Clive Brasier

Rare interspecific hybrids in natural populations of the Dutch elm disease pathogens Ophiostoma ulmi and O. novo-ulmi

Ophiostoma ulmi and O. novo-ulmi are partly reproductively isolated, morphologically, behaviourally and molecularly distinct species responsible for the first and current pandemics of Dutch elm disease, respectively. Among >11000 isolates sampled from Dutch elm disease sites across Eurasia and North America since 1973, nine could not be assigned to O. ulmi or O. novo-ulmi . Of these isolates one (P129) was from Poland and eight (d10, d11, e12, e27, e28, e37, f30 and g3) were from a single bark sample in Portugal. These nine isolates were termed ‘fast-waxy’ isolates because of their unusual cultural characteristics. The possibility that they were interspecific hybrids was investigated. When compared with O. ulmi and O. novo-ulmi for colony pattern, growth-rate, optimum temperature for growth, vascular wilt ability, elm bark colonizing ability, cerato-ulmin toxin production, ability to fertilize (as ♂) O. novo-ulmi , and ability to be fertilized (as ♀) by O. ulmi , they exhibited a combination of O. ulmi -like, O. novo-ulmi -like, intermediate or novel characters (female sterility) consistent with their being hybrids. P129 and representative Portuguese isolates d10 and e27 each exhibited a different combination of characters, indicating each was a different hybrid genotype. When d10 and e27 were independently crossed to the same O. novo-ulmi isolate, differences in their F 1 progeny sets for growth-rate and pathogenicity distributions were consistent with their being different recombinant genotypes. A molecular analysis of P129, d10 and e27 using RAPDs of genomic DNA, rDNA RFLPs and cerato-ulmin gene sequences confirmed that each was a unique interspecific hybrid. The mechanism of origin of these hybrids and their evolutionary significance are discussed. Combined experimental and circumstantial evidence indicates that they are relatively unfit, rare and probably transient, and that they arise when O. novo-ulmi invades territory occupied by O. ulmi and replaces it. Nonetheless, the possibility that the hybrids act as a genetic bridge, facilitating transfer of novel vegetative compatibility loci and other loci form O. ulmi to O. novo-ulmi at recent epidemic fronts, requires investigation.

Naturally occurring non cerato-ulmin producing mutants of Ophiostoma novo-ulmi are pathogenic but lack aerial mycelium

Cerato-ulmin is a protein implicated as a major toxin in the development of Dutch elm disease symptoms in elms infected with Ophiostoma novo-ulmi. O. novo-ulmi isolates typically produce fibrous-striate aerial mycelium on malt extract agar and secrete high levels of cerato-ulmin in liquid medium. However, two different genotypes of O. novo-ulmi (NAN race) from a population sample in Portugal, isolates MAFf8 and PG470, exhibited unusual flat-waxy ‘non-aerial mycelial’ colony types and were found to produce no detectable cerato-ulmin. The two isolates otherwise behaved as normal O. novo-ulmi isolates, including being highly pathogenic to Ulmus procera . When MAFf8 and PG470 were crossed with wild-type NAN O. novo-ulmi isolates, non-aerial mycelial colony phenotype and non cerato-ulmin production was shown in each case to be controlled by a single pleiotropic mutation, termed cu − . A further cross showed the cu − locus was probably allelic in the two isolates. The influence of the cu − locus on colony phenotypes in O. novo-ulmi supports the view that cerato-ulmin is a fungal hydrophobin. The normal pathogenic ability of the two cu − isolates raises questions about the proposed role of cerato-ulmin as a wilt toxin.

De-novo generation of mitochondrial DNA plasmids following cytoplasmic transmission of a degenerative disease in Ophiostoma novo-ulmi

A mitochondrial DNA plasmid was detected in an isolate of Ophiostoma novo-ulmi with a degenerative disease. The DNA plasmid was shown to be derived from the mitochondrial DNA and to map to a region corresponding to the large ribosomal RNA coding region. The DNA plasmid was not transmitted into sexual (ascospore) progeny, irrespective of whether the diseased isolate acted as the female or male parent. Transmission of the disease to healthy, plasmid-free, “recipient” isolates by hyphal anastomosis was not accompanied by transfer of mitochondrial DNA or DNA plasmid from the diseased “donor” isolate, but resulted in de-novo generation of different plasmids, derived from the recipients mitochondrial DNA.

