Publications

4526

A novel aerobic, heterotrophic bacterium, designated BiosLi39T, was isolated from the South East Pacific Ocean. Cells were Gram-negative gliding rods forming yellow colonies on marine agar. The isolate was oxidase-, catalase- and alkaline phosphatase-positive andβ-galactosidase-negative. Strain BiosLi39Tgrew at 20-37 °C (optimum 30 °C), at pH 7.0–9.0 (optimum pH 8.0) and with 20–60 g NaCl l−1(optimum 30–50 g NaCl l−1). The fatty acids (>1 %) comprised iso-C14 : 0, iso-C15 : 1G, iso-C15 : 0, anteiso-C15 : 0, C15 : 1G, C15 : 0, iso-C15 : 02-OH, iso-C16 : 1G, iso-C16 : 0, iso-C16 : 03-OH, iso-C16 : 02-OH, iso-C17 : 03-OH, C17 : 02-OH and three unidentified components with equivalent chain lengths of 17.87, 18.10 and 18.71. A significant proportion of the hydroxylated fatty acids are amide-linked. The lipid pattern indicated the presence of phosphatidylethanolamine, two unidentified aminolipids and three unidentified polar lipids. The strain contained menaquinone 7 as the sole respiratory lipoquinone and did not produce flexirubin-type pigments. The G+C content of the genomic DNA was 37.2 mol%. Comparative 16S rRNA gene sequence analysis indicated that strain BiosLi39Twas distantly related to all of the representatives of the phylumBacteroidetes. Its closest relative wasMarinoscillum furvescensIFO 15994T, with which it shared 92.5 % 16S rRNA gene sequence similarity. On the basis of genotypic, phenotypic and chemotaxonomic characteristics, we propose a novel genus and species,Ekhidnagen. nov., sp. nov., with type strain BiosLi39T(=DSM 19307T=CIP 109600T=OOB 398T).

Citrus huanglongbing, putatively caused by the associated bacterium ‘Candidatus Liberibacter asiaticus’, is the greatest threat to the world citrus industry today. The bacterium is spread locally and regionally by the citrus psyllid Diaphorina citri, and also can be disseminated by propagation of contaminated scion budwood that is grafted to the appropriate rootstock. The planting of ‘Ca. Liberibacter asiaticus’-free trees is a component of a comprehensive strategy to manage huanglongbing. In contrast to the scion budwood, the rootstocks used to produce these trees are grown from seed. This research was undertaken to provide evidence as to whether or not ‘Ca. L. asiaticus’ can be transmitted through seed. Two groups of 360 or more seedlings each of various citrus species were grown from seed removed from fruit on trees that were symptomatic for huanglongbing and confirmed to be infected with ‘Ca. L. asiaticus’ by polymerase chain reaction (PCR) tests. These seedlings were tested multiple times over periods of up to 3 years. No symptoms typical of huanglongbing, such as blotchy leaf mottle, chlorotic shoots, or dieback of branches, were observed in these seedlings, and none of these 723 seedlings tested positive for the presence of ‘Ca. L. asiaticus’ even after repeated testing by sensitive quantitative PCR assays. Some sour orange seedlings did have quite pronounced and atypical growth, including stunting and mild to severe leaf malformation. These atypical growth habits were limited to seedlings that arose from zygotic embryos as determined by expressed-sequence tag simple-sequence repeat analyses. Thus, no evidence of transmission of ‘Ca. L. asiaticus’ via seed was obtained, and an earlier report of transmission of the pathogen through seed was not confirmed.

Pelargonium capitatum (rose pelargonium) is a plant indigenous to southern Africa, originally brought to Western Australia for its ornamental qualities. It has since become naturalized in the Southwest Australian Floristic Region, recognized for its high level of species endemism, where it is a serious invasive weed in bushlands and coastal dunes. Since P. capitatum outcompetes native species it is listed among the top 10 most important coastal weeds of the region (3). In 2008, large patches of stunted, dying, and dead P. capitatum plants were observed within a population covering coastal dunes at Woodman Point, Western Australia (GPS coordinates 32°07′40.51″S, 115°45′28.39″E). Diseased plants had small misshapen leaves in clumps that were often chlorotic or pink, shortened internodes, and exhibited phylloidy typical of infection by a phytoplasma. From August 2009 to January 2010, samples from symptomatic and asymptomatic plants were collected from the site and from plants of an asymptomatic population at another site located on the Murdoch University campus nearby. DNA was extracted from 15 samples collected from symptomatic and asymptomatic plants at the dune site and from five at the campus site. Briefly, 2 to 5 g of leaf and stem tissue was cut into 5-mm pieces and shaken overnight in 30 ml of phosphate-buffered saline buffer. Supernatant was filtered and a pellet was collected by centrifugation. After resuspension in 500 μl of extraction buffer (200 mM Tris-HCl [pH 7.5] 250mM NaCl, 25mM ethylenediaminetetraacetic acid, 0.5% sodium dodecyl sulfate, and 2% polyvinylpyrrolidone), DNA was precipitated in 500 μl of cold isopropanol. Samples were tested for the presence of phytoplasma ribosomal 16S DNA by nested PCR using phytoplasma universal primers P1/P7 followed by amplification with primers Tint, R16mF2, and R16mR1 (1,2,4). Phytoplasma-specific DNA sequences were synthesized directly from amplicons using the above primers. Phytoplasma was detected from both symptomatic and asymptomatic plant samples collected from the dune site but not from the campus site. Analysis of the nine sequences obtained (GenBank Accession Nos. HM583339, HM583340, HM583341, HM583342, HM583343, HM583344, HM583345, HM583346, and HM583347) revealed high sequence identity between isolates (~99%) and with the ‘Candidatus Phytoplasma aurantifolia’ (16SrII) group of phytoplasmas (1,4). Presence of phytoplasma in symptomatic plants was confirmed by histological examination of stem sections stained with Dienes' stain. This finding is significant because there is potential for utilizing this phytoplasma to control P. capitatum where it has invaded ecologically significant sites, although its effect on indigenous plants must be determined first. Although phytoplasmas within the 16SrII group have been identified in Australia previously (1,4), to our knowledge, this is the first report of it infecting P. capitatum. References: (1) K. S. Gibb et al. Phytopathology 85:169, 1995. (2) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) B. M. J. Hussey et al. Western Weeds. A Guide to the Weeds of Western Australia. 2nd ed. Plant Protection Society of Western Australia, Victoria Park, 2007. (4) M. Saqib et al. J. R. Soc. West. Aust. 90:175, 2007.