Agent Of Bacterial Fruit Blotch Research Articles

Type IV Pili Are Required for Virulence, Twitching Motility, and Biofilm Formation of Acidovorax avenae subsp. citrulli

Acidovorax avenae subsp. citrulli is the causal agent of bacterial fruit blotch (BFB), a threatening disease of watermelon, melon, and other cucurbits. Despite the economic importance of BFB, relatively little is known about basic aspects of the pathogen's biology and the molecular basis of its interaction with host plants. To identify A. avenae subsp. citrulli genes associated with pathogenicity, we generated a transposon (Tn5) mutant library on the background of strain M6, a group I strain of A. avenae subsp. citrulli, and screened it for reduced virulence by seed-transmission assays with melon. Here, we report the identification of a Tn5 mutant with reduced virulence that is impaired in pilM, which encodes a protein involved in assembly of type IV pili (TFP). Further characterization of this mutant revealed that A. avenae subsp. citrulli requires TFP for twitching motility and wild-type levels of biofilm formation. Significant reductions in virulence and biofilm formation as well as abolishment of twitching were also observed in insertional mutants affected in other TFP genes. We also provide the first evidence that group I strains of A. avenae subsp. citrulli can colonize and move through host xylem vessels.

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.

Colonization of Female Watermelon Blossoms by Acidovorax avenae ssp. citrulli and the Relationship between Blossom Inoculum Dosage and Seed Infestation

AbstractThe aim of this work was to investigate the ability of Acidovorax avenae ssp. citrulli, the causal agent of bacterial fruit blotch of cucurbits (BFB), to colonize female watermelon blossoms, and to explore the relationship between blossom inoculum dosage and seed infestation. Under greenhouse conditions A. avenae ssp. citrulli colonized stigmas and styles of female watermelon blossoms reaching populations of ≈107 to 108 colony‐forming units (CFU) per blossom for 96 h after inoculation. Acidovorax avenae ssp. citrulli growth on stigmas was slower than that of Pseudomonas syringae Cit7, a non‐pathogenic, foliar epiphyte of tomato. While pollination reduced growth of A. avenae ssp. citrulli, but P. syringae Cit7 was unaffected. Both bacteria colonized style tissues but bacterial growth in the style was significantly less than the stigma. Blossom inoculation with ≈1 × 103A. avenae ssp. citrulli CFU/blossom led to 36–55% infested seedlots within symptomless fruits. On average 14% of the seedlings produced from these seedlots displayed BFB symptoms. There was a strong positive correlation between A. avenae ssp. citrulli inoculum concentration applied to blossoms and the percentage of infested seedlots, as determined by the seedling grow‐out assay (R2 = 0.94). However, this relationship was weaker when seedlot infestation was determined by a polymerase chain reaction‐based assay (R2 = 0.34). There was also a strong positive linear relationship between A. avenae ssp. citrulli blossom inoculum dose and the mean percentage of BFB‐infected seedlings (R2 = 0.99) produced in seedling grow‐out assays. These data support the hypothesis that blossom colonization might be involved in seed infestation under field conditions.

First Report of Infection of Honeydew with Acidovorax avenae subsp. citrulli.

Honeydew fruits (Cucumis melo var. inodorus Jacq. 'Morning Ice,' 'Honeybrew,' and 'Green Flesh') with circular, 3- to 10-mm-diameter lesions that did not extend into the flesh of the fruit were collected from a field in Frio County, TX, in October 1996. Lesions were either water-soaked or had a scabby center with a water-soaked edge. Non-fluorescent, gram-negative bacteria were consistently isolated from lesions. Eleven representative bacterial strains had 0.835 to 0.950 similarity to Acidovorax avenae subsp. citrulli, using Biolog GN Microplates and the MicroLog data base release 3.50 (Biolog, Hayward, CA). Strains were tested for pathogenicity on watermelon (C. lanatus (Thunb.) Matsum. & Nakai 'Royal Sweet') and honeydew seedlings (cv. Morning Ice) by daubing suspensions (approximately 108 CFU/ml) of bacteria onto cotyledons of 1-week-old transplants. Water soaking, followed by necrosis, occurred after 3 to 5 days. A. avenae subsp. citrulli, the causal agent of bacterial fruit blotch of watermelon, was reisolated from lesions. Detached fruit of honeydew (various cultivars) and watermelon (cv. Emperor) were surface disinfested with 70% ethanol, injected sub-epidermally with bacterial suspensions in blemish-free areas, and incubated at 24°C. Water-soaked lesions developed on fruit 10 to 14 days later and the bacteria were reisolated from inoculated fruit. Fruit inoculated with sterile water did not develop symptoms. Female flowers of honeydew plants (cv. Honeybrew) were inoculated at the time of pollination with a suspension of bacteria daubed on the fruit surface. Pollinated fruits were sealed in a moist chamber for 48 h. Circular, water-soaked spots developed 10 to 14 days later. A. avenae subsp. citrulli was reisolated from these lesions. The affected 44-ha field utilized center pivot irrigation and the incidence of diseased fruit exceeded 50%. The source of the pathogen is unknown. Bacterial fruit blotch was identified in watermelon grown under overhead irrigation 0.8 km from this honeydew field 9 weeks earlier. In past years, bacterial fruit blotch has occurred in watermelon fields in Frio County (1). Honeydew from other fields in the vicinity, including one that was also sprinkler irrigated, did not show these symptoms. This is the first report of A. avenae subsp. citrulli causing a disease of honeydew fruit. Reference: M. C. Black et al. Plant Dis. 78:831, 1994.