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Molecular and physiological diversity among Verticillium fungicola var. fungicola
m y c o l o g i c a l r e s e a r c h 1 1 0 ( 2 0 0 6 ) 431 – 440
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Molecular and physiological diversity among Verticillium fungicola var. fungicola Miche`le L. LARGETEAUa,*, Johan P. P. BAARSb, Catherine REGNAULT-ROGERc, Jean-Michel SAVOIEa a
Institut National de la Recherche Agronomique (INRA), BP81, F-33883 Villenave d’Ornon, France Applied Plant Research, Mushroom Research Unit, P.O. Box 6042, NL-5960 AA Horst, The Netherlands c UFR Sciences et Techniques, Univ. Pau et Pays de l’Adour, F-64012 Pau Universite´ cedex, France b
article info
abstract
Article history:
The genetic and physiological variability of Verticillium fungicola var. aleophilum responsible
Received 30 November 2004
for Agaricus bisporus dry bubble disease in North America is well documented but little is
Received in revised form
known about the var. fungicola affecting European crops. Variability was assessed within
26 October 2005
this variety and compared with that reported for the var. aleophilum. Eighteen isolates of
Accepted 30 November 2005
V. fungicola var. fungicola and four var. aleophilum isolates were analysed for DNA polymor-
Published online 17 April 2006
phism, mycelial growth, response to biochemicals produced by A. bisporus, fungicide
Corresponding Editor: Jan I. Lelley
resistance, and pathogenicity assessed by direct inoculation on sporophore or casing contamination. RAPD and AFLP markers delineated three French isolates from a homoge-
Keywords:
neous group containing the other var. fungicola isolates, but no correlation could be drawn
Agaricus bisporus
between DNA polymorphism and the various traits studied. The var. fungicola isolates were
Cultivated mushrooms
more susceptible than the var. aleophilum isolates to the antibiosis effect of A. bisporus. Only
Dry bubble disease
mycelial growth rate at 23 C could explain the variability in aggressiveness among
Fungicolous fungi
the European isolates. The putative effect of the post-incubation temperature on contam-
Mycoparasite
ination during mushroom cultivation was discussed. This work emphasized that, like the American var. aleophilum, the var. fungicola in Europe is genetically homogeneous, but physiological diversity exists, especially in France where it could be related to less standardized cultural practices. ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Verticillium fungicola is responsible for dry bubble disease of the cultivated mushroom Agaricus bisporus. Two varieties affect the mushroom crop, var. aleophilum and var. fungicola. The worldwide distribution of the pathogen incites questions regarding its variability. Bidochka et al. (1999) investigated the production of hydrolytic enzymes and the genetic variability among different species of Verticillium, including two representatives of V. fungicola. Despite physiological similarity,
these two isolates exhibited a 49 % divergence in their rDNA sequence of ITS1. Investigations into the molecular and physiological diversity reported in the literature mainly concern isolates of V. fungicola var. aleophilum collected in North America. Genetic variability within 66 isolates of V. fungicola, the majority originating from North America, was studied by Bonnen and Hopkins (1997). The authors did not find any correlation between RAPD grouping, colony morphology and virulence but they observed a high level of homogeneity among the isolates collected during 1993–1995 for fungicide
* Corresponding author. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2005.11.016
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M. L. Largeteau et al.
response, virulence, colony morphology, geographic origin and RAPD grouping. More recently, Collopy et al. (2001) performed molecular phylogenetic analyses on 40 isolates of V. fungicola gathered at various Pennsylvania mushroom farms in 1999, and 28 isolates of Verticillium spp. collected during the last 50 years from various geographic locations. The authors reported the presence of a clonal population of V. fungicola var. aleophilum among the Pennsylvania isolates and postulated that the lack of diversity observed may be due to a change in the practises regarding the casing of mushroom crops. Recent reports on dry bubble disease in Europe (Desrumeaux & Sedeyn 2001; Gea et al. 2003) refered to the var. fungicola as responsible for the disease, consequently further investigation into the variability of European isolate is required. The four French isolates and the UK isolate included in the work of Collopy et al. (2001) showed an identical rDNA sequence to V. fungicola var. fungicola and did not differ in RAPD patterns. Investigations performed on six French isolates have shown some genetic and physiological variations (Juarez del Carmen et al. 2002). The objective of this study was to examine the genetic variability, physiological dissimilarities and pathogenic diversity within a group of V. fungicola mainly constituted of European isolates in order to compare with var. aleophilum isolates.
geographic origin and source of the 22 isolates screened in this study are listed in Table 1. Isolates VCTC, VF, VK and VV1 belong to the variety fungicola, V01 and V35 to the variety aleophilum based on rDNA sequences (Collopy et al. 2001). Sequencing of the ITS1, 5.8 S and ITS2 rDNA assigned VTAW to the variety aleophilum (Largeteau et al. 2004). CBS440.34, the ex-type strain for var. fungicola (formely V. malthousei) was obtained from the Centraalbureau voor Schimmelcultures (Baarn, the Netherlands). The European isolate VX02, collected in Germany in 1981, identified as var. aleophilum on basis of growth at 30 C, was obtained from a diseased fruit-body of A. bisporus variety Les Miz 60. Voucher cultures are maintained in the INRA (France) or PPO-MRV (NL) culture collection and are publically accessible. Isolates used in this study are listed in Table 1.
Materials and methods
Amplification was performed in a 25 ml reaction mixture containing 0.125 mM dNTPs, 1 DyNAzyme buffer (Finnzymes, Finland), 0.5 U DyNAzyme II polymerase (Finnzymes), 0.2 mM each of primer ITS1 (White et al. 1990) and ALR0 (Collopy et al. 2001), and 75 ng DNA. The thermal cycler (Eppendorf, Hamburg, Germany) was programmed for one cycle of 5 min
Fungi Isolates of Verticillium fungicola were collected from infected fruit-bodies of A. bisporus. The code, date of collection,
DNA extraction Genomic DNA was extracted from freeze-dried mycelia with the Nucleon PhytoPure extraction kit RPN 8510 (Amersham International, Little Chalfort) according to the manufacturer’s instructions.
PCR-RFLP of ITS1–5.8 S–ITS2 regions
Table 1 – Isolate variety, code, date of collection, geographic origin and source of the isolates of Verticillium fungicola used in this study Variety fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola aleophilum aleophilum aleophilum aleophilum
Code
Date
Geographic origin
Source
VCF VCTC VF VK VM R1 VSL S2/VX01 Vff ADAS VV1 V49 V86 440.34 648.80 V9301 V9503 V970716 V9909 VX02 V01 V35 VTAW
1996 1997 1987 1994 1993 1974 2000 1975 Ni 1993 2000 2000 1934 1980 1993 1995 1997 1999 1981 1999 1999 Ni
Chancelade, France St Paterne, France St Paterne, France Nancy, France Me´rignas, France Grashoek, NL Campagne,France NL Adas, UK Villegouge, France Saumur, France Chatellerault, France UK Limburg, NL NL Noordhoek, NL NL NL Germany Avondale, PA, USA Landenberg, PA, USA Taiwan
INRA-Bx INRA-Bx INRA-Bx INRA-Bx INRA-Bx PPO MRU INRA-Bx PPO MRU PPO MRU INRA-Bx INRA-Bx INRA-Bx CBS440.34 CBS648.80 PPO MRU PPO MRU PPO MRU PPO MRU PPO MRU Courtesy Dr D.J. Royse Courtesy Dr D.J. Royse Courtesy Dr J. Peng
INRA-Bx, INRA-UPR 1264 collection; PPO MRU, PPO Mushroom Research Unit collection; CBS, Centraalbureau voor Schimmelcultures. Ni, no information.
Verticillium fungicola diversity
at 95 C, 35 cycles of 1 min at 94 C, 1 min at 55 C, 1 min at 72 C, with a final extension period of 5 min at 72 C. Aliquots of the amplified products were digested for 90 min with BamHI and NdeII restriction endonucleases (Q-BIOgen, Illkirch, France). Restriction products separated on 2 % agarose gel were visualised using a Kodak Digital Science (Courbevoie, France) 1D analyser.
RAPD analyses Amplification was performed in a 25 ml reaction mixture containing 0.1 mM dNTPs, 1 DyNAzyme buffer (Finnzymes Espoo, Finland), 0.8 U DyNAzyme II polymerase, 0.5 mM decamer primer, 5 ng DNA. The Crocodile III Thermal Cycler (Appligene, Illkirch, France) was programmed for one cycle of 6 min at 94 C, 35 cycles of 1 min at 93 C, 2 min at 36 C, 2 min at 72 C, with a final extension period of 6 min at 72 C. Primers OPA02, OPA03, OPA09, OPA11, OPA12, OPA13, OPA17, OPA18, OPA20, OPB06, OPB10, OPD01, OPD04, OPD15, OPD18, OPD20, OPH19, OPH20, OPZ04, OPZ07, OPZ10, OPZ20 (Operon Technologies, CA, USA); UBC28, and UBC30 (University of British Columbia) were screened. Amplified products were separated on ethidium bromide agarose gels (1.5 %, 2.5 V cm1, 4 h), the 1 kb DNA ladder (Life Technologies, Cergy-Pontoise, France) was used as size marker. All RAPD reactions were performed at least twice with at least two batches of genomic DNA and water as control. Numeric images of agarose gels were recorded and the presence or absence of RAPD products was scored with the Kodak Digital Science 1D analyser. Similarity indexes (F) for all pairwise combinations of RAPD amplification patterns were calculated using the following estimation of DNA fragment size (Nei & Li 1979): ÿ F ¼ 2nxy = nx þ ny where nx and ny are the number of fragments in isolates X and Y, respectively, and nxy is the number of fragments shared by the two compared isolates. The matrix of dissimilarity index (1 F) was then subjected to cluster analysis using the NJ method provided by the Phylip (Phylip Software, Seattle, Washington, USA) package 3.5c to draw an unrooted tree.
