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Effect of interaction of Rhizoctonia spp. with other fungi from cereal bare patches on root rot of wheat
Notes and brief articles
256
required before the mechanism of sexual reproduction is fully elucidated. Smith (1983) has reviewed reports of the occurrence of perithecia of M. nivale in Europe and North America. Few observations have been made on cereals, and with the exception of a report by Dennis (1964) on Glyceria fiuitans (L.) R. Br., the teleomorph is unknown on grasses. After years of study, failure by Smith (1983) to induce perithecial production in turfgrass isolates in vitro or to observe perithecia on turfgrasses in Europe or Canada led him to speculate that these isolates may have lost the ability to produce perithecia, a feature that appeared to differentiate turfgrass isolates from cereal isolates. Our results indicate that perithecial production can be induced in turfgrass isolates of M. nivale although the mechanism of induction appears to be different from the mechanism in cereal isolates. As with Smith (1983), we have failed to observe the teleomorph of M. nivale on grasses or cereals in vivo in Canada. This research was supported in part by grant A0449 from the Natural Sciences and Engineering Council of Canada. Additional support from the Ontario Ministry of Agriculture and Food is gratefully acknowledged. REFERENCES
ARX, J. A. VON (1984). Notes on Monographella and Microdochium. Transactions of the British Mycological Society 82, 373-374.
BOOTH, C. (1971). The Genus Fusarium. Commonwealth AgriculturalBureaux, Slough. BRUNDRETT, M. E., PICHE, Y. C. & PETERSON, R. L. (1984). A newmethod for observingthe morphologyof vesicular-arbuscular Mycorrhizae. Canadian Journal of Botany 62, 2128-2134. CUNNINGHAM, J. L. (1972). A miraclemounting fluid for permanentwhole-mountsof microfungi. Mycologia 64, 906--911. DENNIS, R. W. G. (1964). As cited in Smith, J. D. (1983). Fusarium nivale (Gerlachia nivalis) from cereals and grasses: is it the same fungus? Canadian Plant Disease Survey 63, 25-26. GAMS, W. J. & MULLER, E. (1980). Conidiogenesis of Fusarium nivale and Rhynchosporium oryzae and its taxonomic implications. Netherlands Journal of Plant Pathology 86, 45-53. KESSEL, R. G. & SHIH, C. Y. (1974). Scanning Electron Microscopy in Biology: A Student's Atlas on Biological Organization. New York: Springer-Verlag. SAMUELS, G. J. & HALLETT, 1. C. (1983). Microdochium stoueri and Monographella stoveri. New combinations for Fusarium stoveri and Micronectriella stoveri. Transactions of the British Mycological Society 81, 473-483. SMILEY, R. W. (1983). Compendium of Turfgrass Diseases.
St Paul, Minnesota: The American Phytopathological Society. SMITH, J. D. (1983). Fusarium nivale (Gerlachia nivalis) from cereals and grasses: is it the same fungus? Canadian Plant Disease Survey 63, 25-26. SNEDECOR, G. W. & COCHRAN, W. G. (1980). Statistical Methods. Iowa: Iowa State University Press. WIESE, M. W. (1977). Compendium of Wheat Diseases. St Paul. Minnesota: The American Phytopathological Society.
