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A new hyperparasitic fungus, Cladobotryum amazonense, with potential for control of fungal pathogens of cocoa
[ 273 ] Trans. Br. mycol, Soc. 77 (2) 273-278 (1981)
Printed in Great Brita in
A NEW HYPERPARASITIC FUNGUS, CLADOBOTRYUM AMAZONENSE, WITH POTENTIAL FOR CONTROL OF FUNGAL PATHOGENS OF COCOA By C. N . BASTOS
AND
H. C. EVANS*
CEPLAC, Departamento Especial da Amazonia, C.P. 1801, Belem, Pard, Brazil
R. A. SAMSON Centraalbureau voor Schimm elcultures, Baarn, The Netherlands AND
Cladobotryum amazon ense sp.nov., a hyperparasite of the cocoa witches' broom fungus, Crinipellis perniciosa, and other agarics in Amazonian forests, is described. The mycoparasite overgrows host basidiocarps and physically prevents normal hymenial development and spore dissemination. In addition, it produces an extracellular, heat-stable metabolite which induces plasmolysis of basidiospores and is toxic in vitro to a range of fungal pathogens of cocoa. Field application of the toxin protects cocoa tissues from infection by C. perniciosa and Phytophthora palmiuora,
During a survey of the host range of Crinipellis perniciosa (Stahel) Singer, an agaric causing witches' broom disease of cocoa, a hyperparasitic fungus was observed colonizing the basidiocarps which developed on stored liana material originally collected from Amazonian forest. Since C. perniciosa is a major pathogen of cocoa in South America (Baker & Holliday, 1957), any antagonistic organism is of immediate interest and justifies further investigation. This is especiany so in Brazil due to the increasing socioeconomic and ecological role that cocoa cultivation is playing in the recently settled regions of the Brazilian Amazon. The initial approach was to investigate the colonization of basidiocarps by the hyperparasite with a view to biological control of the pathogen. However, it was discovered that basidiospores trapped in the mycelium became plasmolysed and failed to germinate when transferred to water agar (Bastes, 1979), suggesting the presence of an extracellular toxin and possibilities for chemical control. Attention was turned to the production of metabolites by the fungus in culture and to testing toxicity to C. perniciosa and to fungal pathogens of cocoa in general, in vitro and in vivo. The results of these studie s are pre sented together with a taxonomic appraisal of the hyperparasite, a previou sly undescribed species of Cladobotryum, * Present address: Commonwealth Mycological Institute, Kew, Surrey.
ECOLOGY
Pieces of moribund liana with old, dry basidiocarps of C. perniciosa were collected in June 1977 from trash in high forest (mata de cipo) along the Transamazonian highway between Altamira and Itaituba in Para state, Brazil. The material was stored on trestl es in a humid gauzehouse in Belern to await fresh basidiocarp production. However, the first crop was colonized extensively by white mycelial mats of a hyperparasitic fungus which overgrew the hymenial surfaces of older basidiocarps and effectively sealed the caps of young basidiocarps preventing normal development and hymenial exposure. Subsequent crops of basidiocarps, produced up to 3-4 months later, became invaded by the hyperparasite demonstrating its persistence and ability to survive in the absence of host fructifications. In the early part of the 1978-9 wet season a search was conducted in a small forest reserve close to Belem and more isolates of the same fungus were collected on various litterinhabiting agarics including species of Crinipellis and Marasmius. It was observed only sporadicalIy on basidiocarps of C. perniciosa on brooms during a z-year survey of cocoa farms in the Braganca region of Para state. Spraying cocoa broom s with macerated mycelium from agar cultures of the hyperparasite, showed that colonization is rapid, the basidiocarps being covered by a hyphal network within 30 h.
274
Cladobotryum amazonense sp.nov.
lO~m
Fig.
1.
Cladobotryum amazonense (holotype), Conidiophores and conidia from basidiocarp of Crinipellis perniciosa.
