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.

Predicting the risk of cucurbit downy mildew in the eastern United States using an integrated aerobiological model.

Cucurbit downy mildew caused by the obligate oomycete, Pseudoperonospora cubensis, is considered one of the most economically important diseases of cucurbits worldwide. In the continental United States, the pathogen overwinters in southern Florida and along the coast of the Gulf of Mexico. Outbreaks of the disease in northern states occur annually via long-distance aerial transport of sporangia from infected source fields. An integrated aerobiological modeling system has been developed to predict the risk of disease occurrence and to facilitate timely use of fungicides for disease management. The forecasting system, which combines information on known inoculum sources, long-distance atmospheric spore transport and spore deposition modules, was tested to determine its accuracy in predicting risk of disease outbreak. Rainwater samples at disease monitoring sites in Alabama, Georgia, Louisiana, New York, North Carolina, Ohio, Pennsylvania and South Carolina were collected weekly from planting to the first appearance of symptoms at the field sites during the 2013, 2014, and 2015 growing seasons. A conventional PCR assay with primers specific to P. cubensis was used to detect the presence of sporangia in rain water samples. Disease forecasts were monitored and recorded for each site after each rain event until initial disease symptoms appeared. The pathogen was detected in 38 of the 187 rainwater samples collected during the study period. The forecasting system correctly predicted the risk of disease outbreak based on the presence of sporangia or appearance of initial disease symptoms with an overall accuracy rate of 66 and 75%, respectively. In addition, the probability that the forecasting system correctly classified the presence or absence of disease was ≥ 73%. The true skill statistic calculated based on the appearance of disease symptoms in cucurbit field plantings ranged from 0.42 to 0.58, indicating that the disease forecasting system had an acceptable to good performance in predicting the risk of cucurbit downy mildew outbreak in the eastern United States.

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.

Microbial contamination of fuel ethanol fermentations.

Microbial contamination is a pervasive problem in any ethanol fermentation system. These infections can at minimum affect the efficiency of the fermentation and at their worse lead to stuck fermentations causing plants to shut down for cleaning before beginning anew. These delays can result in costly loss of time as well as lead to an increased cost of the final product. Lactic acid bacteria (LAB) are the most common bacterial contaminants found in ethanol production facilities and have been linked to decreased ethanol production during fermentation. Lactobacillus sp. generally predominant as these bacteria are well adapted for survival under high ethanol, low pH and low oxygen conditions found during fermentation. It has been generally accepted that lactobacilli cause inhibition of Saccharomyces sp. and limit ethanol production through two basic methods; either production of lactic and acetic acids or through competition for nutrients. However, a number of researchers have demonstrated that these mechanisms may not completely account for the amount of loss observed and have suggested other means by which bacteria can inhibit yeast growth and ethanol production. While LAB are the primary contaminates of concern in industrial ethanol fermentations, wild yeast may also affect the productivity of these fermentations. Though many yeast species have the ability to thrive in a fermentation environment, Dekkera bruxellensis has been repeatedly targeted and cited as one of the main contaminant yeasts in ethanol production. Though widely studied for its detrimental effects on wine, the specific species-species interactions between D. bruxellensis and S. cerevisiae are still poorly understood.

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.

Systemic Modulation of Gene Expression in Tomato by Trichoderma hamatum 382.

ABSTRACT A light sphagnum peat mix inoculated with Trichoderma hamatum 382 consistently provided a significant (P = 0.05) degree of protection against bacterial spot of tomato and its pathogen Xanthomonas euvesicatoria 110c compared with the control peat mix, even though this biocontrol agent did not colonize aboveground plant parts. To gain insight into the mechanism by which T. hamatum 382 induced resistance in tomato, high-density oligonucleotide microarrays were used to determine its effect on the expression pattern of 15,925 genes in leaves just before they were inoculated with the pathogen. T. hamatum 382 consistently modulated the expression of genes in tomato leaves. We identified 45 genes to be differentially expressed across the replicated treatments, and 41 of these genes could be assigned to at least one of seven functional categories. T. hamatum 382-induced genes have functions associated with biotic or abiotic stress, as well as RNA, DNA, and protein metabolism. Four extensin and extensin-like proteins were induced. However, besides pathogenesis-related protein 5, the main markers of systemic acquired resistance were not significantly induced. This work showed that T. hamatum 382 actively induces systemic changes in plant physiology and disease resistance through systemic modulation of the expression of stress and metabolism genes.

