Factors influencing growth,sporulation and virus transfer in <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> isolates from Castilla and León (Spain)

Biological control with Cryphonectria hypovirus CHV1 of the chestnut blight, caused by the fungus Cryphonectria parasitica, has reduced the impact of the disease in Europe. The virus reduces the virulence of the fungus so that it causes non-lethal cankers, thus enabling the chestnut trees to overcome the disease. The virus can be transmitted horizontally by hyphal anastomosis or vertically to the conidia. In this study, we investigated growth and sporulation of the fungus as well as rates of horizontal transmission of the virus at different temperatures. We used fungal isolates of the vegetative compatibility types (vc types) that are most prevalent in Castilla and León (central northern Spain) to evaluate the effects of fungal strain on the parameters tested. In addition, we infected four isolates of C. parasitica with hypovirus subtypes CHV1-F1 and CHV1-I, to determine the influence of virus subtype on growth, sporulation and virus transfer. We assessed growth of fungal colonies and horizontal transmission of the virus at 15 °C and 25 °C. Colony growth was affected by an interaction between fungal isolates included in vc type EU1 or EU11 and virus at both 15 °C and 25 °C. However, horizontal transmission of the virus was only influenced by the fungal genotype of isolates included in vc type EU1 or EU11, and spore production was only affected by the virus subtype. Vertical transmission was also influenced by the fungal isolate and virus subtype. Growth of the fungal isolates varied depending on the virus subtype with which they were infected. This supports the theory that fungal host and virus subtype influence transmission and dissemination of hypovirulence. The fungal genotype affects colony growth and horizontal transmission of the virus. It is common to expect a good dissemination of the hypovirus with a low vc type diversity but the selection of the best combination of hypovirus and fungal isolate is crucial for the success of biological control not only for small areas but in larger chestnut populations as well.     

Virulence factors analysis of native isolates of Xanthomonas albilineans and Xanthomonas sacchari from Tucumán,Argentina, reveals differences in pathogenic strategies

Xanthomonas albilineans (Xa) and X. sacchari (Xs) are both sugarcane pathogens. Xa is the causal agent of leaf scald disease, but there is limited information about the pathogenicity of Xs. The aim of this work was to study virulence factors of native strains of Xa (Xa32, Xa33, and XaM6) and Xs (Xs14 and Xs15) previously isolated from sugarcane with leaf scald symptoms, to gain insight into the biology of each microorganism. We analysed epiphytic survival, sensitivity to oxidative stress, extracellular degradative enzymes, cell motilities, exopolysaccharide (EPS) characteristics, cell adhesion, biofilm development, and control of stomatal regulation of the five strains. We observed that each species presented similar phenotypes for every factor analysed. Xa strains appeared to be more sensitive to oxidative stress and presented lower epiphytic survival than Xs. All strains presented endoglucanase activity; however, we could only detect protease and amylase activities in Xs strains. Swimming and sliding were higher in Xs, but twitching was variable among species. We also observed that only Xs strains produced a xanthan-like EPS, presented a strong cell adhesion, and structured biofilm. We detected some intraspecific variations showing that higher amounts of EPS produced by Xs14 correlated with its higher sliding motility and its homogenous and more adhesive biofilm. In addition, EPSs of Xs14 and Xs15 presented differences in strand height and acetyl percentage. Finally, we found that strains of both species were able to interfere with stomatal aperture mechanism. All these differences could influence the colonization strategies and/or disease progression in each species.     

Cryptic species and population genetic structure of Plasmopara viticola in São Paulo State,Brazil

Downy mildew (Plasmopara viticola) is one of the most important diseases in grape-growing areas worldwide, including Brazil. To examine pathogen population biology and structure, P. viticola was sampled during the 2015/16 growing season from 516 lesions on nine grape cultivars in 11 locations in subtropical areas of São Paulo State, Brazil. For identification of cryptic species, a subsample of 130 isolates was subjected to cleaved amplified polymorphic sequence (CAPS) analysis, and for 91 of these isolates the ITS1 region was sequenced. These analyses suggest that the population of P. viticola in São Paulo State consists of a single cryptic species, P. viticola clade aestivalis. Seven microsatellite markers were used to determine the genetic structure of all 516 P. viticola isolates, identifying 23 alleles and 55 multilocus genotypes (MLGs). Among these MLGs, 34.5% were clonal and represented 93% of the isolates sampled. Four dominant genotypes were present in at least five different locations, corresponding to 65.7% of the isolates sampled. Genotypic diversity (Ĝ = 0.21–0.89) and clonal fraction (0.58–0.96) varied among locations (populations). Most populations showed significant deviation from Hardy–Weinberg expectations; in addition, excess of heterozygosity was verified for many loci. However, principal coordinate analysis revealed no clusters among locations and no significant isolation by distance was found, suggesting high levels of migration. The results indicate that downy mildew epidemics result from multiple clonal infections caused by a few genotypes of P. viticola, and reproduction of P. viticola in São Paulo State is predominantly asexual.     

