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Selective medium for the isolation from soil of the leaf curl pathogen of anemones
No tes and brief articles Transactions of the Br itish M y cological Society 87, 466-471HORNBY, D . (1984). Akenomy ces costatus sp.no v. and validati on of Akenomy ces Arnaud. Tr ansactions of the B ritish Myco logical S ociety 82, 653-664. MARCHANT, R. (1970). The root surface of A mmophila arenaria as a substrate for micro org an isms. Tr ansactions of the British My cological S ociety 54, 479-482 . OLD, K . M . & N ICOLSON, T. H . (1975). Electron mic roscopi cal studies of the m icroflora of roots of sand dune grasses. New Phytolog ist 74,51-58. ROVIRA, A. D. & CAMPBELL, R. (1974). Scanning electron microscop y of microorganisms on the roots of wint er wheat. M icrobial Ecology 1, 15-23.
553
ROVIRA, A. D. & CAMPBELL, R . (1975). A scanning electron micros cope study of the interaction between micr oogan isms and Gaeumannomy ces graminis (syn . O . graminis) on wheat roots. Microbial Ecology 2 , 177- 18 5. WAID, J. S . (1974). The decomposition of root s. In Th e B iology of Plant L itt er Decomposition, Vol. 1 (ed. C. H . Dick inson & G. J. F. Pugh), pp . 175-210. L ondon , U .K . : Acad emic Press. SALEMA, R. & BRANDAO, 1. (1973). The use of PIPES buffer in the fixat ion of plant cells for electron microscop y. J ournal of Submicroscopic Cy tology 5, 79-96.
SELECTIVE MEDIUM FOR THE ISOLATION FROM SOIL OF THE LEAF CURL PATHOGEN OF ANEMONES BY I. BARKER* AND D. PITT
Department of Biological Sci ences, Washington Singer Laboratories, University of Exeter, Exeter EX4 4QG
A selective medium for the isolation from soil of Colletotrichum acutatum f.sp. pinea was adapted for the isolation of the leaf curl pathogen (Colletotrichum sp.) of anemones in southwest England. The effects of pH and of concentration of pentachloronitrobenzene (PCNB) on the selectivity of the media are described. Leaf curl disease of culti vated anemones in southwest England is caused by a species of Colletotrichum similar to both Colletotrichum acutatum Simmonds and Colletotrichumgloeosporioides (Penz.) Penz . & Sacco (Selley, 1980 ; Price, 1985 ; Doornik, 1985). Isolates of this pathogen have been reported to survive in soil for up to 18 months (Linfield & Price, 1983) and have some ability to infect via the roots of anemone plants (Selley, 1980 ; Doornik, 1985) in artificially infested soil. The question of whether the pathogen can survive in soil in southwest England has important implications in the management of th is disease. This work describes the adaptation of a selective medium for the isolation of C. acutatum f.sp. pinea from soil (Nair & Corbin, 1979) for the isolation of the leaf curl pathogen, also from soil. The need for such a medium has been noted by Doornik (1985), though a selective medium for isolation of the pathogen from corm material has been described (Gullino, Lento & Garibaldi, 1983 ). The pathogen had previously been isolated from art ificially infested fert ile soil using the medium of Na ir & Corbin (1979) (T able 1), but the colonies were difficult to identify and were often overgrown by muco raceous fungi (F ig. 1 A). It was found that
* Pr esent address : Rothamsted Exp erimental Station, Ha rpenden, H erts, AL 5 2JQ. Tr ans. B r. my col. S oc. 88 (4), (1987)
an acidified and weaker corn meal agar (CMA), rather than potato dextrose agar (PD A), basal medium greatl y suppressed contaminant fungal growth and made th e Colletotrichum colonies more distinctive (F ig. 1 B). The concentration of one of the fungicides used, pentachloronitrobenzene (PCN B), and the pH of the medium were optimized by comparing their effects in a factor ial replicated design on the isolation of the pathogen from art ificially infested field soil. A measure of the suppression of other soil fungi was derived from the total number of contaminant colonies isolated from the same source. A suspension containing 1 x 104 conidia was obtained from a PDA plate culture of the pathogen and mixed with 2 g of field soil (brown earth of the Warrington series). The soil was added to 200 ml of sterile distilled water containing 0 '1 % (w Iv) Teepol and shaken for 30 min. Samples (0 '25 ml) were spread on to the various media using a glass rod and the Petri dishes incubated under ' daylight' and ' blacklight' fluorescent light ing with a 1 2 h photoperiod at 25°C. The media were prepared as described by Na ir & Corbin (1979), except that CMA was substituted for PDA and orthophosphoric acid was used for acidification prior to autocla ving . Colonies of C. acutatum and other organ isms isolated were counted separately and analysed by analysis of variance following tests for
Printed in Great B rit ain
554
Notes and brief articles
Table
1.
