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Pseudomonas tolaasii and tolaasin: comparison of symptom induction on a wide range of Agaricus bisporus strains
ELSEVIER
MICROBIOLOGY LETTERS FEMS Microbiology Letters 142 (1996) 99-103
Pseudomonas tolaasii and tolaasin : comparison of symptom induction on a wide range of Agaricus bisporus strains F. Moquet ‘, M. Mamoun bt*, J.M. Olivier b a INRA - CTC, Station de Recherches SW les Champignons, BP 81, 33883 Villenave d’Omon Cedex, France b INRA, Station de Recherches SW les Champignons, BP 81, 33883 Villenaved’OrnonCeakx, France
Received 17 June 1996; accepted 18 June 1996
Abstract
A wide range of Aguricus bisporus, including commercial, wild and hybrid strains, were tested for resistance to brown blotch disease caused by Pseudomonas tolaasii.Effects of toxin and living bacteria were compared. Wild and hybrid A. bisporus ranged in the same order from very poorly to highly susceptible whatever the inoculum type used, tolaasin or bacteria. Symptom aspects induced by both inocula were visually identical, but some differences occurred in response intensity. The data suggest that toxin is probably not the only factor involved in symptom development. Keywords: Agaricus bisporus; Brown blotch disease; Pseudomonas tolaasii; Symptom assessment; Tolaasin
1. Introduction
previously used by Olivier et al. [2], was improved by Rama et al. [8] through the use of tolaasin in place of living bacteria. This work describes the susceptibility to living P. tolaasii and to tolaasin among a wide range of A. bisporus strains using two bacteria strains and their relative toxins. Mushroom susceptibilities and inoculum efficiencies were compared.
The cultivated mushroom Aguricus bisporus (Lange) is susceptible to a variety of pests and diseases [I]. Brown blotch disease, caused by Pseudomonas tolaasii, is responsible for an estimated mushroom crop loss of 8-10% worldwide [2,3]. The threshold level of P. toluusii required to cause visible brown blotch symptoms has been reported to be approximately 10s cells per cap [4,5]. Symptoms are induced by a toxin, tolaasin, first isolated by Peng [6] and described by Nutkins et al. [7]. A test estimating the resistance of A. bisporus strains to P. tolaasii,
2.1. Agaricus bisporus
* Corresponding author. Tel.: +33 56 84 31 71; Fax: +33 56 84 31 78; E-mail: [email protected]
Agaricus bisporus strains included three commercial strains, B62 and C45 from Le Lion (Varrains, France) and HUl from Somycel (Langeais, France), 26 wild strains from the ARP (Agaricus Resource
2. Material and methods
0378-1097/96/$12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Z’ZZSO378-1097(96)00250-9
100
F. Moquet
et al.IFEMS Microbiology Letters 142 (1996) 99-103
Programme, Kerrigan [9]) and INRA - CTC collections, and 86 hybrids between commercial and wild strains. For technical reasons, hybrids were tested in two groups of experiments. Each strain was cultivated on two trays filled with 15 kg of a conventional compost provided by the SCBM-Faleyras Company (France). The casing soil was made of 50% limestone, 50% brown peat (v/v). Fruiting was conducted at 16°C under 88-91% relative humidity. 2.2. Bacterial and toxin suspensions Two Pseudomonas tolaasii strains were used, CNBP 2152 from the National Collection of Phytopathogen Bacteria (Angers, France) and SPC 8907 from the Mushroom Experimental Station (Hors& The Netherlands). Bacterial suspensions, adjusted to 0.3 absorbance at 450 nm (Uvikon 390 spectrophotometer) and providing approximately l-2 x lo8 cfu ml-‘, were prepared as described by Rama et al. [8]. Toxin suspensions were obtained from King’s B agar cultures of P. tolaasii according to the procedure of Nutkins et al. [7] modified by Rama et al. [8] through the use of butanol which removes bacterial slime and improves extraction. Toxin suspension absorbance was measured at 206 nm [7].
