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1. Soil fungal communities as indicator of
soil health in fruit tree orchards
Luisa Manici
Consiglio per la ricerca e la sperimentazione in agricoltura
(CRA)
(Research centre for industrial crops, Bologna, Italy)

2. Background: crop yield decline
European intensive cultivation systems, such as orchards,
vineyards, vegetable and cereal crop are affected by yield
decline of which root rot fungal complex is the main biotic
cause
Complex of soil-borne fungi: Pythium, Rhizoctonia,
Cylindrocarpon, Fusarium, etc.
These pathogens survive on organic residues in soil and
continuous cropping systems (fruit tree orchards, specialized
horticultural crops) can build their inoculum leading to a
progressive soil sickness.
Diagnosis of yield decline/replant disease if very difficult
because clear symptoms are evident solely when infection
level is severe. In any case, they are more evident on young
or weak plant (i.e. after abiotic stress).

3. Yield decline
Root rot caused by Pythium e Fusarium solani
on horticultural crops after monocropping
rotation
Rotation_wheat
Monoculture
monocropping

4. Yield decline
Black root rot of strawberry.
Rhizoctonia, Cylindrocarpon, Pythium
and other weak pathogens

5. Background:
yield decline-replant disease in fruit
tree crops
The decreasing trend of EU fruit production in the last twenty
years has several causes; however replant disorders due to the
eco-functional intensification of growing areas specializing in fruit
production in one of the main causes of this decline.
The biotic component of replant disorders or “soil sikness” of fruit
tree crops have been definitively elucidated in ’80s with the first
application of soil fumigants on replanted orchards.
• May, W. F., Abawi, G. S., 1981. Controlling replant diseased of pone and stone fruit in
northern United States by preplant fumigation Plant Dis. 65, 859-864.
• May, W. F., Merwin, I. A., Abawi, G. S., 1994. Diagnosis, etiology and management of
replant disorders in New York cherry and apple orchards. Acta Hort. 363, 34-41.
• Browne, G. T., Connell, J. H., and Schneider, S. M. 2006. Almond replant disease and
its management with alternative pre-plant soil fumigation treatments and rootstocks.
Plant Dis. 90:869-876

6. Background:
yield decline in fruit tree crops
Pathogenicity of root rot fungal agents ranges in severity
from decreased growth rates to lethality. This depends on
• Specialization level of the pathogens toward the host crop,
which can widely vary.
• plant vigor, physiological state of plants, abiotic factors and
the general status of soil fertility.
The average increase of plant growth on disinfected soil
as compared to replanted soil, is 42%, as inferred from a
recent bioassay on 9 different apple orchards using M9
rootstock (preliminar results of BIO-INCROP project).

7. Background:
yield decline in fruit tree crops
Apple seedlings
control Growth reduction
on replanted soil
Growth reduction of apple seedling on replanted soil using peat as control

8. Plant/pathogens interaction
and Replant disease *
Case Study: Simulation of a post transplant period in South
Tyrol, performed in pot on soil samples taken from five third-
generation orchards, using M9 rootstock plantlets
The trial aimed at
• To evaluate plant response immediately after transplanting on
apple replanted orchards and define soil health indicators.
• To evaluate the effectiveness of replanting in “inter-row” to
minimize replant disease in apple orchards
*Kelderer; Manici; Caputo; Thalheimer 2012 Planting in the ‘inter-row’ to overcome replant disease in apple orchards: a
study on the effectiveness of the practice based on microbial indicators. Plant and Soil 357, 381-393

9. Indicator : Plant growth
Bioassay with
clonal M9
rootstocks
Trial performed at
Laimburg
Research Centre
(BZ,Italy ) in April
2010
Plant growth=
sum of length of
shoots

10. Indicator: Root health
3
9
27 81
Visual scale of four classes of root heath with geometric progression

11. Relationship between plant heath
indicators
Plant growth vr Root health:
Coef. Corr.= 0.82; R2= 66.98; P>0.01.
Plant growth is a good indicator of soil healt in
replanted orchards, evaluated in greenhouse
bioassay

12. Plant growth
Mean separation test (95%) among 5 orchards
and control (fallow)
ANOVA sign P>0.01 29 d
25
Plant growth (cm) 21 bc ab
a
17 a a
13
9
1 2 3 4 5 Fallow
control
Orchards
The general soil health status of replanted orchards is lower
then that of the correspondent fallow control.

