Sandra Denman

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.

Standardizing the Nomenclature for Clonal Lineages of the Sudden Oak Death Pathogen, Phytophthora ramorum

Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P. ramorum based on a consensus established by the P. ramorum research community. Clonal lineages are named with a two letter identifier for the continent on which they were first found (e.g., NA = North America; EU = Europe) followed by a number indicating order of appearance. Clonal lineages known to date are designated NA1 (mating type: A2; distribution: North America; environment: forest and nurseries), NA2 (A2; North America; nurseries), and EU1 (predominantly A1, rarely A2; Europe and North America; nurseries and gardens). It is expected that novel lineages or new variants within the existing three clonal lineages could in time emerge.

Fungal pathogens of proteaceae

Species of Leucadendron, Leucospermum and Protea (Proteaceae) are in high demand for the international floriculture market due to their brightly coloured and textured flowers or bracts. Fungal pathogens, however, create a serious problem in cultivating flawless blooms. The aim of the present study was to characterise several of these pathogens using morphology, culture characteristics, and DNA sequence data of the rRNA-ITS and LSU genes. In some cases additional genes such as TEF 1-α and CHS were also sequenced. Based on the results of this study, several novel species and genera are described. Brunneosphaerella leaf blight is shown to be caused by three species, namely B. jonkershoekensis on Protea repens, B. nitidae sp. nov. on Protea nitida and B. protearum on a wide host range of Protea spp. (South Africa). Coniothyrium-like species associated with Coniothyrium leaf spot are allocated to other genera, namely Curreya grandicipis on Protea grandiceps, and Microsphaeropsis proteae on P. nitida (South Africa). Diaporthe leucospermi is described on Leucospermum sp. (Australia), and Diplodina microsperma newly reported on Protea sp. (New Zealand). Pyrenophora blight is caused by a novel species, Pyrenophora leucospermi, and not Drechslera biseptata or D. dematoidea as previously reported. Fusicladium proteae is described on Protea sp. (South Africa), Pestalotiopsis protearum on Leucospermum cuneiforme (Zimbabwe), Ramularia vizellae and R. stellenboschensis on Protea spp. (South Africa), and Teratosphaeria capensis on Protea spp. (Portugal, South Africa). Aureobasidium leaf spot is shown to be caused by two species, namely A. proteae comb. nov. on Protea spp. (South Africa), and A. leucospermi sp. nov. on Leucospermum spp. (Indonesia, Portugal, South Africa). Novel genera and species elucidated in this study include Gordonomyces mucovaginatus and Pseudopassalora gouriqua (hyphomycetes), and Xenoconiothyrium catenata (coelomycete), all on Protea spp. (South Africa).

Brenneria goodwinii sp. nov., associated with acute oak decline in the UK

A group of nine Gram-negative staining, facultatively anaerobic bacterial strains isolated from native oak trees displaying symptoms of acute oak decline (AOD) in the UK were investigated using a polyphasic approach. 16S rRNA gene sequencing and phylogenetic analysis revealed that these isolates form a distinct lineage within the genus Brenneria, family Enterobacteriaceae, and are most closely related to Brenneria rubrifaciens (97.6 % sequence similarity to the type strain). Multilocus sequence analysis based on four housekeeping genes (gyrB, rpoB, infB and atpD) confirmed their position within the genus Brenneria, while DNA-DNA hybridization indicated that the isolates belong to a single taxon. The isolates can be differentiated phenotypically from their closest phylogenetic neighbours. The phylogenetic and phenotypic data demonstrate that these isolates from oak with symptoms of AOD represent a novel species in the genus Brenneria, for which the name Brenneria goodwinii sp. nov. (type strain FRB 141(T)  = R-43656(T)  = BCC 845(T)  = LMG 26270(T)  = NCPPB 4484(T)) is proposed.

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)).

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.

Isolation studies reveal a shift in the cultivable microbiome of oak affected with Acute Oak Decline.

