Benny D. Bruton

Identification, Phylogenetic Analysis, and Biological Characterization of Serratia marcescens Strains Causing Cucurbit Yellow Vine Disease

ABSTRACT A serious vine decline of cucurbits known as cucurbit yellow vine disease (CYVD) is caused by rod-shaped bacteria that colonize the phloem elements. Sequence analysis of a CYVD-specific polymerase chain reaction (PCR)-amplified 16S rDNA product showed the microbe to be a gamma-proteobacterium related to the genus Serratia. To identify and characterize the bacteria, one strain each from watermelon and zucchini and several noncucurbit-derived reference strains were subjected to sequence analysis and biological function assays. Taxonomic and phylogenetic placement was investigated by analysis of the groE and 16S rDNA regions, which were amplified by PCR and directly sequenced. For comparison, eight other bacterial strains identified by others as Serratia spp. also were sequenced. These sequences clearly identified the CYVD strains as Serratia marcescens. However, evaluation of metabolic and biochemical features revealed that cucurbit-derived strains of S. marcescens differ substantially from strains of the same species isolated from other environmental niches. Cucurbit strains formed a distinct cluster, separate from other strains, when their fatty acid methyl ester profiles were analyzed. In substrate utilization assays (BIOLOG, Vitek, and API 20E), the CYVD strains lacked a number of metabolic functions characteristic for S. marcescens, failing to catabolize 25 to 30 compounds that were utilized by S. marcescens reference strains. These biological differences may reflect gene loss or repression that occurred as the bacterium adapted to life as an intracellular parasite and plant pathogen.

The Influence of Rootstock Selection on Fruit Quality Attributes of Watermelon

Grafting watermelon (Citrullus lanatus) to control Fusarium wilt has been practiced in Europe, the Middle East, and the Far East for decades. Until recently, grafting watermelon has not been practiced in the United States due to labor costs and land availability. There is some disagreement in the literature as to the effects that grafting has on water- melon fruit quality. This study was designed to determine the effects of grafted watermelon on fruit firmness, lycopene content, and total soluble solids (TSS) using five different rootstocks. When using Cucurbita ficifolia or Cucurbita maxima x Cucurbita moschata hybrid as the rootstock, watermelon fruit consistently had higher fruit firmness values. Other C. maxima x C. moschata hybrids or Lagenaria siceraria rootstocks generally produced lower or more varied fruit firmness values. Grafting increased fruit firmness by as much as 25% in some cases, but field and year effects were ob- served. In addition, grafting had no effect on lycopene content or TSS. Furthermore, no off-flavors were detected in fruit from grafted plants, but there was a 5- to 7 day delay in fruit maturity compared to their non-grafted counterpart. Although environment can have a major influence on fruit quality attributes, rootstock selection may be equally important in achieving the desired outcome.

Watermelon juice: a promising feedstock supplement, diluent, and nitrogen supplement for ethanol biofuel production

BackgroundTwo economic factors make watermelon worthy of consideration as a feedstock for ethanol biofuel production. First, about 20% of each annual watermelon crop is left in the field because of surface blemishes or because they are misshapen; currently these are lost to growers as a source of revenue. Second, the neutraceutical value of lycopene and L-citrulline obtained from watermelon is at a threshold whereby watermelon could serve as starting material to extract and manufacture these products. Processing of watermelons to produce lycopene and L-citrulline, yields a waste stream of watermelon juice at the rate of over 500 L/t of watermelons. Since watermelon juice contains 7 to 10% (w/v) directly fermentable sugars and 15 to 35 μmol/ml of free amino acids, its potential as feedstock, diluent, and nitrogen supplement was investigated in fermentations to produce bioethanol.ResultsComplete watermelon juice and that which did not contain the chromoplasts (lycopene), but did contain free amino acids, were readily fermentable as the sole feedstock or as diluent, feedstock supplement, and nitrogen supplement to granulated sugar or molasses. A minimum level of ~400 mg N/L (~15 μmol/ml amino nitrogen) in watermelon juice was required to achieve maximal fermentation rates when it was employed as the sole nitrogen source for the fermentation. Fermentation at pH 5 produced the highest rate of fermentation for the yeast system that was employed. Utilizing watermelon juice as diluent, supplemental feedstock, and nitrogen source for fermentation of processed sugar or molasses allowed complete fermentation of up to 25% (w/v) sugar concentration at pH 3 (0.41 to 0.46 g ethanol per g sugar) or up to 35% (w/v) sugar concentration at pH 5 with a conversion to 0.36 to 0.41 g ethanol per g sugar.ConclusionAlthough watermelon juice would have to be concentrated 2.5- to 3-fold to serve as the sole feedstock for ethanol biofuel production, the results of this investigation indicate that watermelon juice, either as whole juice fermented on-site or as a waste stream from neutraceutical production, could easily integrate with other more concentrated feedstocks where it could serve as diluent, supplemental feedstock, and nitrogen supplement.

