Bacterial fruit blotch of cucurbits at APSnet, http://www.apsnet.org/edcenter/intropp/lessons/prokaryotes/Pages/BacterialBlotch.aspx; bacterial fruit blotch guide from ASTA, http://www.amseed.com/pdfs/DiseaseGuide-BFB-English.pdf; Acidovorax citrulli AAC00-1 genome at JGI, http://genome.jgi-psf.org/aciav/aciav.info.html.

Using DNA fingerprinting by pulse-field gel electrophoresis and repetitive extragenic pallindromic (REP)-polymerase chain reaction (PCR), two distinct groups were confirmed among 64 Acidovorax avenae subsp. citrulli strains collected from a range of cucurbitaceous hosts in the USA, China, Taiwan, Thailand, Canada, Australia, Brazil and Israel. Eighty-two percent of the group I strains were recovered from non-watermelon hosts and the subspecies type strain was the only member of this group that utilized l-leucine as a sole carbon source. On the contrary, 94% of the group II strains were recovered from watermelon and 96% of them utilized l-leucine. Two-week-old watermelon cv. Crimson sweet, cantaloupe cv. Athena, pumpkin cv. Lumina and squash cv. Early yellow crookneck seedlings were susceptible to A. avenae subsp. citrulli strains representing each group with the exception of the subspecies type strain. Overall, seedlings of watermelon cv. Crimson Sweet were most susceptible to A. avenae subsp. citrulli infection followed by cantaloupe, pumpkin and squash. Group II strains were more aggressive watermelon than on other hosts. On the contrary, group I strains were moderately aggressive on all cucurbit hosts tested.

No single strategy will be successful in eliminating contamination of fresh produce and seed by human pathogenic bacteria, but a multi-pronged approach may reduce the risks of outbreaks. An integrated pest management model is likely to work for minimizing the risk of human pathogenic bacteria on seed and fresh produce. Accepted for publication 20 December 2002. Published 21 January 2003.

Although seed production has been moved to semiarid regions to escape seedborne pathogens, seedborne bacterial diseases continue to be problematic and cause significant economic losses worldwide. Infested seeds are responsible for the re-emergence of diseases of the past, movement of pathogens across international borders, or the introduction of diseases into new areas. Considerable attention has been paid to improving the sensitivity and selectivity of seed health assays by using techniques such as flow cytometry and the polymerase chain reaction. There has also been progress in understanding infection thresholds and how they influence seed sample size determination and ultimately the reliability of seed health testing. Disease development and dissemination of pathogens from contaminated seedlots can be predicted using formulas that take into account inoculum density and environmental pressures. In general, seeds infested with bacterial pathogens are distributed within a Poisson distribution. In a subset of contaminated seeds, bacteria are distributed in non-Gaussian distributions, e.g., a lognormal distribution.

An immunomagnetic separation and polymerase chain reaction (IMS-PCR)-based assay was developed for detecting Acidovorax avenae subsp. citrulli in watermelon seed. IMS yielded a 10-fold increase in recovery of A. avenae subsp. citrulli over direct spread-plating on King's Medium B; however, the presence of seed debris reduced IMS efficiency. Synthetic oligonucleotide primers were designed based on the 16S rRNA gene of a known A. avenae subsp. citrulli strain and tested for specific DNA amplification by PCR. The primers amplified DNA from all A. avenae subsp. citrulli strains tested but also yielded amplicons with several closely related bacteria. IMS-PCR resulted in a 100-fold increase in A. avenae subsp. citrulli detection sensitivity over direct PCR and was unaffected by PCR inhibitors in watermelon seed. The threshold of A. avenae subsp. citrulli detection for IMS-PCR was 10 CFU/ml in watermelon seed wash, and seedlots with 0.1% infestation were consistently detected. IMS-PCR represents an efficient and sensitive approach to detecting A. avenae subsp. citrulli in watermelon seedlots.

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.

Acidovorax citrulli causes bacterial fruit blotch of cucurbits, a serious economic threat to watermelon (Citrullus lanatus) and melon (Cucumis melo) production worldwide. Based on genetic and biochemical traits, A. citrulli strains have been divided into two distinct groups: group I strains have been mainly isolated from various non-watermelon hosts, while group II strains have been generally isolated from and are highly virulent on watermelon. The pathogen depends on a functional type III secretion system for pathogenicity. Annotation of the genome of the group II strain AAC00-1 revealed 11 genes encoding putative type III secreted (T3S) effectors. Due to the crucial role of type III secretion for A. citrulli pathogenicity, we hypothesized that group I and II strains differ in their T3S effector repertoire. Comparative analysis of the 11 effector genes from a collection of 22 A. citrulli strains confirmed this hypothesis. Moreover, this analysis led to the identification of a third A. citrulli group, which was supported by DNA:DNA hybridization, DNA fingerprinting, multilocus sequence analysis of conserved genes, and virulence assays. The effector genes assessed in this study are homologous to effectors from other plant-pathogenic bacteria, mainly belonging to Xanthomonas spp. and Ralstonia solanacearum. Analyses of the effective number of codons and gas chromatography content of effector genes relative to a representative set of housekeeping genes support the idea that these effector genes were acquired by lateral gene transfer. Further investigation is required to identify new T3S effectors of A. citrulli and to determine their contribution to virulence and host preferential association.

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.