Draft Genome Sequences of Four Streptomycin-Sensitive Erwinia amylovora Strains Isolated from Commercial Apple Orchards in Ohio.

Erwinia amylovora is the causative agent of fire blight, a devastating disease of apples and pears worldwide. Here, we report draft genome sequences of four streptomycin-sensitive strains of E. amylovora that were isolated from diseased apple trees in Ohio.

Assessing the efficacy of pre-harvest, chlorine-based sanitizers against human pathogen indicator microorganisms and Phytophthora capsici in non-recycled surface irrigation water.

Many factors must be considered in order to develop and implement treatment systems to improve the microbial quality of surface water and prevent the accidental introduction of plant and human pathogens into vegetable crops. The efficacy of chlorine gas (Cl2(g)) and chlorine dioxide (ClO2) injection systems in combination with rapid sand filtration (RSF) was evaluated in killing fecal indicator microorganisms in irrigation water in a vegetable-intensive production area. The efficacy of ClO2 and Cl2(g) was variable throughout the distribution systems and coliform bacteria never dropped below levels required by the United States Environmental Protection Agency for recreational waters. Sampling date and sampling point had a significant effect on the abundance of coliforms in Cl2(g)- and ClO2-treated water. Sampling date and sampling point also had a significant effect on the abundance of generic Escherichia coli in Cl2(g) treated water but only sampling point was significant in ClO2 treated water. Although the waterborne plant pathogen Phytophthora capsici was detected in five different sources of surface irrigation water using baiting and P. capsici-specific PCR, in vitro studies indicated that ClO2 at concentrations similar to those used to treat irrigation water did not reduce mycelial growth or direct germination of P. capsici sporangia and reduced zoospore populations by less than 50%. This study concludes that injection of ClO2 and Cl2(g) into surface water prior to rapid sand filtration is inadequate in reducing fecal indicator microorganism populations and ClO2 ineffectively kills infectious propagules of P. capsici. Additional research is needed to design a system that effectively targets and significantly reduces both plant and human pathogens that are present in surface irrigation water. A model for a multiple barrier approach to treating surface water for irrigation is proposed.

First Report of Xanthomonas gardneri Causing Bacterial Spot of Tomato in Ohio and Michigan.

In 2009 and 2010, outbreaks of bacterial spot characterized by significant fruit spotting occurred in at least 2,000 ha of commercial processing tomatoes in northwest Ohio and southeast Michigan. Losses were estimated at $7.8 million. Diseased fruit and foliage were collected from 32 Ohio and Michigan fields in 2010. Excised lesions from fruit and leaves were dipped briefly in 70% ethanol, air dried, and chopped into pieces in 10 mM potassium phosphate buffer (KPB), pH 7.4. Ten-fold serial dilutions in KPB were plated on yeast dextrose carbonate agar medium and 83 yellow mucoid colonies were purified. All isolates were gram negative and induced a hypersensitive response in tobacco (Nicotiana tabacum) plants 24 h after inoculation with a 108 CFU/ml bacterial suspension in water. All 83 isolates were identified as Xanthomonas spp. using genus-specific primers RST65/69 (2). Of these, 11 were identified as X. euvesicatoria and 8 as X. perforans using the species-specific primers RST27/28 (1) and JJ19/22 (5'-AACCCAACTAATTTCCCTC-3' and 5'-AACGAGATTTGTTACGAACC-3'; J. B. Jones, personal communication), respectively. DNA fingerprint profiles of 62 of the 64 remaining strains generated using BOX-PCR assays (4) were identical to the profile of X. gardneri type strain XCGA2. The DNA profiles of 2 of the 64 Xanthomonas strains did not resemble those of any reference strains. The 16S rDNA and ITS1 genes from two representative strains (SM174-10 and SM230-10) were PCR amplified, direct sequenced, and aligned using nBLAST with the same gene region from XCGA2 (GenBank Accession No. AF123093). Strains SM174-10 and SM230-10 differed from XCGA2 by 2 bp (99% nucleotide similarity). Pathogenicity tests were performed twice on 6-week-old tomato seedlings (cv. Peto 696). Three tomato seedlings were sprayed until runoff with strain SM174-10 (~108 CFU/ml), three seedlings were sprayed similarly with water (control treatment), and all six plants were grown under high relative humidity (24 s of mist per 12 min) at day/night temperatures of 29/23°C for 15 days. Seedlings inoculated with SM174-10 exhibited water-soaked lesions and chlorosis on the foliage, similar to field symptoms, within 14 days. Seedlings sprayed with water did not develop symptoms. Isolates cultured as described above from all three pathogen-inoculated seedlings were similar in morphology to strain SM174-10; no cultures were recovered from water-inoculated plants. The BOX-PCR fingerprint profile of a representative reisolated colony was identical to that of SM174-10. Although bacterial spot of tomato is a common disease in Ohio and Michigan, to our knowledge this is the first report of X. gardneri infecting tomatoes in these states and provides evidence that there may have been a shift in the primary causal agent of bacterial spot from X. euvesicatoria (3) to X. gardneri. References: (1) H. Bouzar et al. Phytopathology 84:39, 1994. (2) A. Obradovic et al. Eur. J. Plant Pathol. 11:285, 2004. (3) F. Sahin. Ph.D. Diss. The Ohio State University, Columbus, 1997. (4) D. J. Versalovic et al. Methods Mol. Cell. Biol. 5:25, 1994.

