisme mellem svampene. Manuskript 3: Det er afgørende af kende til den naturlige forekomst og udbredelse af Metarhizium spp. for at forstå deres bidrag til regulering af insektpopulationer. Der findes dog fortsat kun få studier af den naturlige forekomst af insektpatogene svampe. Formålet med studiet præsenteret i det tredje manuskript var at beskrive forekomst, udbredelse og diversitet af Metarhizium spp. i forbindelse med rødder af forskellige afgrøder i Danmark på tre geografisk afgrænsede lokaliteter. Rødder og rod-associeret jord blev indsamlet på hver lokalitet fra vinterhvede, raps, og fra et uforstyrret græsningsareal. 132 Metarhizium isolater blev indsamlet. Morfologisk og DNA sekventering viste, at 118 af isolaterne tilhørte arten Metarhizium flavoviride, 13 var M. brunneum, og et var M. majus. Ved yderligere karakterisering af intraspecifik variation i M. flavoviride med uspecifikke markører (AFLP) blev diversitet og potentiel lokalitet- eller afgrødespecificitet undersøgt. En høj grad af diversitet blev fundet med de anvendte markører, hvilket indikerer at de ikke alle har samme klonale ophav, men metoden var ikke tilstrækkelig til at identificere lokalitet- eller afgrødespecificitet. Specifikke markører udviklet til M. flavoviride vil være nødvendige for at undersøge dette. Dette studie er det første hvor arten M. flavoviride blev undersøgt mht. molekylær diversitet ved brug af en større samling af isolater. De videnskabelige studier præsenteret i denne afhandling er både vigtige og nye indenfor forskningen i Metarhizium. Studierne øger den nuværende viden om økologisk betydning af Metarhizium spp. som naturlig forekommende mikroorganisme samt ved interaktioner med planter og med andre mikroorganismer. Desuden giver denne afhandling en gennemgang af litteraturen om emnet og betydningen af den præsenterede forskning for insektpatologi.
  • 11. 11 iv. Thesis Objectives The aim of this thesis is to advance our understanding of how members of the entomopathogenic fungal genus Metarhizium interact with other organisms, with an emphasis placed on its role as a biological control agent. To accomplish this goal the following research questions were investigated: ■ Can plant roots act as a vehicle to disseminate Metarhizium in soil? Additionally, while associating with plants, do Metarhizium spp. maintain their pathogenicity to insects? ■ In a seed-treatment biocontrol context, what interactions occur between other organisms, namely: the mycoparasitic fungus Clonostachys rosea, and the plant pathogenic fungus Fusarium culmorum? ■ What is the prominence and species composition of naturally occurring Metarhizium in roots and soil within different agro-ecosystems in Sjælland, Denmark?
  • 12. 12 1. Introduction Agricultural production is incalculably important to human existence both from an economical as well as a nutrient-resources perspective. Worldwide estimates suggest that currently 38% of the Earth’s terrestrial surface is dedicated to agricultural endeavors (Foley et al., 2011). Furthermore, it has been projected that from 2005 to 2050 global crop production will need to increase as much as 110% to meet the caloric and protein demands of the world population (Tilman et al., 2011). We cannot expect to meet the growing agricultural needs by merely dedicating more land to food production; increasing the productivity of the current agricultural systems is vital to our future (Tilman et al., 2011). A significant limiting factor to agriculture yields are pests – this includes herbivores (e.g., arthropods), weeds and plant pathogens. Over the last century advances in chemical pesticide development have greatly mitigated pest related crop losses. However, due to pesticide resistance and an increased awareness of deleterious effects on non-target organisms, including humans, reliable alternative methods for controlling pests are desperately needed. Biological control is an important alternative which has potential to effectively control pest populations with limited risk (Hajek, 2004). Biological control can be defined as “the use of living organisms to suppress the population of a specific pest organism, making it less abundant or less damaging then it would otherwise be” (Eilenberg et al., 2001). There are several types of organisms that have been identified as biological Glossary of Terms Agroecosystem Agricultural ecosystem - Specialized ecosystem which has been manipulated by human activities with the aim to produce high levels of organic output. Includes living and non-living components and their interactions. Anamorph The asexual (conidial or imperfect) stage in the life history of a fungus. BCA Biological control agent – the organism used for Biological control Bioassay The measurement of the potency of any BCA, by means of the response which it produces in a living host. Biological control The use of living organisms to control (usually meaning to suppress) undesirable animals and plants. Entomopathogen A microbe affecting insects (or in a more general sense, other terrestrial arthropods including arachnids), usually causing mortality in the host (as opposed to a more benign relationship). Fungal endophyte An asymptomatic plant-colonizing fungus that lives a portion of its life cycle inside the plant. Pathogenicity The quality or state of being pathogenic. The potential ability to produce disease. Pathogenicity is qualitative, an all-or-none concept Rhizosphere The narrow region around the plant root that is directly influenced by root secretions and associated soil microorganisms. Teleomorph The sexual stage in the life history of a fungus. Virulence The disease-producing power of a microorganism. Virulence can be quantified.
  • 13. 13 control agents (BCAs), including: predators, parasitoids, parasitic nematodes, bacteria, viruses, fungi and microsporidia (Hajek, 2004), the latter group is now recognized and a basal fungal group (James et al., 2006). Each BCA has different characteristics which determine their effectiveness in a particular circumstance. Understanding the environmental factors that contribute to effectiveness will largely determine the success of a BCA. For many years research regarding the fundamental ecology of these organisms has had far less priority than product development; this has likely led to inconsistent results in the field (Vega, 2008). The present thesis contributes in part, to increasing our understanding of the complex interactions involved between a particular fungal BCA and other important organisms in its environment, within a biological control context. 2. The entomopathogenic fungal genus Metarhizium Entomopathogenic fungi are fungal organisms that have the ability to infect and cause disease in an arthropod. The kingdom Fungi is estimated to contain more than 5.1 million species (O'Brien et al., 2005), of those, 750-1000 species are pathogenic to insects (Vega et al., 2012). Fungi of the Ascomycota genus Metarhizium (Hypocreales: Clavicipitaceae) are among one of the most studied groups of entomopathogenic fungi, in fact in the last decade the number of peer- reviewed publications has increased substantially (Figure 1). Metarhizium spp. are ubiquitously found in terrestrial ecosystems worldwide, having been isolated from every continent except Antarctica (Roberts and St. Leger, 2004). Many species of Metarhizium have a wide host range, like M. brunneum and M. robertsii, infecting at least seven insect orders (Veen, 1968; Zimmermann, 1993); however a few species are known to be host specific like M. acridum which only infects some taxa in the order Orthoptera (Driver et al., 2000; Wang et al., 2011). In addition to their host range there are several other key attributes that make Metarhizium spp. ideal candidates for BCAs, including: the infection process is topical (does not need to be ingested by host), infectious conidia can be mass produced on artificial media, remain viable and can be easily 0 300 600 900 1200 1500 1975-1979 1980-1984 1985-1989 1990-1994 1995-1999 2000-2004 2005-2009 2010-2014 Numberof Publications Figure 1. The number of peer-reviewed publications published of 5 year periods for the last 40 years according to a search on web of science with the key word “Metarhizium”.
  • 14. 14 disseminated, and many species of Metarhizium produce secondary metabolites known as destruxins which are toxic to invertebrates and help the pathogen overcome the host immunity quickly (Dorta et al., 1996; Zimmermann, 2007). The infection process of Metarhizium spp. to a suitable host was reviewed by Zimmermann (2007) and is summarized in Figure 2. He describes the process in 6 steps including: 1. Attachment, in which the conidia adheres to the cuticle using a combination of hydrophobic interaction and specialized adhesion proteins; 2. Germination and appressoria formation; 3. Penetration through the cuticle, which is mechanical but aided by the production of enzymes including proteases, chitinases and lipases; 4. Overcoming host defenses, often by the production of novel destruxins; 5. Proliferation within the host, generally via the production of blastospores or hyphae; and lastly, 6. Outgrowth and production of new infective conidia. Figure 2: The infection process, an illustration showing the 6 stages of fungal infection in an insect: 1. Attachment; 2. Germination and appressoria formation; 3. Penetration through the cuticle; 4. Overcoming host defenses; 5. Proliferation within the host; 6. Outgrowth and production of new infective conidia. Illustration by C. A. Keyser.
