A broad spectrum,one-step RT-PCR to detect <Emphasis Type="Italic">Satsuma dwarf virus</Emphasis> variants using universal primers targeting both segmented RNAs 1 and 2

Universal primers to detect Satsuma dwarf virus (SDV), including distantly related strains Citrus mosaic virus (CiMV), Navel orange infectious mottling virus (NIMV), and Hyuganatsu virus (HV), were tested in a convenient one-step RT-PCR assay. SDV was the most broadly detected using uSDVup/uSDVdo primers that specifically targeted a nucleotide sequence in the 3′-noncoding region that is conserved in both segmented RNAs 1 and 2 of SDV among the tested primers. Nucleotide sequence analysis confirmed that the amplified RT-PCR products could be derived from RNAs 1 or 2 of SDV variants, some of which had interesting genetic diversity.     

Quantitative nested real-time PCR detection of <Emphasis Type="Italic">Verticillium longisporum</Emphasis> and <Emphasis Type="Italic">V. dahliae</Emphasis> in the soil of cabbage fields

Verticillium longisporum and V. dahliae, causal agents of Verticillium wilt, are spreading through the cabbage fields of Gunma Prefecture. Using the V. longisporum-specific intron within the 18S rDNA and differences between ITS 5.8S rDNA sequences in Japanese isolates of V. longisporum and V. dahliae, we developed three quantitative nested real-time (QNRT) PCR assays. The QNRT-PCR quantification of V. longisporum or V. dahliae in cabbage field soil was consistent with the severity of Verticillium wilt disease in those fields. In field trials of resistant cultivar YR Ranpo grown for three seasons in soil infested with the pathogen, disease severity and pathogen density in the soil were significantly reduced in a field moderately contaminated by V. dahliae, but only slightly reduced in a highly contaminated field. These results suggest that continuous cultivation of a resistant cultivar is an effective way to reduce the pathogen population. QNRT-PCR assays provide a powerful analytical tool to evaluate the soil population dynamics of V. longisporum and V. dahliae for disease management.     

Subacute chlorpyrifos-induced oxidative stress in rat erythrocytes and the protective effects of catechin and quercetin

This study examined the effects of chlorpyrifos in the rat erythrocyte antioxidant system and evaluated the ameliorating effects of catechin and quercetin on the oxidative damage induced by chlorpyrifos. Sexually mature male Wistar rats were given chlorpyrifos (5.4 mg/kg, 1/25 of the oral LD₅₀), catechin (20 mg/kg), quercetin (20 mg/kg), catechin plus chlorpyrifos, and quercetin plus chlorpyrifos daily via gavage for four weeks. No statistical differences were found in the catechin-only and quercetin-only groups compared with the control group. By the end of the fourth week, chlorpyrifos alone increased the levels of malondialdehyde (MDA) and decreased superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities compared with the control group in rat erythrocytes. In the catechin-plus-chlorpyrifos and quercetin-plus-chlorpyrifos groups, there were statistically significantly decreased MDA levels and increased SOD, CAT, and GPx activities compared with the chlorpyrifos-only group. Thus, it appears that catechin and quercetin ameliorate chlorpyrifos-induced oxidative stress in rat erythrocytes in vivo.     

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.     

Haplotypes of “<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter solanacearum” suggest long-standing separation

Three haplotypes of the recently discovered bacterium species “Candidatus Liberibacter solanacearum” are described and related to geographic ranges. The first two are associated with Zebra Chip/Psyllid Yellows of potatoes and other solanaceous plants, vectored by the tomato/potato psyllid Bactericera cockerelli in North and Central America and New Zealand. The third is associated with diseased carrots in Finland and vectored by the carrot psyllid Trioza apicalis. The haplotypes are described by SNPs on the 16s rRNA, 16s/23s ISR and 50s rplJ and rplL ribosomal protein genes. These SNPs are inherited as a package across the three genes. Haplotype “a” has been found primarily from Honduras and Guatemala through western Mexico to Arizona and California, and in New Zealand. Haplotype “b” is currently known from eastern Mexico and northwards through Texas to south central Washington. These haplotypes show some range overlap in Texas, Kansas and Nebraska. The haplotypes are not yet known to elicit biological differences in the plant or insect hosts. These apparently stable haplotypes suggest separate bacterial populations of long standing.     

