This presentation is to understand the concepts of endophytes that reside within plants & to explore the applications of endophytes for the management of plant diseases.

1. DOCTORAL SEMINAR- I
PRESENTED BY:
VIJAY JOSHI
Ph. D. 2nd year
I.D. No. 48058
ROLE OF ENDOPHYTES FOR THE
MANAGEMENT OF PLANT DISEASES
APP-788

2. 2
CONTENTS
 Introduction
Endophytes??
 Mechanisms of action
Commercialization
Case Studies
Conclusion
Future Prospects

3. GLOBAL PROBLEM
Population growth of Humans!!
CURRENT
POPULATION OF THE
WORLD
7.6 billion
I
N
C
R
E
A
S
I
N
G
9.7 billion
To feed this
huge population
we need
MORE
FOOD
3

4. So how do we get MORE FOOD to feed increasing
population??
Increasing agricultural land
Greater use of chemicals
Safe & Efficient Pesticides
More Farm Mechanization
Greater use of Transgenic Crops
Expanded use of BCA
Limited resource
Against Eco-Friendly Approach
Against Eco-Friendly Approach
Expensive
Restricted due to ethical
concerns
YES !
4

5. Plant disease management faces ever-growing challenges due to-
 Increasing demands for total, safe and diverse foods to support
the booming global population and its improving living
standards.
 Reducing production potential in agriculture due to
competition for land in fertile areas and exhaustion of marginal
arable lands.
 Deteriorating ecology of agro-ecosystems and depletion of
natural resources.
 Increased risk of disease epidemics resulting from agricultural
intensification and monocultures.
5

6. Both abiotic and biotic stresses place limitations and make agricultural yields
unpredictable. For example, fungal pathogens of wheat alone are estimated to
cause losses of up to 29% of the crop (Oerke and Dehne, 2004).
Other groups of pathogens and various abiotic challenges, such as flooding,
drought and soil fertility, place further pressure on production.
Moreover, climate change is predicted to increase the frequency, number of
locations and severity of these threats.
Focus must be directed towards sustainable intensification of agriculture under
fluctuating and unpredictable conditions, as well as the minimization of the threat
of pathogens and abiotic stresses.
6
THE SUSTAINABLE INTENSIFICATION
CHALLENGE !

7. PLANT DISEASE MANAGEMENT
STRATEGIES
7

8. Why we need alternative of chemical
pesticides?
Indiscriminate use
Development of resistant strains in plant pathogens
Resurgence of pest species
Destruction of other beneficial organisms
Direct toxicity to the applicator
Pesticide residue
To cope with the stated problems there is a need to develop ecologically sound,
environmentally safe and economically viable methodologies for plant disease
and pest management.
Biological control has become an utmost important tool for Integrated Pest
Management (IPM).
Endophytic microorganisms offer great-untapped potential as biological agent
for plant disease management, due to their antagonistic properties.
8

9.  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 (Hirch and Broun, 1992).
 Microbes which occur within plant tissue for at least part
of their life cycle without causing disease under any
known circumstances’ Hallmann et al. (1997)
ENDOPHYTES???
9

10. Plant “spheres”
10
Rossmann et al.2017
Plant Microbiome

11. BRIEF HISTORY OF ENDOPHYTES
• Heinrich Friedrich Link –first to describe
endophytes in 1809 “Entophytae”
• De Bary (1866) coined Endophyte
• 1887- Bechame described microzymas-
microorganisms
• 1887- Galippe reported occurance of bacteria
and fungi in interior of vegetables
• 1991- Orlando Petrini defined endophytes as
“all organisms inhabiting plant organs that at
some time in their life cycle can colonize
internal plant tissues without causing apparent
harm to their host”
11
Orlando Petrini
Anton De Bary

12. DIVERSITY OF ENDOPHYTES
12
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.
Leuchtman and Clay (1988) reported endosymbiotic fungal species, viz. Balansia,
Epichloe and Neotyphodium in desert plant, which were protected from the effect of the
desiccating environment since they were contained within the moist internal tissue of the
host. Neotyphodium maintain growth within the basal meristem tissues of the grass plant.