Composition of the selective medium of Nair & Corbin (1979) and the final medium for the isolation of the leaf curl pathogen from soil Concentration of a.i. (p.p.m.) Component
Nair & Corbin (1979)
Final medium
PDA (pH 6'5) 5° 7'5 or 75 100 100 100
CMA (pH 5) 5°
Basal medium Benomyl (Benlate, 50% a.i.) PCNB (Botrilex, 20% a.i.) Streptomycin sulphate Chloramphenicol Chlortetracycline HCl
25 100 100 100
a.i., active ingredient
Table
2.
Effect of pH and PCNB concentration on the mean (five replicate plates) number of contaminant colonies isolated per plate PCNB (p.p.m., a.i.) pH
10
25
50
4'0 5'0 6'0
8·6 12'0 16·6
7'6 10'6 16'4
7"4 5'0 16'2
a.i., active ingredient. Effect of pH significant at P < 0'001. LoS.D. (P
=
0'05) = 2'98.
Table 3. Effect of pH and PCNB concentration on the mean (five replicate plates) number of Colletotrichum sp, colonies isolated per plate PCNB (p.p.m., a.i.) pH
10
25
4'0 5'0 6'0
6'4 8'0 9'0
8'4 12'0 9'0
a.i., active ingredient. LoS.D. (P
50 6,8 7'8 7'8 =
0'05) = 3'76.
B
A
Fig. 1. Agar plates showing isolation of Colletotrichum from fertile soil, (A) using the medium of Nair & Corbin (1979), (B) on the adopted selective medium with an arrow denoting one of several colonies of Colletotrichum.
Trans. Br. mycol. Soc. 88 (4), (1987)
Printed in Great Britain
Notes and brief articles homogeneity of variance, The entire experiment was repeated at a separate date. It can be seen from Table 2 that acidification of the medium from pH 6 to pH 5 or 4 significantly reduced the number of contaminant colonies isolated without affecting the recovery of Colletotrichum (Table 3). However, the media at pH 4 were very soft and impracticable to work with, and hence pH 5 was chosen for the final medium. No such effect was noticed with varying PCNB concentrations (Tables 2, 3), though higher concentrations clearly reduced the radial growth of all the resultant colonies (visual observation). A PCNB concentration of 25 p.p.m. was chosen as a compromise between adequate control of contaminant colony growth and early identification of the Colletotrichum colonies. There was no evidence for an interaction between pH and PCNB concentration. The composition of the final medium is given in Table 1. As a comparison, isolations were also made from the same soil suspension on the original medium of Nair & Corbin (1979) prepared with PCNB at 7'5 and 75 p.p.m. Whilst recovery of Colletotrichum was the same on both of these media, the mean numbers of contaminant colonies were 3'2 and 2'4
555
times greater respectively than on the final adapted medium. The selective medium has proved useful in examining the behaviour of the pathogen in soil and in studying the dispersal of conidia. This work was supported by an M.A.F.F. advanced postgraduate research studentship to I.E.
REFERENCES
DOORNIK, A. W. (1985). Jaarverslag van het Laboratorium voor Bloembollenonderzoek 1984. GULLINO,M. L., LENTO, G. & GARIBALDI, A. (1983). Un substrato semiselettivo per l'isolamento di Colletotrichum gloeosporioides Penz. da organi di anemone infetti. Informatore Fitopatologico 33, 114-116. LINFIELD, C. & PRICE, D. (1983). Anemone leaf curl. Grower 99, 27-29· NAIR, J. & CORBIN, J. B. (1979). A medium for isolation of Colletotrichum acutatum f.sp. pinea from soil. Australasian Plant Pathology 8, 6-7. PRICE, D, (1985). Glasshouse Crops Research Institute Annual Report 1984. SELLEY, A. P. (1980). A new disease of Anemone coronaria caused by Colletotrichum sp. M.Sc. Thesis, University of Exeter.
