Genetic evidence for natural hybridization between the Dutch elm disease pathogens Ophiostoma novo-ulmi ssp. novo-ulmi and O. novo-ulmi ssp. americana

The Dutch elm disease pathogen Ophiostoma novo-ulmi is separated into subspecies novo-ulmi , formerly known as the Eurasian (EAN) race, and subspecies americana , formerly known as the North American (NAN) race. Both subspecies occur in Europe, and hybrids between them are suspected to have emerged in parts of Europe where their ranges overlap. Authenticated isolates of both subspecies were examined in order to determine whether fixed mutations occur in the cerato-ulmin ( cu ) and in the colony type gene ( col1 ). One and six mutations were detected between subspp. novo-ulmi and americana in cu and col1 , respectively. The mutation in cu and one mutation in col1 proved to be located within restriction sites, and were used for PCR-RFLP. This method provides a quick and reliable diagnostic method to differentiate the two subspecies. Seven isolates of O. novo-ulmi from Austria were suspected to be hybrids between subspp. novo-ulmi and americana , and were tested by PCR-RFLP of their cu and col1 genes. Two of the suspected hybrids from Austria (isolates AT73 and AT146) had the cu PCR-RFLP profile of ssp. americana and the col1 PCR-RFLP profile of ssp. novo-ulmi . Furthermore, a ssp. novo-ulmi tester isolate from Poland (P150) showed a similar hybrid pattern. This is the first evidence revealing recombination between two genes and thus hybridization between the two subspecies of O. novo-ulmi in nature.     

Rapid emergence of hybrids between the two subspecies of Ophiostoma novo-ulmi with a high level of pathogenic fitness

During the 1970s Europe was invaded by two subspecies of the Dutch elm disease pathogen Ophiostoma novo-ulmi : subsp . americana from the west and subsp. novo-ulmi from the east. As a result their geographic ranges began to overlap in several areas. Only a weak prezygotic barrier to hybridization exists between the subspecies and in 1980 two hybrids were detected in the Netherlands. A subset of 107 O. novo-ulmi isolates collected in a subspecies overlap zone in Limburg, Netherlands in 1983 was characterized for three phenotypic markers and seven RAPD PCR markers. By phenotype, 33% were shown to be hybrid whereas by RAPD markers 69% were shown to be hybrid. Some isolates shown to be hybrid by phenotype were not revealed to be hybrid by PCR and vice versa. Combining the phenotype and RAPD data the estimated hybrid frequency was ∼78%. The mean growth rate of Limburg hybrid isolates was significantly faster than that of the Limburg subsp. novo-ulmi isolates but not significantly different from Limburg subsp. americana isolates. The Limburg hybrid isolates were just as pathogenic as the parent subspecies on both clonal Ulmus procera and on U.  × Commelin. A subset of 100 isolates collected in another subspecies overlap zone at Orvieto, Italy in 1986 was also assessed with RAPD markers and ∼ 72% were shown to be hybrids. When 20 isolates of a 'pure' subsp. novo-ulmi population in the Baltic Ports area of Poland collected in 1980 were assessed by RAPD markers three isolates exhibited early introgression of subsp. americana DNA. This study therefore demonstrates very rapid emergence of O. novo-ulmi subspecies hybrids and introgressants in Europe in the early 1980s. In terms of two major fitness characters, growth rate and pathogenicity, these early hybrids were as fit as their parent subspecies. It is likely that complex hybrid swarms are now expanding across the continent.     

Assessment of Phaeotheca dimorphospora for biological control of the Dutch elm disease pathogens, Ophiostoma ulmi and O. novo-ulmi

The antagonistic fungus Phaeotheca dimorphospora was tested as a biocontrol agent against the Dutch elm disease pathogens, Ophiostoma ulmi and O. novo-ulmi , on Ulmus americana seedlings in the glasshouse. Curative inoculation of seedlings with P. dimorphospora had no significant effect on disease development. Conditioning inoculation of seedlings with the antagonist protected them against O. ulmi but not against the more aggressive O. novo-ulmi . In seedlings challenged with both the antagonist and either one of the pathogens, P. dimorphospora only spread locally around the inoculation point; however, the pathogens spread systemically throughout the whole plant. O. novo-ulmi survived in the region colonized by P. dimorphospora , whereas O. ulmi did not.     

