- Email: [email protected]
Suppression of Botrytis cinerea causing the grey mould disease of grape-vine by an aggressive mycoparasite, Pythium radiosum
FEMS Microbiology Letters 176 (1999) 25^30
Suppression of Botrytis cinerea causing the grey mould disease of grape-vine by an aggressive mycoparasite, Pythium radiosum Bernard Paul * Laboratoire des Sciences de la Vigne, Institut Jules Guyot, Universiteè de Bourgogne, P.O. Box 138, 21004 Dijon, France Received 2 April 1999; received in revised form 23 April 1999; accepted 26 April 1999
Abstract Pythium radiosum Paul has been found to be an aggressive mycoparasite of Botrytis cinerea which causes grey mould disease on the grape-vine. The mycoparasitic fungus enters the host mycelium, coagulates its protoplasm, empties its contents and finally comes out producing numerous ramifications and sexual structures. When the infected mycelium of B. cinerea is applied to the leaves of the grape-vine, the characteristic grey mould symptoms fail to appear. Since P. radiosum causes no harm to the grape-vine, it can be used as a biological control agent against B. cinerea. A brief account of the mycoparasite and its antagonism towards B. cinerea is discussed here. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Botrytis cinerea; Mycoparasite ; Biological control; Pythium radiosum; Antagonism
1. Introduction Mycoparasites are fungi that can parasitize other fungi. This term is generally used to include parasites that coil around the host hyphae or overgrow other colonies on agar. This may involve either penetration of the host hyphae or antagonism by the production of antibiotics [4], toxic radicals [10], or wall-lytic enzymes [2]. The phenomenon of mycoparasitism is very widespread in nature. A review of the literature on this important subject has already been published [1,8,11,17,18]. Most evidence for the role of mycoparasitism comes from the observation of infected fungal propagules such as spores or other reproduc* Tel.: +33 3 80396143; Fax: +33 3 80396265; E-mail: [email protected]
tive structures. Oospores of Aphanomyces, Phytophthora, Pythium, and Sclerospora have been found to be infected by a wide range of fungi including Fusarium mesmoides, Hyphochytrium catenoides, Olpidiopsis gracilis, Dactyella spp. and Trinacrium subtile [16]. Facultative mycoparasites such as Trichoderma and Gliocladium species have been reported from the sclerotia of di¡erent parasites like Phymatotrichum omnivorum, Rhizoctonia spp., Sclerotinia spp., and Verticillium dahliae [17]. Unfortunately this phenomenon of mycoparasitism has not been much exploited. Some of the mycoparasites that have been used as biological control agents are Ampelomyces quisqualis for the control of cucumber powdery mildew caused by Sphaerotheca fuliginea [7] and Erysiphe cichoracearum [15] under glasshouse conditions. Mycoparasitism of Trichoderma harzianum has been
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 1 3 - X
FEMSLE 8806 17-6-99
26
B. Paul / FEMS Microbiology Letters 176 (1999) 25^30
studied extensively. This mycoparasite is antagonistic and e¡ective in controlling plant pathogenic fungi like Rhizoctonia solani, Sclerotium rolfsii, Fusarium oxysporum, Fusarium culmorum, and Pythium aphanidermatum [3]. It has also been widely studied against Botrytis cinerea causing the grey mould disease of grape-vine and Chondrostereum purpureum, the agent of silver leaf in fruit trees [5]. Pythiaceous fungi are also known to be mycoparasites of a wide range of fungi. The most studied mycoparasite of the genus or perhaps of all mycoparasites known is Pythium oligandrum Drechsler. It has a very wide host range and rapidly attacks the susceptible hosts by lysis or penetration of hyphae [12]. However, it is only one of six known mycoparasitic Pythium species, the others being Pythium acanthicum Drechs., P. acanthophoron Sideris, P. mycoparasiticum Deacon et al., P. periplocum Drechs., and P. nunn Lifshitz et al. [9]. Apart from these well known mycoparasites, one aggressive plant pathogen, P. aphanidermatum (Edson) Fitzpatrick, has also been studied for its mycoparasitic activity [9]. Pythium radiosum was isolated in 1992 from a soil sample collected in the north of France and described as a new species by the author [14]. Since then it has not been reported from anywhere else in the world. It has echinulate oogonia resembling Pythium echinulatum Matthews. When grown together with B. cinerea, it rapidly enters the latter's mycelium and causes extensive damage. This is the ¢rst report of the mycoparasitism of P. radiosum.
