R. D. Gitaitis

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.

Natural Infestation of Onion Seed by Pantoea ananatis, Causal Agent of Center Rot

An immunomagnetic separation and polymerase chain reaction (IMS-PCR) assay was used to detect Pantoea ananatis in naturally infested onion seeds. Using species-specific PCR primers and polyclonal antibodies, IMS-PCR consistently demonstrated detection thresholds of 101 to 103 CFU/ml. There was no significant difference between the numbers of CFU recovered from onion seed wash by IMS (after repeated rinses) and by direct plating, indicating that IMS effectively captured P. ananatis cells from heterogeneous bacterial populations. Using IMS-PCR and IMS followed by plating on nutrient agar, P. ananatis was detected in 19.7% of onion seed samples harvested from two onion fields in which center rot developed naturally in 2000. When planted in germination boxes, 53% of the seed samples that tested positive for P. ananatis produced seedlings with symptoms of center rot. There was no significant difference in germination between infested and noninfested seed samples. This is the first report of natural infestation and transmission of P. ananatis in onion seed.

Molecular characterization of boscalid‐ and penthiopyrad‐resistant isolates of Didymella bryoniae and assessment of their sensitivity to fluopyram

BACKGROUND Didymella bryoniae has a history of developing resistance to single-site fungicides. A recent example is with the succinate-dehydrogenase-inhibiting fungicide (SDHI) boscalid. In laboratory assays, out of 103 isolates of this fungus, 82 and seven were found to be very highly resistant (B(VHR) ) and highly resistant (B(HR) ) to boscalid respectively. Cross-resistance studies with the new SDHI penthiopyrad showed that the B(VHR) isolates were only highly resistant to penthiopyrad (B(VHR) -P(HR) ), while the B(HR) isolates appeared sensitive to penthiopyrad (B(HR) -P(S) ). In this study, the molecular mechanism of resistance in these two phenotypes (B(VHR) -P(HR) and B(HR) -P(S) ) was elucidated, and their sensitivity to the new SDHI fluopyram was assessed. RESULTS A 456 bp cDNA amplified fragment of the succinate dehydrogenase iron sulfur gene (DbSDHB) was initially cloned and sequenced from two sensitive (B(S) -P(S) ), two B(VHR) -P(HR) and one B(HR) -P(S) isolate of D. bryoniae. Comparative analysis of the DbSDHB protein revealed that a highly conserved histidine residue involved in the binding of SDHIs and present in wild-type isolates was replaced by tyrosine (H277Y) or arginine (H277R) in the B(VHR) -P(HR) and B(HR) -P(S) variants respectively. Further examination of the role and extent of these alterations showed that the H/Y and H/R substitutions were present in the remaining B(VHR) -P(HR) and B(HR) -P(S) variants respectively. Analysis of the sensitivity to fluopyram of representative isolates showed that both SDHB mutants were sensitive to this fungicide as the wild-type isolates. CONCLUSION The genotype-specific cross-resistance relationships between the SDHIs boscalid and penthiopyrad and the lack of cross-resistance between these fungicides and fluopyram should be taken into account when selecting SDHIs for gummy stem blight management.

Alternative fumigants for methyl bromide in tobacco and pepper transplant production

Abstract Tobacco and pepper are high-value cash crops in the southeastern USA that require vigorous transplants free of pathogens and insects. Typically soil seedbeds are treated with methyl bromide prior to seeding. Soil fumigants metam-sodium, dichloropropene, chloropicrin and dazomet covered with polyethylene film were evaluated at several rates alone and in combination as alternatives for methyl bromide soil fumigation of tobacco and pepper seedbeds. The studies were conducted over a three-year period, with materials applied in the fall prior to seeding at the beginning of the following year. Nematode and insect pressures were low in each of three sites, but the tests extensively evaluated weeds and soilborne fungi management. Metam-sodium at 935 L ha−1 performed well in all three tests. The combination of metam-sodium (468 L ha−1) plus dichloropropene + 17% chloropicrin (126 L ha−1) provided good control of most of the pests and had high plant yield and vigor when covered with a polyethylene film immediately after treatment. A similar treatment not covered with polyethylene film but sealed with a mechanical soil cultipacker provided poor control of weeds. Stunting of tobacco and pepper was noted, especially in plots treated at the highest rates of metamsodium plus dichloropropene and chloropicrin. These treatments had a pungent odor associated with the treatment, which persisted for several weeks after polyethylene film removal. Many of the treatments, especially metam-sodium and metam-sodium in combination with dichloropropene and chloropicrin, compared well with methyl bromide fumigation for seedbed pest control.