  • 15. 15 2.1 Phylogeny and Taxonomy The taxonomy of the Genus Metarhizium (Family: Clavicipitaceae, Order: Hypocreales, Class: Sordariomycetes Phylum: Ascomycota, Kingdom: Fungi) has been revised many times over the years. In 1883 Russian mycologist Sorokin first introduced the name Metarhizium as the genus name for fungi that are the causal agent of insect disease “green muscardine”, which had originally been called Entomophthora anisopliae by Elia Metchnikoff in 1879 (Zimmermann et al., 1995). For the last 130 years the genus name Metarhizium has persisted, however many of the species names belonging to this genus have changed. Many fungi are pleomorphic, i.e., they have different life stages which are morphologically distinct – often the different stages have been identified as different organisms. To help standardize the descriptions the term teleomorph is used to describe the sexual stage, anamorph to describe the asexual stage, and holomorph when both are present (Hennebert and Weresub, 1977). Species of the genus Metarhizium have historically been considered anamorphic fungi (i.e., only exhibiting the asexual stage and reproduce clonally by mitosporic conidia production) and placed in the former- division Deuteromycota; however the discovery of a sexual and asexual stage (Liang et al., 1991), as well as molecular analysis have facilitated their placement in the phylum Ascomycota (Kepler et al., 2014). In this thesis only the anamorphic stage of Metarhizium is discussed. Another challenge for Metarhizium taxonomy is that many species of Metarhizium are morphologically similar, so identification based on morphological attributes is difficult. Tulloch (1976) made a major revision of the genus Metarhizium based on drawings and the morphological descriptions available, she reduced all known taxa to two speices, M. flavoviride and M. anisopliae. The next major revision of the genus was performed by Driver et al. (2000) using ITS sequence data for phylogenetic analyses; they observed a high level of genetic diversity and were able to identify ten distinct clades, however they restricted their descriptions to varieties rather than species due to limited resolution and support in the sequence analysis. Bischoff et al. (2009) used a multigene phylogenetic approach to resolve the M. anisopliae group which at the time consisted of 4 varieties as defined by Driver et al. (2000); i.e., M. anisopliae var. acridum, M. anisopliae var. anisopliae, M. anisopliae var. lepidiotae, and M. anisopliae var. majus. They describe ten species within the M. anisopliae complex (viz., M. anisopliae, M. acridum, M. brunneum, M. globosum, M. guizhouense, M. lepidiotae, M. majus, M. pingshaense, and M. robertsii) (Bischoff et al., 2009). The taxonomic clarification of the Metarhizium genus was continued by Kepler et al. (2014) and the M. flavoviride complex was resolved into four species (viz., M. flavoviride, M. koreanum, M.
  • 16. 16 minus, and M. pemphigi). Additionally, in this revision an effort was made to reevaluate fungal nomenclature with the intention to give one name to one fungus, regardless of life stage (Taylor, 2011). In doing this Kepler et al. (2014) transferred many taxa from Chamaeleomyces, Metacordyceps and Nomuraea to Metarhizium. The genus Metarhizium now includes 36 species (Table 1). Isolates described as “M. anisopliae” or “M. flavoviride” in studies published prior to (and sometimes after) the 2009 and 2014 taxonomic revisions are now designated as “M. anisopliae sensu lato” (s.l.) or “M. flavoviride s.l.” if the new taxonomic identity according to Bischoff et al. (2009) or Kepler et al. (2014) is unknown or not specified, M. anisopliae and M. flavoviride identified using the current taxonomic criteria are labeled using the sensu stricto (s.s.) designation. As noted by Steinwender (2013) “taxonomy is not set in stone but rather a snapshot of a given moment”, it is likely the genus Metarhizium will continue to develop and change as our understanding of the complexity of life improves.
  • 17. 17 Table 1: Current Species of the Genus Metarhizium, the authorities and year of description – arranged alphabetically. Species name Authorities, year of description M. acridum (Driver & Milner) J.F. Bisch., S.A. Rehner & Humber, 2009 M. album Petch, 1931 M. anisopliae (Metsch.) Sorokin, 1883 M. atrovirens (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber, 2014 M. brasiliense Kepler, S.A. Rehner & Humber, 2014 M. brittlebankisoides (Zuo Y. Liu, Z.Q. Liang, Whalley, Y.J. Yao & A.Y. Liu) Kepler, S.A. Rehner & Humber, 2014 M. brunneum Petch, 1935 M. campsosterni (W.M. Zhang & T.H. Li) Kepler, S.A. Rehner & Humber, 2014 M. carneum (Duché & R. Heim) Kepler, S.A. Rehner & Humber, 2014 M. flavoviride W. Gams & Rozypal, 1973 M. frigidum (Driver & Milner) J.F. Bisch. & S.A. Rehner, 2006 M. globosum J.F. Bisch., S.A. Rehner & Humber, 2009 M. granulomatis (Sigler) Kepler, S.A. Rehner & Humber, 2014 M. guizhouense Q.T. Chen & H.L. Guo, 1986 M. guniujiangense (C.R. Li, B. Huang, M.Z. Fan & Z.Z. Li) Kepler, S.A. Rehner & Humber, 2014 M. indigoticum (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber, 2014 M. khaoyaiense (Hywel-Jones) Kepler, S.A. Rehner & Humber, 2014 M. koreanum Kepler, S.A. Rehner & Humber, 2014 M. kusanagiense (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber, 2014 M. lepidiotae (Driver & Milner) J.F. Bisch., S.A. Rehner & Humber, 2009 M. majus (J.R. Johnst.) J.F. Bisch., S.A. Rehner & Humber, 2009 M. marquandii (Massee) Kepler, S.A. Rehner & Humber, 2014 M. martiale (Speg.) Kepler, S.A. Rehner & Humber, 2014 M. minus (Rombach, Humber & D.W. Roberts) Kepler, S.A. Rehner & Humber, 2014 M. novozealandicum Kepler, S.A. Rehner & Humber, 2014 M. owariense (Kobayasi) Kepler, S.A. Rehner & Humber, 2014 M. owariense f. viridescens (Uchiy. & Udagawa) Kepler, S.A. Rehner & Humber, 2014 M. pemphigi (Driver & R.J. Milner) Kepler, S.A. Rehner & Humber, 2014 M. pingshaense Q.T. Chen & H.L. Guo, 1986 M. pseudoatrovirens (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber, 2014 M. taii Z.Q. Liang & A.Y. Liu, 1991 M. rileyi (Farl.) Kepler, S.A. Rehner & Humber, 2014 M. robertsii J.F. Bisch., S.A. Rehner & Humber, 2009 M. viride (Segretain, Fromentin, Destombes, Brygoo & Dodin ex Samson) Kepler, S.A. Rehner & Humber, 2014 M. viridulum (Tzean, L.S. Hsieh, J.L. Chen & W.J. Wu) B. Huang & Z.Z. Li, 2004 M. yongmunense (G.H. Sung, J.M. Sung & Spatafora) Kepler, S.A. Rehner & Humber, 2014
  • 18. 18 2.2 Ecology Despite having more than 100 years’ worth of research interest, we are only beginning to understand the ecology of Metarhizium spp. and the important role they play in the ecosystem. There are two main reasons why the natural ecology of Metarhizium is important to biological control: first, as a ubiquitous organism infectious to insects, understanding the natural occurrence and distribution, and the contributions of Metarhizium in regulating insect populations is highly relevant; and second, understanding how Metarhizium interacts with other organisms and is affected by abiotic factors will help optimize how to most effectively use them as BCAs. Bruck (2010) pointed out that in plant pathology a concept known as the “disease triangle” is often used to describe the interaction between a host, a pathogen, and the environment; he suggested that this same concept should also be applied to biological control. By emphasizing a total ecological approach to Metarhizium spp. research, which focuses on both the direct and indirect effects of biotic and abiotic factors in the environment, we gain greater clarity of the role Metarhizium spp. play in the ecosystem. In this section I will discuss with regard to Metarhizium: 2.2.1. Abundance and distribution; 2.2.2. Abiotic factors that affect survival and growth; and 2.2.3. Environmental dissemination pathways. In section 3 (below) I will discuss biotic interaction that occur between Metarhizium and other organisms. 2.2.1 Abundance and distribution The United States Department of Agriculture’s (USDA) Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (ARSEF), Ithaca, NY, hosts one of the largest libraries of entomopathogenic fungal isolates collected from all over the world. This collection has over 1500 different isolates of Metarhizium, however most of these isolates, as well as the many others that have been collected over the years, were collected not with the intention to understand abundance and distribution but rather to find new potential products for commercialization: examples include the LUBILOSA project (Roberts and St. Leger, 2004), in which researchers scoured the African and Australian continents searching for entomopathogenic fungi with the intent to develop a BCA for locust control. More recently, an 8 year USDA-APHIS project which surveyed 30,000 soil samples in the western US and collected more than 2,000 new isolates of Metarhizium with a goal to find M. acridum in USA soil (C. A. Keyser, unpublished data). While these “goal-orientated” types of surveys often produce useful information about the ecology and distribution of Metarhizium in nature; they are not designed as ecological studies and so many of the conclusions they can provide are incomplete.