13. Cont…
13
In perennial plants, hyphae grow in the intercellular spaces of the leaf sheath and
blade, typically unbranched and parallel to the leaf axis. (Musgrane, 1984).
Hinton (1996) described the distribution pattern of vegetative hyphe of F.
moniliforme on maize. They found that hyphae were intercellular only and were
present in the roots, stem internodes and leaf sheath.

14. 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.
• The samples are then surface-sterilized by dipping in 70% ethanol for 1 minute.
• Stems and leaves are suface 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 secs followed by rinsing five times in sterilized distilled water.
• The surface sterilized samples are then blot-dried using sterile filter paper.
Patle et al. 2017
14

15. 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 upto 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.
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 upto 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.
Patle et al. 2017
15

16. Identification of Endophytes
Endophyte bacteria and fungi isolates preparation
• All isolates used in the study are collected in microtube 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
16
Mmbga et al. 2018

17. 17
Endophytes: Role and Functions in Crop Health

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

19. Crop Endophytes Activity References
Rice
(Oryza sativa L.)
Pseudomonas, Bacillus,
Enterobacter, and
Micrococcus
PGP activity Mbai et al. (2013)
Enterobacter spp.
Burkholderia spp.
Siderophores Souza et al. (2013)
Peanut
(Arachis hypogaea L.)
B. amyloliquefaciens
Antibiosis against Aspergillus
flavus
Sobolev et al. (2013)
Chilies
(Capsicum annuum L.)
P. fluorescens EBS 20
Antibiosis against Pythium
aphanidermatum
Muthukumar et al. (2010)
Bacillus tequilensis,
Burkholderia cepacia,
Pseudomonas aeruginosa
Antagonistic activity against
Botrytis cinerea, Colleto-
trichum acutatum, Fusarium
oxysporum, and Phytophthora
capsici
Paul et al. (2013)
cont…

20. Crop Endophytes Activity References
Sugarcane
(Saccharum officinarum L.)
Trichoderma virens
Antagonistic against pineapple
disease pathogen, Ceratocystis
paradoxa, owing to the
production of endochitinases
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

21. 21
• The endophytes can be transmitted either horizontally or vertically.
• One of the well-studied examples of vertical transmission via seed has been described for the
asexual Epichloe species. These fungi cannot produce reproductive structures on their hosts
and are naturally propagated by growing into the embryo of a developing seed and,
subsequently, as the seed germinates, hyphae colonize the young seedling.
(Philipson and Christey, 1986)
• In the case of horizontal transmission, propagation is usually dependent on the reproductive
structures of the endophyte, such as spores, that move by wind or rain dispersal, or are
moved by a vector, from plant to plant. This can occur via the soil, through air movement or
by vectors, e.g. insects.
• The horizontal transmission of the sexual species of Epichloe (class 1) via ascospores has
been well documented. (Leuchtmann and Spiering, 2004)
• Ascospores are produced if mating partners are compatible and are ejected into the air and
wind dispersed for mediating contagious infections.
(Chung and Schardl 1997; Brem and Leuchtmann, 1999)
Transmission of endophytes

22. 22
•Reach root surfaces by
chemotactic responses
•Considered subset of the
rhizosphere and/or root-associated
bacterial population
•Root endophytes colonize and
penetrate the epidermis at sites of
lateral root emergence, below the
root hair zone, and in root cracks
•capable of establishing
populations
both inter and intracellularly
•move to other areas by entering
the vascular tissues and spreading
systemically
ENTRY & COLONISATION

23. 23
• With seed germination, amount of carbon and nitrogen compounds are excreted
into the surrounding environment that invites a large population of
microorganisms
(Okon and Labandera-Gonzales, 1994).
• The density of bacteria in the rhizosphere and rhizoplane is always higher than in
the soil which lacks substances secreted from the roots of plants .
• For active invasion, endophytic bacteria must bear the abilities of production of
cellulolytic enzymes to hydrolyze exodermal cell walls of plants.
(Rosenblueth and Martinez-Romero, 2006)
• Apical root zone having thin-walled surface of root cells includes cell elongation
and the root hair zone (zone of active penetration), and the basal root zone with
small cracks are the preferable sites of bacterial attachment and subsequent entry
caused by the emergence of lateral roots (zone of passive penetration).

