Gary R. Bauchan

Methods of developing a core collection of annual Medicago species

A core collection is a subset of a large germplasm collection that contains accessions chosen to represent the genetic variability of the germplasm collection. The purpose of the core collection is to improve management and use of a germplasm collection. Core collections are usually assembled by grouping accessions and selecting from within these groups. The objective of this study was to compare 11 methods of assembling a core collection of the U.S. National collection of annual Medicago species. These methods differed in their use of passport and evaluation data as well as their selection strategy. Another objective was to compare core collections with sample sizes of 5%, 10% and 17% of the germplasm collection. Core collections assembled with evaluation data and cluster analysis better represented the germplasm collection than core collections assembled based solely on passport data and random selection of accessions, The Relative Diversity and the logarithm methods generated better core collections than the proportional method. The 5% and 10% sample size core collection were judged insufficient to represent the germplasm collection.

Role of Curli and Cellulose Expression in Adherence of Escherichia coli O157:H7 to Spinach Leaves

Shiga-toxigenic Escherichia coli O157:H7 outbreaks have been linked to consumption of fresh produce. It is generally recognized that bacterial attachment to vegetal matrices constitutes the first step in contamination of fresh produce. Cellular appendages, such as curli fibers, and cellulose, a constituent of extracellular matrix, have been suggested to be involved in E. coli attachment and persistence in fresh produce. A comparative evaluation was conducted on the ability of Shiga toxin-producing E. coli O157:H7 strains EDL933 and 86-24, linked to two independent foodborne disease outbreaks in humans, and their mutants deficient in curli and/or cellulose expression to colonize and to firmly attach to spinach leaf. Inoculated spinach leaves were incubated at 22°C, and at 0, 24, and 48 h after incubation loosely and strongly attached E. coli O157:H7 populations were determined. Curli-expressing E. coli O157:H7 strains developed stronger association with leaf surface, whereas curli-deficient mutants attached to spinach at significantly (p<0.01) lower numbers. Attachment of cellulose-impaired mutants to spinach leaves was not significantly different from that of curliated strains. The relative attachment strength of E. coli O157:H7 to spinach increased with incubation time for the curli-expressing strains. Laser scanning confocal microscopy (LSCM) analysis of inoculated leaves revealed that curli-expressing E. coli O157:H7 were surrounded by extracellular structures strongly immunostained with anti-curli antibodies. Production of cellulose was not required to develop strong attachment to spinach leaf. These results indicate that curli fibers are essential for strong attachment of E. coli O157:H7 to spinach whereas cellulose is dispensable.

Enhanced Inactivation of Salmonella and Pseudomonas Biofilms on Stainless Steel by Use of T-128, a Fresh-Produce Washing Aid, in Chlorinated Wash Solutions

ABSTRACT The effect of the washing aid T-128 (generally recognized as safe [GRAS] formulation, composed mainly of phosphoric acid and propylene glycol) on inactivation of Salmonella and Pseudomonas populations in biofilms on stainless steel was evaluated under conditions of increasing organic matter loads in chlorinated wash solutions dominated by hypochlorous acid. Biofilms were formed statically on stainless steel coupons suspended in 2% lettuce extract after inoculation with Salmonella enterica serovar Thompson or Newport or with Pseudomonas fluorescens. Coupons with biofilms were washed in chlorine solutions (0, 0.5, 1, 2, 5, 10, or 20 mg/liter at pH 6.5, 5.0 and 2.9), with or without T-128, and with increasing loads of organic matter (0, 0.25, 0.5, 0.75, or 1.0% lettuce extract). Cell populations on coupons were dispersed using intermittent, pulsed ultrasonication and vortexing and enumerated by colony counts on XLT-4 or Pseudomonas agars. Cell responses to fluorescent viability staining of biofilm treatment washing solutions were examined using confocal laser scanning microscopy. Results showed that 0.1% T-128 (without chlorine) reduced P. fluorescens biofilm populations by 2.5 log10 units but did not reduce Salmonella populations. For both Salmonella and Pseudomonas, the sanitizing effect of free chlorine (1.0 to 5.0 mg/liter) was enhanced (P < 0.05) when it was combined with T-128. Application of T-128 decreased the free chlorine depletion rate caused by increasing organic matter in wash waters and significantly (P < 0.05) augmented inactivation of bacteria in biofilms compared to treatments without T-128. Image analysis of surfaces stained with SYTO and propidium iodide corroborate the cultural assay results showing that T-128 can aid in reducing pathogen viability in biofilms and thus can aid in sanitizing stainless steel contact surfaces during processing of fresh-cut produce.