Evaluating Verticillium dahliae for biological control of Ophiostoma novo-ulmi in Ulmus minor

The ability of isolate Vd-48 of Verticillium dahliae to induce resistance against subsequent challenge with Ophiostoma novo-ulmi was examined in Ulmus minor . In the first experiment, conditioning inoculation of 5-year-old elm trees (2–3 m in height) with Vd-48 15 days prior to challenge inoculation with O. novo-ulmi significantly reduced wilting ( P  ≤ 0·05) compared with trees not conditioned with Vd-48. However, in another experiment on 6-year-old trees (2–3·5 m in height), no protection was achieved when the length of time between conditioning and challenge inoculations was 45 or 60 days. In a further experiment, inoculations with Vd-48 alone resulted in severe wilting in 22 out of 118 trees (6–10 years old and 4–7 m in height). Across the 2 years of this last trial, nine trees showed massive feeding wounds made by Scolytus sp., while O. novo-ulmi strains were isolated from six trees. Vd-48 provided a variable prophylactic effect against O. novo-ulmi in U. minor . Major difficulties with this approach to control Dutch elm disease are discussed.     

Differences in cerato-ulmin production between the EAN, NAN and non-aggressive subgroups of Ophiostoma ulmi

A test of comparative in vitro cerato-ulmin wilt toxin production in the aggressive and non-aggressive subgroups of the Dutch elm disease pathogen Ophiostoma ulmi was carried out by turbidity and ELISA methods. Ten non-aggressive, ten EAN aggressive and ten NAN aggressive isolates were tested from a range of geographical sources. In liquid shake cultures the non-aggressive isolates produced the greatest and the NAN aggressives the least mean biomass. Despite considerable variation in cerato-ulmin production by individual isolates in three separate experiments, both the turbidity and ELISA methods showed a clear separation of the non-aggressive and aggressive subgroups. Non-aggressive isolates produced little or no cerato-ulmin (ELISA range of means 0–56.0 ng/ml) and EAN and NAN aggressive isolates moderate to high levels (EAN 1.6–89.0 × 10⁴ ng/ml and NAN 0.2–300 × 10⁴ ng/ml). In the aggressive isolates no correlation was detected between cerato-ulmin production and either biomass or pathogenicity to clonal Commelin elm. The role of cerato-ulmin in the pathogenicity of O. ulmi is discussed.     

Hypovirulence-Associated Double-Stranded RNA from Sclerotinia homoeocarpa Is Conspecific with Ophiostoma novo-ulmi Mitovirus 3a-Ld

ABSTRACT The nucleotide sequence of the hypovirulence-associated double-stranded RNA (dsRNA) in hypovirulent isolate Sh12B of Sclerotinia homoeocarpa, the causal agent of dollar spot of turf grass, was determined. This large dsRNA (L-dsRNA) is 2,632 bp long and is A and U rich (61.0% A+U residues). One strand of this dsRNA contains an open reading frame (ORF) with the potential to encode a protein of 720 amino acids. This ORF contains 12 UGA codons, predicted to encode tryptophan in ascomycete mitochondria, and has a codon bias typical of mitochondrial genes, which is consistent with a mitochondrial localization of this dsRNA. The amino acid sequence contains conserved motifs typical of RNA-dependent RNA polymerases (RdRps). Sequence analyses of the nucleotide and RdRp-like protein revealed that the L-dsRNA is homologous with previously characterized mitochondrial viruses and dsRNAs from other phytopathogenic fungi, and shares 92.4% nucleotide and 95.1% amino acid sequence identities with the Ophiostoma novo-ulmi mitovirus 3a-Ld from Ophiostoma novo-ulmi, the causal agent of Dutch elm disease. The results indicate that these two dsRNAs are conspecific. This is the first report that a hypovirulence-associated dsRNA virus naturally occurs in two taxonomically distinct fungi, and indicates that horizontal transmission of this dsRNA virus may have occurred between these fungi.     

Influence of the d2 factor on survival and infection by the Dutch elm disease pathogen Ophiostoma ulmi

A particular form of mycovirus, the d² factor, which attacks the elm pathogen Ophiostoma ulmi, was found to be extremely deleterious to this fungus both in vitro and in vivo. Most significantly, d²-infected conidia were poor at persisting in feeding grooves, the usual infection court of the pathogen, and also showed a 50-fold reduction in successful xylem infection compared with their healthy counterparts. In addition, transmission of the d² factor from diseased to healthy isolates of O. ulmi was found to take place in feeding grooves, indicating that the fungus exists in a mycelial phase prior to invading xylem vessels.     

