ENDOPHYTES

1. Welcome

2.  European Union (EU) rejects okra
 Higher level of pesticide residue found in shipments
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3. EU bans Indian mangoes
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4. Nature is asking us…………..
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5. Solution is with me………
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6. Plant resident microorganisms in
disease management
S. Ajit kumar
2013-11-195
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7. Outline
 Definition
 Plant resident microorganisms
 Methods of detection
 Common plant resident microorgaisms
 How endophytes enter into plant
 How they attack enemies
 Type of relation and benefits
 Case studies
 Conclusion
 Future line
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8. Plant resident microorganisms
 Those organisms which get established and
adapted on their plant habitat and able to multiply by
themselves
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(Thind, 2012)

9. What are plant resident microorganisms ?
 Broadly classified in to 1) Epiphytes
2) Endophytes
The terms epiphyte and endophyte was coined by
Antony De Bary
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10. Contd..
 Endophyte Endophytic microorganisms are those
that inhabit the interior part of the plants, especially
in leaves, branches, root and stem, without any
harmful effect to the host
 Epiphyte The organisms inhabitating the aerial plant
parts are called as epiphytes
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(Azevedo, 1998)

11. Detection methods…….
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12. Methods for detection of Epiphytes
 Dilution plate method
 Washed disk method
(McInroy and Kloepper, 1995)
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13. Methods for detection of Endophytes
1. Histological observation
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• Cut healthy leaves into small pieces
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• Surface sterilize and incubate on PDA plates
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• Subculture into PDA slants for further studies
12(McInroy and Kloepper, 1995)

14. 2. Surface sterilisation and serial dilution
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• Surface sterilise the healthy samples
• Crush in 0.02 M potassium phosphate buffer
2
• Take 0.1 ml aliquot and pour into sterile
Petri dish (sterility check)
3
• Select samples, dilute serially up to required
dilution
(McInroy and Kloepper, 1995)
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15. Common plant resident microorganisms
Fungi Bacteria
Botryosphaeria sp. Pseudomonas fluorescens
Guignardia sp. Pseudomonas aeruginosa
Xylaria sp. Pseudomonas sepacia
Colletotrichum sp. Bacillus subtilis
Trichoderma sp. Pseudomonas putida
Penicillium sp.
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(Thind, 2012)

16. How endophytes enter into plant ??
 Where is the gate to the party ?
Endophytic microorganisms are transmitted
horizontally by airborne (or) rainborne inoculum
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(Lebron et al., 2001)

17. Mode of colonisation…
A. Hyphae growing across the
leaf
B. Germination of conidia
C. Formation of germ tube
D. Elongation of germ tube
E. Penetration of germ tube in
between the epidermal
cells
(Kuldau and Bacon, 2007) 16

18. How they attack the enemies…?
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19. Contd…..
1. Coiling
Two days after inoculation Six days after inoculation
T-Trichoderma, R-Rhizoctonia (Agrios, 2005)18

20. Contd ……
2. Penetration
 Hyphae of non pathogenic
Pythium nunn
 Penetrating into pathogenic
Phytophthora
(Agrios, 2005)
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21. Contd ……
3. Antibiosis
Trichodermin Gliotoxin
 Antibiotic compounds secreted by biocontrol agents
 Supress the growth of pathogen
(Agrios, 2005) 20

22. Contd ……
4. Siderophore production
Iron chelators
Pseudobactin, Pyoverdins
produced by Pseudomonas
fluorescens
(Agrios, 2005)
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23. Contd ……
5. Competition
 Biocontrol agent and pathogen compete for food
 Biocontrol agent wins the competition and supress
growth of the pathogen
 Eg: Competition between Pythium aphanidermatum
and Pythium ultimum
(Agrios, 2005)
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24. Contd….
6. Lysis
 Inhibit the growth of pathogen at zone of contact
(Agrios, 2005)
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25. Contd….
7. HCN production
Potential microorganisms produce HCN
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(Agrios, 2005)

26. Contd….
8. Ammonia production
 Microorganisms produce ammonia
 Colour of the medium changes to brownish orange
Eg: Pseudomonas fluorescens
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(Agrios, 2005)

27. Type of relation and benefits
Symbiosis
 Mutualism
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28. Microorganisms-benefits
 Source of nutrients
 Protection from the environmental stress
 Place of survival
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(Senthilmurugan et al., 2013)

29. Plant/host -benefits
 Improved growth response
 Drought tolerance
 Antibiotics production
 Activates Induced Systemic Resistance (ISR)
 Enzyme production like ß-1,3- glucanases
and cellulases hydrolyze pathogen cell wall
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(Senthilmurugan et al., 2013)