Polygalacturonase Isozymes Produced by Phomopsis cucurbitae in Relation to Postharvest Decay of Cantaloupe Fruit

ABSTRACT Production of polygalacturonase (PG), a cell wall-degrading enzyme, by Phomopsis cucurbitae (latent infection fungus) was studied in relation to different carbon sources and various stages of cantaloupe fruit development. P. cucurbitae produced multiple PG isozymes both in vitro and in vivo. The fungus produced the highest PG activity and the greatest number of isozymes on pectin compared with those produced on glucose, galactose, and sucrose. Eight P. cucurbitae PG isozymes (pIs 3.7, 4.2, 6.6, 7.0, 7.3, 7.5, 7.8, and 8.6) were detected in extract from inoculated mature fruit (40 days after anthesis) by isoelectric focusing. Isozyme bands with pIs of 4.2, 7.3, and 7.8 were the most prominent. A similar set of PG isozymes was produced by P. cucurbitae in autoclaved mature fruit tissue (mesocarp). When tissue discs taken from 20-, 30-, 40-, and 50-day postanthesis fruit were inoculated with P. cucurbitae, PG activity and the number of PG isozymes extracted from the macerated fruit tissue discs increased with the degree of fruit maturity and ripening. Increases in PG activity and PG isozymes were also correlated with reactivation of latent infections and the beginning of tissue maceration. An anionic PG isozyme (pI 4.2) was only visualized on decayed 50-day-old fruit exocarp, as well as 40- and 50-day-old fruit mesocarp. The experimental results support the hypotheses that P. cucurbitae PG isozymes play an important role in fruit decay once latent infection becomes active following harvest.

Polymerase chain reaction detection and phylogenetic characterization of an agent associated with yellow vine disease of cucurbits

ABSTRACT Diagnosis of yellow vine disease (YVD) in cucurbits, an important disease in the south-central United States, relies on external symptom appearance, phloem discoloration, and the presence of bacterium-like organisms (BLOs) in phloem. Polymerase chain reaction (PCR) amplification of BLO nucleotide sequences was explored as a means to improve diagnostic techniques. PCR, using a primer pair based on sequences of the citrus-greening BLO, amplified a 0.15-kilobase (kb) fragment from the DNA of symptomatic plants, but not from that of asymptomatic plants. Its nucleotide sequence suggested that the DNA amplified was of pro-karyotic origin. A primer pair, designed to amplify nonspecific prokaryotic 16S rDNA, amplified a 1.5-kb DNA fragment in both the symptomatic and asymptomatic plants. The 1.5-kb fragment from the asymptomatic plants corresponded to chloroplast 16S rDNA, and the band from the symptomatic plants was composed of 16S rDNAs from both chloroplasts and a prokaryote. The nucleotide sequence of the prokaryotic DNA was determined and used to design three primers (YV1, YV2, and YV3). Fragments of 0.64 and 1.43 kb were amplified with primers YV1-YV2 and primers YV1-YV3, respectively, from symptomatic plants. Neither primer set yielded fragments from asymptomatic plants, unrelated bacteria, or selected soilborne fungal pathogens of cucurbits. Phylogenetic analysis indicated that the prokaryote is a gamma-3 proteobacterium. The consistent association of the 0.64- and 1.43-kb fragments with symptomatic plants suggests that the gamma-3 proteobacterium may be the causal agent of YVD of cantaloupe, squash, and watermelon.

