Publications

4513

Huanglongbing (HLB), also known as greening, is one of the most important diseases of citrus worldwide. The causal agent is a gram-negative bacterium known to inhabit the phloem of infected plants. Three different candidate species infect citrus: ‘Candidatus Liberibacter africanus’ found in the African continent; ‘Ca. L. asiaticus’ found in Asia, Brazil, and the United States; and ‘Ca. L. americanus’ found in Brazil. (1). Tobacco is an easily transformable plant species that can be used as an experimental host system to quickly screen for candidate genes useful to control plant pathogens. However, no evidence exists on the ability of this plant species to sustain populations of ‘Ca. L. americanus’. With the purpose of transmitting ‘Ca. L. americanus’ from citrus to tobacco, fragments of healthy stems of Cuscuta spp. (dodder) were used to connect an HLB-infected sweet orange plant to each of 10 healthy plants of Nicotiana tabacum L. cv. Xanthi and allowed to remain connected for 30, 45, and 50 days. Three different HLB-infected orange plants and 30 tobacco plants were used in three independent experiments. Most HLB-exposed Xanthi plants exhibited chlorotic leaves after 50 days of exposure probably because of the parasitic effect of dodder; however, an average of 6, 1, and 3 Xanthi plants exhibited a unique blotchy mottle symptom after 30, 45, and 50 days of exposure, respectively. Symptomatic and asymptomatic leaves were collected and analyzed by PCR. The results consistently confirmed the presence of ‘Ca. L. americanus’ only in symptomatic leaves. Sequencing of the PCR product and comparison to the NCBI database also confirmed the identity of the pathogen as ‘Ca. L. americanus’. Electron microscopy analysis of four symptomatic leaves indicated the presence of bacterium-like bodies with round to elongated bacilliform shapes and surrounded by two membranes. These bodies resembled those already described in HLB-infected citrus in Brazil (1). The evidence presented above confirms the successful transmission of ‘Ca. L. americanus’ from citrus to Xanthi using the parasitic plant Cuscuta spp. Reference: (1) F. A. O. Tanaka et al. Fitopatol. Bras. 31:99, 2006.

The objective of this study was to evaluate the use of real-time TaqMan PCR assays for detection of coinfections with “ Candidatus Mycoplasma haemominutum” (Mhm), and Mycoplasma haemofelis (Mhf), in vitro and over time in experimentally infected cats. First, the ability of each real-time PCR assay to detect and quantify mixed infections was determined in vitro by testing mixtures of plasmids containing Mhm and Mhf 16S rDNA with each assay. Subsequently, 4 specific pathogen-free (SPF) cats, 2 of which were splenectomized, were inoculated with blood from a cat infected with both Mhm and Mhf. Sixteen blood samples were then collected from each cat over a 55-day period. Each of the 64 postinoculation samples was tested using both conventional polymerase chain reaction (cPCR) and real-time PCR for the 16S rRNA gene of each organism. When applied to mixtures of plasmid DNA from each species, the results of quantitation with each of the real-time PCR assays approximately reflected the number of plasmid copies present. Forty-nine of 64 post-inoculation samples (77%) were positive using both cPCR and real-time PCR, 4 (6%) were positive using cPCR only, and 3 (5%) were positive using real-time PCR only. Both organisms were detected in 23 samples using real-time PCR. Mixed infections were not detected using cPCR. The size of the corresponding cPCR products suggested infection with Mhm in 4 and Mhf in 18 of these samples. The use of multiple separate real-time PCR assays rather than cPCR alone should thus be considered for epidemiologic studies of hemoplasmosis in cats.

Witches'-broom disease of lime (WBDL) caused by ‘Candidatus Phytoplasma aurantifolia’ is a devastating disease in the Sultanate of Oman, United Arab Emirates, and southern Iran. The disease primarily affects lime (Citrus aurantifolia), but in Iran, it is also found in bakraee, a natural C. reticulata hybrid. The disease has been experimentally transmitted from lime to several citrus cultivars by grafting and to a number of herbaceous hosts by dodder. However, the natural vector of ‘Ca. P. aurantifolia’ has not been determined. The most common phloem-feeding insect associated with lime trees in the area is the leafhopper Hishimonus phycitis. The WBDL phytoplasma has been detected in the body of this leafhopper by ELISA and PCR (1), but previous attempts to establish its vector status have failed. It was recently reported that the leafhopper can release the phytoplasma into a sugar solution by feeding through a Parafilm membrane (4). Here we report successful transmission of WBDL phytoplasma to bakraee seedlings by H. phycitis. The leafhopper nymphs and adults were collected in a WBDL-infected lime orchard in Minab (Hormozgan Province) in May of 2006. Of more than 100 leafhopper samples tested, at least 70% were positive for the phytoplasma by PCR using P1/P7 primer pair (3). Additional field-collected leafhoppers were caged (five per plant) on bakraee seedlings at the two-leaf stage in pots in the greenhouse in Zarghan (Fars Province). After 8 weeks, the remaining leafhoppers were killed with an insecticide. Six months after inoculation, 3 of 10 inoculated plants showed typical symptoms of WBDL, including bud proliferation, general chlorosis, and stunting. Symptomatic plants were strongly positive in PCR assays using primer pair P1/P7. No amplification was obtained with healthy control lime or nonsymptomatic bakraee seedlings. Amplified P1/P7 primed PCR products (1,800 bp) from experimentally vector-challenged bakraee seedlings, captured H. phycitis, and a naturally infected lime tree from Minab were subjected to restriction fragment length polymorphism (RFLP) analysis using AluI, HhaI, HpaII, RsaI, and TaqI enzymes. RFLP patterns from these sources were identical and similar to those reported earlier (2). These analyses verified the identity of WBDL phytoplasma in experimentally infected bakraee seedlings. To our knowledge, this is the first report of natural transmission of ‘Ca. P. aurantifolia’ by H. phycitis. References: (1) J. M. Bové et al. Proc. Conf. IOCV 12:342. 1993. (2) A. J. Khan et al. Phytopathology 92:1038, 2002. (3) B. Schneider et al. Pages 369–380 in: Molecular and Diagnostic Procedures in Mycoplasmology. Vol. 2. S. Razin and J. G. Tully, eds. Academic Press, New York, 1995. (4) M. Siampour et al. Iran. J. Plant Pathol. 41:139 (Farsi) and 35 (English), 2006.

