study of endophytes in abiotic stress management

1. ENDOPHYTIC MICROBES -
THEIR INTERACTION
WITH PLANTS TO
ALLEVIATE ABIOTIC
STRESS

2. Dr. RAJENDRA PRASAD CENTRAL
AGRICULTURAL UNIVERSITY, PUSA,
BIHAR

3. COURSE :
MASTERs'
SEMINAR,
MICRO-
591(1+0)
TO,
Dr. AMAN JAISWAL,
ASSISTANT PROFESSOR CUM SCIENTIST,
DEPARTMENT OF MICROBIOLOGY.
FROM,
SD. VAHIDA REHMAN
M.Sc.(Second semester),
Roll No. :-1905206001,
DEPARTMENT OF MICROBIOLOGY

4. CONTENTS:
 Introduction
 Definition of endophytes
 Source of endophytes
 Transmission of endophytes
 Colonization of endophytes
 Bioprospectives of endophytes
 Endophytes as plant growth promoters
 Endophytes as biocontrol agents
 Endophytes as stress controllers
 Case study
 conclusion

5. INTRODUCTION:
 Variations in the outside environment (abiotic stresses) put the plant metabolism out of homeostasis,
which creates necessity for the plant to harbour some advanced genetic and metabolic mechanisms
within the cellular system.
 Development of stress tolerance in plants is a strategy to cope with the negative effects of adverse
environmental condition.
 Endophytic microorganisms offers great untapped potential to enhance plant growth and
production of natural compounds and as biocontrol agents due to their antagonistic properties
and also induces stress tolerance in plants.

6. WHAT ARE
ENDOPHYTIC
MICROBES?
The term endophyte is derived from two Greek words ‘endo’ =
‘endon’ meaning “within” and ‘phyte’ = ‘phyton’ meaning “ plant”.
The term endophytic was first used by De Bary in 1886 to denote all
those microbes residing inside the living healthy plant.
ENDOPHYTES are microorganisms which lives inside the
healthy plant tissues without causing any apparent harm to host.
(Bacon and White, 2020)
Endophytes are ubiquiotes
They interact biochemically and genetically inducing the beneficial effects in
plant growth and defense without causing pathogenic symptoms
Beneficial effects exhibited by the endophytic bacteria are similar to the
plant growth promoting rhizobacteria (PGPR). Their plant growth promoting
potential was higher than that of PGPR and can survive severe
environmental conditions due to their unique ecological niche.

7.  Many bacterial endophytes belong to the genera, Pseudomonas, Burkholderia and Bacillus.
 Competitive rhizosphere bacteria such as Pseudomonas (Pseudomonas fluorescens),
Azospirillum (Azospirillum brasilense) and Bacillus, were often found as colonizers of
internal tissue of the plants.
 Endophytic stage has been substantiated for many Proteobacteria, Firmicutes, and
filamentous Actinobacteria.
Azospirillum brasilense Burkholderia sp. Pseudomonas fluorescens
BACTERIAL ENDOPHYTES

8. Plant species Bacterial Endophyte Taxa References
Alfalfa (Medicago
sativa L.) roots
γ-proteobacteria: Erwinia sp., Pseudomonas sp.
firmicutes: Bacillus megaterium, B. chosinensis
actinobacteria: Microbacterium trichothecenolyticum
Gagne et al., 1987; Stajkovic
et al., 2009
Soybean (Glycine max
(L.) Merr.) stems,
leaves, roots and
nodules
α-proteobacteria: Erwinia sp., Agrobacterium sp.
γ-proteobacteria: Pseudomonas citronellolis,
Enterobacter sp., Pantoea sp.,
firmicutes: Bacillus fastidiosus
Zinniel et al., 2002;
Kuklinsky-Sobral et al., 2004
Wheat (Triticum
aestivum L.) roots
β-proteobacteria: Burkholderia cepacia
γ-proteobacteria: Klebsiella sp.
firmicutes: Bacillus polymyxa
actinobacteria: Mycobacterium sp.
Mavingui et al., 1992;
Balandreau et al., 2001;
Zinniel et al., 2002;
Iniguez et al., 2004
Wild rice (Oryza
officinalis, O.barthii, O.
rufipogon,) and
cultivated rice
(O.sativaL.) roots and
stems
α-proteobacteria : Azorhizobium sp., Azospirillum sp.,
Azospirillum brasiliense, Bradyrhizobium sp.
β-proteobacteria : Burkholderia graminis,
Firmicutes : Bacillus sp.
Actinobacteria : micrococcus sp.
Engelhard et al., 2000;
Elbeltagy et al., 2001;
Sandhiya et al., 2005;
Mbai et al., 2015