Is there evidence for post-epidemic attenuation in the Dutch elm disease pathogen Ophiostoma novo-ulmi?

The first pandemic of Dutch elm disease (DED) in Europe and North America caused by the introduced Ophiostoma ulmi began in the early 1900s but declined unexpectedly in Europe from the 1930s onwards after killing 30–40% of the elms. Later a second pandemic caused by the much more aggressive Ophiostoma novo-ulmi spread across the same areas and by 1990 most of Britain's c. 30 million mature elms had died. During the second pandemic, O. novo-ulmi acquired debilitating viruses and changed from being largely clonal to highly heterogeneous through horizontal transfer of novel, sometimes deleterious, genes from O. ulmi. In the post-epidemic period a new disease dynamic has emerged, with millions of small recruitment elms repeatedly attacked by sequential cycles of disease. This study investigated the possibility of pathogenic attenuation in O. novo-ulmi as the epidemic has progressed. Isolates collected from the three original outbreak areas in Britain in 1982–3 (early post-epidemic period) and 2011 (advanced post-epidemic) were compared for two fitness components: pathogenic aggressiveness and in vitro growth rate. Mean aggressiveness of the 2011 isolates to clonal English elm (Ulmus procera) proved not to differ significantly from that of the 1982 isolates, either overall or within each outbreak area. Similarly, the mean growth rates of the 1982 and 2011 samples showed no differences. The implications of these findings are discussed in relation to the future of the current DED pandemic in Europe, the decline of the first pandemic, and the difficulty of drawing parallels between different tree–pathogen systems.     

Transmission of double-stranded RNA and a disease factor in Ophiostoma ulmi

A diseased isolate of Ophiostoma ulmi was found to contain 10 segments of double-stranded RNA (dsRNA) with molecular weights ranging from 2.40 x 10⁶ to 0.23 x 10⁶. In contrast seven other healthy isolates in the same vegetative compatibility group contained either no dsRNA or up to four dsRNA segments. Transmission of the disease to healthy isolates by hyphal anastomosis was accompanied by-transmission of the 10 dsRNA segments. In a genetic cross in which the diseased isolate acted as the female parent single-ascospore progeny were healthy and either contained no dsRNA or only one segment of dsRNA. When elm trees were inoculated with diseased isolates, subsequent reisolations were healthy and retained only two to seven of the dsRNA segments or were diseased and retained all 10 dsRNA segments. Following conidiogenesis a diseased isolate gave rise to single-conidial progeny which were either slow growing and diseased, like their parent, with all 10 dsRNA segments, or faster growing like healthy isolates. Some of the faster growing conidial isolates retained only two to seven of the dsRNA segments and were disease-free. However a majority of the faster growing conidial isolates retained all 10 dsRN A segments and were shown to carry the disease in a latent form. The possibility that the disease of O. ulmi is conferred by specific segments of dsRNA and the potential of d-factors for the control of Dutch elm disease are discussed.     

Heritability of Ulmus minor resistance to Dutch elm disease and its relationship to vessel size,but not to xylem vulnerability to drought

Dutch elm disease (DED) is a vascular wilt disease that causes the occlusion and cavitation of xylem vessels. Therefore, it is hypothesized that those elms that are less vulnerable to cavitation by drought might be more resistant to DED. To test this hypothesis, the relationship between xylem vulnerability to cavitation and susceptibility to DED was examined in progenies of crosses between susceptible and resistant individuals of Ulmus minor. Hydraulic conductivity and xylem vulnerability curves were evaluated and anatomical features such as vessel size, length and grouping were measured. Next, elms were inoculated with Ophiostoma novo‐ulmi, the cause of DED, and pre‐dawn and midday water potentials, stomatal conductance and wilting percentages were assessed. Progenies of R × R crosses showed significantly lower mean wilting percentages (30–50%) than the progeny of S × S crosses (75%). Fifty percent conductivity loss was reached at c. ?1 MPa, pointing out a high vulnerability of this species to drought‐induced cavitation. Crown wilting percentage as a result of inoculation and xylem vulnerability to cavitation by water stress did not show any significant correlation. Nevertheless, significant differences in theoretical hydraulic conductivity and vessel size parameters (diameter, length and size distributions) were found among the tested progenies. Susceptible trees had significantly wider and longer vessels. Xylem structure of resistant elms seems to restrict pathogen spread rather than prevent cavitation.     