TAXONOMY
The fungus clearly shows affinities with species of Cladobotryum Nees ex Steud. and Sibirina G. Arnold, but differs sufficiently from the known taxa (Gams & Hoozemans, 1970; Gams, 1973; de Hoog, 1978; Gray & Morgan-Jones, 1980; Garns, 1980) to justify a new species. The fungus cannot be included in Sibirina because this genus has very dense whorls of rather narrowly tapering, strongly plugged phialides. Although no detailed study could be made to determine the conidiogenesis in our isolate due to atypical growth in pure culture, the classification in Cladobotryum is proposed.
Cladobotryum amazonense sp.nov, (Fig. 1) In agaric hospite mycelium album, arachnoideum, stipitern pileumque investiens, deinde grisescens. Hyphae vegetativae hyalinae, septatae, leves, 4-7 pm latae , Conidiophora erecta, recta vel flexuosa, ad 12 Ilm lata, levia, identidem verticillata, rami ultimi 3-6 cellulae conidiogenae verticillatae. Cellulae conidiogenae rectae subulatae, 20-60 pm longae, basi 5-9 I'm latae, ad 2-4 pm sursum angustatae. Conidia 2-3(-7) cellularia, clavata, recta vel nonnumquam modice curvara, levia, tenuitunicata, hyalina, 25-46 x 9-12 tun. Typus IMI 251385, ex basidiomate Crinipellis perniciosae, Belem in Brasilia, C.N. Bastos, June, 1977. Characters on the host: Colonies forming a white,
C. N. Bastos, H. C. Evans and R . A. Samson cobweb-like growth on the stipe and pileus of agaric basidiocarps, in old specimens conspicuous grey tufts or sheets of mycelium occur. Vegetative mycelium hyaline, septate, smooth-walled, 4-7 pm wide. Conidiophores erect, straight or flexuous, up to 12 pm wide, smooth-walled, repeatedly verticillate, whorls on main stalk containing several smaller plagiotropic branches, the ultimate branches bearing terminal verticils of 3 to 6 conidiogenous cells. Conidiogenous cells straight, subulate, 20-60 pm long, 5-9 pm wide at the bases, attenuating toward a tip 2-4 pm wide. Conidia 2-3 (7) celled, smooth- and thin-walled, hyaline, clavate, straight but occasionally slightly curved, 25-46 x 9-12 pm. Chlamydospores or teleomorph not observed. Specimen examined: Ex basidiocarps of Crinipellis perniciosa, Belern, Brazil, C. N. Bastos,June 1977, type
IM1251385. Characters in culture : CBS 471.80 derived from type. Growth on potato dextrose agar (PDA) rapid, colonies attaining 8'2 em diam in 7 days at 25 °C; white at first becoming yellow or tan and finally vinaceous as the agar is stained a deep red. Mycelium loose, floccose, hyaline to pale yellow; hyphae 7-11 pm diam. On standard media and the same supplemented with pieces of agaric basidiocarps sporulation is restricted; on media containing diced fragments of a leathery Amauroderma sp. (Ganodermataceae) fragile, white, lanuginose tufts of erect conidiophores develop at the edges of the culture vessel. Conidiophores robust, erect , hyaline, smooth, thin-walled , septat e every 40-60 (80) pm, reaching 5-8 mm in length, up to 10 pm wide at the base, narrowing to 5-7 pm, producing whorls towards apex, directly or on smaller side branches, each whorl with 4- 5 subulate, conidiogenous cells, 30-40 pm in length, 3'5-4'5 pm wideatthe base, tapering to 1'5-2'0 pm at the truncate apex. Atypical conidia are produced following percurrent proliferation of the conidiogenous cell to form a heel-like extension; conidia cylindrical to filiform, often curved or distorted, hyaline, multiseptate (4-7), variable in length, up to 200 pm averaging 100-120 pm, 40-55 pm diam (F igs 2, 3). Cultural characteristics are similar on malt extra ct, oatmeal, cornmeal and V8 agar. Sporulation is atypical or lacking. Less atyp ical conidia are shown in Fig . 3 and it seems that conidiogenesi s is mod ified by substrate and that type conidia are only produced on the host . Cladobotryum amazonense resembles C. mulliseptatum de Hoog and C. curuatum de Hoog & W. Gams (de Hoog, 1978) but has much larger conidia. C. multiseptatum is the causal agent of
