Low Tunnels Reduce Insect Populations, Insecticide Application, and Chewing Insect Damage on Brussels Sprouts.

Low tunnels covered with spun-bonded fabric (row covers) provide season extension for vegetable production and also afford a physical barrier against airborne insects and other non-soil pests. Brussels sprouts, Brassica oleracea L. group Gemmifera (Brassicaceae), is a popular vegetable in local markets in Virginia; however, unprotected field production is severely affected by insect pest infestation. This study's objective was to determine the level of protection low tunnels provide against insect infestation and leaf herbivory injury. The experiment was conducted at the Virginia Tech Eastern Shore Agricultural Research and Extension Center in Painter, Virginia. The experimental design was split-plot with polyethylene soil mulches (white or black) as whole plot factors and production systems (low tunnel or open field) as subplot factors. In this study, low tunnels reduced insect infestation and chewing herbivory leaf injury to Brussels sprouts. Compared to an unprotected open field, infestations of lepidopteran insects and harlequin bug, Murgantia histrionica (Hahn) (Hemiptera: Pentatomidae) were reduced on plants under low tunnels. However, aphids (Hemiptera: Aphidae) infestation occurred under low tunnels in fall. There was no effect of color mulches (white or black) and no interaction between tunnel and mulch color on insect infestation and chewing injury. Fewer insect infestations and feeding injury indicate that low tunnels can be an effective management tool for sustainable vegetable production.

Quantitative Real-Time PCR Analysis of Individual Flue-Cured Tobacco Seeds and Seedlings Reveals Seed Transmission of Tobacco Mosaic Virus.

Tobacco mosaic virus (TMV) is an extensively studied RNA virus known to infect tobacco (Nicotiana tabacum) and other solanaceous crops. TMV has been classified as a seedborne virus in tobacco, with infection of developing seedlings thought to occur from contact with the TMV-infected seed coat. The mechanism of TMV transmission through seed was studied in seed of the K 326 cultivar of flue-cured tobacco. Cross pollinations were performed to determine the effect of parental tissue on TMV infection in seed. Dissection of individual tobacco seeds into seed coat, endosperm, and embryo was performed to determine TMV location within a seed, while germination tests and separation of the developing seedling into seed coat, roots, and cotyledons were conducted to estimate the percent transmission of TMV. A reverse-transcriptase quantitative PCR (RT-qPCR) assay was developed and used to determine TMV concentrations in individual seed harvested from pods that formed on plants from TMV-infected and noninfected crosses. The results showed maternal transmission of TMV to tobacco seed and seedlings that developed from infected seed, not paternal transmission. RT-qPCR and endpoint PCR assays were also conducted on the separated seed coat, endosperm, and embryo of individual seed and separated cotyledons, roots, and seed coats of individual seedlings that developed from infected tobacco seed to identify the location of the virus in the seed and the subsequent path the virus takes to infect the developing seedling. RT-qPCR and endpoint PCR assay results showed evidence of TMV infection in the endosperm and embryo, as well as in the developing seedling roots and cotyledons within 10 days of initiating seed germination. To our knowledge, this is the first report of TMV being detected in embryos of tobacco seed, demonstrating that TMV is seedborne and seed-transmitted in flue-cured tobacco.

Nicotiana species as surrogate host for studying the pathogenicity of Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbits.

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli is one of the most important bacterial diseases of cucurbits worldwide. However, the mechanisms associated with A. citrulli pathogenicity and genetics of host resistance have not been extensively investigated. We idenitfied Nicotiana benthamiana and Nicotiana tabacum as surrogate hosts for studying A. citrulli pathogenicity and non-host resistance triggered by type III secreted (T3S) effectors. Two A. citrulli strains, M6 and AAC00-1, that represent the two major groups amongst A. citrulli populations, induced disease symptoms on N. benthamiana, but triggered a hypersensitive response (HR) on N. tabacum plants. Transient expression of 19 T3S effectors from A. citrulli in N. benthamiana leaves revealed that three effectors, Aave_1548, Aave_2708, and Aave_2166, trigger water-soaking-like cell death in N. benthamiana. Aave_1548 knockout mutants of M6 and AAC00-1 displayed reduced virulence on N. benthamiana and melon (Cucumis melo L.). Transient expression of Aave_1548 and Aave_2166 effectors triggered a non-host HR in N. tabacum, which was dependent on the functionality of the immune signalling component, NtSGT1. Hence, employing Nicotiana species as surrogate hosts for studying A. citrulli pathogenicity may help characterize the function of A. citrulli T3S effectors and facilitate the development of new strategies for BFB management.

Cetylpyridinium chloride direct spray treatments reduce Salmonella on cantaloupe rough surfaces.

