Phytotoxic polyketides produced by <Emphasis Type="Italic">Phomopsis foeniculi</Emphasis>, a strain isolated from diseased Bulgarian fennel

Recently, a new fungal disease caused by Diaporthe angelicae (anamorph Phomopsis foeniculi) has been found with increasingly frequency on fennel (Foeniculum vulgare) in Bulgaria. Using a bioassay-guided isolation and purification procedure, different metabolites were isolated from the fungal culture filtrates. They were identified by spectroscopic methods as nectriapyrone, a pentaketide monoterpenoid, and altersolanols A and J and macrosporin, three octaketides anthracenones. Leaf puncture bioassay was applied on detached tomato leaves to prove the phytotoxic activity of the fractions and of pure compounds. Nectriapyrone and altersolanols A and J showed a modulated phytotoxicity, while macrosporin was not toxic. Altersolanol A was the most active compound.     

PCR-SSCP analysis of <Emphasis Type="Italic">Fusarium</Emphasis> diversity in asparagus decline in Japan

The diversity of Fusarium populations in asparagus (Asparagus officinalis L.) decline fields in Japan was estimated by PCR-SSCP (single-stranded conformational polymorphism) analysis of the ITS2 regions of the nuclear rRNA genes. This method was used to rapidly and objectively identify pathogens associated with roots of plants showing symptoms of asparagus decline collected from fields in five regions across Japan. Over 651 fusarial isolates were obtained, and were easily differentiated into three principal species. Fusarium oxysporum f. sp. asparagi was most frequently isolated from the domestic five regions (68%), whereas Fusarium proliferatum (28.6%) was less frequent. Fusarium solani was found much rarely (2.5%). The frequency of isolation of Fusarium proliferatum increased gradually from the north to the south of Japan, though considerable differences were found between fields in each region, as well as regional differences among the Fusarium populations. Most of the fusarial isolates were highly pathogenic in vitro. These results reveal that Fusarium oxysporum f. sp. asparagi and Fusarium proliferatum are important biotic factors which lead to asparagus decline in Japan.     

<Emphasis Type="Italic">Cylindrocarpon</Emphasis> species associated with apple tree roots in South Africa and their quantification using real-time PCR

Cylindrocarpon species are known to be a component of the pathogen/pest complex that incites apple replant disease. In South Africa, no information is available on apple associated Cylindrocarpon species and their pathogenicity. Therefore, these aspects were investigated. Among the isolates recovered from apple roots in South Africa, four species (C. destructans, C. liriodendri, C. macrodidymum and C. pauciseptatum) were identified using β-tubulin gene sequencing and phylogenetic analysis. This is the first report of C. liriodendri, C. macrodidymum and C. pauciseptatum on apple trees. Cylindrocarpon macrodidymum was the most prevalent. Isolates within each of the four species were pathogenic towards apple seedlings, but varied in their virulence. With a single exception, all isolates were able to induce lesion development on seedling roots. Only 57% of the isolates, which represented all four species, were able to cause a significant reduction in seedling weight and/or height. The greatest seedling growth reductions were caused by two isolates of C. destructans, and one isolate each of C. liriodendri and C. macrodidymum. A quantitative real-time polymerase chain reaction (qPCR) method was developed for simultaneous detection of all four Cylindrocarpon species. qPCR analyses of Cylindrocarpon from the roots of inoculated seedlings showed that the amount of Cylindrocarpon DNA in roots was not correlated to seedling growth reductions (weight and height) or root rot. The qPCR method is, however, very useful for the rapid identification of apple associated Cylindrocarpon species in roots. The technique may also hold potential for being indicative of Cylindrocarpon disease potential if rhizosphere soil rather than roots are used.     

Morphological and molecular analysis of genetic variability within isolates of <Emphasis Type="Italic">Corynespora cassiicola</Emphasis> from different hosts