9. C – Endophytes /
class – I
Endophytes
NC –Endophytes
/ class – II
Endophytes
Infect some
grasses
Can found in
tissues of non-
vascular plants,
gymnosperms,
angiosperms
Endophytic fungi
The most common Clavicipitaceous (C) - Endophytes studied are Epichloe, or
Neotyphodium species ( Neotyphodium species representing the asexual derivatives of
Epichloe).
Non-clavicipitaceous (NC) endophytes are typically Ascomycotina Fungi.
Fungal endophytes are represented by two major groups

10. Examples of class-I endophytes that confered abiotic stress tolerance :

11. Examples of class-II endophytes confered abiotic stress tolerance:

12. Cntd..

13. BENEFICIAL EFFECTS OF ENDOPHYTES

14.  A large number of mechanisms are proposed to explain the beneficial effects of
endophytes such as
 production of phytohormones ( auxins, gibberellins, cytokinins );
 biological nitrogen fixation;
 mobilization of phosphorus;
 suppression of stress related plant ethylene synthesis by 1-aminocyclopropane-
1carboxylate (ACC) deaminase activity;
 induced systemic resistance/tolerance against abiotic stresses;
 plant defense mechanisms by antagonistic substances (siderophores, Hydrogen
Cyanide [HCN]) or
 through competition for colonization sites and nutrients.
BENEFICIAL EFFECTS

15. SOURCES OF ENDOPHYTES
 Endophytes are found within a wide variety of plant tissues,
including seeds, fruits, stems, roots, tubers, pollen, leaves,
buds, flower and seed tissues.
 live within the intercellular spaces of plants,
 feeding on apoplastic nutrients as non-pathogens without
eliciting defense responses.

16. TRANSMISSION OF ENDOPHYTES

17. ENDOPHYTIC COLONIZATION
 The endophytic colonization ranges from transmission via seeds and vegetative
propagating material to enter into host plants from the surrounding environment
(such as rhizosphere and phyllosphere).
 Successful endophyte-plant interactions require colonization of a plant by the
endophytes.

19. In fungi,
 Endophytic fungi reside entirely within plants, such as root, stem, and leaves.
 C-Endophytic associations are restricted to above ground parts, such as shoot
tissues, and are located intercellularly.
 In contrast NC-Endophytes can grow both in above ground and below ground
tissues or they may be restricted to above ground tissues or roots.
 Fungal entry into plants can occur through wound sites or through stomata, with
good growth in nutrient-rich meristematic tissues, but less so in mature
tissues.
 The mycelium grows systemically aboveground, where they are
intercellularly-localized in recently-formed aerial tissues.

20. In bacteria,
 Bacterial endophytes also occur intercellularly and can be found completely
belowground, aboveground, or both.
 Bacterial produced endoglucanase and endopolygalacturonase were suggested to
be involved in localized cell wall degradation, aiding in bacterial entry through
cracks at root emergence sites or root tips.
 Other reports have also suggested that colonization of the plant can occur via
intercellular space movement as these spaces are mineral rich environments.

