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A new method for the detection of Pleospora bjoerlingii infection of sugar beet seed
Notes and Brief Articles C. R. (1965). Reduction of Sclerotium bataticola infection of peanut kernels by Aspergillus flaous. Phytopathology 55, 934. JOFFE, A. Z. (1969). Aflatoxin production by 1,626 isolates of Aspergillus flaous from groundnut kernels and soil in Israel. Nature, London, lilli, 492. LILLARD, H. S., HANLIN, R. T . & LILLARD, D. A. (1970). Aflatoxigenic isolates of Aspergillusflavus from pecans. Applied Microbiology I9, 12~130 . PONS, W. , CUCULLU, A., FRANZ, A. & GOLDBLATI, L. (1968). Improved objective fluorodensitometric determination of aflatoxins in cottonseed products. Journal of American Society of Oil Chemists 45, 694--699· TABER, R. A. & SCHROEDER, H. W. (1967). Aflatoxin-producing potential of isolates of the Aspergillusfiavus-oryae group from peanuts (Arachis hypogaea). Applied Microbiology JACKSON,
I5, 1{O- 144'
A NEW METHOD FOR THE DETECTION OF PLEOSPORA B]OERLINGII INFECTION OF SUGAR BEET SEED AEDINE MANGAN
Seed Testing Station, Department of Agriculture and Fisheries, Dublin Pleospora bjoerlingii Byford (Phoma betae Frank) infection of sugar beet seed has hitherto been detected by two main methods: incubation of the clusters on moist blotter followed by examination for the presence of pycnidia, or incubation on potato dextrose agar (PDA) for identification of the fungal colonies. The use of specific light and temperature regimes and of pretreatment of the seed in chlorine have improved the reliability of these tests (de Tempe, 1968). However, some seed lots present difficulties under the existing methods. Such a group of samples was received in Dublin from the 1967 and 1968 seed crops in Oregon and British Columbia. These were heavily contaminated with Alternaria spp. and also with eelworms and paramecia. Because of a relatively humid growing season in 1968 it was expected that considerable P. bjoerlingii infection might be present but testing on PDA yielded no more than 13 % detectable P. bjoerlingii colonies. An alternative method to the development of fungal colonies identifiable with the naked eye was sought. P. bjoerlingii forms characteristic holdfast-like structures when infected seedlings or seed clusters are placed in shallow water or on agar in a Petri dish. Within 48 h at room temperature (about 20°C) the holdfasts form where the fungus is in contact with the bottom of the dish. These structures originate as a group of swollen branches at the tip of a hypha (PI. 11, fig. 1) which develop into a clump of swollen cells (PI. 1I, fig. 2). These later become vacuolated (PI. 1 1, fig. 3) but remain conspicuous for a considerable time. In the presence of bacteria the fungal hyphae may be darkened and the clumps reduced in size (PI. 1 1 , fig. 4). The holdfasts form in continuous light or darkness' as well as in normal light conditions in the laboratory. For the purpose of detecting P. bjoerlingii holdfasts formed from beet seed, water agar was found to be a suitable medium. It has the advantage Trans. Br. mycol. Soc. 57 (I ), (1971 ). Printed in Great Britain
Transactions British Mycological Society
170
over PDA that growth of saprophytes is greatly reduced while the pathogen is well developed for identification. Single pycnidiospores and fine debris from heavily infected seed samples were observed to give rise to the holdfasts. As pretreatment of the debris in chlorine almost eliminated the fungus, the desirability of using a chlorine pretreatment of the seed to obtain realistic results was investigated. A number of seed lots were tested on water agar both with and without chlorine pretreatment and the amount of P. bjoerlingii occurring, as shown by the production of holdfasts, was recorded. To evaluate the usefulness of the water agar method as an indicator of seed health these results were compared with the number of diseased seedlings obtained when the same lots were grown either in pasteurized soil or in compost. One hundred untreated clusters from each seed lot were placed, 5 per Petri dish, on water agar (1·6 % Oxoid no, 3), and a second hundred were pretreated for 10 min in 1 % sodium hypochlorite solution before plating. The plates were incubated for 5-6 days at about 20° in normal light conditions. The seed clusters and germinating seedlings were then removed from the dishes to allow unobscured examination. This was done from the reverse side of the dish under a magnification of x 40 on the compound microscope, A higher magnification was required to examine any doubtful structures. Table
I.
