Zucchini shoestring virus: a distinct potyvirus in the papaya ringspot virus cluster.

Zucchini shoestring virus (ZSSV) has been proposed to be a putative potyvirus in the papaya ringspot virus (PRSV) cluster, based on the sequence similarity of its coat protein to those of related potyviruses. ZSSV has been associated with the outbreak of a damaging disease of baby marrow (Cucurbita pepo L.) that had been observed throughout the province of KwaZulu-Natal, in the Republic of South Africa (RSA). We report the genome sequence of ZSSV, determined by next-generation sequencing of total RNA extracted from an infected baby marrow (Cucurbita pepo L.). The ZSSV genome is 10,295 nucleotides long excluding the poly(A) tail and displays a typical potyvirus organization. Algerian watermelon mosaic virus (AWMV; EU410442.1) was identified as the closest relative of ZSSV, sharing the highest nucleotide sequence identity of 65.68%. The nucleotide and amino acid sequence identity values for each protein support the differentiation of ZSSV as a member of a distinct species in the genus Potyvirus. This taxonomic position was also confirmed using the Pairwise Sequence Comparison online tool from the National Center for Biotechnology Information. Phylogenetic analysis of the polyprotein coding sequence of ZSSV grouped ZSSV together with AWMV and Moroccan watermelon mosaic virus, but in different clusters. ZSSV is the second cucurbit-infecting virus in the PRSV cluster present in RSA.

A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies.

Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under variable pressure scanning electron microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05) from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies.

A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies

Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under variable pressure scanning electron microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05) from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies.

In vitro anthelmintic activity of crude extracts of selected medicinal plants against Haemonchus contortus from sheep.

Ethanol extracts of 25 plant species were screened for anthelmintic effects against Haemonchus contortus. Ethanol extracts of each plant were used at various concentrations (10, 20 and 30%) to treat 10-day faecal cultures, incubated at 27°C with control cultures which were treated with ethanol for 48 h. Five plants with high efficacies (Ananas comosus, Aloe ferox, Allium sativum, Lespedeza cuneata and Warburgia salutaris) were selected from the first screening for further investigation using ethanol, dichloromethane and water extracts at four concentrations (2.5, 5, 10 and 20%). Ethanol was the most effective solvent. Larval counts decreased with increasing extract concentrations, of which 10 and 20% had similar effects. Lespedeza cuneata caused more than 70% mortality at all concentrations. However, there remains a need to assess in vivo efficacy of these plants.

Use of mixed cultures of biocontrol agents to control sheep nematodes.

Biological control is a promising non-chemical approach for the control of gastrointestinal nematodes of sheep. Use of combinations of biocontrol agents have been reported to be an effective method to increase the efficacy of biological control effects. In this study, combinations of either two Bacillus thuringiensis (Bt) or Clonostachys rosea (C. rosea) isolates and Bt+C. rosea isolates were evaluated in vitro in microtitre plates for their biocontrol activity on sheep nematodes. The Baermann technique was used to extract the surviving L3 larval stages of intestinal nematodes and counted under a dissecting microscope to determine the larval counts. Results indicate that there was a significant reduction of nematode counts due to combination of biocontrol agents (P<0.001). Combinations of Bt isolates reduced nematodes counts by 72.8%, 64% and 29.8%. The results revealed a control level of 57% when C. rosea isolates P3+P8 were combined. Combination of Bt and C. rosea isolates B10+P8 caused the greatest mortality of 76.7%. Most combinations were antagonistic, with only a few combinations showing an additive effect. None were synergistic. The isolate combinations were more effective than when isolates were used alone.

Laboratory and field evaluation of formulated Bacillus thuringiensis var. israelensis as a feed additive and using topical applications for control of Musca domestica (Diptera: Muscidae) larvae in caged-poultry manure.

Infestations of house flies, Musca domestica L., are a continual problem around poultry establishments. Acute toxicity of two commercial Bacillus thuringiensis variety israelensis (Bti) formulations (water-dispersible granules and bran formulation) was evaluated against larvae in the laboratory and against natural populations of M. domestica larvae in the field applied in feed to chickens and as topical applications in the poultry houses. Bioassay data showed that susceptibility of M. domestica larvae increased to a given concentration of Bti as the duration of exposure increased. In the laboratory studies, the LC(50) values of Bti for the larvae ranged between 65 and 77.4 µg/ml. In the field, a concentration of 10 g Bti/kg of feed resulted in 90% reduction of larvae at 4 wk after treatment. A higher concentration (2 g/liter) of Bti in spray applications was not significantly more effective than the lower concentration of 1 g/liter. Adding Bti to chicken feed is potentially an efficient measure for the management and control of house flies in caged-poultry facilities.

Interaction between Beauveria bassiana and Bacillus thuringiensis var. israelensis for the control of house fly larvae and adults in poultry houses.