  • 19. 19 There have, however, been several studies intended to investigate Metarhizium occurrence in both natural habitats and agroecosystems (Table 2); it is noteworthy that there are a lack of studies from latitudes below 40º (e.g., tropical regions). Many aspects differ between these studies (e.g., sampling and isolation method) which makes direct comparison difficult; however, when viewed as a group we are able to make some generalizations about the abundance and distribution of Metarhizium spp. in different environments, several of which I would like to briefly outline: First, Metarhizium spp. tend to be less abundant in colder regions than in temperate regions (Inglis et al., 2008; Klingen et al., 2002; Vanninen, 1995), this observation is also supported by laboratory work which has demonstrated that most species of Metarhizium , with the exception of M. frigidum, do not grow at cold temperatures (Fernandes et al., 2010a; Fernandes et al., 2008). Second, Metarhizium is more abundant than other entomopathogenic fungi in cultivated fields and open meadows (Bidochka et al., 1998; Quesada-Moraga et al., 2007; Sun et al., 2008; Vanninen, 1995). Third, Metarhizium is primarily found in the soil environment and not on above ground substrates (Meyling et al., 2011; Vega et al., 2012). Lastly, Metarhizium spp. distribution tends to associate with habitats and not with host insects (Bidochka et al., 2001; Fisher et al., 2011; Wyrebek et al., 2011). Traditionally, insect association was thought to be the key factor in determining population structure (Bridge et al., 1997; St. Leger et al., 1992), this shift away from the traditional paradigm has led to many questions about what role these fungi truly play in the environment (Bidochka et al., 2001; Vega, 2008). Another interesting observation we can glean from viewing these studies together is that Metarhizium spp. composition and dominance appears to be location specific. For example, a study in the mid-east part of Canada found M. robertsii to be the most common species of Metarhizium (Wyrebek et al., 2011), while a study in the western part of Canada found M. anisopliae s.l. to be most common (Inglis et al., 2008). Even in Denmark survey studies have yielded inconsistent species compositions. In 1995 Steenberg reported that M. anisopliae s.l. dominated cultivated soils (Vega et al., 2012), also Steinwender et al. (2014) primarily found M. brunneum (a species within the M. anisopliae complex) to be most often isolated from agricultural soil in Denmark. However, Meyling and Eilenberg (2006) found that M. anisopliae s.l. was almost absent in a single organic agroecosystem sampled. Also, as reported in Manuscript 3, we found that in three separate agricultural areas, M. flavoviride was the most frequently isolated species. While further sampling is needed to confirm these observations, it is clear that Metarhizium spp. occurrence is neither random nor ubiquitous.
  • 20. 20 Table 2: Metarhizium abundance and distribution studies. Country (latitude) Reference Isolation method Samples taken Habitat types Metarhizium isolates obtained General Metarhizium results Finland (62º N) (Vanninen, 1995) Insect soil baiting 590 soil samples from 347 sites Forests, agricultural fields 92 isolates Found in lower latitudes, not affected by cultivation Canada (45 ºN) (Bidochka et al., 2001, Bidochka et al., 1998) Insect soil baiting 266 soil samples from 133 sites Natural (forests) and agricultural 357 isolates Most abundant entomopathogen isolated, most frequently recovered from soil baiting at 25ºC, more often in Agricultural soil. Genotype association with habitat, no association with insect host. Norway (66ºN) (Klingen et al., 2002) Insect soil baiting 200 samples Conventional and organic farms 9 isolates Not commonly found. No difference between fields and field margins. Denmark (55 ºN) (Meyling and Eilenberg, 2006) Insect Soil baiting 544 soil samples over two years experimental research farm and hedgerow 134 isolates Surprisingly low occurrence of M. anisopliae s.l. Spain (40ºN) (Quesada- Moraga et al., 2007) Insect soil baiting 244 samples Natural and cultivated areas 71 isolates Most common in cultivated areas. Preferred soil with low clay content. Canada (45 ºN) (Inglis et al., 2008) Insect soil baiting and selective media 250 soil samples Urban, agricultural and forest 250 isolates Metarhizium very widespread and diverse however one genotype dominated. China (40ºN) (Sun et al., 2008) Insect soil Baiting >2300 soil samples cultivated fields and orchards 60% of soil samples More frequent in cultivated fields than orchards. South Africa (33ºS) (Goble et al., 2010) Insect soil baiting 288 soil samples Conventional and organic citrus farms 16 isolates No difference between organic and conventional. USA (44 ºN) (Fisher et al., 2011) Insect baiting with roots 339 root samples Strawberries, blueberry, grapes, and Christmas trees (roots) 94 isolates Species/ plant-root association; M. brunneum = strawberries and blueberries, M. guizhouense = Christmas trees, and M. robertsii = Christmas trees Canada (45 ºN) (Wyrebek et al., 2011) Washed root homogenate on selective media 200 root samples Natural meadows, and forests (roots) 102 isolates Species/ plant-root association; M. brunneum = shrubs and trees, M. guizhouense = tree roots, and M. robertsii = grasses and wildflowers Denmark (55 ºN) (Steinwender et al., 2014) Insect soil baiting 53 soil samples Agricultural field and boarding hedgerow 123+ isolates Five genotypes of M. brunneum , 6 genotypes of M. robertsii. One M. brunneum genotype most dominant. Denmark (55 ºN) Keyser. et al. (2015) – Manuscript 3 Insect soil baiting and selective media 450 soil and root samples Winter wheat, Oilseed rape and natural meadow 132 isolates 118 of the isolates were M. flavoviride, 13 M. brunneum and 1 M. majus. AFLP analysis revealed high level of diversity with the M. flavoviride species
  • 21. 21 2.2.2. Abiotic factors that affect survival and growth Three of the most important abiotic factors that affect entomopathogenic fungal performance are: temperature, humidity, and UV-radiation. Temperature has been shown to affect Metarhizium spp. germination, hyphal growth and infection rates (Keyser, 2010; Keyser et al., 2014a). Growth at high and low temperatures has even been used as phenotypic traits to distinguish certain species of Metarhizium (Fernandes et al., 2010a). Fluctuating ambient temperatures have also been indicted as one of the primary limiting factors in field success (Foster et al., 2010; Foster et al., 2011). Likewise, relative humidity is an important factor in determining growth, infection, sporulation and conidial longevity (Arthurs and Thomas, 2001; Daoust and Roberts, 1983; Milner et al., 1997; Vega et al., 2012). UV radiation, especially UV-B radiation can be highly detrimental to conidia survival (Braga et al., 2001; Rangel et al., 2005) and even sub-lethal UV-B radiation exposure was observed to delay conidial germination (C.A. Keyser, unpublished data). These factors are also highly relevant in understanding the natural distribution and abundance in the field. Some species of Metarhizium have been shown to be less cold tolerant then Beauveria spp. (Fernandes et al., 2008), which may explain why Vanninen (1995) found Metarhizium primarily in the southern Finland areas while Beauveria was isolated all over Finland. In one area of southern Alberta, Canada, Inglis et al. (2008) did not find any Metarhizium isolates, they suggested that a possible explanation for the lack of isolates is that the area surveyed was semi-arid, had very cold winters and short summers. Bidochka et al. (2001) also observed that isolates found in forested areas were more likely to grow at low temperatures while those found in open fields showed a propensity for growth at higher temperatures and UV-B tolerance. Abiotic factors have strong influence on Metarhizium population structure and biological success. One of the challenges that has slowed BCA implementation is that they are sometimes viewed (especially by the end user) to be one-to-one substitutions for chemical pesticides (Cook, 1993). This leads the expectation that they will have a similar shelf life, can be applied in similar conditions, and will have a similar mode of action and time to kill. Distinguishing the differences between applying a living organism and a chemical is vital to successfully integrate BCAs as a viable treatment option. One of the limitations is BCA susceptibility to abiotic environmental factors (Jaronski, 2010; Starnes et al., 1993). Future research that uncovers the mechanisms and limitation regarding responses to abiotic factors will greatly aid in improving BCA effectiveness.
  • 22. 22 2.2.3 Environmental dissemination pathways Fungi are non-motile organisms, in nature the infection propagules (e.g., conidia) of Metarhizium are dispersed passively. Generally dispersal is thought to occur by abiotic factors such as wind and rain (Hajek, 1997; Inglis et al., 2001; Meyling and Eilenberg, 2007). Additionally, it is possible that insects as well as other animals may act as vectors for conidia (Meyling et al., 2006). In Manuscript 1 we show that plants also act as a vector in aiding conidia dispersal through the soil environment (Keyser et al., 2014b), and we speculate that this may explain why entomopathogenic fungi have evolved to associate with plants (see also section 3). Historically, biological control programs utilizing Metarhizium as a bio-pesticide have tried to adapt chemical-pesticide application methods for conidial dispersal (Bateman, 1997) (Figure 3). Often formulating conidia for application in liquid substrate or as a powder, this is then applied topically to vegetation (Booth et al., 2000; Caudwell and Gatehouse, 1996; Griffiths and Bateman, 1997). The effectiveness of biological control programs using Metarhizium have been inconsistent, one factor that may be contributing to the unpredictability of Metarhizium as a BCA is the application method or delivery system. Several studies have shown that application method does greatly influence infection rates and environmental persistence (Farenhorst et al., 2008; Fargues et al., 1997; Jenkins and Thomas, 1996; Kanga et al., 2003); however nearly all of these studies are focused solely on the BCA implementation and thus neglect further investigations to understand the underlying ecological principles contributing to increased infectivity. Understandably, designing Figure 3. Methods of Metarhizium application. (A) In China mortar shells filled with powdered conidia were launched above a forested area using a grenade launcher (photo by Richard Soper), arrow indicates conidial cloud. (B) Conidia formulated in oil is applied to grasshopper infected rangeland in Utah, USA, using a rear-mounted sprayer (photo by C. A. Keyser).
