Erwinia papayae sp. nov., a pathogen of papaya (Carica papaya)

Bacterial canker of papaya (Carica papaya) emerged during the 1980s in different islands of the Caribbean. Nineteen strains of Gram-negative, rod-shaped, non-spore-forming bacteria isolated from papaya were compared to 38 reference and type strains of phytopathogenic Enterobacteriaceae and related bacteria. Phylogenetic analysis of 16S rRNA gene sequences showed that the papaya strains belonged to the genus Erwinia. The DNA G+C content of strain CFBP 5189T, 52·5 mol%, is in the range of the genus Erwinia. The 19 papaya strains were all pathogenic to papaya and were differentiated clearly from type or reference strains of phytopathogenic enterobacteria and related bacteria by phenotypic tests. The papaya strains constituted a discrete DNA hybridization group, indicating that they belonged to a unique genomic species. Thus, strains pathogenic to papaya belong to a novel species for which the name Erwinia papayae sp. nov. is proposed, with the type strain CFBP 5189T (=NCPPB 4294T).

PCR Detection, Characterization, and Distribution of Virulence Genes in Aeromonas spp

ABSTRACT We found 73.1 to 96.9% similarity by aligning the cytolytic enterotoxin gene of Aeromonas hydrophila SSU (AHCYTOEN; GenBank accession no. M84709 ) against aerolysin genes ofAeromonas spp., suggesting the possibility of selecting common primers. Identities of 90 to 100% were found among the eight selected primers from those genes. Amplicons obtained fromAeromonas sp. reference strains by using specific primers for each gene or a cocktail of primers were 232 bp long. Of hybridization group 4/5A/5B (HG4/5A/5B), HG9, and HG12 or non-Aeromonas reference strains, none were positive. PCR-restriction fragment length polymorphism (PCR-RFLP) withHpaII yielded three types of patterns. PCR-RFLP 1 contained two fragments (66 and 166 bp) found in HG6, HG7, HG8, HG10, and HG11. PCR-RFLP 2 contained three fragments (18, 66, and 148 bp) found in HG1, HG2, HG3, and HG11. PCR-RFLP 3, with four fragments (7, 20, 66, and 139 bp), was observed only in HG13. PCR-amplicon sequence analysis (PCR-ASA) revealed three main types. PCR-ASA 1 had 76 to 78% homology with AHCYTOEN and included strains in HG6, HG7, HG8, HG10, and HG11. PCR-ASA 2, with 82% homology, was found only in HG13. PCR-ASA 3, with 91 to 99% homology, contained the strains in HG1, HG2, HG3, and HG11. This method indicated that 37 (61%) of the 61 reference strains were positive with the primer cocktail master mixture, and 34 (58%) of 59 environmental isolates, 93 (66%) of 141 food isolates, and 100 (67%) of 150 clinical isolates from around the world carried a virulence factor when primers AHCF1 and AHCR1 were used. In conclusion, this PCR-based method is rapid, sensitive, and specific for the detection of virulence factors of Aeromonas spp. It overcomes the handicap of time-consuming biochemical and other DNA-based methods.

Incidence and Diversity of Double-Stranded RNAs Occurring in the Chestnut Blight Fungus, Cryphonectria parasitica, in China and Japan

Isolates of the chestnut blight fungus, Cryphonectria parasitica, were randomly sampled from 10 subpopulations in China and 8 subpopulations in Japan and screened for the presence of double-stranded (ds) RNA using an immunoblot procedure with a monoclonal antibody specific for dsRNA. The overall incidence of dsRNA in C. parasitica was 2 and 6% in China and Japan, respectively, much lower than the 28% found previously in North American populations. Genetic relatedness of dsRNAs within and among populations in China and Japan was examined using RNA-RNA hybridizations with labeled-dsRNA probes. The majority of Chinese and Japanese dsRNAs were members of a single hybridization group, related to Cryphonectria hypovirus 1 (CHV1) from Europe, and are referred to as CHV1-type dsRNAs. No evidence was obtained for genetic differentiation between CHV1-type dsRNAs sampled in China and Japan. Five Japanese isolates contained two genetically distinct dsRNAs. The larger segments (approximately 12 kilobases [kb]) were members of the CHV1 hybridization group, while the smaller segments (approximately 3 kb) did not hybridize with any known dsRNA from C. parasitica including the 2.7-kb dsRNA from isolate NB631 from New Jersey or dsRNA from isolate RC1 from Michigan. Two small dsRNA segments (approximately 1.8 and 2 kb) from one isolate sampled from Liaoning Province in northeastern China did not hybridize with any of the dsRNA probes tested including several described dsRNAs of similar size from C. parasitica in North America. Three dsRNAs from Anhui Province, China, hybridized to Cryphonectria hypovirus 2 (CHV2)-specific probes and are thus referred to as CHV2-type dsRNAs. Sequence analysis of 1,627 base pairs of these three CHV2-type dsRNAs from Anhui revealed that they were identical to each other in the region sequenced and very closely related to CHV2-NB58, isolated from New Jersey. We speculate that CHV2-NB58 may have been introduced into North America from this part of China. This is the first record of a North American C. parasitica dsRNA that is genetically related to a dsRNA from Asia.

