Publications

4378

Despite differences in their morphologies, comparative analyses of 16S rRNA gene sequences revealed high levels of similarity (>94 %) between strains of the filamentous bacterium ‘CandidatusNostocoida limicola’ and the cocciTetrasphaera australiensisandTetrasphaera japonicaand the rodTetrasphaera elongata, all isolated from activated sludge. These sequence data and their chemotaxonomic characters, including cell wall, menaquinone and lipid compositions and fingerprints of their 16S–23S rRNA intergenic regions, support the proposition that these isolates should be combined into a single genus containing six species, in the familyIntrasporangiaceaein theActinobacteria. This suggestion receives additional support from DNA–DNA hybridization data and when partial sequences of therpoC1gene are compared between these strains. Even though few phenotypic characterization data were obtained for these slowly growing isolates, it is proposed, on the basis of the extensive chemotaxonomic and molecular evidence presented here, that ‘CandidatusN. limicola’ strains Ben 17, Ben 18, Ben 67, Ben 68 and Ben 74 all be placed into the speciesTetrasphaera jenkinsiisp. nov. (type strain Ben 74T=DSM 17519T=NCIMB 14128T), ‘CandidatusN. limicola’ strain Ben 70 intoTetrasphaera vanveeniisp. nov. (type strain Ben 70T=DSM 17518T=NCIMB 14127T) and ‘CandidatusN. limicola’ strains Ver 1 and Ver 2 intoTetrasphaera veronensissp. nov. (type strain Ver 1T=DSM 17520T=NCIMB 14129T).

Conventional PCR and two real‐time PCR (RTi‐PCR) methods were developed and compared using the primer pairs CQULA03F/CQULA03R and CQULA04F/CQULA04R, and TaqMan probe CQULAP1 designed from a species‐specific sequence of the rplJ/rplL ribosomal protein gene, for diagnosis of citrus huanglongbing (HLB) disease in southern China. The specificity and sensitivity of the three protocols for detecting ‘ Candidatus Liberibacter asiaticus’ in total DNA extracts of midribs collected from infected citrus leaves with symptoms in Guangxi municipality, Jiangxi Province and Zhejiang Province, were tested. Sensitivities using extracted total DNA (measured as copy number, CN per µ L of recombinant plasmid solution) were 439·0 (1·30 × 10 5 CN µ L −1 ), 4·39 (1·30 × 10 3 CN µ L −1 ) and 0·44 fg µ L −1 (1·30 × 10 2 CN µ L −1 ) for conventional PCR, TaqMan and SYBR Green I (SGI) RTi‐PCR, respectively. SGI RTi‐PCR was the most sensitive, but its specificity needed to be confirmed by running a melt‐curve assay. The TaqMan RTi‐PCR assay was rapid and had the greatest specificity. Concerning the correlation of PCR detection results with the various HLB symptoms, uneven mottling of leaves had the highest positive rate (96·50%), indicating that leaf mottling was the most reliable symptom for field surveys. Dynamic analysis results from the TaqMan assays showed that the titre (CN) g −1 citrus tissue of ‘ Ca. L. asiaticus’ was highest between October and December (threshold cycle ( C t ) average = 29·3, CN = 3·35 × 10 7 ) and lowest between March and May ( C t average = 32·0, CN = 5·10 × 10 6 ) in 2004 and 2005. The optimized molecular‐based assays should prove useful for presymptom diagnosis of HLB disease, monitoring and identification of ‘ Ca. L. asiaticus’, and field epidemic regulation.

