Characterization of Finger Millet Blast Pathogen (Pyricularia grisea) and Its Management Using Biocontrol Agents and Fungicides

1. First Bio-Innovate Regional Scientific Conference
United Nations Conference Centre (UNCC-ECA)
Addis Ababa, Ethiopia, 25-27 February 2013
Characterization of Finger Millet Blast Pathogen
(Pyricularia grisea) and Its Management Using
Biocontrol Agents and Fungicides
Getachew Gashaw, Tesfaye Alemu and Kassahun Tesfaye

2. Content outline
Introduction
Objectives
Materials and methods
Results and Discussions
Conclusions and Recommendations
Acknowledgment

3. 1. Introduction
1

4. 2

5. 2. Specific Objectives
To isolate and characterize finger millet blast pathogen from
various areas
To study the effect of different cultural and growth factors of
Pyricularia grisea in the laboratory
To carry out pathogenicity test and estimate the yield losses
caused by Pyricularia grisea
To evaluate In Vitro antagonistic activities of Trichoderma and
Pseudomonas species and fungicides against P. grisea
3

6. 3. Materials and Methods
Experimental site
Mycological Research Laboratory
In Vivo experimental conducted in the Department of Microbial,
Cellular and Molecular Biology, Addis Ababa University
Study areas and samples collection
(East and West Welega, Metekel, Awi, and West Gojam)
The survey route followed major roads to towns and localities
in
45 districts separated by 10-12 km from each other 4

7. Isolation of Pyricularia grisea
5

8. Cultural and morphological characterizations of P. grisea
 Surface texture, pigmentation and mycelial growth
on different solid media
All the media were sterilized, at 121°C for 15 minutes
Colony diameters of each isolate in plates were measured in
millimeter, at two days intervals for 10 days
Mycelial colour, type of margin and sporulation were recorded
Shape, colour, size (length & width), septation of conidia
6

9. Effect of temperature levels on growth of P. grisea isolates
Six isolates of Pyricularia grisea were grown on malt extract
agar, at six temperatures (15, 20, 25, 30, 35 and 40O
C)
Colony diameters of each isolate were measured in millimeter
Effect of hydrogen ion concentration(pH) on P. grisea
isolates
Potato dextrose broth medium was used
pH levels (3, 3.5, 4, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0)
The dry weight of each isolate was measured
Carbon and Nitrogen utilizations P. grisea isolates 7

10. Pathogenicity test
Evaluation of Finger millet varieties under green house condition
 Boneya, Local Check and Tadesse obtained from BARC
 sown in 21cm plastic pots filled with 5kg of autoclaved soil
When the seedlings were six weeks old the leaves were;
 cleaned with sterile distilled water & predisposed to nearly 95%
humidity for 24 hours (Sreenivasaprasad et al., 2005)
Spore suspensions of 15 days old culture were adjusted to the
concentration of 105
spore/ml for all isolates
Six weeks old finger millet seedlings were inoculated by spraying
on leaves by using hand sprayer (Han et al., 2003) with controls 8

11. Pathogenicity test
9
a. Conidia spray on the foliage b. Incubation of seedlings

12. Blast disease assessment
10

13. Disease assessment cont’d….
Blast DS and DI was assessed according to the scale of Waller
et al. (2002); DS% = nxv/9N x100; Where:
(n)= Number of plants in each category,
 (v) = Numerical values of symptoms category.
(N)= Total number of plants,
(9) = Maximum numerical value of symptom category.
DI (%) = Number of infected plant units X 100
Total number (healthy and infected of units assessed)
11

14. Assessment of yield losses
Finger millet yield loss was calculated using the equation
developed by Mousanejad et al. (2010).
12

15. In Vitro evaluation of biocontrol agents and fungicides
against P. grisea isolates
T. harzianum (AUT1), T. viride (AUT2), and P. fluorescens
Dual culture method used (Rao, 2003, and Rangajaran et al., 2003)
Percentage of radial growth inhibition was calculated by
Riungu et al. (2008)
The poisoned food technique (Nine and Thapliyal, 1993)
Stock concentrations of the fungicides (a.i) were used
Bayleton 50%, Curzate 43.95%, Ridomil 68%, Sancozeb 80%
Relative growth reduction for each rate of fungicide was calculated
by Riungu et al. 2008.
One way ANOV procedures of SPSS statistical analysis software 13

16. 4. RESULTS AND DISCUSSIONS
Isolation of finger millet blast (Pyricularia grisea) isolates
42 isolates of P. grisea isolated from diseased leaf, neck and
finger/seed of finger millet weed and wild relative species from
different locations
14

17. Cultural and morphological characteristics of P. grisea isolates
Cultural characteristics
The colony of the isolates showed significant differences
Growth rate and slight variations in colour
Colony colors, isolates imparted on the different growth media
Gray, greyish black, black and buff white colors
Awoderu et al. (1991); Meena (2005)
15
Pg.11 Pg.20 Pg.22
Pg.26 Pg.40 Pg. 41
HSEA

18. 16
Table 1. Evaluation of culture media for the growth of P. grisea

19. Conidial characteristics of P. grisea isolates
17

20. Shape of conidia
18
Pg.11 Pg.20
Fig. 1. Microscopic observation of morphology of P. grisea isolates

21. 19
Fig.2 Conidia and conidiophores of Pyricularia grisea isolates
Mijan (2000)