Comparison of Pleospora bjoerlingii infection levels obtained in seed and seedling tests Average % P. bjoerlingii
,
Year of harvest
Origin of seed British Columbia British Columbia Oregon Oregon France Italy Ireland
1967 1968 1967 1968 1969 1969 1968-9
* Multigerm seed.
No, of samples tested
,
Seed on water agar
3* 7* 3* 4*
0'7 68 0 59 45 13 95
lOt lOt 4*
t
,
Untreated Cl pretreated
0'3 32 0'3 48 19 1'2 52
Seedlings
3'3 38 4"3 38 20 8 58
Genetic monogerm seed.
Seedling tests on Irish seed lots consisted of planting 100 clusters in DeEE compost (moss peat based) in 'multipots'. The foreign lots were grown by L. D. Leach in pasteurized soil using 160 monogerm and 120 multigerm seed units. All seedlings were grown in the glasshouse for 3-4 weeks. Any seedlings with damping-off and those with blackleg at lifting time were checked either in water or on water agar for the presence of the pathogen as shown by production of holdfasts. Results are given in Table 1 of average % P. bjoerlingii found on lots of seed from various countries for different seasons using the water agar method and the seedling method. Good agreement is shown between pretreated seeds on water agar and Trans. Br. mycol. Soc. 57 (1), (1971). Printed in Great Britain
Notes and Brief Articles
171
seedling infection for moderately and heavily infected seeds, but for the healthier lots agreement is poor. When comparing the agar results with the seedling results it must be borne in mind that the latter do not take into account any diseased clusters failing to produce seedlings, or any pre-emergence damping-off. In the application of the water agar test for detection of P. bJoerlingii on beet seed clusters the results obtained with chlorine pretreated seeds show closer agreement generally with the seedling infection levels in the soil tests than do those with untreated seeds. As the chlorine treatment eliminated most infection from the fine debris of a seed sample it could be expected to reduce surface infection of seed clusters. Such surface infection may not give rise to seedling infection in soil. The reason for the lack of agreement between the agar and soil tests in the case of the British Columbia and Oregon 1967 samples is not known. The larger percentages of the pathogen detected on untreated seed compared with pretreated seed is contrary to the observation of de Tempe (1968) when using PDA. However, his lower figures obtained for untreated seed may have been due to saprophytes obscuring the presence of the pathogen. The main advantage of the water agar test over existing tests is the ability to identify P. bJoerlingii even in the presence of heavy contamination of the clusters with saprophytic organisms. The latter, in addition to producing less profuse growth on water agar than on an enriched medium, generally grow on or near the surface of the agar while the pathogen spreads down into the medium to produce its characteristic structures on the dish surface. The detection of high levels of infection in the 1968 American lots, which were unsatisfactory on PDA, demonstrates this advantage. The function of the holdfasts or hyphal clumps produced by P. bjoerlingii is not known. They have only been observed where the fungus has come into contact with the petri dish surface. They are not considered to be young or abortive pycnidia as these can be observed developing in the medium simultaneously and at various levels. Cultures of Phoma exigua Desm. and Plenodomas lingam (Tode ex Fr.) Hohnel when examined under similar conditions produced pycnidia but no holdfasts. The detection of P. bJoerlingii on sugar beet seed by identification of the characteristic hyphal clumps or holdfasts is suggested as a routine method of estimating infection levels. It is an extremely sensitive test and with a small amount of experience is easy and quick to carry out. No special incubation conditions are necessary. This method was found successful where the standard potato dextrose agar method was unsatisfactory due to competing micro-organisms. The author wishes to thank Professor L. D. Leach and the Irish Sugar Company for permission to use his unpublished results (June 1970) of seedling infection levels in soil.