Twenty-two isolates of Corynespora cassiicola obtained from cucumber, papaya, eggplant, tomato, bean, Vigna, sesame and Hevea rubber (Hevea brasiliensis) were analysed by morphological features, the differences of the ribosomal DNA internal transcribed spacer (rDNA-ITS) region sequence and the inter simple sequence repeat (ISSR) technique. Variability of morphological features was observed among the isolates. Pathogenicity tests showed that isolates from different hosts attacked Hevea rubber. Sequences of two outgroup taxa, C. proliferata and C. citricola, were downloaded from GenBank. The phylogenetic trees were constructed by using the rDNA-ITS region sequences from 24 Corynespora spp. isolates. In this analysis, the 24 sequences grouped into two clusters (A and B). Cluster A consists of sequences from all isolates of C. cassiicola; whereas cluster B consists of the two outgroup taxa, C. proliferata and C. citricola. However, the ITS region is conservative, and is not fit for studying differences among isolates. A total of 114 DNA fragments was amplified with 16 ISSR primers, among which 102 were polymorphic (89.5%). A dendrogram was created by the unweighted pair-group method with arithmetic averaging (UPGMA) analysis, and 22 isolates grouped into three clusters (C, D and E). Cluster C is composed of all of the Hevea rubber isolates, whereas cluster D is composed of nine isolates: four from papaya, five from cucumber, eggplant, bean, vigna and sesame. Cluster E is composed of two isolates from cucumber and tomato. These analyses showed that the genetic diversity was very rich among the tested isolates. There are no correlations between the morphological characteristics or rDNA-ITS region sequences of the 22 isolates and their host or geographical origin, but there is a link between ISSR clusters and their host origins. ISSR markers appear to be useful for intra-species population study in C. cassiicola.     

Screening of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> isolates from vineyards in Israel for resistance to fungicides

Plots in two vineyards in the Golan Heights, Israel were treated with six botryticides during three growing seasons with 3 applications per season. Applications of fenhexamid, pyrimethanil and cyprodinil + fludioxonil were effective, resulting in 52–65% and 53–63% mean reduction in grey mould incidence and severity, respectively. Carbendazim, fluazinam and iprodione were ineffective or slightly effective. Five hundred and sixteen B. cinerea isolates were collected from infected berries or trapped from the air in the vineyards, and profiles of sensitivity to benomyl, fenhexamid, fluazinam, fludioxonil, iprodione and pyrimethanil were established for each of the isolates based on a mycelial growth test. Seventy-four percent of the isolates were sensitive to the six tested fungicides, and the other 26% of the isolates were classified into 10 phenotypes characterized by resistance to one or more fungicides. Resistant isolates showed fitness parameters similar or reduced in comparison to sensitive isolates. Resistance to benzimidazoles and to dicarboximides was the most frequent (up to 25%) and apparently pre-existed in the populations tested. Increased frequency of benzimidazole resistance, but not dicarboximide resistance, was observed following the 3 years of applications of the fungicides. High level resistance to pyrimethanil was present at a frequency of about 2% in both vineyards in the first 2 years of the sampling survey and reached 10% in the third year at Site 2. A few isolates were resistant to fenhexamid or fludioxonil (0.8 or 0.2%, respectively). No strong resistance to fluazinam was detected, although numerous, less sensitive isolates, presumably possessing multi-drug resistance traits, were recovered at higher frequency from the plots treated with fluazinam than from the untreated plots.     

Improving sooty blotch and flyspeck severity estimation on apple fruit with the aid of standard area diagrams

Sooty blotch and flyspeck is caused by numerous species of fungi that colonize the surface of apple fruit and thereby lower its market value. Although this disease poses a substantial threat to apple growers’ profitability in some regions, reliable and cost-effective methods for epidemiological and disease control studies have not been validated, nor are they widely available. We modified a standard area diagram to aid sooty blotch and flyspeck severity assessments and quantified its impact on accuracy and precision of visual estimates. Samples of ‘Fuji’ and ‘Mutsu’ fruit were photographed both from the top and laterally. Severity was assessed from a sub-sample of 160 images using image analysis software. Validation of the diagram was performed by eight raters who independently assessed severity in two series of selected images representing the lateral view and the top view, initially unaided and subsequently with the aid of the scale. Severity estimates ranged from 0.4% to 98% (most fruit had <10% severity). Accuracy and precision of the estimates were significantly improved when using the diagrammatic scale; concordance correlation coefficient values increased from 0.81 to 0.95. A strong tendency to underestimate severity for the mid-range to high levels was minimized when using the aid, which also improved reproducibility of the estimates among raters. In addition to strengthening evidence that a standard area diagram can be used reliably in sooty blotch and flyspeck studies, we expanded its application to disease assessment in the peduncle region, which enhances the usefulness of the method for evaluating efficacy of management practices.     

