Publications

4537

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.

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.