This document discusses screening potential biocontrol agents against Alternaria leaf spot disease in Rauwolfia serpentina. Potential antagonistic fungi were isolated from healthy Rauwolfia serpentina leaves and tested against the pathogenic Alternaria alternata using dual culture and well-agar techniques. Trichoderma harzianum ISO-1, ISO-2 and Trichoderma piluliferum were the most effective in inhibiting the growth of A. alternata. Aspergillus niger and Penicillium sublateritium also showed moderate antagonism. These antagonists show potential for controlling leaf spot disease in Rauwolfia serpentina.

1. Screening potential of biocontrol agents
against Alternaria leaf spot disease of
Rauwolfia serpentina
Presented by: Shikha Thakur
Forest Research Institute
Dehradun

2. .
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

3. Medicinal Properties
Roots are used as a valuable remedy
High blood pressure
Insomnia
 Anxiety
Excitement
 Schizophrenia
Epilepsy
Hypochondria
Disorders of the central nervous system
Leaves juice is applied for the removal of opacities of the cornea of the
eyes.

4. Fig,b. Leaf spots on Rauwolfia
serpentina (Sarpagandha) caused by
Alternaria alternata
Fig,c. Conidia of Alternaria
alternata
Disease of Rauwolfia serpentina
Leaf spots in Rauwolfia serpentina were found to be caused by Alternaria
alternata.
Symptoms
The disease appears as minute yellow spots which gradually increase in size,
turns into dark brown concentric rings and finally the leaves fall off .

5. “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”.
Biological Control

6. Objective
To assess the efficacy of the antagonistic
fungi through in vitro Dual culture and
Well-in-agar techniques.

7. 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)

8. 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 .

9. 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

10. 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, Where
a= % of area of test pathogen sp. with antagonist in the
same Petri plate (cm2)
b= % of area of antagonist, and
c= % area of inhibition zone between antagonist and
pathogen or overgrowth of antagonist over test fungus.

11. Screening – Dual Culture Technique
Interactions between antagonistic and pathogenic fungi
T.harzianum
ISO-1
T.harzianum
ISO-2
T.piluliferum A.niger
Penicillium
sublateritium
P.citreo-viride P.frequentans P.herquei Cladosporium
cladosporioides

12. S.No. Pathogenic Fungus Antagonistic Fungi Antagonistic
Efficacy
(Mean)
1. Alternaria alternata Trichoderma
harzianum 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 Penicillium
sublateritium
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 Cladosporium
cladosporioides
34.7
SEM±12.8
CD 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
91.4
34.7
70.7
60.8 59.9
87.0
100 100 100
0
20
40
60
80
100
120
PercentInhibition
Antagonist Fungi

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

14. Trichoderma
harzianum ISO-1
T.harzianum ISO-2 T.piluliferum A.niger
Penicillium
sublateritium
P.citreo-viride P.frequentans P.herquei Cladosporium
cladosporioides
Alternaria alternata
Screening – Well-in-Agar Technique
Radial growth inhibition of pathogenic fungi

15. S.No. Pathogenic
Fungi
Antagonistic Fungi Percentage
Inhibition
(Mean)
1. Alternaria
alternata
Trichoderma
harzianum ISO-1
78.2
2. Alternaria
alternata
T.harzianum ISO-2
80.8
3. Alternaria
alternata
T.piluliferum
81.4
4. Alternaria
alternata
A.niger
70.6
5. Alternaria
alternata
P.sublateritium
71.3
6. Alternaria
alternata
P.citreo-viride
37.4
7. Alternaria
alternata
P.frequentans
31.8
8. Alternaria
alternata
P.herquei
42.2
9. Alternaria
alternata
Cladosporium
cladosporioides
42.5
SEM ± 11.45
CD 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
70.6
42.5
37.4
31.8
42.2
71.3
78.2
80.8
81.4
0
10
20
30
40
50
60
70
80
90
PercentInhibition
Antagonist Fungi

16. 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.