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European Union (EU) rejects okra

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Nature is asking us…………..

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Solution is with me………

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Plant resident microorganisms in disease management

S. Ajit kumar

2013-11-195

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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 6

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)

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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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

9(Azevedo, 1998)

Detection methods…….

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Methods for detection of Epiphytes

Dilution plate method

Washed disk method

(McInroy and Kloepper, 1995)

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Methods for detection of Endophytes1. Histological observation

1• Cut healthy leaves into small pieces

2• Surface sterilize and incubate on PDA plates

3• Subculture into PDA slants for further studies

12(McInroy and Kloepper, 1995)

2. Surface sterilisation and serial dilution

1• 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)13

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.

14 (Thind, 2012)

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)

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

How they attack the enemies…?

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Contd….. 1. Coiling

Two days after inoculation Six days after inoculation

T-Trichoderma, R-Rhizoctonia (Agrios, 2005)18

Contd …… 2. Penetration

Hyphae of non pathogenic

Pythium nunn

Penetrating into pathogenic

Phytophthora

(Agrios, 2005)19

Contd …… 3. Antibiosis

Trichodermin Gliotoxin

Antibiotic compounds secreted by biocontrol agents

Supress the growth of pathogen

(Agrios, 2005) 20

Contd …… 4. Siderophore production

Iron chelators

Pseudobactin, Pyoverdins

produced by Pseudomonas

fluorescens

(Agrios, 2005)

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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)22

Contd….

6. Lysis Inhibit the growth of pathogen at zone of contact

(Agrios, 2005)

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Contd….

7. HCN production Potential microorganisms produce HCN

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(Agrios, 2005)

Contd….

8. Ammonia production

Microorganisms produce ammonia

Colour of the medium changes to brownish orange Eg: Pseudomonas fluorescens

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(Agrios, 2005)

Type of relation and benefits

Symbiosis

Mutualism

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Microorganisms-benefits

Source of nutrients

Protection from the environmental stress

Place of survival

27 (Senthilmurugan et al., 2013)

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

28 (Senthilmurugan et al., 2013)

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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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)30

Screening- epiphytic fungiS.NO Fungal isolate Per cent

inhibitionAntagonistic 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)31

Contd…

S.NO Fungal isolate Per cent inhibition Antagonistic reaction

9 10F (Trichoderma sp.) 100.0 Overgrowth10 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 Overgrowth14 21F(Asperigillus flavus) 58.56 Cessation of growth15 22F(Unidentified) 100.0 Overgrowth16 25F(Unidentified) 22.54 Aversion

17 T. harzianum 100.0 Overgrowth

(Bhavani, 2004)32

Contd…

1F, 8F, 10F, 20F, 22F showed cent per cent inhibtion 20F strain is selected for further studies

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Screening-epiphytic bacteria

S.NO Bacterial isolate Per cent inhibition

1 1B 55.15

2 2B 9.503 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

Contd…S.NO Bacterial

isolatePer cent inhibition

10 12B 27.6211 13B 33.2112 14B 8.5713 15B 60.0014 16B 4.7715 17B 43.8216 18B 42.8517 19B 46.6818 20B 32.4019 23B 62.8520 24B 60.5221 Pf(T) 78.1122 Pf(K) 77.14 (Bhavani, 2004)

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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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•.

1F 4F 8F 9F10F

20F22F

T.harzian

um 3B15B

21B23B

24BPf(t

)Pf(K

)

Control

0

20

40

60

80

100

120

Mea

n di

amet

er o

f the

col

ony

(mm

)

Percent inhibition over control37

(Bhavani, 2004)

Mean diameter of colony

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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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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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)

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)

CE-1 CE-2 CE-3 CE-4 CE-5 CE-6 CE-7 CE-8 CONTROL0

20

40

60

80

100

120

Mycelial dry wt (cg)

Myc

elia

l dry

wt (

cg)

Per cent inhibition over control42

(Hima et al., 2011)

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)

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)

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

Rachel Carson

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