275
'mildew' or 'cobweb' disease of Agaricus brunnescens Peck . FUNGITOXICITY
In v itro Cladobotryum amazonense was grown on potato
dextrose liquid medium (PDL) for 10-12 days at c. 25° and filtered through cotton-wool to remove mycelium. Initially the crude, conspicuously yellow filtrate was sterilized using a Seitz bacterial filter but then routinely autoclaved when the toxin was found to be heat stable. Germination of Crinipel/is perniciosa basidiospores was tested as described by Evans & Bastos (1980). No germination was recorded in the culture filtrate after 24-48 h, due to spore plasmolysis, compared with almost 100 % germination in sterile PDL. Similar culture filtrates were incorporated into PDA plates (1 :4) which were centrally inoculated with agar discs (3 mm diam) containing one of a range of fungal pathogens isolated from cocoa in the Brazilian Amazon. Growth of all fungi was completely inhibited or markedly reduced compared with the controls, PDA with no filtrate, 6 days after incubation at c. 25° (Table 1). In vivo S eedlings: Newly emerging flushes of 1- to 2-
month-old cocoa seedlings (selfed ICS-39) were sprayed to run-off with culture filtrates of C. amaz onense containing 0'5 % commercial sticker, and 8 h later inoculated with basidiospores of C. perniciosa (Evans, 1978). The treated seedlings were assessed after 2 months and compared with inoculated but unsprayed seedling s. Pods : Hanging, 30 to ao-day-old cocoa pods (clone ICS-39) were sprayed and inoculated as above and examined 4 months later. Large green pods (c. 5 months old) of the same clone were treated with filtrate; inoculated with a fine spray of a zoospore suspension of P. palmivora, the black pod pathogen of cocoa; enclosed in plastic bags containing moist cotton-wool pads and recorded 10 days later. Pods of similar ages were selected and inoculated with the appropriate pathogen without prior treatment with filtrate. The results (T able 2) demonstrate that culture filtrates of C. amazonense are highly toxic to spores of C. perniciosa and P . palmiuora under field conditions; infection being significantly reduced compared with the untreated, inoculated controls. DISCUSSION
Cladobotryum amazonense appears to be a common hyperparasite of agarics in forests in the
276
Cladobotryum amazonense sp.nov.
Fig. 2. Cladobotryum amazonense. Atypical conidiophores and conidia on PDA supplemented with polypore.
Brazilian Amazon, but is rarely found in cocoa farms, possibly due to the less favourable microclimate and/or the presence of commercial fungicides. The mycoparasite is able to persist on woody tissues and colonize basidiocarps when they appear, hindering normal basidiocarpdevelopment and basidiospore release. Host spores trapped in the mycoparasite mycelium are plasmolysed by an extracellular metabolite. Thus two alternative methods of control of the witches' broom pathogen through the use of C. amazonense are feasible: direct biological control, by treatment of brooms with mycelial inoculum of the hyperparasite,
and chemical control, by treatment of healthy cocoa tissues with mycotoxin from culture filtrates. Both approaches are novel in concept but suffer from the usual drawbacks of more conventional methods. Adequate foliage protection is difficult with an irregularly flushing tree crop such as cocoa, and, in addition, C. perniciosa is a difficult target, surviving systemically within modified host tissues hidden in a dense canopy, hence the previous lack of success of fungicides for the control of witches' broom disease of cocoa (Baker & Holliday, 1957).
c. N. Bastos, H. C. Evans and R. A. Samson
277
IOJ.l.ffi
Fig. 3. Cladobotryum amazonense. Atypical conidiophores and conidia from oatmeal agar after 24 days at 25 0.