Table I General linear model for strain. inoculum, A. bisporus
Commercial
Wild
Hybrid (group
1)
Hybrid (group 2)
suspension
and sporophore
2.3.
Resistance test
For each A. bisporus strain, 40 mushrooms at stage [lo] were randomly harvested from the two trays, and arranged into moist chambers. The inoculum consisted of a 20 ul droplet of bacterial or toxin suspension placed at the top of the cap. From each P. tolaasii strain, bacterial suspensions were prepared in duplicate, each one applied to five caps. Similarly, two groups of five caps received toxin suspension duplicates. The experiment was repeated two or three times. 2.4. Data assessment Symptoms were evaluated after 48 h of incubation at 16°C according to a rating scale of 0 (no symptom) to 2.5 (extensive dark sunken lesions) [8]. Mean scores reported by the Student-Newman-Keuls test represented average symptoms. 2.5. Statistical analyses Results were analysed by the general linear models and correlations procedures provided by the SAS system (SAS Institute Inc., Cary, NC, USA).
effects on susceptibility
of commercial,
wild and hybrid strains of
Inoculum
Suspension
Sporophore
Strain X inoculum
Strain X suspension
1
4 0.110 0.58
6 3.1892 16.74’
2 0.086 0.45
4 0.278 0.70
76 1.758 4.45*
26 0.219 0.55 82 0.272 1.31 37 0.112 0.58
df Mean square F value
2 102.271 536.85’
3 22.939 102.42’
0.391 2.05
df Mean square F value
26 30.28’
3 31.392 79.42+
0.333 0.84
df Mean square F value
82 3.529 17.02’
1 34.272 166.29’
1 0.224 1.08
4 0.359
1.73
82 1.141 5.51’
df Mean square F value
37 1.854 9.52’
3 8.088 41.54
I 0.128 0.66
4 0.033 0.17
110 0.341 1.75
*Significant at the 1% level
11.967
I
F. Moquet et al. IFEMS Microbiology Letters 142 (1996) 99-103
Investigations concerned differences in A. bisporus susceptibility (strain effect), in toxin and bacteria aggressivity (inoculum effect), and between suspension duplicates (suspension effect).
Table 2 Range of susceptibility Agaricus strain
Commercial Wild Hybrid
3. Results Differences in commercial strain susceptibility were observed whatever the inoculum type, living bacteria or toxin, and the P. tolaasii strain used (Table 1). A great diversity in symptom intensity was observed among wild and hybrid A. bisporus strains (Table 2). No significant differences occurred between sporophore replicates and suspension duplicates of the same inoculum type (Table 1). Inocula exhibited differences in symptom induction, whatever the Agaricus strain tested (Table 1). Toxin from CNBP 2152 and living bacteria SPC 8907 were more aggressive than bacteria CNBP 2152, throughout this study. Variations in aggressivity classification were observed between bacteria SPC 8907 and its toxin (Table 3). All Pearson correlation coefficients between all types of inocula were highly significant (Table 4). Coefficients between both bacteria and both toxins were higher than those between bacteria and toxin.
Table 3 Student-Newman-Keuls
test for classification
of inocula aggressivity
(min.-max.)
101
among Agaricus strains
Range of mean scores bacteria
toxin
OS&l.55 0.1&2.15 O.lc2.35
0.55-1.65 0.05-2.50 0.2c2.60
4. Discussion The present work reports the first known data concerning the wide range of susceptibility observed among wild and hybrid strains of A. bisporus. Bacteria and toxin inoculum induced similar ranges in susceptibility. No absolute resistance was found. Absence of intra-strain variations (sporophore replicate effect) is not surprising for commercial strains because of their stability related to the breeding process. Wild and hybrid A. bisporus exhibited similar intra-strain homogeneity in susceptibility. Sporophore response to P. tolaasii appears as a stable character under the same experimental conditions. This aspect and the absence of a suspension effect confirm the reproducibility and reliability of the test carried out with either living bacteria or toxin for A. bisporus classification.
