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Characterization of finger millet blast pathogen (Pyricularia grisea) and Its management using biocontrol agents and fungicides
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Characterization of finger millet blast pathogen (Pyricularia grisea) and Its management using biocontrol agents and fungicides

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Presented by Getachew Gashaw, Tesfaye Alemu and Kassahun Tesfaye at the First Bio-Innovate Regional Scientific Conference, Addis Ababa, Ethiopia, 25-27 February 2013 …

Presented by Getachew Gashaw, Tesfaye Alemu and Kassahun Tesfaye at the First Bio-Innovate Regional Scientific Conference, Addis Ababa, Ethiopia, 25-27 February 2013

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  • 1. Characterization of Finger Millet Blast Pathogen (Pyricularia grisea) and Its Management Using Biocontrol Agents and FungicidesGetachew Gashaw, Tesfaye Alemu and Kassahun Tesfaye First Bio-Innovate Regional Scientific Conference United Nations Conference Centre (UNCC-ECA) Addis Ababa, Ethiopia, 25-27 February 2013
  • 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 40OC) Colony diameters of each isolate were measured in millimeterEffect 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 measuredCarbon and Nitrogen utilizations P. grisea isolates 7
  • 10. Pathogenicity testEvaluation 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
  • 11. Pathogenicity testa. Conidia spray on the foliage b. Incubation of seedlings 9
  • 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 fungicidesagainst 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 DISCUSSIONSIsolation 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) Pg.11 Pg.20 Pg.22 Pg.26 Pg.40 Pg. 41 HSEA 15
  • 18. Table 1. Evaluation of culture media for the growth of P. grisea 16
  • 19. Conidial characteristics of P. grisea isolates 17
  • 20. Shape of conidiaFig. 1. Microscopic observation of morphology of P. grisea isolates Pg.11 Pg.20 18
  • 21. Fig.2 Conidia and conidiophores of Pyricularia grisea isolates Mijan (2000) 19
  • 22. Table 2. Conidial size of the six isolates of the pathogen after 10 days incubation, at 27±1oCNo. Isolate Range(μm) Average (μm)1 Pg.11 21.05-28.80 x 6.55-9.54 19.35 x 6.852 Pg.20 18.01-24.03 x 6.84-10.28 20.50 x 8.773 Pg.22 15.66-24.37 x 7.70-12.90 18.32 x 10.574 Pg.26 22.79-29.77 x 6.87-12.13 23.98 x 9.505 Pg.40 24.36-29.48 x 8.35-11.92 25.72 x 10.146 Pg.41 26.91-35.43 x 6.35-9.20 31.17 x 7.78 20
  • 23. 21
  • 24. Table 3. Evaluation of mycelial growth of P.grisea at differenttemperature levels Similarly, Arunkumar and Singh (1995) ;Suryanarayanan, (1996) 22
  • 25. Table 4. Dry mycelial weight of the isolates of Pyricularia griseaat different pH level 23
  • 26. Table 5. Effects carbon sources on mycelial growth of P. grisea isolatesTochinai and Nakano (1940); Otsuka et al. (1965); Onofeghara et al. (1973) 24
  • 27. Table 6. Effect of nitrogen sources on mycelial growth of P. grisea isolates Apparao (1956); Otsuka et al. (1965) 25
  • 28. Table 7. Average disease incidences and severities and their ranges in different agro climatic Regions of Ethiopia.Ecological Disease Av. disease Disease Av. disease Altitude zone incidence incidence severity severity Mean±SD (%) (% ) ±SD (%) (%)±SD East 35-68.3 54.1±10.30a 18-35.8 28.6±6.7ab 1862.1±165.9ab Wellega West 45-76.7 63.03±11.04a 22.5-50 34.6±8.4a 1726.2±129.7b Wellega 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 37-95 57.9±16.5a 5-69 23.7±16.3ab 1990.4±185.9a Gojjam 28 Values in the same letters are not significantly different 26
  • 29. Table 8. Percentage of disease incidence and severity under green house condition 27
  • 30. 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) 28
  • 31. Table10. In vitro evaluation of antagonistic activity of Trichodermaspecies and P. fluorescens against P. grisea isolates Harish et al. (2007) ; Rosales et al. (1995) 29
  • 32. In vitro testing cont’d……..Pg.11 Pg.20 Pg.22Pg.26 Pg.40 Pg.41 AAUT1 AUT2 Pseudomonas fluorescens Control 30
  • 33. Table 10. Mean percent of mycelial growth inhibition of the test isolateson PDA amended with fungicides after 7days of incubation, at 27 ±1oC 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±9.6aMean ±SD 71.9±5.6 70.7 ±10.7 78.1±4.3 87.1±2.1 76.9±9.2 Percich et al. (1997) 31
  • 34. In vitro evaluation of fungicides cont’d…. Sancozeb 200PPM 500PPM 800PPM 1000PPM 32
  • 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. 34
  • 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. 36