Jiahuai Hu

Internal Colonization of Salmonella enterica Serovar Typhimurium in Tomato Plants

Several Salmonella enterica outbreaks have been traced back to contaminated tomatoes. In this study, the internalization of S. enterica Typhimurium via tomato leaves was investigated as affected by surfactants and bacterial rdar morphotype, which was reported to be important for the environmental persistence and attachment of Salmonella to plants. Surfactants, especially Silwet L-77, promoted ingress and survival of S. enterica Typhimurium in tomato leaves. In each of two experiments, 84 tomato plants were inoculated two to four times before fruiting with GFP-labeled S. enterica Typhimurium strain MAE110 (with rdar morphotype) or MAE119 (without rdar). For each inoculation, single leaflets were dipped in 109 CFU/ml Salmonella suspension with Silwet L-77. Inoculated and adjacent leaflets were tested for Salmonella survival for 3 weeks after each inoculation. The surface and pulp of ripe fruits produced on these plants were also examined for Salmonella. Populations of both Salmonella strains in inoculated leaflets decreased during 2 weeks after inoculation but remained unchanged (at about 104 CFU/g) in week 3. Populations of MAE110 were significantly higher (P<0.05) than those of MAE119 from day 3 after inoculation. In the first year, nine fruits collected from one of the 42 MAE119 inoculated plants were positive for S. enterica Typhimurium. In the second year, Salmonella was detected in adjacent non-inoculated leaves of eight tomato plants (five inoculated with strain MAE110). The pulp of 12 fruits from two plants inoculated with MAE110 was Salmonella positive (about 106 CFU/g). Internalization was confirmed by fluorescence and confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can move inside tomato plants grown in natural field soil and colonize fruits at high levels without inducing any symptoms, except for a slight reduction in plant growth.

Sclerotinia blight resistance in Virginia-type peanut transformed with a barley oxalate oxidase gene.

Transgenic peanut lines expressing oxalate oxidase, a novel enzyme to peanut, were evaluated for resistance to Sclerotinia blight in naturally infested fields over a 5-year period. Area under the disease progress curve (AUDPC) for transgenic lines in single rows planted with seed from single-plant selections averaged 78, 83, and 90% lower than nontransgenic parents in 2004, 2005, and 2006, respectively. In addition, AUDPC in 14 transgenic lines planted with bulked seed in two-row plots averaged 81% lower compared with nontransgenic parents in 2005 and 86% lower in 16 transgenic lines in 2006. Six transgenic lines yielded 488 to 1,260 kg/ha greater than nontransgenic parents in 2005, and 10 lines yielded 537 to 2,490 kg/ha greater in 2006. Fluazinam (0.58 kg a.i./ha) fungicide sprays in 2008 and 2009 reduced AUDPC in transgenic and nontransgenic lines but AUDPC was lowest in transgenic lines. Without fluazinam, yields of transgenic lines averaged 1,133 to 1,578 kg/ha greater than nontransgenic lines in 2008 and 1,670 to 2,755 kg/ha greater in 2009. These results demonstrated that the insertion of barley oxalate oxidase in peanut conveyed a high level of resistance to Sclerotinia blight, and negated the need for costly fungicide sprays.

qPCR Quantification of Pathogenic Guignardia citricarpa and Nonpathogenic G. mangiferae in Citrus

Citrus black spot, a major citrus disease caused by Guignardia citricarpa, was recently introduced in Florida. The nonpathogenic fungal endophyte G. mangiferae is commonly found in the same citrus tissues as G. citricarpa. Quantitative polymerase chain reaction (qPCR) assays based on internal transcribed spacer (ITS)-1 genes were developed to detect, quantify, and distinguish between these morphologically similar organisms in environmental samples. The primer/probe sets GCITS and GMITS were more than 95% efficient in single-set reactions in complex environmental DNA samples. Detection of 10 fg of G. citricarpa and G. mangiferae DNA was possible. Pycnidiospore disruption resulted in detection of single pycnidiospores with 78 (59 to 102; 95% confidence interval [CI]) and 112 (92 to 136; 95% CI) ITS copies for G. citricarpa and G. mangiferae, respectively. Detection was from partially decomposed leaves where fruiting bodies cannot be morphologically distinguished. Temperature and wetting period have significant effects on Guignardia spp. pseudothecia production in leaf litter. Based on relative biomass or the proportion of nuclei detected, G. citricarpa and G. mangiferae respond more strongly to wetting period than temperature. This qPCR assay will provide additional epidemiological data on black spot in tissues where G. citricarpa and G. mangiferae are not easily distinguished.

Survival of Salmonella enterica Typhimurium in Water Amended with Manure

Outbreaks of Salmonella enterica have been associated with water sources. Survival of S. enterica in various environments has been studied but survival in water has rarely been attempted. In two separate experiments, we examined the survival of S. enterica Typhimurium in clean spring water at various eutrophication levels and temperatures. In the first experiment, lasting for 135 days, survival of S. enterica (10(10) CFU/ml) in water with 0, 50, 100, 500, and 1,000 mg/liter of added carbon at 7, 17, and 27°C was monitored weekly. In the second experiment, lasting for 3 weeks, survival of S. enterica in water at 0, 100, and 200 mg/ liter of added carbon and 27°C was studied daily. Each experiment had four replicates. Dissolved organic carbon was measured daily in each experiment. At the beginning, midpoint, and end of the survival study, microbial communities in both experiments were assessed by denaturing gradient gel electrophoresis (DGGE). Even at minimal carbon concentrations, S. enterica survived for at least 63 d. Survival of Salmonella was highly dependent on eutrophication levels (as measured by dissolved organic carbon) and temperature, increasing at high eutrophication levels, but decreasing at high temperatures. Survival was also strongly affected by microbial competition or predation.