on cultivated,
wild and hybrid strains of A. bisporus
A. bisporus
Inoculum
Mean score
SNK grouping
Commercial
Bacteria SPC 8907 Toxin CNBP 2152 Toxin SPC 8907 Bacteria CNBP 2152
1.07 0.79 0.60 0.49
A B C D
Wild
Toxin CNBP 2152
1.49
Bacteria SPC 8907 Toxin SPC 8907 Bacteria CNBP 2152
1.26 1.23 0.77
A B B C
Toxin CNBP 2152
0.92
Bacteria CNBP 2152
0.74
Toxin SPC 8907
0.84
Bacteria SPC 8907 Toxin CNBP 2152 Bacteria CNBP 2152
0.78 0.74 0.58
Hybrid
(group
1)
Hybrid (group 2)
A B A B B C
F. Moquet et ul. IFEMS
102 Table 4 Pearson correlation
coefficients between each inoculum
Bacteria CNBP 2152
Bacteria SPC 8907
Microbiology
type for symptom
Letters I42 (1996)
induction
on the overall A. bisporus strain collection
Bacteria CNBP 2152
Bacteria SPC 8907
Toxin CNBP 2152
Toxin SPC 8907
I .ooooo
0.72353 0.0001 137
0.63827 0.0001 242
0.63765 0.0001 138
1.ooooo 0.0000 143
0.65041 0.0001 117
0.68493 0.0001 142
1.oOOOO 0.0000 248
0.69661 0.0001 119
0.0000’ 276”
Toxin CNBP 2152
Toxin SPC 8907 *P>
99-103
1.ooooO 0.0000 145
Irl under Ho: p=O.
a Number
of observations.
Toxin induced more prominent symptoms than living bacteria did, with some exceptions concerning toxin SPC 8907. Bacteria SPC 8907 produced very large quantities of slime, which interfere with the extraction process and could explain variations in toxin effect. Agaricus bisporus strain responses to each inoculum type were not exactly the same. However, because of significant Pearson coefficients the classification of fungal strains remained identical whatever the inoculum type. Nutkins et al. [7] reported that partially purified toxin reproduces disease symptoms of the intact organism. Under our conditions, all A. bisporus strains tested developed symptoms of the same visual aspect (brown, sunken) after bacteria or toxin inoculation; nevertheless, some differences occurred in symptom intensity. Closer relationships were obtained between both bacteria and both toxins than between bacteria and toxin, which suggest that toxin is not the only factor responsible for symptoms. Similar results have been reported by Patil et al. [l I] about Pseudomonas syringae pv. phaseolicola and phaseolotoxin effects on bean. The use of toxin in place of living bacteria is convenient for breeding programmes based on resistance assessment and avoid variations in bacteria aggressivity related to the instability between rough and smooth forms of P. tolaasii colonies [2,12]. Mechan-
isms involved in browning must be clarified. Further experiments are in progress to detect factors different from tolaasin and involved in symptom development.