Table 1. Effect of culture filtrate of Cladobotryum amazonense on growth of some fungal pathogens of cocoain vitro Radial growth (mm) of colony* ,----"'--~
Phytophthora palmivora Butl. Ceratocystis fimbriata Ell. & Haist. Botryodiplodia theobromae Pat. Colletotrichum gloeosporioides (Penz.) Sacco Corticium salmonicolor Berk. & Br. Phellinus noxius (Corner) G.H. Cunn.
PDA
PDA plus filtrate
75
0
60
0
80 76
0 21
75
10
70
20
* Mean of four replicates, zero signifies no growth onto the medium although growth may have occurred on the inoculum plug.
Table 2. Effect of culture filtrate of Cladobotryum amazonense on infectivity of two fungal pathogens of cocoa Inoculation plus Inoculation filtrate Crinipellis perniciosa Pods 0/15 *15/15 Seedlings 18/20 3/20 Phytophthora palmivora Pods 18/20 4/20 Total infectivity (%) 12"7 92"7 * No. of infections/no. of inoculations. Nevertheless, C. amazonense offers certain advantages over conventional chemicals, not least the ease and cheapness of production of mycelium or toxin and it is planned to include the hyperparasite in field trials to compare performance with standard fungicide treatments. Also, work is in progress to identify the toxin which is evidently not enzymatic in nature, although it may aid fungal nutrition by provoking lysis of host tissues. There
Cladobotryum amazonense sp.nov. is some evidence that autolysis occurs since filtrates from old cultures are significantly less toxic than to-day-old cultures (C. N . Bastos, unpubl.). In conclusion, C. amazonense produces a broad spectrum mycotoxin which is effective against some of the major pathogens of cocoa and has potential both for their control and the control of plant pathogenic fungi in general. The two senior authors were supported by the Comissao Executiva do Plano da Lavoura Cacaueira (Brazil) and the Overseas Development Administration (U K) respectively. Drs W. Gams and G. S. de Hoog kindly reviewed the manuscript. The Latin diagnosis and Fig. 3 were provided by Dr Gams. REFERENCES BAKER, R. E. D. & HOLLIDAY, P. (1957). Witches' broom disease of cacao (Marasmius perniciosus Stahel). Phytopathological Papers 2. Commonwealth Mycological Institute. BASTOS, C. N. (1979). Hiperparisitismo do fungo
Dactylium sp. a Crinipellis perniciosa (Stahel) Singer em vassoura-de-bruxa do cacaueiro. Revista Theobroma 9,197-200. EVANS, H. C. (1978). Wit<:hes' broom disease of cocoa (Crinipellis perniciosa) in Ecuador. 1. The fungus. Annals of Applied Biology 89, 185-192. EVANS, H. C. & BASTOS, C. N . (1980). Basidiospore germination as a mean s of assessing resistance to Crinipellis perniciosa (witches' broom disease) in cocoa cultivars. Transactions of the British M ycological Society 74, 525-536. GAMS, W. (1973). Phialides with solitar y conidia? Remarks on conidium ontogeny in some hyphomycetes. Persoonia 7, 161-169. GAMS, W. (1980). Cladobotryum penicillatum sp.nov. Persoonia 11, 71-74. GAMS, W. & HOOZEMANS, A. C. M. (1970). Cladobotryum-konidienformen von Hypomyces-Arten. Persoonia 6, 95-110. GRAY, D. J. & MORGAN-JONES, G. (1980). Notes on Hyphomycetes XXXIV. Some mycoparasitic species. Mycotaxon 10, 375-404. HOOG, G. S. DE (1978). Notes on some fungicolous Hyphomycetes and their relatives. Persoonia 10, 33-8 1.