DETECTION AND MORPHOLOGY OF HYPHAE OF COMMON BUNT FUNGI (TILLETIA LAEVIS AND T. TRITICI) IN WHEAT SEEDLINGS BY J. F. KOLLMORGEN AND D. J. BALLINGER Victorian Crops Research Institute, Private Bag 260, Horsham, Victoria, Australia 3400
A technique was developed to detect hyphae of the common bunt fungi (Tilletia laevis and T. triticii in wheat seedlings. This accurately predicted the subsequent incidence of bunted heads in two field trials. Details of the morphology of Tilletia hyphae in leaf-sheath tissues are given.
Tilletia laevis Kuhn and Tilletia tritici (Bjerk.) R. Wolff cause common bunt of wheat. In Australia, as in other parts of the world, the disease is controlled by treating seed with fungicides. An estimated 80 % of Australian wheat seed is treated before sowing (Jones, 1981) and disease outbreaks are uncommon. Nevertheless, each year new chemicals are tested for efficacy against common bunt in an attempt to replace current products with materials that are less expensive, more fungicidal and more environmentally acceptable. Alternative chemicals are also sought because, in the past, some recommended products were phytotoxic under particular environmental conditions and were withdrawn at short notice (Tuohey, Mullett & Easton, 1972; Kuiper, 1974; Kollmorgen & Ballinger, 1975). Furthermore, it is essential to have Trans. Br. mycol. Soc. 88 (4), (1987)
alternative products in case strains of the bunt fungi resistant to current chemicals are encountered or selected, as happened with hexachlorobenzene (Kuiper, 1965). Most of the chemicals that have been used to control common bunt in Australia, e.g. carboxin, fenaminosulf, prevent germination of seed-borne teliospores. Although the ability of a chemical to inhibit teliospore germination or restrict development of the promycelium and sporidia can be assessed on agar plates it is extremely difficult from these data alone to predict the concentration necessary to control common bunt in the field. Bunticides are rarely evaluated on plants grown under controlled-environment conditions. Thus in Australia chemicals are routinely tested for control of common bunt in the field. Seed is artificially
Printed in Great Britain
553
ROVIRA, A. D. & CAMPBELL, R . (1975). A scanning electron micros cope study of the interaction between micr oogan isms and Gaeumannomy ces graminis (syn . O . graminis) on wheat roots. Microbial Ecology 2 , 177- 18 5. WAID, J. S . (1974). The decomposition of root s. In Th e B iology of Plant L itt er Decomposition, Vol. 1 (ed. C. H . Dick inson & G. J. F. Pugh), pp . 175-210. L ondon , U .K . : Acad emic Press. SALEMA, R. & BRANDAO, 1. (1973). The use of PIPES buffer in the fixat ion of plant cells for electron microscop y. J ournal of Submicroscopic Cy tology 5, 79-96.
