First Bio-Innovate Regional Scientific ConferenceUnited 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

Content outline

Introduction

Objectives

Materials and methods

Results and Discussions

Conclusions and Recommendations

Acknowledgment

1. Introduction

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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. grisea3

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

Isolation of Pyricularia grisea

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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  

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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 40OC)

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

Richards’s medium was used (Otsuka et al., 1957)7

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 105spore/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

Pathogenicity test

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a. Conidia spray on the foliage b. Incubation of seedlings

Blast disease assessment

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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)

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Assessment of yield losses

Finger millet yield loss was calculated using the equation

developed by Mousanejad et al. (2010).

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In Vitro evaluation of biocontrol agents and fungicides against P. grisea isolatesT. 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

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

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Cultural and morphological characteristics of P. grisea isolates

Cultural characteristicsThe 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)

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Pg.11 Pg.20 Pg.22Pg.26 Pg.40 Pg. 41

HSEA

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

Conidial characteristics of P. grisea isolates

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Shape of conidia

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Pg.11 Pg.20

Fig. 1. Microscopic observation of morphology of P. grisea isolates

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Fig.2 Conidia and conidiophores of Pyricularia grisea isolates

Mijan (2000)

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 7.7820

Table 2. Conidial size of the six isolates of the pathogen after 10 days incubation, at 27±1oC

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Table 3. Evaluation of mycelial growth of P.grisea at different temperature levels

Similarly, Arunkumar and Singh (1995) ;Suryanarayanan, (1996)

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

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Table 5. Effects carbon sources on mycelial growth of P. grisea isolates

Tochinai and Nakano (1940); Otsuka et al. (1965); Onofeghara et al. (1973)

Table 6. Effect of nitrogen sources on mycelial growth of P. grisea isolates

25Apparao (1956); Otsuka et al. (1965)

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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

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

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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)

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)

In vitro testing cont’d……..

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Pg.11 Pg.20 Pg.22

Pg.41Pg.26 Pg.40

Control

AAUT1 AUT2 Pseudomonas fluorescens

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

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Table 10. Mean percent of mycelial growth inhibition of the test isolates on PDA amended with fungicides after 7days of incubation, at 27 ±1oC

Percich et al. (1997)

In vitro evaluation of fungicides cont’d….

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200PPM

500PPM

800PPM

1000PPM

Sancozeb

5. Conclusions and recommendations

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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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Recommendation

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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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