Ross E. Beever

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea.

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea–specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.

Mycoviruses of filamentous fungi and their relevance to plant pathology

Mycoviruses (fungal viruses) are reviewed with emphasis on plant pathogenic fungi. Based on the presence of virus-like particles and unencapsidated dsRNAs, mycoviruses are common in all major fungal groups. Over 80 mycovirus species have been officially recognized from ten virus families, but a paucity of nucleic acid sequence data makes assignment of many reported mycoviruses difficult. Although most of the particle types recognized to date are isometric, a variety of morphologies have been found and, additionally, many apparently unencapsidated dsRNAs have been reported. Until recently, most characterized mycoviruses have dsRNA genomes, but ssRNA mycoviruses now constitute about one-third of the total. Two hypotheses for the origin of mycoviruses of plant pathogens are discussed: the first that they are of unknown but ancient origin and have coevolved along with their hosts, the second that they have relatively recently moved from a fungal plant host into the fungus. Although mycoviruses are typically readily transmitted through asexual spores, transmission through sexual spores varies with the host fungus. Evidence for natural horizontal transmission has been found. Typically, mycoviruses are apparently symptomless (cryptic) but beneficial effects on the host fungus have been reported. Of more practical interest to plant pathologists are those viruses that confer a hypovirulent phenotype, and the scope for using such viruses as biocontrol agents is reviewed. New tools are being developed based on host genome studies that will help to address the intellectual challenge of understanding the fungal-virus interactions and the practical challenge of manipulating this relationship to develop novel biocontrol agents for important plant pathogens.

An osmosensing histidine kinase mediates dicarboximide fungicide resistance in Botryotinia fuckeliana (Botrytis cinerea)

A two-component histidine protein kinase gene, homologous to os-1 from Neurospora crassa, was cloned and sequenced from a single ascospore isolate of Botryotinia fuckeliana. A series of nine spontaneous mutants resistant to dicarboximide fungicides was selected from this strain and characterized with respect to fungicide resistance and osmotic sensitivity. Genetic crosses of the mutants with an authentic Daf1 strain showed that the phenotypes mapped to this locus. Single point mutations (seven transitions, one transversion, and one short deletion) were detected in the alleles of the nine mutants sequenced. The mutational changes were shown to cosegregate with the dicarboximide resistance and osmotic sensitivity phenotypes in progeny obtained from crossing selected resistant strains with a sensitive strain. All mutations detected are predicted to result in amino acid changes in the coiled-coil region of the putative Daf1 histidine kinase, and it is proposed that dicarboximide fungicides target this domain.

Taxonomy and Genetic Variation of Botrytis and Botryotinia

The species of the anamorphic genus Botrytis and its associated Botryotinia teleomorphs are briefly assessed. Recent progress in understanding the genetics of variation in the polyphagous B. cinerea (teleomorph Bt. fuckeliana) is summarised, with emphasis on chromosome complement and extrachromosomal elements. Sexual and vegetative compatibility studies are reviewed in relation to the limited evidence of clonality revealed by DNA population markers. It is concluded that in contrast to the traditional view of this species, sexual reproduction plays a major role in determining variation whereas heterokaryosis plays only a limited role. Evidence supporting the existence of a second polyphagous species within B. cinerea sensu lato is discussed. The limited knowledge of the genetics of the host- restricted species is briefly described.

Genome characterization of a flexuous rod-shaped mycovirus, Botrytis virus X, reveals high amino acid identity to genes from plant ‘potex-like’ viruses

Summary.This study reports the molecular characterization of a flexuous rod-shaped mycovirus, Botrytis virus X (BVX), infecting the plant-pathogenic fungus, Botrytis cinerea. BVX contains a ssRNA genome of 6966 nucleotides, and a poly(A) tract at or very near the 3′ terminus. Computer analysis of the genomic cDNA sequence of BVX revealed five potential open reading frames (ORFs). ORF1 showed significant amino acid sequence identity to the replicase proteins of plant ‘potex-like’ viruses, including 73% identity to the RNA-dependent RNA polymerase (RdRp) region of the allexivirus, garlic virus A (GarV-A). The C-terminal region of ORF3 shared amino acid homology with plant ‘potex-like’ coat proteins. The remaining ORFs did not reveal significant homology with known protein sequences. BVX differs substantially from Botrytis virus F (BVF), another flexuous rod-shaped mycovirus characterized from the same B. Cinerea isolate. It is proposed that the mycovirus BVX belongs to a new, as yet unassigned genus in the plant ‘potex-like’ virus group, distinct from BVF.

Presence of double-stranded RNA and virus-like particles in Botrytis cinerea

Double-stranded RNA (dsRNA) was demonstrated in mycelial extracts in 143 of 200 isolates of Botrytis cinerea from a range of hosts in New Zealand. The dsRNA profiles differed widely between isolates in the number, size and relative concentration of dsRNA species present. Virus-like particles (VLPs) were observed by electron microscopy in partly purified virus preparations in three of five dsRNA-containing isolates examined. Morphological types present included isometric particles of varying size classes (approx. 30, 35, 40 and 45 nm) and bacilliform particles (approx. 25 × 63 nm). Such particles were not observed in five isolates apparently lacking dsRNA but flexuous rods with a modal length of approx. 720 nm were present in one. Total RNA extraction of partly purified VLP preparations gave similar profiles to those obtained from mycelial dsRNA extractions of the same isolates, suggesting that the VLPs represent encapsidated dsRNA. Comparison of 12 dsRNA-containing and 12 dsRNA-free isolates indicated slight differences between the two groups in radial growth rate, virulence on bean leaves and sclerotial number but the differences were minor and the ranges overlapped.