AFLP analyses Isolates VCTC, VF, VV1, V9503, R1, 440.34 and 648.80 were submitted to AFLP analyses as described by Vos et al. (1995). In short, DNA was digested using EcoRI and MseI restriction enzymes. Restriction fragments were ligated to EcoRI and MseI adapters. Pre-amplifications were performed with EcoRI þ0 and MseI þ0. Amplifications were performed with Eco þ AT and Mse þAA, AC, AG, CA, CG, CT, GA, GC, GG, and GT (and partially TT). Amplification products were separated using capillary electrophoresis on an ABI 3700 equipment (Perkin Elmer, Boston, MA). Banding patterns were analysed using Genographer (Montana State University, Bozenam, Montana, USA) (Benham et al. 1999) and converted to the presence or absence of bands. A distance matrix was made using the program Restdist (Phylip Software) and converted to a tree using NJ method from the Phylip package 3.6.
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Mycelial growth rate Inoculum plugs (5 mm diam) were removed from the edge of 10-d-old cultures and placed at the centre of Petri dishes filled with MEA medium (10 g l1 malt extract and 15 g l1 agar). A batch of cultures was allowed to grow at 23 C, another batch at 30 C. Mycelial growth of each colony was recorded on two perpendicular diameters and the mycelial growth rate between d 3 and d 12 (period of linear growth) was calculated for each colony. Two experiments were performed, each with five Petri dishes per isolate and temperature of incubation.
In vitro antibiosis Putative variability in germination and colony extension of Verticillium fungicola in response to diffusible or volatile compounds produced by Agaricus bisporus was investigated using double layer cultures. The effect of V. fungicola on the development of the A. bisporus colony was also assessed with this method. Agaricus bisporus Amycel 2100 was cultivated on MEA medium (9 cm-diam Petri dishes) until the colony reached 4 cm in diameter. Then 0.5 ml PN solution (1.2 g l1 bactopeptone Difco (Detroit, USA), 6 g l1 Natrium pyrophosphate and 15 g l1 agar) containing a total amount of 106 spores of V. fungicola was mixed with 6.5 ml water agar (1.5 % agar) maintained at 50 C and the obtained suspension was poured over a growing colony of A. bisporus. The cultures were incubated at 23 C for 9 d before the surface of the culture medium covered with V. fungicola colonies and the surface of the colony of A. bisporus were recorded. Five replicates were prepared, each with A. bisporus from a different preculture and V. fungicola from a different suspension of conidiae.
Susceptibility to chlorothalonil The commercial fungicide Banko (Calliope, Pau) was added to the MEA medium just before it was poured into Petri dishes at quantities corresponding to 0, 20, 200, 400, 800, and 4000 ppm chlorothalonil. Cultures were grown at 23 C for 19 d and mycelial growth of each colony was recorded on two perpendicular diameters. Growth in percent of control (unamended medium) was calculated, and isolates were assigned to four classes, sensitive (0–15 % of the control), slightly resistant (>15–40 %), moderately resistant (>40–65 %) and highly resistant (>65 %) (Bonnen & Hopkins 1997). Two different experiments were performed, each with five replicates per isolate and fungicide concentration.
Virulence assays For each Verticillium fungicola isolate, eight freshly harvested sporophores of Agaricus bisporus 2100 (Amycel, Vendoˆme, France) were placed into a plastic box used as a moist chamber. Twenty microlitres of a conidial suspension of V. fungicola at 106 conidia ml1 were placed at the cap surface. The diameter of the necrosis was recorded after 5 d of incubation at 20 C. The experiment was repeated twice.
434
Pathogenicity in cropping experiments Agaricus bisporus 2100 was grown in 0.9 m2 trays filled with commercial mushroom compost (Renaud S.A., Pons, France) spawned at 0.8 %. The incubation was for 13 d in a climatic room set at 24 C, with 92 % relative humidity before casing. Nine days after casing the room temperature was decreased to 16 C. Eleven days after casing a conidial suspension of Verticillium fungicola var. fungicola was sprayed onto the surface of the casing layer at a rate of 106 conidia m2. Each V. fungicola isolate (VCF, VCTC, VF, VK, V9503, and 440.34) was supplied to six trays. To assess contamination caused by spores originating from bubbles grown on contaminated trays, six uncontaminated trays were used as bait cultures. The experiment was performed twice with two different batches of compost. Healthy and diseased mushrooms were harvested for four weeks and weighed separately. Data reported were diseased mushrooms in percent of the total crop (weight) but statistical analyses were performed on transformed data (arcsinus of the square root of the percentages).
M. L. Largeteau et al.
the var. fungicola isolates into a distinct cluster, remote from the var. aleophilum isolates. All primers distinguished between VX02, VTAW, and the American isolates, shown to be remote on the dendrogram, but V01 and V35 grouped together. Fifteen of the 18 European var. fungicola isolates did not show any RAPD polymorphism and can be considered clonal. VCF, VV1, and VCTC showed polymorphism with one (OPH20), two (OPA12 and OPH20) and nine primers (OPA09, OPA11, OPA12, OPA13, OPA17, OPA18, OPD04, OPH19, and UBC30) out of 24, respectively. Variability was characterised by the presence or absence of a single band. (Fig 1). Analysis of the AFLP banding pattern yielded 198 bands of which 71 were informative. All seven strains screened could be identified from each other but genetic distances were very small. Four ill-defined groups (VCTC; 648.80; 440.34 þ V9503 þ R1; VV1 þ VF) were observed on the dendrogram (Fig 2).
Physiological variation Statistical analyses Data were analysed by the general linear model provided by the SAS system (SAS Institute Inc., Cary, NC) and SYSTAT 10 (SPSS Chicago IL). Variance analyses (ANOVA) were performed and followed, when necessary, by the Student– Newmans–Keuls test. The Pearson correlation was used to determine correlations between the parameters. A principal component analysis was performed on data recorded for mycelial growth at 23 C, Verticillium fungicola surface in antibiosis test, necrosis in virulence assays, fungicide resistance and pathogenicity in cultures. Added data concerning VCTC, VM, and VV1 evaluated for pathogenicity in other crops (Juarez del Carmen et al. 2002) were placed on the principal components identified in the present work. They were shown with different marks on the PCA.
Significant physiological dissimilarities (mycelial growth, antibiosis effect and fungicide resistance) were observed among all isolates, within the group of var. aleophilum isolates, the group of var. fungicola, and between French and Dutch isolates. ANOVA with contrast revealed significant differences between var. aleophilum and var. fungicola isolates for all the traits studied, whereas the French and Dutch isolates only differed significantly for development in presence of Agaricus bisporus (Tables 2 and 3). The Student–Newman–Keuls’s test was used to classify the isolates for the various traits. Isolate 440.34, collected in 1934 in the UK, did not differ from seven other European isolates of var. fungicola collected over years 1974 to 2000 for mycelial growth at 23 C. Only the var. aleophilum isolates were able to develop at 30 C, with significant differences in mycelial growth (Table 4).
Results Vfvf (15 isolates)
DNA polymorphisms
VCF V01
Amplification of the ITS region with ITS1 and ALR0 primers yielded a single 560 bp PCR product for all the Verticillium fungicola isolates. The amplified products obtained with the four var. aleophilum isolates (V01, V35, VX02 and VTAW) were not digested by BamHI whereas a single restriction site was present in the amplicons of the other isolates. All isolates showed a single restriction site for NdeII, but fragment lengths discriminated between the four var. aleophilum isolates (315 and 245 bp) and the 14 other isolates, including four already identified V. fungicola var. fungicola (350 and 210 bp). The latter were all assigned to the variety fungicola based on this result, their inability to grow at 30 C, and the RAPD analyses reported herein. The DNA polymorphism detected by RAPD patterns was consistent in replicate experiments, each performed with a different batch of DNA. Cluster analysis of pairwise dissimilarity indexes between isolates using the NJ method placed
VV1
V35
VX02
VCTC
VTaw
0.1
Vfvf VCF VV1 VCTC
Fig 1 – Dendrogram showing the relationship among the 22 isolates of Verticillium fungicola in the NJ analysis based on dissimilarity indexes calculated from the RAPD patterns. Vfvf, V. fungicola var. fungicola.
Verticillium fungicola diversity
435
648.80 VF VCTC
440.34
VV1 V9503 R1
0.001 Fig 2 – Dendrogram showing the relationship among isolates of Verticillium fungicola in the NJ analysis based on the distance matrix calculated from the AFLP patterns.
20 ppm, growths were 76–45 % of control after 5 d of contact and 84–53 % after 19 d (data not shown). A concentration of 400 ppm (¼150 mg cm2, comparable with doses used at mushroom farms) discriminated between isolates. Growths ranged from 34.3–66.8 % of the controls. This concentration was used for the classification of the strains. V49, V9909 and 440.34, each from a different country, were the less resistant (Table 4). The distribution of the isolates in classes of resistance showed that even at high concentrations the mycelial growth was not totally inhibited for any strain, most were moderately resistant. Comparison of distributions on d 5 and d 19 revealed that some isolates adapted after a few days of contact with the fungicide (Table 5). No correlation could be drawn between RAPD grouping and fungicide resistance. Significant differences were observed among isolates collected in 1993 and among those collected in 2000 but the two groups did not differ significantly. No correlation could be drawn between the various physiological traits studied except for antibiosis effects for which the surfaces covered by V. fungicola and A. bisporus were significantly correlated (Table 6).
