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Isolation of Ascochyta rabiei and a convenient method for copious inoculum production
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MYCOTECHNOLOGY ISOLATION OF Ascochyto robiei AND A CONVENIENT METHOD FOR COPIOUS INOCULUM PRODUCTION BY S SARWAR ALAM, R N STRANGE Dept. Botany and Microbiology, University CoIIege, Gower Street, London WCIE 6BT AND S H QURESHI Nat. Agricultural Res. Centre, Islamabad, Pakistan Blight of chickpea (Cicer arietinum L) caused by Ascochyta rabiei (Pass.)Labr. is a serious problem in many areas. In the 1979-1980 season, 700/0 of the Pakistan crop was lost (Nene, 1982). The parasite attacks all aerial parts of the plant giving rise to dark necrotic spots on leaves, stems and pods. As the infection progresses pycnidia are formed in concentric rings (Hafiz . 1951). Where stems are infected girdling commonly occurs causing breakage at this point. Plant parts above the lesion die rapidly . In order to study the disease reliable methods are required for the routine isolation of the fungus from infected plant tissue as well as the production of inoculum for infection experiments and screening trials . Isolation: We have found the following procedure gives about 90% success rate . Infected pods, stems or leaflets were sterilized in 5% sodium hypochlorite for 1 minute and dried on sterile filter paper. The material was plated on 2% water agar and incubated at 20°C for 5-7 day s. Fungal colonies growing from the plant material were subcultured onto chickpea seed agar, consisting of an extract from 60g chickpea seed made by boiling the seed in water for 30 minutes. Sucrose (20g) and agar (20g) were added to the extract and the volume made up to 1 litre with water. Incubation on this medium for 1-2 weeks resulted in the development of colonies of the fungus with pycnidia . Inoculum multiplicatian: Chickpea seed was softened by boiling for 15-30 minutes in water, drained and autoclaved for 30 minutes at 121° in a conical flask . Spore suspension was
prepared from a slant of the fungus growing on chickpea seed agar by the addition of sterile distilled water and agitation with a sterile loop . Enough spore suspension was added to wet the seeds and the flask was shaken to ensure distribution of the inoculum. After incubating for 7-10 days at 20° , abundant pycnidia were present on the seed . Agitation with sterile distilled water resulted in a spore suspension uncontaminated by mycelium. Three isolates of the fungus gave the following numbers of spores/seed: 8.89 x 10' ±- 1.16 x 10', 11.00 X 10' ±- 2.93 x 10' and 7.88 x 10' ±- 2.68 x 10'. These high numbers together with absence of mycelium make the method most appropriate for the production of inoculum for field screening since the inoculum may be applied by means of a spray without the hazard of nozzle blockage by wefts of mycelium. These methods have worked extremely well in our laboratory and may also be of value to others working with pycnidial fungi. This works was supported by the Commission of the European Communities Contract TSD.A.201 UK(H).
REFERENCES
H AFI Z, A (1951). Cultural studies on Ascochyto
robiei with special reference to zonation. Transactions of the British MycologicolSociety 34, 259-269. NENE, Y L (1982) . A review of Ascochyta blight of chickpea. Trop icol Pest Management 28, 61 -70 .