Acknowledgments This work was supported by a grant from the European Community (AIR2-CT93-0953) and a contract between INRA and CTC, Saint-Paterne, France. We wish to thank J. Laffitte for toxin extraction and inoculation procedure. We are grateful to M. Lafargue for statistical advice. We thank P. Castant, C. Coldefy and J. Lamarque for their technical assistance. References [l] Fletcher, J.T., White, P.F. and Gaze, R.H. (1989) Mushrooms: Pest and Disease Control, 2nd edn., 174 pp. Intercept Ltd., Andover, MA. [2] Olivier, J.M., Guillaumes, J. and Martin, D. (1978) Study of a bacterial disease of mushroom caps. In: Proc. 4th Int. Conf. Plant Path. Bact. (INRA Ed.), pp. 903-916, Angers. [3] Rainey, P.B., Brodey, C.L. and Johnstone, K. (1992) Biology of Pseudomonas tolaasii, cause of brown disease of the cultivated mushroom. Adv. Plant Pathol. 8, 95-117. [4] Nair, N.G. and Bradley, J.K. (1980) Mushroom blotch bacterium during cultivation. Mushroom J. 90, 201-203. [5] Wong, W.C. and Preece, T.F. (1982) Pseudomonas tolaasii in
E Moquet et al. IFEMS Microbiology Letters 142 (1996) 99-103 cultivated mushroom (Agaricus bisporus) crops: numbers of bacterium and symptom development on mushrooms grown in various environments after artificial inoculation. J. Appl. Bacterial. 53, 87-96. [6] Peng, J.C. (1986) Resistance to disease in Agaricus bisporus (Lange) Imbach. Ph.D. Thesis, University of Leeds. [7] Nutkins, J.C., Mortishire-Smith, R.J., Packman, L.C., Brodey, C.L., Rainey, P.B., Johnstone, K. and Williams, D.H. (1991) Structure determination of Tolaasin, an extracellular lipodepsipeptide produced by the mushroom pathogen Pseudomonas tolaasii Paine. J. Am. Chem. Sot. 113, 2661-2627. [S] Rama, T., Mamoun, M. and Olivier, J.M. (1995) Rapid extraction of a bacteria free fraction inducing blotch symptoms on Agaricw bisporus. Mushroom Sci. 14, 571-577.
103
[9] Kerrigan, R.W. (1991) What on earth is the Agaricus Recovery Program? Mycologist 5, 22. [lo] Hammond, J.B.W. and Nichols, R. (1976) Carbohydrates metabolism in Agaricus bisporus (Lange) Sing. : Changes in soluble carbohydrates during growth of mycelium and sporophore. J. Gen. Microbial. 93, 309-320. [ll] Patil, S.S., Hayward, A.C. and Emmons, R. (1974) An uhraviolet-induced non-toxigenic mutant of Pseudomonas phaseolicola of altered pathogenicity. Phytopathology 64, 590-595. [12] Cutri, S.S., Macauley, P.J. and Roberts, W.P. (1984) Characteristics of pathogenic non-fluorescent (smooth) and nonpathogenic fluorescent (rough) forms of Pseudomonas tolaasii and Pseudomonas gingeri. J. Appl. Bacterial. 57, 291-298.
MICROBIOLOGY LETTERS FEMS Microbiology Letters 142 (1996) 99-103
Pseudomonas tolaasii and tolaasin : comparison of symptom induction on a wide range of Agaricus bisporus strains F. Moquet ‘, M. Mamoun bt*, J.M. Olivier b a INRA - CTC, Station de Recherches SW les Champignons, BP 81, 33883 Villenave d’Omon Cedex, France b INRA, Station de Recherches SW les Champignons, BP 81, 33883 Villenaved’OrnonCeakx, France
Received 17 June 1996; accepted 18 June 1996
Abstract
A wide range of Aguricus bisporus, including commercial, wild and hybrid strains, were tested for resistance to brown blotch disease caused by Pseudomonas tolaasii.Effects of toxin and living bacteria were compared. Wild and hybrid A. bisporus ranged in the same order from very poorly to highly susceptible whatever the inoculum type used, tolaasin or bacteria. Symptom aspects induced by both inocula were visually identical, but some differences occurred in response intensity. The data suggest that toxin is probably not the only factor involved in symptom development. Keywords: Agaricus bisporus; Brown blotch disease; Pseudomonas tolaasii; Symptom assessment; Tolaasin
1. Introduction
previously used by Olivier et al. [2], was improved by Rama et al. [8] through the use of tolaasin in place of living bacteria. This work describes the susceptibility to living P. tolaasii and to tolaasin among a wide range of A. bisporus strains using two bacteria strains and their relative toxins. Mushroom susceptibilities and inoculum efficiencies were compared.
