Microbial endophytes are microorganisms that live within plant tissues without causing disease or harm. They have been isolated from many plant species worldwide and show diversity across environments. Endophytes are isolated from surface-sterilized plant tissues and identified based on genetic sequencing. Bacterial endophytes provide benefits like nitrogen fixation, plant growth promotion, biocontrol of pathogens, and abiotic stress resistance. Fungal endophytes also produce secondary metabolites and biocontrol pathogens. Future work aims to develop endophytes as biofertilizers and biopesticides through improved isolation techniques and delivery methods.

1. Microbial
Endophytes
By :Iqra Malik

2. ENDOPHYTES ???
The word endophyte came from two Greek
words, “endon” = within “phyton” =plant.
Microorganisms, which colonize symptomless
in living plant tissue without causing any
immediate, overt, negative effect on the plant .

3. Microbes which occur
within plant tissue for at
least part of their life
cycle without causing
disease under any known
circumstances.

4. DIVERSITY OF
ENDOPHYTES
• Endophytes are widespread and can
survive in tropical, temperate and arid
areas -
• The first report related to the isolation of
endophytic fungi from tropical host plants
belonging to the families Araceae,
Bromiliaceae and Orchidiceae was made
by Petrini and Dreyfuss (1981).
• In an extensive survey of fungal
endophytes in four cultivars on winter
wheat, Sieber et al. (1988) reported
Fusarium culmorum and F. graminearum
as endophyte in all the parts of plant,
except leaf and glumes.

6. Isolation of Endophytes
Collection of plant sample for isolation
• The bacterial and fungal endophytes are
isolated from roots, stems and leaves of the
healthy plants.
• The isolation is done from the plant
immediately after collection.
• The plant samples are washed in running tap
water for 10-15 mins. to remove adhering soil
particles, air-dried and roots, stems and leaves
are separated out.
• The separated plant roots, stems and leaves
are weighed up to one gram on a weighing
balance. And samples are then soaked in
distilled water and drained.

7. Steps
• The samples are then surface-
sterilize by dipping in 70%
ethanol for 1 minute.
• Stems and leaves are surface sterilized with
4% sodium hypochlorite for 5 minutes and
• roots with 2% sodium hypochlorite for 10
minutes and then treated with 70% ethanol
for 30 sec followed by rinsing five times in
sterilized distilled water.
• The surface sterilized samples are then
blot-dried using sterile filter paper.

8. Isolation of Fungal Endophytes
• The surface sterilized samples are
macerated in one ml of distilled water in
pestle and mortar.
• For each macerated sample that is roots, stems
and leaves serial dilutions are made up to 10-5
dilutions.
• 100μl from each dilution of the respective sample
are then poured in their respective petri plates so
labeled from 10-1 to 10-5 containing Potato
Dextrose Agar Medium and then spread with
spreader for the isolation of fungal endophytes.
• The plating is done in triplicate for each dilution.
The plates are then incubated at 28°C for two
weeks.

9. Isolation of Bacterial Endophytes
• The surface sterilized samples are macerated
in one ml of sterile distilled water in pestle and
mortar.
• For each macerated sample that is root, stem and
leaves serial dilutions are made up to 10-5 dilutions.
• 100μl from each dilution of the respective sample
is then poured in their respective petri plates so
labeled from 10-1 to 10-5 containing Nutrient Agar
Medium and then spread with spreader for the
isolation of the bacterial endophytes.
• The plating is done in triplicate for each dilution.
The plates are then incubated at 37°C for 72 – 96
hours.

10. Identification of Endophytes
• Endophyte bacteria and fungi isolates preparation
• All isolates used in the study are collected in
micro tube 1.5 mL.
• All isolates re-cultured in Nutrient Agar (NA) media
in petri-dish by streaking the collection to the
plates and
• incubated for 72 h in room temperature.
• The growth culture was then regrowth in the
same media and incubated for 48 h. The pure
• colony growth in the plates then used for further
study.
• Bacteria endophyte isolates are identified based
on its 16SrRNA gene & 18SrRNA gene in case of
fungal endophyte