Spinacia oleracea L. Leaf Stomata Harboring Cryptosporidium parvum Oocysts: a Potential Threat to Food Safety

ABSTRACT Cryptosporidium parvum is a cosmopolitan microscopic protozoan parasite that causes severe diarrheal disease (cryptosporidiosis) in mammals, including humans and livestock. There is growing evidence of Cryptosporidium persistence in fresh produce that may result in food-borne infection, including sporadic cases as well as outbreaks. However, drinking and recreational waters are still considered the major sources of Cryptosporidium infection in humans, which has resulted in prioritization of studies of parasite etiology in aquatic environments, while the mechanisms of transmission and parasite persistence on edible plants remain poorly understood. Using laser scanning confocal microscopy together with fluorescein-labeled monoclonal antibodies, C. parvum oocysts were found to strongly adhere to spinach plants after contact with contaminated water, to infiltrate through the stomatal openings in spinach leaves, and to persist at the mesophyll level. These findings and the fact that this pathogenic parasite resists washing and disinfection raise concerns regarding food safety.

Identification and utilization of a sow thistle powdery mildew as a poorly adapted pathogen to dissect post-invasion non-host resistance mechanisms in Arabidopsis

To better dissect non-host resistance against haustorium-forming powdery mildew pathogens, a sow thistle powdery mildew isolate designated Golovinomyces cichoracearum UMSG1 that has largely overcome penetration resistance but is invariably stopped by post-invasion non-host resistance of Arabidopsis thaliana was identified. The post-invasion non-host resistance is mainly manifested as the formation of a callosic encasement of the haustorial complex (EHC) and hypersensitive response (HR), which appears to be controlled by both salicylic acid (SA)-dependent and SA-independent defence pathways, as supported by the susceptibility of the pad4/sid2 double mutant to the pathogen. While the broad-spectrum resistance protein RPW8.2 enhances post-penetration resistance against G. cichoracearum UCSC1, a well-adapted powdery mildew pathogen, RPW8.2, is dispensable for post-penetration resistance against G. cichoracearum UMSG1, and its specific targeting to the extrahaustorial membrane is physically blocked by the EHC, resulting in HR cell death. Taken together, the present work suggests an evolutionary scenario for the Arabidopsis–powdery mildew interaction: EHC formation is a conserved subcellular defence evolved in plants against haustorial invasion; well-adapted powdery mildew has evolved the ability to suppress EHC formation for parasitic growth and reproduction; RPW8.2 has evolved to enhance EHC formation, thereby conferring haustorium-targeted, broad-spectrum resistance at the post-invasion stage.

Ectopic expression of AtPAD4 broadens resistance of soybean to soybean cyst and root-knot nematodes

BackgroundThe gene encoding PAD4 (PHYTOALEXIN-DEFICIENT4) is required in Arabidopsis for expression of several genes involved in the defense response to Pseudomonas syringae pv. maculicola. AtPAD4 (Arabidopsis thaliana PAD4) encodes a lipase-like protein that plays a regulatory role mediating salicylic acid signaling.ResultsWe expressed the gene encoding AtPAD4 in soybean roots of composite plants to test the ability of AtPAD4 to deter plant parasitic nematode development. The transformed roots were challenged with two different plant parasitic nematode genera represented by soybean cyst nematode (SCN; Heterodera glycines) and root-knot nematode (RKN; Meloidogyne incognita). Expression of AtPAD4 in soybean roots decreased the number of mature SCN females 35 days after inoculation by 68 percent. Similarly, soybean roots expressing AtPAD4 exhibited 77 percent fewer galls when challenged with RKN.ConclusionsOur experiments show that AtPAD4 can be used in an economically important crop, soybean, to provide a measure of resistance to two different genera of nematodes.

External mouthpart morphology in the Tenuipalpidae (Tetranychoidea): Raoiella a case study

The use of low-temperature scanning electron microscopy (LTSEM) to study external mouthpart morphology in the Tenuipalpidae, in particular the genus Raoiella, has brought some aspects of the mechanics of feeding in this group into question. In addition, an LTSEM study on the specialized feeding behaviour of Raoiellaindica Hirst (Tetranychoidea: Tenuipalpidae) revealed host plant use in this species could be affected by stomatal complex morphology.