EFFECT OF INTERACTION OF RHIZOCTONIA SPP. WITH OTHER FUNGI FROM CEREAL BARE PATCHES ON ROOT ROT OF WHEAT BY F. A. ROBERTS AND K. SIVASITHAMPARAM
Soil Science and Plant Nutrition, School of Agriculture, University of Western Australia, Nedlands, Western Australia, 6009 Rhizoetonia solani (Ag 8), binucleate Rhizoctonia sp. (Ag C), Fusariumgraminearum, Bipolaris sorokiniana, Waitea circinata, Mortierella sp. and Pythium irregulare, from bare patches of
wheat and barley were evaluated as root pathogens of wheat. Combinations of these fungi caused more severe root disease than the same fungi used singly. A complex of these fungi rather than anyone pathogen is proposed as the cause of the bare patch disease of cereals. Rhizoctonia spp. attack and cause root rots of
subterranean clover (Wong & Sivasithamparam, 1985) and white clover (Maughan & Barbetti, 1983) and cause bare patch of wheat and barley (Roberts & Sivasithamparam, 1986) in Western Australia. In some diseases, R. solani Kuhn has been found in complexes with a range of associated fungi and sometimes has synergistic relationships with nematode or virus infections (Harris & Moen, Trans. Br. mycol. Soc. 89 (2), (1987)
1985a). There are a number of root diseases in which R. solani is but one member of a complex of fungi which incite disease with more or less the same pattern of symptoms (Bateman, 1970). These include corn root rot (Rao et al., 1978), cotton stunt (Reynolds & Hanson, 1957), strawberry root rot (Wilhelm et al., 1972) and cutting rots of Peperomia obtusifolia (Munnecke & Chandler, 1953). A complex offungi (Pythium irregulare Buisman,
Printed in Great Britain
Notes and brief articles Phytophthora clandestina Greenhalgh, Pascoe & Taylor, Fusarium oxysporum Schlecht., R. solani and F. avenaceum (Fr.) Sacc.) have been associated with root rots of subterranean clover in the south western region of Western Australia (Barbett i et al., 1986). Work by Harris & Moen (1985a) has shown that a complex of organisms may be responsible for the Rhizoctonia disease complex of wheat in South Australia, Scott et al. (1979) implicated Periconia macrospinosa Lefebvre & Johnson, Pythium oligandrum Drechsler, Rhizoctonia cerealis van der Hoeven and R. solani as the main fungi associated with the ' crater ' disease of summer wheat in South Africa. Neate (1985) also reported the existence of Rhizoetonia spp, other than R. solani in South Australian wheat fields; the species include Ceratobasidium cornigerum (Bourdot) Rogers, Waitea circinata Warcup & Talbot and Iodophanus carneus (Pers.) Korf. Neate & Warcup (1985) designated a new anastomosis group, Ag 8, comprising the most common strain of R. solani pathogenic to roots of cereals in South Australia. Roberts & Sivasithamparam (1986) proposed that a complex of fungi may be involved in the production of bare patches of cereals in Western Australia. We report here the results of a glasshouse pathogenicity test to determine the effects of the interaction of certain root rot fungi isolated from bare patches of cereals (Roberts & Sivasithamparam, 1986), on the development of root rot in wheat plants and to examine their potential involvement in the bare patch disease. Isolates of Rhizoctonia solani (Ag 8), binucleate Rhizoctonia sp. (Ag C), Fusarium gramin earum Schwabe, Bipolaris sorokiniana (Sacc.) Shoern., Waitea circinata , Mortierella sp. (IM I 310812) and Pythium irregulare were obtained from roots of wheat plants
Table
1.
257
collected in July , 1984 from bare patches at Lake Grace, Western Australia (Roberts & Sivasithamparam, 1986). Fusarium gramin earum had been grown on Potato Dextrose Agar (PD A) and stored on ceramic beads (Lange & Boyd, 1968 ), All other fungi had been stored on PDA slants under sterile mineral soil for 7-8 months at 4-5 0. Inoculum was prepared by growing the test fungi on moist sterile millet seeds (Panicum miliaceum L.) for 3-4 weeks at 23-25°. The colonized millet seeds were thoroughly mixed with pasteurized field soil from Lake Grace which had a history of severe bare patch of wheat . Control pots contained uncolonized sterile millet seeds. The soil had (p.p.rn.) nitrate-nitrogen 10'0, phosphorus 12'0, sulphate-sulfur 18'0, calcium 460, magnesium 73'0, potassium 80'0, copper 0'5, zinc 0'2, manganese 7'0, iron 65'0, total salts 74'0, sodium 190 '0, boron 0'05 and molybdenum 0'11. It also had a fertility index of 3 '94 ME (%), organic matter 2'1 %, calcium/magnesium ratio 3'78, exchangeable sodium ESP (%) 20'97 and a pH of 5'7. Plastic pots (7'5 ern diam) containing 200 g of inoculum-supplemented soil were sown with 10 surface-sterilized pregerminated seeds of wheat ( Triticum aestivum L. cv, Gamenya) and covered with a thin layer of inert plastic beads to reduce dry ing and cross-contamination between treatments . There were ten replicate pots for each treatment. The pots were placed in temperature controlled water-cooled tanks at 15 ± 1° and maintained at 65 % water-holding capacity (Piper, 1944). Treatments are shown in Table 1. Four weeks after sowing, surviving wheat seedlings were counted, washed and rated for root rot severity as described in Roberts & Sivasithamparam (1986). To establish Koch 's postulates, the fungi were re-isolated. Ten root pieces
