Exploitation of endophytic fungi for plant disease management

Division of Entomology
ICAR- Indian Agricultural Research Institute
New Delhi
Seminar Leaders: Dr. Robin Gogoi
Dr. M.S. Gurjar
Chairman: Dr. Bishwajeet Paul
Speaker: Devendra K. Meena
11487
Exploitation of endophytic fungi for plant
disease management
Credit Seminar: Pl PATH 691

 Introduction
 Plant- Endophytic fungi interaction
 Diversity of endophytic fungi in plants
 Colonization
 Endophytic fungi : Mechanism
 Case studies
 Conclusion
 Future aspects
Outline of Seminar

 Endophytes represent an endosymbiotic
association of microbial population intricately
associated with plants either intercellularly or
intracellularly.
The Concept of
 Endophytes & term coined by the German
botanist, Anton de Bary in 1886 to describe
microorganisms that colonize internal tissues of
stems and leaves.
 Endophytes have evoked great interests among
scientists around the world in the 1970s, such as the
discovery of the clavicipitaceous endophytic
fungus Neotyphodium coenophialum, which is
toxic to cattle fed with tall fescue.
Introduction
Anton De Bary
(Cline et al., 2018)

Plant- Endophytic fungi interaction
(Yan et al., 2019)

 The host plant bearing endophytic
community may occupy competent,
obligate, passenger types of
microbial population or residing in
facultative association.
 Among fungi, the members of
different phyla such as
Ascomycota, Basidiomycota,
Mucoromycota, and Oomycota
have been identified as endophytic
from various crops. Figure: Abundance of endophytic fungi
belonging to diverse phyla isolated from
various plants. (Rana et al., 2019)
Diversity of endophytic fungi in plants

Figure: Distribution of predominant genera of
endophytic fungi isolated from various host
plants.

Figure: Relative abundance of endophytic fungi in the root of Dendrobium
moniliforme after isolation in synthetic media. (Shah et al., 2018).

 The endophytic fungi colonization in host
plants could occur inside leaf segments,
petioles, root tissues, seeds, fruits, buds,
over stems, and/or sometimes
inflorescences of weed plants.
 Many species of fungi colonize on living
bark or twigs and small branches of
conifers and broad leaved trees.
 Live either intercellularly or
intracellularly within plant tissues.
Colonization Mechanism
(Mengistu, 2020)

Schematic representation of the colonization strategies of fungal endophytes.
(Fesel and Zuccaro, 2016 )

Endophytic fungi in disease resistance (Latz et al., 2018)

(Yadav et al., 2015)
 Total of 18 endophytic fungi were isolated from leaves, stem and root of
Ocimum sanctum and Aloe vera .
 On the basis of different morphological characterstics isolates were
categorized.
 The results of the antifungal activity showed that AVR1 and AVR3 were
able to suppress mycelial growth of F. oxysporum by producing antibiotic
compounds (inhibition zone ).

Antibiotics produced by fungal endophytes
Fungal endophyte Host Compounds Activity References
Acremonium zeae Maize Pyrrocidines A, B Antifungal Wicklow et al.
(2005)
Verticillium sp. Chinese
foxglove
Massariphenone, ergosterol
peroxide
Antifungal You et al.
(2009)
Phomopis cassiae Cassia
spectabilis
Cadinane sesquiterpenes Antifungal Silva et al.
(2006)
Muscodor albus Tropical tree Tetrohydofuran, 2-methyl
furan, 2-butanone, aciphyllene
Antibacterial Atmosukarto
et al. (2005)
Pestalotiopsis
maculans
Euphorbia
hirta
Citreoisocoumarin, paxilline,
nigricinol, sceptrin, cladosporin
Antibacterial
antifungal
Akpotu et al.,
2017
Chaetomium
globosum
Ginkgo
biloba
Chaetomugilin A and D Antifungal Qin et al.,
2009

Figure: Light micrographs of the hyphal interactions between three endophytic fungi
and different soil borne pathogenic fungi in dual cultures.
 A Hypha of Choiromyces aboriginum (Ca) coiled around hyphae of R. solani (Rs).
 B Coiling around a hypha of P. aphanidermatum (Pa) by C. aboriginum (Ca).
 C Growth of Stachybotrys elegans (Se) within a hypha of P. aphanidermatum (Pa).
 D Hyphae of Cylindrocarpon sp. (C) inside hyphal cells of P. aphanidermatum
(Pa).