Development of a Microemulsion Formulation for Antimicrobial SecA Inhibitors

In our previous study, we have identified five antimicrobial small molecules via structure based design, which inhibit SecA of Candidatus Liberibacter asiaticus (Las). SecA is a critical protein translocase ATPase subunit and is involved in pre-protein translocation across and integration into the cellular membrane in bacteria. In this study, eleven compounds were identified using similarity search method based on the five lead SecA inhibitors identified previously. The identified SecA inhibitors have poor aqueous solubility. Thus a microemulsion master mix (MMX) was developed to address the solubility issue and for application of the antimicrobials. MMX consists of N-methyl-2-pyrrolidone and dimethyl sulfoxide as solvent and co-solvent, as well as polyoxyethylated castor oil, polyalkylene glycol, and polyoxyethylene tridecyl ether phosphate as surfactants. MMX has significantly improved the solubility of SecA inhibitors and has no or little phytotoxic effects at concentrations less than 5.0% (v/v). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the SecA inhibitors and streptomycin against eight bacteria including Agrobacterium tumefaciens, Liberibacter crescens, Rhizobium etli, Bradyrhizobium japonicum, Mesorhizobium loti, and Sinorhizobium meliloti phylogenetically related to Las were determined using the broth microdilution method. MIC and MBC results showed that the 16 SecA inhibitors have antibacterial activities comparable to that of streptomycin. Overall, we have identified 11 potent SecA inhibitors using similarity search method. We have developed a microemulsion formulation for SecA inhibitors which improved the antimicrobial activities of SecA inhibitors.

Assessment of Peanut Quality and Compositional Characteristics among Transgenic Sclerotinia Blight-Resistant and Non-Transgenic Susceptible Cultivars

This study presents the results of a comparison that includes an analysis of variance and a canonical discriminant analysis to determine compositional equivalence and similarity between transgenic, sclerotinia blight-resistant and non-transgenic, susceptible cultivars of peanut in 3 years of field trials. Three Virginia-type cultivars (NC 7, Wilson, and Perry) and their corresponding transgenic lines (N70, W73, and P39) with a barley oxalate oxidase gene were analyzed for differences in key mineral nutrients, fatty acid components, hay constituents, and grade characteristics. Results from both analyses demonstrated that transgenic lines were compositionally similar to their non-transgenic parent cultivar in all factors as well as market-grade characteristics and nutritional value. Transgenic lines expressing oxalate oxidase for resistance to sclerotinia blight were substantially equivalent to their non-transgenic parent cultivar in quality and compositional characteristics.

Inheritance of mefenoxam resistance in Phytophthora nicotianae populations from a plant nursery

Three sexual crosses involving isolates of P. nicotianae with differing sensitivity to mefenoxam were established to study the inheritance of mefenoxam resistance. Mefenoxam sensitivity was determined by measuring the mycelial growth on both mefenoxam-amended clarified V8 agar and non-amended agar and then calculating the relative growth. When both parents had the same phenotype (both were resistant or both were sensitive), all F1 progeny had the parental phenotype and no segregation for a major effect gene was observed in sensitivity to mefenoxam. However, variations in the mycelial growth of progeny indicated the segregation of minor-effect genes. When the cross involving the mefenoxam-resistant isolate 3A4 and a sensitive parent, the F1 progeny segregated for mefenoxam resistance in a ratio of 1:1 (resistant: sensitive), indicating that the mefenoxam resistance is controled by a single dominant gene. Mating type was not linked to the mefenoxam-resistance gene locus. One RAPD marker linked in trans to the MEX locus was obtained by bulked segregant analysis using RAPD markers and was converted to a sequence characterized amplified region marker (SCAR). The SCAR maker identified in this study is a limited but useful tool for differentiating homozygous resistant isolates from sensitive isolates of P. nicotianae.

Comparative susceptibility of peanut genetically engineered for sclerotinia blight resistance to non-target peanut pathogens

Field trials were conducted from 2006 to 2008 at the Tidewater Agricultural Research and Extension Center (TAREC) in Suffolk, Virginia to determine whether Blight Blocker transgenic peanut lines showed possible increased or decreased susceptibility to common peanut pathogens. Disease susceptibility was evaluated for seven transgenic lines containing a barley oxalate oxidase gene and their corresponding parental cultivars (Perry, Wilson, NC 7). In addition to Sclerotinia blight, the peanut diseases evaluated included: i) early leaf spot caused by Cercospora arachidicola, ii) Cylindrocladium black rot caused by Cylindrocladium parasiticum, iii) southern stem rot caused by Sclerotium rolfsii, iv) tomato spotted wilt virus, and v) aflatoxin levels in seeds caused by Aspergillus flavus or A. parasiticus. Results demonstrated that the susceptibility of Blight Blocker transgenic lines to common peanut pathogens was similar to that of non-transgenic cultivars, while transgenic lines provided resistance to Sclerotinia blight caused by S. minor. Transgenic lines consistently provided high levels of resistance to S. minor in all three years, however, the barley oxalate oxidase had little or no effect on the disease susceptibility to other organisms on peanut. The results of this research should provide additional evidence needed to petition for deregulation of Blight Blocker peanut lines.