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An efficient method of detecting Cercospora canescens in bean seeds
Notes and brief articles J. (1961 ). Electron microscope observations on the flagella of the zoosporangial zoospores of Plasmodiophora brassicae and Spongospora subterranea. Proceedings of Koninklijk Nederlandse Akademie van Wetenschappen C 64, 157-161.
KOLE, A. P. & GIELlNK, A.
tomato seedlings are readily available; and the use of a nutrient solution eliminates the slow process of washing roots free from soil. REFERENCES
HEWITT, E.
J. (1966). Sand and Water Culture
Methods , znd ed. Commonwealth Agricultural Bureaux, U .K.
AN EFFICIENT METHOD OF DETECTING CERCOSPORA CANESCENS IN BEAN SEEDS BY O. D. DHINGRA AND G. L. ASMUS
CENTREINAR, Unioersidade Federal de Vifosa, 36.570, Vifosa, MG, Brasil Seed-borne Cercospora canescens was detected more readily on seedlings of Phaseolus vulgaris which had been killed with paraquat. Out of 172 samples tested 32 % were found to be infected, at levels up to 13'5 %.
Cercospora canescens Ellis & Martin, the cause of leaf-spot and blotch of beans, Phaseolus vulgaris L., has been reported to be seed-borne (Menezes et al., 1978), but in our routine studies of seed pathology of beans, the recovery of C. canescens was sporadic and at very low levels. Standard blotter-tests with or without 2-40, have been shown to be more efficient than agar tests for detecting Cercospora spp. in seeds of other crops (Mathur & Kabbere, 1975; Neergaard, 1973), because Cercospora was masked by fast growing fungi on agar. The blotter test was not satisfactory for detection of C. canescens in beans, since Phomopsis and Fusarium spp. overgrew the Cercospora. Attempts to detect C. canescens using seedling-symptom tests failed since no symptoms appeared. It was possible that seed-borne C. canescens was causing latent infection in the seedlings. Cerkauskas & Sinclair (1980) reported that by treating soybean tissues with paraquat (1,1' -dimethyl-a.a'-bipyridylium dichloride), latent infection could be detected. As a consequence of this report, the following technique, termed •seedling-paraquat', was developed for efficient detection of C. canescens in bean seeds. One hundred and seventy-two samples of 20 cultivars of beans grown under different agroclimatic conditions were tested . Two hundred seeds of
Table
1.
each sample were surface disinfected by immersing in 1'7 % NaOCl for 1 min, followed by 1 min in 70 % ethanol and washed in sterile water. The seeds were then planted in methyl bromide-treated sand in a greenhouse. About 5 days after emergence, the seedlings were sprayed with a 10 % solution of paraquat and then covered with a plastic tent to maintain high humidity. Seedlings died within 24 h and after 4-{) days the dead seedlings were examined for fungal growth. Comparisons were made with results from the standard blotter test, with 2-4D, using 200 seeds from each sample . When examined, profuse growth of fungi, especially Fusarium spp., was observed on the leaves and hypocotyl, and sometimes on the cotyledons of the seedlings. Cercospora, when present, formed colonies only on the upper side of the cotyledons. They were felty, dark olivaceous green to dark brown; and spored profusely. Initially colonies on the cotyledons where examined microscopically to confirm the identity of the fungus. With experience, however they could be identified in situ with the naked eye or with the help of a hand lens. The fast-growing fungi did not interfere with the detection of this pathogen. Using this technique the pathogen was detected in 32 % of the samples, compared to 11 % using the
Comparative frequency levels of seed infection with Cercospora canescens in samples, using two techniques
172
Infection ('Yo) Detection technique Seedling paraquat Z-4D-blotter Trans. Br . mycol. Soc. 81 (z), (1983)
0
0'5- 1
l'5- z '5
3-5
117 153
34 10
12
5
7
2
Printed in Great Britain
5'S-10t 10
z o
2 0
bean-seed
4 26
Notes and brief articles
2-4D blotter test (Table 1). The overall mean level of infection was 1'9 % of the seeds using' seedlingparaquat' technique as opposed to only 0'63 % with the 2-4D-blotter method. The difference between the two methods was significant at the 5 % level using a paired t test. The percentage of C. canescens-infected seeds varied between 0 and 13' 5 % as determined by the new technique and between 0 and 4 % when determined by the 2-4D-blotter method.