A Satellite RNA of Ophiostoma novo-ulmi Mitovirus 3a in Hypovirulent Isolates of Sclerotinia homoeocarpa

ABSTRACT Two genetically distinct double-stranded RNA (dsRNA) elements were identified in hypovirulent isolates of Sclerotinia homoeocarpa, the causal agent of dollar spot of turfgrass. The large dsRNA (L-dsRNA) was consistently present in all hypovirulent isolates, whereas the small dsRNA (S-dsRNA) was found only in some hypovirulent isolates. Virulence comparisons revealed that there was no significant difference between isolates containing one or both dsRNAs. Therefore, the L-dsRNA appears to be the genetic determinant of hypovirulence, while the S-dsRNA is not essential for hypovirulence in S. homoeocarpa. The L-dsRNA in hypovirulent isolate Sh12B of S. homoeocarpa was previously characterized as a fungal mitochondrial virus and designated Ophiostoma novo-ulmi mitovirus 3a-Sh12B (OnuMV3a-Sh12B) because it was conspecific with O. novo-ulmi mitovirus 3a-Ld from O. novo-ulmi, the causal agent of Dutch elm disease. In the present study, the nucleotide sequences of the S-dsRNAs (738 to 767 nucleotides) in hypovirulent isolates Sh12B, Sh279B, and Sh286B were determined. Nucleotide sequence analysis indicated that the S-dsRNA was not derived from the OnuMV3a dsRNA and it could not encode an RNA-dependent RNA polymerase. These results are consistent with biological data that the S-dsRNA was always associated with the L-dsRNA and was never found independently. Therefore, the S-dsRNA can be regarded as a satellite RNA of OnuMV3a in S. homoeocarpa. Northern blotting analysis indicated that nucleic acid extracts from isolate Sh12B of S. homoeocarpa contained more single (+) stranded RNA than dsRNA for this satellite RNA. The 5'- and 3'-terminal sequences of the positive strand of the S-dsRNA each could be folded into a stem-loop structure and the terminal 21 nucleotides were complementary to each other, potentially forming a panhandle structure.     

Monoclonal antibodies to specific components of the Dutch elm disease pathogen Ophiostoma ulmi

The potential of polyclonal antisera and monoclonal antibodies to differentiate the EAN and NAN aggressive subgroups of Ophiostoma ulmi was explored. Polyclonal antisera, when tested by ELISA, cross-reacted widely with unrelated species and failed to distinguish between the two aggressive subgroups but small quantitative differences were found, particularly between antigens secreted overnight, by EAN and NAN germlings. Monoclonal antibodies were raised in mice against mycelial homogenates. From two fusions, 33 cell lines were raised that secreted antibodies positive for O. ulmi. Approximately one third were non-specific; 11 were specific either to species or subspecies. Two cell lines differentiated mycelial antigens of the aggressive isolates of O. ulmi from those of the non-aggressive subgroup, but not antigens from surface washings. Only quantitative differences were detected between the EAN and NAN aggressive subgroups. Almost all the monoclonal antibodies and antiserum recognized antigens present in surface washings of cultures on solid medium, in cell-free extracts of mycelial homogenates, in cell-free culture fluids, and in substances secreted overnight by germinating spores. Specific detection of such molecules promises to provide a highly sensitive mechanism for studying early pathogen/host plant interactions. Most of the monoclonal antibodies appeared to have potential diagnostic value; they gave readings twofold to tenfold higher with extracts from diseased than from healthy tissue. However, one cell line that secreted antibodies specific to O. ulmi cross-reacted strongly with extracts of healthy tissue.     

Antifungal effect and reduction of <Emphasis Type="Italic">Ulmus minor</Emphasis> symptoms to <Emphasis Type="Italic">Ophiostoma novo-ulmi</Emphasis> by carvacrol and salicylic acid