Role Bending: Complex Relationships Between Viruses, Hosts, and Vectors Related to Citrus Leprosis, an Emerging Disease

Citrus leprosis complex is an emerging disease in the Americas, associated with two unrelated taxa of viruses distributed in South, Central, and North America. The cytoplasmic viruses are Citrus leprosis virus C (CiLV-C), Citrus leprosis virus C2 (CiLV-C2), and Hibiscus green spot virus 2, and the nuclear viruses are Citrus leprosis virus N (CiLV-N) and Citrus necrotic spot virus. These viruses cause local lesion infections in all known hosts, with no natural systemic host identified to date. All leprosis viruses were believed to be transmitted by one species of mite, Brevipalpus phoenicis. However, mites collected from CiLV-C and CiLV-N infected citrus groves in Mexico were identified as B. yothersi and B. californicus sensu lato, respectively, and only B. yothersi was detected from CiLV-C2 and CiLV-N mixed infections in the Orinoco regions of Colombia. Phylogenetic analysis of the helicase, RNA-dependent RNA polymerase 2 domains and p24 gene amino acid sequences of cytoplasmic leprosis viruses showed a close relationship with recently deposited mosquito-borne negevirus sequences. Here, we present evidence that both cytoplasmic and nuclear viruses seem to replicate in viruliferous Brevipalpus species. The possible replication in the mite vector and the close relationship with mosquito borne negeviruses are consistent with the concept that members of the genus Cilevirus and Higrevirus originated in mites and citrus may play the role of mite virus vector.

Mutation of a chloroplast-targeting signal in Alternanthera mosaic virus TGB3 impairs cell-to- cell movement and eliminates long-distance virus movement

Cell-to-cell movement of potexviruses requires coordinated action of the coat protein and triple gene block (TGB) proteins. The structural properties of Alternanthera mosaic virus (AltMV) TGB3 were examined by methods differentiating between signal peptides and transmembrane domains, and its subcellular localization was studied by Agrobacterium-mediated transient expression and confocal microscopy. Unlike potato virus X (PVX) TGB3, AltMV TGB3 was not associated with the endoplasmic reticulum, and accumulated preferentially in mesophyll cells. Deletion and site-specific mutagenesis revealed an internal signal VL(17,18) of TGB3 essential for chloroplast localization, and either deletion of the TGB3 start codon or alteration of the chloroplast-localization signal limited cell-to-cell movement to the epidermis, yielding a virus that was unable to move into the mesophyll layer. Overexpression of AltMV TGB3 from either AltMV or PVX infectious clones resulted in veinal necrosis and vesiculation at the chloroplast membrane, a cytopathology not observed in wild-type infections. The distinctive mesophyll and chloroplast localization of AltMV TGB3 highlights the critical role played by mesophyll targeting in virus long-distance movement within plants.

Drought Responses of Foliar Metabolites in Three Maize Hybrids Differing in Water Stress Tolerance

Maize (Zea mays L.) hybrids varying in drought tolerance were treated with water stress in controlled environments. Experiments were performed during vegetative growth and water was withheld for 19 days beginning 17 days after sowing. Genotypic comparisons used measured changes of leaf water potential or results were expressed by time of treatment. Total dry matter of the drought tolerant hybrid on the final harvest was 53% less than that of the intermediate and susceptible maize hybrids when plants were water sufficient. This showed that maize hybrids selected for extreme drought tolerance possessed a dwarf phenotype that affected soil water contents and leaf water potentials. Changes of shoot and root growth, leaf water potential, net photosynthesis and stomatal conductance in response to the time of water stress treatment were diminished when comparing the drought tolerant to the intermediate or susceptible maize hybrids. Genotypic differences were observed in 26 of 40 total foliar metabolites during water stress treatments. Hierarchical clustering revealed that the tolerant maize hybrid initiated the accumulation of stress related metabolites at higher leaf water potentials than either the susceptible or intermediate hybrids. Opposite results occurred when changes of metabolites in maize leaves were expressed temporally. The above results demonstrated that genotypic differences were readily observed by comparing maize hybrids differing in drought tolerance based on either time of treatment or measured leaf water potential. Current findings provided new and potentially important insights into the mechanisms of drought tolerance in maize.