(Received for publication 4 October t980)
Printed in Great Brita in
A NEW HYPERPARASITIC FUNGUS, CLADOBOTRYUM AMAZONENSE, WITH POTENTIAL FOR CONTROL OF FUNGAL PATHOGENS OF COCOA By C. N . BASTOS
AND
H. C. EVANS*
CEPLAC, Departamento Especial da Amazonia, C.P. 1801, Belem, Pard, Brazil
R. A. SAMSON Centraalbureau voor Schimm elcultures, Baarn, The Netherlands AND
Cladobotryum amazon ense sp.nov., a hyperparasite of the cocoa witches' broom fungus, Crinipellis perniciosa, and other agarics in Amazonian forests, is described. The mycoparasite overgrows host basidiocarps and physically prevents normal hymenial development and spore dissemination. In addition, it produces an extracellular, heat-stable metabolite which induces plasmolysis of basidiospores and is toxic in vitro to a range of fungal pathogens of cocoa. Field application of the toxin protects cocoa tissues from infection by C. perniciosa and Phytophthora palmiuora,
During a survey of the host range of Crinipellis perniciosa (Stahel) Singer, an agaric causing witches' broom disease of cocoa, a hyperparasitic fungus was observed colonizing the basidiocarps which developed on stored liana material originally collected from Amazonian forest. Since C. perniciosa is a major pathogen of cocoa in South America (Baker & Holliday, 1957), any antagonistic organism is of immediate interest and justifies further investigation. This is especiany so in Brazil due to the increasing socioeconomic and ecological role that cocoa cultivation is playing in the recently settled regions of the Brazilian Amazon. The initial approach was to investigate the colonization of basidiocarps by the hyperparasite with a view to biological control of the pathogen. However, it was discovered that basidiospores trapped in the mycelium became plasmolysed and failed to germinate when transferred to water agar (Bastes, 1979), suggesting the presence of an extracellular toxin and possibilities for chemical control. Attention was turned to the production of metabolites by the fungus in culture and to testing toxicity to C. perniciosa and to fungal pathogens of cocoa in general, in vitro and in vivo. The results of these studie s are pre sented together with a taxonomic appraisal of the hyperparasite, a previou sly undescribed species of Cladobotryum, * Present address: Commonwealth Mycological Institute, Kew, Surrey.
ECOLOGY
Pieces of moribund liana with old, dry basidiocarps of C. perniciosa were collected in June 1977 from trash in high forest (mata de cipo) along the Transamazonian highway between Altamira and Itaituba in Para state, Brazil. The material was stored on trestl es in a humid gauzehouse in Belern to await fresh basidiocarp production. However, the first crop was colonized extensively by white mycelial mats of a hyperparasitic fungus which overgrew the hymenial surfaces of older basidiocarps and effectively sealed the caps of young basidiocarps preventing normal development and hymenial exposure. Subsequent crops of basidiocarps, produced up to 3-4 months later, became invaded by the hyperparasite demonstrating its persistence and ability to survive in the absence of host fructifications. In the early part of the 1978-9 wet season a search was conducted in a small forest reserve close to Belem and more isolates of the same fungus were collected on various litterinhabiting agarics including species of Crinipellis and Marasmius. It was observed only sporadicalIy on basidiocarps of C. perniciosa on brooms during a z-year survey of cocoa farms in the Braganca region of Para state. Spraying cocoa broom s with macerated mycelium from agar cultures of the hyperparasite, showed that colonization is rapid, the basidiocarps being covered by a hyphal network within 30 h.
274
Cladobotryum amazonense sp.nov.
lO~m
Fig.
1.
Cladobotryum amazonense (holotype), Conidiophores and conidia from basidiocarp of Crinipellis perniciosa.