REFERENCES
CERKAUSKAS, R. F. & SINCLAIR, J. B. (1980). Use of paraquat for detection of fungi in soybean tissues. Phytopathology 70, 1036-1038. MATHUR, S. B. & KABBERE, F. (1975). Seed-borne fungi of sesame in Uganda. Seed Science and Technology 3, 665-660. MENEZES, J. R., MOHAN, S. K., ROSSETTO, E. A. & BIANCHINI, A. (1978). Qualidade sanitaria de sementes de feijjio na regiao norte do Estado do Parana. Fitopatologia Brasileira 3, 122-123 (Abstr.) NEERGAARD, P. (1973). Detection of seed-borne pathogens by culture tests. Seed Scienceand Technology 1,217-254.
CHEMICAL CONTROL OF FUSARIUM CULMORUM ON RYEGRASS, LOLIUM PERENNE BY ERIC J. GUSSIN AND JAMES M. LYNCH*
Agricultural Research Council, Letcombe Laboratory, Wantage, Oxon. OXI2 9JT A formulation containing calcium peroxide compared favourably with carbendazim and drazoxolon in controlling Fusarium culmorum on ryegrass. Direct drilling of grass, where old swards are desiccated by herbicides, offers considerable economic advantages over traditional methods of reseeding (Allen, 1981). However, the abundant decaying organic matter that this leaves on the soil surface can sometimes be detrimental to the newly sown grass. There is evidence for the production of phytotoxins from the organic matter (Gussin & Lynch, 1981; Lynch, 1977, 1978; Patrick, 1971). Fusarium culmorum (W.G.Sm.) Sacco has been shown to be a major colonizer of couch grass (Agropyron repensL.) rhizomes after they have been killed prior to direct drilling (Penn & Lynch, 1982), and this fungus is a well-known pathogen ofgrasses (Bennett, 1935), causing both pre- and postemergence death (Michail & Carr, 1966). It has been isolated from establishing ryegrass seedlings and causes symptoms in the laboratory similar to those found in the field. We have now compared the efficacy of a number of fungicides with a formulation containing calcium peroxide which has been shown to have some antifungal properties (Lynch, Harper & Sladdin, 1981). Fusarium culmorum was isolated from diseased ryegrass (Lolium perenne L. cv. S24) seedlings and cultured in sterile soil to maintain pathogenicity. When required, crumbs of soil were inoculated on to potato sucrose agar and used within 14 days. Spore suspensions were prepared by adding
* Glasshouse Crops Research Institute, Littlehampton, W. Sussex BN 16 3PU. (Address for correspondence). Trans. Br. mycol. Soc. 81 (2), (1983)
deionized water and brushing the Petri dishes with a camel-hair brush. The suspension was filtered through a muslin cloth and adjusted to 106 spores rnl" with sterile distilled water as necessary. Ryegrass seed (cv. S24) was dressed in 5 g batches by adding chemicals to the seed in glass jars on a roller (30 rev.jmin) for 1 h to ensure a uniform dressing. The chemicals were formulated in inert talc (Table 1) and screened initially at 250 mg a.i. kg- 1 seed unless stated otherwise. Seeds were dressed about 1 week in advance of being sown. Dressing rates for subsequent testing varied depending upon the level of activity found in the initial screen. The phytotoxicity of each of the compounds used was determined at the start of the investigation by sowing seeds (to) dressed with 250 mg a.i. kg" into pots (2'5 ern diam, 4'5 em deep) containing vermiculite (14 em") in the absence of the fungus. The initial screen determined which compounds had activity against Fusarium spp. and the best of these were then compared with a calcium peroxide formulation containing 60 % (wjw) Ca0 2 and 40% (wjw) CaOH 2 bound to the seed using a 4 % (v/w) solution of polyvinyl alcohol at a range of dressing rates. In all tests, dressed seeds (to) were sown into pots (2'5 em diam) containing vermiculite (14 ern") inoculated with a F. culmorum spore suspension (3 ml x 106 spores mr'). Non-dressed seed was sown into infected vermiculite to check the pathogenicity ofthe fungus on each occasion. Non-dressed seed sown into uninfected vermiculite served as the control and all
Printed in Great Britain
KOLE, A. P. & GIELlNK, A.
tomato seedlings are readily available; and the use of a nutrient solution eliminates the slow process of washing roots free from soil. REFERENCES
HEWITT, E.