Treatments and inoculum levels used to test pathogenicity on wheat
Treatment (1) Fusarium graminearum (2) Pythium irregulare (3) Bipolaris sorokiniana (4) Mort ierel/a sp. (5) Waitea circinata (6) Rhizocton ia solani (Ag 8) (7) R . solani (Ag 8)+ Rhizoctonia sp . (Ag C) (binucleate) + F. graminearum (8) As (7)+ P. irregulare (9) As (8)+ B. sorokiniana ( to) As (9) + Mortierella sp. (11) As (10) + Waitea circinata (12) Control (uncolonized sterile millet seeds)
Inoculum level (%)* 0'5 0'5 2 '0 2 '0 2'0 0 -2
0 -2+0'2+0'5
As (7)+ 0 '5 As (8)+ 2'0 As (9)+ 2-0 As ( 10)+ 2'0
* Weight of inoculum/ weight of air-dried soil. Trans. Br. mycol. S oc. 89 (2), (1987)
Printed in Great Br itain
Notes and brief articles ern long) were selected from the ten replicate pots of each treatment, surface sterilized for 10 min with 0'125 % sodium hypochlorite, rinsed in three changes of sterile distilled water, blotted dry and plated on PDA containing streptomycin sulfate (100 units ml"). Plates were incubated at 25°. All fungi tested were re-isolated from rotted tissues. The effect of fungal interaction on disease expression measured as root disease is shown in Table 2. Plants inoculated with the P. irregulare showed a mild root rot, moderate stunting of seedlings, slight discolouration of lower leaves and no vascular browning. Roots of plants infected with R. solani were discoloured from light to dark brown with soft tips, and were often nipped-off near the crown. The plants were stunted and leaves chlorotic. Roots of plants infected with F. graminearum were slightly necrotic, while those inoculated with B. sorokiniana showed a moderate root rot with black mycelial pads on infected roots and dark brown lesions on primary roots which coalesced to become black streaks. Roots of plants inoculated with W. circinata were filled with orange sclerotium-like bodies and had small orange to orange brown lesions. Plants inoculated with Mortierella sp. were healthy, free of root rot and (0'5-1'0
Table
2.
similar to uninoculated controls. Combinations of fungi (treatments 7, 8, 9, 10 and 11 of Table 1) produced significantly more disease than the same fungi used singly, A combination of Rhizoctonia solani (Ag 8) + binucleate Rhizoctonia sp. (Ag C) + F. graminearum + P. irregulare + B. sorokiniana resulted in the highest root disease index (96 %) recorded (Table 2). Shoot weight, plant size and root weight were found to be negatively correlated with root disease index. Wong, Barbetti & Sivasithamparam (1984) observed a similar effect with Rhizoetonia spp. affecting roots of subterranean clover in Western Australia. Ag 8, which has been recognized as a major pathogen in South Australia (Neate & Warcup, 1985), is more pathogenic with other fungi than by itself. Other fungi appeared to have a significant role in the rotting of roots, for instance the addition of P. irregulare to a complex containing Ag 8 and F. graminearum nearly doubled the amount of disease. Pythium sp. has been proposed as a serious pathogen of wheat in South Australia (Pittaway & Rathjen, 1983) and the U.S.A. (Cook & Haglund, 1982). It is also possible that the introduced P. irregulare would have multiplied on unutilized portions of millet seeds incorporated with the inoculum of other fungi. Increase in activity within
Effects of fungi singly and in combination on root disease index and survival of wheat
Treatments Uninoculated control Mortierella sp. Rhizoctonia solani (Ag 8) P. irregulare Waitea circinata Bipolaris sorokiniana R. solani (Ag 8) + Rhizoctonia (Ag C) + F. graminearum F. graminearum R. solani (Ag 8)+ Rhizoctonia (Ag C) + F. graminearum + P. irregulare-e B. sorokiniana+ Mortierella sp, + W. circinata R. solani (Ag 8) + Rhizoctonia (Ag C) + F. graminearum + P. irregulare-e B, sorokiniana+ Mortierella sp. R. solani (Ag 8)+ Rhizoctonia (Ag C) + F. graminearum + P. irregulare R. solani (Ag 8)+ Rhizoetonia (Ag C) + F. graminearum + P, irregulare + B. sorokiniana
Mean root disease index (%)* 7'6
at
6'5 a 41'5 b 45'1 b 47'0 b 56'1 b 57'1 b
Mean survival (%) 80 bt 98 a 98 a 62 c 75 b 97 a 100 a
77'3 c 89'3 d
93'2 d
94'5 d
32 d
56 c
* Disease index was computedaccording to McKinney (1923) and incorporated results for mean survival.