Spatio-temporal association of GLRaV-3-infected grapevines,and effect of insecticidal treatments on mealybug populations in Virginia vineyards

Grapevine leafroll disease (GLD) is caused by a number of viruses in the grapevine leafroll-associated virus complex (GLRaVs). GLRaV-3 is the most commonly found GLRaV and is transmitted by mealybugs (Hemiptera: Pseudococcidae) and soft scale insects (Hemiptera: Coccidae). A series of investigations were conducted to understand spatio-temporal patterns of GLRaV-3 and mealybugs, and to examine management strategies of mealybugs under the environmental conditions of Virginia, USA. During the study, a rapid increase (185 % per year) of GLRaV-3 positive vines was observed at an experimental vineyard. Spatial Analysis by Distance IndicEs (SADIE) indicated a mix of aggregated, random, and uniform distributions of GLRaV-3 within six surveyed vineyards, and a temporal association of GLRaV-3-positive vines over three years at the experimental vineyard. Results from a field experiment in 2009–2011 showed the effect of a delayed dormant application of acetamiprid (Assail, 0.182 L/ha) to be not significantly differing in mealybug abundance from the unsprayed check in 2010 and 2011. Moreover, an application of the contact insecticide beta-cyfluthrin (Baythroid XL, 0.219 L/ha) resulted in significantly higher numbers of mealybugs counted in 2011. This study also demonstrated that GLRaV-3 was spread from mature vines to newly planted sentinel vines during the first growing season, with no apparent effect of prevailing wind direction. Results from another insecticide trial conducted in 2010–2011 demonstrated that both dinotefuran (Scorpion, 0.292 L/ha) and spirotetramat (Movento, 0.439 L/ha) treatments significantly reduced mealybug counts when mealybug populations were high, but only spirotetramat significantly reduced mealybug counts when mealybug populations were low.     

SYBR Green real-time quantitative PCR for the specific detection and quantification of ‘Candidatus Liberibacter solanacearum’ in field samples from New Zealand

Diseases of solanaceous crops caused by the phloem-limited bacterium ‘Candidatus Liberibacter solanacearum’ (Lso), vectored by the tomato potato psyllid Bactericera cockerelli, pose a major economic threat to crop production. Lso is yet to be cultured and, therefore, effective control strategies depend heavily on the early detection of the pathogen via polymerase chain reaction (PCR) assays. In this study, two new assays for the detection of Lso in New Zealand field samples were developed, and compared with previously available assays. Firstly, a single-tube semi-nested gel-based PCR assay was developed for the genus-specific detection of liberibacter species, and shown to provide increased sensitivity over standard and nested PCR. Secondly, a single-tube semi-nested SYBR Green real-time PCR (qPCR) assay was developed for the specific detection of Lso in field samples from New Zealand, with a limit of detection of five copies of the target gene per reaction. Semi-nested qPCR showed similar sensitivity compared with TaqMan qPCR with the primer-probe combination LsoF-HLBpr and was 10- to 50-fold more sensitive than the conventional PCR assays tested. Quantification of titre in Lso-affected tubers by SYBR Green qPCR revealed a positive relationship between pathogen titre and the discolouration of fried tuber slices, a symptom indicative of Lso infection. Quantification of Lso in field samples of potato and tomato also revealed many samples with titres below the limit of detection of conventional PCR. The observation of low-titre samples demonstrated the utility of SYBR Green qPCR for detection of Lso, as in addition to increased sensitivity melt-curve analysis enables confirmation of qPCR data by identifying false positive results.     

