Publications

4475

Abstract Marine pelagic redoxclines are areas of enhanced biogeochemical cycling inhabited by distinct functional groups of prokaryotes. In this study, the diversity and abundance of archaeal and bacterial nitrifying populations throughout a pelagic redoxcline in the central Baltic Sea were examined using a suite of molecular methods. 16S rRNA/rRNA gene as well as bacterial and archaeal amoA mRNA/amoA gene fingerprints and clone libraries revealed that the putative nitrifying assemblages consisted solely of one crenarchaeotal subcluster, named GD2, which was closely related to Candidatus Nitrosopumilus maritimus. Neither distinct differences between transcript- and gene-based fingerprints nor pronounced differences in the crenarchaeotal composition throughout the whole redoxcline were detected. The abundance of this GD2 subgroup, as determined by the oligonucleotide probe Cren537 and the newly developed and more specific probe Cren679 showed that GD2 and total crenarchaeotal cell numbers were nearly identical throughout the redoxcline. The highest GD2 abundance (2.3 × 105 cells ml−1) occurred in the suboxic zone, accounting for around 26% of total prokaryotic cells. Below the chemocline, GD2 abundance was relatively stable (1.5–1.9 × 105 cells ml−1). Archaeal amoA expression was detected only in the putative nitrification zone and formed a narrow band in the suboxic layer, where ammonium, oxygen, nitrate, nitrite and phosphate concentrations were below 5 μmol l−1. To our knowledge this is the first study to show the dominance of only one crenarchaeotal nitrifying key cluster in a natural habitat. The metabolic properties and survival mechanisms present in this cluster inside and outside the nitrification zone remain to be determined.

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.

Crotalaria spectabilis Roth. (Fabaceae), commonly known as showy rattlebox, is an herbaceous legume mainly used as a green manure crop to improve soil properties and as a source of durable fiber. However, the plant is toxic to mammals and birds because of the presence of pyrrolidizine alkaloids. A native of India and the Malay Peninsula, the species has been introduced into other areas such as the United States and Pacific Islands where the plant is an invader of cultivated lands. Fasciated rattlebox plants were sighted in fields in New Delhi in February 2010, with approximately 99% of the examined plants symptomatic. Symptoms included flattening of stems in a descending gradient of severity from the apex to the base of each affected branch. Shoots showed longitudinal undulations bearing highly reduced leaves and uneven distribution of flowers and fruits. To identify the causal agent, 10 symptomatic plants and 8 asymptomatic plants (latter sampled from a field approximately 1.5 km away) were collected for nucleic acid analysis. Total genomic DNA was extracted from flattened stems as well as the roots of symptomatic plants, and from the same tissues of asymptomatic plants, and subjected to nested-PCR using phytoplasma 16S ribosomal DNA universal primer pair P1/P7, followed by R16F2/R2 (4). A known aster yellows-infected Catharanthus roseus plant was used as a control sample. Results depicted a characteristic phytoplasma amplicon of 1.25 kb in all samples from symptomatic plants and the control plant. No amplification was observed from asymptomatic plants. To obtain a full-length sequence, a representative amplicon was purified with the QIAquick gel extraction kit (QIAGEN, Valencia, CA), cloned into pGEM-T Easy vector (Promega, Madison, WI), and sequenced. Comparison of the 1,243-bp sequence (Genbank Accession No. HM137557) using BLAST analysis of the NCBI database showed 99% homology with sequences of members of 16SrI group phytoplasmas, i.e., ‘Candidatus Phytoplasma asteris’ such as Japanese spurge yellows (AB551736.1), Mulberry yellow dwarf (GQ249410.1), and Bamboo witches'-broom (FJ853161.1) phytoplasmas. The profiles of in vitro restriction fragment length polymorphism (RFLP) analysis obtained by digestion of the nested-PCR products with HhaI, KpnI, and AluI were similar to those of in silico RFLP analyses and coincided with the pattern of the 16SrI-B subgroup. Phylogenetic analysis of phytoplasma 16S rDNA sequences based on the maximum likelihood method using MEGA Version 4.1 also placed the Crotalaria fasciation (CF) phytoplasma within the 16SrI-B cluster. In India, C. tetragona plants infected with 16SrI phytoplasma (FJ185141) causing witches'-broom symptoms (1) showed 98% similarity with the CF phytoplasma. However, the results support previous molecular investigations associating 16SrI phytoplasma with fasciation of herbaceous plants, including Lilium spp. (2) and Asparagus officinalis (3). To our knowledge, C. spectabilis represents a new host that can be fasciated as a result of phytoplasma infection. Because of the weedy nature of C. spectabilis, this host could facilitate spread of the phytoplasma. References: (1) P. Baiswar et al. Plant Pathol. 19:17, 2009. (2) A. Bertaccini et al. FEMS Microbiol. Lett. 249:79, 2005. (3) J. Franova and K. Petrzik. J. Phytopathol. 158:317, 2010.K. (4) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996.

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.