Discrete lineages within Alternaria alternata species group: Identification using new highly variable loci and support from morphological characters.

The Alternaria alternata species group is ubiquitous in the environment acting as saprotrophs, human allergens, and plant pathogens. Many morphological species have been described within the group and it is unclear whether these represent re-descriptions of the same species or discrete evolutionary taxa. Sequencing of five loci identified three major lineages within the A. alternata species group. These loci included three new phylogenetic loci (TMA22, PGS1, and REV3) identified as highly variable based on publically available genome sequence data for Dothideomycete species. Lineages were identified as A. alternata ssp. arborescens, A. alternata ssp. tenuissima, and A. alternata ssp. gaisen in accordance with the placement of reference isolates. The phylogenetic results were supported by morphological analysis, which differentiated strains in A. alternata ssp. arborescens and A. alternata ssp. tenuissima and also aligned with previous morphological species descriptions for A. arborescens and A. tenuissima. However, phylogenetic analysis placed the morphologically described species A. alternata and A. mali within the A. alternata ssp. tenuissima and did not support them as discrete taxa. As A. alternata are of phytosanitary importance, the molecular loci used in this study offer new opportunities for molecular identification of isolates by national plant protection organizations.

Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens.

The vascular wilt fungi Verticillium dahliae and V. albo-atrum infect over 200 plant species, causing billions of dollars in annual crop losses. The characteristic wilt symptoms are a result of colonization and proliferation of the pathogens in the xylem vessels, which undergo fluctuations in osmolarity. To gain insights into the mechanisms that confer the organisms' pathogenicity and enable them to proliferate in the unique ecological niche of the plant vascular system, we sequenced the genomes of V. dahliae and V. albo-atrum and compared them to each other, and to the genome of Fusarium oxysporum, another fungal wilt pathogen. Our analyses identified a set of proteins that are shared among all three wilt pathogens, and present in few other fungal species. One of these is a homolog of a bacterial glucosyltransferase that synthesizes virulence-related osmoregulated periplasmic glucans in bacteria. Pathogenicity tests of the corresponding V. dahliae glucosyltransferase gene deletion mutants indicate that the gene is required for full virulence in the Australian tobacco species Nicotiana benthamiana. Compared to other fungi, the two sequenced Verticillium genomes encode more pectin-degrading enzymes and other carbohydrate-active enzymes, suggesting an extraordinary capacity to degrade plant pectin barricades. The high level of synteny between the two Verticillium assemblies highlighted four flexible genomic islands in V. dahliae that are enriched for transposable elements, and contain duplicated genes and genes that are important in signaling/transcriptional regulation and iron/lipid metabolism. Coupled with an enhanced capacity to degrade plant materials, these genomic islands may contribute to the expanded genetic diversity and virulence of V. dahliae, the primary causal agent of Verticillium wilts. Significantly, our study reveals insights into the genetic mechanisms of niche adaptation of fungal wilt pathogens, advances our understanding of the evolution and development of their pathogenesis, and sheds light on potential avenues for the development of novel disease management strategies to combat destructive wilt diseases.

A genetic linkage map of Venturia inaequalis, the causal agent of apple scab.

BACKGROUND: Venturia inaequalis is an economically-important disease of apple causing annual epidemics of scab worldwide. The pathogen is a heterothallic ascomycete with an annual cycle of sexual reproduction on infected apple leaf litter, followed by several cycles of asexual reproduction during the apple growing season. Current disease control is achieved mainly through scheduled applications of fungicides. Genetic linkage maps are essential for studying genome structure and organisation, and are a valuable tool for identifying the location of genes controlling important traits of interest such as avirulence, host specificity and mating type in V. inaequalis. In this study, we performed a wide cross under in vitro conditions between an isolate of V. inaequalis from China and one from the UK to obtain a genetically diverse mapping population of ascospore progeny isolates and produced a map using AFLP and microsatellite (SSR) markers. FINDINGS: Eighty-three progeny were obtained from the cross between isolates C0154 (China) x 01/213 (UK). The progeny was screened with 18 AFLP primer combinations and 31 SSRs, and scored for the mating type locus MAT. A linkage map was constructed consisting of 294 markers (283 AFLPs, ten SSRs and the MAT locus), spanning eleven linkage groups and with a total map length of 1106 cM. The length of individual linkage groups ranged from 30.4 cM (Vi-11) to 166 cM (Vi-1). The number of molecular markers per linkage group ranged from 7 on Vi-11 to 48 on Vi-3; the average distance between two loci within each group varied from 2.4 cM (Vi-4) to 7.5 cM (Vi-9). The maximum map length between two markers within a linkage group was 15.8 cM. The MAT locus was mapped to a small linkage group and was tightly linked to two AFLP markers. The map presented is over four times longer than the previously published map of V. inaequalis which had a total genetic distance of just 270 cM. CONCLUSION: A genetic linkage map is an important tool for investigating the genetics of important traits in V. inaequalis such as virulence factors, aggressiveness and mating type. The linkage map presented here represents a significant improvement over currently published maps for studying genome structure and organisation, and for mapping genes of economic importance on the V. inaequalis genome.

Population Variation of Apple Scab (Venturia inaequalis) Isolates from Asia and Europe.