t
Meansin the samecolumnfollowed by the sameletter are not significantly different from eachother at 5 % level (Duncan's Multiple RangeTest). Trans. Br. mycol. Soc. 89 (2), (1987)
Printed in Great Britain
Notes and brief articles soil of P . irregulare in the pre sence of uncolonized millet seeds has been noted with root rot of subterranean clover (Barberti & Sivasithamparam, 1987)·
Harris & Moen (1985b) found that a complex of fungi were associated with root rot of wheat in South Australia. In a pot experiment, they observed that through different stages of plant growth a succession of fungi were involved in the root rot, where R. solani was the primary pathogen. There is also evidence that soil and climatic effects, induding the soil structure down the profile, may be of overriding importance in Rhizoctonia stunting of wheat (Naiki, 1985; Deacon & Scott, 1985). However, we propose that the bare patch disease which is a seedling disease in W. Australia is caused by a complex of fungi. We thank the Western Australian Wheat Industry Committee for the award of a studentship for one of us (F. A. Roberts). REFERENCES
BARBETTl, M. J. & SIVASITHAMPARAM, K . (1987). Effects of soil pasteurization on root rot, seedling survival and plant dry weight of subterranean clover inoculated with six fungal root pathogens. Australian Journal of Agricultural Re search 38, 317-327. BARBETTI, M. J., SIVASITHAMPARAM, K . & WONG, D . H . (1986). Root rot of subterranean clover . Review ofPlant Pathology 65, 287-295. BATEMAN, D. F. (1970). Pathogens and disease. In Rhizoctonia solani : Biology and Pathology (ed. J. R. Parmeter), pp. 161-171. Berkeley : University of California Pre ss. COOK, R. J . & HAGLUND, W. A. (1982). Pythium root rot : A barrier to yield of Pacific Northwest wheat . Wash ington State University Agricultural R esearch Center Bulletin No. XB0913. pp. 20. DEACON, J . W . & SCOTT, D. B. (1985). Rhizoetonia solani associated with crater disease (stu nt ing) of wheat in South Africa. Transactions of the British Mycological Society 85, 319-327. HARRIS, J . R. & MOEN, R. (1985a). Secondary infect ion and fertilizer response of wheat initially infected with Rhizoctonia solani. Transactions of the British Mycological Society 84, 307-315. HARRIS, J . R. & MOEN, R. (1985 b). Replacement of Rhizoctonia solani on wheat seedlings by a succession of root rot fungi. Transactions of the British M ycological Society 84, 11-20. LANGE, B. & BoYD, W. J. R. (1968). Preservation of fungal spores by dr ying on porcela in beads . Phytopathology 58, 1711-1712. MAUGHAN, R. D . & BARBETTl, M . J . (1983). Rhiz octonia
Tra ns. Br. my col. S oc. 89 (2), (1987)
259
root rot of white clover. Australian Plant Pathology 12, 13-14· McKINNEY, H . A. (1923). Influence of soil temperature and moisture on infection of wheat seedlings by H elmintho sporium satiuum . Journal of A gricultural Research %6, 195-218. MUNNECKE, D. E. & CHANDLER, P. A. (1953). Some diseases of variegated Peperomia. Plant Disease R eporter 37,434-435· NAIKI, T. (1985). Population and survival of sclerotia of Rhizoctonia solani in soil. In Ecology and Management of So il-Borne Plant Pathogens (ed . C. A. Parker, A. D . Rovira , K . J. Moore, P . T. W . Wong & J. F . Kollmorgen), pp . 