2. Materials and methods P. radiosum (F-12) was re-cultured from the author's personal collection of pythiaceous fungi, maintained at the `Institut Jules Guyot', in Dijon, France. B. cinerea (B-3) was also obtained from the culture collection of this institute. Both fungi were maintained on PDA (potato dextrose agar), and PCA (potato carrot agar) media. P. radiosum was also maintained on boiled hemp-seed halves in sterile distilled water. Inter-hyphal interactions were studied by placing both fungi on the same PDA plate on opposite ends (Fig. 2a) and also on a thin ¢lm of PDA on a glass slide (Fig. 2b) which was obtained as follows.
A glass rod was heated and bent in a `V' shape in such a way that it could enter a glass petri dish of 10 cm diameter. It was introduced into the petri dish and a glass slide was placed on the top of the V rod. The whole was autoclaved. A thin layer of PDA was poured aseptically on the glass slide and the two fungi were inoculated at the opposite ends of the PDA ¢lm. To maintain humidity, enough sterile distilled water was poured in the petri dish so that the water remained well below the slide. Three replicates were prepared in the same manner and all were incubated at 25³C for 15 days. From time to time sterile distilled water was added to the petri dishes in order to avoid dehydration of the thin PDA ¢lm. After 15 days a small square of the PDA ¢lm was aseptically cut and placed on a fresh slide in a drop of sterile water. A coverslip was placed on the ¢lm and this was sealed with nail polish to avoid dehydration. The slide was then observed by microscope. Inoculation experiments with B. cinerea were carried out on the leaves of 1-month-old Vitis vinifera vitro cultures maintained in our laboratory. The leaves were taken out aseptically and were introduced into three sterile petri dishes having a sterile ¢lter paper. Sterile distilled water was used to humidify the ¢lter paper. One leaf was placed in each petri dish. The leaf of the ¢rst petri dish was inoculated with 50 Wl sterile distilled water in which B. cinerea mycelium from a 1-week-old PDA culture was introduced. The leaf of the second petri dish was inoculated with 50 Wl of sterile distilled water in which the parasitized mycelium (with P. radiosum) was placed, and the leaf in the third petri dish was inoculated with 50 Wl of sterile distilled water in which the mycelium of P. radiosum was introduced. All three petri dishes were maintained at room temperature (20³C). Occasionally the ¢lter papers of the three dishes were humidi¢ed with sterile distilled water. This experiment was repeated thrice.
3. Results 3.1. Morphological description P. radiosum (Fig. 1a^e): mycelia are well branched at times bearing conical spines measuring 3^7 Wm in
FEMSLE 8806 17-6-99
B. Paul / FEMS Microbiology Letters 176 (1999) 25^30
27
Fig. 1. Morphological structures of P. radiosum. a^c : Oogonia ornamented with conical to mammiform spines. d: Smooth-walled sporangia. e: Scanning electron micrograph of an ornamented oogonium. Bar: a^d = 25 Wm, e = 10 Wm.
FEMSLE 8806 17-6-99
28
B. Paul / FEMS Microbiology Letters 176 (1999) 25^30
Fig. 2. Interaction between B. cinerea (B-3) and P. radiosum (F-12). a: Antagonism between B-3 and F-12 on PDA plate. b: Antagonism between B-3 and F-12 on a ¢lm of PDA on a slide.
Fig. 3. Hyphal interactions between B. cinerea and P. radiosum. a: Normal hypha of B. cinerea. b: Hypha of P. radiosum (mycoparasite) inside the mycelium of B. cinerea (host). c: Coagulated protoplasm in the hypha of B. cinerea. d: Proliferation and exit of P. radiosum hyphae from the hypha of B. cinerea. e: Emptied hypha of B. cinerea. f: Damaged hypha of B. cinerea with sexual structure of P. radiosum. Bar : a^c, e, f = 35 Wm, d=100 Wm.