Diversity in virulence and pathogenicity The antibiosis experiments distinguished between both varieties of Verticillium fungicola for germination ability on a growing colony of A. bisporus (Table 4). Conidiae of the four isolates of var. aleophilum germinated on the whole surface of the agar, including over the colony of A. bisporus, whereas conidiae of var. fungicola germinated around the mushroom colony but only on very small areas above. Little variation occurred within this variety. Colonies of V. fungicola covered the surface of the culture medium on controls without A. bisporus. After the supply of conidiae the A. bisporus colony continued to grow, and significant variations, depending on the isolate of V. fungicola supplied, were observed. Growth of A. bisporus was limited under the four isolates of var. aleophilum, as intensive growth was detected under developing colonies of VV1, V86 and VSL (Table 4). A low dose of chlorothalonil was enough to affect the mycelial development of the pathogen. In the presence of
All Verticillium fungicola isolates screened in the virulence assays induced necrosed brown tissues around and under the inoculation site and profuse sporulation. Inoculation of a suspension of conidiae of V. lecanii caused a level, yellow– beige spot, restricted to the surface of the pilei surface and no symptoms were observed after inoculation with a Verticillium sp. isolate distinct from V. fungicola by ITS amplification (data not shown). The French and Dutch isolates differed significantly for the diameter of the necrosis (Table 2). The correlation coefficient of 0.30 (df ¼ 17) between the diameter of the necrosis and the year of collection was not significant (P ¼ 0.05). Isolates chosen for evaluation of pathogenicity consisted of the ex-type strain 440.34, isolates VCTC and VCF polymorphic with RAPD markers, and three isolates (VF, VK, and V9503) from the homogeneous RAPD group. The percentages of diseased mushrooms in the two experiments were significantly correlated (r ¼ 0.91, P < 0.02) but higher percentages were
Table 2 – Analysis of variance for in vitro mycelial growth, fungicide resistance and necrosis induction in virulence assays measured for the 22 Verticillium fungicola isolates Source
D.F.
d
All isolates Intra var. aleophilum Intra var. fungicola Intra French isolates Intra Dutch isolates French vs Dutch isolates
21 3 17 8 6 1
Mycelial growth rate at 23 Ca
Fungicide resistanceb
Necrosis diameterc
Mean square
F value
Mean square
F value
Mean square
F value
5.1709 11.6463 3.6912 4.2733 1.8997 0.1906
43.23** 120.53** 31.12** 30.60** 17.50** 1.67ns
296.00 144.40 326.90 209.50 367.76 33.98
31.82** 45.32** 30.04** 15.66** 83.19** 3.65ns
0.2860 0.1391 0.3228 0.3383 0.0889 1.4821
16.62** 4.12* 23.95** 16.87** 18.50** 86.14**
*Significant at P < 0.05; **significant at P <0.01; ns, not significant at P ¼ 0.05. a Mycelial growth from d 3 to d 12. b Growth on medium amended with 400 ppm of chlorothalonil. c Diameter of the necrosis produced by the deposition of a conidial suspension on sporophore caps. d Isolates are listed in Table 1.
436
M. L. Largeteau et al.
Table 3 – Analysis of variance for antibiosis effects of Agaricus bisporus on the 22 isolates of Verticillium fungicola Source
V. fungicolaa
D.F.
All isolates Intra var. aleophilum Intra var. fungicola Intra French isolates Intra Dutch isolates French vs Dutch isolates
21 3 17 8 6 1
A. bisporusb
Mean square
F value
Mean square
F value
282.43 8.49 51.47 69.00 7.07 121.69
44.29** 0.58 ns 10.35** 8.81** 2.48 ns 19.08**
318.54 1.18 254.90 487.10 61.71 26.08
28.44** 0.70 ns 19.19** 30.65** 4.14** 2.33 ns
**Significant at P < 0.01; ns, not significant at P ¼ 0.05. a Surface covered by colonies of V. fungicola. b Surface of the A. bisporus colony recorded on the double layer cultures on d 9 after the supply of the conidial suspension.
Clustering
obtained in the second experiment. Contaminated trays, including that with the less aggressive isolate VCF, gave significantly higher percentages of diseased mushrooms compared with bait trays. The four French isolates were significantly different (Table 7). Data concerning VCTC, VM, and VV1 assessed for pathogenicity in other crops (Juarez del Carmen et al., 2002) were included to look for putative correlations. There was no correlation between the percentages of diseased mushrooms and the year of collection (P ¼ 0.05). The mycelial growth at 23 C was the only physiological trait that correlated significantly with pathogenicity (Table 8).
A principal component analysis was performed on data recorded for mycelial growth at 23 C, Verticillium fungicola surface in antibiosis test, fungicide resistance, necrosis in virulence assays and percentages of diseased mushrooms. The distribution of the isolates on components 1 (39.3 % of total variance explained) and 2 (28.4 %), and on components 1 and 3 (19.2 % explained) is shown in Fig 3. The major contributions to component 1 loading were the percentage of diseased mushrooms and the surface covered with V. fungicola on
Table 4 – Classification of the isolates of Verticillium fungicola for physiological traits and necrosis induction in virulence assays Variety/ geographic origin
Code
Mycelial growth rate (cm 9 d1)a
Antibiosisb
Fungicide resistancea (% growth)
Necrosis diametera (mm)
23 C
30 C
V. fungicola (cm2)
A. bisporus (cm2)
1.6 a 0.6 c 1.0 b 1.5 a
47.4 a 48.5 a 48.2 a 49.0 a
18.9 e 20.0 e 19.8 e 19.4 e
54.6 cde 50.2 e 54.3 cde 40.5 f
12.4 b 12.9 b 12.0 bc 9.9 de
aleophilum
VTaw VX02 V01 V35
4.6 ac 4.5 a 2.9 e 3.8 c
fungicola/Fr
VCF VCTC VF VK VM VSL VV1 V49 V86
3.2 d 4.4 a 3.2 d 3.1 de 3.0 de 3.2 d 4.2 b 3.7 c 3.6 c
0 0 0 0 0 0 0 0 0
d d d d d d d d d
23.2 g 29.7 cdef 33.9 bc 26.6 efg 32.1 bcd 25.3 fg 29.1 cdef 32.8 bcd 26.1 efg
29.8 d 19.3 e 17.6 e 38.3 bc 24.1 e 40.7 ab 44.7 a 34.2 cd 40.1 ab
53.0 de 62.1 ab 52.7 de 55.2 cde 63.3 ab 60.3 abc 52.7 de 36.7 fg 54.9 cde
15.2 a 12.6 b 15.2 a 11.4 bcd 9.6 de 9.7 de 13.0 b 13.0 b 11.4 bcd
fungicola/NL
R1 S2/VX01 648.80 V9301 V9503 V970716 V9909
3.6 c 3.6 c 3.6 c 3.8 c 3.3 d 3.6 c 2.8 e
0 0 0 0 0 0 0
d d d d d d d
32.9 bcd 32.6 bcd 30.7 bcde 30.8 bcde 30.9 bcde 29.4 cdef 31.3 bcde
31.2 d 29.7 d 31.8 cd 32.2 cd 28.7 e 34.8 cd 33.1 cd
55.9 cde 58.1 bcd 49.4 e 62.9 ab 66.9 a 62.8 ab 36.3 fg
10.2 cde 9.3 de 10.2 cde 12.2 b 10.2 cde 10.2 cde 8.9 e
fungicola/UK
Vff ADAS 440.34
2.5 f 3.6 c
0d 0d
28.2 def 35.3 b
33.4 cd 29.3 d
62.7 ab 34.3 g
9.0 e 9.2 e
a See legend on Table 2. b See legend on Table 3. c Values within a column followed by the same letter do not differ significantly by the Student–Newman–Keuls test (P ¼ 0.05).
Verticillium fungicola diversity
437
Table 5 – Effect of chlorothalonil on the mycelial growth of the 22 isolates of Verticillium fungicola Date
Classes of resistancea
Numbers of isolates per class for each concentration of fungicide 20 ppm
200 ppm
400 ppm
800 ppm
4000 ppm
d5
Sensitive Slightly resistant Moderately resistant Highly resistant
0 1 11 13
0 3 19 3
0 3 21 1
0 4 21 0
0 10 15 0
d 19
Sensitive Slightly resistant Moderately resistant Highly resistant
0 0 11 11
0 1 14 7
0 2 18 2
0 2 18 2
0 4 16 2
a Sensitive, 0–15 % of the control; slightly resistant, >15–40 %; moderately resistant, >40–65 %; highly resistant, >65 %.
double-layer cultures. Components 1 and 2 grouped the isolates but VCF (which was less aggressive to mushroom crops) and 440.34 (which was better developed in the antibiosis test) differed by component 1. The distribution of the isolates on components 1 and 3 was not informative.
Discussion Genetic variability within Verticillium fungicola was recently investigated by Collopy et al. (2001) and Juarez del Carmen et al. (2002) but with only few European isolates. The present investigation including 18 European isolates of V. fungicola var. fungicola and four isolates of V. fungicola var. aleophilum from various origins strengthened the previous findings and provided additional information about the level of genetic variability. We observed a high level of DNA polymorphism among the var. aleophilum isolates from different geographic origins. Beside differences in cultivation practises, the mushroom strain must be taken into consideration as a factor influencing the
variability of the pathogen. The high level of genetic homogeneity of the commercial mushroom was proposed by Bonnen and Hopkins (1997) as being one of the factors responsible for the level of homogeneity in American populations of V. fungicola. We have no information concerning the mushroom strain from which VTAW was isolated, but VX02 was from Les Miz 60, an old variety of A. bisporus, while V01 and V35 were from recent crops. But as Bonnen and Hopkins (1997) reported, this factor is not alone in explaining the lack of variability within the V. fungicola populations because the ex-type strain 440.34, collected in 1934, did not differ from the group of 15 isolates showing no variability. Similar results were reported concerning old North American isolates. Bonnen and Hopkins (1997) placed in the same RAPD group an isolate from 1950 with 55 isolates collected during years 1970–1995. Collopy et al. (2001), who performed RAPD analysis on V. fungicola var. aleophilum isolates, observed identical patterns for 40 Pennsylvania isolates collected during 1999 and 13 North American isolates collected during the last 50 y. The uniformity of the casing material between mushroom farms through the adoption of pasteurized peat moss was proposed to explain the lack of diversity in V. fungicola var. aleophilum responsible for recent dry bubble outbreaks in North America (Collopy et al. 2001). Obornik et al. (2000) proposed that RAPD-based phylogeny and genetic variability of
Table 6 – Correlation between the various traits measured on the 22 isolates of Verticillium fungicola Necrosis Fungicide diameter resistance
Mycelial growth at 23 C Necrosis diameter Fungicide resistance (400 ppm)
0.45098a 0.0402b
Antibiosis V. fungicola A. bisporus surface surface 0.35316 0.1163
0.07633 0.7423
0.23071 0.3143
Table 7 – Classification of six isolates of Verticillium fungicola var. fungicola according to their pathogenicity in cultures Verticillum isolate
Diseased mushrooms (%)a Experiment 1
0.01006 0.9655
0.21473 0.3499
0.27527 0.2272
0.32434 0.1515
0.04030 0.8623
V. fungicola surface a Pearson correlation coefficients, N ¼ 21. b Prob > jrj under H0: r ¼ 0.
0.74115 0.0001
VCTC V9503 440.34 VF VK VCF Control
b
30.4 a 36.2 a 30.0 a 19.4 b 13.9 c 8.2 d 3.8 e
Experiment 2 46.7 a 38.4 b 46.4 a 29.8 c 19.9 d 12.4 e 4.7 f
a Diseased mushrooms in percent of the total crop (weight). b Values within a column following by the same letter do not differ significantly by the Student–Newman–Keuls test (P ¼ 0.05).