24. Fig. Endophytic bacterial colonization in plants. Bacteria can enter a plant at several root
zones as indicated above. Endophytes can either remain at the site of entry (indicated in blue)
or move deeper inside or occupy the intercellular space of the cortex and xylem vessels
(indicated in green). Red and yellow represent rhizospheric bacteria which are unable to
colonize inner plant tissues.
24Muthukumar et al. 2017

25. Interactions Between Endophytes and Plants
25
1. Endophytic Fungi and Plants
 Biotic stresses from which endophytes can provide protection include plant pathogens, insects and
nematodes.
 Abiotic stresses include nutrient limitation, drought, salination and extreme pH values and
temperatures.
 In return, plants provide spatial structure, protection from desiccation, nutrients and, in the case of
vertical transmission, dissemination to the next generation of hosts.
(Schulz, 2006; Aly et al. 2011)
 Endophytes may also play a role in the ecosystem by affecting plant growth through antagonistic
fungal–fungal interactions.
Example- Interaction between the pathogen Ustilago maydis and the endophyte Fusarium verticillioides
within their shared plant host (maize, Zea mays), whereby the endophyte is capable of reducing the rate of
pathogen growth, possibly by secreting metabolites that break down plant compounds that limit U. maydis
growth.
(Estrada et al. 2012)
The root-colonizing facultative endophyte Piriformospora indica forms beneficial symbioses with crop
plants.
Fakhro et al. (2010) studied the effects of inoculation of tomato with P. indica and observed that in soil, P.
indica colonizes the roots of tomato, increases the biomass of the leaves by up to 20% and reduces the
disease severity caused by Verticillium dahliae, the causal agent of Verticillium wilt, by more than 30%.

26. Fig.- Illustration and comparison of endophytic colonisation and pathogenic disease development
in plants
26
(Chowdhary and Sharma, 2017)

27. 2. Endophytic bacteria and Plant Interaction
• Bacteria are able to trigger signalling 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.
(Chakraborty et al., 2006)
• 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 defence proteins).
James et al., (2002)
27

28. Secondary metabolites production by fungal endophytes
28
 Epicorazines A-B
 Flavipin
 Epipiridones
 Ambuic Acid
 Cordycepsidone
 Colletonoic acid
 Anti-bacterial, anti-fungal and anti-algal activities
 Biocontrol activity
(Li et al. 2001, Varughese et al. 2012)

29. 29
FUNCTIONS OF BACTERIAL ENDOPHYTES
Phytostimulation
•Indole acetic acid production
•Ehylene production
•Cytokinin and giberrelins production
Biofertilization
•Nitrogen fixation
•Phosphate solubilization
•Siderophore production
Biocontrol
•Antibiotics production
•Hydrolytic enzymes production
•Siderophore production
•Induced Systemic Resistance (ISR)
•Exopolysaccharide production

30. 30
Exploring the potential of endophytes in agriculture

31. 31(Muthukumar et al. 2017)

32. 32
Khare et al. 2018

33. How Endophytes attack Plant Pathogens:
Mechanisms
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
33
Ecological effect
• Occupation of ecological niche
• Hyperparasites and predation

