Publications

4981

Changes in carbohydrate metabolism in sweet orange ( Citrus sinensis ) infected with ‘ Candidatus Liberibacter asiaticus’, a purported cause of citrus Huanglongbing (HLB), were investigated. Starch levels in HLB‐infected leaves with and without symptoms increased 3·1‐ and 7·9‐fold, respectively, compared to healthy controls. In symptomless leaves, sucrose and fructose accumulated significantly ( P  <   0·05) in both midribs and lobes, and glucose only in the midribs (greater than fivefold); whereas maltose levels were reduced to 64·6% and 86·8% in the midribs and foliar lobes, respectively, of the values in healthy leaves. In leaves with symptoms, sucrose and glucose remained at high levels compared to healthy leaves, whilst no accumulation of fructose was observed; by contrast, the maltose content decreased to as low as 49·6% of that in healthy leaves. Fourfold induction of cell‐wall‐bound invertase activity was detected in both types of leaves on diseased plants. Additionally, the expression profiles of starch breakdown genes suggested that the transcription of DPE2 and MEX1 was down‐regulated. Together with the reduction of maltose, it is suggested that the impairment of starch breakdown may contribute to the starch accumulation in infected leaves. The imbalance of carbohydrate partitioning and its relation to disease physiology are discussed.

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).

ABSTRACT Mealybugs (Coccoidea: Pseudococcidae) are sap-sucking plant parasites that harbor bacterial endosymbionts within specialized organs. Previous studies have identified two subfamilies, Pseudococcinae and Phenacoccinae, within mealybugs and determined the primary endosymbionts (P-endosymbionts) of the Pseudococcinae to be Betaproteobacteria (“ Candidatus Tremblaya princeps”) containing Gammaproteobacteria secondary symbionts. Here, the P-endosymbionts of phenacoccine mealybugs are characterized based on 16S rRNA from the bacteria of 20 species of phenacoccine mealybugs and four outgroup Puto species (Coccoidea: Putoidae) and aligned to more than 100 published 16S rRNA sequences from symbiotic and free-living bacteria. Phylogenetic analyses recovered three separate lineages of bacteria from the Phenacoccinae, and these are considered to be the P-endosymbionts of their respective mealybug hosts, with those from (i) the mealybug genus Rastrococcus belonging to the Bacteroidetes , (ii) the subterranean mealybugs, tribe Rhizoecini, also within Bacteroidetes , in a clade sister to cockroach endosymbionts ( Blattabacterium ), and (iii) the remaining Phenacoccinae within the Betaproteobacteria , forming a well-supported sister group to “ Candidatus Tremblaya princeps.” Names are proposed for two strongly supported lineages: “ Candidatus Brownia rhizoecola” for P-endosymbionts of Rhizoecini and “ Candidatus Tremblaya phenacola” for P-endosymbionts of Phenacoccinae excluding Rastrococcus and Rhizoecini. Rates of nucleotide substitution among lineages of Tremblaya were inferred to be significantly faster than those of free-living Betaproteobacteria . Analyses also recovered a clade of Gammaproteobacteria , sister to the P-endosymbiont lineage of aphids (“ Candidatus Buchnera aphidicola”), containing the endosymbionts of Putoidae, the secondary endosymbionts of pseudococcine mealybugs, and the endosymbionts of several other insect groups.

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.