Spatial heterogeneity,incidence-incidence and incidence-lesion density relationship of apple scab (<Emphasis Type="Italic">Venturia inaequalis</Emphasis>) in managed orchards

The spatial pattern of apple scab was characterized using 10 years of disease incidence and lesion density data collected in managed orchards located in Quebec, Canada. Distributional analyses indicated that scab incidence was better characterized by the beta-binomial than the binomial distribution in 53 and 65% of the data sets at the leaf and shoot scales, respectively. Median values of the beta-binomial parameter θ, a measure of small-scale aggregation, were near 0 (0.003 and 0.028) at both sampling scales, indicating that disease incidence was close to being randomly distributed (low degree of aggregation). For lesion density, the negative binomial distribution fitted the data better than the Poisson distribution in 86% of the data sets at the leaf scale. The median value of the index of dispersion k was 0.068, indicating that aggregation was present. For all apple scab measurements, the power law models provided a good fit to the data. The estimated slope and intercept parameters were significantly greater than 1 and 0, respectively, suggesting that spatial heterogeneity changed systematically with disease incidence. Results of a covariance analysis showed that spatial heterogeneity of scab incidence at both scales and lesion density was not dependent upon shoot type but that spatial heterogeneity of scab incidence and lesion density at the leaf scale was influenced by the sampling period. A hierarchical analysis showed that scab incidence at the tree scale increased as a saturation-type curve with respect to incidence at the leaf or shoot scales. A similar relationship was observed for incidences at the shoot and leaf scales. An effective sample size model based on the binary power law parameters (Madden and Hughes, Phytopathology 89:770–781, 1999) gave the best fit to the leaf and shoot data, respectively. The incidence-lesion density relationship at both scales was well described by a complementary log-log (CLL) and log transformation model ( R_adj² = 0.97 and R_adj² = 0.94 ) \left( {R_{{adj}}^2 = 0.97\,and\,R_{{adj}}^2 = 0.94} \right) , however, the models tended to underestimate lesion density. The information of the spatial relations of apple scab within and between hierarchical scales acquired from this study can be used in developing and evaluating practical disease management strategies and to improve apple scab assessments for fungicide or cultivar susceptibility trials.     

PCR-based detection of sunflower white blister rust (<Emphasis Type="Italic">Pustula helianthicola</Emphasis> C. Rost &; Thines) in soil samples and asymptomatic host tissue

Sequencing of partial cox2 (part of the mitochondrial cytochrome-c-oxydase (COX) gene) was performed with samples from the oomycete genus Pustula, the white blister rusts of Asteraceae and related families. Sequence comparison uncovered several single nucleotide polymorphisms (SNPs) between P. spinulosa and host specific strains of Pustula isolated from Senecio vulgaris, Tragopogon pratensis and cultivated sunflower, Helianthus annuus. Based on these differences, specific primers were designed for PCR-based detection of white blister rust strains pathogenic to sunflower. The specificity of the primers was confirmed by cross testing with DNA from various oomycetes occurring in the same locality. The limit of detection for DNA of P. helianthicola was 10 pg. This allowed detection with DNA from single sporangia and single oospores. The PCR-based experiments allowed detection of the presence of sunflower white blister rust in soil samples from fields on which infected plants had been cultivated several months before. Moreover, the molecular tools were successfully applied to trace the pathogen in asymptomatic tissue of infected plants, demonstrating the systemic nature of Pustula on sunflower.     

Advances in sunflower downy mildew research

This review summarises the progress in research on sunflower downy mildew as reported in publications of the past 10 to 15 years, the period since the last comprehensive review on Plasmopara halstedii. Particular attention is paid to subjects that showed much progress and may be of particular interest to sunflower pathologists, mycologists or molecular biologists. Accordingly, single sections are devoted to the problems of taxonomic and phylogenetic aspects, host specificity, the host—pathogen interaction including resistance phenomena, as well as epidemiology and disease management. Reflecting the progress achieved in some fields and illuminating the deficits in others should stimulate the reader's interest in this very significant pathosystem.     

Effect of host and inoculum patterns on take-all disease of wheat incidence,severity and disease gradient

The importance of the spatial aspect of epidemics has been recognized from the outset of plant disease epidemiology. The objective of this study was to determine if the host spatial structure influenced the spatio-temporal development of take-all disease of wheat, depending on the inoculum spatial structure. Three sowing patterns of wheat (broadcast sowing, line sowing and sowing in hills) and three patterns of inoculum (uniform, aggregated and natural infestation) were tested in a field experiment, repeated over 2 years. Disease (severity, root disease incidence, plant disease incidence and, when applicable, line and hill incidences) was assessed seven times during the course of each season and the spatial pattern was characterized with incidence-incidence relationships. In the naturally infested plots, disease levels at all measurement scales were significantly higher in plots sown in hills, compared to plots sown in line, which were in turn significantly more diseased than plots with broadcast sowing. Disease aggregation within roots and plants was stronger in line and hill sowing than in broadcast sowing. Analysis of the disease gradient in the artificially infested plots showed that the disease intensified (local increase of disease level) more than it extensified (spatial spread of the disease), the effect of the introduced inoculum was reduced by 95% at a distance of 15 cm away from the point of infestation. Yield was not significantly affected by sowing pattern or artificial infestation.