CLASSIFICATION OF SWEET ONIONS BASED ON INTERNAL DEFECTS USING IMAGE PROCESSING AND NEURAL NETWORK TECHNIQUES

Maintaining product quality is the key to success in the fresh fruit and vegetable market. Quality assessment with computer vision techniques is possible; however, two basic issues need to be addressed before an automatic sorting system can be developed: (1) which image features best correlate with the product quality, and (2) which classifier should be used for optimal classification. To address these issues, sweet onions were line–scanned for internal defects using x–ray imaging. Spatial and transform features were evaluated for their contributions to product classification based on internal defects. The Bayesian method was used for selecting the salient features. Spatial edge features combined with selected discrete cosine transform (DCT) coefficients proved to be good indicators of internal defects. A neural classifier performed better than the Bayesian classifier for sorting onions into two classes (good or defective) by achieving an overall accuracy of 90%. Losses and false positives were limited to 6% and 10%, respectively. The accuracy, losses, and false positives for the Bayesian classifier were 80%, 16%, and 17%, respectively.

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.

First Report of a Fruit Rot of Pumpkin Caused by Acidivorax avenae subsp. citrulli in Georgia

In September 1998, a fruit rot was reported affecting pumpkin (Cucurbita pepo) in a commercial field in Terrell Co., Georgia. Symptoms on the surface of fruit occurred as round, necrotic spots or cracks a few millimeters in diameter. With age, the tissue surrounding these lesions became soft and wrinkled. A soft rot expanded into the flesh of the pumpkin, originating from the lesions observed on the surface. In time, infected pumpkins totally collapsed. V-shaped, necrotic lesions occurred at the margin of the leaf and extended inward toward the mid-rib. Samples were collected from the field and bacteria were isolated from fruit and leaf lesions onto Kings medium B (1). The bacterium isolated was rod shaped, gram negative, nonflourescent, oxidase positive, Tween 80 positive, carboxymethyl cellulose positive, β-OH butyrate positive, and malonate negative. The bacterium reacted positively with polyclonal antibodies specific for the watermelon fruit blotch pathogen Acidivorax avenae subsp. citrulli and was identified as A. avenae subsp. citrulli by MIDI (Microbial Identification System, Newark, DE) according to statistical analysis of fatty acid data. Results from polymerase chain reaction (PCR) amplification of the bacterium isolated from pumpkin yielded 360-bp fragments that, when digested with the restriction enzyme HaeIII, had DNA banding patterns identical to those of stock A. avenae subsp. citrulli DNA. Kochs postulates were completed successfully with 2-week-old watermelon seedlings. This is the first report of A. avenae subsp. citrulli causing fruit rot of pumpkin in Georgia. Reference: (1) E. O. King et al. J. Lab. Clin. Med. 44:301, 1954.

Pantoea allii sp. nov., isolated from onion plants and seed

Eight yellow-pigmented, Gram-negative, rod-shaped, oxidase-negative, motile, facultatively anaerobic bacteria were isolated from onion seed in South Africa and from an onion plant exhibiting centre rot symptoms in the USA. The isolates were assigned to the genus Pantoea on the basis of phenotypic and biochemical tests. 16S rRNA gene sequence analysis and multilocus sequence analysis (MLSA), based on gyrB, rpoB, infB and atpD sequences, confirmed the allocation of the isolates to the genus Pantoea. MLSA further indicated that the isolates represented a novel species, which was phylogenetically most closely related to Pantoea ananatis and Pantoea stewartii. Amplified fragment length polymorphism analysis also placed the isolates into a cluster separate from P. ananatis and P. stewartii. Compared with type strains of species of the genus Pantoea that showed >97 % 16S rRNA gene sequence similarity with strain BD 390(T), the isolates exhibited 11-55 % whole-genome DNA-DNA relatedness, which confirmed the classification of the isolates in a novel species. The most useful phenotypic characteristics for the differentiation of the isolates from their closest phylogenetic neighbours are production of acid from amygdalin and utilization of adonitol and sorbitol. A novel species, Pantoea allii sp. nov., is proposed, with type strain BD 390(T) ( = LMG 24248(T)).