Cetylpyridinium chloride (CPC) solutions (0, 0.5, or 1.0%) were applied to cantaloupe ("Athena" and "Hale's Best Jumbo" cultivars) rind plugs, either before or after inoculation with a broth culture of Salmonella Michigan (109 CFU/mL) and held at 37°C for 1 or 24 hr. Rind plugs were diluted, shaken, and sonicated, and solutions were enumerated. Texture quality and color were evaluated over 14 days storage at 4°C after 0 and 1% CPC spray applications. A 0.5 or 1.0% (vol/vol) application of CPC after Salmonella reduced the pathogen levels between 2.34 log CFU/mL and 5.16 log CFU/mL in comparison to the control (p < .01). No differences were observed in the firmness and color of 1% CPC treated cantaloupes. Salmonella concentrations on cantaloupes, treated with 1.0% CPC, were lower after 1 hr storage as compared to 24 hr. And, Salmonella on "Athena" surfaces were more susceptible to CPC spray treatments than on "Hale's Best Jumbo." PRACTICAL APPLICATIONS: Cetylpyridinium chloride (CPC) is the active ingredient of some antiseptic oral mouth rinses, and has a broad antimicrobial spectrum with a rapid bactericidal effect on gram-positive pathogens. The spray application of CPC solutions to cantaloupe may reduce the level of Salmonella surface contamination during production from irrigation water and manure fertilizers and, during food processing by contaminated equipment and food handlers. Since the surfaces of cantaloupes are highly rough or irregular, bacteria can easily attach to these surfaces and become difficult to remove. Appropriate postharvest washing and sanitizing procedures are needed that can help control Salmonella and other pathogens on melons, especially on cantaloupes with nested surfaces. A direct surface spray application of CPC may be an alternative antimicrobial postharvest treatment to reduce pathogen contamination of cantaloupe melons, while providing an alternative to chlorine-based solutions.

Delmopinol hydrochloride reduces Salmonella on cantaloupe surfaces.

Since the surfaces of cantaloupes are highly rough or irregular, bacteria can easily attach and become difficult to remove. Appropriate postharvest washing and sanitizing procedures can help control Salmonella and other pathogens on cantaloupe or other melons during postharvest operations. Delmopinol hydrochloride (delmopinol) is a cationic surfactant that is effective for treating and preventing gingivitis and periodontitis. The application of delmopinol to two cantaloupe cultivars was evaluated for reducing the level of inoculated Salmonella. Athena and Hale's Best Jumbo (HBJ) cantaloupe rind plugs (2.5 cm. dia.) were inoculated with nalidixic acid-resistant Salmonella Michigan (approx. 1.0 × 109 CFU/ml). After 15 min, rind plugs were sprayed with 10 ml of a delmopinol spray solution (0% or 1.0% vol/vol) and held at 35°C for 1 hr or 24 hr. Rind plugs were diluted with Butterfield's phosphate buffer, shaken and sonicated, and solutions were enumerated on 50 ppm nalidixic acid-tryptic soy agar. The texture quality and color of additional cantaloupes were evaluated, after 1% delmopinol spray treatment, over 14-day storage at 4°C. A 1.0% application of delmopinol after 1 hr reduced Salmonella concentration by ~3.1 log CFU/ml for both "HBJ" skin rind plugs and "Athena" stem scar rind plugs in comparison to the control (p < .05). No differences were observed in the texture and color (L*, a*, b* values) of 1% delmopinol-treated cantaloupes as compared to control. Storage of cantaloupes treated with 1.0% delmopinol solution for 1 hr had a greater effect on reducing concentration of Salmonella compared to 24-hr treatment. A surface spray application of 1% delmopinol on cantaloupes could be an alternative antimicrobial postharvest treatment that could make surface bacteria more susceptible to sanitizers or physical removal.

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes.

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a series of incubators. A temperature gradient is passively established across the surface of the table between the heated and cooled ends and is lost quickly at distances above the surface. Since temperature is only controlled on the table surface, experiments are restricted to shallow containers, such as Petri dishes, placed on the table. Welding continuous aluminum vertical strips or gussets perpendicular to the surface of a table enables temperature control in depth via convective heat flow. Soil in the channels between gussets was maintained across a gradient of temperatures allowing a greater diversity of experimentation. The gusseted design was evaluated by germinating oat, lettuce, tomato, and melon seeds. Soil temperatures were monitored using individual, battery-powered dataloggers positioned across the table. LED lights installed in the lids or along the sides of the gradient table create a controlled temperature chamber where seedlings can be grown over a range of temperatures. The gusseted design enabled accurate determination of optimum temperatures for fastest germination rate and the highest percentage germination for each species. Germination information from gradient table experiments can help predict seed germination and seedling growth under the adverse soil conditions often encountered during field crop production. Temperature effects on seed germination, seedling growth, and soil ecology can be tested under controlled conditions in a laboratory using a gusseted thermogradient table.

Shelf Life Determination of Fresh Blueberries (Vaccinium corymbosum) Stored under Controlled Atmosphere and Ozone.