21. BIOPROSPECTING OF ENDOPHYTES
 Endophytes as plant growth promotors
 Endophytes as biocontrol agents against pathogens
 Endophytes as stress controller in plants

22. Endophytes as plant
growth promoters :
Endophytes release active metabolites by utilizing nutrients
secreted by the plants and enhances plant development.
Plant growth promotion of endophytes is achieved through
the production of plant growth enhancing substances
such as
Siderophore
s
production,
indole
acetic acid
(IAA),
cytokinins,
gibberellins
phosphorus
solubilizati
on,
atmospheric
N fixation,
supplying
essential
vitamins
and
enzymes,
and
helping
the plants
in nutrient
acquisition

23. root development
ability of plants to absorb
phosphorus from the soil
Obtain nitrogen from
biological nitrogen fixation
(BNF)
have nitrogen fixation
genes (nifH)
phosphate assimilation
IAA production
Saccharum officinarum
(sugarcane)
Glycine max
(Soybean )
Capsicum annuum L.
Additionally, endophytes help symbiotic rhizobia to form nodules with non-specific
hosts, aiding in plant growth promotion.

24.  Other beneficial effects on plant growth attributed to endophytes includes
 osmotic adjustment,
 stomatal regulation,
 modification of root morphology,
 enhanced uptake of minerals and
 alteration of nitrogen accumulation and metabolism.
 Nevertheless, endophytes can indirectly promote growth by stimulating the
production of growth hormones by the plants.
 Class 2 endophytes (non-clavicipitaceous endophytes) increased host shoot
and/or root biomass, because of the induction of plant hormones.

25. Endophytes as
biocontrol
agents:
 Endophyte associated microbes induces jasmonic acid
(JA) and/or ethylene mediated Induced Systemic
Resistance (ISR)
 produce and secrete one or more compounds with
antibiotic activity
 produce toxins or structural compounds
 increased efficiency in iron uptake by the beneficial
microorganism through siderophores contributes to
their ability to colonize the plant roots,
which helps in removal of the deleterious organisms from
potential sites of infection.

26.  Other biocontrol by-product is HCN, which effectively blocks the cytochrome oxidase
pathway and is highly toxic agent.
 Endophytic bacteria like Achromobacter, Acinetobacter, Pseudomonas and Actinomycetes
like Streptomyces, Microbispora protected plants against root pathogens as potential
biocontrol agents.
 Bacterial diazotrophs such as Azospirillum sp. and Rhizobium sp. Were suitable as
biocontrol agents due to their colonization of ecological niche like that of phytopathogens.

27. ENDOPHYTESAS STRESSCONTROLLER IN PLANTS:
 Endophytic microbes aid in plant health by deterring herbivory and
pathogenesis while also facilitating
 plant growth through nutrient uptake ( modification of root
morphology and alteration of nitrogen accumulation and
metabolism),
 water use efficiency(osmotic adjustment and stomatal
regulation)and
 curtailing of environmental stresses.
 The endophytes in return, obtain access to the host plant nutrients
and dissemination to the next generation.

29. ABIOTIC STRESS
TOLERANCE VIA
ENDOPHYTES:
Symbiotically conferred abiotic
stress tolerance involves two
mechanisms,
• Activation of host stress response systems
soon after exposure to stress, allowing the
plants to avoid or mitigate the impacts of
stress and
• Biosynthesis of anti-stress biochemicals by
endophytes.

30. ENHANCEMENT OF WATER USE EFFICIENCY DURING
DROUGHT:
 The drought tolerance phenomenon is explained by
 enhanced accumulation of solutes(osmolytes like proline) in tissues of
endophyte infected plants.
 Production of anti oxidant molecules for ROS scavenging
 Production of phytohormones like ABA,IAA..etc.,
 Use less water and increase biomass as compared to non-infected plants.

32.  The grass and Neotyphodium spp. endophyte mutualistic symbiosis could enhance
the tolerance to drought stress.
 In addition to grass species, the evergreen tree Theobroma cacao infected with the
endophytic fungus Trichoderma hamatum isolate DIS 219b, exhibited delayed
drought-induced changes in stomatal conductance and net photosynthesis.

33. NUTRIENT
STRESS
( NUTRIENT
UPTAKE AND
RECYCLING):
 Endophytes enable supply of macro nutrients and micro
nutrients to their host plants.
 Plant root exudates are metabolized by nitrogen-fixing bacteria
and in turn provide nitogen to the plants for amino acid
synthesis.
 Endophytes promote plant growth by phosphate solubilization.
 Biodegradation of litter of its host plant.
 Endophytes have the ability to decompose organic components
including lignin, cellulose, hemicelluloses which facilitate in
nutrient cycling.