FEMSLE 8806 17-6-99
B. Paul / FEMS Microbiology Letters 176 (1999) 25^30
29
Fig. 4. Inoculation experiments on V. vinifera. a: Symptoms of grey mould disease on V. vinifera caused by B. cinerea. b: Healthy leaf of V. vinifera inoculated with parasitized B. cinerea with P. radiosum.
length. Sporangia are mostly intercalary measuring 6^30 Wm in diameter and are mostly non-sporulating (Fig. 1d). Oogonia are mostly intercalary and catenulate and are covered with conical to mammiform spines (Fig. 1a,b,c,e). Oogonia (excluding spines) are 10^35 Wm in diameter and are provided with spines which can measure up to 15 Wm long and up to 6 Wm thick at the base. Antheridia are rare and when present are hypogynous. After fertilization the oospores produced are aplerotic with a relatively thin wall. In the hyphal interaction experiments, the colonies of P. radiosum (F-12) which are whitish in color spread into the greyish colonies of B. cinerea (B-3). The latter does not spread into the former's zone (Fig. 2a,b). Microscopic observation of the mycelium in the zone of contact reveals that P. radiosum enters freely into the mycelium of B. cinerea (Fig. 3b^d) and once inside it rami¢es (Fig. 3b), the rami¢cations enter the septa of the fungal hyphae thus passing from one cell to another without any hindrance. At ¢rst the cytoplasm of the host cell is coagulated (Fig. 3c) and then the mycoparasite consumes the cytoplasm leaving an empty host mycelium (Fig. 3e) and ¢nally the mycoparasite comes out forming here and there an entangled mass of its mycelium around the host (Fig. 3d) and reproductive structures (Fig. 3f).
After inoculation on the leaf the vitro plants of V. vinifera showed typical grey mould symptoms on the leaf of the ¢rst petri dish (Fig. 4a), no disease on the leaf of the second petri dish (Fig. 4b) and also no infection on the third. All three replicates showed the same results.
4. Discussion The experiments show that the mycelium of P. radiosum can rapidly enter the hyphae of B. cinerea. Upon entry, the fungus rami¢es within and provokes large-scale destruction of the host mycelium. Hence the former is undoubtedly a mycoparasite of the latter. Since the parasitized mycelium of B. cinerea fails to produce the characteristic grey mould symptoms on the leaves of V. vinifera, and since the mycoparasite does not harm the leaves of the grape-vine in any way, P. radiosum can be used as a biocontrol agent against B. cinerea. The mode of action of P. radiosum di¡ers from that of P. oligandrum which often causes explosive lysis of host mycelium [12], whereas P. radiosum brings about cytoplasmic coagulation like that observed in the case of P. aphanidermatum [9]. The fact that P. radiosum penetrated the host mycelium and grew extensively within it may be useful in the
FEMSLE 8806 17-6-99
30
B. Paul / FEMS Microbiology Letters 176 (1999) 25^30
analysis of the mechanisms of mycoparasitism, especially the suggested roles of cell wall-degrading enzymes [2,6,13].
[10]
[11]
References [1] Adams, P.B. (1990) The potential of mycoparasites for biological control of plant diseases. Annu. Rev. Phytopathol. 72, 58^73. [2] Chet, I. (1987) Trichoderma ^ application, mode of action, and potential as a biocontrol of soil-borne plant pathogenic fungi. In: Innovative Approaches to Plant Disease Control (Chet, I., Ed.), pp. 137^160. Wiley, New York. [3] Chet, I. and Inbar, J. (1994) Biological control of fungal pathogens. Appl. Biochem. Biotechnol. 48, 37^43. [4] Denis, C. and Webster, J. (1971) Antagonistic properties of species-groups of Trichoderma. I. Production of non-volatile antibiotics. Trans. Br. Mycol. Soc. 57, 363^369. [5] Dubos, B. (1987) Fungal antagonism in aerial agrobiocenoses. In : Innovative Approaches to Plant Disease Control (Chet, I., Ed.), pp. 107^136. Wiley, New York. [6] Elad, Y., Lifshitz, R. and Baker, R. (1985) Enzymatic activity of the mycoparasite Pythium nunn during interaction with host and non-host fungi. Physiol. Plant Pathol. 27, 131^ 148. [7] Jarvis, W.R. and Slingby, K. (1977) The control of powdery mildew of greenhouse cucumber by water sprays and Ampelomyces quisqualis. Plant Dis. Rep. 61, 728^730. [8] Je¡eries, P. and Young, T.W.K. (1994) Interfungal Parasitic Relationship, CAB International, Wallingford. [9] Jones, E.E. and Deacon, J.W. (1995) Comparative physiology and behaviour of the mycoparasites Pythium acanthophoron,
