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.     

Antimicrobial activity of essential oils and their components against the three major pathogens of the cultivated button mushroom, Agaricus bisporus

Essential oils of Matricaria chamommilla, Mentha piperita, M. spicata, Lavandula angusti folia, Ocimum basilicum, Thymus vulgaris, Origanum vulgare, Salvia officinalis, Citrus limon and C. aurantium and their components; linalyl acetate, linalool, limonene, α-pinene, β-pinene, 1,8-cineole, camphor, carvacrol, thymol and menthol were assayed for inhibitory activity against the three major pathogens of the button mushroom, Agaricus bisporus, i.e. the fungi Verticillium fungicola and Trichoderma harzianum and the bacterium Pseudomonas tolaasii. The highest and broadest activity was shown by the Origanum vulgare oil. Carvacrol possessed the highest antifungal activity among the components tested.     

Effect of compost and casing treatments of insecticides against the sciarid Bradysia ocellaris (Diptera: Sciaridae) and on the total yield of cultivated mushrooms,Agaricus bisporus

BACKGROUND: Bradysia ocellaris (Comstock) is one of the major pests of cultivated mushrooms, Agaricus bisporus (Lange) Imbach. The feeding of sciarid fly larvae causes damage to the cultivation substrates, compost and casing. A comparative study of insecticidal treatments incorporated into compost and casing against B. ocellaris was conducted during 2003–2004. Treatments with an organophosphate, diazinon, a pyrazole, fipronil, and an insect growth regulator (IGR), triflumuron, were incorporated into casing. Another IGR, cyromazine, was incorporated into compost. Insecticidal incorporation into compost and casing has been associated with reductions in mushroom yield. Therefore, the effect of these insecticidal treatments on the overall yield was evaluated. RESULTS: A combined treatment, incorporation of cyromazine at 10 mg kg^?1 into compost and of triflumuron at 20 mg kg^?1 into casing, provided 100% control against B. ocellaris. There was no apparent reduction in yield as a result of incorporation of the tested chemicals into either compost or casing. CONCLUSION: The results obtained from this study suggest that incorporation of insecticides into both compost and casing is required to control a moderate to high pressure of insect pests. There were no significant yield reductions as a result of the incorporation of pesticides in the Australian mushroom‐growing system. Copyright © 2009 Society of Chemical Industry     

Serological and Molecular Characterization of Peanut chlorotic fan-spot virus, a New Species of the Genus Tospovirus

ABSTRACT To clarify the serological relationship of Peanut chlorotic fan-spot virus (PCFV) with other tospoviruses, antisera were produced against the nucleocapsid (N) proteins of this virus and tospoviruses from four serogroups including Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Groundnut ringspot virus (GRSV), and Watermelon silver mottle virus (WSMoV). In immunodiffusion tests, the antisera only reacted with their homologous antigens. Similar results were noticed in indirect enzyme-linked immunosorbent assay and immunoblot tests, with the exception that strong cross-reactions were observed in heterologous combinations between TSWV and GRSV. The results indicated that the N protein of PCFV is not serologically related to those of the tospoviruses from the four serogroups. To further characterize the virus, viral S double-stranded RNA was extracted from PCFV-infected Chenopodium quinoa and used for cDNA cloning and sequencing. The full-length viral strand of the S RNA was determined to be 2,833 nucleotides, with an inverted repeat at the 5' and 3' ends and two open reading frames in an ambisense arrangement. The 3'-terminal sequence (5'-AUUGCUCU-3') of the viral S RNA is identical to those of other tospoviruses, indicating that PCFV belongs to the genus Tospovirus. The N and the NSs proteins of PCFV share low amino acid identities (22.3 to 67.5% and 19.3 to 54.2%) with those of reported tospoviruses, respectively. The phylogenetic dendrogram of the N gene of PCFV compared with those of other tospoviruses indicates that PCFV is distinct from other tospoviruses. In hybridization analyses, an N gene cDNA probe of PCFV did not react with viral RNAs of TSWV, GRSV, INSV, and WSMoV, and vice versa. Thus, based on these results, we conclude that PCFV is a new tospovirus species.     

Phylogenetic Analysis of 16S rRNA Genes and PCR Analysis of the nec1 Gene from Streptomyces spp. Causing Common Scab, Pitted Scab, and Netted Scab in Finland