  • 23. 23 BCAs to fit seamlessly into insecticide application methods greatly enhances their usability and facilitates an easy transition away from chemical pesticides. Unfortunately, without an understanding of the natural dispersal mechanisms which contribute to efficacy, consistent biological control results will be more a matter of luck than design. Only by investigating the ecological principles can we hope to improve reliability. For example, in a study to evaluate the M. brunneum biocontrol product Met52 for control of Black vine weevil, Otiorhychus sulcatus (Col. Curculionidae) in field grown strawberries, Ansari and Butt (2013) tested several application methods using the granular formulation of the commercial product. The methods of application included: 1. Premixed in soil; 2. Drench application to the base of each plant; and 3. Plant roots being dipped in an aqueous suspension of the conidial product and then planted. They found that Black vine weevil control was more efficient when the drench application method was used. Further studies revealed that plant roots were colonized by significantly more conidia than when the drench method was used (Ansari and Butt, 2013). This additional information allowed them to conclude that the conidia concentration in the rhizosphere was a key component to improving infectivity. It was recently shown, however that M. brunneum population dynamics in the rhizosphere may depend on adaptations to the local environmental conditions; a Norwegian M. brunneum isolate proliferated more in strawberry rhizopheres at ambient temperatures in Norway than an isolate similar to Met52 which originated from Austria (Klingen et al., 2015). Clearly, understanding how infectious propagules are dispersed naturally can improve BCA performance in the field. 3. Trophic interaction One of the greatest challenges to fully understanding the ecology of the world we live in is accounting for all the trophic interactions that influence an organism; however for BCAs it is crucial that we account for these interactions as they will greatly affect efficiency. If we truly hope to exploit biological control to suppress pests, we must view insect pathology not as one organism acting on another, but rather as a node in a complex web of intertwined organisms and response variables which have co-evolved and adapted to each other. This can only be achieved by studying the organisms in combination and not individually and valuing both direct and indirect interactions. The following sections will highlight several key interactions that occur between Metarhizium and other important organisms (Figure 4). Some of these interactions have been well studied (e.g., Metarhizium ↔ Insects), while for others very little is known (e.g., Metarhizium ↔ Other microorganisms ↔ Plants). The overall message that I hope to convey is the importance of
  • 24. 24 not only understanding the various responses associated with individual bi-trophic interactions, but also a broad perspective of how they affect each other. Figure 4: Illustration depicting several trophic interactions that may occur between Metarhizium spp. and other organisms. Blue arrows represent direct interactions and orange arrows represent indirect interactions. Diagram by C. A. Keyser 3.1 Metarhizium ↔ Insects Among the millions of fungal species in the world, Metarhizium spp. have garnered significant scientific and economic interest primarily because of how they interact with arthropods. A large portion of the research involving Metarhizium centers on its use as a BCA and consequently
  • 25. 25 deals with its relationship to various insects. As a generalist, many Metarhizium spp. are able to infect a wide range of hosts (Veen, 1968; Zimmermann, 1993), however the virulence can vary considerably between different host species (Butt et al., 1992), making it necessary to test virulence towards each insect species of interest. Furthermore, virulence varies between isolates even within the same fungal species (Keyser, 2010), which further complicates the selection of appropriate isolates for BCA use. The variation in virulence should not necessarily be looked at as a negative aspect for a potential BCA; it is important to remember that a very small percentage of all insect species are important pests – many herbivores do not cause any economic damage and some predators, parasitoids and pollinators are important beneficial insects. The variation in host virulence allows selection for a BCA that is highly virulent to a particular pest insect while being less virulent to non-target insects. For example, Falagiarda (2014) observed that a commercial isolate of M. brunneum, the same isolate used in Manuscript 1 and 2, to be highly virulent to the grain-pest insect T. molitor but having very low virulence to the beneficial coleopteran, Atheta coriaria (Figure 5). Her results suggest that this isolate of M. brunneum could therefore be considered a “low risk” substance under the proposed EU legislation. Further testing might investigate whether there is an additive or synergistic effect when M. brunneum and A. coriaria are used in combination as BCAs. Many factors from both the host and the pathogen contribute to virulence; it is a give and take relationship with each attempting to maximize their own fitness. Each step in the infection process (see Figure 2) is controlled by specific cues and responses which determine the success of the infection. For example, ambient pH levels on and around the insect cuticle regulate the secretion of proteolytic and chitinolytic enzymes which aid in the penetration process (St. Leger et al., 1998). In response to the infections, insects have evolved different immune responses, both physiological and behavioral, to mitigate the pathogenic effects. For example, after exposure to Metarhizium there are changes in insect biochemistry, including melanization of the cuticle and antimicrobial defenses of the haemolymph (Dubovskiy et al., 2013; Gillespie et al., 2000). Also, behavioral adaptations have been noted, including: increased grooming among infected ants and termites (Hughes et al., 2002; Qiu et al., 2014; Yanagawa et al., 2008), or basking in the sun in order to elicit a “behavioral fever” which restricts or kills the infecting pathogen (Blanford and Thomas, 2001; Kemp, 1986; Ouedraogo et al., 2004). These immune responses can severely limit the effects of a pathogen; however some species of Metarhizium have developed traits to more efficiently overcome insect immunity. For example, to increase virulence many of the generalist
  • 26. 26 species of Metarhizium produce cyclic peptides known as destruxins (Roberts and St. Leger, 2004); these insecticidal toxins cause muscle paralysis and suppress immunoresponses in the host (Pedras et al., 2002; Roberts, 1981). These examples represent only a small fraction of the many interactions that take place between insects and Metarhizium; however, they begin to illustrate the complex dance that occurs between host and pathogen. The level of complexity can be even greater when multiple trophic levels of insects are considered. For example, Rännbäck et al. (2015) observed a reduction in parasitoid egg laying when hosts were infected with Metarhizium. While the complexity of these interactions can seem overwhelming, it is highly relevant that we understand what is occurring if we hope to successfully implement BCA to control pest insects. 3.2 Metarhizium ↔ Plants Most land plants form symbiotic relationships with soil fungi (van der Heijden et al., 1998). It is therefore not surprising that several species of Metarhizium have been observed to interact with some plants. A fungal endophyte is defined as an asymptomatic plant-colonizing fungus that lives a portion of its life cycle inside the plant (Behie and Bidochka, 2014a). Metarhizium spp. have a dual life cycle, persisting in the environment both as an insect pathogen and as a facultative saprophyte (Wang et al., 2005). While soil has long been considered a reservoir for naturally occurring Metarhizium, it has not been clear whether the recovered isolates were from dormant conidia in the soil or hyphae actively growing on plant material (St Leger, 2008). Metarhizium has been viewed for many years as only an insect pathogen – the realization that it also actively interacts with plants a b Figure 5. Bioassay survival curves after exposure to Metarhizium brunneum or Beauveria bassiana of (a) Tenebrio molitor larvae and (b) Atheta coriaria after exposure to M. brunneum (Falagiarda, 2014).