The cultivated mushroom Aguricus bisporus (Lange) is susceptible to a variety of pests and diseases [I]. Brown blotch disease, caused by Pseudomonas tolaasii, is responsible for an estimated mushroom crop loss of 8-10% worldwide [2,3]. The threshold level of P. toluusii required to cause visible brown blotch symptoms has been reported to be approximately 10s cells per cap [4,5]. Symptoms are induced by a toxin, tolaasin, first isolated by Peng [6] and described by Nutkins et al. [7]. A test estimating the resistance of A. bisporus strains to P. tolaasii,
2.1. Agaricus bisporus
* Corresponding author. Tel.: +33 56 84 31 71; Fax: +33 56 84 31 78; E-mail: [email protected]
Agaricus bisporus strains included three commercial strains, B62 and C45 from Le Lion (Varrains, France) and HUl from Somycel (Langeais, France), 26 wild strains from the ARP (Agaricus Resource
2. Material and methods
0378-1097/96/$12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Z’ZZSO378-1097(96)00250-9
100
F. Moquet
et al.IFEMS Microbiology Letters 142 (1996) 99-103
Programme, Kerrigan [9]) and INRA - CTC collections, and 86 hybrids between commercial and wild strains. For technical reasons, hybrids were tested in two groups of experiments. Each strain was cultivated on two trays filled with 15 kg of a conventional compost provided by the SCBM-Faleyras Company (France). The casing soil was made of 50% limestone, 50% brown peat (v/v). Fruiting was conducted at 16°C under 88-91% relative humidity. 2.2. Bacterial and toxin suspensions Two Pseudomonas tolaasii strains were used, CNBP 2152 from the National Collection of Phytopathogen Bacteria (Angers, France) and SPC 8907 from the Mushroom Experimental Station (Hors& The Netherlands). Bacterial suspensions, adjusted to 0.3 absorbance at 450 nm (Uvikon 390 spectrophotometer) and providing approximately l-2 x lo8 cfu ml-‘, were prepared as described by Rama et al. [8]. Toxin suspensions were obtained from King’s B agar cultures of P. tolaasii according to the procedure of Nutkins et al. [7] modified by Rama et al. [8] through the use of butanol which removes bacterial slime and improves extraction. Toxin suspension absorbance was measured at 206 nm [7].
Table I General linear model for strain. inoculum, A. bisporus
Commercial
Wild
Hybrid (group
1)
Hybrid (group 2)
suspension
and sporophore
2.3.
Resistance test
For each A. bisporus strain, 40 mushrooms at stage [lo] were randomly harvested from the two trays, and arranged into moist chambers. The inoculum consisted of a 20 ul droplet of bacterial or toxin suspension placed at the top of the cap. From each P. tolaasii strain, bacterial suspensions were prepared in duplicate, each one applied to five caps. Similarly, two groups of five caps received toxin suspension duplicates. The experiment was repeated two or three times. 2.4. Data assessment Symptoms were evaluated after 48 h of incubation at 16°C according to a rating scale of 0 (no symptom) to 2.5 (extensive dark sunken lesions) [8]. Mean scores reported by the Student-Newman-Keuls test represented average symptoms. 2.5. Statistical analyses Results were analysed by the general linear models and correlations procedures provided by the SAS system (SAS Institute Inc., Cary, NC, USA).
effects on susceptibility
of commercial,
wild and hybrid strains of
Inoculum
Suspension
Sporophore
Strain X inoculum
Strain X suspension
1
4 0.110 0.58
6 3.1892 16.74’
2 0.086 0.45
4 0.278 0.70
76 1.758 4.45*
26 0.219 0.55 82 0.272 1.31 37 0.112 0.58
df Mean square F value
2 102.271 536.85’
3 22.939 102.42’
0.391 2.05
df Mean square F value
26 30.28’
3 31.392 79.42+
0.333 0.84
df Mean square F value
82 3.529 17.02’
1 34.272 166.29’
1 0.224 1.08
4 0.359
1.73
82 1.141 5.51’
df Mean square F value
37 1.854 9.52’
3 8.088 41.54
I 0.128 0.66
4 0.033 0.17
110 0.341 1.75
*Significant at the 1% level
11.967
I
F. Moquet et al. IFEMS Microbiology Letters 142 (1996) 99-103
Investigations concerned differences in A. bisporus susceptibility (strain effect), in toxin and bacteria aggressivity (inoculum effect), and between suspension duplicates (suspension effect).
