John H. Carder’s research while affiliated with East Malling Research and other places

The relationships of two host-adapted pathotypes of Verticillium dahliae have been examined at the molecular level using restriction fragment length polymorphisms. Isolates obtained from and adapted to Mentha×piperita (peppermint), which were presumed to be haploid, formed a distinct subspecific group (referred to as M) related to the previously described non-host-adapted subspecific group A of V. dahliae. The limited molecular variation found among the four group M isolates was not related to geographic origin. Isolates from several cruciferous hosts (and one from Beta vulgaris (sugar beet)), which are thought to be natural, stable diploids, formed another distinct group (referred to as D) that was markedly different from all previously described subspecific groupings in both V. dahliae and V. alboatrum. This group of isolates might better be regarded as a separate species. Again, only limited variation was found within the D group. Polymorphisms revealed by two probes distinguished two isolates derived from Brassica rapa (Chinese cabbage) from the six other isolates (four from Brassica napus (oilseed rape) and one each from Raphanus raphanistrum (wild radish) and Beta vulgaris).

Eight isolates of Verticillium dahliae from Japan, classified into four groups based on pathogenicity to differential hosts, were compared with isolates in previously defined RFLP groups. Within each of two of the pathogenicity groups (JB and JC) the pairs of haploid isolates were closely related but those in a third group (JA; isolates not pathogenic to sweet pepper or tomato) were not. Only one of the six haploid isolates (one of the two in the JA group) could be placed in an existing RFLP group. The two diploid isolates (the JD pathogenicity group) were similar to RFLP group D and only distantly related to the six haploid isolates. Of the Japanese pathogenicity groups, only JD corresponded to an existing RFLP group.

The sequences of the internal transcribed spacers (ITS) 1 and 2 of the rRNA genes of 38 Verticillium alboatrum and V. dahliae isolates have been determined. The isolates represented RFLP groups of both species, V. dahliae vegetative compatibility groups and pathotypes, and V. alboatrum‘group 2’(the majority of the V. alboatrum isolates studied were in‘group 1′). The ITS sequences of a single V. tricorpus isolate were also determined. RFLP groups L and NL of V. atboatrum were distinct, with a maximum of three nucleotide differences between any isolate. Most haploid V. dahliae isolates were identical and separated from most L/NL V. alboatrum isolates by differences at five or six positions. A few haploid V. dahtiae isolates, not forming any obvious grouping, differed from the majority, each at a single position. Diploid isolates of V. dahtiae were identical but only one nucleotide difference separated them from some RFLP group L V. alboatrum isolates. At least six differences separated the diploid isolates of V. dahliae from the haploid ones. The‘group 2’V. atboatrum isolates were more distantly related to‘group 1’isolates (at least 17 positions different) than was V. tricorpus. The possibility of defining specific primers for use in PCR to discriminate species and subspecific groups is discussed.

The extracellular subtilisin-like protease activity of several fungal pathogens of nematodes, insects and plants was recorded after batch cultivation. Nematophagous and entomophagous isolates of Verticillium, Paecilomyces, Beauveria and Metarhizium produced Suc-(Ala)2-Pro-Phe-pNA hydrolysing enzymes that differed in serological properties based on Western blot analysis with polyclonal antibodies. The antibodies used were raised against the main subtilisin-like proteases from one species from each of the four genera. From each strain of V. chlamydosporium tested, between one and four isoforms with different pI values were separated by isoelectric focusing and visualized by enzymoblots. The occurrence of multiple isoforms with unique N-terminal sequences in single strains might suggest the presence of subtilisin gene families. Using as a probe a fragment of the Pr1 gene from M. anisopliae, RFLPs were observed that confirmed qualitative differences between subtilisin-like genes within and between species. In contrast to these pathogens of invertebrates, plant-pathogenic species of Verticillium did not produce subtilisin-like proteases, nor were homologous genes detected. Such activity, however, and the corresponding genes, were detected in species weakly pathogenic to plants, or saprotrophs.

Two fungal isolates, formerly classified as Verticillium alboatrum and proposed as forming the basis of a new sub-group ('Group 2') within the species, have been shown to be non-pathogenic to known hosts of V. alboatrum and, on the basis of molecular evidence, to be closely related to Verticillium psalliotae and Verticillium fungicola. We propose that the taxon V. alboatrum be confined to those closely related isolates, usually plant pathogenic and usually producing dark resting mycelium, referred to by other authors as Group 1. The only sub-specific groupings which appear valid (based on pathological and molecular evidence) comprise: (1) host-adapted isolates from lucerne; and (2) all other isolates.

A method is described for identifying strains of Metarhizium suitable for use in field experiments. It involves the restriction endonuclease digestion of a PCR product derived from the PrI protease gene and the analysis of the fragments by electrophoresis. Using this technique, 40 Metarhizium strains produced 15 different profile types and were clustered into four groups. Correlation between the profile of restriction fragments and geographic origin was observed for certain groups of strains. This PCR strategy allowed the identification of fungal strains, using as samples spores scraped from the surface of single insects killed by the fungus or single whole dead insects with external mycelium. The sequence of the PrI PCR product from two strains revealed that Prl gene has at least three introns.