SELECTIVE MEDIUM FOR THE ISOLATION FROM SOIL OF THE LEAF CURL PATHOGEN OF ANEMONES BY I. BARKER* AND D. PITT
Department of Biological Sci ences, Washington Singer Laboratories, University of Exeter, Exeter EX4 4QG
A selective medium for the isolation from soil of Colletotrichum acutatum f.sp. pinea was adapted for the isolation of the leaf curl pathogen (Colletotrichum sp.) of anemones in southwest England. The effects of pH and of concentration of pentachloronitrobenzene (PCNB) on the selectivity of the media are described. Leaf curl disease of culti vated anemones in southwest England is caused by a species of Colletotrichum similar to both Colletotrichum acutatum Simmonds and Colletotrichumgloeosporioides (Penz.) Penz . & Sacco (Selley, 1980 ; Price, 1985 ; Doornik, 1985). Isolates of this pathogen have been reported to survive in soil for up to 18 months (Linfield & Price, 1983) and have some ability to infect via the roots of anemone plants (Selley, 1980 ; Doornik, 1985) in artificially infested soil. The question of whether the pathogen can survive in soil in southwest England has important implications in the management of th is disease. This work describes the adaptation of a selective medium for the isolation of C. acutatum f.sp. pinea from soil (Nair & Corbin, 1979) for the isolation of the leaf curl pathogen, also from soil. The need for such a medium has been noted by Doornik (1985), though a selective medium for isolation of the pathogen from corm material has been described (Gullino, Lento & Garibaldi, 1983 ). The pathogen had previously been isolated from art ificially infested fert ile soil using the medium of Na ir & Corbin (1979) (T able 1), but the colonies were difficult to identify and were often overgrown by muco raceous fungi (F ig. 1 A). It was found that
* Pr esent address : Rothamsted Exp erimental Station, Ha rpenden, H erts, AL 5 2JQ. Tr ans. B r. my col. S oc. 88 (4), (1987)
an acidified and weaker corn meal agar (CMA), rather than potato dextrose agar (PD A), basal medium greatl y suppressed contaminant fungal growth and made th e Colletotrichum colonies more distinctive (F ig. 1 B). The concentration of one of the fungicides used, pentachloronitrobenzene (PCN B), and the pH of the medium were optimized by comparing their effects in a factor ial replicated design on the isolation of the pathogen from art ificially infested field soil. A measure of the suppression of other soil fungi was derived from the total number of contaminant colonies isolated from the same source. A suspension containing 1 x 104 conidia was obtained from a PDA plate culture of the pathogen and mixed with 2 g of field soil (brown earth of the Warrington series). The soil was added to 200 ml of sterile distilled water containing 0 '1 % (w Iv) Teepol and shaken for 30 min. Samples (0 '25 ml) were spread on to the various media using a glass rod and the Petri dishes incubated under ' daylight' and ' blacklight' fluorescent light ing with a 1 2 h photoperiod at 25°C. The media were prepared as described by Na ir & Corbin (1979), except that CMA was substituted for PDA and orthophosphoric acid was used for acidification prior to autocla ving . Colonies of C. acutatum and other organ isms isolated were counted separately and analysed by analysis of variance following tests for
Printed in Great B rit ain
554
Notes and brief articles
Table
1.
Composition of the selective medium of Nair & Corbin (1979) and the final medium for the isolation of the leaf curl pathogen from soil Concentration of a.i. (p.p.m.) Component
Nair & Corbin (1979)
Final medium
PDA (pH 6'5) 5° 7'5 or 75 100 100 100
CMA (pH 5) 5°
Basal medium Benomyl (Benlate, 50% a.i.) PCNB (Botrilex, 20% a.i.) Streptomycin sulphate Chloramphenicol Chlortetracycline HCl
25 100 100 100
a.i., active ingredient
Table
2.
Effect of pH and PCNB concentration on the mean (five replicate plates) number of contaminant colonies isolated per plate PCNB (p.p.m., a.i.) pH
10
25
50
4'0 5'0 6'0
8·6 12'0 16·6
7'6 10'6 16'4
7"4 5'0 16'2
a.i., active ingredient. Effect of pH significant at P < 0'001. LoS.D. (P
=
0'05) = 2'98.
Table 3. Effect of pH and PCNB concentration on the mean (five replicate plates) number of Colletotrichum sp, colonies isolated per plate PCNB (p.p.m., a.i.) pH
10
25
4'0 5'0 6'0
6'4 8'0 9'0
8'4 12'0 9'0
a.i., active ingredient. LoS.D. (P
50 6,8 7'8 7'8 =
0'05) = 3'76.
B
A
Fig. 1. Agar plates showing isolation of Colletotrichum from fertile soil, (A) using the medium of Nair & Corbin (1979), (B) on the adopted selective medium with an arrow denoting one of several colonies of Colletotrichum.