Huanglongbing (greening) disease caused by a nonculturable, phloem-limited bacterium is a severe disease of citrus. On the basis of the influence of temperature on host symptoms and the causal agent, this disease can be categorized as Asian caused by “Candidatus Liberibacter asiaticus”, African caused by “Ca. L. africanus”, and American caused by “Ca. L. americanus”. Kumquat (Fortunella margarita (Lour.) Swingle), a member of the Rutaceae is an economically important crop for export and local consumption in Taiwan. Recently, a Huanglongbing-like disease was found on kumquat in Yilan County, the largest kumquat-producing area in northeastern Taiwan. Even though the disease has been reported on Citrus spp. from Taiwan, it has never been reported on kumquat. Symptoms of infected kumquat were mottling, yellowing, hardening, and curling of leaves followed by premature defoliation, twig dieback, decay of feeder rootlets and lateral roots, and ultimately the death of the entire plant. Typical sieve-tube-restricted bacteria were observed in kumquat cells by electron microscopy (1). In addition, psyllid-transmission tests demonstrated that the Asian psyllid (Diaphorina citri) could transmit this bacterium to healthy kumquats. Positive bud graft transmissions were obtained to F. margarita, F. japonica (Thunb.) Swingle, F. obovata Hort. ex Tanaka, Luchen sweet orange (Citrus sinensis (L.) Osb.), and Wentan pummelo (C. maxima f. sp. butan Hay.). These inoculated plants showed symptoms in 3 to 8 months, and bacteria could be detected by polymerase chain reaction (PCR) using a common primer pair that amplified a 226-bp specific DNA fragment (2). For further molecular identification, the bacterial DNA was extracted from the inoculated plants and PCR was performed by using two sets of primers selected from the 16S rRNA region (GenBank Accession No. L22532) and 16S/23S intergenic spacer region (GenBank Accession No. AB019793). The expected DNA fragments of 1,389 bp and 862 bp were, respectively, amplified from symptomatic plants but not from healthy plants. The PCR products were cloned and sequenced (GenBank Accession Nos. DQ302750 and DQ207841). The 16S rRNA has 98 to 99% identity and 16S/23S intergenic spacer region has 99% identity to the corresponding region of “Ca. L. asiaticus” in GenBank. These molecular analyses confirm the presence of “Ca. L. asiaticus” in kumquat. Since Huanglongbing has been rarely reported naturally on kumquat, further analysis of this bacterium as a special strain of “Ca. L. asiaticus” is needed. References: (1) M. Garnier et al. Ann. Microbiol. 135A:169, 1984. (2) T. H. Hung et al. J. Phytopathol. 147:599, 1999.

Summary Twelve strains of filamentous bacteria morphologically identified as ‘ Microthrix parvicella ’ were isolated from industrial activated sludge wastewater treatment plants. 16S rRNA gene sequences analysis showed that these strains were all closely related to ‘ Candidatus Microthrix parvicella’. Six of them, however, had a 16S rRNA gene similarity of only 95.7% and 96.7% to ‘ Candidatus Microthrix parvicella’ suggesting the presence of a new species. The name ‘ Candidatus Microthrix calida’ is proposed for this new microorganism. The physiological properties of these six isolates supported the description of a new taxon. The ‘ Candidatus Microthrix calida’ strains produced thin filaments (0.3–0.7 µm diameter), they did not grow on the media supporting the growth of ‘ Candidatus Microthrix parvicella’ and could be cultivated at higher temperature (up to 36.5°C). Preliminary data on substrate uptake were obtained by microautoradiography on pure culture. Two new fluorescence in situ hybridization (FISH) probes, Mpa‐T1‐1260 specific for ‘ Candidatus Microthrix calida’ and Mpa‐all‐1410 targeting both Microthrix species, were designed. The presence of Microthrix spp. was investigated in 114 activated sludge plants. ‘ Microthrix parvicella ’ morphotype was detected in 23% of the analysed samples and FISH analysis revealed that ‘ Candidatus Microthrix calida’ was present in 5% of them. The remaining ‘ M. parvicella ’ filaments were positive with probe Mpa‐all‐1410 but could not all be identified as ‘ Candidatus Microthrix parvicella’ suggesting the presence of more hitherto undescribed biodiversity within this morphotype.