screening potential of biocontrol agents

Screening potential of biocontrol agents against Alternaria leaf spot disease of

Rauwolfia serpentina

Presented by: Shikha ThakurForest Research Institute

Dehradun

.

Introduction

The plants that possess therapeutic properties or exert beneficial pharmacological effects on the human body are generally designated as “Medicinal Plants”.Medicinal Plants considered as a rich compositor of the secondary metabolites such as ◊ Vitamins◊ Minerals◊ Trace elements◊ Volatile oils◊ Tannins ◊ Alkaloids ◊ Flavonoids

Rauwolfia serpentina (L). Benth. ex Kurz. (Apocynaceae) commonly known as Sarpagandha Medicinal PropertiesRoots are used as a valuable remedy High blood pressure Insomnia AnxietyExcitement SchizophreniaEpilepsyHypochondria Disorders of the central nervous systemLeaves juice is applied for the removal of opacities of the cornea of the eyes.

Fig,a. Rauwolfia serpentina

Fig,b. Leaf spots on Rauwolfia serpentina (Sarpagandha) caused by

Alternaria alternata

Fig,c. Conidia of Alternaria alternata

Disease of Rauwolfia serpentinaLeaf spots in Rauwolfia serpentina were found to be caused by Alternaria alternata. SymptomsThe disease appears as minute yellow spots which gradually increase in size, turns into dark brown concentric rings and finally the leaves fall off .

“It is the reduction of inoculum density or disease producing activities of a pathogen or parasite in its active or dormant state, by one or more organisms, accomplished naturally or through manipulation of

the environment, host or antagonists”.

Baker and Cook,1974

Biological Control

ObjectiveTo assess the efficacy of the antagonistic fungi through in

vitro Dual culture and Well-in-agar techniques.

Methodology

Isolation Screening

φ Isolation of pathogenic fungi from diseased leaves (Dhingra and Sinclair,1985)φ Isolation of antagonistic fungi from phylloplane through serial dilution plate method (Aneja,2005 ; Dickinson,1967)

φ Dual Culture Technique (Dhingra and Sinclair,1985)

φ Well -in-Agar Technique (Dhingra and Sinclair;1985)

Results Isolation◊Alternaria alternata was the pathogenic fungus isolated from the diseased leaves of Rauwolfia serpentina.

◊Trichoderma harzianum ISO-1, T.harzianum ISO-2, T.piluliferum, Aspergillus niger, Penicillium sublateritium, P.citreo-viride, P.herquei, P.frequentans and Cladosporium cladosporioides were the antagonist fungi isolated from the phylloplane of the healthy leaves of Rauwolfia serpentina .

Trichoderma harzianum ISO-1

T.harzianum ISO-2 T.piluliferum A.niger

Penicillium sublateritium

P.citreo-viride P.frequentans P.herquei Cladosporium cladosporioides

Antagonistic fungi isolated from the phylloplane of the healthy leaves of Rauwolfia serpentina

(Alternaria alternata) Pathogenic fungus

Dual Culture Technique

Antagonistic efficacy of the antagonistic fungi against the pathogenic fungi was evaluated according to the formula described by (Ojha, 2000):Antagonistic efficacy = b+c-a, Wherea= % of area of test pathogen sp. with antagonist in the same Petri plate (cm2)b= % of area of antagonist, andc= % area of inhibition zone between antagonist and pathogen or overgrowth of antagonist over test fungus.

Screening – Dual Culture TechniqueInteractions between antagonistic and

pathogenic fungi

T.harzianum ISO-1

T.harzianum ISO-2

T.piluliferum A.niger


P.citreo-viride P.frequentans P.herquei Cladosporium cladosporioides

S.No. Pathogenic Fungus Antagonistic Fungi Antagonistic Efficacy(Mean)

1. Alternaria alternata Trichodermaharzianum ISO-1

100

2. Alternaria alternata T.harzianum ISO-2 100

3. Alternaria alternata T.piluliferum 100

4. Alternaria alternata A.niger 91.4

5. Alternaria alternata Penicilliumsublateritium

87

6. Alternaria alternata P.citreo-viride 70.7

7. Alternaria alternata P.frequentans 60.8

8. Alternaria alternata P.herquei 59.9

9. Alternaria alternata Cladosporiumcladosporioides

34.7

SEM±12.8CD at 5% = 4.56

Table 1. In vitro Screening of Phylloplane

antagonists against A.alternata under dual

culture technique

Graphical Representation Of Percent Inhibition of

Antagonists against A.alternata

A.nige

r

C.clad

ospo

rioi

P.citr

eovir

ide

P.fre

quen

tans

P.her

quei

P.sub

later

itiu

T.har

zianu

m ISO-2

T.har

zianu

m ISO-1

T.pilu

liferu

m0

20

40

60

80

100

120

91.4

34.7

70.7

60.8 59.9

87.0

100 100 100

Antagonist Fungi

Perc

ent

Inhi

biti

on

Antagonistic efficacy of the antagonistic fungi against the pathogenic fungus was evaluated according to the formula % Inhibition = R2-R1/R2 * 100Where, R2= Radial growth of test pathogen in controlR1= Radial growth of test pathogen in treatment

Well-in-agar Technique

Trichodermaharzianum ISO-1

T.harzianum ISO-2 T.piluliferum A.niger


P.citreo-viride P.frequentans P.herquei Cladosporiumcladosporioides

Alternaria alternata

Screening – Well-in-Agar Technique Radial growth inhibition of pathogenic fungi

S.No. Pathogenic Fungi

Antagonistic Fungi Percentage Inhibition(Mean)

1. Alternaria alternata

Trichodermaharzianum ISO-1 78.2


T.harzianum ISO-2 80.8


T.piluliferum 81.4


A.niger 70.6


P.sublateritium 71.3


P.citreo-viride 37.4


P.frequentans 31.8


P.herquei 42.2


Cladosporiumcladosporioides 42.5

SEM ± 11.45CD at 5% = 3.31

Table 2. In vitro Screening of Phylloplane

antagonistic fungi against A.alternata under well -in -agar

technique

Graphical Representation Of Percent Inhibition of

Antagonists against A.alternata

A.nige

r

C.clad

ospo

rioi

P.citr

eovir

ide

P.fre

quen

tans

P.her

quei

P.sub

later

itiu

T.har

zianu

m ISO -1

T.har

zianu

m ISO - 2

T.pilu

liferu

m0

10

20

30

40

50

60

70

80

90

70.6

42.537.4

31.8

42.2

71.3

78.2 80.8 81.4

Antagonist Fungi

Perc

ent

Inhi

biti

on

Conclusion

Thus, the study revealed that Trichoderma harzianum ISO-1, ISO-2 and Trichoderma piluliferum were most effective in inhibiting the growth of the test pathogen (Alternaria alternata) while Aspergillus niger and P. sublateritium were found to be moderately effective.

So, they can be recommended as potential antagonists for field application to control Leaf spots disease in Rauwolfia serpentina.

THANK YOU !!!

screening potential of biocontrol agents

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