There are still no effective means to control Dutch elm disease (DED), caused by the vascular fungi Ophiostoma ulmi and O. novo-ulmi. Plant phenolics may provide a new strategy for DED control, given their known antifungal activity against pathogens and their involvement in plant defence mechanisms. The in vitro antifungal activity of salicylic acid, carvacrol, thymol, phenol, o-cresol, m-cresol, p-cresol, and 2,5-xylenol against the DED pathogens was tested. Also, the protective effect of watering Ulmus minor seedlings with these compounds was tested against O. novo-ulmi. Salicylic acid, carvacrol, and thymol showed the strongest antifungal in vitro activity, while carvacrol and salicylic acid provided the strongest in vivo protection against O. novo-ulmi (63 and 46% reduction of leaf wilting symptoms with respect to controls, respectively). The effect of the treatments on tree phenology was low, and a significant negative relation was observed between the number of days to bud burst and the leaf wilting symptoms after inoculation, probably determined by genetic differences among the elm tree progenies used. The treatments with salicylic acid, carvacrol and thymol induced the highest shift in phenolic metabolite profile with respect to control trees. The protective effect of carvacrol and salicylic acid is discussed in terms of their combined activity as antifungal compounds and as inductors of tree defence responses.     

China and the origins of Dutch elm disease: an appraisal

Circumstantial evidence has led to the widespread assumption that Dutch elm disease (caused by Ophiostoma ulmi ) spread to Europe and North America from China. The author's recent surveys indicate that the disease is probably absent from China, and only a relatively recent arrival in the Soviet Tien Shan mountain region on the western Chinese border. Alternative origins for the disease include introduction from the Himalayas, and recent rapid evolution within Europe from a fungus such as Ophiostoma piceae . Elucidation of the various possibilities is complicated by the need to account for the spread of at least three genetically divergent subgroups of the pathogen.     

Linkage of the genes determining mating type and fungicide tolerance in Ophiostoma ulmi

Two allelic mutations conferring high and low levels of in vitro tolerance to the fungicide methyl benzimidazole-2-ylcarbamate (MBC) were identified in the elm pathogen Ophiostoma ulmi. Variants expressing a third and intermediate level of tolerance were also identified. The locus for fungicide tolerance was linked to the mating type locus in both the aggressive and non-aggressive strains of the fungus.     

Internal Transcribed Spacer Regions 1 and 2 and Random Amplified Polymorphic DNA Analysis of Didymella bryoniae and Related Phoma Species Isolated from Cucurbits

ABSTRACT Didymella bryoniae (anamorph Phoma cucurbitacearum) is the causal agent of gummy stem blight, although other Phoma species are often isolated from cucurbit plants exhibiting symptoms of the disease. The molecular and phylogenetic relationships between D. bryoniae and these Phoma species are unknown. Isolates of D. bryoniae and Phoma obtained from cucurbits grown at various geographical locations in the United States were subjected to random amplified polymorphic DNA (RAPD) analysis and internal transcribed spacer (ITS) sequence analysis (ITS-1 and ITS-2) to determine the molecular and phylogenetic relationships within and between these fungi. Using RAPD fingerprinting, 59 isolates were placed into four phylogenetic groups, designated RAPD group (RG) I, RG II, RG III, and RG IV. D. bryoniae isolates clustered in either RG I (33 isolates), RG II (12 isolates), or RG IV (one isolate), whereas all 13 Phoma isolates clustered to RG III. There was greater than 99% sequence identity in the ITS-1 and ITS-2 regions between isolates in RG I and RG II, whereas isolates in RG III, P. medicaginis ATCC 64481, and P. exigua ATCC 14728 clustered separately. On muskmelon seedlings, a subset of RG I isolates were highly virulent (mean disease severity was 71%), RG II and RG IV isolates were slightly virulent (mean disease severity was 4%), and RG III isolates were nonpathogenic (disease severity was 0% for all isolates). The ITS sequences indicate that RG I and RG II are both D. bryoniae, but RAPD fingerprints and pathogenicity indicate that they represent two different molecular and virulence subgroups.     

Hypovirulence and Double-Stranded RNA in Botrytis cinerea

ABSTRACT Twenty-one strains of Botrytis cinerea isolated from 13 species of plants grown in China were compared for pathogenicity on Brassica napus, mycelial growth on potato dextrose agar, and presence of double-stranded (ds)RNA. The results showed that the strain CanBc-1 was severely debilitated in pathogenicity and mycelial growth, compared with the 20 virulent strains. A dsRNA of approximately 3.0 kb in length was detected in CanBc-1 and 4 hypovirulent single-conidium (SC) isolates of CanBc-1, but was not detected in the 20 virulent strains of B. cinerea and 4 virulent SC isolates of CanBc-1. Results of the horizontal transmission experiment showed that the hypovirulent trait of CanBc-1 was transmissible and the 3.0-kb dsRNA was involved in the transmission of hypovirulence. Analysis of a 920-bp cDNA sequence generated from the 3.0-kb dsRNA of CanBc-1 indicated that the dsRNA element was a mycovirus, designated as B. cinerea debilitation-related virus (BcDRV). Further analyses showed that BcDRV is closely related to Ophiostoma mitovirus 3b infecting O. novo-ulmi, the causal agent of Dutch elm disease. Mitochondria and cytoplasm in hyphal cells of CanBc-1 became degenerated, compared with the virulent isolate CanBc-1c-66 of B cinerea. This is the first report on the occurrence of Mitovirus-associated hypovirulence in B. cinerea.     