[12]
[13]
[14] [15]
[16]
[17] [18]
P. oligandrum, and P. mycoparasiticum. Biocontrol Sci. Technol. 5, 27^39. Kim, K.K., Fravel, D.R. and Papvizas, G.C. (1990) Glucose oxidase as the antifungal principle of talaron from Talaromyces £avus. Can. J. Microbiol. 36, 760^764. Kranz, J. (1981) Hyperparasitism of biotrophic fungi. In: Microbial Ecology of the Phylloplane (Blackman, Ed.), pp. 327^ 352. Academic Press, New York. Laing, S.A.K. and Deacon, J.W. (1991) Video microscopical comparison of mycoparasitism by Pythium oligandrum, P. nunn, and an unnamed Pythium species. Mycol. Res. 95, 469^479. Lewis, K., Whipps, J.M. and Cooke, R.C. (1989) Mechanisms of biological disease control with special reference to the case study of Pythium oligandrum as an antagonist. In : Biotechnology of Fungi for Improving Plant Growth (Whipps, J.M. and Lumsden, R.D., Eds.), pp. 191^217, Cambridge University Press, Cambridge. Paul, B. (1992) Pythium radiosum, a new species with ornamented oogonia from France. Mycol. Helv. 5, 1^8. Sundheim, L. (1983) L'hyperparasite Ampelomyces quisqualis dans la lutte contre O|ëdium du concombre. Colloq. INRA 18, 145^154. Whipps, J.M. (1991) E¡ects of mycoparasites on sclerotiaforming fungi. In: Biotic Interactions and Soil-borne Diseases (Beemster, A.B.R., Bollen, G.J., Gerlagh, M., Ruissen, M.A., Schippers, B. and Tempel, A., Eds.), pp. 129^140. Elsevier, Amsterdam. Whipps, J.M. (1997) Developments in the biological control of soil-borne plant pathogens. Adv. Bot. Res. 26, 1^134. Whipps, J.M., Lewis, K. and Cooke, R.C. (1988) Mycoparasitism and plant disease control. In: Fungi in Biological Control Systems (Burge, M.N., Ed.), pp. 161^187. Manchester University Press, Manchester.
FEMSLE 8806 17-6-99
Suppression of Botrytis cinerea causing the grey mould disease of grape-vine by an aggressive mycoparasite, Pythium radiosum Bernard Paul * Laboratoire des Sciences de la Vigne, Institut Jules Guyot, Universiteè de Bourgogne, P.O. Box 138, 21004 Dijon, France Received 2 April 1999; received in revised form 23 April 1999; accepted 26 April 1999
Abstract Pythium radiosum Paul has been found to be an aggressive mycoparasite of Botrytis cinerea which causes grey mould disease on the grape-vine. The mycoparasitic fungus enters the host mycelium, coagulates its protoplasm, empties its contents and finally comes out producing numerous ramifications and sexual structures. When the infected mycelium of B. cinerea is applied to the leaves of the grape-vine, the characteristic grey mould symptoms fail to appear. Since P. radiosum causes no harm to the grape-vine, it can be used as a biological control agent against B. cinerea. A brief account of the mycoparasite and its antagonism towards B. cinerea is discussed here. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Botrytis cinerea; Mycoparasite ; Biological control; Pythium radiosum; Antagonism
1. Introduction Mycoparasites are fungi that can parasitize other fungi. This term is generally used to include parasites that coil around the host hyphae or overgrow other colonies on agar. This may involve either penetration of the host hyphae or antagonism by the production of antibiotics [4], toxic radicals [10], or wall-lytic enzymes [2]. The phenomenon of mycoparasitism is very widespread in nature. A review of the literature on this important subject has already been published [1,8,11,17,18]. Most evidence for the role of mycoparasitism comes from the observation of infected fungal propagules such as spores or other reproduc* Tel.: +33 3 80396143; Fax: +33 3 80396265; E-mail: [email protected]
tive structures. Oospores of Aphanomyces, Phytophthora, Pythium, and Sclerospora have been found to be infected by a wide range of fungi including Fusarium mesmoides, Hyphochytrium catenoides, Olpidiopsis gracilis, Dactyella spp. and Trinacrium subtile [16]. Facultative mycoparasites such as Trichoderma and Gliocladium species have been reported from the sclerotia of di¡erent parasites like Phymatotrichum omnivorum, Rhizoctonia spp., Sclerotinia spp., and Verticillium dahliae [17]. Unfortunately this phenomenon of mycoparasitism has not been much exploited. Some of the mycoparasites that have been used as biological control agents are Ampelomyces quisqualis for the control of cucumber powdery mildew caused by Sphaerotheca fuliginea [7] and Erysiphe cichoracearum [15] under glasshouse conditions. Mycoparasitism of Trichoderma harzianum has been