First Report of 'Candidatus Liberibacter solanacearum' Naturally Infecting Tomatoes in the State of Mexico, Mexico.

In January 2011, tomato (Solanum lycopersicum) plants exhibiting stunting, yellow mosaic, short, chlorotic leaves, aborted flowers, and reduced-size fruits, symptoms similar to those exhibited by plants infected by 'Candidatus Liberibacter solanacearum' (2), were observed in approximately 5% of tomato plants in greenhouses in Jocotitlan in the State of Mexico, Mexico. Occasional plant recovery was also observed. Tomato plants in this facility were previously shown to be infected by Mexican papita viroid (MPVd), Pepino mosaic virus (PepMV), and aster yellows phytoplasma. Eight symptomatic leaf samples (designated MX11-01 to MX11-08) were collected and screened against selected tomato viruses and pospiviroids by reverse transcription (RT)-PCR using purified plant RNA or for 'Ca. L. solanacearum' by PCR using purified plant DNA. As expected, both PepMV and MPVd were detected in these samples. However, two 'Ca. L. solanacearum'-specific PCR products (1,168 and 669 bp) were also amplified in two samples (MX11-02 and MX11-05) using primers OA2 (2) and OI2c (1) or CL514F/CL514R (3), respectively. Each 'Ca. L. solanacearum'-specific PCR product was gel purified with Geneclean (Q-Biogene, Carlsbad, CA) and cloned into pCR2.1 using TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced (Functional Biosciences, Madison, WI). Sequences of 16S rRNA (1,168 bp) in both isolates (GenBank Accession Nos. JF811596 and JF811597) were identical. However, the 669-bp 50S rRNA sequences in these two isolates (GenBank Accession Nos. JF811598 and JF811599) contained two single nucleotide polymorphism (SNP) mutations. BLASTn searches showed that both 16S rRNA and 50S gene sequences in MX11-05 were identical to the 'Ca. L. solanacearum' previously identified on potato in Chihuahua (GenBank Accession Nos. FJ829811 and FJ829812) and Saltillo (GenBank Accession Nos. FJ498806 or FJ498807) in eastern Mexico. These 'Ca. L. solanacearum' isolates were recently classified as the "b" haplotype (4). Alignment analysis of the 'Ca. L. solanacearum' 16S rRNA sequences also revealed the conserved SNP mutations (g.212T > G and g.581T > C) in MX11-02 and MX11-05 as previously identified for other "b" haplotype isolates (4). 'Ca. L. solanacearum' was first identified in greenhouse tomatoes in 2008 in New Zealand (2). It has also been identified in greenhouse and field tomatoes in the United States. 'Ca. L. solanacearum' was previously reported to infect field tomatoes in Sinaloa, Mexico (3), which was recently considered as the "a" haplotype (4). To our knowledge, this is the first report of 'Ca. L. solanacearum' naturally infecting tomatoes in Jocotitlan in the State of Mexico, Mexico. The greenhouse tomato 'Ca. L. solanacearum' may be transmitted from infected solanaceous plants by potato psyllids (Bactericera cockerelli), which were observed in this facility. References: (1) S. Jagoueix et al. Int. J. Syst. Bacteriol. 44:379, 1994. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza et al. Plant Dis. 93:1076, 2009 (4) W. R. Nelson et al. Eur. J. Plant Pathol. 130:5, 2011.