Plant immune defense system Yen et al., 2019
MTI ETI

 Many endophytic fungi produce and release lytic
enzymes that can hydrolyze a wide variety of polymeric
compounds, including chitin, proteins, cellulose etc.
 β-1,3glucanases, chitinases, proteases etc, are the
examples of lytic enzyme produced by endophytic
microorganisms.
 For instance: Trichoderma virens, antagonistic against
pineapple disease pathogen, Ceratocystis paradoxa,
owing to the production of endochitinases.
(Dumaresq et al., 2012)
Lytic enzyme secretion

Mitigation of salinity stress through application of phytohormones using
endophytic fungi (Hamayun et al., 2017)
Endophytic fungi in stress tolerance

Endophytic fungi Isolation source Biological roles
Alternaria alternate Leaves of potato Produced IAA & GA
Aspergillus fumigatus,
Phoma glomerata,
Rice In rice promoting shoot length, chlorophyll
contents, and biomass
Piriformospora indica Tomato, Root of Sebacinales Protect plants against drought tolerance
and salt tolerance
Chaetomium globosum LK4 Capsicum Annuum Increased photosynthetic response under
water stress condition
Paecilomyces formosus Cucumbers Growth promotion under high salinity and
Temperature stress
Penicillium minioluteum Chenopodium quinoa Biotic stress Mitigation
Exophiala pisciphila Root of Zea mays Decrease in cadmium phytotoxicity and
a significant increase in maize growth
Beauveria bassiana Seeds of tomato and cotton Space competition with pathogens
Rhizoctonia solani and Pythium myriotylum
Trichoderma hamatum UoM
13
Root of Pennisetum glaucum Enhancing systemic immunity against the
downy mildew pathogen
Biological roles played by endophytic fungi in association with host plants
(Yan et al., 2019)

Objective: To know how P. indica help the infected chickpea plants to overcome
disease load of B. cinerea, tripartite interaction of P. indica, B. cinerea and chickpea
plants and the role of antioxidant enzymes.
Case Study - I

1. Plant and fungal culture and growth conditions
 P. indica was cultured in the laboratory routinely on solidified Aspergillus
modified medium and were incubated for 7–10 days at 30- 32 °C.
 B. cinerea was cultured on solidified potato dextrose agar (PDA) solid media at
20–22 °C for 15–20 days.
 For germination purpose, Petri plate contacting seeds of chickpea was kept in the
incubation at 23- 25°C.
2. Role of P. indica in bioprotection: Following sets were made
 Chickpea plants grown for additional 30 days without any fungus (C).
 Plants were inoculated with P. indica at day 0 and then grown for 30 days (P).
 Plants were infected with B. cinerea at day 0 and grown for 30 days (B).
 Plants were first inoculated with P. indica at day 0 and later infected with B. cinerea
at day 10 and grown for additional 20 days (P-B).
 Plants were first infected with B. cinerea at day 0 and then inoculated with P. indica
at day 10 and grown for additional 20 days (B-P).
 Plants were simultaneously exposed to both fungi at day 0 and then grown for 30
days (P+B).
Materials and Methods

3. PCR analyses:
 To quantify the presence of P. indica and B. cinerea in chickpea plants
and analysed the expression of EF-1-alpha (tef) gene (AJ249912.1) of
P. indica, the cpr1 gene (AJ609393.1) of B. cinerea and the elongation
factor 1-alpha (EF1-α) (LOC101488243) of the chickpea plants by
using the primer pairs.
4. Antioxidant enzyme activities in chickpea plants:
 The roots and shoots were separately frozen in liquid nitrogen and
homogenized with an ice-chilled mortar and liquid nitrogen in QB
buffer without 1,4-dithiothreitol [for SOD, CAT and GST assays].
 For the GR assay, 50 mg polyvinyl pyrrolidone per gram of tissue was
added.
Cont..

Table: Percent colonization of P.
indica in chickpea roots.
Figure: Microscopic view of P. indica
intracellular densely packed pear shaped
chlamydospores in the plant roots..
Results

Figure: Morphological root growth pattern analysis in case of chickpea plant
inoculated with P. indica and infected with B. cinerea in different experimental
condition. (A) Change in length of crown roots determined (B) Number of secondary
roots determined.
A B

Figure: Impact of colonization of P. indica on biomass yield. Impact of simultaneous
and alternate inoculation of P. indica and B. cinerea on biomass yield (dry weight)
relative to plants exposed to P. indica or B. cinerea alone or not exposed to any
fungus at 30 days.

Figure: Interaction of P. indica and B. cinerea with chickpea plants. Amplification
of DNA from chickpea roots after 5, 15 and 30 days of alternate inoculation of P.
indica and B. cinerea

Figure: Antioxidant enzyme activities in chickpea roots. Activity of antioxidant enzymes in root
of plants with alternate (P→B) and (B→P) and simultaneous (P+B) colonization/infection of P.
indica (P) and B. cinerea (B), (A) GST, (B) GR, (C) CAT and (D) SOD activities.

Figure: Antioxidant enzyme activities in chickpea shoots. Activity of antioxidant enzymes in
shoot of plants with alternate (P→B and B→P) and simultaneous (P+B) colonization/infection of
P. indica (P) and B. cinerea (B). (A) GST, (B) GR, (C) CAT and (D) SOD activities.