Table 2 Range of susceptibility Agaricus strain
Commercial Wild Hybrid
3. Results Differences in commercial strain susceptibility were observed whatever the inoculum type, living bacteria or toxin, and the P. tolaasii strain used (Table 1). A great diversity in symptom intensity was observed among wild and hybrid A. bisporus strains (Table 2). No significant differences occurred between sporophore replicates and suspension duplicates of the same inoculum type (Table 1). Inocula exhibited differences in symptom induction, whatever the Agaricus strain tested (Table 1). Toxin from CNBP 2152 and living bacteria SPC 8907 were more aggressive than bacteria CNBP 2152, throughout this study. Variations in aggressivity classification were observed between bacteria SPC 8907 and its toxin (Table 3). All Pearson correlation coefficients between all types of inocula were highly significant (Table 4). Coefficients between both bacteria and both toxins were higher than those between bacteria and toxin.
Table 3 Student-Newman-Keuls
test for classification
of inocula aggressivity
(min.-max.)
101
among Agaricus strains
Range of mean scores bacteria
toxin
OS&l.55 0.1&2.15 O.lc2.35
0.55-1.65 0.05-2.50 0.2c2.60
4. Discussion The present work reports the first known data concerning the wide range of susceptibility observed among wild and hybrid strains of A. bisporus. Bacteria and toxin inoculum induced similar ranges in susceptibility. No absolute resistance was found. Absence of intra-strain variations (sporophore replicate effect) is not surprising for commercial strains because of their stability related to the breeding process. Wild and hybrid A. bisporus exhibited similar intra-strain homogeneity in susceptibility. Sporophore response to P. tolaasii appears as a stable character under the same experimental conditions. This aspect and the absence of a suspension effect confirm the reproducibility and reliability of the test carried out with either living bacteria or toxin for A. bisporus classification.
on cultivated,
wild and hybrid strains of A. bisporus
A. bisporus
Inoculum
Mean score
SNK grouping
Commercial
Bacteria SPC 8907 Toxin CNBP 2152 Toxin SPC 8907 Bacteria CNBP 2152
1.07 0.79 0.60 0.49
A B C D
Wild
Toxin CNBP 2152
1.49
Bacteria SPC 8907 Toxin SPC 8907 Bacteria CNBP 2152
1.26 1.23 0.77
A B B C
Toxin CNBP 2152
0.92
Bacteria CNBP 2152
0.74
Toxin SPC 8907
0.84
Bacteria SPC 8907 Toxin CNBP 2152 Bacteria CNBP 2152
0.78 0.74 0.58
Hybrid
(group
1)
Hybrid (group 2)
A B A B B C
F. Moquet et ul. IFEMS
102 Table 4 Pearson correlation
coefficients between each inoculum
Bacteria CNBP 2152
Bacteria SPC 8907
Microbiology
type for symptom
Letters I42 (1996)
induction
on the overall A. bisporus strain collection
Bacteria CNBP 2152
Bacteria SPC 8907
Toxin CNBP 2152
Toxin SPC 8907
I .ooooo
0.72353 0.0001 137
0.63827 0.0001 242
0.63765 0.0001 138
1.ooooo 0.0000 143
0.65041 0.0001 117
0.68493 0.0001 142
1.oOOOO 0.0000 248
0.69661 0.0001 119
0.0000’ 276”
Toxin CNBP 2152
Toxin SPC 8907 *P>
99-103
1.ooooO 0.0000 145
Irl under Ho: p=O.
a Number
of observations.