Co-transmission of <Emphasis Type="Italic">Tomato yellow leaf curl virus</Emphasis> (TYLCV)-Mld and TYLCV-IL by the whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

The ability of the whitefly Bemisia tabaci to transmit two strains of Tomato yellow leaf curl virus, the Israel and Mild strains, was studied after serial transfers of individual whiteflies that were viruliferous for both strains to tomato plants. After single whiteflies had successive acquisition feedings first on a single plant infected with one strain and then on a plant infected with the other strain, the single whiteflies later transmitted intermittently one, the other, or both strains to the test plants during serial transfers at 1-day intervals. Because both strains were found in the head, abdomen, and legs dissected from whiteflies during the retention period after the two successive acquisition feedings, both strains apparently circulate from midgut cells to salivary glands through the hemolymph.     

<Emphasis Type="Italic">In silico</Emphasis> genomic subtraction guides development of highly accurate,DNA-based diagnostics for <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> race 3 biovar 2 and blood disease bacterium

New rapid diagnostic methods are urgently needed to discriminate the quarantine pathogen Ralstonia solanacearum (Rs) race 3 biovar 2 (R3B2) from other populations of Rs that lack the adaptation to cause bacterial wilt disease in temperate regions. We used an in silico bioinformatic approach to identify several genome sequences potentially specific to R3B2 strains. Primer sets were designed to PCR-amplify sequences in these regions, and four sets were ultimately shown to be >99% accurate for detection of R3B2 strains. On the basis of these results, several primers were designed to enable development of a loop-mediated isothermal amplification assay that was rapid, technologically simple, and essentially 100% accurate for identification of R3B2 when applied to a comprehensive collection of geographically diverse Rs strains. We fortuitously found that a sequence in one of the “R3B2-specific” regions has ~90% identity to a sequence present in strains of the blood disease bacterium (BDB), a member of the Rs species complex that infects banana. Alignments of these sequences allowed design of a second PCR primer set that proved 100% accurate for identification of BDB strains when tested on the 22 BDB strains available to us. These results demonstrate the power of in silico genomic subtraction for rapid identification of population-specific DNA sequences and for the development of simple, reliable detection methods for Rs subpopulations.     

Extracts from <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> induce effective resistance to the pathogen in both <Emphasis Type="Italic">Arabidopsis</Emphasis> and solanaceous plants

Plants defend themselves against microbial invasions by detecting conserved molecules, collectively called pathogen-associated molecular patterns (PAMPs). PAMPs-triggered basal resistance is the first inducible layer of plant defense. Here we found that Ralstonia solanacearum strain RS1002 can efficiently grow and cause disease in ecotype Columbia-0 of the model plant Arabidopsis thaliana in a manner dependent on the Hrp type III secretion system (T3SS). The extent of disease symptoms caused by R. solanacearum was reduced in plants pretreated with ΔhrpY mutant deficient in the functional Hrp T3SS. Pretreatment with a boiled extract (BE) from R. solanacearum had a similar inhibitory effect on disease development or bacterial multiplication in both Arabidopsis and several solanaceous plants. Simultaneous inoculations with BE and R. solanacearum did not induce BE-mediated resistance, nor did a BE treatment with proteinases. These results indicate that host plants recognize an unknown proteinaceous PAMP in the BE to induce disease resistance and that the Hrp T3SS of R. solanacearum can suppress it. From an analysis using Arabidopsis mutants lacking PAMP receptors, the elongation factor Tu of R. solanacearum was shown to partially contribute to BE-mediated basal resistance in Arabidopsis plants.     

High Performance Thin Layer Chromatography (HPTLC) profile of an insecticidal compound of <Emphasis Type="Italic">Trochus radiatus</Emphasis> and assessment of mucus-associated bacterial extracts

The current study is based on the screening of novel insecticides from new sources that have not being exploited hitherto. The major objectives of this research work were to extract marine molluscs, Lambis lambis, Trochus radiatus and Chicoreus ramosus, from Tuticorin coastal waters using different solvents to test their insecticidal properties and partially purify the active components. The ethyl acetate extracts of L. lambis and T. radiatus showed 100% mortality of Sitophilus oryzae, at a concentration of 200 μg ml⁻¹. The LC₅₀ values for ethyl acetate extracts of L. lambis, T. radiatus and C. ramosus were found to be 67.08, 348.86 and 571.42 μg ml⁻¹, respectively. With regard to bacterial metabolites, at a concentration of 200 μg ml⁻¹ the ethyl acetate extract of A3 was able to elicit an activity of 40% and that of strain A1 – 20%. The LC₅₀ values of the bacterial metabolites were also investigated. Upon chromatographic separation of active ethyl acetate extracts of T. radiatus, the 100% methanol column-purified fraction was found to have an activity of 30% at a concentration of 10 μg against S. oryzae. The purity of the partially pure active compound was observed to be 72.78% on analysis with high pressure thin layer chromatography.