Most eukaryotic organisms have sub-repeat sequences in the intergenic regions (IGRs) of their rRNA gene clusters but such subrepeats are unusual in fungi. Verticillium alboatrum and V. dahliae (hyphomycetes) have been shown to have imperfect sub-repeat sequences in their IGRs which probably did not arise by simple reiteration. Although the consensus sequences derived from the subrepeats of V. alboatrum, of haploid isolates of V. dahliae and of diploid isolates of this species were all related, the overall structures of the sub-repeat regions in the three were clearly distinct. Considerable variation in the total length of these regions was found between haploid isolates of V. dahliae but this was not correlated with any identified character, including restriction fragment length polymorphism (RFLP) group. Earlier work on RFLPs, using random genomic clones as probes, showed that the isolates of V. dahliae studied, most of which came from the U.K., could be divided into clearly defined groups. Within-isolate length variation was seen only in a few cases. A single base difference, occurring at the same position in each repeat, was characteristic for V. dahliae RFLP group B or groups A and M combined. A second difference gave similar differentiation but was not present in all repeats. No length variation was seen between V. alboatrum isolates or between two diploid isolates of V. dahliae. The extent of the differences in structures of sub-repeat regions suggest that haploid and diploid isolates of V. dahliae would be better regarded as separate species, along with V. alboatrum, but the lack of correlation between sub-repeat length and any other known character means that sub-repeat length cannot be used for rapid identification of sub-specific taxa.

Eight isolates of Verticillium chlamydosporium Goddard, which have been recently reclassified into two species and two varieties of each species, were studied for biochemical and genetic evidence to support subdivision of the species aggregate. A commercial test system (LRA ZYM, bio-Mérieux UK Ltd) was used to assay for the presence of 87 enzyme activities (glycosidases, esterases, arylamidases, and one transferase) in culture media. Heterologous DNA probes, most derived from the plant pathogenic species V. dahliae and V. alboatrum, were used to examine RFLPs. Most of the RFLPs seen were probably associated with the ribosomal RNA gene cluster. Although cluster analysis of the LRA ZYM results indicated that some subspecific groupings of isolates may exist it did not support their division into two species. Similarly, RFLPs were found which distinguished between isolates but these were not correlated with the proposed species classilication.

On the basis of molecular variation, as revealed by RFLPs, isolates of both Verticillium dahliae and V. alboatrum were divided into two major groups, designated A and B in V. dahliae and L and NL in V. alboatrum. There was very limited variation within the groups. A few isolates of V. dahliae did not fall consistently into either of the two groups; when tested with a range of the probe/enzyme pairs which distinguished the groups, these isolates showed combinations of the polymorphisms which distinguished A and B (i.e. an isolate of this type appeared like group A isolates with some pairs and like group B isolates with others). No isolates sharing characters from the L and NL groups were seen in V. alboatrum. In V. alboatrum the groups correlated with original hosts of the isolates (all L isolates being originally from lucerne; isolates from all other hosts were in NL). There was no clear correlation between grouping and original host in V. dahliae but the relative frequency of occurrence of isolates in the groups did vary with host. Cellulase isozyme pattern was correlated with fungal species and in V. dahliae with isolate group; in V. alboatrum most isolates (whether from the L or NL group) gave an identical pattern, but two of the four L group isolates tested gave a second, distinct pattern.

Molecular variation within and between six species belonging to the fungal genus Verticillium has been examined, either by direct visualization of strongly fluorescing bands in DNA digests or by probing Southern blots with rDNA, mtDNA or random genomic clones derived from an isolate of V. albo-atrum. The two main plant pathogenic species studied, V. albo-atrum and V. dahliae, could be differentiated from each other and from the other four species by the direct visualization approach, although the bands were not always easily seen against the background smear of single or low copy number fragments. Variation was also revealed by probing with rDNA or mtDNA but this variation was generally not correlated with identified interspecific or intraspecific taxonomic groupings. When Southern blots were probed with random genomic clones, all isolates of V. albo-atrum originating from lucerne were clearly differentiated from all other isolates of this species tested which originated from a range of other hosts. There was, however, little variation within these two groups of V. albo-atrum isolates. V. dahliae was clearly differentiated from V. albo-atrum by probing with random genomic clones and considerable variation within this species was evident. Only a few other taxon-specific polymorphisms were identified when using the limited set of probes tested but others may be located when more probes have been examined. The location of genes involved in important attributes such as pathogenicity will require the construction of linkage maps of molecular markers and genes of interest. The results suggest that for the analysis of V. albo-atrum the most useful parasexual cross would be between a lucerne and a non-lucerne isolate and that for the analysis of V. dahliae sufficiently disparate pairs of isolates exist.