R. R. Walcott

Role of Blossoms in Watermelon Seed Infestation by Acidovorax avenae subsp. citrulli

ABSTRACT The role of watermelon blossom inoculation in seed infestation by Acidovorax avenae subsp. citrulli was investigated. Approximately 98% (84/87) of fruit developed from blossoms inoculated with 1 x 10(7) or 1 x 10(9) CFU of A. avenae subsp. citrulli per blossom were asymptomatic. Using immunomagnetic separation and the polymerase chain reaction, A. avenae subsp. citrulli was detected in 44% of the seed lots assayed, despite the lack of fruit symptoms. Furthermore, viable colonies were recovered from 31% of the seed lots. Of these lots, 27% also yielded seedlings expressing bacterial fruit blotch symptoms when planted under conditions of 30 degrees C and 90% relative humidity. A. avenae subsp. citrulli was detected and recovered from the pulp of 33 and 19%, respectively, of symptomless fruit whose blossoms were inoculated with A. avenae subsp. citrulli. The ability to penetrate watermelon flowers was not unique to A. avenae subsp. citrulli, because blossoms inoculated with Pantoea ananatis also resulted in infested seed and pulp. The data indicate that watermelon blossoms are a potential site of ingress for fruit and seed infestation by A. avenae subsp. citrulli.

Investigating intraspecific variation of Acidovorax avenae subsp. citrulli using DNA fingerprinting and whole cell fatty acid analysis.

ABSTRACT To assess the diversity of Acidovorax avenae subsp. citrulli, 121 strains from watermelon, cantaloupe, and pumpkin were compared using pulse field gel electrophoresis of SpeI-digested DNA and gas chromatographic analysis of fatty acid methyl esters. Twenty-nine unique DNA fragments resulted from DNA digestion, and 14 distinct haplotypes were observed. Based on cluster analysis, two subgroups, I and II, were recognized, which accounted for 84.8% (eight haplotypes) and 15.2% (six haplotypes) of the strains, respectively. Results of cellular fatty acid analysis varied quantitatively and qualitatively for the A. avenae subsp. citrulli strains and supported the existence of the two subgroups. Group I includes strains from cantaloupe and pumpkin as well as the ATCC type strain, which was first described in the United States in 1978, whereas group II represents the typical watermelon fruit blotch-causing strains that appeared in the mainland United States in 1989. Knowledge of the two A. avenae subsp. citrulli groups may be useful in screening for watermelon fruit blotch resistance.

Simultaneous Detection of Acidovorax avenae subsp. citrulli and Didymella bryoniae in Cucurbit Seedlots Using Magnetic Capture Hybridization and Real-Time Polymerase Chain Reaction

To improve the simultaneous detection of two pathogens in cucurbit seed, a combination of magnetic capture hybridization (MCH) and multiplex real-time polymerase chain reaction (PCR) was developed. Single-stranded DNA hybridization capture probes targeting DNA of Acidovorax avenae subsp. citrulli, causal agent of bacterial fruit blotch, and Didymella bryoniae, causal agent of gummy stem blight, were covalently attached to magnetic particles and used to selectively concentrate template DNA from cucurbit seed samples. Sequestered template DNAs were subsequently amplified by multiplex real-time PCR using pathogen-specific TaqMan PCR assays. The MCH multiplex real-time PCR assay displayed a detection threshold of A. avenae subsp. citrulli at 10 CFU/ml and D. bryoniae at 10(5) conidia/ml in mixtures of pure cultures of the two pathogens, which was 10-fold more sensitive than the direct real-time PCR assays for the two pathogens separately. Although the direct real-time PCR assay displayed a detection threshold for A. avenae subsp. citrulli DNA of 100 fg/microl in 25% (1/4 samples) of the samples assayed, MCH real-time PCR demonstrated 100% detection frequency (4/4 samples) at the same DNA concentration. MCH did not improve detection sensitivity for D. bryoniae relative to direct real-time PCR using conidial suspensions or seed washes from D. bryoniae-infested cucurbit seed. However, MCH real-time PCR facilitated detection of both target pathogens in watermelon and melon seed samples (n = 5,000 seeds/sample) in which 0.02% of the seed were infested with A. avenae subsp. citrulli and 0.02% were infested with D. bryoniae.

Development of an improved isolation approach and simple sequence repeat markers to characterize Phytophthora capsici populations in irrigation ponds in southern Georgia.