Nucleotide-sequence analysis indicates that a DNA plasmid in a diseased isolate of Ophiostoma novo-ulmi is derived by recombination between two long repeat sequences in the mitochondrial large subunit ribosomal RNA gene.

The nucleotide sequence of a mitochondrial plasmid (2234 bp) in a diseased isolate of Ophiostoma novo-ulmi, and sequences of the mitochondrial DNA that overlap and flank the plasmid end-points, have been determined. The plasmid was shown to be derived from the O. novo-ulmi mitochondrial large subunit ribosomal RNA gene and contained most of intron 1, the whole of exon 2, and probably the first part of intron 2. Within intron 1 there is an open reading frame with the potential to encode a 323 amino-acid polypeptide which contained dodecapeptide sequences typical of RNA maturases and DNA endonucleases. The endpoints of the plasmid in the mtDNA were located within two 90-bp direct imperfect repeat sequences, one of which comprised the last 7 bp of exon 1 and the first 83 bp of intron 1 whilst the other comprised the last 7 bp of exon 2 and the first 83 bp of intron 2. It is proposed that the Ld plasmid was generated by intramolecular recombination between these two repeats with the crossover point probably within the last 15 bp.

Sequence of RNA-dependent RNA polymerase genes provides evidence for three more distinct mitoviruses in Ophiostoma novo-ulmi isolate Ld.

Three of the twelve double-stranded (ds) RNAs, dsRNAs 1a, 1b and 3b, which are located in the mitochondria of a diseased isolate, Ld, of the Dutch elm disease fungus, Ophiostoma novo-ulmi have been cDNA cloned and sequenced. Examination of the sequences of the RdRp genes predicted from the nucleotide sequences of the three dsRNAs suggest that they constitute the genome of three new mitoviruses.

Multiple insertions and deletions determine the size differences between the mitochondrial DNAs of the EAN and NAN races of Ophiostoma novo-ulmi

Restriction endonuclease fragment maps of the mitochondrial DNAs (mtDNAs) of two isolates of the North American (NAN) race and two isolates of the Eurasian (EAN) race of the Dutch elm disease pathogen, Ophiostoma novo-ulmi , were constructed and compared. Over half of the restriction maps of all four isolates were indistinguishable. Furthermore, the relative map positions of the fragments that hybridized with cloned fragments of Podospora anserina mtDNA containing genes of known function were the same in all the isolates. These results indicate a close relationship between the NAN and EAN mtDNAs. The mtDNAs of the two NAN isolates W2 tol1 (55·7 kb) and PG402 (56·7 kb), differed by only a single 1·0 kb insertion. However, the sizes of the mtDNAs of the two EAN isolates, H2023 (63·6 kb) and H277 (65·6 kb), were significantly greater than those of the two NAN isolates, due to the presence of additional insertions at a variety of locations in the mtDNA genome. Compared to the NAN PG402 mtDNA, the EAN H2023 and H277 mtDNAs contained four and five additional insertions, respectively, only two of which were in common. Unique restriction sites due to very small insertions/deletions or point mutations were also found in H2023 mtDNA. The implications of these results with regard to the evolution of O. novo-ulmi and its NAN and EAN races are discussed.

Additional file 10: of Host-induced aneuploidy and phenotypic diversification in the Sudden Oak Death pathogen Phytophthora ramorum

Diverse CCNVs revealed by BIC-seq analysis (upper graph for each panel) and a read-depth analysis for heterozygous allele ratios using 10 Kb long non-overlapping sliding window (lower graph). A concatenated view of the 52 largest scaffolds with the total length of 300 MB, which corresponding to approximately a half of the total genome of Phytophthora ramorum, are shown. Scaffolds numbers for large CCNV regions are indicated with pink bars, and those for LOH are shown with green bars. Scales show log (base 2) fold difference between sample isolates and reference isolates for BIC-seq analysis and log (base 2) ratios of alleles of sample isolates for the heterozygous allele ratio analysis. At each heterozygous locus, a read count ratio (more-abundant allele/less-abundant allele) was calculated. A) Pr-102, the genome sequence isolate is trisomic. B) Oak isolate Pr-16 is trisomic as well as cnLOH. C) Trisomy persists in a re-isolate of Pr-102 from California bay. D) A re-isolate Pr745#4 and E) an oak isolate Pr-140.7 are CCNV heterokaryons. F) nwt EU1 isolate P2386 revealed CCNVs and LOH when wt EU1 isolate P2363 was used as a reference. (PDF 1014 kb)