Spatial pattern analysis of citrus canker-infected plantings in são paulo, Brazil, and augmentation of infection elicited by the asian leafminer

ABSTRACT Eradication of Asiatic citrus canker (ACC) has become increasingly difficult over the last decade, following the introduction of the Asian leafminer into Brazil and Florida, which has led to changes in the eradication protocols. The present study, undertaken in Brazil, was aimed at characterizing the spatial patterns of ACC in commercial citrus plantings to gain better understanding of the dynamics of the disease subsequent to introduction of the leafminer. The spatial patterns of ACC were mapped in 326 commercial citrus plantings and statistically assessed at various spatial dimensions. The presence of "within-group" aggregation in each plot was examined via beta-binomial analysis for groups of trees parsed into three-by-three-tree quadrats. The relative intensity of aggregation was expressed as a binomial index of dispersion (D) and heterogeneity among plots expressed as the intracluster correlation coefficient, rho. The population of data sets was found to fall into three D categories, D < 1.3, 1.3 3.5. These categories then were related to other spatial characteristics. The binary form of Taylor's power law was used to assess the overdispersion of disease across plots and was highly significant. When the overall population of plots was parsed into D categories, the Taylor's R (2) improved in all cases. Although these methods assessed aggregation well, they do not give information on the number of foci or aggregations within each plot. Therefore, the number of foci per 1,000 trees was quantified and found to relate directly to the D categories. The lowest D category could be explained by a linear relationship of number of foci versus disease incidence, whereas the higher two categories were most easily explained by a generalized beta function for the same relationship. Spatial autocorrelation then was used to examine the spatial relationships "among groups" composed of three-by-three-tree quadrats and determine common distances between these groups of ACC-infected trees. Aggregation was found in >84% of cases at this spatial level and there was a direct relationship between increasing D category and increasing core cluster size, and aggregation at the among-group spatial hierarchy was generally stronger for the within-row than for the across-row orientation. Clusters of disease were estimated to average between 18 and 33 tree spaces apart, and the presence of multiple foci of infection was commonplace. The effectiveness of the eradication protocol of removing all "exposed" trees within 30 m surrounding each "ACC-infected tree" was examined, and the distance of subsequent infected trees beyond this 30-m zone from the original focal infected tree was measured for each plot. A frequency distribution was compiled over all plots to describe the distance that would have been needed to circumscribe all of these outliers as a theoretical alternative protocol to the 30-m eradication protocol. The frequency distribution was well described by a monomolecular model (R(2) = 0.98) and used to determine that 90, 95, and 99% of all newly infected trees occurred within 296, 396, and 623 m of prior-infected trees in commercial citrus plantings, respectively. These distances are very similar to previously reported distances determined for ACC in residential settings in Florida.     

Need for flagella for complete virulence of Pseudomonas syringae pv. tabaci: genetic analysis with flagella-defective mutants ?fliC and ?fliD in host tobacco plants

The flagellins purified from Pseudomonas syringae pv. tabaci induce a hypersensitive reaction in nonhost tomato cells. To investigate the role of flagella and flagellin in the compatible interaction, we generated two types of flagella-defective mutant. The fliC mutant lost the fliC gene that encodes flagellin protein, whereas the fliD mutant lost the fliD gene that encodes HAP2-capping protein. The two mutants had markedly reduced ability to cause disease symptoms in tobacco leaves. Furthermore, propagation of the mutants in tobacco leaves was less than that in wild-type pv. tabaci. Compared to the inoculation with wild-type pv. tabaci, inoculation with the two mutants did not markedly induce the expression of typical defense response-related genes such as PAL and hsr203J. Complementation of each fliC and fliD gene to the corresponding deficient mutant restored motility and virulence. These results indicate that flagella of P. syringae pv. tabaci are indispensable organelles for complete virulence on host tobacco plants.     

An Evaluation of the Genetic Diversity of Xylella fastidiosa Isolated from Diseased Citrus and Coffee in São Paulo, Brazil

