Janet L. Taylor

Comparison of the 5.8s rDNA and internal transcribed spacer sequences of isolates of Leptosphaeria maculans from different pathogenicity groups

The regions coding for the 5.8s rRNA and the flanking internal transcribed spacers (ITS1 and ITS2) from nine isolates of the blackleg pathogen Leptosphaeria maculans and one isolate of Sclerotinia sclerotiorum were amplified by the polymerase chain reaction and sequenced. Five of the L. maculans isolates were highly virulent to Brassica plants, two were weakly virulent and two were isolated from the cruciferous weed Thlaspi arvense. The 5.8s DNA sequences of all L. maculans isolates were identical. However, there were major differences in both ITS1 and ITS2 sequences that correlated with the pathogenicity grouping. Phylogenetic analysis of the ITS sequences by both parsimony and maximum-likelihood methods indicated that each pathogenicity group was statistically different from each other with the weaklyvirulent isolates being more closely related to the Thlaspi than to the highly-virulent isolates. The relationships of L. maculans to other fungi, based on a comparison of the 5.8s rDNA sequences, are discussed.

Phylogenetic relationship among several Leptosphaeria species based on their ribosomal DNA sequences

The sequences coding for the 18 s and 5·8 s ribosomal RNAs and their internal transcribed spacers (ITS1 and ITS2) from isolates of five Leptosphaeria species were amplified by PCR and sequenced. Phylogenetic analysis of these sequences indicated that, with the exception of L. bicolor, Leptosphaeria species are closely related. There were two distinct lineages among species of Leptosphaeria , one represented by two weakly virulent isolates of L. maculans and the other by L. microscopica, L. nodorum and L. doliolum . The highly virulent isolate of L. maculans was not placed definitively in either lineage and may represent an additional branch of the genus. The sugarcane pathogen L. bicolor was found to be less closely related to the other Leptosphaeria species than to Alternaria alternata and Pleospora herbarum , and is likely to be a representative of a different genus among the Pleosporales.

The phytoalexin camalexin is not metabolized by Phoma lingam, Alternaria brassicae, or phytopathogenic bacteria

Abstract The metabolism of the cruciferous phytoalexin camalexin by the blackleg (Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm), and blackspot (Alternaria brassicae (Berk.) Sacc.) fungi, as well as the phytopathogenic bacteria Pseudomonas syringae, P. cichorii, Erwinia carotovora, and Xanthomonas campestris was evaluated. The micro-organisms were incubated with camalexin for different time periods and the cultures were extracted and analyzed by high performance liquid chromatography and proton nuclear magnetic resonance. The results of these analyses indicated that camalexin was not metabolized by any of the micro-organisms. Importantly, camalexin appeared to inhibit the production of the phytotoxin destruxin B by A. brassicae, but did not affect the phytotoxins produced by P. lingam. Furthermore, the growth of bacteria and fungi was inhibited almost completely in the presence of camalexin at concentrations ≥0.40 mM.

PHOMAPYRONES : THREE METABOLITES FROM THE BLACKLEG FUNGUS

Abstract Three new 2-pyrones were isolated from the phytopathogenic fungus Leptosphaeria maculans, the asexual stage Phoma lingam, and their structures were elucidated by spectroscopic methods.

Phomalairdenone: a new host-selective phytotoxin from a virulent type of the blackleg fungus Phoma lingam.

The chemical structure and bioactivity of phomalairdenone (7), a new sesquiterpenic host-selective phytotoxin produced by an unusual virulent type isolate of the blackleg fungus [Phoma lingam, perfect stage Leptosphaeria maculans (Desm.) Ces. et de Not.] are reported.

Phomaligin a and other yellow pigments in Phoma lingam and P. wasabiae

Abstract The structure determination of a new yellow pigment obtained from weakly virulent isolates of Phoma lingam , the blackleg fungus, is described. Investigations of the secondary metabolite profile and the molecular genetic characteristics of weakly virulent isolates of P. lingam indicate it is closely related to P. wasabiae .

Phomaligols and phomaligadiones: New metabolites from the blackleg fungus

Abstract The isolation and structure elucidation of four new metabolites (1–4) are described. The structures were determined employing 2D NMR spectroscopy. The isolation of these unusual metabolites from Phoma lingam may have chemotaxonomic implications.

Isolation and characterization of novel defence‐related genes induced by copper, salicylic acid, methyl jasmonate, abscisic acid and pathogen infection in Brassica carinata

Summary To examine the defence response in Brassica carinata we differentially screened a cDNA library made from CuCl(2)-treated (Cu) leaves. The sequence of 17 of the 27 cDNA clones examined that showed Cu-induction had a high similarity to defence genes from other plant species. Among other clones that showed higher expression in the Cu leaves were two cDNAs encoding polypeptides of 351 and 250 amino acids, designated BcCJS1 and BcCJAS1. BcCJS1 had similarity to S-adenosyl-l-methionine: salicylic acid carboxyl methyltransferase from Clarkia breweri. However, the enzyme activity was not found in extracts from E. coli expressing BcCJS1. BcCJAS1 did not show extensive similarity to any genes with known function in the databases but it did contain three regions of amino acid sequence that are frequently found in amidotransferases. A third Cu-induced mRNA, Bcp6PGL, showed very high (86%) similarity to a putative 6-phosphogluconolactonase (6PGL) from Arabidopsis thaliana. In addition to Cu induction, BcCJS1 expression was induced by methyl jasmonate (MeJA) and salicylic acid (SA), BcCJAS1 expression by MeJA, SA and abscisic acid and Bcp6PGL expression by MeJA. The expression of all three genes increased after Alternaria brassicae infection. BcCJS1 and BcCJAS1 were induced within 1 h after MeJA- but not until 3 h after SA-treatment. The expression of both genes was systemically induced after infection with a compatible or incompatible fungal pathogen. SA systemically induced only BcCJAS1. The effects of various inhibitors of signalling pathways on expression of the three genes were studied.

Production of 2,5-dioxopiperazine by a new isolate type of the blackleg fungus Phoma lingam

New isolates of the blackleg fungus [Phoma lingam, perfect stage Leptosphaeria maculans (Desm.) Ces. et de Not.], virulent to the traditionally resistant brown mustard (Brassica juncea), produce L-valyl-L-tryptophan anhydride, a 2,5-dioxopiperazine different from those produced by blackleg isolates virulent to canola (B. napus and B. rapa).

Small scale functional genomics of the blackleg fungus, Leptosphaeria maculans: analysis of a 38 kb region

A small-scale, functional genomics approach was applied to Leptosphaeria maculans, the dothideomycete that causes blackleg disease of oilseed Brassicas. Analysis of 126 Expressed Sequence Tags identified a putative Zn[II]t2Cys6 gene, znr1, most similar to qflR, the gene regulating aflatoxin production in Aspergillus species. Eight other genes were identified within a 38.1 kb region of a cosmid clone surrounding this gene. A novel monomodular amino acyl activator gene (maa1), most similar to bacterial non-ribosomal peptide synthetases involved in antibiotic and toxin production was adjacent to znr1. Additionally, homologues for genes encoding a major facilitator transporter, a DNA mismatch repair protein and an endo/exoxylanase found in other organisms in clusters of functionally related genes involved in antibiotic or toxin production were present on the clone. The linkage of these genes suggested that they may function in metabolite production in L. maculans. Nevertheless, targeted disruption of znr1 and maa1 did not result in any alteration in growth in vitro, metabolite production, pathogenicity on Brassica napus or B. juncea, or changes in transcription of the other genes on the cosmid.