  • 27. 27 is both novel and exciting for the field of Metarhizium research. For this reason and because it is of special interest to the research contained in this thesis, I will give a more comprehensive outline of the current status on Metarhizium ↔ Plants interactions. As mentioned in section 2.2.1, specific species of Metarhizium have been shown to associate closely with different plants. In a Canadian-field survey Wyrebek et al. (2011) observed that M. robertsii was the only species associating with grass roots, while M. guizhouense tended to associate with roots of trees and M. brunneum was found in the roots of shrubs and trees. In a similar study Fisher et al. (2011) found that in Oregon, M. brunneum associated strongly with roots of Strawberry and Blueberry plants while M. robertsii and M. guizhouense were isolated predominantly from Christmas tree roots. The plant specificity observed in these two studies could indicate a history of co-evolution between Metarhizium and certain plants. The rhizosphere is a region of soil where plant-root exudate influences soil microorganisms. M. anisopliae s.s was first discovered to be a rhizosphere competent isolate by Hu and St Leger (2002) in a field study designed to evaluate the fate of the BCA after application. In a cabbage field soil samples were collected 4-5 cm from the plant as well as directly next to the taproot. The researchers observed that even after a year the population of M. anisopliae s.s. remained high in the rhizosphere area while it declined over time in the bulk soil (Hu and St Leger, 2002). M. brunneum was also observed to be rhizosphere competent in soilless potting media (Bruck, 2005) and several Metarhizium spp. isolates have been shown to germinate and grow in root exudates (Pava-Ripoll et al., 2011; Wang et al., 2005). Additionally, specialized genes which become active when Metarhizium interacts with plants or plant compounds have been identified; like, the Mad2 gene which is involved in adhesion to plants during colonization (Barelli et al., 2011; Wang and St Leger, 2007), or the Mrt and Mlrnv genes which transport oligosaccharides found in root exudate (Fang and St Leger, 2010; Liao et al., 2013). These studies clearly show that Metarhizium is more than just an entomopathogen but that it has evolved to propagate while interacting with plants. In an effort to determine if Metarhizium had a localized plant-tissue preference when associating with plants in the field Behie et al. (2015) performed a field survey of grasses and forbs in Canada. They found that endophytic Metarhizium spp. (95% of which were M. robertsii) associations were exclusively with plant roots and not with hypocotyl or the stem and leafs of a plant. In vitro laboratory studies with Haricot beans also showed endophytic root preference of Metarhizium spp. (Behie et al., 2015). However, Batta (2013) was able to re-isolate M. anisopliae s.s from untreated leaves, petioles and stems of the oilseed rape plants when other leaves were
  • 28. 28 sprayed with a high dose of M. anisopliae. Furthermore, Golo et al. (2014) observed that cowpea and cucumber plants resulting from M. robertsii or M. acridum inoculated seeds had endophytic association in both the roots and leaves of the plants after 12 days. They further observed the production of destruxins by M. robertsii in the cowpea plants. Akello and Sikora (2012) showed that M. anisopliae s.s. could live for at least a month inside V. faba roots as an endophyte after seeds were soaked for 4 hours in a conidial suspension. Interestingly, in a similar study Akutse et al. (2013) did not observe M. anisopliae s.s. colonizing any part of V. faba plant when seeds were inoculated with a conidial suspension even though the same fungal isolate and plant were used as well as similar methods as Akello and Sikora (2012); although in addition to treating the seeds with conidia (as was done in Manuscript 1), Akello and Sikora (2012) also drenched soil with a conidial suspension, the additional load of conidia in the soil might explain why an endophytic interaction was observed in one and not the other. Further implications regarding the importance of Metarhizium ↔ Plants interactions were suggested when, in a proof of concept study, Behie et al. (2012) showed that nitrogen from a M. robertsii-infected insect can be transferred to a plant via an endophytic hyphal connection. In a follow-up, in-depth study Behie and Bidochka (2014b) tested the insect-derived nitrogen- transferring abilities of five species of Metarhizium [i.e., M. acridum, M. brunneum, M. pemphigi (=flavoviride var. pemphigi), M. guizhouense, and M. robertsii] to four types of plants (haricot bean, soybean, switchgrass and wheat). They showed that all five Metarhizium species had the capacity to transfer nitrogen to plants, although in varying degrees. In addition, they showed that nitrogen was also transferred in the field, despite other competing microorganisms (Behie and Bidochka, 2014b). Metarhizium-plant associations have also been observed to promote plant growth. Sasan and Bidochka (2012) observed that after inoculation with M. robertsii, switchgrass and haricot bean plants both had increased root hair growth. Also, increased plant growth has been observed in several agricultural crops, including soy bean (Khan et al., 2012), tomato (Elena et al., 2011), and corn (Liao et al., 2014). In soy bean, Khan et al. (2012) observed endophytic interaction with M. anisopliae s.l. increased biomass, chlorophyll contents, transpiration rate, photosynthetic rates and leaf area compared to untreated control plants. While wild type M. robertsii, M. brunneum and M. anisopliae s.s. increased many aspects of corn growth, Liao et al. (2014) observed that when Metarhizium genes that are associated with either adhesion to the plant or the utilization of plant exudates were knocked out, no plant growth promotion was observed.
  • 29. 29 The ecological significances of delving in to the complex systems of the Metarhizium ↔ Plants interactions are clear. Furthermore, these fundamental research studies also have important implications for biological control. Nearly all the studies so far have described either beneficial or neutral effects on the plant resulting from Metarhizium associations. Based on the results of these studies, Metarhizium based BCA may do more than just protect crops from insect pests, they may also aid in nutrient acquisitions and plant-growth promotion. However, more research is necessary to illuminate the mechanisms involved in these processes and whether the benefits observed in the laboratory are also seen in the field. Thus far, the majority of the published studies involving Metarhizium ↔ Plants interaction have focused on responses exhibited by the plant. This is most likely because traditionally underlying justification for Metarhizium research has been in the interest of plant protection, however, research into beneficial or deleterious effects to the fungal organism should also be of interest. In Manuscript 1, after demonstrating that species of Metarhizium will disperse along a growing root though soil, I suggest that a possible benefit that Metarhizium derives from associating with plants is mobility and proximity to potential hosts. It is likely that the fungus is also affected in other ways while interacting with plants so it is important that questions like, “Why would a Metarhizium organism give up a limited resource such as nitrogen and what does it receive in return”, continue to be investigated. Metarhizium ↔ Plants interactions are an important part of the future of biological control research. 3.3 Metarhizium ↔ Other microorganisms Very few studies have been conducted to investigate the interaction between Metarhizium and other microorganisms. This is an important aspect of Metarhizium ecology that should be addressed more thoroughly. Metarhizium spp. are abundantly found in the soil environment (Tkaczuk et al., 2014). It is likely that an actively growing saprophyte will have developed antimicrobial strategies to survive and compete in an environment teaming with natural microorganisms. I observed evidence of this while performing isolation on nutrient agar. Inadvertently, a petri plate which had been inoculated with a new isolate of M. flavoviride also became contaminated with a fast growing unknown fungus, however a very clear inhibition zone was present between the M. flavoviride and the contaminate (Figure 6a). Additionally, while developing the methodology for Manuscript 2, media plates with M. brunneum, M. robertsii and M. flavoviride were also inoculated with plugs of Fusarium culmorum. A small zone of inhibition was observed on the plates with M. brunneum and M. robertsii and a much larger zone was present on
  • 30. 30 Figure 6: Inhibition zones observed between Metarhizium spp. isolates and other fungi on PDAY media. (A) M. flavoviride isolate from bait insect on petri plate with un-known fungal contaminant. (B) M. brunneum, M. robertsii, and M. flavoviride on petri plate with Fusarium culmorum. those with M. flavoviride (Figure 6b). Sasan and Bidochka (2013) also observed a zone of inhibition when M. robertsii and Fusarium solani were cultured on the same petri plate and they noted a significant reduction of the colony size of the F. solani. Furthermore, Sasan and Bidochka (2013) showed that liquid media, which had had M. robertsii growing in it but was then passed through a filter to remove all fungal material suppressed F. solani germination. They suggested that this indicates that M. robertsii secretes an anti-fungal compound that inhibits F. solani growth. Krauss et al. (2004) tested the interaction between several entomopathogenic fungi, including two strains of M. anisopliae s.l., and mycoparasitic fungi by completely colonizing a petri plate with the entomopathogen and then placing an agar plug of the mycoparasite on top of the entomopathogen colony. They claimed that because the mycoparasite was not in contact with the media any growth indicated it was receiving nutrients from the fungus and not the media. They found that of the isolates they tested M. anisopliae s.l. was the most susceptible to mycroparasitism and allowed growth of all the mycoparasites tested including Clonostachys byssicola, C. rosea and Lecanicillium lecanii (Krauss et al., 2004). These two studies demonstrate that Metarhizium spp. are both affected by and effectors of other microorganisms. Further studies with regard to the mechanisms involved in these interactions would greatly expand our understanding of the ecology of Metarhizium spp. in the field and identify potential benefits and challenges to their use as BCAs.