22. No. Isolate Range(μm) Average (μm)
1 Pg.11 21.05-28.80 x 6.55-9.54 19.35 x 6.85
2 Pg.20 18.01-24.03 x 6.84-10.28 20.50 x 8.77
3 Pg.22 15.66-24.37 x 7.70-12.90 18.32 x 10.57
4 Pg.26 22.79-29.77 x 6.87-12.13 23.98 x 9.50
5 Pg.40 24.36-29.48 x 8.35-11.92 25.72 x 10.14
6 Pg.41 26.91-35.43 x 6.35-9.20 31.17 x 7.78
20
Table 2. Conidial size of the six isolates of the pathogen after
10 days incubation, at 27±1o
C

23. 21

24. 22
Table 3. Evaluation of mycelial growth of P.grisea at different
temperature levels
Similarly, Arunkumar and Singh (1995) ;Suryanarayanan, (1996)

25. Table 4. Dry mycelial weight of the isolates of Pyricularia grisea
at different pH level
23

26. 24
Table 5. Effects carbon sources on mycelial growth of P. grisea isolates
Tochinai and Nakano (1940); Otsuka et al. (1965); Onofeghara et al. (1973)

27. Table 6. Effect of nitrogen sources on mycelial growth of P. grisea isolates
25Apparao (1956); Otsuka et al. (1965)

28. 28
Table 7. Average disease incidences and severities and their ranges
in different agro climatic Regions of Ethiopia.
Values in the same letters are not significantly different
Ecological
zone
Disease
incidence
(%)
Av. disease
incidence
(% ) ±SD
Disease
severity
(%)
Av. disease
severity
(%)±SD
Altitude
Mean±SD
East
Wellega
35-68.3 54.1±10.30a
18-35.8 28.6±6.7ab
1862.1±165.9ab
West
Wellega
45-76.7 63.03±11.04a
22.5-50 34.6±8.4a
1726.2±129.7b
Metekel 10-75 50.8±26.5a
0-25 22.3±23.1ab
1152±153.8c
Awi 20-65 46.7±15.00a
5-27 15.7±8.1b
1913.3±277.9ab
West
Gojjam
37-95 57.9±16.5a
5-69 23.7±16.3ab
1990.4±185.9a
26

29. Table 8. Percentage of disease incidence and severity under
green house condition
27

30. 28
Table 9. Assessment of yield losses caused by P. grisea isolates on
the three finger millet varieties under green house condition
Takan et al. (2004); Adipala (1989); Ahmad et al. (2011)

31. Table10. In vitro evaluation of antagonistic activity of Trichoderma
species and P. fluorescens against P. grisea isolates
29Harish et al. (2007) ; Rosales et al. (1995)

32. In vitro testing cont’d……..
30
Pg.11 Pg.20 Pg.22
Pg.41Pg.26 Pg.40
Control
AAUT1 AUT2 Pseudomonas fluorescens

33. Isolates Mean inhibition percentage by*
Bayleton Curzate Ridomil Sancozeb Mean ±SD
Pg.11 73.9±4.6a
69.5±13.4a
72.2±0.7c
85.5±2.0a
75.3±9.0a
Pg.20 70.7±7.0a
69.4±10.3a
80.5±3.0a
87.3±2.1a
77.0±9.5a
Pg.22 68.1±7.0a
67.0±12.1a
79.4±3.1ab
88.4±1.8a
75.7±11.1a
Pg.26 74.9±3.2a
73.3±11.3a
78.3±1.2ab
86.9±1.5a
78.3±7.6a
Pg.40 73.6±5.9a
75.3±9.9a
82.5±3.4a
88.0±3.3a
79.8±8.2a
Pg.41 70.1±5.4a
69.6±12.7a
75.7±4.3bc
86.6±1.4a
75.5±9.6a
Mean ±SD 71.9±5.6 70.7 ±10.7 78.1±4.3 87.1±2.1 76.9±9.2
31
Table 10. Mean percent of mycelial growth inhibition of the test isolates
on PDA amended with fungicides after 7days of incubation, at 27 ±1o
C
Percich et al. (1997)

34. In vitro evaluation of fungicides cont’d….
32
200PPM
500PPM
800PPM
1000PPM
Sancozeb

35. 5. Conclusions and recommendations
33

36. In Vitro evaluation of the effectiveness of biological agents on the
mycelia growth of the isolates, in general, showed that the
 Pseudomonas fluorescence was less effective than the two
Trichoderma species (AUT1 and AUT2)
The most effective fungicide was found to be Sancozeb followed by
Ridomil, Bayleton, and Curzate.
The interspecific relative susceptibility of P. grisea isolates showed
a pattern of Pg.11 > Pg.22 > Pg.41> Pg.20> Pg.26> Pg.40 on all
fungicides.
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37. Recommendation
35

38. ACKNOWLEDGEMENT
 BioInnovate Eastern Africa
 Bako Agricultural Research Center
 Department of Microbial Cellular Molecular Biology,
Collage of Natural Sciences, Addis Ababa University.
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