13. Comunities of root fungal endophytes
sites 1 2 3 4 5
species R IR R IR R IR R IR R IR F
Cylindrocarpon obtusisporum
Cylindrocarpon olidum
Rhizoctonia sp. AG- F
Rhizoctonia solani AG-6
Phoma sp.
Alternaria alternata
Fusarium culmorum
Rhizoctonia sp. AG-P
Humicola grisea
Fusarium acuminatum
Fusarium compactum
Fusarium oxysporum
Cylindrocarpon destructans
Fusarium solani
Fusarium equiseti
Trichoderma sp.
Rhizoctonia sp. AG-A
Fusarium lateritium
Rhizoctonia sp. AG-G
Fusarium semitectum
Abundance classes: <5% 5-10% <10-25% <25-50%

14. Pathogenicity/
functionality of root
endophytes
interaction plant
endophyte of 3
population of root
endophytes ( 74
isolates) was evaluated
by growing M9
plantlets on artificially
inoculated peat.

15. Pathogenicity/functionality of root endophytes
Scatterplot by Level Code
6,2
Dray weight (4 plants)
Mutual
5,2
Commensal
4,2
3,2 Parassitic
2,2
Cylindroc Rhizoctonia Control Fusarium sp
This figure shows the variability of plant-endophyte relationship and
suggests its potentiality in soil health.

16. Soil suppressiveness
Soil suppressiveness is the natural ability of soil to control pest
and pathogens of plants
The two main components of soil suppressiveness are:
1. Microbial biomass (SOM and evapotranspiration)
2. Microbial diversity (cropping practices)
• Functional diversity, by increasing several beneficial mechanisms:
• by increasing indigenous antagonists to soil borne pathogens and
nematodes
• by protecting roots with mutual relationship (antagonism toward
pathogens e.i competition for colonization, antibiosis toward
pathogens).
• by increasing plant vigor with positive effects such as plant growth
promotion, inducing acquired resistance etc.

17. Soil suppressiveness - Biomass
Case study on comparing soil health in conventional and
organic apple orchards replanted orchards <50 years in
apple growing area of South Tyrol*
Parameters:
• Growth test with apple seedling assay
• Fungal communities (endophytic fungi on apple seedling after
bioassay)
• Soil chemical analysis (N, K, humic substances, TOC etc)
* Manici L. M., Ciavatta C., Kelderer M., Erschbauber G.2003Replant problems in South Tyrol: role of fungal pathogens and
microbial populations in conventional and organicapple orchards. Plant and Soil 256, 315-324

18. Conventional Control Organic
• Growth score significantly differred for management: organic >conventional
• Root endophytic communities did not differ (Rhizoctonia sp. Cylindrocarpon
spp. and Pythium spp. and Fusarium spp. were shared by both cropping
systems)

19. Conventional Organic
Growth stcore
14 (Growth score)
12
10
8
6
4
2
0
Sites
1 2 3
CFU g-1 soil
60000 Coeff. Corr =0.94**
50000
Total fungi in soil ( MIC)
40000
30000
20000
10000
0
1 2 3
Growth score and soil inhabiting fungi significantly differred both for site (3)
and management (organic>conventional) and they were highly corelated

20. Plant growth/soil helath- Biomass
Root
COL MIC TOC HA+FA Nmineral
% cfu g-1 soil % %
Growth -0.8* 0.94** -0.023 -0.03 -0.04
score
Total fungi -0.73 1 0.09 0.01 -0.13
(MIC)
Colonization 1 -0.73 0.5 0.5 0.02
frequency
(COL)
** P>0.01 * P>0.05
MIC, indicator of fungal biomass was the only parameter positively
and significantly related to apple growth score and negatively
correlated to root colonization. TOC, SOM, salinity, HA+FA and N
availability did not result correlated with GS.