Acute Oak Decline is a syndrome within the Oak Decline complex in Britain. Profuse stem bleeding and larval galleries of the native buprestid, Agrilus biguttatus characterize the disease. A systematic study comparing healthy with diseased trees was undertaken. This work reports the result of isolations from healthy trees, diseased and non-symptomatic tissue within AOD affected trees, at five sites in England. Bacteria and fungi were identified using the DNA gyrase B gene, or ITS 1 sequencing. A significantly higher proportion of diseased tissues (82%) yielded more bacteria than either healthy (18%) or non-symptomatic tissue in diseased trees (33%). Overall bacterial community compositions varied at each site, but significant similarities were evident in diseased tissues at all sites. Enterobacteriaceae dominated in diseased trees whereas Pseudomonadaceae dominated healthy trees. Significant associations between diseased tissues and certain bacterial species occurred, implying that the cause of tissue necrosis was not due to random microbiota. Brenneria goodwinii and Gibbsiella quercinecans were key species consistently isolated from diseased tissue; Rahnella victoriana and an un-named Pseudomonas taxon were also frequently isolated from both healthy and diseased trees. Most fungi isolated were from the outer bark and had no significant association with tree health status. It was concluded that there was a shift in the cultivatable bacterial microbiome of diseased trees, with Enterobacteriaceae strongly represented in symptomatic but not healthy tissues. No single species dominated the isolations from diseased tissues and the tissue degradation in AOD is therefore likely to have a polymicrobial cause.

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.

Responses of the two‐spotted oak buprestid, Agrilus biguttatus (Coleoptera: Buprestidae), to host tree volatiles

Abstract BACKGROUND Agrilus bigutattus (Fabricius) is a forest pest of increasing importance in the United Kingdom. The larvae damage weakened native oaks and are thought to contribute to premature tree death. Suspected links with acute oak decline (AOD) are not yet confirmed, but AOD‐predisposed trees appear to become more susceptible to A. biguttatus attack. Thus, management may be necessary for control of this insect. To explore the possibility of monitoring beetle populations by baited traps, the host tree volatiles regulating A. biguttatus–oak interactions were studied. RESULTS Biologically active volatile organic compounds in dynamic headspace extracts of oak foliage and bark were identified initially by coupled gas chromatography–electroantennography (GC‐EAG) and GC–mass spectrometry (GC‐MS), and the structures were confirmed by GC coinjection with authentic compounds. Of two synthetic blends of these compounds comprising the active leaf volatiles, the simpler one containing three components evoked strongly positive behavioural responses in four‐arm olfactometer tests with virgin females and males, although fresh leaf material was more efficient than the blend. The other blend, comprising a five‐component mixture made up of bark volatiles, proved to be as behaviourally active for gravid females as bark tissue. CONCLUSIONS These initial results on A. biguttatus chemical ecology reveal aspects of the role of attractive tree volatiles in the host‐finding of beetles and underpin the development of semiochemically based surveillance strategies for this forest insect.

Microbiome and infectivity studies reveal complex polyspecies tree disease in Acute Oak Decline

Decline-diseases are complex and becoming increasingly problematic to tree health globally. Acute Oak Decline (AOD) is characterized by necrotic stem lesions and galleries of the bark-boring beetle, Agrilus biguttatus, and represents a serious threat to oak. Although multiple novel bacterial species and Agrilus galleries are associated with AOD lesions, the causative agent(s) are unknown. The AOD pathosystem therefore provides an ideal model for a systems-based research approach to address our hypothesis that AOD lesions are caused by a polymicrobial complex. Here we show that three bacterial species, Brenneria goodwinii, Gibbsiella quercinecans and Rahnella victoriana, are consistently abundant in the lesion microbiome and possess virulence genes used by canonical phytopathogens that are expressed in AOD lesions. Individual and polyspecies inoculations on oak logs and trees demonstrated that B. goodwinii and G. quercinecans cause tissue necrosis and, in combination with A. biguttatus, produce the diagnostic symptoms of AOD. We have proved a polybacterial cause of AOD lesions, providing new insights into polymicrobial interactions and tree disease. This work presents a novel conceptual and methodological template for adapting Koch’s postulates to address the role of microbial communities in disease.