51-53 . St . Paul: American Phytopathological Societ y. NEATE, S. M. (1985). Rhiz octonia in South Australian wheat fields. In Ecology and Management of S oil-Borne Plant Pathogens (ed. C. A. Parker, A. D. Rov ira, K . J . Moore, P. T . W . Wong & J . F. Kollmorgen), pp. 54-56. St Paul: American Phytopathological Society . NEATE, S. M . & WARCUP, J . H . (1985). Anastomosis grouping of some isolates of Thanatephorus cucumeris from agricultural soils in South Australia. Transactions of the British M ycological S ociety 85, 615--620. PIPER, C. S. (1944). S oil and Plant Analysis. Universit y of Adelaide, Adelaide. PITTAWAY, P. & RATHJEN, A. (1983). Are fungal root pathogens major components of the site selection pressures on wheat yield ? Proceedings of the Fourth International Congress of Plant Pathology, Melbourne, 1983, pp . 167. RAo, B. A., SCHMITTHENNER, A. F., CALDWELL, R. & ELLETT, C. W. (1978). Prevalence and virulence of Pythium species associated with root rot of com in poorly drained soil. Phytopathology 68, 1557-1563. REYNOLDS, H. W . & HANSON, R. G. (1957). Rhizoctonia disease of cotton in presence or absence of the cotton root-knot nematode in Arizona. Phytopathology 47, 256-261. ROBERTS, F. A. & SIVASITHAMPARAM, K. (1986). Identity and pathogenicity of Rhizoctonia spp . associated with bare patch of cereals at a field site in Western Australia. Netherlands J ournal of Plant Pathology 9%, 185-195. SCOTT, D. B., VISSER, C. P . N. & RUFENACHT, E. M. C. (1979). Crater disease of summer wheat in African dry lands . Plant Disease Reporter 63, 836-840. WILHELM, S., NELSON, P. E., THOMAS, H. E. & JOHNSON, H. (1972). Pathogenicity of strawberry root rot caused by Ceratobasidium species. Phytopathology 6%, 700--705. WONG, D. H ., BARBETTI, M . J. & SIVASITHAMPARAM, K . (1984). Effects of soil temperature and moisture on the pathogenicity of fung i associated with root rot of subterranean clover. Australian Journal of Agricultural Research 35, 675--684. WONG, D . H . & SIVASITHAMPARAM, K. (1985). Rhizoctonia spp . associated with root rots of subterranean clover in Western Australia. Transa ctions of the British M y cological S ociety 85, 21-27.
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required before the mechanism of sexual reproduction is fully elucidated. Smith (1983) has reviewed reports of the occurrence of perithecia of M. nivale in Europe and North America. Few observations have been made on cereals, and with the exception of a report by Dennis (1964) on Glyceria fiuitans (L.) R. Br., the teleomorph is unknown on grasses. After years of study, failure by Smith (1983) to induce perithecial production in turfgrass isolates in vitro or to observe perithecia on turfgrasses in Europe or Canada led him to speculate that these isolates may have lost the ability to produce perithecia, a feature that appeared to differentiate turfgrass isolates from cereal isolates. Our results indicate that perithecial production can be induced in turfgrass isolates of M. nivale although the mechanism of induction appears to be different from the mechanism in cereal isolates. As with Smith (1983), we have failed to observe the teleomorph of M. nivale on grasses or cereals in vivo in Canada. This research was supported in part by grant A0449 from the Natural Sciences and Engineering Council of Canada. Additional support from the Ontario Ministry of Agriculture and Food is gratefully acknowledged. REFERENCES
ARX, J. A. VON (1984). Notes on Monographella and Microdochium. Transactions of the British Mycological Society 82, 373-374.