J. (1966). Sand and Water Culture
Methods , znd ed. Commonwealth Agricultural Bureaux, U .K.
AN EFFICIENT METHOD OF DETECTING CERCOSPORA CANESCENS IN BEAN SEEDS BY O. D. DHINGRA AND G. L. ASMUS
CENTREINAR, Unioersidade Federal de Vifosa, 36.570, Vifosa, MG, Brasil Seed-borne Cercospora canescens was detected more readily on seedlings of Phaseolus vulgaris which had been killed with paraquat. Out of 172 samples tested 32 % were found to be infected, at levels up to 13'5 %.
Cercospora canescens Ellis & Martin, the cause of leaf-spot and blotch of beans, Phaseolus vulgaris L., has been reported to be seed-borne (Menezes et al., 1978), but in our routine studies of seed pathology of beans, the recovery of C. canescens was sporadic and at very low levels. Standard blotter-tests with or without 2-40, have been shown to be more efficient than agar tests for detecting Cercospora spp. in seeds of other crops (Mathur & Kabbere, 1975; Neergaard, 1973), because Cercospora was masked by fast growing fungi on agar. The blotter test was not satisfactory for detection of C. canescens in beans, since Phomopsis and Fusarium spp. overgrew the Cercospora. Attempts to detect C. canescens using seedling-symptom tests failed since no symptoms appeared. It was possible that seed-borne C. canescens was causing latent infection in the seedlings. Cerkauskas & Sinclair (1980) reported that by treating soybean tissues with paraquat (1,1' -dimethyl-a.a'-bipyridylium dichloride), latent infection could be detected. As a consequence of this report, the following technique, termed •seedling-paraquat', was developed for efficient detection of C. canescens in bean seeds. One hundred and seventy-two samples of 20 cultivars of beans grown under different agroclimatic conditions were tested . Two hundred seeds of
Table
1.
each sample were surface disinfected by immersing in 1'7 % NaOCl for 1 min, followed by 1 min in 70 % ethanol and washed in sterile water. The seeds were then planted in methyl bromide-treated sand in a greenhouse. About 5 days after emergence, the seedlings were sprayed with a 10 % solution of paraquat and then covered with a plastic tent to maintain high humidity. Seedlings died within 24 h and after 4-{) days the dead seedlings were examined for fungal growth. Comparisons were made with results from the standard blotter test, with 2-4D, using 200 seeds from each sample . When examined, profuse growth of fungi, especially Fusarium spp., was observed on the leaves and hypocotyl, and sometimes on the cotyledons of the seedlings. Cercospora, when present, formed colonies only on the upper side of the cotyledons. They were felty, dark olivaceous green to dark brown; and spored profusely. Initially colonies on the cotyledons where examined microscopically to confirm the identity of the fungus. With experience, however they could be identified in situ with the naked eye or with the help of a hand lens. The fast-growing fungi did not interfere with the detection of this pathogen. Using this technique the pathogen was detected in 32 % of the samples, compared to 11 % using the
Comparative frequency levels of seed infection with Cercospora canescens in samples, using two techniques
172
Infection ('Yo) Detection technique Seedling paraquat Z-4D-blotter Trans. Br . mycol. Soc. 81 (z), (1983)
0
0'5- 1
l'5- z '5
3-5
117 153
34 10
12
5
7
2
Printed in Great Britain
5'S-10t 10
z o
2 0
bean-seed
4 26
Notes and brief articles
2-4D blotter test (Table 1). The overall mean level of infection was 1'9 % of the seeds using' seedlingparaquat' technique as opposed to only 0'63 % with the 2-4D-blotter method. The difference between the two methods was significant at the 5 % level using a paired t test. The percentage of C. canescens-infected seeds varied between 0 and 13' 5 % as determined by the new technique and between 0 and 4 % when determined by the 2-4D-blotter method.