Toxin induced more prominent symptoms than living bacteria did, with some exceptions concerning toxin SPC 8907. Bacteria SPC 8907 produced very large quantities of slime, which interfere with the extraction process and could explain variations in toxin effect. Agaricus bisporus strain responses to each inoculum type were not exactly the same. However, because of significant Pearson coefficients the classification of fungal strains remained identical whatever the inoculum type. Nutkins et al. [7] reported that partially purified toxin reproduces disease symptoms of the intact organism. Under our conditions, all A. bisporus strains tested developed symptoms of the same visual aspect (brown, sunken) after bacteria or toxin inoculation; nevertheless, some differences occurred in symptom intensity. Closer relationships were obtained between both bacteria and both toxins than between bacteria and toxin, which suggest that toxin is not the only factor responsible for symptoms. Similar results have been reported by Patil et al. [l I] about Pseudomonas syringae pv. phaseolicola and phaseolotoxin effects on bean. The use of toxin in place of living bacteria is convenient for breeding programmes based on resistance assessment and avoid variations in bacteria aggressivity related to the instability between rough and smooth forms of P. tolaasii colonies [2,12]. Mechan-
isms involved in browning must be clarified. Further experiments are in progress to detect factors different from tolaasin and involved in symptom development.
Acknowledgments This work was supported by a grant from the European Community (AIR2-CT93-0953) and a contract between INRA and CTC, Saint-Paterne, France. We wish to thank J. Laffitte for toxin extraction and inoculation procedure. We are grateful to M. Lafargue for statistical advice. We thank P. Castant, C. Coldefy and J. Lamarque for their technical assistance. References [l] Fletcher, J.T., White, P.F. and Gaze, R.H. (1989) Mushrooms: Pest and Disease Control, 2nd edn., 174 pp. Intercept Ltd., Andover, MA. [2] Olivier, J.M., Guillaumes, J. and Martin, D. (1978) Study of a bacterial disease of mushroom caps. In: Proc. 4th Int. Conf. Plant Path. Bact. (INRA Ed.), pp. 903-916, Angers. [3] Rainey, P.B., Brodey, C.L. and Johnstone, K. (1992) Biology of Pseudomonas tolaasii, cause of brown disease of the cultivated mushroom. Adv. Plant Pathol. 8, 95-117. [4] Nair, N.G. and Bradley, J.K. (1980) Mushroom blotch bacterium during cultivation. Mushroom J. 90, 201-203. [5] Wong, W.C. and Preece, T.F. (1982) Pseudomonas tolaasii in
E Moquet et al. IFEMS Microbiology Letters 142 (1996) 99-103 cultivated mushroom (Agaricus bisporus) crops: numbers of bacterium and symptom development on mushrooms grown in various environments after artificial inoculation. J. Appl. Bacterial. 53, 87-96. [6] Peng, J.C. (1986) Resistance to disease in Agaricus bisporus (Lange) Imbach. Ph.D. Thesis, University of Leeds. [7] Nutkins, J.C., Mortishire-Smith, R.J., Packman, L.C., Brodey, C.L., Rainey, P.B., Johnstone, K. and Williams, D.H. (1991) Structure determination of Tolaasin, an extracellular lipodepsipeptide produced by the mushroom pathogen Pseudomonas tolaasii Paine. J. Am. Chem. Sot. 113, 2661-2627. [S] Rama, T., Mamoun, M. and Olivier, J.M. (1995) Rapid extraction of a bacteria free fraction inducing blotch symptoms on Agaricw bisporus. Mushroom Sci. 14, 571-577.
103
[9] Kerrigan, R.W. (1991) What on earth is the Agaricus Recovery Program? Mycologist 5, 22. [lo] Hammond, J.B.W. and Nichols, R. (1976) Carbohydrates metabolism in Agaricus bisporus (Lange) Sing. : Changes in soluble carbohydrates during growth of mycelium and sporophore. J. Gen. Microbial. 93, 309-320. [ll] Patil, S.S., Hayward, A.C. and Emmons, R. (1974) An uhraviolet-induced non-toxigenic mutant of Pseudomonas phaseolicola of altered pathogenicity. Phytopathology 64, 590-595. [12] Cutri, S.S., Macauley, P.J. and Roberts, W.P. (1984) Characteristics of pathogenic non-fluorescent (smooth) and nonpathogenic fluorescent (rough) forms of Pseudomonas tolaasii and Pseudomonas gingeri. J. Appl. Bacterial. 57, 291-298.