438
M. L. Largeteau et al.
Table 8 – Correlation between the pathogenicity in cultures and the various physiological traits measured on the eight isolates of Verticillium fungicola Mycelial growth (23 C) Aggressiveness
a
0.71192 0.0476b
Necrosis diameter
Fungicide resistance (400 ppm)
0.1387 0.5691
0.1633 0.6993
Antibiosis V. fungicola surface
A. bisporus surface
0.4677 0.2425
0.06423 0.8799
a Pearson correlation coefficients, N ¼ 8. b Prob > jrj under H0: r ¼ 0.
character studied was taken into consideration separately, significant physiological dissimilarities and pathogenicity variability were observed among the European var. fungicola isolates. Diversity in tolerance to hydrogen peroxide was previously observed among a group of six French var. fungicola isolates (Juarez del Carmen et al. 2002). The var. fungicola population screened appeared less homogeneous than the var. aleophilum populations from North America studied by Bonnen and Hopkins (1997). European isolates gathered in 1993 and 2000 were too few to provide confirmation, but based on the whole var. fungicola sample, it did not seem that the physiological variability is progressing. In our work on antibiosis, conidiae of V. fungicola germinated around the mushroom colony, which is in accordance with the observation of Calonje et al. (2000) in dual-cultures on Raper medium. These authors also reported the germination of conidiae inoculated on a colony of A. bisporus transferred to an empty Petri dish. The absence of nutrients could explain the lack of defense of A. bisporus. In our study, the mushroom was fed, and consequently maintained an active metabolism which allowed it to produce defensive compounds preventing conidiae of var. fungicola to germinate on the colony. Although, the restriction of the inhibition to the surface of the mushroom colony showed that inhibitors do not diffuse well and/or were rapidly inactivated and were
3 2
Component 2
Paecilomyces sp. and Lecanicillium lecanii (syn.) Verticillium lecanii reflect the life strategy of the entomopathogenic fungi, and that the mobility of spores is a major influence on the evolution of these organisms. The common origin of some cultivation materials, especially peat moss, in the three European countries from which the isolates originated could be a factor of dispersion of V. fungicola spores favourable to the genetic homogeneity of the pathogen. In the present study, Dutch isolates chosen at random except for date in the PPO-MRU collection, were from restricted geographic sites and from mushroom farms cultivating with standardised practices, and there was no genetic variability observed. Although all V. fungicola var. fungicola isolates we screened fall into a single cluster, some genetic variability exists inside the present sample of European isolates. The AFLP analysis performed on randomly selected var. fungicola isolates showed some variability but the groups were ill-defined, which confirmed the RAPD results. Within the var. fungicola, VCTC, VV1, VK, and VF were identical regarding the ITS sequences (Collopy et al. 2001), but they differed with RAPD and AFLP markers in our investigations. These results confirmed that RAPD reveals a higher level of polymorphism, as shown in other pathogen populations such as Fusarium verticillioides (Voigt et al. 1995) and Colletotrichum lindemuthianum (Sicard et al. 1997). The three V. fungicola var. fungicola isolates found polymorphous in the present study were from various French locations, corresponding to variations in compost, casing and cultural practices. This could explain why genetic variability was only detected within the group of French isolates, but more samples are necessary before a final conclusion can be drawn. Within the var. fungicola isolates we identified a homogeneous population comprising 13 isolates collected in France and the Netherlands over a period of 27 y (1974–2000) and one isolate collected in 1934 in the UK. VV1 and VCTC isolated at French mushroom farms in 1993 and 1997, respectively, were distinguished from this population, whereas the Dutch isolates collected during the same years were not. Such an absence of correlation between geographic location and RAPD pattern was also observed for Verticillium dahliae (Messner et al. 1996; Lachquer & Sedra 2002) and it was generally the same concerning L. lecanii (Mor et al. 1996). A clonal population was identified for the var. aleophilum in North America (Collopy et al. 2001). A major result emerging from our investigation is that similarly, the var. fungicola in Europe is also genetically homogeneous. Despite of the high genetic homogeneity observed with different tools, significant differences for the expression of physiological and pathogenicity traits were observed. When each
-4
VCTC
VCTC VV1
1
VCF 0 -2
0
VF -1
VK VM
2
4
V9503
-2
440.34
-3
Component 1 Fig 3 – Distribution of isolates of V. fungicola var. fungicola after principal component analysis. (C) Isolates used to determine the principal components; (:) additional isolates placed on the principal component plan.
Verticillium fungicola diversity
moderately efficient as they allow some germination of var. fungicola and do not prevent that of var. aleophilum. In addition to the homogeneity of the casing material used to grow the mushrooms the use of fungicides was proposed as another cause of the genetic homogeneity observed among the V. fungicola isolates due to the selection of fungicide resistant strains. Various opinions emerge from the literature on the efficiency of chlorothalonil to reduce dry bubble disease on mushroom crops. van Zaayen (1977), Gandy and Spencer (1981), and Maszkiewicz (2001) considered the fungicide efficient to control dry bubble disease on mushroom crops. Fletcher and Hims (1981) and Coutinho et al. (2000) observed that chlorothalonil failed to control the disease. We observed a high level of fungicide resistance in vitro for all but three isolates. The susceptibility of 440.34 and V49 was not surprising because chlorothalonil was not used at the time the former was collected and the mushroom farm where the latter was collected used organic farming practises. The susceptible V9909 was isolated when chlorothalonil was commercially unavailable in the Netherlands. But some moderately to highly resistant isolates were collected before the fungicide was authorized (1996 in France, 1980 in the Netherlands) for mushroom cropping. A relatively high level of tolerance to chlorothalonil even before its introduction was previously reported by Bonnen and Hopkins (1997) for isolates of V. fungicola mainly in North America. The authors related that the level of tolerance varied little over the period of collect. Gea et al. (1997) observed that the majority of the 20 isolates of V. fungicola var. fungicola collected from various fruit-bodies of A. bisporus from prochloraz-treated crops were very resistant to chlorothalonil in vitro. Considering the high level of resistance of the isolates we screened, we can question the impact of the common use of prochloraz–Mn complex at mushroom farms before the use of chlorothalonil on the resistance of V. fungicola to the latter fungicide. In the work of Bonnen and Hopkins (1997) little correlation could be drawn between RAPD grouping and fungicide response. Similarly, we did not find any correlation between RAPD polymorphism and fungicide resistance. This absence of correlation is not restricted to the fungicola species. No or little correlation was observed between RAPD polymorphism and virulence for isolates of L. lecanii (Mor et al. 1996) and V. dahliae (Ramsay et al. 1996). The symptoms observed in the present virulence assays corresponded to those observed by Bonnen and Hopkins (1997) on most isolates collected between 1971 and 1995 and to all isolates collected in 1993–1995 in North America. The two Verticillium isolates distinct from the species V. fungicola failed to induce necrosis which conferred the virulence assays specificity. Mushroom strains showing mainly spotted mushrooms could be considered developing resistance compared with strains producing mainly bubbles. Inoculation directly on the pilei gives information on the virulence rather than on the level of aggressiveness of V. fungicola isolates and was more valuable in classifying A. bisporus strains than Verticillium isolates. These observations could explain the absence of correlation between the percentages of diseased mushrooms in culture and the necrosis diameter in virulence assays observed here. A significant correlation was found between aggressiveness in cultures and mycelial
439
growth rate at 23 C, which suggests that the high temperature maintained for a week after casing, which is favourable to colonisation of the casing layer by V. fungicola, may have considerable incidence on infection. Although no significant correlation was observed between aggressiveness and in vitro antibiosis, the recognition mechanisms between V. fungicola and A. bisporus cell walls described by Bernardo et al. (2004) are a putative source of variation in aggressiveness during cropping. Genetic and physiological investigations revealed a higher level of diversity within the group of var. fungicola isolates we studied than within North American populations of var. aleophilum isolates (Bonnen & Hopkins 1997; Collopy et al. 2001) but neither DNA polymorphism nor dissimilarities in physiological traits but mycelial growth rate at 23 C could explain the variability in aggressiveness among the European isolates. The comparison of French and Dutch isolates, the latter cultivated under more standardized practises, emphasised the effect of cultivation standardisation on the genetic evolution of the variety fungicola toward a clonal population.
Acknowledgements We thank Thierry Gibard and Patrick Hendrickx for technical assistance, and Patrick Castant and Christiane Coldefy for mushroom cultivation. M.L.L. is grateful to Olivier Le Gall for his help in molecular analyses. J.P.P.B. gratefully acknowledges financial support of the Dutch Ministry for Agriculture.
references
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Desrumeaux B, Sedeyn P, 2001. Resistance of dry bubbles (Verticillium fungicola var. fungicola) to prochloraz – 2nd series. Champignonberichten 191: 8–11. Fletcher JT, Hims MJ, 1981. Dry bubble disease control. Mushroom Journal 100: 138. Gandy DG, Spencer DM, 1981. Fungicide evaluation for control of dry bubble, caused by Verticillium fungicola, on commercial mushroom strains. Scientia Horticulturae 14: 107–115. Gea FJ, Tello JC, Honrubia M, 1997. In vitro sensitivity of Verticillium fungicola to selected fungicides. Mycopathologia 136: 133–137. Gea FJ, Tello JC, Navarro MJ, 2003. Occurrence of Verticillium fungicola var. fungicola on Agaricus bitorquis mushroom crops in Spain. Phytopathology 151: 98–100. Juarez del Carmen S, Largeteau-Mamoun ML, Rousseau T, Regnault-Roger C, Savoie J-M, 2002. Genetic and physiological variation in isolates of Verticillium fungicola causing dry bubble disease of the cultivated button mushroom, Agaricus bisporus. Mycological Research 106: 1163–1170. Lachquer K, Sedra MH, 2002. Characterization of Verticillium dahliae Kleb. isolates from Olea europea using RAPD markers. Phytopathologia Mediterranea 41: 170–178. Largeteau ML, Mata G, Savoie J-M, 2004. Verticillium fungicola affects Agaricus bisporus cultivation in Mexico. FEMS Microbiology Letters 236: 191–196. Maszkiewicz J, 2001. Efficacy of chlorothalonil fungicide (Gwarant 500 SC) against wet bubble of champignon (Mycogone perniciosa), dry bubble of champignon (Verticillium fungicola) and dactylium (Dactylium dendroides). Vegetable Crops Research Bulletin 55: 131–135. Messner R, Schweigkofler W, Ibl M, Berg G, Prillinger H, 1996. Molecular characterization of the plant pathogen Verticillium dahliae Kleb. using RAPD-PCR and sequencing of the 18SrRNAgene. Journal of Phytopathology 144: 347–354.