Cacti (Opuntia spp.) are perennial, evergreen, succulent plants native to arid areas of the Americas. Because of their aesthetic appearance, many cacti have been cultivated and introduced to other parts of the world as ornamentals. Cacti are susceptible to phytoplasma infections and develop witches'-broom (WB) disease. Currently, all reported cactus WB cases are associated with infections by phytoplasmas in the peanut witches'-broom group (16SrII) (1,2,4). During a phytoplasma diversity survey carried out during 2004 in Yunnan, China, we collected 29 malformed and 14 healthy-looking naturally occurring cactus plants from 14 locations representing five geographical regions. Each of the 29 malformed plants exhibited stunted growth and possessed clusters of highly proliferating cladodia, typical symptoms of cactus WB disease. Nested-PCR was carried out on the DNA samples extracted from young cladodia of these plants using phytoplasma-universal 16S rDNA primers P1A/P7A and R16F2n/R16R2 (3). Results revealed that all 29 diseased plants that were examined were infected by phytoplasmas, whereas all 14 healthy-looking plants were negative for phytoplasmas. Subsequent restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 1.25-kb 16S rDNA fragments indicated that 28 diseased plants were infected by a phytoplasma of group 16SrII, whereas one plant (from Suan Village) was infected by a ‘Candidatus Phytoplasma asteris’-related (group 16SrI) phytoplasma designated as strain YN26. Nucleotide sequence analysis of the strain YN26 partial rRNA operon (GenBank Accession No. EF190970), covering a near full-length 16S rRNA gene, a 16S-23S rRNA intergenic spacer, a tRNA-Ile gene, and a partial 23S rRNA gene, suggested that this phytoplasma is most closely related to an ash witches'-broom phytoplasma (GenBank Accession No. AY566302, 99.7% identity) and an epilobium phyllody phytoplasma (GenBank Accession No. AY101386, 99.7% identity), both members of subgroup16SrI-B. This YN26-infected cactus plant was transferred to a greenhouse and maintained for more than 2 years, during which time DNA samples were extracted and tested two additional times. The same 16S rDNA RFLP pattern type was consistently obtained in these tests, confirming that the plant remained infected by the 16SrI phytoplasma. To our knowledge, this is the first report of a natural infection of a cactus species by a group 16SrI phytoplasma. Since this 16SrI-cactus WB phytoplasma was found in the same geographical location where 16SrII-cactus WB phytoplasma was detected both in this and a previous study (1), the findings raised the question whether 16SrI- and 16SrII-cactus WB phytoplasmas have overlapping geo- and bioecological niches. References: (1) H. Cai et al. Plant Pathol. 51:394, 2002. (2) E. Choueiri et al. Plant Dis. 89:1129, 2005. (3) I. M. Lee et al. Int. J. Syst. Evol. Microbiol 54:337, 2004. (4) N. Leyva-Lopez et al. Phytopathology (Abstr.) 89(suppl):S45, 1999.

Four formate-utilizing methanogens were isolated from ovine (strain KM1H5-1PT) and bovine (strains AK-87, OCP and ZA-10T) rumen contents. Based on 16S rRNA gene sequence analysis, the methanogen strains were found to belong to the orderMethanobacterialesin the genusMethanobrevibacter. Strains ZA-10Tand KM1H5-1PTgained energy for growth by the reduction of CO2to CH4using H2or formate exclusively as electron donors. Increasing formate concentrations to 220 mM in batch cultures increased the growth of strain KM1H5-1PTbut did not affect the growth of strain ZA-10T. Substrate specificity and resistance to cell-wall lysis supported the affiliation of the strains to the genusMethanobrevibacter. Strains ZA-10Tand KM1H5-1PTshowed 16S rRNA gene sequence similarity of 98.0 and 98.6 % to their closest recognized relatives,Methanobrevibacter thaueriCWTandMethanobrevibacter ruminantiumM1T, respectively. DNA–DNA hybridization experiments indicated that the strains were not affiliated at the species level to their closest recognized relatives, with DNA reassociation values of only 28 % between strains ZA-10TandMethanobrevibacter thaueriCWTand <25 % between strains KM1H5-1PTandMethanobrevibacter ruminantiumM1T. Based on the data presented, the new strains are considered to represent two novel species of the genusMethanobrevibacter, for which the namesMethanobrevibacter milleraesp. nov. (type strain ZA-10T=DSM 16643T=OCM 820T) andMethanobrevibacter olleyaesp. nov. (type strain KM1H5-1PT=DSM 16632T=OCM 841T) are proposed.