Purification and characterisation of a prominent polygalacturonase isozyme produced by Phomopsis cucurbitae in decayed muskmelon fruit

Phomopsis cucurbitae is a latent infection fungus which causes muskmelon fruit decay only after harvest. It can cause severe losses during fruit storage and marketing in the U.S.A., Japan and some Central American countries. Previous studies showed that P. cucurbitae produced polygalacturonase (PG) in both culture and muskmelon fruit tissue. In this study, a prominent PG isozyme (PG1) produced by P. cucurbitae in decayed fruit was purified to homogeneity by a procedure of extraction, ultrafiltration, preparative isoelectric focusing, anion exchange, and gel filtration chromatography. The isozyme had a primary endo-activity, a relative molecular weight of 54 kD according to SDS-PAGE, and a pl of 4.2 based on IEF-PAGE. The isozyme was active in a low pH range from 2 to 6, and was optimum at pH 5. Optimum temperature for the activity of PG1 was 40—45 °C. The K m and V max on polygalacturonic acid were 44.7 μg ml −1 and 0.14 μg −1 enzyme, respectively. The purified PG1 effectively macerated mature fruit tissue, suggesting that the isozyme may be involved in the pathogenesis of Phomopsis fruit rot of muskmelon.

Genotyping of Serratia marcescens Strains Associated with Cucurbit Yellow Vine Disease by Repetitive Elements-Based Polymerase Chain Reaction and DNA-DNA Hybridization

ABSTRACT The bacterium that causes cucurbit yellow vine disease (CYVD) has been placed in the species Serratia marcescens based on 16S rDNA and groE sequence analysis. However, phenotypic comparison of the organism with S. marcescens strains isolated from a variety of ecological niches showed significant heterogeneity. In this study, we compared the genomic DNA of S. marcescens strains from different niches as well as type strains of other Serratia spp. through repetitive elements-based polymerase chain reaction (rep-PCR) and DNA-DNA hybridization. With the former, CYVD strains showed identical banding patterns despite the fact that they were from different cucurbit hosts, geographic locations, and years of isolation. In the phylogenetic trees generated from rep-PCR banding patterns, CYVD strains clearly were differentiated from other strains but formed a loosely related group with S. marcescens strains from other niches. The homogeneity of CYVD strains was supported further by the DNA relatedness study, in that labeled DNA from the cantaloupe isolate, C01-A, showed an average relative binding ratio (RBR) of 99%, and 0.33% divergence to other CYVD strains. Used as a representative strain of CYVD, the labeled C01-A had a RBR of 76%, and a 4.5% divergence to the S. marcescens type strain. These data confirm the previous placement of CYVD strains in S. marcescens. Our investigations, including rep-PCR, DNA-DNA hybridization, and previous phenotyping experiments, have demonstrated that CYVD-associated strains of S. marcescens cluster together in a group significantly different from other strains of the species.

Retention of Cucurbit Yellow Vine Disease Bacterium Serratia marcescens Through Transstadial Molt of Vector Anasa tristis (Hemiptera: Coreidae)

Abstract Retention or loss of transmissibility after molting was tested for adult and nymphal squash bug, Anasa tristis (De Geer), a natural vector of the plant pathogen Serratia marcescens Bizio, the causal agent of cucurbit yellow vine disease. Squash bug adults and nymphs fed from bacteria-infiltrated squash cubes were caged on squash test plants and transferred weekly to new plants for eight consecutive weeks. Twelve percent of the bugs that acquired as adults transmitted the bacterium to at least one of the test plants; 75% of these transmitters inoculated more than one plant. Transmission to plants occurred as late as 3 to 8 weeks postacquisition. Ten percent of squash bugs that fed on S. marcescens as fifth instars inoculated plants after molting to the adult stage; 77% of these transmitters inoculated more than one plant. Two insects that fed on S. marcescens as third instars inoculated squash plants. When examined by scanning electron microscopy, the foregut cibaria of transmitting insects were free of bacteria-like structures. The ability of A. tristis to transmit S. marcescens after molting to the adult stage suggests that the hemocoel acts as the site of retention of transmissible bacteria.

A Review of Ipomoviruses and Watermelon Vine Decline in Florida

The genus Ipomovirus is a small group of whitefly-transmitted viruses within the family Potyviridae, the largest group of RNA plant viruses, which are mostly aphid transmitted (Berger et al. 2005). Under current taxonomic guidelines there are three accepted members [Cucumber vein yellowing virus (CVYV), Cassava brown streak virus (CBSV) and Sweet potato mild mottle virus (SPMMV)] and one tentative member [Sweet potato yellow dwarf virus (SPYDV)] in the genus Ipomovirus (Berger et al. 2005; Colinet et al. 1996, 1998; Janssen et al. 2005; Lecoq et al. 2000).