Inducible β-Lactam Resistance in Aeromonas hydrophila: Therapeutic Challenge for Antimicrobial Therapy

Despite the abundant amount of knowledge about inducible chromosomally mediated β-lactamases among Aeromonasspecies, extended-spectrum β-lactam-resistant A. hydrophila strains selected in clinical practice were rarely reported. In the present study, two strains of A. hydrophila, A136 and A139, with markedly different susceptibilities to extended-spectrum cephalosporins were isolated from blood and the tip segment of an arterial catheter of a burn patient. Another strain (A136m) was selected in vitro by culturing A136 in a subinhibitory concentration of cefotaxime, the β-lactam agent administered for the treatment of Aeromonas bacteremia in this patient. Typing studies by arbitrarily primed PCR and pulsed-field gel electrophoresis indicated a clonal relationship among strains A136, A136m, and A139. These strains were identified to be of DNA hybridization group 1. Wild-type strain A136 was resistant only to ampicillin and cephamycins, but A136m and A139 were highly resistant to the expanded- and broad-spectrum cephalosporins. The presence of increased β-lactamase activity in A139 suggests that A139 is a derepressed mutant which overexpresses β-lactamases. These results call attention to the use of β-lactam agents for the treatment of invasive Aeromonas infections.

Diversity of Hypoviruses and Other Double-Stranded RNAs in Cryphonectria parasitica in North America

Double-stranded (ds) RNAs in Cryphonectria parasitica were randomly sampled from nine subpopulations in North America using an antibody-based detection system for dsRNA. dsRNA was detected in 166 (28%) of a total of 595 C. parasitica isolates sampled by immunoblotting. Incidence of dsRNA infection within subpopulations ranged from 0% in samples from New Hampshire and Ontario to 100% in County Line, MI. Most of the dsRNAs sampled were approximately 9 to 13 kb in size. dsRNAs from 72 isolates analyzed by probing Northern blots with 32P-labeled dsRNAs were in one of three hybridization groups. One hybridization group was widespread throughout eastern North America, being found in New York, New Jersey, Maryland, West Virginia, Kentucky, and Michigan. These dsRNAs hybridized to dsRNA from the previously described C. parasitica isolate SR2 from Maryland and are referred to as SR2-type dsRNAs. The second hybridization group was found almost exclusively in Michigan. The Michigan dsRNAs cross-hybridized to Cryphonectria hypovirus 3-GH2 (CHV3-GH2) and are referred to as CHV3-type dsRNAs.One dsRNA sampled from Kentucky hybridized to CHV3-type dsRNAs from Michigan. This dsRNA was probably derived from a fungal isolate that had been intentionally released for biological control at this same site 10 years previously and had become established in Kentucky. The third hybridization group was found only in New Jersey. These dsRNAs were much smaller than all other dsRNAs (3 and 5 kb) and were found in all 11 isolates that were probed; two of these isolates also had SR2-type dsRNA in mixed infections. None of the North American dsRNAs hybridized to CHV1 from Europe, even though CHV1 has been released in numerous locations in eastern North America for biological control of chestnut blight. Similarly, no dsRNAs hybridized to CHV2-NB58, a hypovirus found previously in New Jersey. Mixed infections of SR2-type and CHV3-type dsRNAs were found in 13 of 15 isolates from Frankfort, MI, while another nearby subpopulation (County Line) was infected with only CHV3-type dsRNAs. The distribution of dsRNA hybridization groups in C. parasitica thus presents a mixed picture, since one hybridization group is widespread, whereas two others are primarily restricted to smaller geographic areas.