BOOTH, C. (1971). The Genus Fusarium. Commonwealth AgriculturalBureaux, Slough. BRUNDRETT, M. E., PICHE, Y. C. & PETERSON, R. L. (1984). A newmethod for observingthe morphologyof vesicular-arbuscular Mycorrhizae. Canadian Journal of Botany 62, 2128-2134. CUNNINGHAM, J. L. (1972). A miraclemounting fluid for permanentwhole-mountsof microfungi. Mycologia 64, 906--911. DENNIS, R. W. G. (1964). As cited in Smith, J. D. (1983). Fusarium nivale (Gerlachia nivalis) from cereals and grasses: is it the same fungus? Canadian Plant Disease Survey 63, 25-26. GAMS, W. J. & MULLER, E. (1980). Conidiogenesis of Fusarium nivale and Rhynchosporium oryzae and its taxonomic implications. Netherlands Journal of Plant Pathology 86, 45-53. KESSEL, R. G. & SHIH, C. Y. (1974). Scanning Electron Microscopy in Biology: A Student's Atlas on Biological Organization. New York: Springer-Verlag. SAMUELS, G. J. & HALLETT, 1. C. (1983). Microdochium stoueri and Monographella stoveri. New combinations for Fusarium stoveri and Micronectriella stoveri. Transactions of the British Mycological Society 81, 473-483. SMILEY, R. W. (1983). Compendium of Turfgrass Diseases.
St Paul, Minnesota: The American Phytopathological Society. SMITH, J. D. (1983). Fusarium nivale (Gerlachia nivalis) from cereals and grasses: is it the same fungus? Canadian Plant Disease Survey 63, 25-26. SNEDECOR, G. W. & COCHRAN, W. G. (1980). Statistical Methods. Iowa: Iowa State University Press. WIESE, M. W. (1977). Compendium of Wheat Diseases. St Paul. Minnesota: The American Phytopathological Society.
EFFECT OF INTERACTION OF RHIZOCTONIA SPP. WITH OTHER FUNGI FROM CEREAL BARE PATCHES ON ROOT ROT OF WHEAT BY F. A. ROBERTS AND K. SIVASITHAMPARAM
Soil Science and Plant Nutrition, School of Agriculture, University of Western Australia, Nedlands, Western Australia, 6009 Rhizoetonia solani (Ag 8), binucleate Rhizoctonia sp. (Ag C), Fusariumgraminearum, Bipolaris sorokiniana, Waitea circinata, Mortierella sp. and Pythium irregulare, from bare patches of
wheat and barley were evaluated as root pathogens of wheat. Combinations of these fungi caused more severe root disease than the same fungi used singly. A complex of these fungi rather than anyone pathogen is proposed as the cause of the bare patch disease of cereals. Rhizoctonia spp. attack and cause root rots of
subterranean clover (Wong & Sivasithamparam, 1985) and white clover (Maughan & Barbetti, 1983) and cause bare patch of wheat and barley (Roberts & Sivasithamparam, 1986) in Western Australia. In some diseases, R. solani Kuhn has been found in complexes with a range of associated fungi and sometimes has synergistic relationships with nematode or virus infections (Harris & Moen, Trans. Br. mycol. Soc. 89 (2), (1987)
1985a). There are a number of root diseases in which R. solani is but one member of a complex of fungi which incite disease with more or less the same pattern of symptoms (Bateman, 1970). These include corn root rot (Rao et al., 1978), cotton stunt (Reynolds & Hanson, 1957), strawberry root rot (Wilhelm et al., 1972) and cutting rots of Peperomia obtusifolia (Munnecke & Chandler, 1953). A complex offungi (Pythium irregulare Buisman,
Printed in Great Britain
Notes and brief articles Phytophthora clandestina Greenhalgh, Pascoe & Taylor, Fusarium oxysporum Schlecht., R. solani and F. avenaceum (Fr.) Sacc.) have been associated with root rots of subterranean clover in the south western region of Western Australia (Barbett i et al., 1986). Work by Harris & Moen (1985a) has shown that a complex of organisms may be responsible for the Rhizoctonia disease complex of wheat in South Australia, Scott et al. (1979) implicated Periconia macrospinosa Lefebvre & Johnson, Pythium oligandrum Drechsler, Rhizoctonia cerealis van der Hoeven and R. solani as the main fungi associated with the ' crater ' disease of summer wheat in South Africa. Neate (1985) also reported the existence of Rhizoetonia spp, other than R. solani in South Australian wheat fields; the species include Ceratobasidium cornigerum (Bourdot) Rogers, Waitea circinata Warcup & Talbot and Iodophanus carneus (Pers.) Korf. Neate & Warcup (1985) designated a new anastomosis group, Ag 8, comprising the most common strain of R. solani pathogenic to roots of cereals in South Australia. Roberts & Sivasithamparam (1986) proposed that a complex of fungi may be involved in the production of bare patches of cereals in Western Australia. We report here the results of a glasshouse pathogenicity test to determine the effects of the interaction of certain root rot fungi isolated from bare patches of cereals (Roberts & Sivasithamparam, 1986), on the development of root rot in wheat plants and to examine their potential involvement in the bare patch disease. Isolates of Rhizoctonia solani (Ag 8), binucleate Rhizoctonia sp. (Ag C), Fusarium gramin earum Schwabe, Bipolaris sorokiniana (Sacc.) Shoern., Waitea circinata , Mortierella sp. (IM I 310812) and Pythium irregulare were obtained from roots of wheat plants
Table
1.