  • 31. 31 3.4 Multi-trophic interactions with Metarhizium Each additional trophic level included in a study greatly increases the size and complexity of the experiment necessary to evaluate all the variable permutations. It is therefore not surprising that most studies focus on bi-trophic interactions. Nevertheless, multi-trophic interactions which include three or more levels are necessary to both better grasp what occurs in nature and provide greater predictive power to BCA employment. Generally bi-trophic interactions are concerned with direct effects while multi-trophic interactions must account for both direct and indirect effects. In the following section I will review several Metarhizium studies which evaluate multi-trophic interactions. The trophic levels that I focus on are those of different types of organisms (i.e., other microorganisms, insect and plants); however multi-trophic interactions that involve multiple species of the same type of organism (e.g., insect ↔ insect ↔ microorganism) – as was seen above in the study by Rännbäck et al. (2015) in which interaction between a parasitoid insect as pest insect and M. brunneum were evaluated – should not be neglected. 3.4.1 Metarhizium ↔ Other microorganisms ↔ Insects There are a few examples of tri-trophic interaction involving Metarhizium, other microbes and insects; generally they are geared towards evaluating how the biological control capacity of Metarhizium is affected when other microorganisms are present. Hughes and Boomsma (2004) found that the normally avirulent, opportunistic fungal pathogen Aspergillus flavus would out- compete a highly virulent isolate of M. anisopliae s.l. when applied as a mixture on a leaf-cutting ant host. They explained that alone A. flavus is generally unable to overcome the host immune defenses, however, when coupled with a second pathogen like Metarhizium, which is more adept at suppressing the immune responses, the avirulent fungus is then able to grow much faster and better utilize the pilfered resources. Geetha et al. (2012) demonstrated that co-application of multiple fungi on the same insect host negatively influence the virulence and sporulation of M. anisopliae s.l. Also, Krauss et al. (2004) found that when applied in combination with mycoparasitic fungi on three different species of insects, the virulence of M. anisopliae s.l. was not significantly affected, despite the authors having found that M. anisopliae s.l. was highly susceptible to mycroparasitism. In Manuscript 2, an insect bioassay was conducted in which Tenebrio molitor larvae were inoculated with one of three species of Metarhizium (M. brunneum, M. flavoviride s.s., or M. robertsii) combined with either the mycoparasite Clonostachys rosea or the plant pathogen Fusarium culmorum or both. We observed that, when compared to the individual treatments, for some of the combined treatments there was a slight reduction in virulence; however, in general,
  • 32. 32 virulence remained similar to the individual Metarhizium treatments. Based on this study and the others mentioned it is obvious that interaction which affect the BCA performance of Metarhizium occur when other microorganisms are present. Since microbes are always present in the field these interactions are highly relevant – especially those with other BCA like C. rosea which are also often applied for crop protection. 3.4.2 Metarhizium ↔ Other microorganisms ↔ Plants In light of the recent emphasis placed on Metarhizium ↔ plant interactions, the tri-trophic interaction between Metarhizium, other microorganisms and plants are highly relevant. There have been very few studies which have looked at these interactions. Sasan and Bidochka (2013) found Haricot bean seeds had higher germination when in the presence of M. robertsii and F. solani than when only F. solani was present. The resulting plants also showed significantly less F. solani infection symptoms when M. robertsii was also present. In Manuscript 2 we also evaluate the interactions between Metarhizium (M. brunneum or M. flavoviride), C. rosea, F. culmorum and Winter wheat. We observed that the presence of either of the Metarhizium species did not reduce the F. culmorum infection or hinder the biocontrol efficacy of C. rosea. The two beneficial BCAs may therefore potentially be used in concert against both insect pests and plant pathogens without obstructing each other’s effects. But no additive nor synergistic effects should be expected (Manuscript 2). Several important differences exist between Manuscript 2 and the study by Sasan and Bidochka (2013) which might account for the different observations. For example, the species of Metarhizium and Fusarium that were used are different, as was the plant species and inoculation method; also Sasan and Bidochka (2013) allowed an establishment period for M. robertsii and F. solani in the soil before introducing the bean seeds, where as in Manuscript 2, un-germinated seeds were exposed to F. culmorum first and then Metarhizium spp. before germination. 3.4.3 Metarhizium ↔ Plants ↔ Insects The indirect effects to plants are nearly always implied in Metarhizium ↔ insect studies which have a biological control aspect to them. Nevertheless these indirect effects (i.e. increased plant productivity caused by reduced effects of pest insects after BCA application) are often not measured. One example is a study by Kabaluk and Ericsson (2007) in which they found that corn seeds treated with the M. brunneum BCA F52 (called M. anisopliae by the authors) resulted in an increased yield in comparison to untreated control plants when planted in fields with wireworm. On the other hand, indirect effects may also be observed to occur to the insect when the plant interacts with Metarhizium. For example, Akello and Sikora (2012) showed that aphids feeding on V. faba
  • 33. 33 plants which had been treated as seeds with different fungi including Metarhizium spp. had reduced survival, offspring fitness, development and fecundity compared to untreated control. They demonstrated that Metarhizium spp. isolates along with other endophytic isolates indirectly protected the plant from aphid damage. However, the mechanisms behind this remain unknown. 3.4.4 Metarhizium ↔ Other microorganisms ↔ Plants ↔ Insects The last type of interaction that I would like to mention is that which involves four or more trophic levels. Very few studies have attempted to incorporate so many different trophic levels; in fact I was unable to identify any other studies, involving Metarhizium, which evaluated response variables for four trophic levels. One example, which uses another entomopathogenic fungus (B. bassiana) tested whether insect pollinators could be utilized as delivery vehicles to vector two BCAs to plants in order to control both an insect pest and a plant pathogen (Kapongo et al., 2008). They found in laboratory greenhouse trials that the bees successfully vectored the BCAs to the flowering plants and they observed a reduction in both plant pathogen and insect pest occurrence. Manuscript 2 also describes a system with up to five trophic levels are evaluated. F. culmorum infected seeds were treated with C. rosea, a mycoparasitic fungus which can be used as a BCA against plant disease, and two Metarhizium spp. isolates. After the plants had grown for 14 days the roots were feed to T. molitor larvae which were then monitored for mortality and fungal disease. There were two main response variables assessed in this study, plant disease incidence and insect mortality. Combination effects could be assessed by comparing the data of the combination treatments with those of the non-combined treatments. Prior to this experiment we had several hypotheses on what combination effects we might expect. Metarhizium has been shown to have antifungal properties (Sasan and Bidochka, 2013) and therefore might aid in Fusarium disease suppression. As a potential mycoparasite of Metarhizium spp. (Krauss et al., 2004), C. rosea may severely reduce the ability of Metarhzium to disperse along the root and subsequently infect the larvae. On the other hand, isolates of Fusarium spp. and C. rosea have been observed to be pathogenic to insects (Teetor-Barsch and Roberts, 1983; Vega et al., 2008), and may therefore have an additive effect on insect virulence when in combination with Metarhizium spp. As reported in Manuscript 2, no effect on plant-disease reduction by C. rosea was observed when Metarhizium was added, however when C. rosea was present, alone or combined with Metarhizium spp. there was little to no disease symptoms in the plants; the high efficiency of the C. rosea treatment may however have masked any positive effects of Metarhizium. In most of the treatments there was a significant reduction in virulence to the insect when the seeds
  • 34. 34 had been treated with Metarhizium spp. combined with another fungus. This indicates that there is an indirect effect on insect virulence when other fungi are present. Future studies should evaluate if this effect is due to direct interaction between the combined fungi or due to an indirect effect like resource competition. It is clear however, that multi-trophic interactions are important and may yield unexpected results. 4. Methodology In fulfilling the research requirements of this thesis it was necessary that I develop and test various experimental methodologies and expand my analytical skills. The following section will briefly summarize and describe a few important methodological techniques I worked with. 4.1 Selective media Selective media is often used to assist in the isolation of entomopathogenic fungi. The basic principle of selective media is to provide a substrate that allows the growth of a desired organism while discouraging the growth of others. This can be accomplished in several ways, for example, many bacteria are inhibited by low pH levels while fungi are not. Also the nutrient composition of many growth media favor particular organisms, e.g., Potato dextrose media and Sabouraud dextrose media are better suited for fungal growth than bacterial. While usually not included as a selective medium, insects used for the soil baiting method (Zimmermann, 1986) are essentially a “ready- made” selective medium that screens out any non-pathogenic organisms and selects for those that can infect the host. Media are often amended with more specific growth inhibiting components to further select for desired organisms. Antibiotics, like streptomycin or chloramphenicol, can be added to help to reduce contaminating bacteria. Contaminating fungi are more problematic. For many species of Beauveria and Metarhizium some fungicides, like dodine or cyclohexamide, in low concentration help to eliminate the fast growing contaminants like Rhizopus or Trichoderma while allowing Metarhizium and Beauveria growth (Fernandes et al., 2010b; Rangel et al., 2010) (Figure 7). Many different media have been developed for the isolating of Metarhizium spp.; Table 3 lists some of the major component combinations that have been found effective as well as the studies in which they were presented.
  • 35. 35 Figure 7: Selective media test with several types of media (columns) to evaluate effectivness at supressing naturally occuring microbes while allowing the growth of entomopathogenic fungi. Both natural and Metarhizium “spiked soil” was used (rows). Soil was diluted in ddH2O and then 200µl was pipetted onto each plate. Plate incubated for 21 days at ~21ºC.