Trans. Br. mycol. Soc. 57 (1), (1971). Printed in Great Britain
Transactions British Mycological Society
172
REFERENCE
( 1968). Some experiments on test ing beet seed for Phoma betae, Proceedings of the International Seed Testing Association 33, 567-472.
TEMPE,]. DE
EXPLANATION OF PLATE II
Pleospora bjoerlingii holdfast-like structures in water agar (all x 250) Fig. I . Young holdfasts forming from an infected beet seed. Fig. 2. Holdfasts formed from pycnidiospores incubated at 20° for 48 h. Fig. 3. Vacuolated cells of older holdfasts. Fig . 4. Darkened and restricted holdfasts in the presence of bacteria.
ELECTROPHORETIC PATTERNS OF ENZYMES FROM ISOLATES OF FUSARIUM GRAMINEARUM R. B. DRYSDALE AND P. M. BRATT
Department oj Microbiology, University oj Birmingham The genus Fusarium contains many species pathogenic to important agricultural and horticultural crops. Thus the rapid and accurate identification of taxa within the genus is of potential economic importance. The genus, however, is notoriously difficult to classify using the methods of classical mycology because of the lack of morphological stability and uniformity of species within the group. Because the identification of Fusaria may be difficult using orthodox techniques Booth (1966) suggested the use of biochemical methods. To investigate the potential of such methods we have used the technique of polyacrylamide gel electrophoresis of soluble protein extracted from mycelium in an attempt to distinguish five strains of F. graminearum, a fungus being used in this department in a study of tissue specificity in cereals (Strange & Smith, 1971 ) • Cultures of F. graminearum, obtained from the Commonwealth Mycological Institute and designated CMI 103220, 103225, 116240, 116244, 129482, were maintained on potato dextrose agar slopes. The fungi were grown at 25 °C for 7 days on a shaker in 250 ml conical flasks containing 100 ml malt extract broth. The mycelium was harvested by filtration, washed with distilled water, rolled dry in paper towels and either stored at - 20° or used immediately. After grinding with powdered glass the mycelium was extracted with 0·85 %saline (4 ml per g mycelium), centrifuged (30000 g, 30 min) and the concentration of protein in the supernatant measured (Lowry, Rosebrough, FaIT & Randall, 1951). If necessary the extract was concentrated by dialysis against solid sucrose at 4° overnight. The protein concentration in each extract was adjusted to 10 mg/ml and 0'05 ml samples were subjected to polyacrylamide gel electrophoresis. Trans. Br. mycol, Soc. 57 (I), (1971). Printed in Great Britain
I5, 1{O- 144'
A NEW METHOD FOR THE DETECTION OF PLEOSPORA B]OERLINGII INFECTION OF SUGAR BEET SEED AEDINE MANGAN
Seed Testing Station, Department of Agriculture and Fisheries, Dublin Pleospora bjoerlingii Byford (Phoma betae Frank) infection of sugar beet seed has hitherto been detected by two main methods: incubation of the clusters on moist blotter followed by examination for the presence of pycnidia, or incubation on potato dextrose agar (PDA) for identification of the fungal colonies. The use of specific light and temperature regimes and of pretreatment of the seed in chlorine have improved the reliability of these tests (de Tempe, 1968). However, some seed lots present difficulties under the existing methods. Such a group of samples was received in Dublin from the 1967 and 1968 seed crops in Oregon and British Columbia. These were heavily contaminated with Alternaria spp. and also with eelworms and paramecia. Because of a relatively humid growing season in 1968 it was expected that considerable P. bjoerlingii infection might be present but testing on PDA yielded no more than 13 % detectable P. bjoerlingii colonies. An alternative method to the development of fungal colonies identifiable with the naked eye was sought. P. bjoerlingii forms characteristic holdfast-like structures when infected seedlings or seed clusters are placed in shallow water or on agar in a Petri dish. Within 48 h at room temperature (about 20°C) the holdfasts form where the fungus is in contact with the bottom of the dish. These structures originate as a group of swollen branches at the tip of a hypha (PI. 11, fig. 1) which develop into a clump of swollen cells (PI. 1I, fig. 2). These later become vacuolated (PI. 1 1, fig. 3) but remain conspicuous for a considerable time. In the presence of bacteria the fungal hyphae may be darkened and the clumps reduced in size (PI. 1 1 , fig. 4). The holdfasts form in continuous light or darkness' as well as in normal light conditions in the laboratory. For the purpose of detecting P. bjoerlingii holdfasts formed from beet seed, water agar was found to be a suitable medium. It has the advantage Trans. Br. mycol. Soc. 57 (I ), (1971 ). Printed in Great Britain