First Report of Bacterial Canker of Pepper in Ohio.

Light brown, raised lesions were observed on the leaves of bell pepper (Capsicum annuum L.) plants throughout a commercial field in northwest Ohio in 1999. Bacterial streaming from the lesions was observed microscopically. Five representative, pale yellow colonies isolated on yeast dextrose carbonate medium were selected and purified. All isolates induced a hypersensitive response in Mirabilis jalapa L. plants 24 h after inoculation with a 1 × 108 CFU/ml bacterial suspension. All five were identified as Clavibacter michiganensis subsp. michiganensis by fatty acid methyl ester analysis (mean similarity index [S.I.] = 0.76; MIDI, Newark, DE). Identity was confirmed at the University of Hawaii (W. Kaneshiro and A. Alvarez) by carbon substrate utilization pattern (mean S.I. = 0.75; Biolog, Hayward, CA) and positive reactions with C. michiganensis subsp. michiganensis-specific monoclonal antibodies (clones 103-142-1-1 and 103-148-2-1) in ELISA. DNA extracted from lesions and pure cultures was used as template in a polymerase chain reaction (PCR) assay with primers specific for C. michiganensis subsp. michiganensis (CMM-5 and CMM-6) (1). DNA from a known strain of C. michiganensis subsp. michiganensis served as a positive control, while water and DNA from healthy tomato plants were used as negative controls. A 0.6-kb PCR product was amplified from lesions, pure cultures of all five strains, and positive control DNA, but not from the negative controls. Pathogenicity tests were performed twice on 5- to 6-week-old bell pepper (cvs. Collossal, Lafayette, King Arthur, Brigadier, and Commandant) and tomato (cv. Peto 696) plants. Pepper plants were inoculated with each strain by clipping the lowest petioles with scissors that had been dipped into a bacterial suspension (1 × 108 CFU/ml) or by spray inoculation (approximately 5 ml/plant). Tomato plants were inoculated by clipping. Both inoculation methods included a water control. All five strains caused water-soaked lesions on leaves of all pepper varieties within 7 days after spray inoculation. Pepper plants inoculated by clipping did not develop symptoms. DNA extracts from lesions of challenged pepper plants were positive in PCR. All inoculated tomato seedlings exhibited wilting, streaks, and cankers in the stems and necrosis of leaf margins within 15 days after inoculation. None of the control plants developed symptoms. All five strains were re-isolated from inoculated tomato and pepper plants. The original pepper strains and the strains re-isolated from tomatoes were compared using rep-PCR with ERIC primers (4). DNA fingerprints of the re-isolated strains were identical to those of the original strains and were characteristic of C. michiganensis subsp. michiganensis type C. Bacterial canker is a common disease of tomatoes worldwide and has occurred in Ohio for at least 70 years. However, this is the first report of C. michiganensis subsp. michiganensis infecting peppers in Ohio. While the pathogen does not appear to cause systemic disease in peppers, it may serve as a source of inoculum for tomatoes, which are highly susceptible to the disease and often produced in the same greenhouse as peppers or planted in adjacent fields. Bacterial canker has been reported previously from commercial pepper fields in California (2) and Indiana (3). References: (1) J. Dreier et al. Phytopathology 85:462, 1995. (2) M. Lai. Plant Dis. Rep. 60:339, 1976. (3) R. Latin et al. Plant Dis. 79:860, 1995. (4) F. J. Louws et al. Appl. Environ. Microbiol. 60:2286, 1994.