34.  Choi et.al.(2008) have reported pseudomonas sp. mediating phosphate
solubulisation , in rice and wheat by producing gibberellic acid.
 Piriformospora indica and Azotobacter chroococcum improved uptake of
mineral nutrients especially Zn in wheat.
 Endophytes Pseudomonas, Stenotrophomonas and Burkholderia
contributed N to the grasses growing in nutrient-poor sand dunes, as
evident by the detection of nitrogenase with antibodies in roots, within
cell walls of stems and rhizomes.

35. SALINITY AND ALKALINITY TOLERANCE:
 Tolerance in plants to salt stress is associated with the alleviation of antioxidant
enzymes.
 The ROS scavengers include glutathione, ascorbate, tocopherol, and the
ENZYMES,
 Superoxide dismutase(SOD)
 catalases(CAT),
 ascorbate or thiol-dependent peroxidases(APX),
 glutathione reductases(GR),
 dehydroascorbate reductases(DHAR), and mono-dehydroascorbate
reductases(MDHAR).
 These are involved in removal of ROS directly or indirectly in the cell via
regeneration of ascorbate and glutathione

36.  Piriformospora indica colonized barley had higher ascorbate
concentrations enhancing the tolerance of salt-sensitive plant.
 Aspergillus fumigatus sp. LH02, Penicillium minioluteum LHL09,
Metarhizium anisopliae LHL07, and Penicillium funiculosum LHL06
from the roots of Glycine max L. resulted in higher plant biomass and
improved growth under moderate and high salinity stress.

37. TEMPERATURE STRESS TOLERANCE:
 High and low temperatures inhibit plant growth by destroying the photosynthetic apparatus and
cell membrane.
 Endophytes have been reported to protect host plants from the extreme temperature damages.
 Dichanthelium lanuginosum plants inoculated with fungal endophyte Curvularia showed
tolerance to soil temperature at 50˚C for 3 days and intermittent as high as 65˚C for 10 days
while noninfected plants died at 50˚C.
 Bacterial endophyte Burkholderia phytofirmans strain PsJN inoculation could increase grapevine
growth and keep physiological activity at a low temperature, and improved its ability to
withstand cold stress with 10.7-fold increases for root weight at 4˚C and 2.2-fold increases for
total plant biomass at 4˚C.

38. PHYTOREMEDIATION:
 Endophytes have been used at phytoremediation sites to degrade the toxic compounds .
 In addition, herbicide residue is toxic to plants and needs to be reduced or removed. Pea
(Pisum sativum) plants were employed to degrade 2,4-D in cropland.
 When infected with a genetically tagged bacterial endophyte that possesses the natural
ability to degrade 2,4-D, the plants showed a higher capacity to remove 2,4-D from the
contaminated soil with no 2,4-D accumulation in aboveground plant parts.

39.  For phytoremediation of toxic metals, endophytes may have a metal-resistance or
sequestration system and could reduce metal toxicity and influence metal translocation
to the aboveground plant parts.
 The Root Associated Dark Septate Endophyte(DSE), Exophiala pisciphila isolated
from zeya mays showed enhanced antioxidant enzyme activity under increased soil Cd
stress.
 Alteration in the levels of 1-aminocyclopropane-1-carboxylate (ACC) by
Pseudomonas and Gigaspora can alter the tolerance of heavy metals directly through
the manipulation of plant ethylene levels.
Cntd…