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 1 3 - X
FEMSLE 8806 17-6-99
26
B. Paul / FEMS Microbiology Letters 176 (1999) 25^30
studied extensively. This mycoparasite is antagonistic and e¡ective in controlling plant pathogenic fungi like Rhizoctonia solani, Sclerotium rolfsii, Fusarium oxysporum, Fusarium culmorum, and Pythium aphanidermatum [3]. It has also been widely studied against Botrytis cinerea causing the grey mould disease of grape-vine and Chondrostereum purpureum, the agent of silver leaf in fruit trees [5]. Pythiaceous fungi are also known to be mycoparasites of a wide range of fungi. The most studied mycoparasite of the genus or perhaps of all mycoparasites known is Pythium oligandrum Drechsler. It has a very wide host range and rapidly attacks the susceptible hosts by lysis or penetration of hyphae [12]. However, it is only one of six known mycoparasitic Pythium species, the others being Pythium acanthicum Drechs., P. acanthophoron Sideris, P. mycoparasiticum Deacon et al., P. periplocum Drechs., and P. nunn Lifshitz et al. [9]. Apart from these well known mycoparasites, one aggressive plant pathogen, P. aphanidermatum (Edson) Fitzpatrick, has also been studied for its mycoparasitic activity [9]. Pythium radiosum was isolated in 1992 from a soil sample collected in the north of France and described as a new species by the author [14]. Since then it has not been reported from anywhere else in the world. It has echinulate oogonia resembling Pythium echinulatum Matthews. When grown together with B. cinerea, it rapidly enters the latter's mycelium and causes extensive damage. This is the ¢rst report of the mycoparasitism of P. radiosum.
2. Materials and methods P. radiosum (F-12) was re-cultured from the author's personal collection of pythiaceous fungi, maintained at the `Institut Jules Guyot', in Dijon, France. B. cinerea (B-3) was also obtained from the culture collection of this institute. Both fungi were maintained on PDA (potato dextrose agar), and PCA (potato carrot agar) media. P. radiosum was also maintained on boiled hemp-seed halves in sterile distilled water. Inter-hyphal interactions were studied by placing both fungi on the same PDA plate on opposite ends (Fig. 2a) and also on a thin ¢lm of PDA on a glass slide (Fig. 2b) which was obtained as follows.
A glass rod was heated and bent in a `V' shape in such a way that it could enter a glass petri dish of 10 cm diameter. It was introduced into the petri dish and a glass slide was placed on the top of the V rod. The whole was autoclaved. A thin layer of PDA was poured aseptically on the glass slide and the two fungi were inoculated at the opposite ends of the PDA ¢lm. To maintain humidity, enough sterile distilled water was poured in the petri dish so that the water remained well below the slide. Three replicates were prepared in the same manner and all were incubated at 25³C for 15 days. From time to time sterile distilled water was added to the petri dishes in order to avoid dehydration of the thin PDA ¢lm. After 15 days a small square of the PDA ¢lm was aseptically cut and placed on a fresh slide in a drop of sterile water. A coverslip was placed on the ¢lm and this was sealed with nail polish to avoid dehydration. The slide was then observed by microscope. Inoculation experiments with B. cinerea were carried out on the leaves of 1-month-old Vitis vinifera vitro cultures maintained in our laboratory. The leaves were taken out aseptically and were introduced into three sterile petri dishes having a sterile ¢lter paper. Sterile distilled water was used to humidify the ¢lter paper. One leaf was placed in each petri dish. The leaf of the ¢rst petri dish was inoculated with 50 Wl sterile distilled water in which B. cinerea mycelium from a 1-week-old PDA culture was introduced. The leaf of the second petri dish was inoculated with 50 Wl of sterile distilled water in which the parasitized mycelium (with P. radiosum) was placed, and the leaf in the third petri dish was inoculated with 50 Wl of sterile distilled water in which the mycelium of P. radiosum was introduced. All three petri dishes were maintained at room temperature (20³C). Occasionally the ¢lter papers of the three dishes were humidi¢ed with sterile distilled water. This experiment was repeated thrice.
