Botryodiplodia sp. - Crops for the Future

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9.1.4 Isolation of the pathogen Materials: • Bunsen burner • 4 beakers • Scalpel and forceps • 1 empty petri-dish • Measuring cylinder (10 ml) • Pure PDA plates Chemicals: • 100 ml ethanol solution (70 %) • Sodium hypochlorite solution (5 %) • 250 ml distilled water For the isolation of the pathogen partially diseased fruit are required. The fruit should have enough healthy tissue. The fruit which were therefore bought either from local markets or collected during the field trips. From the fruit tissue squares were taken with the size of 1 cm² and the thickness of 3 mm. It was important that only 1/3 of the tissue was diseased. The tools which were necessary for this had to be sterilized after every cut (scalpel and forceps). The cuttings took place in an empty petri-dish. The pieces obtained were surface sterilized by dipping them for three minutes in a 5 % sodium hypochlorite solution. The solution was prepared immediately before the surface sterilization, because the effectiveness of the solution decelerates with running time. After that the pieces were washed twice by dipping them into Figure 23: Isoloation of the distilled water for three minutes to remove the rests of the pathogen on rambutan (five sterilization solution. In each case two washed fruit pieces days old plate) were put into one PDA plate and incubated. The mycelium of the pathogen covered the whole plate after a few days (Figure 23) and could be subcultivated. The pathogenicity of the isolated organism was proved using Koch’s postulates. Koch’s postulates can be summarised in four steps (FREDRICKS and RELMAN, 1996): • The organism must be found in all plants suffering from the disease, but not in healthy plants. • The organism must be isolated from a diseased plant and grown in pure culture. • The cultured organism should cause disease when introduced into a healthy plant. • The organism must be reisolated from the experimentally infected plant. 28
If the Koch’s postulates are fulfilled it is proven that the isolated pathogen and the pathogen which causes the disease of the fruit are identical. 9.1.5 Bio-Assays Materials: • Bunsen burner • Cork borer (number 5) • Spatula • Micropipette and pipette-tips For the spore suspension: • Small flask • Loop • Measuring cylinder (10 ml) • Funnel with cotton wool Chemicals: • 150 ml Ethanol (70 %) • 50 ml distilled water The so called “bio-assays” are in vitro experiments and should give information about the activity of the bio-control agents (BC-agent) against the pathogens. Therefore several experimental series were undertaken, which respectively proved two BC-agents against four pathogens. The activity of the BC-agents was proved in petri-dishes filled with 15 ml PDA. Near the outer border of the Petri-dish four wells has been cut into the medium with a constant distance (Figure 24). The wells were cut with a cork borer number 5 (1 cm in diameter). Afterwards 0.8 ml of a spore suspension of a BCagent was filled in each well. Following this a mycelia disk of the pathogen, which was cut with the same cork borer (No. 5) from a 7 days old culture, was placed in the middle Figure 24: Preparation of a petri-dish for a bioassay of the petri-dish (Figure 24). The spore suspension was won out of 5 to 10 days old cultures of the BC-agents according to the time of full sporulation. Therefore 20 mm distilled water was filled into the culture plate and the upper layer of the fungus was scraped off the medium. This mix of distilled water and fungus tissue was filtered to receive a pure spore suspension of the BC-agent. For the controls the wells were filled with distilled water. In this manner five replications per pathogen took place, so that one experimental series consisted out of 60 culture plates. 29
Page 1: Control of Post Harvest Disease (Bo
Page 4 and 5: List of Figures Figure 1: The Sri L
Page 6 and 7: 1 Summary Experiments were undertak
Page 8 and 9: 3 Sri Lanka The Democratic Socialis
Page 10 and 11: 3.2 Climate The climate of Sri Lank
Page 12 and 13: valuable consultations in industria
Page 14 and 15: Rambutan trees are either dioecious
Page 16 and 17: However, the most important genus i
Page 18 and 19: 6.2.1.2 Annona muricata Annona muri
Page 20 and 21: Figure 9: Annona squamosa Figure 10
Page 22 and 23: Characteristically for this fungus
Page 24 and 25: Figure 15: Phialides of Trichoderma
Page 26 and 27: autoclave is used for liquid steril
Page 28 and 29: Figure 21: Soil sampling in a rambu
Page 32 and 33: The effectivity of the BC-agent was
Page 34 and 35: Figure 29: Experimental series of a
Page 36 and 37: Two of the Trichoderma sp. with a h
Page 38 and 39: Results of the sporulation experime
Page 40 and 41: 12 References ALAM, M.S., BEGUM, M.
Page 42 and 43: ONG, P.K.C., ACREE, T.E. and LAVIN,
Page 44 and 45: Figure 36: List of bio-control agen
Page 46: Tmu III Galagedera • Mycelial gro

Botryodiplodia sp. - Crops for the Future

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