Field trials were carried out in the poultry houses to determine if a spray formulation of Beauveria bassiana (Balsamo) Vuillemin isolate R444 would enhance the control potential of Bacillus thuringiensis var. israelensis (Bti) applied in chicken feed as a larvicide against house fly larvae and the emergence of adult house flies. The trials compared larval mortalities and adult house fly emergence that resulted from the Bti plus B. bassiana treatments with those resulting from a commercial larvicide, Larvadex, plus B. bassiana treatments. All treatments significantly reduced the house fly larval densities and adult house fly emergence compared with any of the agents acting alone. After 6 wk of application, house fly larvae decreased by 11% as a result of B. bassiana treatment alone, 41% for 250 mg.kg(-1) Bti alone, and 42% for 500 mg.kg(-1) Bti alone. Larval mortalities as a result of the combination treatments were 45 and 52% as a result of 250 mg.kg(-1) Bti plus B. bassiana and 500 mg.kg(-1) Bti plus B. bassiana treatments respectively. House fly larval mortalities as a result of Larvadex and B. bassiana were 30% for Larvadex alone and 38% as a result of Larvadex plus B. bassiana. The Bti treatments were more effective at inhibiting the emergence of adult house flies than Larvadex, even when Larvadex was applied together with B. bassiana. The interaction effects of Bti plus B. bassiana and Larvadex plus B. bassiana were additive. These trials suggested that in the control of house fly larvae, the efficacy of Bti, applied as a larvicide, may be improved with frequent spray applications of B. bassiana to the chicken manure.

Colonization of Bacillus spp. on seeds and in plant rhizoplane.

Seed coating, dipping and Scanning Electron Microscopy (SEM) were employed to study bacterial and fungal colonization of the seeds and rhizoplane of maize (Zea mays L.) during the early stages of growth. Isolation of Bacillus spp. entailed screening soil bacteria with potential growth stimulation and plant pathogen suppressive abilities isolated from the rhizospheres and rhizoplanes of vegetable crops. The bacterial colonization of the spermosphere was 90%. When the coated seeds were fully germinated, bacteria moved to the emerging radicle. Virtually no bacteria occurred on the root tip both for the treated and untreated. However, colonization was 20% in the basal portion of the roots close to the seed-root junction. SEM observations showed that the bacterial cells were arranged linearly and laterally on the growing root axis. This phenomenon was more noticeable in the seedlings dipped in the bacterial culture on the 3rd day after germination. The results indicate that attachment to the seed coat and the rhizoplane by the plant growth-promoting rhizobacterium (PGPR) is an important factor in the successful colonization of the rhizoplane. The significance of the work is to ascertain that the inoculated Bacillus spp. adhered to and established in the rhizoplane of maize. It can therefore be used as a PGPR and as a biocontrol agent.

Assessment of the Effects of Fertilizer Applications on Gray Leaf Spot and Yield in Maize.

The effects of the application of 0, 60, and 120 kg N ha-1 and of 0, 25, 50, and 150 kg K ha-1 on final disease severity, standardized area under disease progress curve, and grain yield were investigated at Cedara, South Africa, on a maize (Zea mays) hybrid susceptible to gray leaf spot (GLS), caused by Cercospora zeae-maydis. The trial was a randomized 3 × 4 factor design, split for fungicide treatments, and replicated three times. With increased N and K levels, final percent leaf blighting and the standardized area under disease progress curve were higher. In fungicide-treated maize, grain yields increased with increasing levels of N and K, as expected. In non-fungicide-treated maize, grain yield increased significantly with increased levels of N, despite increased disease severity. This was in contrast to small increases in grain yields from non-fungicide-treated maize with increased K levels, which were not significant. This was probably because grain yield response, which should have occurred at higher K applications, was reduced by increased disease severity. The effect of N, P, and K on GLS wasinvestigated at Ahrens. Maize was grown in a 4 × 4 × 4 N-P-K factorial, in a randomized complete block design. Fertilizer was applied at 0, 60, 120, and 180 kg N ha-1, 0, 30, 60, and 120 kg P ha-1, and 0, 50, 100, and 150 kg K ha-1. No fungicides were applied. A single disease assessment at physiological maturity showed that final disease severity increased with increasing levels of N, P, and K. These results have implications for small-scale farmers who are encouraged to fertilize for increased grain yields but may not have the resources to apply fungicide sprays to control fungal diseases.

Frequency and Timing of Fungicide Applications for the Control of Gray Leaf Spot in Maize.

Timing and frequency of fungicide treatments for management and control of gray leaf spot of maize, caused by Cercospora zeae-maydis, were quantified with the logistic model and area under disease progress curve (AUDPC). Control was most effective when spraying commenced as disease severity levels reached 2 to 3% of the leaf area blighted and when lesions were restricted to the basal five leaves of the maize plant. Highest grain yields were achieved with treatments providing disease control until the crop was physiologically mature. To provide this length of control, the frequency and number of fungicide applications varied with the stage of host development when disease was first apparent; with early infections, more fungicide treatments were necessary to provide protection until physiological maturity. Yield responses to fungicides appeared to be a function of the growth stage of the host when sprays were initiated, the amount of disease at spray date, the length of fungicide control, and effective control through to physiological maturity.