3. Results 3.1. Morphological description P. radiosum (Fig. 1a^e): mycelia are well branched at times bearing conical spines measuring 3^7 Wm in
FEMSLE 8806 17-6-99
B. Paul / FEMS Microbiology Letters 176 (1999) 25^30
27
Fig. 1. Morphological structures of P. radiosum. a^c : Oogonia ornamented with conical to mammiform spines. d: Smooth-walled sporangia. e: Scanning electron micrograph of an ornamented oogonium. Bar: a^d = 25 Wm, e = 10 Wm.
FEMSLE 8806 17-6-99
28
B. Paul / FEMS Microbiology Letters 176 (1999) 25^30
Fig. 2. Interaction between B. cinerea (B-3) and P. radiosum (F-12). a: Antagonism between B-3 and F-12 on PDA plate. b: Antagonism between B-3 and F-12 on a ¢lm of PDA on a slide.
Fig. 3. Hyphal interactions between B. cinerea and P. radiosum. a: Normal hypha of B. cinerea. b: Hypha of P. radiosum (mycoparasite) inside the mycelium of B. cinerea (host). c: Coagulated protoplasm in the hypha of B. cinerea. d: Proliferation and exit of P. radiosum hyphae from the hypha of B. cinerea. e: Emptied hypha of B. cinerea. f: Damaged hypha of B. cinerea with sexual structure of P. radiosum. Bar : a^c, e, f = 35 Wm, d=100 Wm.
FEMSLE 8806 17-6-99
B. Paul / FEMS Microbiology Letters 176 (1999) 25^30
29
Fig. 4. Inoculation experiments on V. vinifera. a: Symptoms of grey mould disease on V. vinifera caused by B. cinerea. b: Healthy leaf of V. vinifera inoculated with parasitized B. cinerea with P. radiosum.
length. Sporangia are mostly intercalary measuring 6^30 Wm in diameter and are mostly non-sporulating (Fig. 1d). Oogonia are mostly intercalary and catenulate and are covered with conical to mammiform spines (Fig. 1a,b,c,e). Oogonia (excluding spines) are 10^35 Wm in diameter and are provided with spines which can measure up to 15 Wm long and up to 6 Wm thick at the base. Antheridia are rare and when present are hypogynous. After fertilization the oospores produced are aplerotic with a relatively thin wall. In the hyphal interaction experiments, the colonies of P. radiosum (F-12) which are whitish in color spread into the greyish colonies of B. cinerea (B-3). The latter does not spread into the former's zone (Fig. 2a,b). Microscopic observation of the mycelium in the zone of contact reveals that P. radiosum enters freely into the mycelium of B. cinerea (Fig. 3b^d) and once inside it rami¢es (Fig. 3b), the rami¢cations enter the septa of the fungal hyphae thus passing from one cell to another without any hindrance. At ¢rst the cytoplasm of the host cell is coagulated (Fig. 3c) and then the mycoparasite consumes the cytoplasm leaving an empty host mycelium (Fig. 3e) and ¢nally the mycoparasite comes out forming here and there an entangled mass of its mycelium around the host (Fig. 3d) and reproductive structures (Fig. 3f).
After inoculation on the leaf the vitro plants of V. vinifera showed typical grey mould symptoms on the leaf of the ¢rst petri dish (Fig. 4a), no disease on the leaf of the second petri dish (Fig. 4b) and also no infection on the third. All three replicates showed the same results.
4. Discussion The experiments show that the mycelium of P. radiosum can rapidly enter the hyphae of B. cinerea. Upon entry, the fungus rami¢es within and provokes large-scale destruction of the host mycelium. Hence the former is undoubtedly a mycoparasite of the latter. Since the parasitized mycelium of B. cinerea fails to produce the characteristic grey mould symptoms on the leaves of V. vinifera, and since the mycoparasite does not harm the leaves of the grape-vine in any way, P. radiosum can be used as a biocontrol agent against B. cinerea. The mode of action of P. radiosum di¡ers from that of P. oligandrum which often causes explosive lysis of host mycelium [12], whereas P. radiosum brings about cytoplasmic coagulation like that observed in the case of P. aphanidermatum [9]. The fact that P. radiosum penetrated the host mycelium and grew extensively within it may be useful in the
FEMSLE 8806 17-6-99
30
B. Paul / FEMS Microbiology Letters 176 (1999) 25^30
analysis of the mechanisms of mycoparasitism, especially the suggested roles of cell wall-degrading enzymes [2,6,13].