Evolution of an Osmosensing Histidine Kinase in Field Strains of Botryotinia fuckeliana (Botrytis cinerea) in Response to Dicarboximide Fungicide Usage

ABSTRACT DNA sequence polymorphisms in the putative two-component histidine protein kinase encoded by the Daf1 gene have been identified within a sample of 5 sensitive and 27 dicarboximide-resistant field strains of Botryotinia fuckeliana (anamorph Botrytis cinerea). The gene of 3948 bp is predicted to encode a 1315-amino acid protein comprising an N-terminal region, an amino acid repeat region, which has been hypothesized to be the binding site for dicarboximide fungicide, and a C-terminal region encompassing kinase and response regulator domains. Two amino acid variants were distinguished among the sensitive strains characterized by alanine (group 1), or threonine (group 2), at position 1259 in the C-terminal region. All resistant strains could be classified into either group 1 or group 2 but, in addition, all showed changes in the second amino acid repeat region. On the basis of the differences in this repeat region, four classes of resistant strains were recognized; class 1 characterized by an isoleucine to serine mutation, class 2 by an isoleucine to asparagine mutation, class 3 by an isoleucine to arginine mutation (all at position 365), and class 4 by an isoleucine to serine mutation (position 365) as well as a glutamine to proline mutation (position 369). All classes showed similar low levels of resistance to iprodione and to vinclozolin, except for class 3 and class 4 strains, which show low resistance to iprodione but moderate (class 3) or high (class 4) resistance to vinclozolin. The classes as a group did not differ from sensitive strains in osmotic sensitivity measured as mycelial growth response, but some class 1 strains showed an abnormal morphology on osmotically amended medium. The evolution of the amino acid differences is discussed in relation to field observations. It is proposed that class 1 and class 2 strains arose by single mutations within the sensitive population, whereas classes 3 and 4 arose by single mutations within a resistant population.

'Candidatus Phytoplasma australiense' is the phytoplasma associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases

Sequence comparisons and phylogenetic analysis of the 16S rRNA genes and the 16S/23S spacer regions of the phytoplasmas associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases revealed minimal nucleotide differences between them resulting in the formation of a monophyletic group. Therefore, along with Australian grapevine yellows, the phytoplasmas associated with Phormium yellow leaf and papaya dieback should also be considered as ‘Candidatus Phytoplasma australiense’.

A review of the plant virus, viroid and mollicute records for New Zealand

Considering New Zealand’s small size and remoteness, a remarkable number of plant pathogens have been recorded. A complete review of the records of plant viruses, viroids and mollicutes in New Zealand found good documentary evidence for 186 viral diseases, 5 viroids and 6 mollicutes, of which 59 viruses, 3 viroids, and 4 mollicutes have been recorded since publication of the previous complete review in 1989. Based on a lack of sufficient documented evidence, a further 27 viruses, 5 viroid and 3 mollicute records were considered unconfirmed. The confirmed pathogens not only infect agricultural and horticultural crops but some also affect the native flora.

Comparison of the complete genome sequence of two closely related isolates of ‘Candidatus Phytoplasma australiense’ reveals genome plasticity

Background‘Candidatus Phytoplasma australiense’ is associated with at least nine diseases in Australia and New Zealand. The impact of this phytoplasma is considerable, both economically and environmentally. The genome of a NZ isolate was sequenced in an effort to understand its pathogenicity and ecology. Comparison with a closely related Australian isolate enabled us to examine mechanisms of genomic rearrangement.ResultsThe complete genome sequence of a strawberry lethal yellows (SLY) isolate of ‘Candidatus Phytoplasma australiense’ was determined. It is a circular genome of 959,779 base pairs with 1126 predicted open reading frames. Despite being 80 kbp larger than another ‘Ca. Phytoplasma australiense’ isolate PAa, the variation between housekeeping genes was generally less than 1% at a nucleotide level. The difference in size between the two isolates was largely due to the number and size of potential mobile units (PMUs), which contributed to some changes in gene order. Comparison of the genomes of the two isolates revealed that the highly conserved 5′ UTR of a putative DNA-directed RNA polymerase seems to be associated with insertion and rearrangement events. Two types of PMUs have been identified on the basis of the order of three to four conserved genes, with both PMUs appearing to have been present in the last common ancestor of ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma australiense’. Comparison with other phytoplasma genomes showed that modification methylases were, in general, species-specific. A putative methylase (xor IIM) found in ‘Ca. Phytoplasma australiense’ appeared to have no analogue in any other firmicute, and we believe has been introduced by way of lateral gene transfer. A putative retrostransposon (ltrA) analogous to that found in OY-M was present in both isolates, although all examples in PAa appear to be fragments. Comparative analysis identified highly conserved 5′ and 3′ UTR regions of ltrA, which may indicate how the gene is excised and inserted.ConclusionsComparison of two assembled ‘Ca. Phytoplasma australiense’ genomes has shown they possess a high level of plasticity. This comparative analysis has yielded clues as to how rearrangements occur, and the identification of sets of genes that appear to be associated with these events.