ABSTRACT Strains of Xylella fastidiosa, isolated from sweet orange trees (Citrus sinensis) and coffee trees (Coffea arabica) with symptoms of citrus variegated chlorosis and Requeima do Café, respectively, were indistinguishable based on repetitive extragenic palindromic polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus PCR assays. These strains were also indistinguishable with a previously described PCR assay that distinguished the citrus strains from all other strains of Xylella fastidiosa. Because we were not able to document any genomic diversity in our collection of Xylella fastidiosa strains isolated from diseased citrus, the observed gradient of increasing disease severity from southern to northern regions of S?o Paulo State is unlikely due to the presence of significantly different strains of the pathogen in the different regions. When comparisons were made to reference strains of Xylella fastidiosa isolated from other hosts using these methods, four groups were consistently identified consistent with the hosts and regions from which the strains originated: citrus and coffee, grapevine and almond, mulberry, and elm, plum, and oak. Independent results from random amplified polymorphic DNA (RAPD) PCR assays were also consistent with these results; however, two of the primers tested in RAPD-PCR were able to distinguish the coffee and citrus strains. Sequence comparisons of a PCR product amplified from all strains of Xylella fastidiosa confirmed the presence of a CfoI polymorphism that can be used to distinguish the citrus strains from all others. The ability to distinguish Xylella fastidiosa strains from citrus and coffee with a PCR-based assay will be useful in epidemiological and etiological studies of this pathogen.     

Effect of cropping systems and crop successions on fumonisin levels in corn from Northern Paraná State,Brazil

In this study the effect of different cropping systems and crop successions was evaluated on natural Fusarium sp. contamination and fumonisin levels in corn. The cropping systems consisted of a conventional and no-tillage area cultivated with corn in the summer following either oats or fallow in the winter (2006 and 2007 growing seasons). In addition, the effect of applying nitrogen fertilizer (0, 22.5, 45.0, 90.0 and 90.0 kg ha⁻¹ nitrogen supplemented with potassium oxide) on fumonisin contamination was evaluated in the 2006 growing season. Fusarium sp. was detected in 90% samples in 2006 and in 100% samples in 2007. In both growing seasons, no-till corn following oats showed the highest mean fumonisin levels and differed significantly (P < 0.05) from all the others (2006) and from conventional till corn following either oats or fallow in the winter (2007). Fumonisin levels ranged from 0.13 to 19.52 μg g⁻¹ (mean 6.97 μg g⁻¹) and from 3.70 to 7.75 μg g⁻¹ (mean 6.29 μg g⁻¹) in no-till corn following oats from the 2006 and 2007 growing seasons, respectively. Plots treated with 0 kg ha⁻¹ and 22.5 kg ha⁻¹ nitrogen showed the highest mean fumonisin levels and differed significantly from those with 45.0 and 90 kg ha⁻¹ nitrogen. Fumonisin levels correlated negatively (P < 0.05) with the nitrogen fertilization rates. Although no-till is advantageous from a soil conservation standpoint, it may enhance the potential for fumonisin contamination in corn.     

Dissipation of quinalphos (O,O, diethyl O‐quinaxalin‐2yl phosphorothioate) residues in and/or on cauliflower (Brassica oleracea Var. Botrytis)

Abstract

A field trial was conducted to study the magnitude of quinalphos residues in and/or cauliflower (Brassica oleracea Var Botrytis. The cauliflower crop was sprayed once with quinalphos 25 e.c. at 0.25 and 0.5 kg a.i./ha 104 days after transplanting. The samples of cauliflower drawn at different time intervals were analysed colorimetrically. The initial deposits of quinalphos on cauliflower were found to be 5.79 and 9.52 ppm, an the half life values 1.61 and 2.12 days, respectively, at 0.25 and 0.5 kg a.i./ha. The waiting periods determined were 11 days at the higher and 7 days at the lower dose of quinalphos.     

PCR–RFLP identifies differences in hrpZ sequences to distinguish two genetic groups of Pseudomonas syringae pv. syringae strains from barley and wheat with bacterial black nod

Differences in hrpZ sequences determined by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) were used to investigate the molecular epidemiology of Pseudomonas syringae pv. syringae (PSS) strains that were isolated from diseased barley and wheat plants in Okayama, Japan. PCR–RFLP using HhaI separated PSS strains into two groups (A and B). Although specific PCR–RFLP groups of PSS strains were not always isolated from specific cultivars or seeds produced in a specific area, many strains isolated from barley and wheat belonged to PCR–RFLP groups A and B, respectively.     