  • 36. 36 Table 3: Selective media ingredients that have been observed to be effective for the isolation of Metarhizium spp isolates from soil. All concentratoins represent grams/liter unless otherwise specified. Media Ingredients Veen&Ferron,(1966) Beilharzetal.,(1982) Chaseetal.,(1986) Liuetal.,(1993) Shimazu&Sato,(1996) Strasseretal.,(1997) Hughesetal.,(2004) Shinetal.,(2010) Rengaletal.,(2010) Fernandesetal.,(2010b) Wyrebeketal.,(2011) Rocha&Luz,(2012) Steinwender,(2013) Keyseretal.,(2014b) SelectiveIngredients Dodine 0.065 0.46* 0.01* 1ml* 0.1 0.05 0.02 0.325 0.300 0.29 0.2 Cycloheximide 0.25 0.25 0.05 0.25 0.5 0.06 0.25 Chloramphenicol 0.5 0.5 0.05 0.5 0.2 0.014 0.5 Thiabendazole 0.001 Chlortetracycline 0.005 0.005 Copper chloride (CuCl2) 0.02 Gentamicin Streptomycin 0.6 0.1 1 Penicillin 0.4 Tetracycline 0.05 Crystal violet 0.01 0.01 0.01 0.01 NutrientIngredients PDA X X X X SDA 30% X X X Yeast extract 1 1 X Glucose 10 10 20 Peptone 10 10 10 Oxgall 15 15 Oatmeal infusion 20 20 20 Agar 35 20 20 35 18 20 pH 10+ 6.9 6.9
  • 37. 37 4.2 Bioassay statistics A bioassay is a scientific study with the aim to determine the biological activity or potency of a substance by testing its effect on the growth of an organism. In insect pathology an infection bioassay is a study which evaluates the infectivity of a particular pathogen on a host insect. The ability to cause infection in insects is one of the most basic and interesting traits of entomopathogenic fungi like Metarhizium spp. It is for this reason that many studies of these fungi, among other things include infection bioassays. Infection bioassays are often designed to evaluate pathogenicity, the ability to cause infection, and virulence, the killing power of a pathogen. The most common type of studies that include infection bioassays are those aimed at determining the biological control potential of a pathogen for a particular host. However, infection bioassays are regularly included in other types of studies as a phenotypic trait that can be used to compare different genotypes to each other or measure changes within the same isolate after a mutation or stress. For example Jin et al. (2012) used infection bioassays to show the effects of the Hog1-type mitogen-activated protein kinase gene in M. acridum, or Lopes et al. (2013) who while performing a diversity study of Beauveria spp. and Metarhizium spp. in banana fields included a bioassay and interpreted low virulence to explain the low occurrence of epizootics. For any insect pathologist an understanding of how to appropriately design and execute an infection bioassay is a vital skill. 4.2.1 Experimental design There are several factors that influence the outcome of an infection bioassay; however, the two most important factors are time and dose. Therefore, experiments are usually designed around these factors either as dose-mortality or time-mortality experiments. In a dose-mortality experiment the pathogen dose or number of conidia administered to each host is varied while the time at which the response is evaluated is kept constant. Often in a dose-mortality experiment the response is reported as a value of LD50 or LD90 (dose at which 50% or 90% of individuals exhibit the desired response, i.e., mortality) for a particular treatment. The most important factors are sample size and dose range. It has been recommended that sample size of 120 to 240 insects is necessary to obtain a reliable response (Robertson et al., 1984). Ideally the dose range will include three to eight doses that result in 25 to 75% responses at the time of observations (Robertson et al., 1984), however, the experiment can be checked at several time periods and then the most appropriate observation time for the analysis can be selected afterwards.
  • 38. 38 In a time-mortality experiment, dose is kept constant (or multiple doses are considered separately) and the response is measured over a period of time, often results are reported as LT50 or LT90. To avoid data being correlated in a time-mortality experiment, either different groups of insects must be used for each time measurement so that the data are not correlated or the method of analysis must allow for correlated data. The bioassay performed in Manuscript 1 and 2 were designed as time-mortality experiments with the data for each dose being analyzed separately. I conducted a survey of 30 randomly selected scientific publications selected from a Google Scholar search with the key words “Metarhizium” + “Bioassay” and limited to papers published between Jan 2010 and April 2014. Of these 30 papers, 15 included time-mortality bioassay designs, 7 dose-mortality experiments and 8 with both (Table 4). 4.2.2 Types of Statistical Analyses 4.2.2.1 Abbot correction: Random mortality not related to a treatment but rather experimental conditions being tested in an infection bioassay can be adjusted for in the analysis using the control group. The most common method employed in insect pathology is the Abbot’s formula (Abbot, 1925). The following formula is used to estimate the percentage of insects killed by a particular treatment: 𝑃 = ( 𝐶−𝑇 𝐶 ) ∗ 100. Where P is the estimated percentage of insect mortality due to treatment, C is the percentage dead in the control and T is the percentage of dead in treatment. A few considerations before implementing an Abbot adjustment include: First, the Abbot formula assumes pathogenicity of a treatment, and therefore should not be used if the researcher wishes to determine if a particular treatment differs from the control. Second, if a control has greater mortality than a treatment an Abbot correction will result in negative percent mortality and is not appropriate. Lastly, an Abbot correction cannot be used when performing a survival analysis because the binomial data used for the analyzed is of the individual and not the group. 4.2.2.2 Probit analysis: Infection bioassays are typically analyzed in one of two ways, the first is using a probit analysis model. A probit analysis is most appropriate for dose-response designs but can be used in a time-response design if different groups of insects are used for each time measurement otherwise the data are correlated and the model is not valid; however, examples have been shown where correlated data can be analyzed using a probit transformation of proportion of insects killed (Goettel and Inglis, 1997). Probit analysis is a type of regression used to analyze binomial response variables. It transforms the sigmoid dose-response curve to a straight line that can then be analyzed by linear
  • 39. 39 regression analysis. Probit analysis was first published in 1934 by Chester Bliss, an entomologist working with pesticides and their effectiveness for pest control (Bliss, 1934). Later in 1952 David Finney, a professor of statistics, published a book called Probit Analysis (Finney, 1952). Probit analysis continues to be one of the preferred statistical methods in understanding dose-response relationships. 4.2.2.3 Survival analysis: The survival analysis is historically one of the oldest fields of statistics dating as far back as the 17th century (Aalen et al., 2009). As suggested in the name, a survival analysis traditionally is used to analyze survival or death rates, however today it is often used in engineering to predict failure times of machines. A survival analysis is used to analyze data in which time duration until an event is of interest occurs. The response is often referred to as a failure time, survival time, or event time. One of the primary advantages of using a survival model is that it has the ability to account for censored data, most statistical models don’t. Censored data is data in which the response is unknown in the window of observation, for example, if an experiment ends before all the individuals are dead those individuals are considered censored. An ordinary linear regression model cannot handle censored data. Despite its early beginnings, it was not until the late 1950s that the field of survival analysis was significantly advanced. A publication in 1958 by Kaplan and Meier which allowed a survival curve estimation was presented and then later in 1972 Cox published a method of comparing survival curves (Aalen et al., 2009). Both of these modeling approaches advanced the usefulness and the applicability of survival analysis to the current state. In Manuscripts 1 and 2 bioassays were performed. The experimental design of these bioassays in each study was based on a survival analyses. A Cox proportional hazard model was used to compare the survival curves between the various treatments. This model was most appropriate for the type of data that I collected because it reduced the number of dose-treatments needed, allowed for the same batch of insects to be checked daily and accounted for censored data (insects surviving beyond the time frame of the experiment). Of the 30 studies surveyed included in table 4, 13 used a probit analysis, 8 a survival analysis, 3 used both, and 6 used either another type of analysis or did not specify.