REFERENCES
CERKAUSKAS, R. F. & SINCLAIR, J. B. (1980). Use of paraquat for detection of fungi in soybean tissues. Phytopathology 70, 1036-1038. MATHUR, S. B. & KABBERE, F. (1975). Seed-borne fungi of sesame in Uganda. Seed Science and Technology 3, 665-660. MENEZES, J. R., MOHAN, S. K., ROSSETTO, E. A. & BIANCHINI, A. (1978). Qualidade sanitaria de sementes de feijjio na regiao norte do Estado do Parana. Fitopatologia Brasileira 3, 122-123 (Abstr.) NEERGAARD, P. (1973). Detection of seed-borne pathogens by culture tests. Seed Scienceand Technology 1,217-254.
CHEMICAL CONTROL OF FUSARIUM CULMORUM ON RYEGRASS, LOLIUM PERENNE BY ERIC J. GUSSIN AND JAMES M. LYNCH*
Agricultural Research Council, Letcombe Laboratory, Wantage, Oxon. OXI2 9JT A formulation containing calcium peroxide compared favourably with carbendazim and drazoxolon in controlling Fusarium culmorum on ryegrass. Direct drilling of grass, where old swards are desiccated by herbicides, offers considerable economic advantages over traditional methods of reseeding (Allen, 1981). However, the abundant decaying organic matter that this leaves on the soil surface can sometimes be detrimental to the newly sown grass. There is evidence for the production of phytotoxins from the organic matter (Gussin & Lynch, 1981; Lynch, 1977, 1978; Patrick, 1971). Fusarium culmorum (W.G.Sm.) Sacco has been shown to be a major colonizer of couch grass (Agropyron repensL.) rhizomes after they have been killed prior to direct drilling (Penn & Lynch, 1982), and this fungus is a well-known pathogen ofgrasses (Bennett, 1935), causing both pre- and postemergence death (Michail & Carr, 1966). It has been isolated from establishing ryegrass seedlings and causes symptoms in the laboratory similar to those found in the field. We have now compared the efficacy of a number of fungicides with a formulation containing calcium peroxide which has been shown to have some antifungal properties (Lynch, Harper & Sladdin, 1981). Fusarium culmorum was isolated from diseased ryegrass (Lolium perenne L. cv. S24) seedlings and cultured in sterile soil to maintain pathogenicity. When required, crumbs of soil were inoculated on to potato sucrose agar and used within 14 days. Spore suspensions were prepared by adding
* Glasshouse Crops Research Institute, Littlehampton, W. Sussex BN 16 3PU. (Address for correspondence). Trans. Br. mycol. Soc. 81 (2), (1983)
deionized water and brushing the Petri dishes with a camel-hair brush. The suspension was filtered through a muslin cloth and adjusted to 106 spores rnl" with sterile distilled water as necessary. Ryegrass seed (cv. S24) was dressed in 5 g batches by adding chemicals to the seed in glass jars on a roller (30 rev.jmin) for 1 h to ensure a uniform dressing. The chemicals were formulated in inert talc (Table 1) and screened initially at 250 mg a.i. kg- 1 seed unless stated otherwise. Seeds were dressed about 1 week in advance of being sown. Dressing rates for subsequent testing varied depending upon the level of activity found in the initial screen. The phytotoxicity of each of the compounds used was determined at the start of the investigation by sowing seeds (to) dressed with 250 mg a.i. kg" into pots (2'5 ern diam, 4'5 em deep) containing vermiculite (14 em") in the absence of the fungus. The initial screen determined which compounds had activity against Fusarium spp. and the best of these were then compared with a calcium peroxide formulation containing 60 % (wjw) Ca0 2 and 40% (wjw) CaOH 2 bound to the seed using a 4 % (v/w) solution of polyvinyl alcohol at a range of dressing rates. In all tests, dressed seeds (to) were sown into pots (2'5 em diam) containing vermiculite (14 ern") inoculated with a F. culmorum spore suspension (3 ml x 106 spores mr'). Non-dressed seed was sown into infected vermiculite to check the pathogenicity ofthe fungus on each occasion. Non-dressed seed sown into uninfected vermiculite served as the control and all
Printed in Great Britain