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journal homepage: www.elsevier.com/locate/mycres
Molecular and physiological diversity among Verticillium fungicola var. fungicola Miche`le L. LARGETEAUa,*, Johan P. P. BAARSb, Catherine REGNAULT-ROGERc, Jean-Michel SAVOIEa a
Institut National de la Recherche Agronomique (INRA), BP81, F-33883 Villenave d’Ornon, France Applied Plant Research, Mushroom Research Unit, P.O. Box 6042, NL-5960 AA Horst, The Netherlands c UFR Sciences et Techniques, Univ. Pau et Pays de l’Adour, F-64012 Pau Universite´ cedex, France b
article info
abstract
Article history:
The genetic and physiological variability of Verticillium fungicola var. aleophilum responsible
Received 30 November 2004
for Agaricus bisporus dry bubble disease in North America is well documented but little is
Received in revised form
known about the var. fungicola affecting European crops. Variability was assessed within
26 October 2005
this variety and compared with that reported for the var. aleophilum. Eighteen isolates of
Accepted 30 November 2005
V. fungicola var. fungicola and four var. aleophilum isolates were analysed for DNA polymor-
Published online 17 April 2006
phism, mycelial growth, response to biochemicals produced by A. bisporus, fungicide
Corresponding Editor: Jan I. Lelley
resistance, and pathogenicity assessed by direct inoculation on sporophore or casing contamination. RAPD and AFLP markers delineated three French isolates from a homoge-
Keywords:
neous group containing the other var. fungicola isolates, but no correlation could be drawn
Agaricus bisporus
between DNA polymorphism and the various traits studied. The var. fungicola isolates were
Cultivated mushrooms
more susceptible than the var. aleophilum isolates to the antibiosis effect of A. bisporus. Only
Dry bubble disease
mycelial growth rate at 23 C could explain the variability in aggressiveness among
Fungicolous fungi
the European isolates. The putative effect of the post-incubation temperature on contam-
Mycoparasite
ination during mushroom cultivation was discussed. This work emphasized that, like the American var. aleophilum, the var. fungicola in Europe is genetically homogeneous, but physiological diversity exists, especially in France where it could be related to less standardized cultural practices. ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Verticillium fungicola is responsible for dry bubble disease of the cultivated mushroom Agaricus bisporus. Two varieties affect the mushroom crop, var. aleophilum and var. fungicola. The worldwide distribution of the pathogen incites questions regarding its variability. Bidochka et al. (1999) investigated the production of hydrolytic enzymes and the genetic variability among different species of Verticillium, including two representatives of V. fungicola. Despite physiological similarity,
these two isolates exhibited a 49 % divergence in their rDNA sequence of ITS1. Investigations into the molecular and physiological diversity reported in the literature mainly concern isolates of V. fungicola var. aleophilum collected in North America. Genetic variability within 66 isolates of V. fungicola, the majority originating from North America, was studied by Bonnen and Hopkins (1997). The authors did not find any correlation between RAPD grouping, colony morphology and virulence but they observed a high level of homogeneity among the isolates collected during 1993–1995 for fungicide
* Corresponding author. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2005.11.016
432
M. L. Largeteau et al.
response, virulence, colony morphology, geographic origin and RAPD grouping. More recently, Collopy et al. (2001) performed molecular phylogenetic analyses on 40 isolates of V. fungicola gathered at various Pennsylvania mushroom farms in 1999, and 28 isolates of Verticillium spp. collected during the last 50 years from various geographic locations. The authors reported the presence of a clonal population of V. fungicola var. aleophilum among the Pennsylvania isolates and postulated that the lack of diversity observed may be due to a change in the practises regarding the casing of mushroom crops. Recent reports on dry bubble disease in Europe (Desrumeaux & Sedeyn 2001; Gea et al. 2003) refered to the var. fungicola as responsible for the disease, consequently further investigation into the variability of European isolate is required. The four French isolates and the UK isolate included in the work of Collopy et al. (2001) showed an identical rDNA sequence to V. fungicola var. fungicola and did not differ in RAPD patterns. Investigations performed on six French isolates have shown some genetic and physiological variations (Juarez del Carmen et al. 2002). The objective of this study was to examine the genetic variability, physiological dissimilarities and pathogenic diversity within a group of V. fungicola mainly constituted of European isolates in order to compare with var. aleophilum isolates.
geographic origin and source of the 22 isolates screened in this study are listed in Table 1. Isolates VCTC, VF, VK and VV1 belong to the variety fungicola, V01 and V35 to the variety aleophilum based on rDNA sequences (Collopy et al. 2001). Sequencing of the ITS1, 5.8 S and ITS2 rDNA assigned VTAW to the variety aleophilum (Largeteau et al. 2004). CBS440.34, the ex-type strain for var. fungicola (formely V. malthousei) was obtained from the Centraalbureau voor Schimmelcultures (Baarn, the Netherlands). The European isolate VX02, collected in Germany in 1981, identified as var. aleophilum on basis of growth at 30 C, was obtained from a diseased fruit-body of A. bisporus variety Les Miz 60. Voucher cultures are maintained in the INRA (France) or PPO-MRV (NL) culture collection and are publically accessible. Isolates used in this study are listed in Table 1.
Materials and methods
Amplification was performed in a 25 ml reaction mixture containing 0.125 mM dNTPs, 1 DyNAzyme buffer (Finnzymes, Finland), 0.5 U DyNAzyme II polymerase (Finnzymes), 0.2 mM each of primer ITS1 (White et al. 1990) and ALR0 (Collopy et al. 2001), and 75 ng DNA. The thermal cycler (Eppendorf, Hamburg, Germany) was programmed for one cycle of 5 min
Fungi Isolates of Verticillium fungicola were collected from infected fruit-bodies of A. bisporus. The code, date of collection,
DNA extraction Genomic DNA was extracted from freeze-dried mycelia with the Nucleon PhytoPure extraction kit RPN 8510 (Amersham International, Little Chalfort) according to the manufacturer’s instructions.
PCR-RFLP of ITS1–5.8 S–ITS2 regions
Table 1 – Isolate variety, code, date of collection, geographic origin and source of the isolates of Verticillium fungicola used in this study Variety fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola fungicola aleophilum aleophilum aleophilum aleophilum
Code
Date
Geographic origin
Source
VCF VCTC VF VK VM R1 VSL S2/VX01 Vff ADAS VV1 V49 V86 440.34 648.80 V9301 V9503 V970716 V9909 VX02 V01 V35 VTAW
1996 1997 1987 1994 1993 1974 2000 1975 Ni 1993 2000 2000 1934 1980 1993 1995 1997 1999 1981 1999 1999 Ni
Chancelade, France St Paterne, France St Paterne, France Nancy, France Me´rignas, France Grashoek, NL Campagne,France NL Adas, UK Villegouge, France Saumur, France Chatellerault, France UK Limburg, NL NL Noordhoek, NL NL NL Germany Avondale, PA, USA Landenberg, PA, USA Taiwan
INRA-Bx INRA-Bx INRA-Bx INRA-Bx INRA-Bx PPO MRU INRA-Bx PPO MRU PPO MRU INRA-Bx INRA-Bx INRA-Bx CBS440.34 CBS648.80 PPO MRU PPO MRU PPO MRU PPO MRU PPO MRU Courtesy Dr D.J. Royse Courtesy Dr D.J. Royse Courtesy Dr J. Peng
INRA-Bx, INRA-UPR 1264 collection; PPO MRU, PPO Mushroom Research Unit collection; CBS, Centraalbureau voor Schimmelcultures. Ni, no information.
Verticillium fungicola diversity
at 95 C, 35 cycles of 1 min at 94 C, 1 min at 55 C, 1 min at 72 C, with a final extension period of 5 min at 72 C. Aliquots of the amplified products were digested for 90 min with BamHI and NdeII restriction endonucleases (Q-BIOgen, Illkirch, France). Restriction products separated on 2 % agarose gel were visualised using a Kodak Digital Science (Courbevoie, France) 1D analyser.
RAPD analyses Amplification was performed in a 25 ml reaction mixture containing 0.1 mM dNTPs, 1 DyNAzyme buffer (Finnzymes Espoo, Finland), 0.8 U DyNAzyme II polymerase, 0.5 mM decamer primer, 5 ng DNA. The Crocodile III Thermal Cycler (Appligene, Illkirch, France) was programmed for one cycle of 6 min at 94 C, 35 cycles of 1 min at 93 C, 2 min at 36 C, 2 min at 72 C, with a final extension period of 6 min at 72 C. Primers OPA02, OPA03, OPA09, OPA11, OPA12, OPA13, OPA17, OPA18, OPA20, OPB06, OPB10, OPD01, OPD04, OPD15, OPD18, OPD20, OPH19, OPH20, OPZ04, OPZ07, OPZ10, OPZ20 (Operon Technologies, CA, USA); UBC28, and UBC30 (University of British Columbia) were screened. Amplified products were separated on ethidium bromide agarose gels (1.5 %, 2.5 V cm1, 4 h), the 1 kb DNA ladder (Life Technologies, Cergy-Pontoise, France) was used as size marker. All RAPD reactions were performed at least twice with at least two batches of genomic DNA and water as control. Numeric images of agarose gels were recorded and the presence or absence of RAPD products was scored with the Kodak Digital Science 1D analyser. Similarity indexes (F) for all pairwise combinations of RAPD amplification patterns were calculated using the following estimation of DNA fragment size (Nei & Li 1979): ÿ F ¼ 2nxy = nx þ ny where nx and ny are the number of fragments in isolates X and Y, respectively, and nxy is the number of fragments shared by the two compared isolates. The matrix of dissimilarity index (1 F) was then subjected to cluster analysis using the NJ method provided by the Phylip (Phylip Software, Seattle, Washington, USA) package 3.5c to draw an unrooted tree.