TAXONOMY
The fungus clearly shows affinities with species of Cladobotryum Nees ex Steud. and Sibirina G. Arnold, but differs sufficiently from the known taxa (Gams & Hoozemans, 1970; Gams, 1973; de Hoog, 1978; Gray & Morgan-Jones, 1980; Garns, 1980) to justify a new species. The fungus cannot be included in Sibirina because this genus has very dense whorls of rather narrowly tapering, strongly plugged phialides. Although no detailed study could be made to determine the conidiogenesis in our isolate due to atypical growth in pure culture, the classification in Cladobotryum is proposed.
Cladobotryum amazonense sp.nov, (Fig. 1) In agaric hospite mycelium album, arachnoideum, stipitern pileumque investiens, deinde grisescens. Hyphae vegetativae hyalinae, septatae, leves, 4-7 pm latae , Conidiophora erecta, recta vel flexuosa, ad 12 Ilm lata, levia, identidem verticillata, rami ultimi 3-6 cellulae conidiogenae verticillatae. Cellulae conidiogenae rectae subulatae, 20-60 pm longae, basi 5-9 I'm latae, ad 2-4 pm sursum angustatae. Conidia 2-3(-7) cellularia, clavata, recta vel nonnumquam modice curvara, levia, tenuitunicata, hyalina, 25-46 x 9-12 tun. Typus IMI 251385, ex basidiomate Crinipellis perniciosae, Belem in Brasilia, C.N. Bastos, June, 1977. Characters on the host: Colonies forming a white,
C. N. Bastos, H. C. Evans and R . A. Samson cobweb-like growth on the stipe and pileus of agaric basidiocarps, in old specimens conspicuous grey tufts or sheets of mycelium occur. Vegetative mycelium hyaline, septate, smooth-walled, 4-7 pm wide. Conidiophores erect, straight or flexuous, up to 12 pm wide, smooth-walled, repeatedly verticillate, whorls on main stalk containing several smaller plagiotropic branches, the ultimate branches bearing terminal verticils of 3 to 6 conidiogenous cells. Conidiogenous cells straight, subulate, 20-60 pm long, 5-9 pm wide at the bases, attenuating toward a tip 2-4 pm wide. Conidia 2-3 (7) celled, smooth- and thin-walled, hyaline, clavate, straight but occasionally slightly curved, 25-46 x 9-12 pm. Chlamydospores or teleomorph not observed. Specimen examined: Ex basidiocarps of Crinipellis perniciosa, Belern, Brazil, C. N. Bastos,June 1977, type
IM1251385. Characters in culture : CBS 471.80 derived from type. Growth on potato dextrose agar (PDA) rapid, colonies attaining 8'2 em diam in 7 days at 25 °C; white at first becoming yellow or tan and finally vinaceous as the agar is stained a deep red. Mycelium loose, floccose, hyaline to pale yellow; hyphae 7-11 pm diam. On standard media and the same supplemented with pieces of agaric basidiocarps sporulation is restricted; on media containing diced fragments of a leathery Amauroderma sp. (Ganodermataceae) fragile, white, lanuginose tufts of erect conidiophores develop at the edges of the culture vessel. Conidiophores robust, erect , hyaline, smooth, thin-walled , septat e every 40-60 (80) pm, reaching 5-8 mm in length, up to 10 pm wide at the base, narrowing to 5-7 pm, producing whorls towards apex, directly or on smaller side branches, each whorl with 4- 5 subulate, conidiogenous cells, 30-40 pm in length, 3'5-4'5 pm wideatthe base, tapering to 1'5-2'0 pm at the truncate apex. Atypical conidia are produced following percurrent proliferation of the conidiogenous cell to form a heel-like extension; conidia cylindrical to filiform, often curved or distorted, hyaline, multiseptate (4-7), variable in length, up to 200 pm averaging 100-120 pm, 40-55 pm diam (F igs 2, 3). Cultural characteristics are similar on malt extra ct, oatmeal, cornmeal and V8 agar. Sporulation is atypical or lacking. Less atyp ical conidia are shown in Fig . 3 and it seems that conidiogenesi s is mod ified by substrate and that type conidia are only produced on the host . Cladobotryum amazonense resembles C. mulliseptatum de Hoog and C. curuatum de Hoog & W. Gams (de Hoog, 1978) but has much larger conidia. C. multiseptatum is the causal agent of