30. Plant resident microorganisms-translocation
 Endophytes from the cocoa crop has proved the mode of
colonisation
 EB-35, EB-65 were choosed for radiotracer study
 Bacteria labelled with 32P(Radioactive) and applied on cocoa
seedlings
 EB-35, EB-65 gave positive results when applied on leaves
and pods
 Bacillus subtilis (EB-35), Pseudomonas aeruginosa (EB-65)
( Kurian, 2011)
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31. Biological management of Phytophthora pod
rot of cocoa
 Epiphytic microflora in healthy cocoa pods were
isolated
Biocontol agents isolated
 Total 17 epiphytic fungi and 22 epiphytic bacteria
 One epiphytic fungal strain (20F) and two
epiphytic bacterial strains (23B and 24B) selected
 Trichoderma viride (20F) and Pseudomonas
fluorescens (23B and 24B)
(Bhavani, 2004)
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32. Screening- epiphytic fungi
S.NO Fungal isolate Per cent
inhibition
Antagonistic reaction
1 1F(Trichoderma sp.) 100.0 Overgrowth
2 3F(Penicillium sp.) 26.13 Homogenous
3 4F (Unidentified) 100.0 Overgrowth
4 5F(Asperigillus niger) 41.45 Cessation of growth
5 6F(Penicillium sp.) 54.05 Homogenous
6 7F(Unidentified) 64.86 Homogenous
7 8F (Trichoderma sp.) 100.0 Overgrowth
8 9F (Unidentified) 100.0 Overgrowth
(Bhavani, 2004)
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33. Contd…
S.NO Fungal isolate Per cent inhibition Antagonistic
reaction
9 10F (Trichoderma sp.) 100.0 Overgrowth
10 12F (Unidentified) 57.67 Aversion
11 14F (Asperigillus niger) 70.27 Cessation of growth
12 17F (Asperigillus flavus) 85.59 Cessation of growth
13 20F(Trichoderma sp.) 100.0 Overgrowth
14 21F(Asperigillus flavus) 58.56 Cessation of growth
15 22F(Unidentified) 100.0 Overgrowth
16 25F(Unidentified) 22.54 Aversion
17 T. harzianum 100.0 Overgrowth
(Bhavani, 2004)
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34. Contd…
 1F, 8F, 10F, 20F, 22F showed cent per cent
inhibtion
 20F strain is selected for further studies
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35. Screening-epiphytic bacteria
S.NO Bacterial isolate Per cent inhibition
1 1B 55.15
2 2B 9.50
3 3B 61.21
4 4B 58.78
5 5B 25.71
6 6B 21.91
7 7B 10.48
8 10B 57.14
9 11B 30.48
(Bhavani, 2004) 34

36. Contd…
S.NO Bacterial
isolate
Per cent inhibition
10 12B 27.62
11 13B 33.21
12 14B 8.57
13 15B 60.00
14 16B 4.77
15 17B 43.82
16 18B 42.85
17 19B 46.68
18 20B 32.40
19 23B 62.85
20 24B 60.52
21 Pf(T) 78.11
22 Pf(K) 77.14
(Bhavani, 2004)
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37. Contd…
 Epiphytic bacteria and standard cultures of P.
fluorescens evaluated against P. palmivora
23B, 24B showed more than 60 per cent inhibition
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38. •.
0
20
40
60
80
100
120
Meandiameterofthecolony(mm)
Percent inhibition over control37
(Bhavani, 2004)
Mean diameter of colony

39. Compatibility-20F with fungicides
 20F(Trichoderma viride) showed no inhibition with
(0.2 per cent) Akomin-40
Akomin-40 (0.3 per cent) and Indofil-M-45 (0.2 per
cent) showed inhibition of 11.85 per cent
Cent per cent inhibition noticed with Bordeaux
mixture and Bavistin
(Bhavani, 2004)
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40. Compatibility- 23B and 24B -Fungicides
 23B, 24B are compatible with concentration (0.2
per cent) of Indofil-M-45
Bordeaux mixture and kocide were highly inhibitory
to growth of bacteria
(Bhavani, 2004)
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41. Endophyte for early leaf spot management
in groundnut
 Endophytic microflora of healthy groundnut
leaves were isolated
 Biocontrol agents isolated
Total 8 promising endophytes (CE-1 to CE-8)
CE-6 is selected for further studies
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(Hima et al., 2011)

42. Screening- bacteria
Bacterial
antagonist
Mycelial dry
weight (cg)
Per cent inhibition over control
CE-1 60.0 43.4
CE-2 46.0 56.6
CE-3 54.3 48.8
CE-4 45.8 56.8
CE-5 45.3 57.3
CE-6 41.0 61.3
CE-7 55.0 48.1
CE-8 61.0 42.5
Control 106.0 0
41(Hima et al., 2011)

43. 0
20
40
60
80
100
120
CE-1 CE-2 CE-3 CE-4 CE-5 CE-6 CE-7 CE-8 CONTROL
Mycelial dry wt (cg)
Mycelialdrywt(cg)
Per cent inhibition over control
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(Hima et al., 2011)

44. contd…
The bacterial endophyte CE-6-61.3 per cent
inhibition over pathogen
CE-6 bacterial endophyte was identified as
Pseudomonas aeruginosa
43(Hima et al., 2011)

45. Compatibility-CE-6 -Fungicides
 CE-6 compatible with Saaf (1.7) per cent inhibition
with control
 CE-6 less compatible with hexaconazole (47.6) per
cent inhibition
44(Hima et al., 2011)

46. Trichoderma- as endophyte
 Endophytic isolates of Trichoderma viride and
Trichoderma pseudokoningii from black pepper
 Showed 64.4 and 65.6 per cent inhibition of mycelial
growth of Phytophthora capsici
(Mathew et al., 2011)
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47. Conclusion…
 It is association based control
 Eco friendly
 No adverse effects on natural enemies
 Host specific control approach
 Its own spreading ability
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48. Future line
 Use of plant resident microorganisms for disease
management is an unexploited area of research
Interactions among the epiphytic and endophytic
microorganisms are completely unexploited
Understanding these interactions and manipulating
them genetically will improve the plant health
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49. 48

50. “Man is the part of the nature, and his war against
nature is invetibaly a war against himself”
Rachel Carson
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