ABSTRACT The sequences of the 16S rRNA genes (nucleotides 29 to 1,521) from various Streptomyces strains pathogenic to potato were compared. These included 10 pathogenic Streptomyces strains isolated from potato scab lesions in Finland, the type strains of S. aureofaciens NRRL 2209(T) and S. lydicus ATCC 25470(T), 'S. griseus subsp. scabies' ATCC 10246, and two S. griseus strains that were originally deposited to the collection as pathogens. The nucleotide sequence (>94.5% sequence identity [SI]) and length (1,469 to 1,481 nucleotides) of the analyzed region varied. Phylogenetic analysis of 16S rRNA genes placed Finnish strains into three species, supported by previously characterized morphological and physiological traits. Six Finnish strains, including two strains that deviated from the others in one trait (no spiral sporophores or D-xylose utilization), had identical 16S rRNA genes and were identified as S. scabies (99.9% SI to S. scabies ATCC 49173). Three Finnish strains were identified as S. turgidiscabies, a species previously described only in Japan (99.9% SI to S. turgidiscabies ATCC 700248). Finnish strain 317 and S. aureofaciens NRRL 2209 (99.8% SI) were placed in a distinct phylogenetic cluster together with Kitosatospora spp., which suggests that S. aureofaciens may belong to the recently revived genus Kitosatospora. In pathogenicity tests, S. scabies caused characteristic symptoms of common scab, S. turgidiscabies caused mainly pitted scab, and S. aureofaciens caused netted scab and necrotic lesions on stolons of potato cultivars Bintje and Matilda in the greenhouse. The nec1 gene and the intergenic region between nec1 and the 5' transposase pseudogene ORFtnp were successfully amplified by polymerase chain reaction from S. scabies ATCC 49173 and the pathogenic Finnish strains of S. scabies, but not from a nonpathogenic strain of S. scabies, three pathogenic and two nonpathogenic strains of S. turgidiscabies, and S. aureofaciens.     

Molecular Diagnostics, Taxonomy, and Phylogeny of the Stem Nematode Ditylenchus dipsaci Species Complex Based on the Sequences of the Internal Transcribed Spacer-rDNA

ABSTRACT The stem nematode Ditylenchus dipsaci is of great economic importance worldwide as a parasite of agricultural crops and horticultural plants. The internal transcribed spacer (ITS) of rDNA from 23 populations of the D. dipsaci complex from various host plants were amplified and sequenced. Seven previously studied populations were also included in the study. The phylogenetic analysis of the full ITS and ITS2 sequence alignments using minimum evolution, maximum parsimony, and Bayesian inference under the complex model of DNA evolution revealed trees with two main clades: (i) D. dipsaci sensu stricto with diploid chromosome numbers and comprising most isolates from agricultural, ornamental, and several wild plants, and (ii) Ditylenchus spp. with polyploid chromosome numbers, reproductively isolated from diploid populations, and subdivided into six subclades ("giant race" from Vicia faba, Ditylenchus species parasitizing various Asteraceae, and a Ditylenchus sp. from Plantago maritima). Using the energy minimization approach and comparative sequence analysis, it has been found that the secondary structure of ditylenchid ITS2 is organized in three main domains. The importance of knowledge on the RNA structure for phylogenetic analysis is discussed. Conventional polymerase chain reaction (PCR) and real-time PCR with SYBR green dye I with a species specific primer have been developed for detection and quantification of D. dipsaci sensu stricto Validation tests revealed a rather high correlation between real numbers of fourth-stage juveniles of the stem nematodes in a sample and expected numbers detected by real-time PCR. Problems of accuracy of quantification are discussed.     

Molecular Detection of Phytophthora ramorum, the Causal Agent of Sudden Oak Death in California, and Two Additional Species Commonly Recovered from Diseased Plant Material

ABSTRACT Sudden oak death is a disease currently devastating forest ecosystems in several coastal areas of California. The pathogen causing this is Phy-tophthora ramorum, although species such as P. nemorosa and P. pseudo-syringae often are recovered from symptomatic plants as well. A molecular marker system was developed based on mitochondrial sequences of the cox I and II genes for detection of Phytophthora spp. in general, and P. ramorum, P. nemorosa, and P. pseudosyringae in particular. The first-round multiplex amplification contained two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA was of sufficient quality to allow for polymerase chain reaction (PCR) amplification and the other specific for amplification of sequences from Phytophthora spp. The plant primers amplified the desired amplicon size in the 29 plant species tested and did not interfere with amplification by the Phytophthora genus-specific primer pair. Using DNA from purified cultures, the Phytophthora genus-specific primer pair amplified a fragment diagnostic for the genus from all 45 Phytophthora spp. evaluated, although the efficiency of amplification was lower for P. lateralis and P. sojae than for the other species. The genus-specific primer pair did not amplify sequences from the 30 Pythium spp. tested or from 29 plant species, although occasional faint bands were observed for several additional plant species. With the exception of one plant species, the resulting amplicons were smaller than the Phytophthora genus-specific amplicon. The products of the first-round amplification were diluted and amplified with primer pairs nested within the genus-specific amplicon that were specific for either P. ramorum, P. nemorosa, or P. pseudo-syringae. These species-specific primers amplified the target sequence from all isolates of the pathogens under evaluation; for P. ramorum, this included 24 isolates from California, Germany, and the Netherlands. Using purified pathogen DNA, the limit of detection for P. ramorum using this marker system was approximately 2.0 fg of total DNA. However, when this DNA was spiked with DNA from healthy plant tissue extracted with a commercial miniprep procedure, the sensitivity of detection was reduced by 100- to 1,000-fold, depending on the plant species. This marker system was validated with DNA extracted from naturally infected plant samples collected from the field by comparing the sequence of the Phytophthora genus-specific amplicon, morphological identification of cultures recovered from the same lesions and, for P. ramorum, amplification with a previously published rDNA internal transcribed spacer species-specific primer pair. Results were compared and validated with three different brands of thermal cyclers in two different laboratories to provide information about how the described PCR assay performs under different laboratory conditions. The specificity of the Phytophthora genus-specific primers suggests that they will have utility for pathogen detection in other Phytophthora pathosystems.