  • 40. 40 Table 4: A survey of experimental design and statistical methods used in 30 studies; selected from a Google Scholer search with key words “Metarhizium + Bioassay”, limited to 2010-2014. Time – Response Method of analysis Host Treatment method Gao et al. (2013) Survival analysis Bombyx mori Larvae immersed in 5 ×10 6 suspension for 1 min or injected with 10µl of 10 6 conidia/ml. 15 insect (×3) checked every 12h. Garza- Hernandez et al. (2013) Survival analysis Aedes aegypti Infection of mosquitos co-infected with virus. 25 adults exposed to filter paper impregnated with 1 × 10 8 conidia/ml. Checked daily until all dead. Goble et al. (2014) Survival analysis Anoplophora glabripennis For 36 individuals the ventral surfaces were pressed on to two agar plugs with conidia for 5 sec. Also, for 24 beetles were submerged for 10 sec in a 15ml suspension of 1 × 10 7 conidia/ml. Checked daily for 50 days. Howard et al. (2010) Survival analysis Anopheles gambiae 25 adult mosquitos were placed in a tube with netting that had been treated in a fungus. Checked daily until all fungal treated individuals died. Jin et al. (2012) Method not specified Locusta migratoria Adults were either: dipped up to the head-thorax junction in a soybean-oil suspension containing 1×10 7 conidia/ml; or, injected with 5 µl of a 1 × 10 6 conidia/ml in the haemocoel cavity through. Twenty insect per rep, checked every 12h. Lopes et al. (2013) Survival analysis Cosmopolites sordidus Adults were submerged in conidial treatment of 1 × 10 8 conidia/ml for 90 sec. Sixteen-eighteen insects per treatments, mortality assessed every other day for 14 or 16 days. Orduno-Cruz et al. (2011) Logistic regression Metamasius spinolae Twenty adults were immersed in 500ml suspension with 1 ×10 8 conidia/ml for 10 second. Checked for 41 days. Peng et al. (2011) Survival analysis Anoplophora glabripennis Females exposed to fiber bands impregnated with fungi. Males held on conidia-covered surface for 30 seconds. Checked daily for 50 days. Quinelato et al. (2012) Probit analysis & non-parametric Kruskal–Wallis test Rhipicephalus microplus Larvae (~100/tube) were immersed in 1.2 × 10 8 , 10 7 , 10 6 and 10 5 conidia/ml suspensions for 3 min. The suspension was absorbed out of test tube, mortality checked every 5 days for 30 days. Mortality was estimated as a percentage. Quintela et al. (2013) Probit analysis and Survival analysis Tibraca limbativentris Adults were inoculated with 10µl of 5 × 10 7 conidia/mL suspension on the dorsal region. Checked daily for 12 days. Reyes- Villanueva et al. (2011) Survival analysis Aedes aegypti Females exposed to filter paper impregnated with 6 × 10 8 conidia/ml for 48 hours and checked for survival. Also, females exposed to exposed males that had been exposed to impregnated filter paper for 48h and watched for survival. San Andrés et al. (2014) Probit analysis Ceratitis capitata Adults exposed for 30s to an “infective dish”, a dish with 3.1 × 10 8 conidia spread uniformly over the dish. 10 host per treatment checked daily for 10 days. Tavassoli et al. (2012) General linear model and repeated measure analysis Ornithodoros lahorensis Eggs, larvae, and adult ticks were immersed in either 1 × 10 5 or 1 × 10 7 conidia/ml for 5s. Survival was checked every 3 days for 21days. Wang et al. (2012) Survival analysis Bombyx mori & Locusta migratoria Silkworms injected with 10 μL of suspension of 5 × 10 5 conidia/ml, locust injected with 10 µl of 1 × 10 7 . Ten insects in each repetition. Mortality checked every 12 hours. Wang et al. (2014) Probit analysis Galleria mellonella & Tenebrio molitor Each individual larva (30 per repetition) was sprayed with 1 ml of suspension of 10 7 conidia/ml. Monitored daily for mortality.
  • 41. 41 Dose – Response Ansari et al. (2010) Probit analysis Culicoides nubeculosus Eggs suspended in 10 8 conidia/ml until hatch. Larvae suspended in 10 4 , 10 5 , 10 6 , 10 7 , or 10 8 conidia/ml – checked for 3 days. Contreras et al. (2014) Probit analysis Tuta absoluta Eight pupae suspended in one of six concentrations (0.34- 11.15 × 10 9 viable conidia per liter), checked every 3 days for up to 3 weeks. Leemon and Jonsson (2012) Probit analysis Rhipicephalus microplus & Lucilia cuprina Six fungal concentrations ranging from 1 × 10 9 to 1 × 10 4 conidia/ml. Ticks were either topically inoculated with 2µl or submerged in inoculum. Blowflies were either topically treated with 2µl suspension or fungi was mixed with food source. 20 insects per treatment, checked for 10 days. Luz et al. (2011) Probit analysis Anopheles gambiae and A. arabiensis Eggs were topically treated with 5 × 10 6 conidia/cm 2 . Also, eggs in soil treated with oil formulation of 10 5 , 3.3 × 10 5 , 10 6 , 3.3 × 10 6 , and 10 7 conidia/cm 2 . Checked every 5 d for 30 d. Nussenbaum and Lecuona (2012) Probit analysis Anthonomus grandis Lots of isolates tested by submerging adults for 15 sec in a 5 × 10 8 conidia/ml, checked daily for 20 d. Also, 40 individuals immersed treatments of: 1 × 10 6 , 5 × 10 6 , 1 × 10 7 , 5 × 10 7 , 1 × 10 8 and 5 × 10 8 conidia/ml; checked daily for 15 d. Zayed et al. (2013) Probit analysis Phlebotomus papatasi Suspension of 1 × 10 6 , 5 × 10 6 , 1 × 10 7 , 5 × 10 7 , 1 × 10 8 , and 5 × 108 conidia/ml prepared. 0.5g of each was mixed with 0.15g finely ground larval diet. 10 larvae added to each vile. 9 replicates. Mortality based on failure for adult to emerge. Behle and Jackson (2014) Method not specified Alphitobius diaperinus Larvae were exposed to treated soil. Conidial treatment ranged from 1.69 × 10 7 to 2.08 × 10 5 conidia/ml. 30 larvae per dosage was used. Mortality was monitored for 14 days. Both Dose and Time Response Jin et al. (2011) Method not specified Nilaparvata lugens Thirty-forty nymphs were sprayed at 5 different rates on with one of three fungal concentrations (2 × 10 8 , 2 × 10 7 and 2 × 10 6 conidia/mL). Checked daily. Kirubakaran et al. (2013) Probit analysis and ANOVA Cnaphalocrocis medinalis Leaves sprayed with 5ml of 1 × 10 3 , 1 × 10 4 , 1 × 10 5 , 1 × 10 6 , 1 × 10 7 and 1 × 10 8 conidia/ml in water or oil formulation, 20 larvae exposed to treatments, checked daily for 8 days. Maldonado- Blanco et al. (2013) Probit analysis Aedes aegypti Submersion of 25 larvae in fungal concentrations of 1 × 10 5 , 3 × 10 5 , 5 × 10 5 , 8 × 10 5 or 1 × 10 6 submerged spores/ml. Insects checked daily for 5 days. Mishra et al. (2011) Probit analysis Musca domestica One ml fungal suspension of 10 3 , 10 5 , 10 6 , 10 7 , and 10 9 conidia/ml were applied to fly diet. Checked daily for 5 days. Also done in an arena setup. Ortiz-Urquiza et al. (2013) Probit analysis and Survival analysis Ceratitis capitata & G. mellenella Adult flies were treated with 1 of 6 spore concentrations from 10 3 - 10 8 conidia/ml by spraying. Galleria larvae treated by injecting 8µl of suspension. 10 insect per treatment. Shan and Feng (2010). Method not specified Myzus persicae A leaf with 40 adult aphids was sprayed with 1 ml suspension, 1 × 10 6 , 1 × 10 7 and 1 × 10 8 conidia/ml), mortality was checked daily for 8 days. Vázquez- Martínez et al. (2013) Probit analysis Anopheles albimanus Larvae were placed in cups with 200 ml of 2.6×10 7 conidia/mL. Checked daily until dead or adult emergence. Adults were immobilized with cold and inoculated with 5µl 2.7×10 8 conidia/mL suspension. Checked daily for 7 days. Yousef et al. (2013) Probit analysis and Survival analysis Bactrocera oleae Adults were treated with 1 ml of a 1.0 × 10 8 conidia/ml by spraying. Checked daily for 12 d. Two assays for puparia. 1. 1 ml of a10 8 conidia/ml added to 30g soil with 63 puparia; 2. Puparia immersed in fungal suspension for 10 seconds.
  • 42. 42 v. Conclusion and future perspectives The use of living organisms to control pest insects is an important part of current and future crop protection. Understanding the fundamental ecology of these organisms is vital to their success as BCAs. For example, research regarding their natural occurrence and effect on host populations greatly enhances their potential for more efficient utilization in pest regulation; e.g., conservation biological control. Additionally, research which investigates how these organisms interact with other organisms, viz. plants, plant pathogens and other BCAs, is crucial to predicting their effectiveness and maintaining consistency as BCAs. This thesis advances the current scientific knowledge regarding the ecology and biological control use of Metarhizium spp. fungi in several areas, namely:  M. flavoviride is the dominant species of the Metarhizium community found in some agroecosystems in Denmark.  Significant intra-species diversity exists within the understudied species M. flavoviride.  M. brunneum, M. flavoviride and M. robertsii, applied as a seed treatment of conidia, will disperse through soil with a growing plant root. Furthermore, these species of Metarhizium will maintain pathogenicity to insects while interacting with plant roots.  Dual BCA seed treatments of C. rosea and M. brunneum or M. flavoviride will control F. culmorum infection in wheat seedlings.  A reduction in virulence occurs when M. brunneum or M. flavoviride and C. rosea are applied as a seed treatment on F. culmorum infected plants. However, virulence to T. molitor was still observed at a significant level when compared to untreated controls. Perhaps one of the most valuable components of any research is not the conclusion provided but rather the new questions that remain unanswered. Based on the observations of this thesis there are several research questions that I think should be addressed, including:  Worldwide survey studies that endeavor to elucidate the distribution and occurrence of Metarhizium spp. in agriculture. These studies should emphasize habitat associations as well seeking to understand what characteristics promote abundance in particular areas. Highly important to these studies will be the continued development of molecularly based ecological tools, like isolating SSR markers (microsatellites) that can explicitly discriminate genotypic diversity within the M. flavoviride species.