Brian W. Bainbridge

Morphological and molecular characterization of Verticillium longisporum comb. nov., pathogenic to oilseed rape

Verticillium wilt in winter-sown oilseed rape (Brassica napus L. ssp. oleifera), which has not yet been reported in the U.K. but is widespread in Europe, was shown to be caused by the host-specific, ‘near-diploid’ Verticillium longisporum comb. nov. Thirty-one cruciferous isolates (plus one from sugarbeet) of V. longisporum, mainly from oilseed rape from Germany, Sweden, Japan, France and Poland, were characterized and compared with nine typical isolates of V. dahliae and five of V. albo-atrum. Isolates of V. longisporum were distinguished from those of V. dahliae by three morphological characters, i.e. elongate microsclerotia, long conidia (7.1—8.8 μm) and mainly three phialides per node on conidiophores, whereas those of V. dahliae had ± spherical microsclerotia short conidia (3.5–5.5 μm), and 4–5 phialides per node. Isolates of V. longisporum lacked extracellular polyphenol oxidase activity (p.p.o.); they showed mean conidial nuclear diam. (DAPI fluorescence) of ca 1.85 μm, and ‘near-diploid’ standardized arbitrary DNA values (Feulgen DNA microdensitometry) of 0.89–1.17 (mean, 1.02). For isolates of V. dahliae, extracellular p.p.o. activity was detectable, and the corresponding figures for conidial nuclear diam. were ca 1.16 μm and DNA values of 0.45–0.65 (mean, 0.57), respectively. Using three oligonucleotide primers, isolates of V. longisporum were clearly distinguishable from those of V. dahliae and V. albo-atrum by their RAPD band profiles. Greenhouse pathogenicity, tests, employing five winter oilseed rape cvs, confirmed the pathogenicity of V. longisporum, whereas V. dahliae was non-pathogenic. On the basis of all the above characters, this host-specific pathotype, V. longisporum, should now be considered as a distinct species. Evidence is presented to suggest that it may have evolved by parasexual hybridization between a strain of V. albo-atrum and a strain of V. dahliae, thus explaining its ‘near diploid’ state and the origin of four recombinants detected.

Restriction fragment length polymorphisms in the ribosomal RNA gene complex of isolates of the entomopathogenic fungus Metarhizium anisopliae

Restriction fragment length polymorphisms (RFLPs) in DNA coding for ribosomal RNA (rDNA) were analysed from different species and isolates of Metarhizium. M. anisopliae var. anisopliae, M. anisopliae var. majus, M. album and M. flavoviride could be differentiated by the presence of restriction enzyme sites in the rDNA repeat unit. Isolates of M. anisopliae could be differentiated according to their geographical origin after cluster analysis of rDNA RFLPs, but there was no apparent correlation between RFLPs and original insect host. Some M. anisopliae var. majus isolates were shown to be heterozygous diploids.

Modern Approaches to the Taxonomy of Aspergillus

Techniques from molecular biology have provided a series of new tools for the analysis of diversity in the fungi. These techniques have been applied to a variety of fungal groups but only rather limited work has been done on the genus Aspergillus, which is surprising considering the economic importance of species within the genus. The availability of a detailed molecular genetic systems in Aspergillus nidulans has been of considerable help in providing a scientific base, but it appears on the whole that molecular geneticists have not been very interested in the taxonomy of the genus. However, a need to study the epidemiology, detection, diagnosis, identification, classification, characterisation and quantification of Aspergillus has resulted in an increasing interest in the taxonomic basis for differences within the genus. This has made it essential that closer links are forged between molecular biologists and taxonomists.

Ribosomal, mitochondrial and amplified DNA polymorphisms in Verticillium albo-atrum pathogenic to hops, lucerne and other plants.

Forty-seven isolates of Verticillium albo-atrum, 35 from hop (Humulus lupulus), seven from lucerne (alfalfa, Medicago sativa) and five from four other hosts, were analysed for DNA polymorphisms. Restriction fragment length polymorphisms (RFLPs) were detected in ribosomal RNA genes (rDNA) using Southern hybridization. Polymorphisms in mitochondrial DNA (mtDNA) were detected in ethidium bromide stained gels after digestion of total genomic DNA with restriction enzymes which recognize four bases containing only G and C. Amplified polymorphic DNA (APD) was analysed using primers based on rDNA sequences from the intergenic spacer (IGS) and 25S regions. These data were used to construct phenograms using either squared Euclidean dissimilarity coefficients (SEDC) and cluster analysis, or unweighted pair grouping with arithmetic averaging (UPGMA). rDNA RFLPs revealed one group with 44 isolates, a second group with two atypical hop isolates, and a third group containing a single avirulent lucerne isolate. mtDNA RFLPs separated rDNA group one into two subgroups, one group containing 38 isolates from different hosts and the other containing all six virulent lucerne isolates. APD analysis divided the isolates into 16 phenotypes, 12 of which contained most of the hop isolates, but there was no correlation with origin, hop cultivar, pathogenicity or year of isolation. One APD phenotype contained all six virulent lucerne isolates, indicating the genetic differentiation between hop and lucerne isolates. Two further APD phenotypes coincided with the second atypical group containing two hop isolates and a distinct avirulent lucerne isolate, respectively. The three methods revealed that three isolates differed markedly from those of the main group.