Diversity Among Strains of Xanthomonas campestris pv. vitians from Lettuce.

ABSTRACT Diversity in host range, pathogenicity, phenotypic characteristics, repetitive extragenic palindromic polymerase chain reaction (rep-PCR) profiles, and sequence of the 16S-23S rDNA spacer region was examined among 44 Xanthomonas strains isolated from lettuce. Forty-two of the strains were divided into two groups, designated A and B. Seventy percent were Group A, and most of the remaining strains including a reference strain (LMG 938) were Group B. Group A strains induced both local and systemic symptoms, whereas Group B strains caused only distinct necrotic spots. Two strains, including the X. campestris pv. vitians type strain, were distinct from the Group A and B strains and were not pathogenic on lettuce. Analysis of fatty acid profiles, serotype, carbon substrate utilization patterns, and protein fingerprints confirmed this grouping. The Group A and B strains also formed two unique clusters (I and II) by rep-PCR profiling that corresponded to the two groups. Direct sequencing of a PCR-amplified DNA fragment (680 bp) from the 16S-23S rDNA spacer region of four representative strains, however, did not differentiate these groups. Serology and rep-PCR fingerprinting can be used to diagnose and identify X. campestris pv. vitians strains, while the other analyses evaluated are useful for strain characterization.

Report of Bacterial Leaf Spot on Collards and Turnip Leaves in Ohio.

In 2000, circular water-soaked lesions typical of bacterial leaf spot were observed on leaves of collards (Brassica oleracea L. var. viridis) throughout commercial fields in northwest Ohio. Light brown, rectangular, water-soaked lesions were observed on turnip leaves (Brassica rapa L.). Bacterial streaming from lesions on both crops was observed microscopically. Cream colored, fluorescent colonies were isolated from diseased tissues on Pseudomonas F medium, and eight representative colonies (four from collards and four from turnip) were selected and purified. Fatty acid methyl ester analysis was performed on all of the isolates. Two from collards and two from turnip were identified as Pseudomonas syringae pv. maculicola (mean similarity index = 0.82 [MIDI Inc., Newark, DE]). DNA extracts from pure cultures of the P. syringae pv. maculicola strains were used as template in a polymerase chain reaction (PCR) assay with primers derived from the region of the coronatine gene cluster controlling synthesis of the coronafacic acid moiety found in P. syringae pv. tomato and P. syringae pv. maculicola (CorR and CorF2) (D. Cuppels, personal communication). DNA from P. syringae pv. tomato strain DC3000 and P. syringae pv. maculicola strain 88-10 (2) served as positive controls, while water and DNA from Xanthomonas campestris pv. vesicatoria strain Xcv 767 were used as negative controls. The expected 0.65-kb PCR product was amplified from three of four strains (two from turnip and one from collards) and the positive control DNA, but not from the negative controls. Pathogenicity tests were performed twice on 6-week-old turnip ('Forage Star', 'Turnip Topper', 'Turnip Alamo', 'Turnip 7'), collard ('Champion') and mustard (Brassica juncea L. 'Southern Giant Curl') seedlings using the three PCR-positive strains. Premisted seedlings were spray-inoculated separately with each of the three strains (2 × 108 CFU/ml, 5 ml per plant) and a water control. Greenhouse temperatures were maintained at 20 ± 1°C. For both tests, all strains caused characteristic lesions on all of the crucifer cultivars within 5 days after inoculation; the control plants did not develop symptoms. To satisfy Koch's postulates, one of the turnip strains was reisolated from 'Turnip Topper' plants, and the collard strain was reisolated from 'Champion' plants. The three original and two reisolated strains induced a hypersensitive response in Mirabilis jalapa L. and Nicotiana tabacum L. var. xanthia plants 24 h after inoculation with a bacterial suspension (1 × 108 CFU/ml). The original and reisolated strains were compared using rep-PCR with the primer BOXA1R (1). The DNA fingerprints of the reisolated strains were identical to those of the original strains. To our knowledge, this is the first report of bacterial leaf spot on commercially grown collards and turnip greens in Ohio. References: (1) B. Martin et al. Nucleic Acids Res. 20:3479, 1992. (2) R. A. Moore et al. Can. J. Microbiol. 35:910, 1989.

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.