AFLP analyses Isolates VCTC, VF, VV1, V9503, R1, 440.34 and 648.80 were submitted to AFLP analyses as described by Vos et al. (1995). In short, DNA was digested using EcoRI and MseI restriction enzymes. Restriction fragments were ligated to EcoRI and MseI adapters. Pre-amplifications were performed with EcoRI þ0 and MseI þ0. Amplifications were performed with Eco þ AT and Mse þAA, AC, AG, CA, CG, CT, GA, GC, GG, and GT (and partially TT). Amplification products were separated using capillary electrophoresis on an ABI 3700 equipment (Perkin Elmer, Boston, MA). Banding patterns were analysed using Genographer (Montana State University, Bozenam, Montana, USA) (Benham et al. 1999) and converted to the presence or absence of bands. A distance matrix was made using the program Restdist (Phylip Software) and converted to a tree using NJ method from the Phylip package 3.6.
433
Mycelial growth rate Inoculum plugs (5 mm diam) were removed from the edge of 10-d-old cultures and placed at the centre of Petri dishes filled with MEA medium (10 g l1 malt extract and 15 g l1 agar). A batch of cultures was allowed to grow at 23 C, another batch at 30 C. Mycelial growth of each colony was recorded on two perpendicular diameters and the mycelial growth rate between d 3 and d 12 (period of linear growth) was calculated for each colony. Two experiments were performed, each with five Petri dishes per isolate and temperature of incubation.
In vitro antibiosis Putative variability in germination and colony extension of Verticillium fungicola in response to diffusible or volatile compounds produced by Agaricus bisporus was investigated using double layer cultures. The effect of V. fungicola on the development of the A. bisporus colony was also assessed with this method. Agaricus bisporus Amycel 2100 was cultivated on MEA medium (9 cm-diam Petri dishes) until the colony reached 4 cm in diameter. Then 0.5 ml PN solution (1.2 g l1 bactopeptone Difco (Detroit, USA), 6 g l1 Natrium pyrophosphate and 15 g l1 agar) containing a total amount of 106 spores of V. fungicola was mixed with 6.5 ml water agar (1.5 % agar) maintained at 50 C and the obtained suspension was poured over a growing colony of A. bisporus. The cultures were incubated at 23 C for 9 d before the surface of the culture medium covered with V. fungicola colonies and the surface of the colony of A. bisporus were recorded. Five replicates were prepared, each with A. bisporus from a different preculture and V. fungicola from a different suspension of conidiae.
Susceptibility to chlorothalonil The commercial fungicide Banko (Calliope, Pau) was added to the MEA medium just before it was poured into Petri dishes at quantities corresponding to 0, 20, 200, 400, 800, and 4000 ppm chlorothalonil. Cultures were grown at 23 C for 19 d and mycelial growth of each colony was recorded on two perpendicular diameters. Growth in percent of control (unamended medium) was calculated, and isolates were assigned to four classes, sensitive (0–15 % of the control), slightly resistant (>15–40 %), moderately resistant (>40–65 %) and highly resistant (>65 %) (Bonnen & Hopkins 1997). Two different experiments were performed, each with five replicates per isolate and fungicide concentration.
Virulence assays For each Verticillium fungicola isolate, eight freshly harvested sporophores of Agaricus bisporus 2100 (Amycel, Vendoˆme, France) were placed into a plastic box used as a moist chamber. Twenty microlitres of a conidial suspension of V. fungicola at 106 conidia ml1 were placed at the cap surface. The diameter of the necrosis was recorded after 5 d of incubation at 20 C. The experiment was repeated twice.
434
Pathogenicity in cropping experiments Agaricus bisporus 2100 was grown in 0.9 m2 trays filled with commercial mushroom compost (Renaud S.A., Pons, France) spawned at 0.8 %. The incubation was for 13 d in a climatic room set at 24 C, with 92 % relative humidity before casing. Nine days after casing the room temperature was decreased to 16 C. Eleven days after casing a conidial suspension of Verticillium fungicola var. fungicola was sprayed onto the surface of the casing layer at a rate of 106 conidia m2. Each V. fungicola isolate (VCF, VCTC, VF, VK, V9503, and 440.34) was supplied to six trays. To assess contamination caused by spores originating from bubbles grown on contaminated trays, six uncontaminated trays were used as bait cultures. The experiment was performed twice with two different batches of compost. Healthy and diseased mushrooms were harvested for four weeks and weighed separately. Data reported were diseased mushrooms in percent of the total crop (weight) but statistical analyses were performed on transformed data (arcsinus of the square root of the percentages).
M. L. Largeteau et al.
the var. fungicola isolates into a distinct cluster, remote from the var. aleophilum isolates. All primers distinguished between VX02, VTAW, and the American isolates, shown to be remote on the dendrogram, but V01 and V35 grouped together. Fifteen of the 18 European var. fungicola isolates did not show any RAPD polymorphism and can be considered clonal. VCF, VV1, and VCTC showed polymorphism with one (OPH20), two (OPA12 and OPH20) and nine primers (OPA09, OPA11, OPA12, OPA13, OPA17, OPA18, OPD04, OPH19, and UBC30) out of 24, respectively. Variability was characterised by the presence or absence of a single band. (Fig 1). Analysis of the AFLP banding pattern yielded 198 bands of which 71 were informative. All seven strains screened could be identified from each other but genetic distances were very small. Four ill-defined groups (VCTC; 648.80; 440.34 þ V9503 þ R1; VV1 þ VF) were observed on the dendrogram (Fig 2).
Physiological variation Statistical analyses Data were analysed by the general linear model provided by the SAS system (SAS Institute Inc., Cary, NC) and SYSTAT 10 (SPSS Chicago IL). Variance analyses (ANOVA) were performed and followed, when necessary, by the Student– Newmans–Keuls test. The Pearson correlation was used to determine correlations between the parameters. A principal component analysis was performed on data recorded for mycelial growth at 23 C, Verticillium fungicola surface in antibiosis test, necrosis in virulence assays, fungicide resistance and pathogenicity in cultures. Added data concerning VCTC, VM, and VV1 evaluated for pathogenicity in other crops (Juarez del Carmen et al. 2002) were placed on the principal components identified in the present work. They were shown with different marks on the PCA.
Significant physiological dissimilarities (mycelial growth, antibiosis effect and fungicide resistance) were observed among all isolates, within the group of var. aleophilum isolates, the group of var. fungicola, and between French and Dutch isolates. ANOVA with contrast revealed significant differences between var. aleophilum and var. fungicola isolates for all the traits studied, whereas the French and Dutch isolates only differed significantly for development in presence of Agaricus bisporus (Tables 2 and 3). The Student–Newman–Keuls’s test was used to classify the isolates for the various traits. Isolate 440.34, collected in 1934 in the UK, did not differ from seven other European isolates of var. fungicola collected over years 1974 to 2000 for mycelial growth at 23 C. Only the var. aleophilum isolates were able to develop at 30 C, with significant differences in mycelial growth (Table 4).
Results Vfvf (15 isolates)
DNA polymorphisms
VCF V01
Amplification of the ITS region with ITS1 and ALR0 primers yielded a single 560 bp PCR product for all the Verticillium fungicola isolates. The amplified products obtained with the four var. aleophilum isolates (V01, V35, VX02 and VTAW) were not digested by BamHI whereas a single restriction site was present in the amplicons of the other isolates. All isolates showed a single restriction site for NdeII, but fragment lengths discriminated between the four var. aleophilum isolates (315 and 245 bp) and the 14 other isolates, including four already identified V. fungicola var. fungicola (350 and 210 bp). The latter were all assigned to the variety fungicola based on this result, their inability to grow at 30 C, and the RAPD analyses reported herein. The DNA polymorphism detected by RAPD patterns was consistent in replicate experiments, each performed with a different batch of DNA. Cluster analysis of pairwise dissimilarity indexes between isolates using the NJ method placed
VV1
V35
VX02
VCTC
VTaw
0.1
Vfvf VCF VV1 VCTC
Fig 1 – Dendrogram showing the relationship among the 22 isolates of Verticillium fungicola in the NJ analysis based on dissimilarity indexes calculated from the RAPD patterns. Vfvf, V. fungicola var. fungicola.
Verticillium fungicola diversity
435
648.80 VF VCTC
440.34
VV1 V9503 R1
0.001 Fig 2 – Dendrogram showing the relationship among isolates of Verticillium fungicola in the NJ analysis based on the distance matrix calculated from the AFLP patterns.
20 ppm, growths were 76–45 % of control after 5 d of contact and 84–53 % after 19 d (data not shown). A concentration of 400 ppm (¼150 mg cm2, comparable with doses used at mushroom farms) discriminated between isolates. Growths ranged from 34.3–66.8 % of the controls. This concentration was used for the classification of the strains. V49, V9909 and 440.34, each from a different country, were the less resistant (Table 4). The distribution of the isolates in classes of resistance showed that even at high concentrations the mycelial growth was not totally inhibited for any strain, most were moderately resistant. Comparison of distributions on d 5 and d 19 revealed that some isolates adapted after a few days of contact with the fungicide (Table 5). No correlation could be drawn between RAPD grouping and fungicide resistance. Significant differences were observed among isolates collected in 1993 and among those collected in 2000 but the two groups did not differ significantly. No correlation could be drawn between the various physiological traits studied except for antibiosis effects for which the surfaces covered by V. fungicola and A. bisporus were significantly correlated (Table 6).