257
collected in July , 1984 from bare patches at Lake Grace, Western Australia (Roberts & Sivasithamparam, 1986). Fusarium gramin earum had been grown on Potato Dextrose Agar (PD A) and stored on ceramic beads (Lange & Boyd, 1968 ), All other fungi had been stored on PDA slants under sterile mineral soil for 7-8 months at 4-5 0. Inoculum was prepared by growing the test fungi on moist sterile millet seeds (Panicum miliaceum L.) for 3-4 weeks at 23-25°. The colonized millet seeds were thoroughly mixed with pasteurized field soil from Lake Grace which had a history of severe bare patch of wheat . Control pots contained uncolonized sterile millet seeds. The soil had (p.p.rn.) nitrate-nitrogen 10'0, phosphorus 12'0, sulphate-sulfur 18'0, calcium 460, magnesium 73'0, potassium 80'0, copper 0'5, zinc 0'2, manganese 7'0, iron 65'0, total salts 74'0, sodium 190 '0, boron 0'05 and molybdenum 0'11. It also had a fertility index of 3 '94 ME (%), organic matter 2'1 %, calcium/magnesium ratio 3'78, exchangeable sodium ESP (%) 20'97 and a pH of 5'7. Plastic pots (7'5 ern diam) containing 200 g of inoculum-supplemented soil were sown with 10 surface-sterilized pregerminated seeds of wheat ( Triticum aestivum L. cv, Gamenya) and covered with a thin layer of inert plastic beads to reduce dry ing and cross-contamination between treatments . There were ten replicate pots for each treatment. The pots were placed in temperature controlled water-cooled tanks at 15 ± 1° and maintained at 65 % water-holding capacity (Piper, 1944). Treatments are shown in Table 1. Four weeks after sowing, surviving wheat seedlings were counted, washed and rated for root rot severity as described in Roberts & Sivasithamparam (1986). To establish Koch 's postulates, the fungi were re-isolated. Ten root pieces
Treatments and inoculum levels used to test pathogenicity on wheat
Treatment (1) Fusarium graminearum (2) Pythium irregulare (3) Bipolaris sorokiniana (4) Mort ierel/a sp. (5) Waitea circinata (6) Rhizocton ia solani (Ag 8) (7) R . solani (Ag 8)+ Rhizoctonia sp . (Ag C) (binucleate) + F. graminearum (8) As (7)+ P. irregulare (9) As (8)+ B. sorokiniana ( to) As (9) + Mortierella sp. (11) As (10) + Waitea circinata (12) Control (uncolonized sterile millet seeds)
Inoculum level (%)* 0'5 0'5 2 '0 2 '0 2'0 0 -2
0 -2+0'2+0'5
As (7)+ 0 '5 As (8)+ 2'0 As (9)+ 2-0 As ( 10)+ 2'0
* Weight of inoculum/ weight of air-dried soil. Trans. Br. mycol. S oc. 89 (2), (1987)
Printed in Great Br itain
Notes and brief articles ern long) were selected from the ten replicate pots of each treatment, surface sterilized for 10 min with 0'125 % sodium hypochlorite, rinsed in three changes of sterile distilled water, blotted dry and plated on PDA containing streptomycin sulfate (100 units ml"). Plates were incubated at 25°. All fungi tested were re-isolated from rotted tissues. The effect of fungal interaction on disease expression measured as root disease is shown in Table 2. Plants inoculated with the P. irregulare showed a mild root rot, moderate stunting of seedlings, slight discolouration of lower leaves and no vascular browning. Roots of plants infected with R. solani were discoloured from light to dark brown with soft tips, and were often nipped-off near the crown. The plants were stunted and leaves chlorotic. Roots of plants infected with F. graminearum were slightly necrotic, while those inoculated with B. sorokiniana showed a moderate root rot with black mycelial pads on infected roots and dark brown lesions on primary roots which coalesced to become black streaks. Roots of plants inoculated with W. circinata were filled with orange sclerotium-like bodies and had small orange to orange brown lesions. Plants inoculated with Mortierella sp. were healthy, free of root rot and (0'5-1'0
Table
2.