Transactions British Mycological Society
170
over PDA that growth of saprophytes is greatly reduced while the pathogen is well developed for identification. Single pycnidiospores and fine debris from heavily infected seed samples were observed to give rise to the holdfasts. As pretreatment of the debris in chlorine almost eliminated the fungus, the desirability of using a chlorine pretreatment of the seed to obtain realistic results was investigated. A number of seed lots were tested on water agar both with and without chlorine pretreatment and the amount of P. bjoerlingii occurring, as shown by the production of holdfasts, was recorded. To evaluate the usefulness of the water agar method as an indicator of seed health these results were compared with the number of diseased seedlings obtained when the same lots were grown either in pasteurized soil or in compost. One hundred untreated clusters from each seed lot were placed, 5 per Petri dish, on water agar (1·6 % Oxoid no, 3), and a second hundred were pretreated for 10 min in 1 % sodium hypochlorite solution before plating. The plates were incubated for 5-6 days at about 20° in normal light conditions. The seed clusters and germinating seedlings were then removed from the dishes to allow unobscured examination. This was done from the reverse side of the dish under a magnification of x 40 on the compound microscope, A higher magnification was required to examine any doubtful structures. Table
I.
Comparison of Pleospora bjoerlingii infection levels obtained in seed and seedling tests Average % P. bjoerlingii
,
Year of harvest
Origin of seed British Columbia British Columbia Oregon Oregon France Italy Ireland
1967 1968 1967 1968 1969 1969 1968-9
* Multigerm seed.
No, of samples tested
,
Seed on water agar
3* 7* 3* 4*
0'7 68 0 59 45 13 95
lOt lOt 4*
t
,
Untreated Cl pretreated
0'3 32 0'3 48 19 1'2 52
Seedlings
3'3 38 4"3 38 20 8 58
Genetic monogerm seed.
Seedling tests on Irish seed lots consisted of planting 100 clusters in DeEE compost (moss peat based) in 'multipots'. The foreign lots were grown by L. D. Leach in pasteurized soil using 160 monogerm and 120 multigerm seed units. All seedlings were grown in the glasshouse for 3-4 weeks. Any seedlings with damping-off and those with blackleg at lifting time were checked either in water or on water agar for the presence of the pathogen as shown by production of holdfasts. Results are given in Table 1 of average % P. bjoerlingii found on lots of seed from various countries for different seasons using the water agar method and the seedling method. Good agreement is shown between pretreated seeds on water agar and Trans. Br. mycol. Soc. 57 (1), (1971). Printed in Great Britain
Notes and Brief Articles
171
seedling infection for moderately and heavily infected seeds, but for the healthier lots agreement is poor. When comparing the agar results with the seedling results it must be borne in mind that the latter do not take into account any diseased clusters failing to produce seedlings, or any pre-emergence damping-off. In the application of the water agar test for detection of P. bJoerlingii on beet seed clusters the results obtained with chlorine pretreated seeds show closer agreement generally with the seedling infection levels in the soil tests than do those with untreated seeds. As the chlorine treatment eliminated most infection from the fine debris of a seed sample it could be expected to reduce surface infection of seed clusters. Such surface infection may not give rise to seedling infection in soil. The reason for the lack of agreement between the agar and soil tests in the case of the British Columbia and Oregon 1967 samples is not known. The larger percentages of the pathogen detected on untreated seed compared with pretreated seed is contrary to the observation of de Tempe (1968) when using PDA. However, his lower figures obtained for untreated seed may have