275
'mildew' or 'cobweb' disease of Agaricus brunnescens Peck . FUNGITOXICITY
In v itro Cladobotryum amazonense was grown on potato
dextrose liquid medium (PDL) for 10-12 days at c. 25° and filtered through cotton-wool to remove mycelium. Initially the crude, conspicuously yellow filtrate was sterilized using a Seitz bacterial filter but then routinely autoclaved when the toxin was found to be heat stable. Germination of Crinipel/is perniciosa basidiospores was tested as described by Evans & Bastos (1980). No germination was recorded in the culture filtrate after 24-48 h, due to spore plasmolysis, compared with almost 100 % germination in sterile PDL. Similar culture filtrates were incorporated into PDA plates (1 :4) which were centrally inoculated with agar discs (3 mm diam) containing one of a range of fungal pathogens isolated from cocoa in the Brazilian Amazon. Growth of all fungi was completely inhibited or markedly reduced compared with the controls, PDA with no filtrate, 6 days after incubation at c. 25° (Table 1). In vivo S eedlings: Newly emerging flushes of 1- to 2-
month-old cocoa seedlings (selfed ICS-39) were sprayed to run-off with culture filtrates of C. amaz onense containing 0'5 % commercial sticker, and 8 h later inoculated with basidiospores of C. perniciosa (Evans, 1978). The treated seedlings were assessed after 2 months and compared with inoculated but unsprayed seedling s. Pods : Hanging, 30 to ao-day-old cocoa pods (clone ICS-39) were sprayed and inoculated as above and examined 4 months later. Large green pods (c. 5 months old) of the same clone were treated with filtrate; inoculated with a fine spray of a zoospore suspension of P. palmivora, the black pod pathogen of cocoa; enclosed in plastic bags containing moist cotton-wool pads and recorded 10 days later. Pods of similar ages were selected and inoculated with the appropriate pathogen without prior treatment with filtrate. The results (T able 2) demonstrate that culture filtrates of C. amazonense are highly toxic to spores of C. perniciosa and P . palmiuora under field conditions; infection being significantly reduced compared with the untreated, inoculated controls. DISCUSSION
Cladobotryum amazonense appears to be a common hyperparasite of agarics in forests in the
276
Cladobotryum amazonense sp.nov.
Fig. 2. Cladobotryum amazonense. Atypical conidiophores and conidia on PDA supplemented with polypore.
Brazilian Amazon, but is rarely found in cocoa farms, possibly due to the less favourable microclimate and/or the presence of commercial fungicides. The mycoparasite is able to persist on woody tissues and colonize basidiocarps when they appear, hindering normal basidiocarpdevelopment and basidiospore release. Host spores trapped in the mycoparasite mycelium are plasmolysed by an extracellular metabolite. Thus two alternative methods of control of the witches' broom pathogen through the use of C. amazonense are feasible: direct biological control, by treatment of brooms with mycelial inoculum of the hyperparasite,
and chemical control, by treatment of healthy cocoa tissues with mycotoxin from culture filtrates. Both approaches are novel in concept but suffer from the usual drawbacks of more conventional methods. Adequate foliage protection is difficult with an irregularly flushing tree crop such as cocoa, and, in addition, C. perniciosa is a difficult target, surviving systemically within modified host tissues hidden in a dense canopy, hence the previous lack of success of fungicides for the control of witches' broom disease of cocoa (Baker & Holliday, 1957).
c. N. Bastos, H. C. Evans and R. A. Samson
277
IOJ.l.ffi
Fig. 3. Cladobotryum amazonense. Atypical conidiophores and conidia from oatmeal agar after 24 days at 25 0.