34. Plant Protection Mechanism by Endophytic Fungus
• Broadly there are three different ways of defensive interactions between endophytic fungi
and pathogens in plants.
 In direct effect inhibition, protection is primarily localised and conferred by antibiosis
(antibacterial, antifungal secondary metabolites or lytic enzymes) to endophyte-inflicted
plant segments.
(Arnold et al. 2003).
 Indirect inhibition inherent plant defence (SAR and ISR) pathways are elicited. For
instance, endophytic Fusarium solani induced systemic resistance proteins (PR5 and PR7)
towards tomato leaf pathogen Septoria lycopersici in root tissues.
(Kavroulakis et al. 2007)
• Furthermore, endophytic microbes have been known to promote host plant growth and
physiology. Colletotrichum sp., an endophyte of A. aninua, regulated plant processes by
producing indole acetic acid.
(Lu et al. 2000)
 The third way of pathogen suppression is by way of ecological niche occupation.
Endophytes colonise host tissues faster than corresponding pathogens leading to depletion
of resources.
(Pal and Gardener, 2006)
34

35. • Innumerable compounds such as hydrocyanic acids (HCN), DAPG, phenazines, pyrrolnitrin,
enzymes and phytohormones to protect plant from toxic effect of fungal pathogens are
considered as the significant products to help endophytes to be colonized in rhizosphere
(Castro-Sowinski et al. 2007; Ramette. et al. 2011; Jousset et al. 2011).
• While induced disease resistance activities are allied with the abilities to produce secondary
metabolites, such as antibiotics or chitinase enzyme, which can inhibit growth of plant
pathogens. Hence they act as biocontrol agents.
(Christina et al. 2013; Wang et al. 2014).
• Endophytic bacteria are correlated with the enhanced plant growth by the production of
hormones that increase accessibility of nutrients, such as nitrogen, potassium and phosphorus
(Glick, 2012).
• Endophytic bacteria can also induce seedling emergence and stimulate plant growth (under
stress conditions (Bent and Chanway, 1998)
• The direct positive effects are production of phytohormones such as IAA, GA, etc. non-
symbiotic nitrogen fixation, and biofortification of phosphorous and other essential nutrients
include the trace elements to plants for phytostimulation and to the soil for increasing
fertilization power of soil. (Burdman et al. 2000).
• Besides, under iron-stress conditions in the soil and on the surface plant, endophytes produce
iron-chelator molecule called siderophores used to transport iron in a competitive way and
deprived for the pathogenic fungi as essential bioavailable element.
(Pedraza et al. 2007)
35
Plant Protection Mechanism by Endophytic Bacteria

36. 36

37. From Concept to Commerce: Developing a Successful Fungal Endophyte
Inoculant for Agricultural Crops
37
Murphy et al. 2018

38. 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 and,
• Must colonize plant parts naturally obligately with
species specificness.
38

39. Commercially Available Products
Marrone Bioinnovations has licensed Muscodor albus and EPA approval for the
release of this organism for use in agriculture is expected soon. Trade Name
Ennoble.
 It literally sterilizes the soil in which it has been placed It is a potential
replacement for methyl bromide which is now restricted from use in many
countries.
Adaptive Symbiotic Technologies have commercialized endophytes to improve
agriculture in relation to climate changes, which may give rise to water, drought
and salination stresses. The products include BioEnsureR -Corn and
BioEnsureR -Rice.
39

40. CASE
STUDIES
40

41. CASE STUDY-1
41
Endophytic Bacillus cereus Effectively Controls Meloidogyne incognita on Tomato Plants
Through Rapid Rhizosphere Occupation and Repellent Action.
Hai-Jing HU, Ya-Li Chen, Yu-Fang Wang, Yun-Yun Tang, Shuang-Lin Chen, and Shu-Zhen Yan
Plant Disease- 2017- 101: 448-455
Published Online:13 Dec 2016 https://doi.org/10.1094/PDIS-06-16-0871-RE
OBJECTIVES –
 Screening for an efficient endophytic bacteria strain contributing to reduction of the
use of pesticides in the control of root knot nematodes.
 To study colonization dynamics of endophytic bacteria BCM2, SZ5, and CCM7.
 To study biocontrol potential of endophytes on M. incognita in tomato.
 To study second stage juvenile repelling action when tomato roots were treated
with endophytic bacteria.