been due to saprophytes obscuring the presence of the pathogen. The main advantage of the water agar test over existing tests is the ability to identify P. bJoerlingii even in the presence of heavy contamination of the clusters with saprophytic organisms. The latter, in addition to producing less profuse growth on water agar than on an enriched medium, generally grow on or near the surface of the agar while the pathogen spreads down into the medium to produce its characteristic structures on the dish surface. The detection of high levels of infection in the 1968 American lots, which were unsatisfactory on PDA, demonstrates this advantage. The function of the holdfasts or hyphal clumps produced by P. bjoerlingii is not known. They have only been observed where the fungus has come into contact with the petri dish surface. They are not considered to be young or abortive pycnidia as these can be observed developing in the medium simultaneously and at various levels. Cultures of Phoma exigua Desm. and Plenodomas lingam (Tode ex Fr.) Hohnel when examined under similar conditions produced pycnidia but no holdfasts. The detection of P. bJoerlingii on sugar beet seed by identification of the characteristic hyphal clumps or holdfasts is suggested as a routine method of estimating infection levels. It is an extremely sensitive test and with a small amount of experience is easy and quick to carry out. No special incubation conditions are necessary. This method was found successful where the standard potato dextrose agar method was unsatisfactory due to competing micro-organisms. The author wishes to thank Professor L. D. Leach and the Irish Sugar Company for permission to use his unpublished results (June 1970) of seedling infection levels in soil.
Trans. Br. mycol. Soc. 57 (1), (1971). Printed in Great Britain
Transactions British Mycological Society
172
REFERENCE
( 1968). Some experiments on test ing beet seed for Phoma betae, Proceedings of the International Seed Testing Association 33, 567-472.
TEMPE,]. DE
EXPLANATION OF PLATE II
Pleospora bjoerlingii holdfast-like structures in water agar (all x 250) Fig. I . Young holdfasts forming from an infected beet seed. Fig. 2. Holdfasts formed from pycnidiospores incubated at 20° for 48 h. Fig. 3. Vacuolated cells of older holdfasts. Fig . 4. Darkened and restricted holdfasts in the presence of bacteria.
ELECTROPHORETIC PATTERNS OF ENZYMES FROM ISOLATES OF FUSARIUM GRAMINEARUM R. B. DRYSDALE AND P. M. BRATT
Department oj Microbiology, University oj Birmingham The genus Fusarium contains many species pathogenic to important agricultural and horticultural crops. Thus the rapid and accurate identification of taxa within the genus is of potential economic importance. The genus, however, is notoriously difficult to classify using the methods of classical mycology because of the lack of morphological stability and uniformity of species within the group. Because the identification of Fusaria may be difficult using orthodox techniques Booth (1966) suggested the use of biochemical methods. To investigate the potential of such methods we have used the technique of polyacrylamide gel electrophoresis of soluble protein extracted from mycelium in an attempt to distinguish five strains of F. graminearum, a fungus being used in this department in a study of tissue specificity in cereals (Strange & Smith, 1971 ) • Cultures of F. graminearum, obtained from the Commonwealth Mycological Institute and designated CMI 103220, 103225, 116240, 116244, 129482, were maintained on potato dextrose agar slopes. The fungi were grown at 25 °C for 7 days on a shaker in 250 ml conical flasks containing 100 ml malt extract broth. The mycelium was harvested by filtration, washed with distilled water, rolled dry in paper towels and either stored at - 20° or used immediately. After grinding with powdered glass the mycelium was extracted with 0·85 %saline (4 ml per g mycelium), centrifuged (30000 g, 30 min) and the concentration of protein in the supernatant measured (Lowry, Rosebrough, FaIT & Randall, 1951). If necessary the extract was concentrated by dialysis against solid sucrose at 4° overnight. The protein concentration in each extract was adjusted to 10 mg/ml and 0'05 ml samples were subjected to polyacrylamide gel electrophoresis. Trans. Br. mycol, Soc. 57 (I), (1971). Printed in Great Britain