Table 1. Effect of culture filtrate of Cladobotryum amazonense on growth of some fungal pathogens of cocoain vitro Radial growth (mm) of colony* ,----"'--~
Phytophthora palmivora Butl. Ceratocystis fimbriata Ell. & Haist. Botryodiplodia theobromae Pat. Colletotrichum gloeosporioides (Penz.) Sacco Corticium salmonicolor Berk. & Br. Phellinus noxius (Corner) G.H. Cunn.
PDA
PDA plus filtrate
75
0
60
0
80 76
0 21
75
10
70
20
* Mean of four replicates, zero signifies no growth onto the medium although growth may have occurred on the inoculum plug.
Table 2. Effect of culture filtrate of Cladobotryum amazonense on infectivity of two fungal pathogens of cocoa Inoculation plus Inoculation filtrate Crinipellis perniciosa Pods 0/15 *15/15 Seedlings 18/20 3/20 Phytophthora palmivora Pods 18/20 4/20 Total infectivity (%) 12"7 92"7 * No. of infections/no. of inoculations. Nevertheless, C. amazonense offers certain advantages over conventional chemicals, not least the ease and cheapness of production of mycelium or toxin and it is planned to include the hyperparasite in field trials to compare performance with standard fungicide treatments. Also, work is in progress to identify the toxin which is evidently not enzymatic in nature, although it may aid fungal nutrition by provoking lysis of host tissues. There
Cladobotryum amazonense sp.nov. is some evidence that autolysis occurs since filtrates from old cultures are significantly less toxic than to-day-old cultures (C. N . Bastos, unpubl.). In conclusion, C. amazonense produces a broad spectrum mycotoxin which is effective against some of the major pathogens of cocoa and has potential both for their control and the control of plant pathogenic fungi in general. The two senior authors were supported by the Comissao Executiva do Plano da Lavoura Cacaueira (Brazil) and the Overseas Development Administration (U K) respectively. Drs W. Gams and G. S. de Hoog kindly reviewed the manuscript. The Latin diagnosis and Fig. 3 were provided by Dr Gams. REFERENCES BAKER, R. E. D. & HOLLIDAY, P. (1957). Witches' broom disease of cacao (Marasmius perniciosus Stahel). Phytopathological Papers 2. Commonwealth Mycological Institute. BASTOS, C. N. (1979). Hiperparisitismo do fungo
Dactylium sp. a Crinipellis perniciosa (Stahel) Singer em vassoura-de-bruxa do cacaueiro. Revista Theobroma 9,197-200. EVANS, H. C. (1978). Wit<:hes' broom disease of cocoa (Crinipellis perniciosa) in Ecuador. 1. The fungus. Annals of Applied Biology 89, 185-192. EVANS, H. C. & BASTOS, C. N . (1980). Basidiospore germination as a mean s of assessing resistance to Crinipellis perniciosa (witches' broom disease) in cocoa cultivars. Transactions of the British M ycological Society 74, 525-536. GAMS, W. (1973). Phialides with solitar y conidia? Remarks on conidium ontogeny in some hyphomycetes. Persoonia 7, 161-169. GAMS, W. (1980). Cladobotryum penicillatum sp.nov. Persoonia 11, 71-74. GAMS, W. & HOOZEMANS, A. C. M. (1970). Cladobotryum-konidienformen von Hypomyces-Arten. Persoonia 6, 95-110. GRAY, D. J. & MORGAN-JONES, G. (1980). Notes on Hyphomycetes XXXIV. Some mycoparasitic species. Mycotaxon 10, 375-404. HOOG, G. S. DE (1978). Notes on some fungicolous Hyphomycetes and their relatives. Persoonia 10, 33-8 1.
(Received for publication 4 October t980)