ABSTRACT Phytophthora capsici, the causal agent of Phytophthora blight, is a major concern in vegetable production in Georgia and many other states in the United States. Contamination of irrigation water sources by P. capsici may be an important source of inoculum for the pathogen. A simple method was developed in this study to improve the efficiency of recovering P. capsici from fruits used as baits in irrigation ponds. In contrast to direct isolation on agar plates, infected fruit tissues were used to inoculate stems of pepper seedlings, and the infected pepper stems were used for isolation on agar plates. With isolation through inoculation of pepper stems, the frequency of recovering P. capsici from infected eggplant and pear fruits increased from 13.9% to 77.7% and 8.1% to 53.5%, respectively, compared with direct isolation on agar plates. P. capsici was isolated from seven out of nine irrigation ponds evaluated, with most of the ponds containing both A1 and A2 mating types and a 4:5 ratio of A1 to A2 when isolates from all ponds were calculated. All P. capsici isolates were pathogenic on squash plants, and only a small proportion (8.2%) of the isolates were resistant or intermediately sensitive to mefenoxam. Simple sequence repeats (SSRs) were identified through bioinformatics mining of 55,848 publicly available expressed sequence tags of P. capsici in dbEST GenBank. Thirty-one pairs of SSR primers were designed, and SSR analysis indicated that the 61 P. capsici isolates from irrigation ponds were genetically distinct. Cluster analysis separated the isolates into five genetic clusters with no more than two genetic groups in one pond, indicating relatively low P. capsici genetic diversity in each pond. The isolation method and SSR markers developed for P. capsici in this study could contribute to a more comprehensive understanding of the genetic diversity of this important pathogen.

Recovery of Pantoea ananatis, causal agent of center rot of onion, from weeds and crops in Georgia, USA

Abstract Center rot of onion, caused by Pantoea ananatis, has been a problem of sweet onions in Georgia since 1997. A polymerase chain reaction (PCR) protocol was developed to screen for populations of P. ananatis on plant surfaces. Plant samples producing a positive PCR reaction were targeted for further processing to culture the bacterium. In a survey of the Vidalia onion-growing region of Georgia, we detected and cultured P. ananatis from 25 asymptomatic weed species, which included commonly occurring weeds such as crabgrass (Digitaria sanguinalis), Florida beggarweed (Desmodium tortuosum), Florida pusley (Richardia scabra), sicklepod (Cassia obtusifolia), Texas Millet (Panicum texanum), tall verbena (Verbena bonariensis) and yellow nutsedge (Cyperus esculentus). In addition, the bacterium was recovered from crop plants such as Bermuda grass (Cynodon dactylon), cowpea (Vigna unguiculata) and soybean (Glycine max). Based on the field survey and tests with strains held in storage in the Coastal Plain Experiment Station culture collection, we concluded that P. ananatis was in Georgia prior to 1997, and was distributed widely on weeds and crops throughout southern Georgia.

The type VI protein secretion system contributes to biofilm formation and seed-to-seedling transmission of Acidovorax citrulli on melon.

The type VI protein secretion system (T6SS) is essential for the virulence of several Gram-negative bacteria. In this study, we identified a T6SS gene cluster in Acidovorax citrulli, a plant-pathogenic bacterium that causes bacterial fruit blotch (BFB) of cucurbits. One T6SS cluster, of approximately 25 kb in length and comprising 17 genes, was found in the A. citrulli AAC00-1 genome. Seventeen A. citrulli mutants were generated, each with a deletion of a single T6SS core gene. There were significant differences in BFB seed-to-seedling transmission between wild-type A. citrulli strain, xjl12, and ΔvasD, ΔimpK, ΔimpJ and ΔimpF mutants (71.71%, 9.83%, 8.41%, 7.15% and 5.99% BFB disease index, respectively). In addition, we observed that these four mutants were reduced in melon seed colonization and biofilm formation; however, they were not affected in virulence when infiltrated into melon seedling tissues. There were no significant differences in BFB seed-to-seedling transmission, melon tissue colonization and biofilm formation between xjl12 and the other 13 T6SS mutants. Overall, our results indicate that T6SS plays a role in seed-to-seedling transmission of BFB on melon.

Simultaneous Detection of Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola in Rice Seed Using a Padlock Probe-Based Assay

Based on 16S-23S internal transcribed spacer ribosomal DNA sequence data, two padlock probes (PLPs), P-Xoo and P-Xoc, were designed and tested to detect Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola, respectively. These PLPs were combined with dot-blot hybridization to detect X. oryzae pv. oryzae and X. oryzae pv. oryzicola individually in rice seed. Using this technique, a detection sensitivity of 1 pg of X. oryzae pv. oryzae genomic DNA was observed. The technique also facilitated the detection of X. oryzae pv. oryzae in rice seedlots with 2% artificially infested seed. With regards to X. oryzae pv. oryzicola a detection threshold of 1 pg genomic DNA was observed and the pathogen was successful detected in rice seedlots with 0.2% artificially infested seed. The PLP assays detected X. oryzae pv. oryzae and X. oryzae pv. oryzicola in 39.3% (13 of 33) and 21.3% (10 of 47) of naturally infested commercial rice seedlots, respectively. In contrast, conventional polymerase chain reaction using OSF1/OSR1 and XoocF/XoocR primers sets detected X. oryzae pv. oryzae and X. oryzae pv. oryzicola in 9.1% (3 of 33) and 8.5% (4 of 47) of the same rice seedlots, respectively. We also detected both pathogens simultaneously in two seedlots, which successfully proved that PLPs (P-Xoo and P-Xoc) combined with reverse dotblot hybridization can be used to simultaneously detect multiple pathogens in naturally infested commercial rice seedlots. This approach has the potential to be an important tool for detecting multiple pathogens in seed and thereby preventing the spread of important pathogens.