Trans. Br. mycol. Soc. 88 (4), (1987)
Printed in Great Britain
Notes and brief articles homogeneity of variance, The entire experiment was repeated at a separate date. It can be seen from Table 2 that acidification of the medium from pH 6 to pH 5 or 4 significantly reduced the number of contaminant colonies isolated without affecting the recovery of Colletotrichum (Table 3). However, the media at pH 4 were very soft and impracticable to work with, and hence pH 5 was chosen for the final medium. No such effect was noticed with varying PCNB concentrations (Tables 2, 3), though higher concentrations clearly reduced the radial growth of all the resultant colonies (visual observation). A PCNB concentration of 25 p.p.m. was chosen as a compromise between adequate control of contaminant colony growth and early identification of the Colletotrichum colonies. There was no evidence for an interaction between pH and PCNB concentration. The composition of the final medium is given in Table 1. As a comparison, isolations were also made from the same soil suspension on the original medium of Nair & Corbin (1979) prepared with PCNB at 7'5 and 75 p.p.m. Whilst recovery of Colletotrichum was the same on both of these media, the mean numbers of contaminant colonies were 3'2 and 2'4
555
times greater respectively than on the final adapted medium. The selective medium has proved useful in examining the behaviour of the pathogen in soil and in studying the dispersal of conidia. This work was supported by an M.A.F.F. advanced postgraduate research studentship to I.E.
REFERENCES
DOORNIK, A. W. (1985). Jaarverslag van het Laboratorium voor Bloembollenonderzoek 1984. GULLINO,M. L., LENTO, G. & GARIBALDI, A. (1983). Un substrato semiselettivo per l'isolamento di Colletotrichum gloeosporioides Penz. da organi di anemone infetti. Informatore Fitopatologico 33, 114-116. LINFIELD, C. & PRICE, D. (1983). Anemone leaf curl. Grower 99, 27-29· NAIR, J. & CORBIN, J. B. (1979). A medium for isolation of Colletotrichum acutatum f.sp. pinea from soil. Australasian Plant Pathology 8, 6-7. PRICE, D, (1985). Glasshouse Crops Research Institute Annual Report 1984. SELLEY, A. P. (1980). A new disease of Anemone coronaria caused by Colletotrichum sp. M.Sc. Thesis, University of Exeter.
DETECTION AND MORPHOLOGY OF HYPHAE OF COMMON BUNT FUNGI (TILLETIA LAEVIS AND T. TRITICI) IN WHEAT SEEDLINGS BY J. F. KOLLMORGEN AND D. J. BALLINGER Victorian Crops Research Institute, Private Bag 260, Horsham, Victoria, Australia 3400
A technique was developed to detect hyphae of the common bunt fungi (Tilletia laevis and T. triticii in wheat seedlings. This accurately predicted the subsequent incidence of bunted heads in two field trials. Details of the morphology of Tilletia hyphae in leaf-sheath tissues are given.
Tilletia laevis Kuhn and Tilletia tritici (Bjerk.) R. Wolff cause common bunt of wheat. In Australia, as in other parts of the world, the disease is controlled by treating seed with fungicides. An estimated 80 % of Australian wheat seed is treated before sowing (Jones, 1981) and disease outbreaks are uncommon. Nevertheless, each year new chemicals are tested for efficacy against common bunt in an attempt to replace current products with materials that are less expensive, more fungicidal and more environmentally acceptable. Alternative chemicals are also sought because, in the past, some recommended products were phytotoxic under particular environmental conditions and were withdrawn at short notice (Tuohey, Mullett & Easton, 1972; Kuiper, 1974; Kollmorgen & Ballinger, 1975). Furthermore, it is essential to have Trans. Br. mycol. Soc. 88 (4), (1987)
alternative products in case strains of the bunt fungi resistant to current chemicals are encountered or selected, as happened with hexachlorobenzene (Kuiper, 1965). Most of the chemicals that have been used to control common bunt in Australia, e.g. carboxin, fenaminosulf, prevent germination of seed-borne teliospores. Although the ability of a chemical to inhibit teliospore germination or restrict development of the promycelium and sporidia can be assessed on agar plates it is extremely difficult from these data alone to predict the concentration necessary to control common bunt in the field. Bunticides are rarely evaluated on plants grown under controlled-environment conditions. Thus in Australia chemicals are routinely tested for control of common bunt in the field. Seed is artificially
Printed in Great Britain