α-Amylase inhibitor in local Himalyan collections of Colocasia: Isolation, purification, characterization and selectivity towards α-amylases from various sources

The α-amylase inhibitor from corms of Colocasia collected from Bhota village of Hamirpur district, Himachal Pradesh was purified to 17.21 folds with 61.61% recovery using ammonium sulfate precipitation, gel filtration chromatography (sephadex G-200) and ion exchange chromatography (DEAE-sephadex). A single band of the purified inhibitor was obtained by Native-PAGE. SDS-PAGE revealed the purified inhibitor to be a monomer with molecular weight of 13,900 daltons. The nature of inhibition was found to be of non-competitive type as determined by Lineweaver-Burk plot and a K_i value of 0.54 nmole was obtained by Dixon’s plot. The inhibitor was found to be heat stable and retained 81.50% activity at 70 °C temperature. Inhibitor was found to have pH optima of 6.9. The purified inhibitor was found to have inhibitory activity against α-amylases extracted from the larvae of Callosobruchus chinensis, Tribolium castaneum, Corcyra cephalonica and midgut α-amylase of Spodoptera littoralis. 100% larval mortality of C. cephalonica was observed when fed on wheat flour mixed with 0.0036% (w/w) of purified inhibitor. Purified α-amylase inhibitor was found to inhibit the activity of human salivary α-amylase. It also had inhibitory activity against potato α-amylases and reduced sugar content in treated potato slices. The purified inhibitor was found to be a glycoprotein. In the present study, the ability of the inhibitor to inhibit insect amylases highlights its possible role in pest resistance and post harvest decay of crop plants. Inhibitory activity of α-amylase inhibitor against mammalian amylases could suggest its potential in treatment of diabetes and cure of nutritional problems, which result in obesity.     

Weed or not a weed? Density,perceptions and management of Chromolaena odorata (Asteraceae) in West Africa: Voices from Ghana

The status (weed or beneficial shrub) of Chromolaena odorata in West Africa has been a major subject of debate since its introduction in the 1930s. Its negative impacts on agriculture and biodiversity are clear. However, indigenous people have harnessed several benefits of the plant, making management strategies less straightforward. Using indigenous voices in Ghana, the current perceptions of C. odorata in West Africa were assessed. Here, 90 respondents in three regions of Ghana were interviewed, using questionnaires, on aspects relating to the abundance, perceptions and management of C. odorata in their communities. Overall, results showed that the density of C. odorata had declined. The majority of the respondents considered C. odorata as a fallow shrub, a medicinal plant or both. However, impacts on crop production and injuries incurred during physical control were also highlighted. The local uses of the plant in Ghana included treating fevers and injuries, and as a fallow shrub for soil nutrient replenishment. Although most of the respondents classified C. odorata as a useful shrub in Ghana, they also highlighted the use of local farming tools such as cutlasses for the slash of the plant. The use of herbicides for the control of C. odorata was rare and not applied specifically for C. odorata. In conclusion, local awareness programmes on the deleterious impacts of C. odorata on ecosystem functioning and livelihoods are required for its effective management in West Africa.     

Diversity and spatial distribution of vegetative compatibility types and mating types of <Emphasis Type="BoldItalic">Cryphonectria parasitica</Emphasis> in the Aydın Mountains,Turkey

In this study, the population structure of the chestnut blight fungus Cryphonectria parasitica in the Aydın Mountains was investigated to make inferences about fungal reproduction and population diversity. A total of 213 C. parasitica isolates from eight subpopulations were used to determine vegetative compatibility (vc) and mating types of the population. Furthermore geostatistical analysis was performed to define the spatial structure of the population. The results showed that the isolates were vegetatively compatible with the European vc types of either EU-1 or EU-12. Both vc types were found in almost all subpopulations, but their frequencies varied depending on location. The results of a PCR assay showed that both mating types of C. parasitica (MAT-1 and MAT-2) exist in the population. MAT-1 comprised 65% of the total isolates, and the ratio of mating types was significantly skewed from 1:1. Genotyping based on combined vc and mating type data revealed four genotypes: EU-1/MAT-1 (28.6%), EU-1/MAT-2 (34.7%), EU-12/MAT-1 (36.2%) and EU-12/MAT-2 (0.5%). Geostatistatical analysis indicated that vc types, mating types and vc/mating genotypes were spatially autocorrelated and clustered in their distributions. Results suggested that C. parasitica could have a clonal population structure that is generated by asexual reproduction. Low vc-type diversity suggests that the C. parasitica population in the Aydın Mountains may be highly suitable to hypovirus invasion, thereby providing a high potential for successful biological control. However, co-occurrence of sexually compatible strains of EU-1 and EU-12 at the same locations in close proximity creates a high risk of increase in vc-type diversity.