11. Culturing – isolation and identification

12. Crop
and Colletotrichumgraminicola
Endophytes Activity
Nitrogen fixation, production o f IAA, siderophores,
lytic enzymes. Antagonistic to the pathogenic
References
Corn
(Zea mays L.)
Bacillus spp.
fungi Fusarium
Colletotrichum
verticillioides,
graminicola,
Bipolaris maydis, and Cercospora
zeae-maydis
Zecchin et al. (2014)
Enterobacter spp.
Nitrogen fixation
IAA production,
Turmeric
(Curcuma longa L.)
Bacillus
thuringiensis,
cereus,
Bacillus
Bacillus solubilization antagonism against
sp., Escherichia coli, Klebsiella
Bacillus pumilus, Pseudomonas pneumoniae, and some of the
putida, Clavibacter michiganensis
phosphate
fungi like Fusarium solani and
Alternaria alternata
Kumar et al. (2016)
Black pepper
(Piper nigrum L.)
P. aeruginosa, P. putida, Bacillus
Antagonistic
megaterium
to Phytophthora
capsici, the causal agent offoot rot
of black pepper
Aravind et al. (2009)
Banana
(Musa spp.)
B. amyloliquefaciens, B. subtilis Antagonistic
subsp. subtilis, B. thuringiensis
activity against
Fusarium oxysporum f.sp cubense Souja et al. (2014)
BACTERIAL ENDOPHYTES

13. Crop Endophytes Activity References
Sugarcane
(Saccharum officinarumL.)
Trichoderma virens
Antagonistic against pineapple
disease pathogen, Ceratocystis
paradoxa, owing to the
production ofendochitinases
RomaoDumaresq et al. (2012)
Aspergillus niger,
Trichoderma atroviride,
Alternaria sp.,
Annulohypoxylon stygium,
Talaromyces wortmannii
Excellent producers of
hydrolytic enzymes
(hemicellulases and related
enzymes) to be used as part of
blends to decompose
sugarcane biomass at
industrial level
Robl et al. (2013)
Tomato
(Lycopersicum esculentum)
Chinese cabbage
(Brassica campestris)
Scolecobasidium humicola
Improve plant growth under
organic nitrogen conditions
Mahmoud and Narisawa
(2013)
Cotton
(Gossypium hirsutum)
Drechslerella dactyloides,
Exserohilum rostratum
Alternaria tenuissima
Epicoccum nigrum,
Acremonium alternatum
Cladosporium
cladosporioides, Chaetomium
globosum, Paecilomyces sp.
Antagonists against plant
pathogens
Ek-Ramos et al. (2013)
FUNGAL ENDOPHYTES

14. Benefits of endophytic bacteria
• Bacteria are able to trigger signaling
pathways to produce extracellular
metabolites with higher toxicity for other
microorganism that leads to destruction of
higher pathogen, called induced systemic
resistance(ISR).
• Myriad of bacteria has been documented for
beneficial effects, alleviation of several abiotic
and biotic stresses. Pseudomonas and Bacillus
sp., have been studied as potential candidate to
provide ISR to plants.

15. Benefits
• A compatible host plant is
necessarily needed for successful
colonization.
• An endophyte Azoarcus sp. Strain BH72
expressed Nif genes in rice roots evaluated
using proteomic approaches and jasmonic
acid treatment to dissect rice roots
responsed for colonization (which induces
plant defense proteins).

16. Secondary metabolites production by fungal endophytes
• Epicorazines A-B
• Flavipin
• Epipiridones
• Ambuic Acid
• Cordycepsidone
• Colletonoic acid
• Anti-bacterial, anti-fungal and anti-algal
activities
• Biocontrol activity

17. FUNCTIONS OF BACTERIAL
ENDOPHYTES
Biocontrol
•Antibiotics production
•Hydrolytic enzymes production
•Siderophore production
•Induced Systemic Resistance (ISR)
•Exopolysaccharide production

18. Functions
Phytostimulation
•Indole acetic acid production
•Ehylene production
•Cytokinin and giberrelins production
Biofertilization
•Nitrogen fixation
•Phosphate solubilization
•Siderophore production

19. How Endophytes attack Plant Pathogens:
Mechanisms
Ecological effect
 Occupation of ecological niche
 Hyperparasites and predation
Direct inhibition of plant pathogens
 Antibiotics production
 Secretion of Lytic enzymes
Indirect inhibition of plant pathogens
 Induction of plant resistance
 Stimulation of plants secondary metabolites
 Promotion of plant growth and physiology

20. NEED &ATTRIBUTES OF
ENDOPHYTES
• The endophytes must possess
following attributes for agricultural
exploitation-
• They must not induce plant disease,
• Should be capable to spread inside
plant parts,
• Should be culturable
• Must colonize plant parts naturally
obligately with species specificness.

22. Future prospects
• Development of specific culture
medium to isolate endophytic microbes
• Isolation and identification of more efficient
endophytic plant growth promotion and plant
disease microbes for management
• Detailed studies on multiple interactions between
endopohytes, plants and pathogens
• Appropriate delivery methods need to be
developed and tested for agronomic use