Aspects of Fungal Genetics

The study of fungal genetics has made a number of significant contributions to our knowledge of genetic processes. The early idea of the connection between genes and enzymes was based on nutritional mutants of Neurospora, and rapid progress in bacterial genetics occurred when this approach and selective techniques were extended to Escherichia coli. One major advantage which some fungi have over bacteria is the occurrence of meiosis in a closed sac, the ascus in the ascomycetes. This allows the genetic effects of a single meiotic event to be studied in detail. There is nothing comparable in bacteria and very often selective techniques are necessary even to detect bacterial recombination. In addition, fusion of nuclei followed by meiosis does not occur in the bacteria. The result of this has been that fungal genetics has made a significant contribution to basic ideas on mechanisms of recombination. In general bacteria are easier to handle for biochemical analysis, and therefore the biochemical basis of recombination is better understood in bacteria than in fungi. This chapter will examine some of the important experiments on recombination in fungi and will include details of the parasexual cycle which occurs in a variety of filamentous fungi.

Genetic Analysis of Bacteriophages

The study of bacteriophages has had a crucial influence on many areas of genetics and molecular biology. Part of the evidence for DNA as the genetic material came from phage T4, and the study of φX174 has played a significant role in the analysis of DNA replication. Phages show great variation in structure, and examples of some of these are shown in Table 4.1.

The Genetics of Streptomycetes

The streptomycetes are bacteria which produce small granular colonies with very thin hyphae and spores. In some ways they resemble the filamentous fungi, but their genetic systems and absence of a nuclear membrane firmly classifies them with the eubacteria. They are rarely dealt with in genetics textbooks, which is surprising as industrially they are very important. They are responsible for the production of 60% of all known antibiotics, including erythromycin, tetracycline and streptomycin. The filamentous habit and production of spores has prevented the analysis of recombination by some of the elegant techniques available for E. coli, but selective techniques showed that recombination could occur. Early work concentrated on Streptomyces coelicolor (Hopwood, 1967; Hopwood et al., 1973) but other species have now been studied (Hopwood and Merrick, 1977). In many ways the early literature is confusing, as the details of the various systems were not known. Strains will be described by the recent nomenclature which has been made possible by the discovery of two plasmids, SCP1 and SCP2, in 5. coelicolor.

Recent Advances in the Genetics of Filamentous Fungi

The rapid progress in molecular genetics has been extended to the analysis of the filamentous fungi (Bennett and Lasure, 1985). The reasons for this have been that they are technically easy to work with, have well-characterized genetic systems, can be characterized biochemically and a number of genera produce commercially important compounds such as antibiotics. Progress has been made in isolating temperature-sensitive mutants, in extending the parasexual cycle to industrial fungi, in detecting protoplast fusion and transformation, in analysis of regulation and in the cloning of fungal genes.

Basic Principles of Microbial Genetics

The genetic study of microbes has played a highly significant role in the recent developments in molecular biology, recombinant DNA technology and the preparation of useful products such as insulin, human growth hormone and blood clotting factors. It was no coincidence that the first artificially-produced hybrid DNA was constructed using bacterial plasmids, and many of the spectacular advances and discoveries have been dependent on microbial systems or on microbial models. This success can be traced back to the first experiments on the molecular genetics of DNA in the genetic transformation of bacteria, as well as to the first isolation of metabolic mutants in fungi. Microbes are ideally suited to the combined biochemical and genetic approach which had early successes in the solution of the genetic code and the regulation of gene activity. The discovery and analysis of plasmid and bacteriophage systems laid the foundation for the exploitation of recombinant DNA techniques, which in their turn were dependent on the discovery of highly specific enzymes, also in bacteria. These techniques have revealed details of genetic organization which traditional genetic methods could not have brought to light. However, this should not be allowed to overshadow the contribution which microbial genetics has made to our understanding of natural variation, in studies on the origin of antibiotic resistance in pathogenic bacteria and the control of antibiotic synthesis in the streptomycetes and the fungi. Later chapters will review the recent extension of modern techniques to the yeasts, filamentous fungi and streptomycetes.

Recombinant DNA Technology

The basic techniques of genetics have relied heavily on spontaneous or induced mutation, followed by standard recombination methods using sexual or asexual processes. There are natural barriers to limit recombination between different organisms, although some of these can be circumvented. Much interest has recently centred around techniques which avoid natural barriers to recombination. A wide variety of techniques has been used, including production of hybrids by cell or protoplast fusion, transfer of genes between different genera of bacteria by promiscuous plasmids, and extracellular manipulation of DNA from widely different sources.