Differential Attachment of Salmonella enterica and Enterohemorrhagic Escherichia coli to Alfalfa, Fenugreek, Lettuce, and Tomato Seeds

ABSTRACT Vegetable seeds have the potential to disseminate and transmit foodborne bacterial pathogens. This study was undertaken to assess the abilities of selected Salmonella and enterohemorrhagic Escherichia coli (EHEC) strains to attach to fungicide-treated versus untreated, and intact versus mechanically damaged, seeds of alfalfa, fenugreek, lettuce, and tomato. Surface-sanitized seeds (2 g) were exposed to four individual strains of Salmonella or EHEC at 20°C for 5 h. Contaminated seeds were rinsed twice, each with 10 ml of sterilized water, before being soaked overnight in 5 ml of phosphate-buffered saline at 4°C. The seeds were then vortexed vigorously for 1 min, and pathogen populations in seed rinse water and soaking buffer were determined using a standard plate count assay. In general, the Salmonella cells had higher attachment ratios than the EHEC cells. Lettuce seeds by unit weight had the highest numbers of attached Salmonella or EHEC cells, followed by tomato, alfalfa, and fenugreek seeds. In contrast, individual fenugreek seeds had more attached pathogen cells, followed by lettuce, alfalfa, and tomato seeds. Significantly more Salmonella and EHEC cells attached to mechanically damaged seeds than to intact seeds (P < 0.05). Although, on average, significantly more Salmonella and EHEC cells were recovered from untreated than fungicide-treated seeds (P < 0.05), fungicide treatment did not significantly affect the attachment of individual bacterial strains to vegetable seeds (P > 0.05), with a few exceptions. This study fills gaps in the current body of literature and helps explain bacterial interactions with vegetable seeds with differing surface characteristics. IMPORTANCE Vegetable seeds, specifically sprout seeds, have the potential to disseminate and transmit foodborne bacterial pathogens. This study investigated the interaction between two important bacterial pathogens, i.e., Salmonella and EHEC, and vegetable seeds with differing surface characteristics. This research helps understand whether seed surface structure, integrity, and fungicide treatment affect the interaction between bacterial cells and vegetable seeds.

Reliable and Sensitive Detection of Acidovorax citrulli in Cucurbit Seed Using a Padlock-Probe-Based Assay

A method was developed using a padlock probe (PLP) and dot-blot hybridization for detecting Acidovorax citrulli in cucurbit seed. The PLP was designed based on the 16S-23S internal transcribed spacer ribosomal DNA sequence from A. citrulli. The detection threshold for the PLP assay was 100 fg of genomic DNA, and A. citrulli was detected in 100% of artificially infested seedlots with 0.1% infestation or greater. In addition, using the PLP assay, 4 of 8 melon seedlots collected from Xinjang province and 15 of 47 watermelon seedlots collected from Ningxia province were positive for A. citrulli. In contrast, a conventional polymerase chain reaction (PCR) assay that relied on primers WFB1 and WFB2 facilitated A. citrulli detection in 1 of 8 and 5 of 47 seedlots from Xinjiang and Ningxia provinces, respectively. These data indicate that the PLP and dot-blot hybridization technique was more effective than conventional PCR for seed health testing.

Integrated management of bacterial streak and bulb rot of onion.

Significantly higher levels of bacterial streak and bulb rot of onion (BSBR), caused by Pseudomonas viridiflava, occurred in onions receiving excessive applications of fertiliser and were correlated with higher nitrogen content in plant tissues. Based on previous work that weeds, particularly cutleaf evening primrose (Oenothera laciniata), were the primary source of initial inoculum, oxyfluorfen was evaluated as a post-emergence herbicide after transplanting. The herbicide not only reduced weed levels as expected, but also reduced the incidence of BSBR. Lower levels of BSBR also occurred in plots treated with weekly applications of cupric hydroxide and maneb when compared to non sprayed plots, or plots treated only with the fungicide chlorothalonil. Harvesting onions with cutting shears contaminated with P. viridiflava increased the incidence of postharvest rots in onions clipped immediately after uprooting but not in onions allowed to cure in the field for 48 h. Finally, lower levels of postharvest disease occurred when onion bulbs were stored under controlled-atmospheric (CA) conditions (3% oxygen, 5% carbon dioxide, and 92% nitrogen) at 1°C. The various control procedures were combined into an integrated management strategy for the control of BSBR in Georgia.