SOLUBLE CARBOHYDRATES OF VESICULAR-ARBUSCULAR MYCORRHIZAL FUNGI By F AMIJEE AND P STRIBLEY Soil Microbiology Department, Rothamsted Experim ental Station, Harpenden , Herts AL5 2JQ In ectomycorrhizas, erico id mycorrhizas, carbon passes from the host to the mycobiont where it is converted into specific fungal sugars, such as mannitol and trehalose (Harley & Smith. 1983) . In vesiculararbuscular (VA) mycorrhizas carbon also
moves from host to fungus (Cox et al., 1975). but attempts to detect mannitol and trehalose in the mycorrhizas have been unsuccessful (Bevege et aI., 1975). Lewis (1975)commented that mannitol is unlikely to be present in VA mycorrhizal fungi but concluded that
MYCOTECHNOLOGY ISOLATION OF Ascochyto robiei AND A CONVENIENT METHOD FOR COPIOUS INOCULUM PRODUCTION BY S SARWAR ALAM, R N STRANGE Dept. Botany and Microbiology, University CoIIege, Gower Street, London WCIE 6BT AND S H QURESHI Nat. Agricultural Res. Centre, Islamabad, Pakistan Blight of chickpea (Cicer arietinum L) caused by Ascochyta rabiei (Pass.)Labr. is a serious problem in many areas. In the 1979-1980 season, 700/0 of the Pakistan crop was lost (Nene, 1982). The parasite attacks all aerial parts of the plant giving rise to dark necrotic spots on leaves, stems and pods. As the infection progresses pycnidia are formed in concentric rings (Hafiz . 1951). Where stems are infected girdling commonly occurs causing breakage at this point. Plant parts above the lesion die rapidly . In order to study the disease reliable methods are required for the routine isolation of the fungus from infected plant tissue as well as the production of inoculum for infection experiments and screening trials . Isolation: We have found the following procedure gives about 90% success rate . Infected pods, stems or leaflets were sterilized in 5% sodium hypochlorite for 1 minute and dried on sterile filter paper. The material was plated on 2% water agar and incubated at 20°C for 5-7 day s. Fungal colonies growing from the plant material were subcultured onto chickpea seed agar, consisting of an extract from 60g chickpea seed made by boiling the seed in water for 30 minutes. Sucrose (20g) and agar (20g) were added to the extract and the volume made up to 1 litre with water. Incubation on this medium for 1-2 weeks resulted in the development of colonies of the fungus with pycnidia . Inoculum multiplicatian: Chickpea seed was softened by boiling for 15-30 minutes in water, drained and autoclaved for 30 minutes at 121° in a conical flask . Spore suspension was
prepared from a slant of the fungus growing on chickpea seed agar by the addition of sterile distilled water and agitation with a sterile loop . Enough spore suspension was added to wet the seeds and the flask was shaken to ensure distribution of the inoculum. After incubating for 7-10 days at 20° , abundant pycnidia were present on the seed . Agitation with sterile distilled water resulted in a spore suspension uncontaminated by mycelium. Three isolates of the fungus gave the following numbers of spores/seed: 8.89 x 10' ±- 1.16 x 10', 11.00 X 10' ±- 2.93 x 10' and 7.88 x 10' ±- 2.68 x 10'. These high numbers together with absence of mycelium make the method most appropriate for the production of inoculum for field screening since the inoculum may be applied by means of a spray without the hazard of nozzle blockage by wefts of mycelium. These methods have worked extremely well in our laboratory and may also be of value to others working with pycnidial fungi. This works was supported by the Commission of the European Communities Contract TSD.A.201 UK(H).
REFERENCES
H AFI Z, A (1951). Cultural studies on Ascochyto
robiei with special reference to zonation. Transactions of the British MycologicolSociety 34, 259-269. NENE, Y L (1982) . A review of Ascochyta blight of chickpea. Trop icol Pest Management 28, 61 -70 .
SOLUBLE CARBOHYDRATES OF VESICULAR-ARBUSCULAR MYCORRHIZAL FUNGI By F AMIJEE AND P STRIBLEY Soil Microbiology Department, Rothamsted Experim ental Station, Harpenden , Herts AL5 2JQ In ectomycorrhizas, erico id mycorrhizas, carbon passes from the host to the mycobiont where it is converted into specific fungal sugars, such as mannitol and trehalose (Harley & Smith. 1983) . In vesiculararbuscular (VA) mycorrhizas carbon also
moves from host to fungus (Cox et al., 1975). but attempts to detect mannitol and trehalose in the mycorrhizas have been unsuccessful (Bevege et aI., 1975). Lewis (1975)commented that mannitol is unlikely to be present in VA mycorrhizal fungi but concluded that