21. Soil suppressiveness - biodiversity
Case study: soil fungi as indicator relationship between soil
biodiversity on soil health in fruit tree crops*
Two cropping systems were compared :
Peach: sod system with permanent vegetative cover and massive
animal manure before planting new orchards
Horticultural: continuous cropping based on chemical fertilization.
Soil health for peach (target crop) was evaluated in a
greenhouse bioassay with micropropagated plantlets of
peach rootstock GF677
Manici, L. M.; Caputo, F. 2010 Soil fungal communities as indicators for replanting new peach orchards in intensively
cultivated areas. European Journal of Agronomy 33: 188-196

22. Location: Piana del Sele (Campania region), an
intensively grown area, southern Italy, SOM
varying from 1.8 to 2.5 %

23. Comparison of biodiversity
Indicator: root endophytic fungal communities
Indicator:
Diversity profile, graphical method two compare biodiversity of two communities
(endophite fungal comm. in Peach and horticultural sites)
27
24
21
18
Peach >
Diversity
15 Soil from Peach Syst Horticultural
12
9
6
3 Soil from Horticultural Syst
0
0 1 2
alpha
alpha=0, this function gives the total species number.
alpha=1 gives an index proportional to the Shannon index, (richness)
alpha=2 gives an index which behaves like the Simpson index. (eveness)

24. Fungal community composition of root endophytic fungi
Functional meaning of higher biodiversity
P1 P2 P3 P4 P5 H1 H2 H3 H4 H5
Fusarium semitectum
Trichoderma aureovoride
Trichoderma harzianum
Others
Mucor hiemalis
Fusarium acuminatum +
Pythium sp.
Penicillium sp
Macrophomina phaseolina
Humicola grisea
Aspergillus ochraceus
Chaetomium funicola
Alternaria sp
Phoma medicaginis
Fusarium compactum
Fusarium equiseti
Fusarium oxysporum
Fusarium dimerum
Fusarium solani
Cylindrocarpon destructans * +
Sterile fungus 1
Rhizoctonia sp.
Mortie rella sp
Phomopsis sp.
Penicillium semplicissimum
Thielaviopsis basicola* +
*root Pathogen of horticultural crops

25. Pathogenicity/functionality of root endophytes
Thielaviopsis basicola
GF677 grown on peach soil 20 GF677 on hoticolt. soil.
No pethogenic species 20 Growth reduction if
Growth score
15
compared to peach samples
10
5
0
Thie. 7 Thie. 3 Thie. 2 Thie. 1 Thie. 4 control
Cylindrocarpon destructans
20
Growth score 15
10
Thilaviopsis basicola e
Cylindrocarpon destructans
5
0
Cyl. 17 Cyl. 10 Cyl. 19 Cyl. 9 Cyl. 13 control

26. How to induce soil suppressiveness in
organic fruit tree orchards
By increasing microbial biomass and microbial diversity
with their multifunctional actions.
By reducing the specialization level of root rot fungal agents
toward the crops
• Break before replanting new orchards
• Use of cover crops to reduce specialization of soil borne
fungi and pests toward the host crop
By increasing plant vigour (root emission)
• Plant breeding for rootstock improvement
• agronomic tools (improving nutrition, physical aspects and
increasing growth promoting compounds such as humic
acids)
INNOVATIVE CROPPING PRACTICES

27. BIO-INCROP, a project on organic fruit
tree cropping systems
General aim: exploitation of natural resources to
increase soil health
Research Actions based on
1. Biological resources indigenous to the orchard soil
system (beneficial microbial communities)
2. Natural resources exogenous to orchards (waste-
deriving material, bio-formulates, cover crops)
Output: Innovative cropping practices developed on
response of microbial factors involved in
suppressiveness and biological fertility of soil

28. Thank you for your
attention