Fresh blueberries are commonly stored and transported by refrigeration in controlled atmospheres to protect shelf life for long periods of storage. Ozone is an antimicrobial gas that can extend shelf life and protect fruit from microbial contamination. Shelf life of fresh highbush blueberries was determined over 10-day storage in isolated cabinets at 4°C or 12°C under different atmosphere conditions, including air (control); 5% O2 : 15% CO2 : 80% N2 (controlled atmosphere storage (CAS)); and ozone gas (O3) 4 ppm at 4°C or 2.5 ppm at 12°C, at high relative humidity (90-95%). Samples were evaluated for yeast and molds growth, weight loss, and firmness. CAS and O3 did not delay or inhibit yeast and molds growth in blueberries after 10 days at both temperatures. Fruit stored at 4°C showed lower weight loss values compared with 12°C. Blueberries stored under O3 atmosphere showed reduced weight loss at 12°C by day 10 and loss of firmness when compared to the other treatments. Low concentrations of ozone gas together with proper refrigeration temperature can help protect fresh blueberries quality during storage.

Diversity of the spinach (Spinacia oleracea) spermosphere and phyllosphere bacterial communities.

The bacterial diversity of seeds, transmission of bacteria from seed to phyllosphere, and fate of seed-transmitted bacteria on mature plants are poorly characterized. Understanding the dynamics of microbial communities is important for finding bio-control or mitigation strategies for human and plant pathogens. Bacterial populations colonizing spermosphere and phyllosphere of spinach (Spinacia oleracea) seedlings and plants were characterized using pyrosequencing of 16S rRNA gene amplicons. Spinach seed microbiota was composed of three bacterial phyla: Proteobacteria, Firmicutes and Actinobacteria, belonging to > 250 different operational taxonomic units (OTUs). Seed and cotyledon bacterial communities were similar in richness and diversity. Richness of 3-4 leaf-stage of development plants increased markedly to > 850 OTUs classified within 11 phyla. Although some bacterial OTUs were detected on seeds, cotyledons and plants, the breadth of new sequences indicates the importance of multiple sources outside the seed in shaping phyllosphere community. Most classified sequences were from previously undescribed taxa, highlighting the benefits of pyrosequencing in describing seed diversity and phyllosphere bacterial communities. Bacterial community richness increased from 250 different OTUs for spinach seeds and cotyledons, to 800 OTUs for seedlings. To our knowledge this is the first comprehensive characterization of the spinach microbiome, complementing previous culture-based and clone library studies.

Seed ultrastructure and water absorption pathway of the root-parasitic plant Phelipanche aegyptiaca (Orobanchaceae).

BACKGROUND AND AIMS: Obligate root parasitic plants of the Orobanchaceae do not germinate unless they chemically detect a host plant nearby. Members of this family, like Orobanche, Phelipanche and Striga, are noxious weeds that cause heavy damage to agriculture. In spite of their economic impact, only a few light microscopical studies of their minute seeds have been published, and there is no knowledge of their ultrastructure and of the role each tissue plays during the steps preceding germination. This paper describes the ultrastructure of Phelipanche seeds and contributes to our understanding of seed tissue function. METHODS: Seeds of P. aegyptiaca were examined under light, scanning electron, transmission electron and fluorescence microscopy following various fixations and staining protocols. The results were interpreted with physiological data regarding mode of water absorption and germination stimulation. KEY RESULTS AND CONCLUSIONS: The endothelium, which is the inner layer of the testa, rapidly absorbs water. Its interconnected cells are filled with mucilage and contain labyrinthine walls, facilitating water accumulation for germination that starts after receiving germination stimuli. Swelling of the endothelium leads to opening of the micropyle. The perisperm cells underneath this opening mediate between the rhizosphere and the embryo and are likely to be the location for the receptors of germination stimuli. The other perisperm cells are loaded with lipids and protein bodies, as are the endosperm and parts of the embryo. In the endosperm, the oil bodies fuse with each other while they are intact in the embryo and perisperm. Plasmodesmata connect the perisperm cells to each other, and the cells near the micropyle tightly surround the emerging seedling. These perisperm cells, and also the proximal embryo cells, have dense cytoplasmic contents, and they seem to represent the two seed components that are actively involved in transfer of reserve nutrients to the developing seedling during germination.

A gel diffusion assay for visualization and quantification of chitinase activity.

Higher plants, bacteria, fungi, insects, and crustaceans all produce chitinases. Chitinase genes in many organisms are currently under investigation. Chitinase activity is usually assayed with radiolabeled or fluorogenic substrates. We developed a simple, inexpensive, nonradioactive gel-diffusion assay for chitinase that can be used to screen large numbers of samples. In this assay, chitinase diffuses from a small circular well cut in an agarose or agar gel containing the substrate glycol chitin, a soluble, modified form of chitin. Chitinase catalyzes the cleavage of glycol chitin as it diffuses through the gel, leaving a dark, unstained circular zone around the well, because the fluorescent dye calcofluor binds only to undigested chitin. Sample activities can be determined from linear regression of log-standard enzyme concentration versus the zone diameter of internal standards on each Petri dish used for a diffusion assay.