Susan Kirk

Phytophthora alni sp. nov. and its variants: designation of emerging heteroploid hybrid pathogens spreading on Alnus trees

In 1993 a destructive new Phytophthora pathogen of riparian Alnus trees was discovered in the UK and subsequently shown to be present in other parts of Europe. The new Phytophthora comprised a group of emergent heteroploid hybrids, probably between P. cambivora and a species related to P. fragariae. These included a common, near tetraploid standard hybrid, the presumptive allopolyploid; and four scarcer major variant types with chromosome numbers intermediate between diploid and tetraploid, named the Swedish, Dutch, German and UK variants. The standard hybrid type is formally designated here as Phytophthora alni subsp. alni. The Swedish variant is designated as P. alni subsp. uniformis; and the Dutch, German and UK variants collectively as P. alni subsp. multiformis. The properties of the Dutch, German and UK variants within subsp. multiformis are informally described. The problems of designating emergent species hybrids under the International Code of Botanical Nomenclature and the reasons for the taxonomic choices made are discussed.

Phytophthora kernoviae sp. nov., an invasive pathogen causing bleeding stem lesions on forest trees and foliar necrosis of ornamentals in the UK

A new Phytophthora pathogen of trees and shrubs, previously informally designated Phytophthora taxon C, is formally named here as P. kernoviae. P. kernoviae was discovered in late 2003 during surveys of woodlands in Cornwall, south-west England, for the presence of another invasive pathogen, P. ramorum. P. kernoviae is self-fertile (homothallic), having plerotic oogonia, often with distinctly tapered stalks and amphigynous antheridia. It produces papillate sporangia, sometimes markedly asymmetric with medium length pedicels. Its optimum temperature for growth is ca 18 degrees C and upper limit ca 26 degrees. Currently, P. kernoviae is especially noted for causing bleeding stem lesions on mature Fagus sylvatica and foliar and stem necrosis of Rhododendron ponticum. P. kernoviae is the latest of several invasive tree Phytophthoras recently identified in the UK. Its geographical origins and the possible plant health risk it poses are discussed.

Designation of the EAN and NAN races of Ophiostoma novo-ulmi as subspecies

The two subpopulations of the Dutch elm disease pathogen Ophiostoma novo-ulmi , previously known as the Eurasian (EAN) and North American (NAN) races, are redesignated as subspecies novo-ulmi and americana. In addition to their partial reproductive isolation, wide range of physiological and molecular differences and different geographic ranges, the two subspecies can be discriminated by their perithecial form and dimensions. These perithecial differences are described, using perithecia produced in multiple intra-subspecies crosses. Perithecia of subsp. novo-ulmi have an average neck length of ca 450 μm, base width of ca 103 μn and neck length:base width ratio ca 4.4. Perithecia of subsp. americana have an average neck length of ca 295 μn, base width ca 116 μm and neck length:base width ratio ca 2.6. The average shape and dimensions of subsp. americana perithecia is similar to that of O. ulmi. The average perithecial form of subsp. novo-ulmi , as well as of O. himal-ulmi , is rather distinctive. The current known geographical distribution of subspecies novo-ulmi and americana , based on > 6500 samples, is presented.

Sibling species within Ophiostoma piceae

Fifty-five hardwood or conifer derived isolates of the heterothallic fungus Ophiostoma piceae from Europe and North America were separated into two inter-sterile mating groups. One group, designated OPH, came almost exclusively from hardwood hosts while the other isolates, designated OPC, came predominantly (75%) from conifers. The A and B sexual compatibility types occurred within both groups, though seven OPH isolates from North America were all B types. The OPH and OPC forms of O. piceae are considered here to be distinct reproductively isolated sibling species. The OPH group may equate to O. querci and the OPC group to O. piceae . Each sibling species occurs in both Europe and North America. Their possible evolutionary origins are discussed.

Production of gametangia by Phytophthora ramorum in vitro.

Until now gametangia have not been obtained between paired European A1 and American A2 isolates of Phytopthora ramorum in vitro. Their production in artificial culture relies on interspecific pairings. Using P. drechsleri and P. cambivora testers, 51 of 110 P. ramorum isolates from across Europe were all shown to be A1s; while 32 of 38 American isolates from across California and southwest Oregon were shown to be A2s. However, these interspecific pairings are complex, unusually slow and unpredictable. A range of culture media and conditions are described that were tested, unsuccessfully, with a view to enhancing the efficiency of the interspecific pairings. In further tests, gametangia were obtained between A1 and A2 isolates of P. ramorum when juvenile, pre-chlamydospore producing mycelia were mixed together on carrot agar. The gametangia formed in 3-10 d, sparsely to frequently, initially only within the boundaries of the mixed inocula but subsequently in the extended mycelial growth. Chlamydospores were also produced. This inoculum-mixing method, though again sometimes unpredictable, should enhance efficiency of testing for compatibility types and facilitate further studies on whether the sexual outcrossing system of P. ramorum is functional. Differences between sexual reproduction of P. ramorum and that of other heterothallic Phytophthora species are discussed.

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.