[10]
[11]
References [1] Adams, P.B. (1990) The potential of mycoparasites for biological control of plant diseases. Annu. Rev. Phytopathol. 72, 58^73. [2] Chet, I. (1987) Trichoderma ^ application, mode of action, and potential as a biocontrol of soil-borne plant pathogenic fungi. In: Innovative Approaches to Plant Disease Control (Chet, I., Ed.), pp. 137^160. Wiley, New York. [3] Chet, I. and Inbar, J. (1994) Biological control of fungal pathogens. Appl. Biochem. Biotechnol. 48, 37^43. [4] Denis, C. and Webster, J. (1971) Antagonistic properties of species-groups of Trichoderma. I. Production of non-volatile antibiotics. Trans. Br. Mycol. Soc. 57, 363^369. [5] Dubos, B. (1987) Fungal antagonism in aerial agrobiocenoses. In : Innovative Approaches to Plant Disease Control (Chet, I., Ed.), pp. 107^136. Wiley, New York. [6] Elad, Y., Lifshitz, R. and Baker, R. (1985) Enzymatic activity of the mycoparasite Pythium nunn during interaction with host and non-host fungi. Physiol. Plant Pathol. 27, 131^ 148. [7] Jarvis, W.R. and Slingby, K. (1977) The control of powdery mildew of greenhouse cucumber by water sprays and Ampelomyces quisqualis. Plant Dis. Rep. 61, 728^730. [8] Je¡eries, P. and Young, T.W.K. (1994) Interfungal Parasitic Relationship, CAB International, Wallingford. [9] Jones, E.E. and Deacon, J.W. (1995) Comparative physiology and behaviour of the mycoparasites Pythium acanthophoron,
[12]
[13]
[14] [15]
[16]
[17] [18]
P. oligandrum, and P. mycoparasiticum. Biocontrol Sci. Technol. 5, 27^39. Kim, K.K., Fravel, D.R. and Papvizas, G.C. (1990) Glucose oxidase as the antifungal principle of talaron from Talaromyces £avus. Can. J. Microbiol. 36, 760^764. Kranz, J. (1981) Hyperparasitism of biotrophic fungi. In: Microbial Ecology of the Phylloplane (Blackman, Ed.), pp. 327^ 352. Academic Press, New York. Laing, S.A.K. and Deacon, J.W. (1991) Video microscopical comparison of mycoparasitism by Pythium oligandrum, P. nunn, and an unnamed Pythium species. Mycol. Res. 95, 469^479. Lewis, K., Whipps, J.M. and Cooke, R.C. (1989) Mechanisms of biological disease control with special reference to the case study of Pythium oligandrum as an antagonist. In : Biotechnology of Fungi for Improving Plant Growth (Whipps, J.M. and Lumsden, R.D., Eds.), pp. 191^217, Cambridge University Press, Cambridge. Paul, B. (1992) Pythium radiosum, a new species with ornamented oogonia from France. Mycol. Helv. 5, 1^8. Sundheim, L. (1983) L'hyperparasite Ampelomyces quisqualis dans la lutte contre O|ëdium du concombre. Colloq. INRA 18, 145^154. Whipps, J.M. (1991) E¡ects of mycoparasites on sclerotiaforming fungi. In: Biotic Interactions and Soil-borne Diseases (Beemster, A.B.R., Bollen, G.J., Gerlagh, M., Ruissen, M.A., Schippers, B. and Tempel, A., Eds.), pp. 129^140. Elsevier, Amsterdam. Whipps, J.M. (1997) Developments in the biological control of soil-borne plant pathogens. Adv. Bot. Res. 26, 1^134. Whipps, J.M., Lewis, K. and Cooke, R.C. (1988) Mycoparasitism and plant disease control. In: Fungi in Biological Control Systems (Burge, M.N., Ed.), pp. 161^187. Manchester University Press, Manchester.
FEMSLE 8806 17-6-99