Apple scab, caused by Venturia inaequalis, is one of the most of damaging diseases worldwide on apple and currently is managed mainly by scheduled applications of fungicides. Understanding pathogen population structure is important for breeding and deployment of resistant cultivars. Isolates of V. inaequalis were sampled from a number of cultivars in China, India, and the United Kingdom to estimate differences in pathogen populations. Amplified fragment length polymorphism (AFLP) markers were used to genotype isolates, mostly from China and the United Kingdom. The AFLP data indicated that, overall, there were significant differences in V. inaequalis populations from China and the United Kingdom. Within China, there was no significant differentiation associated with their geographical or cultivar origins. In contrast, populations from four cultivars in two U.K. orchards (monoculture of Gala and a mixture orchard of Bramley, Cox, and Worcester) differed significantly. Furthermore, populations from Gala and Worcester were more homogenous than expected but those from Cox were more diverse than expected. In total, 80 isolates were selected randomly from three countries for virulence testing: 20 from the United Kingdom (10 from Gala and 10 from Cox), 30 from China (10 from Gala, 10 from Fuji, and 10 from Qingquan), and 30 from India (10 from Gala, 10 from Golden Delicious, and 10 from Black Ben Davis); of these 80 isolates, 41, 47, and 59 were inoculated against each of these cultivars in the United Kingdom, India, and China, respectively. The two local cultivars from India (Black Ben Davis) and the United Kingdom (Cox) were more resistant against non-indigenous isolates, particularly those from China, than they were against indigenous isolates; the Chinese local cultivar (Qingguan) showed a higher general level of resistance against isolates regardless of their origin.

Isolates of Verticillium dahliae Pathogenic to Crucifers Are of at Least Three Distinct Molecular Types.

ABSTRACT Diverse isolates of the soilborne wilt fungi Verticillium dahliae and V. albo-atrum were studied to understand the nature and origins of those infecting cruciferous hosts. All isolates from cruciferous crops produced microsclerotia, and the majority produced long conidia with a high nuclear DNA content; these isolates were divided into two groups by amplified fragment length polymorphism (AFLP) analysis. One group could be subdivided by other criteria such as rRNA sequences and mitochondrial DNA restriction fragment length polymorphism (RFLP) analysis. Two crucifer isolates were short spored and had a low nuclear DNA content. The results are consistent with the crucifer isolates being interspecific hybrids. The long-spored isolates are best regarded as amphihaploids (or allodiploids) with the AFLP groups probably each representing separate interspecific hybridization events. The short-spored crucifer isolates appear to be derived from interspecific hybrids and are here called 'secondary haploids'. Molecular evidence suggests that one parent in the crosses was similar to V. dahliae. The other parent of the amphihaploids seems to have been more similar to V. albo-atrum than to V. dahliae, but was distinct from all isolates of either species so far studied. The implications for the taxonomy of crucifer isolates are discussed and the use of the name V. longisporum, proposed elsewhere for just some of these isolates, is discouraged.

Plant pathogenic Verticillium species: how many of them are there?

SUMMARY Two of the currently widely accepted species in the section Nigrescentia of the genus Verticillium are major plant pathogens inducing wilt diseases in a wide range of mainly dicotyledonous hosts. Three species closely related to these two are less important wilt pathogens and soil saprophytes. A sixth species, V. theobromae, causes the cigar end of banana. Molecular and genetic studies have shown that these species represent a complex pool of discrete lineages of varying degrees of relatedness with unknown levels of gene flow between them. Most isolates are haploid, but some are thought to be amphihaploid interspecific hybrids. Until our understanding of this complex is much improved, it seems most appropriate to add only one new species, for wilt isolates primarily associated with potato and producing dark-resting-mycelium in bundles (currently known as V. albo-atrum Grp2). It is suggested that the following be retained: (i) V. dahliae to include all isolates which produce only microsclerotia, (ii) V. albo-atrum to cover the majority of isolates producing only dark-resting-mycelium (and not in bundles), and (iii) V. nigrescens, V. nubilum, V. tricorpus and V. theobromae for the minor wilt pathogens/saprophytes and the non-wilt pathogen.

An immunodominant membrane protein gene from the Western X-disease phytoplasma is distinct from those of other phytoplasmas.

Membrane proteins mediate several important processes, including attachment, in several Mollicute species. Phytoplasmas are non-culturable plant pathogenic mollicutes that are transmitted in a specific manner by certain phloem-feeding insect vectors. Because it is likely that phytoplasma membrane proteins are involved with some aspect of the transmission process, their identification, isolation and characterization are important first steps in understanding phytoplasma transmission. A 32 kDa immunodominant protein (IDP) from the Western X-disease (WX) phytoplasma was purified from infected plants by immunoprecipitation using monoclonal antibodies, and two peptides from a tryptic digest were sequenced. PCR primers designed from these sequences amplified a 145 bp product which hybridized with WX-related phytoplasmas in Southern blots. This PCR product was used to identify a 2.5 kbp ECO:RI-HIN:dIII fragment that was cloned and sequenced. A complete 864 bp ORF (idpA) was identified for which the putative translation product contained both of the tryptic digest peptide sequences that were used to design the PCR primers. Analysis of the predicted IdpA sequence indicated two transmembrane domains but no cleavage point. The amino acid sequence had no significant homology with other known phytoplasma IDP genes. The idpA ORF was cloned into an Escherichia coli expression vector and a fusion protein of the predicted size was identified in Western blots using a WX-specific antiserum. A rabbit polyclonal antiserum was prepared to the purified expression protein and this reacted with both the E. coli-expressed and native WX phytoplasma proteins. This newly identified WX IDP (IdpA) is distinct from other known mollicute membrane proteins.