Summary
 Biomass and root development were found to be significantly
improved in chickpea plants colonized with P. indica as compared
to the plants grown without P. indica as well as from the plants
infected with the B. cinerea.
 PCR analyses showed that gradual increase in the colonization of P.
indica in the plants result in the inhibition of the colonization of B.
cinerea.
 P. indica colonized plants showed increased antioxidant enzyme
activities

Objective: Aims to isolate W. somnifera-associated fungal endophytes, to
evaluate their ability to suppress Fusarium crown & root rot (FCCR) severity, to
promote tomato growth, and to inhibit Fusarium oxysporum f. sp. radicis
lycopersici (FORL) in vitro growth.
Case study-II

1. Withania somnifera plant sampling and fungal isolation
2. Preparation of conidial suspensions and cell-free culture filtrates
of endophytic fungi
 Conidia were harvested from 7-day-old cultures, suspended in 100 mL of PDB
medium incubated at 25 °C for 12 days at 150 rpm, filtered through Whatman no. 1
filter paper and the concentration of the conidial suspension was adjusted to 106
conidia/mL.
 Fungal cultures, previously grown for 15 days at 28 °C in a PDB medium, were
filtered through Whatman no. 1. filter paper and centrifuged thrice at 10,000 rpm
for 10 min.
3.Assessment of endophytic colonization ability
 For each individual fungal isolate, five tomato seedlings were dipped for 30 min
into 25 mL of the fungal conidial suspension and controls were dipped in equal
volume of SDW.
 They were maintained for 60 days at 20–25 °C, 70–85% relative humidity, and for a
12-h photoperiod.
Materials and Method

CONT…
4. Assessment of FCRR suppression ability
 Conidial suspensions and cell-free culture filtrates of fungal isolates
were tested for their ability to suppress the FCRR disease on tomato
under greenhouse conditions.
5. Assessment of growth-promoting ability
 Conidial suspensions or cell-free culture filtrates from tested fungal
isolates were tested in vivo for their ability to enhance tomato growth.
6. Assessment of in vitro antagonistic activity
 The endophytic isolates were tested for their antagonistic activity
against FORL using the dual culture technique.

Table: Diversity of endophytic fungi recovered from Withania somnifera
on PDA medium and their relative isolation frequency.
Result

NC, untreated control:no isolation; I1, isolate from flowers; I2, I6, I4, isolates
from leaves; I5, isolates from stems; and I3, I7, isolates from fruits.
Table: Re-isolation frequency (%) of endophytic fungal isolates from tomato
roots, crowns, and stems noted 60 days post inoculation.

Table: Effects of endophytic fungi recovered from Withania somnifera on Fusarium
crown and root rot severity, as compared to controls, noted 60 days post-inoculation.
NC, uninoculated and untreated; IC, inoculated with Fusarium oxysporum f. sp. radicis-
lycopersici and untreated; FC, inoculated with FORL and treated with hymexazol-based
fungicide.

Table: Effects of conidia-based preparations and cell-free culture filtrates from endophytic
fungi on tomato growth parameters recorded 60 days post-inoculation with Fusarium
oxysporum f. sp. radicis-lycopersici as compared to controls.

Table: Antifungal activity of endophytic fungal isolates recovered from Withania
somnifera and their cell-free culture filtrates toward Fusarium oxysporum f. sp.
Radicis lycopersici noted after 5 days of incubation at 25 °C compared to control.

 All isolates enhanced treated tomato growth parameters by 21.5–90.3% over
FORL-free control and by 27.6–93.5% over pathogen-inoculated control.
 All tested isolates significantly decreased by 28.5–86.4% disease severity over
FORL-inoculated control.
 The highest disease suppression, by 86.4–92.8% over control and by 81.3–
88.8% over hymexazol-treated control, was achieved by the I6 isolate.
 FORL radial growth was suppressed by 58.5–82.3%versus control when dual
cultured with tested isolates and by 61.8–83.2%using their cellfree culture
filtrates.
Summary

 The use of endophytic fungi for the plant disease control is relatively new
and unexplored area of research.
 Fungal endophytes are much diversified & important components of
sustainable agriculture in view of their ability to produce phytohormones
and solubilize phosphates, siderophore production, inhibiting plant
pathogens, have a crucial role in plant defense and promoting plant
growth.
 The major benefit of embracing such beneficial microorganisms in the field
of agriculture is to bring about reduction in the use of different
agrochemicals such as pesticides, chemical fertilizers and this would
make agriculture more productive and sustainable.
Conclusion

 Keeping in view the various beneficial activities carried
out by fungal endophytes, therefore in future it is needed
to enhance biocontrol efficiency of endophytes.
 Future research will need to take into account the
development of genomic tools allowing for further studies
on the life of endophytes inside plants and plant– microbe
interactions.
 Future research in this field will have significant
environmental and economic implications.
56
Future prospects

Exploitation of endophytic fungi for plant disease management

Exploitation of endophytic fungi for plant disease management

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