similar to uninoculated controls. Combinations of fungi (treatments 7, 8, 9, 10 and 11 of Table 1) produced significantly more disease than the same fungi used singly, A combination of Rhizoctonia solani (Ag 8) + binucleate Rhizoctonia sp. (Ag C) + F. graminearum + P. irregulare + B. sorokiniana resulted in the highest root disease index (96 %) recorded (Table 2). Shoot weight, plant size and root weight were found to be negatively correlated with root disease index. Wong, Barbetti & Sivasithamparam (1984) observed a similar effect with Rhizoetonia spp. affecting roots of subterranean clover in Western Australia. Ag 8, which has been recognized as a major pathogen in South Australia (Neate & Warcup, 1985), is more pathogenic with other fungi than by itself. Other fungi appeared to have a significant role in the rotting of roots, for instance the addition of P. irregulare to a complex containing Ag 8 and F. graminearum nearly doubled the amount of disease. Pythium sp. has been proposed as a serious pathogen of wheat in South Australia (Pittaway & Rathjen, 1983) and the U.S.A. (Cook & Haglund, 1982). It is also possible that the introduced P. irregulare would have multiplied on unutilized portions of millet seeds incorporated with the inoculum of other fungi. Increase in activity within
Effects of fungi singly and in combination on root disease index and survival of wheat
Treatments Uninoculated control Mortierella sp. Rhizoctonia solani (Ag 8) P. irregulare Waitea circinata Bipolaris sorokiniana R. solani (Ag 8) + Rhizoctonia (Ag C) + F. graminearum F. graminearum R. solani (Ag 8)+ Rhizoctonia (Ag C) + F. graminearum + P. irregulare-e B. sorokiniana+ Mortierella sp, + W. circinata R. solani (Ag 8) + Rhizoctonia (Ag C) + F. graminearum + P. irregulare-e B, sorokiniana+ Mortierella sp. R. solani (Ag 8)+ Rhizoctonia (Ag C) + F. graminearum + P. irregulare R. solani (Ag 8)+ Rhizoetonia (Ag C) + F. graminearum + P, irregulare + B. sorokiniana
Mean root disease index (%)* 7'6
at
6'5 a 41'5 b 45'1 b 47'0 b 56'1 b 57'1 b
Mean survival (%) 80 bt 98 a 98 a 62 c 75 b 97 a 100 a
77'3 c 89'3 d
93'2 d
94'5 d
32 d
56 c
* Disease index was computedaccording to McKinney (1923) and incorporated results for mean survival.
t
Meansin the samecolumnfollowed by the sameletter are not significantly different from eachother at 5 % level (Duncan's Multiple RangeTest). Trans. Br. mycol. Soc. 89 (2), (1987)
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Notes and brief articles soil of P . irregulare in the pre sence of uncolonized millet seeds has been noted with root rot of subterranean clover (Barberti & Sivasithamparam, 1987)·
Harris & Moen (1985b) found that a complex of fungi were associated with root rot of wheat in South Australia. In a pot experiment, they observed that through different stages of plant growth a succession of fungi were involved in the root rot, where R. solani was the primary pathogen. There is also evidence that soil and climatic effects, induding the soil structure down the profile, may be of overriding importance in Rhizoctonia stunting of wheat (Naiki, 1985; Deacon & Scott, 1985). However, we propose that the bare patch disease which is a seedling disease in W. Australia is caused by a complex of fungi. We thank the Western Australian Wheat Industry Committee for the award of a studentship for one of us (F. A. Roberts). REFERENCES
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