Rahnella victoriana sp. nov., Rahnella bruchi sp. nov., Rahnella woolbedingensis sp. nov., classification of Rahnella genomospecies 2 and 3 as Rahnella variigena sp. nov. and Rahnella inusitata sp. nov., respectively and emended description of the genus Rahnella ☆

Isolations from oak symptomatic of Acute Oak Decline, alder and walnut log tissue, and buprestid beetles in 2009-2012 yielded 32 Gram-negative bacterial strains showing highest gyrB sequence similarity to Rahnella aquatilis and Ewingella americana. Multilocus sequence analysis (using partial gyrB, rpoB, infB and atpD gene sequences) delineated the strains into six MLSA groups. Two MLSA groups contained reference strains of Rahnella genomospecies 2 and 3, three groups clustered within the Rahnella clade with no known type or reference strains and the last group contained the type strain of E. americana. DNA-DNA relatedness assays using both the microplate and fluorometric methods, confirmed that each of the five Rahnella MLSA groups formed separate taxa. Rahnella genomospecies 2 and 3 were previously not formally described due to a lack of distinguishing phenotypic characteristics. In the present study, all five Rahnella MLSA groups were phenotypically differentiated from each other and from R. aquatilis. Therefore we propose to classify the strains from symptomatic oak, alder and walnut and buprestid beetles as: Rahnella victoriana sp. nov. (type strain FRB 225(T)=LMG 27717(T)=DSM 27397(T)), Rahnella variigena sp. nov. (previously Rahnella genomosp. 2, type strain CIP 105588(T)=LMG 27711(T)), Rahnella inusitata sp. nov. (previously Rahnella genomosp. 3, type strain DSM 30078(T)=LMG 2640(T)), Rahnella bruchi sp. nov. (type strain FRB 226(T)=LMG 27718(T)=DSM 27398(T)) and Rahnella woolbedingensis sp. nov. (type strain FRB 227(T)=LMG 27719(T)=DSM 27399(T)).

Survival of clones of NAN Ophiostoma novo-ulmi around its probable centre of appearance in North America.

275 isolates of Ophiostoma novo-ulmi , sampled across the southern Great Lakes region of North America from Wisconsin to Ohio in 1996, were analysed for vegetative compatibility (vc) types. Over 60% of the sample was a clonal vc component, comprising only two vc types (the AMSG and EUSG); the remainder of the sample was highly heterogeneous for vc types. Vc diversity was highest in Indiana and Michigan; close to where NAN O. novo-ulmi probably first appeared in North America. Each vc clone exhibited a uniform RAPD haplotype and a lower frequency of the A compared with the B mating type. This is consistent with the clones being mainly asexually spread, but with a potential for inbreeding, an unusual feature in a ’genetic clone’. The heterogeneous component, however, exhibited diverse RAPD haplotypes and a near 1:1 ratio of A:B mating types, indicating that the mainly novel vc types of this component arise via sexual recombination. In Europe, dominant vc clones have been quickly replaced by novel vc types. In the Great Lakes region, however, vc clones appear to have survived for over 50 years despite a high potential for emergence of new vc types via sexual recombination. No differences were found between the clonal and heterogeneous components with regard to growth rate and pathogenicity, two important fitness parameters. Two other factors, a low level of selection imposed by deleterious d-factor viruses and a density dependent effect associated with vegetative incompatibility, may have favoured prolonged survival of the clones in North America.

Description of Brenneria roseae sp. nov. and two subspecies, Brenneria roseae subspecies roseae ssp. nov and Brenneria roseae subspecies americana ssp. nov. isolated from symptomatic oak ☆

Gram-negative, facultatively anaerobic bacteria were isolated from symptomatic oak tissue in the UK and USA. Partial gyrB sequencing placed ten strains in the genus Brenneria, with B. goodwinii as the closest phylogenetic relative. The strains were investigated further using a polyphasic approach including MLSA (based on partial gyrB, rpoB, infB and atpD gene sequences), 16S rRNA gene sequencing, DNA-DNA relatedness studies and both phenotypic and chemotaxonomic assays. The MLSA and 16S rRNA gene analyses separated the strains into two groups based on origin, suggesting that they belong to Brenneria as two novel species. However, the DNA-DNA relatedness values revealed a closer relationship between the groups and indicated that they should belong to the same species. As the two groups of strains from the UK and USA can be differentiated from each other phenotypically and by ERIC PCR fingerprints, it is proposed to classify them as novel subspecies of a novel Brenneria species. The name Brenneria roseae sp. nov. (FRB 222(T)=LMG 27714(T)=NCPPB 4581(T)) is proposed, with Brenneria roseae subsp. roseae ssp. nov. (FRB 222(T)=LMG 27714(T)=NCPPB 4581(T)) for the strains from the UK and Brenneria roseae subsp. americana ssp. nov. (FRB 223(T)=LMG 27715(T)=NCPPB 4582(T)) for the strains from the USA.

Gibbsiella greigii sp. nov., a novel species associated with oak decline in the USA.

In 2010, cream-coloured, Gram-negative staining, facultatively anaerobic enterobacteria were isolated from a single black oak tree (Quercus kelloggii) exhibiting decline symptoms in southern California, USA. These 12 isolates were tentatively identified as Gibbsiella quercinecans based on partial gyrB sequencing. Closer examination of the strains using multilocus sequence analysis, based on partial sequences of gyrB, rpoB, infB and atpD genes, and almost complete 16S rRNA gene sequencing suggested that the isolates belong to a novel taxon within the genus Gibbsiella with G. quercinecans as their closest phylogenetic relative. DNA-DNA relatedness studies confirmed that the strains belong to a single taxon in Gibbsiella, which can be differentiated from other members of the genus by several phenotypic traits. Therefore, the name Gibbsiella greigii sp. nov. is proposed for this novel species isolated from symptomatic Q. kelloggii in the USA with FRB 224(T) (=LMG 27716(T)=NCPPB 4583(T)) as the type strain.