A rapid virulence assay for the Dutch elm disease fungus Ophiostoma novo‐ulmi by inoculation of apple (Malus × domestica ‘Golden Delicious’) fruits

Large‐scale virulence tests using trees or saplings are expensive, time‐consuming and require a considerable amount of space. The suitability of using ‘Golden Delicious’ apples as a rapid screen for identifying Ophiostoma novo‐ulmi transformants with reduced virulence was thus evaluated. When a collection of O. novo‐ulmi field isolates belonging to subspecies novo‐ulmi or americana was inoculated to apples, members of subsp. novo‐ulmi induced, on average, larger necrotic lesions than subsp. americana isolates. The size of the lesions on apples was not correlated with mycelial growth rate of isolates on nutrient agar. Insertional mutants from O. novo‐ulmi subsp. novo‐ulmi isolate H327 were inoculated to ‘Golden Delicious’ apples and Ulmus parvifolia × U. americana saplings in parallel experiments. Results clearly indicated that the O. novo‐ulmi transformants included several exhibiting significantly altered levels of virulence. Variability among replicates within a treatment was reduced in apple inoculation data compared to elm sapling data. Overall, the ‘Golden Delicious’ apple assay was found to be an excellent means for rapidly assessing the virulence level of O. novo‐ulmi isolates.     

Genetic Analysis of Isolates of Botrytis cinerea Sensitive and Resistant to Benzimidazole and Dicarboximide Fungicides

ABSTRACT A total of 56 isolates of B. cinerea collected from ornamental crops from commercial greenhouses were examined by random amplified polymorphic DNA (RAPD) fingerprint analyses. Isolates were examined as two independent sets of 35 and 36 isolates, with 15 isolates common to both sets. The isolates had four phenotypes: 17 were sensitive to two commonly used fungicides, thiophanate-methyl (a benzimidazole) and vinclozolin (a dicarboximide) (S(T)S(V)); 18 were resistant to both fungicides (R(T)R(V)); 16 were resistant to thiophanate-methyl but sensitive to vinclozolin (R(T)S(V)); and 5 were sensitive to thiophanate-methyl but resistant to vinclozolin (S(T)R(V)). Relationships among the isolates were determined by cluster analyses of mean character differences using the unweighted pair group method using arithmetic average and cladograms were constructed. Isolates were clustered primarily by fungicide-sensitivity phenotype. In one set of greenhouse isolates, 6 of 10 S(T)S(V) isolates clustered together with a bootstrap confidence value of 91%. In the other fingerprint set of greenhouse isolates, 9 of 11 S(T)S(V) isolates clustered together and had a bootstrap confidence value of 98%. Isolates resistant to thiophanate-methyl, vinclozolin, or both fungicides usually were not clustered with other isolates or were clustered with isolates of the same phenotype. To further elucidate these relationships, variant isolates resistant to one or both fungicides were produced on fungicide-amended agar medium from 14 S(T)S(V) greenhouse isolates. These 14 S(T)S(V) parent isolates, 57 resistant variant isolates, and 11 resistant greenhouse isolates were analyzed as three independent RAPD fingerprint sets. Variants selected from eight S(T)S(V) parent isolates were resistant to both thiophanate-methyl and vinclozolin even though parent isolates were exposed to only one of the fungicides. Isolates resistant only to vinclozolin (S(T)R(V)) had fingerprint patterns similar to and clustered with those of parent isolates, while fingerprint patterns of isolates resistant to thiophanate-methyl (i.e., R(T)R(V) or R(T)S(V)), regardless of sensitivity to vinclozolin, clustered differently from both those of S(T)S(V) parent isolates and those of S(T)R(V) isolates derived from the same parent. R(T)R(V) and R(T)S(V) variant isolates derived from the same fungicide-sensitive parents only clustered with other variants having the same phenotype.