40. CASE STUDY

41.  Aim : To isolate the plant hormone (GAs and IAA) secreting endophytic fungal strain from the roots of
cucumber plant and access its role in host plant physiology under salinity stress.
 Materials and methods:
 120 root samples of cucumber plants
 2.5% sodium hypoclorite
 Autoclaved distilled water
 Root pieces are inoculated in petri plates containing Hagem Media (0.5%glucose, 0.05%KH2PO4, 0.05% MgSO4.7H2O,
0.05%NH4Cl, 0.1% FeCl3, 80ppm streptomycin, 1.5% agar, pH 5.6)
 Newly emerged fungal spots from the roots were isolated and grown on PDA medium under sterilized condition.
 These fungal strains were inoculated in Czapek broth to separate the liquid culture medium and fungal mycelia (centrifuge at
2500rpm at 4C for 15 min.
 The cultural filtrate 50 ml and mycelia 5.4gm were immediately shifted to -70C freezer and then freeze dried for 4 days.
 For bioassay experiment , rice seeds were surface sterilized with 2.5% sodium hypochlorite for 30 min and rinsed
with autoclaved distilled water and then incubated for 24 hrs with 20 ppm uniconazol to obtain equally germinated
seeds
 Pre germinated seeds were transferred to pots having water, agar medium under aseptic conditions grown in growth
chamber for 10days.
 10 micro litre fungal culture filtrate was applied at the apex of the rice seedlings

42. TOTAL 120 ROOT SAMPLES WERE COLLECTED FROM FIELD
STERILISED AND WASHED WITH AUTOCLAVED DISTILLED
WATER
ROOT PIECES OF 0.2cm WERE INOCULATED IN HAGEM
MEDIA
NEWLY EMERGED FUNGAL SPOTS WERE ISOLATED AND
GROWN IN PDA MEDIA
TOTAL NINE DIFFERENT FUNGAL STRAINS WERE ISOLATED
AND WERE GROWN ON CZAPEK BROTH

43. THE CULTURAL FILTRATE WERE IMMEDIATELY SHIFTED
TO -70C FREEZER AND THEN FREEZE DRIED FOR 4 DAYS
FUNGAL STRAINS WERE ISOLATED BASED ON
MORPHOLOGICAL ANALYSIS
ISOLATED FUNGAL STRAINS WERE SCREENED ON GA’s
DEFICIENT MUTANT RICE CULTIVAR(Waito-C) AND GA’s
BIOSYNTHESIS CULTIVAR(Dongjin-byeo) SEEDLINGS
GROWTH ATRRIBUTES OF BOTH THE CULTIVARS
WERE RECORDED AFTER THE WEEK OF
TREATMENT

44. CSH-6H applied cultural filtrates showed higher chlorophyll content increased shoot
length and fresh weights

45. CSH-6H CF showed that increased chlorophyll content, shoot length and
fresh weight in Dongjin-byeo rice seedlings

46. The cultural filtrate of CSH-6H has shown significant growth compared to
others and identified as Paecilomyces formosus LHL10

47. Results
Presence of Paecilomyces formosus association in plants have showed significant increase in the
production of phytohormones (GAs and IAA) have promoted the host-plant growth even in saline
conditions.

48. a) The higher Relative Water Content (RWC) showed by endophyte inoculated plants indicates the beneficial
association and rescuing role of Paecilomyces formosus to curtail the adverse affect of salinity stress.
b) Lower electrolyte discharge in endophyte associated plants indicated a lower permeability of plasma membrane
attributed to the integrity and stability of cellular tissues due to endophyte-plant interaction.

49. c) MDA content is further decreased in endophyte associated plants growing in saline conditions.
d) Increased free proline content provides an energy source for plant growth and survival and also suggesting a
decline in the ionic influx inside the cellular masses preventing Paecilomyces formosus associated plants from osmotic
stress.

50. e) Higher nitrogen assimilation in Paecilomyces formosus inoculated plants than endophyte free control plants
with or without stress.
f) Accumulation of antioxidants inside endophyte associated plants shows higher oxidant radical scavenging and
also extend greater resistance to oxidative damage in the salinity stress conditions.

51. CONCLUSION:
 Paecilomyces formosus LH10 produced many physiologically active
and inactive GAs and IAA, which helped the Waito-C and Dongjin-
byeo rice plants to grow well and significantly mitigated the negative
effects of salinity stress in cucumber plants.

52. CONCLUSION:
 Microbe mediated stress tolerance in plants is an ecofriendly approach for
the better crop yield. They can increase the crop land and species
diversity.
 Endophytes are good tool for enhancement of plant growth, improvement
of quality, and yield of plant products, through genetic engineering and
tissue culture
 The investigation is still going on to understand the endophytic nature of
micro organisms.
 It is still under the scan that how a pathogenic microbe is switching over
the endophytic lifestyle.