42. Results
42

43. 43
Fig.1. Population of Bacillus cereus BCM2 (str′), B. cereus SZ5 (str′), and B.
altitudinis CCM7 (str′) on tomato roots. Population is expressed in log x (CFU/g) of roots (y
axis) and days (x axis).

44. 44

45. 45
Fig.2. A, Fluorescence micrograph (Zeiss A1) observe of green fluorescent protein (GFP)-
tagged Bacillus cereus BCM2. B. cereusBCM2-str′-gfp after incubation in Luria-Bertani medium
(LB) for 12 h. The bar represents 10 μm.
B, Population of B. cereus BCM2-str′ and B. cereus BCM2 (str′)-pBCgfp-1 represented by
optical density at 600 nm observed during 1 to 108 h after incubation in LB.

46. 46
Fig. 4.Confocal laser-scanning microscopy analysis of endophytic bacteria Bacillus cereus BCM2 (str′)-
pBCgfp-1 in tomato root tissue after inoculated for 7 days. The bar indicates 200 μm. A, Distribution of
green fluorescent protein (GFP)-tagged BCM2 in new root; B, distribution of GFP-tagged BCM2 in
lateral root; C, GFP signal was gathered in lateral root junctions; D, GFP-tagged BCM2 gathered small
clusters to intercellular spaces from cortex to vascular cylinder; E, endophytic bacteria occupied the space
of root hair and epidermal cells in tomato roots; F, more clusters were detected in giant cells in the root of
disease tomato caused by Meloidogyne incognita; and G, tomato root uninoculated.

47. 47
Fig. 5.Meloidogyne incognita second-stage juvenile (J2) repelling action when tomato roots were treated
with three endophytic bacteria. A, Endophytic bacteria-treated and sterile water-treated tomato roots on
water agar plate; B, 100 M. incognita J2 in 20 μl of water; C, more M. incognita J2 invaded tomato roots
not inoculated with endophytic bacteria; and D, the number of M. incognita J2 in different tomato roots;
asterisks (**) indicate significantly different (P ≤ 0.001, Dunnett test, n = 10) from control (no endophytic
bacteria).

48. CASE STUDY-2
48
OBJECTIVES-
 To isolate endophytic bacteria from six healthy Citrus spp. to inhibit the pathogen.
 Screening of effective endophytic bacteria+ isolate to control citrus canker on lime
plants.

49. Results
49

50. 50

51. 51

52. 52

53. 53

54. 54
• B. subtilis LE24, B. amyloliquefaciens LE109 and B. tequilensis PO80, which are
endophytic bacteria isolated from healthy citrus plants, displayed an ability to inhibit
the growth of X. citri subsp. citri.
• The most effective strains used to control citrus canker disease on lime were B.
subtilis LE24 and B. amyloliquefaciens LE109.
Conclusion
• The benefits of the endophytic bacteria include the ability to multiply inside the host
plant tissues.
• The genus Bacillus forms endospores that can survive in the environment when
applied to control plant diseases.
• The Bacillus strains thus play important roles in the biological control of citrus
canker disease on lime plants.

55. Advantages
• Easy to culture invitro
• Applied as seed treatments
• Reduce initial root damage
• Escape microbial competition
• Capable of influencing host response to pathogen attack
• Do not produce any toxic symptoms
• Instead promote plant growth
• Depend on root exudates for multiplicaion
• Environmental friendly
• Activate soil biology and restore soil fertility.
55

56. FUTURE PROSPECTS
56

57. Future prospects
• Development of specific culture medium to isolate endophytic
microbes
• Isolation and identification of more efficient endophytic
microbes for plant growth promotion and plant disease
management
• Detailed studies on multiple interactions between endopohytes,
plants and pathogens
• Appropriate delivery methods need to be developed and tested
for agronomic use
57

58. CONCLUSION
58
CONCLUSION

59. 59