Diversity in virulence and pathogenicity The antibiosis experiments distinguished between both varieties of Verticillium fungicola for germination ability on a growing colony of A. bisporus (Table 4). Conidiae of the four isolates of var. aleophilum germinated on the whole surface of the agar, including over the colony of A. bisporus, whereas conidiae of var. fungicola germinated around the mushroom colony but only on very small areas above. Little variation occurred within this variety. Colonies of V. fungicola covered the surface of the culture medium on controls without A. bisporus. After the supply of conidiae the A. bisporus colony continued to grow, and significant variations, depending on the isolate of V. fungicola supplied, were observed. Growth of A. bisporus was limited under the four isolates of var. aleophilum, as intensive growth was detected under developing colonies of VV1, V86 and VSL (Table 4). A low dose of chlorothalonil was enough to affect the mycelial development of the pathogen. In the presence of
All Verticillium fungicola isolates screened in the virulence assays induced necrosed brown tissues around and under the inoculation site and profuse sporulation. Inoculation of a suspension of conidiae of V. lecanii caused a level, yellow– beige spot, restricted to the surface of the pilei surface and no symptoms were observed after inoculation with a Verticillium sp. isolate distinct from V. fungicola by ITS amplification (data not shown). The French and Dutch isolates differed significantly for the diameter of the necrosis (Table 2). The correlation coefficient of 0.30 (df ¼ 17) between the diameter of the necrosis and the year of collection was not significant (P ¼ 0.05). Isolates chosen for evaluation of pathogenicity consisted of the ex-type strain 440.34, isolates VCTC and VCF polymorphic with RAPD markers, and three isolates (VF, VK, and V9503) from the homogeneous RAPD group. The percentages of diseased mushrooms in the two experiments were significantly correlated (r ¼ 0.91, P < 0.02) but higher percentages were
Table 2 – Analysis of variance for in vitro mycelial growth, fungicide resistance and necrosis induction in virulence assays measured for the 22 Verticillium fungicola isolates Source
D.F.
d
All isolates Intra var. aleophilum Intra var. fungicola Intra French isolates Intra Dutch isolates French vs Dutch isolates
21 3 17 8 6 1
Mycelial growth rate at 23 Ca
Fungicide resistanceb
Necrosis diameterc
Mean square
F value
Mean square
F value
Mean square
F value
5.1709 11.6463 3.6912 4.2733 1.8997 0.1906
43.23** 120.53** 31.12** 30.60** 17.50** 1.67ns
296.00 144.40 326.90 209.50 367.76 33.98
31.82** 45.32** 30.04** 15.66** 83.19** 3.65ns
0.2860 0.1391 0.3228 0.3383 0.0889 1.4821
16.62** 4.12* 23.95** 16.87** 18.50** 86.14**
*Significant at P < 0.05; **significant at P <0.01; ns, not significant at P ¼ 0.05. a Mycelial growth from d 3 to d 12. b Growth on medium amended with 400 ppm of chlorothalonil. c Diameter of the necrosis produced by the deposition of a conidial suspension on sporophore caps. d Isolates are listed in Table 1.
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M. L. Largeteau et al.
Table 3 – Analysis of variance for antibiosis effects of Agaricus bisporus on the 22 isolates of Verticillium fungicola Source
V. fungicolaa
D.F.
All isolates Intra var. aleophilum Intra var. fungicola Intra French isolates Intra Dutch isolates French vs Dutch isolates
21 3 17 8 6 1
A. bisporusb
Mean square
F value
Mean square
F value
282.43 8.49 51.47 69.00 7.07 121.69
44.29** 0.58 ns 10.35** 8.81** 2.48 ns 19.08**
318.54 1.18 254.90 487.10 61.71 26.08
28.44** 0.70 ns 19.19** 30.65** 4.14** 2.33 ns
**Significant at P < 0.01; ns, not significant at P ¼ 0.05. a Surface covered by colonies of V. fungicola. b Surface of the A. bisporus colony recorded on the double layer cultures on d 9 after the supply of the conidial suspension.
Clustering
obtained in the second experiment. Contaminated trays, including that with the less aggressive isolate VCF, gave significantly higher percentages of diseased mushrooms compared with bait trays. The four French isolates were significantly different (Table 7). Data concerning VCTC, VM, and VV1 assessed for pathogenicity in other crops (Juarez del Carmen et al., 2002) were included to look for putative correlations. There was no correlation between the percentages of diseased mushrooms and the year of collection (P ¼ 0.05). The mycelial growth at 23 C was the only physiological trait that correlated significantly with pathogenicity (Table 8).
A principal component analysis was performed on data recorded for mycelial growth at 23 C, Verticillium fungicola surface in antibiosis test, fungicide resistance, necrosis in virulence assays and percentages of diseased mushrooms. The distribution of the isolates on components 1 (39.3 % of total variance explained) and 2 (28.4 %), and on components 1 and 3 (19.2 % explained) is shown in Fig 3. The major contributions to component 1 loading were the percentage of diseased mushrooms and the surface covered with V. fungicola on
Table 4 – Classification of the isolates of Verticillium fungicola for physiological traits and necrosis induction in virulence assays Variety/ geographic origin
Code
Mycelial growth rate (cm 9 d1)a
Antibiosisb
Fungicide resistancea (% growth)
Necrosis diametera (mm)
23 C
30 C
V. fungicola (cm2)
A. bisporus (cm2)
1.6 a 0.6 c 1.0 b 1.5 a
47.4 a 48.5 a 48.2 a 49.0 a
18.9 e 20.0 e 19.8 e 19.4 e
54.6 cde 50.2 e 54.3 cde 40.5 f
12.4 b 12.9 b 12.0 bc 9.9 de
aleophilum
VTaw VX02 V01 V35
4.6 ac 4.5 a 2.9 e 3.8 c
fungicola/Fr
VCF VCTC VF VK VM VSL VV1 V49 V86
3.2 d 4.4 a 3.2 d 3.1 de 3.0 de 3.2 d 4.2 b 3.7 c 3.6 c
0 0 0 0 0 0 0 0 0
d d d d d d d d d
23.2 g 29.7 cdef 33.9 bc 26.6 efg 32.1 bcd 25.3 fg 29.1 cdef 32.8 bcd 26.1 efg
29.8 d 19.3 e 17.6 e 38.3 bc 24.1 e 40.7 ab 44.7 a 34.2 cd 40.1 ab
53.0 de 62.1 ab 52.7 de 55.2 cde 63.3 ab 60.3 abc 52.7 de 36.7 fg 54.9 cde
15.2 a 12.6 b 15.2 a 11.4 bcd 9.6 de 9.7 de 13.0 b 13.0 b 11.4 bcd
fungicola/NL
R1 S2/VX01 648.80 V9301 V9503 V970716 V9909
3.6 c 3.6 c 3.6 c 3.8 c 3.3 d 3.6 c 2.8 e
0 0 0 0 0 0 0
d d d d d d d
32.9 bcd 32.6 bcd 30.7 bcde 30.8 bcde 30.9 bcde 29.4 cdef 31.3 bcde
31.2 d 29.7 d 31.8 cd 32.2 cd 28.7 e 34.8 cd 33.1 cd
55.9 cde 58.1 bcd 49.4 e 62.9 ab 66.9 a 62.8 ab 36.3 fg
10.2 cde 9.3 de 10.2 cde 12.2 b 10.2 cde 10.2 cde 8.9 e
fungicola/UK
Vff ADAS 440.34
2.5 f 3.6 c
0d 0d
28.2 def 35.3 b
33.4 cd 29.3 d
62.7 ab 34.3 g
9.0 e 9.2 e
a See legend on Table 2. b See legend on Table 3. c Values within a column followed by the same letter do not differ significantly by the Student–Newman–Keuls test (P ¼ 0.05).
Verticillium fungicola diversity
437
Table 5 – Effect of chlorothalonil on the mycelial growth of the 22 isolates of Verticillium fungicola Date
Classes of resistancea
Numbers of isolates per class for each concentration of fungicide 20 ppm
200 ppm
400 ppm
800 ppm
4000 ppm
d5
Sensitive Slightly resistant Moderately resistant Highly resistant
0 1 11 13
0 3 19 3
0 3 21 1
0 4 21 0
0 10 15 0
d 19
Sensitive Slightly resistant Moderately resistant Highly resistant
0 0 11 11
0 1 14 7
0 2 18 2
0 2 18 2
0 4 16 2
a Sensitive, 0–15 % of the control; slightly resistant, >15–40 %; moderately resistant, >40–65 %; highly resistant, >65 %.
double-layer cultures. Components 1 and 2 grouped the isolates but VCF (which was less aggressive to mushroom crops) and 440.34 (which was better developed in the antibiosis test) differed by component 1. The distribution of the isolates on components 1 and 3 was not informative.
Discussion Genetic variability within Verticillium fungicola was recently investigated by Collopy et al. (2001) and Juarez del Carmen et al. (2002) but with only few European isolates. The present investigation including 18 European isolates of V. fungicola var. fungicola and four isolates of V. fungicola var. aleophilum from various origins strengthened the previous findings and provided additional information about the level of genetic variability. We observed a high level of DNA polymorphism among the var. aleophilum isolates from different geographic origins. Beside differences in cultivation practises, the mushroom strain must be taken into consideration as a factor influencing the
variability of the pathogen. The high level of genetic homogeneity of the commercial mushroom was proposed by Bonnen and Hopkins (1997) as being one of the factors responsible for the level of homogeneity in American populations of V. fungicola. We have no information concerning the mushroom strain from which VTAW was isolated, but VX02 was from Les Miz 60, an old variety of A. bisporus, while V01 and V35 were from recent crops. But as Bonnen and Hopkins (1997) reported, this factor is not alone in explaining the lack of variability within the V. fungicola populations because the ex-type strain 440.34, collected in 1934, did not differ from the group of 15 isolates showing no variability. Similar results were reported concerning old North American isolates. Bonnen and Hopkins (1997) placed in the same RAPD group an isolate from 1950 with 55 isolates collected during years 1970–1995. Collopy et al. (2001), who performed RAPD analysis on V. fungicola var. aleophilum isolates, observed identical patterns for 40 Pennsylvania isolates collected during 1999 and 13 North American isolates collected during the last 50 y. The uniformity of the casing material between mushroom farms through the adoption of pasteurized peat moss was proposed to explain the lack of diversity in V. fungicola var. aleophilum responsible for recent dry bubble outbreaks in North America (Collopy et al. 2001). Obornik et al. (2000) proposed that RAPD-based phylogeny and genetic variability of
Table 6 – Correlation between the various traits measured on the 22 isolates of Verticillium fungicola Necrosis Fungicide diameter resistance
Mycelial growth at 23 C Necrosis diameter Fungicide resistance (400 ppm)
0.45098a 0.0402b
Antibiosis V. fungicola A. bisporus surface surface 0.35316 0.1163
0.07633 0.7423
0.23071 0.3143
Table 7 – Classification of six isolates of Verticillium fungicola var. fungicola according to their pathogenicity in cultures Verticillum isolate
Diseased mushrooms (%)a Experiment 1
0.01006 0.9655
0.21473 0.3499
0.27527 0.2272
0.32434 0.1515
0.04030 0.8623
V. fungicola surface a Pearson correlation coefficients, N ¼ 21. b Prob > jrj under H0: r ¼ 0.
0.74115 0.0001
VCTC V9503 440.34 VF VK VCF Control
b
30.4 a 36.2 a 30.0 a 19.4 b 13.9 c 8.2 d 3.8 e
Experiment 2 46.7 a 38.4 b 46.4 a 29.8 c 19.9 d 12.4 e 4.7 f
a Diseased mushrooms in percent of the total crop (weight). b Values within a column following by the same letter do not differ significantly by the Student–Newman–Keuls test (P ¼ 0.05).
438
M. L. Largeteau et al.
Table 8 – Correlation between the pathogenicity in cultures and the various physiological traits measured on the eight isolates of Verticillium fungicola Mycelial growth (23 C) Aggressiveness
a
0.71192 0.0476b
Necrosis diameter
Fungicide resistance (400 ppm)
0.1387 0.5691
0.1633 0.6993
Antibiosis V. fungicola surface
A. bisporus surface
0.4677 0.2425
0.06423 0.8799
a Pearson correlation coefficients, N ¼ 8. b Prob > jrj under H0: r ¼ 0.
character studied was taken into consideration separately, significant physiological dissimilarities and pathogenicity variability were observed among the European var. fungicola isolates. Diversity in tolerance to hydrogen peroxide was previously observed among a group of six French var. fungicola isolates (Juarez del Carmen et al. 2002). The var. fungicola population screened appeared less homogeneous than the var. aleophilum populations from North America studied by Bonnen and Hopkins (1997). European isolates gathered in 1993 and 2000 were too few to provide confirmation, but based on the whole var. fungicola sample, it did not seem that the physiological variability is progressing. In our work on antibiosis, conidiae of V. fungicola germinated around the mushroom colony, which is in accordance with the observation of Calonje et al. (2000) in dual-cultures on Raper medium. These authors also reported the germination of conidiae inoculated on a colony of A. bisporus transferred to an empty Petri dish. The absence of nutrients could explain the lack of defense of A. bisporus. In our study, the mushroom was fed, and consequently maintained an active metabolism which allowed it to produce defensive compounds preventing conidiae of var. fungicola to germinate on the colony. Although, the restriction of the inhibition to the surface of the mushroom colony showed that inhibitors do not diffuse well and/or were rapidly inactivated and were
3 2
Component 2
Paecilomyces sp. and Lecanicillium lecanii (syn.) Verticillium lecanii reflect the life strategy of the entomopathogenic fungi, and that the mobility of spores is a major influence on the evolution of these organisms. The common origin of some cultivation materials, especially peat moss, in the three European countries from which the isolates originated could be a factor of dispersion of V. fungicola spores favourable to the genetic homogeneity of the pathogen. In the present study, Dutch isolates chosen at random except for date in the PPO-MRU collection, were from restricted geographic sites and from mushroom farms cultivating with standardised practices, and there was no genetic variability observed. Although all V. fungicola var. fungicola isolates we screened fall into a single cluster, some genetic variability exists inside the present sample of European isolates. The AFLP analysis performed on randomly selected var. fungicola isolates showed some variability but the groups were ill-defined, which confirmed the RAPD results. Within the var. fungicola, VCTC, VV1, VK, and VF were identical regarding the ITS sequences (Collopy et al. 2001), but they differed with RAPD and AFLP markers in our investigations. These results confirmed that RAPD reveals a higher level of polymorphism, as shown in other pathogen populations such as Fusarium verticillioides (Voigt et al. 1995) and Colletotrichum lindemuthianum (Sicard et al. 1997). The three V. fungicola var. fungicola isolates found polymorphous in the present study were from various French locations, corresponding to variations in compost, casing and cultural practices. This could explain why genetic variability was only detected within the group of French isolates, but more samples are necessary before a final conclusion can be drawn. Within the var. fungicola isolates we identified a homogeneous population comprising 13 isolates collected in France and the Netherlands over a period of 27 y (1974–2000) and one isolate collected in 1934 in the UK. VV1 and VCTC isolated at French mushroom farms in 1993 and 1997, respectively, were distinguished from this population, whereas the Dutch isolates collected during the same years were not. Such an absence of correlation between geographic location and RAPD pattern was also observed for Verticillium dahliae (Messner et al. 1996; Lachquer & Sedra 2002) and it was generally the same concerning L. lecanii (Mor et al. 1996). A clonal population was identified for the var. aleophilum in North America (Collopy et al. 2001). A major result emerging from our investigation is that similarly, the var. fungicola in Europe is also genetically homogeneous. Despite of the high genetic homogeneity observed with different tools, significant differences for the expression of physiological and pathogenicity traits were observed. When each
-4
VCTC
VCTC VV1
1
VCF 0 -2
0
VF -1
VK VM
2
4
V9503
-2
440.34
-3
Component 1 Fig 3 – Distribution of isolates of V. fungicola var. fungicola after principal component analysis. (C) Isolates used to determine the principal components; (:) additional isolates placed on the principal component plan.
Verticillium fungicola diversity
moderately efficient as they allow some germination of var. fungicola and do not prevent that of var. aleophilum. In addition to the homogeneity of the casing material used to grow the mushrooms the use of fungicides was proposed as another cause of the genetic homogeneity observed among the V. fungicola isolates due to the selection of fungicide resistant strains. Various opinions emerge from the literature on the efficiency of chlorothalonil to reduce dry bubble disease on mushroom crops. van Zaayen (1977), Gandy and Spencer (1981), and Maszkiewicz (2001) considered the fungicide efficient to control dry bubble disease on mushroom crops. Fletcher and Hims (1981) and Coutinho et al. (2000) observed that chlorothalonil failed to control the disease. We observed a high level of fungicide resistance in vitro for all but three isolates. The susceptibility of 440.34 and V49 was not surprising because chlorothalonil was not used at the time the former was collected and the mushroom farm where the latter was collected used organic farming practises. The susceptible V9909 was isolated when chlorothalonil was commercially unavailable in the Netherlands. But some moderately to highly resistant isolates were collected before the fungicide was authorized (1996 in France, 1980 in the Netherlands) for mushroom cropping. A relatively high level of tolerance to chlorothalonil even before its introduction was previously reported by Bonnen and Hopkins (1997) for isolates of V. fungicola mainly in North America. The authors related that the level of tolerance varied little over the period of collect. Gea et al. (1997) observed that the majority of the 20 isolates of V. fungicola var. fungicola collected from various fruit-bodies of A. bisporus from prochloraz-treated crops were very resistant to chlorothalonil in vitro. Considering the high level of resistance of the isolates we screened, we can question the impact of the common use of prochloraz–Mn complex at mushroom farms before the use of chlorothalonil on the resistance of V. fungicola to the latter fungicide. In the work of Bonnen and Hopkins (1997) little correlation could be drawn between RAPD grouping and fungicide response. Similarly, we did not find any correlation between RAPD polymorphism and fungicide resistance. This absence of correlation is not restricted to the fungicola species. No or little correlation was observed between RAPD polymorphism and virulence for isolates of L. lecanii (Mor et al. 1996) and V. dahliae (Ramsay et al. 1996). The symptoms observed in the present virulence assays corresponded to those observed by Bonnen and Hopkins (1997) on most isolates collected between 1971 and 1995 and to all isolates collected in 1993–1995 in North America. The two Verticillium isolates distinct from the species V. fungicola failed to induce necrosis which conferred the virulence assays specificity. Mushroom strains showing mainly spotted mushrooms could be considered developing resistance compared with strains producing mainly bubbles. Inoculation directly on the pilei gives information on the virulence rather than on the level of aggressiveness of V. fungicola isolates and was more valuable in classifying A. bisporus strains than Verticillium isolates. These observations could explain the absence of correlation between the percentages of diseased mushrooms in culture and the necrosis diameter in virulence assays observed here. A significant correlation was found between aggressiveness in cultures and mycelial
439
growth rate at 23 C, which suggests that the high temperature maintained for a week after casing, which is favourable to colonisation of the casing layer by V. fungicola, may have considerable incidence on infection. Although no significant correlation was observed between aggressiveness and in vitro antibiosis, the recognition mechanisms between V. fungicola and A. bisporus cell walls described by Bernardo et al. (2004) are a putative source of variation in aggressiveness during cropping. Genetic and physiological investigations revealed a higher level of diversity within the group of var. fungicola isolates we studied than within North American populations of var. aleophilum isolates (Bonnen & Hopkins 1997; Collopy et al. 2001) but neither DNA polymorphism nor dissimilarities in physiological traits but mycelial growth rate at 23 C could explain the variability in aggressiveness among the European isolates. The comparison of French and Dutch isolates, the latter cultivated under more standardized practises, emphasised the effect of cultivation standardisation on the genetic evolution of the variety fungicola toward a clonal population.
Acknowledgements We thank Thierry Gibard and Patrick Hendrickx for technical assistance, and Patrick Castant and Christiane Coldefy for mushroom cultivation. M.L.L. is grateful to Olivier Le Gall for his help in molecular analyses. J.P.P.B. gratefully acknowledges financial support of the Dutch Ministry for Agriculture.
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