The taxonomic position of Xanthomonas ampelina1

The internal taxonomic structure and the suprageneric relations of the causal agent of bacterial blight and canker of grapevine, the misnamed Xanthomonas ampelina, were examined. By studying more than 30 strains of different geographical origin by polyacrylamide gel electrophoresis and DNA: DNA hybridization, we established the homogeneity of X. ampelina. [¹⁴C]-labelled ribosomal RNA (rRNA) was prepared from the type strain of X. ampelina (NCPPB 2217) and hybridized with DNA from different representative strains from the Pseudomonas acidovorans rRNA complex in rRNA superfamily III. X. ampelina was found to be a separate branch in rRNA superfamily III, related at a suprageneric level to P. acidovorans, Alcaligenes paradoxus and the other organisms of the P. acidovorans rRNA complex.     

Detection of Pseudomonas syringae pv. aptata in Irrigation Water Retention Basins by Immunofluorescence Colony-staining

Bacterial blight of cantaloupe (Cucumis melo) caused by Pseudomonas syringae pv. aptata was first observed in south-western France and has since spread to all cantaloupe-growing areas of this country. Use of pesticides registered for this disease has proved ineffective and no commercial cultivars of cantaloupe are resistant to this blight. To develop control strategies for this disease, the principal sources of inoculum were investigated. Among the different sources of inoculum studied, we report the isolation of P. syringae pv. aptata from irrigation water retention basins in south-western France using the immunofluorescence colony-staining (IFC) method. In this study, the pathogen was detected at a low concentration (12 and 70cful^–1) in two different retention basins. These results suggest that P. syringae pv. aptata can survive in water used to irrigate cantaloupe crops and could be a source of inoculum for epidemics of bacterial blight. To develop control strategies for this bacterial disease, the importance of water retention basins as sources of inoculum for bacterial blight of cantaloupe needs to be evaluated relative to other potential sources such as seeds, plants from nurseries and plant debris in the soil.     

Current status of Xanthomonas ampelina in Spain and susceptibility of Spanish cultivars to bacterial necrosis1

The causal agent of bacterial necrosis of vine, Xanthomonas ampelina, was first recorded in Spain in 1978 on grapevines in the region of Zaragoza, where the total infested area is now around 9000 ha. The disease was later recorded in Rioja where small foci of around 50 ha were found, and in Galicia, where it exists in the area of Ribeiro and Pontevedra. Surveys done in other grapevine-growing areas of the country have not led to the detection of the disease. The symptoms described in Spain are the same as those in other countries and bacterial necrosis has not, at present, been identified on table grapes. Losses resulting from the disease are closely linked to climatic conditions and the drought of these last few years has practically eliminated symptoms of X. ampelina in Aragon and the area of Rioja. Chemical control of the disease through the use of cupric formulations is not sufficient during rainy years with favourable conditions, and the best method for preventing the disease seems to be the use of resistant cultivars or those displaying weak susceptibility. A 3-year experimental field study of the varietal susceptibility of 30 Spanish cultivars has shown that Airén, Garnacha tintorera, Juan Ibáñez and Vidadillo are the most susceptible. Cardinal, Garnacha negra, Mazuela, Merseguera, Monastrell and Trepat are the least susceptible of all the cultivars studied. A parallel study of the susceptibility of plants grown in vitro has been undertaken and may be useful for rapid testing of varietal susceptibility.     

Assessment of Pierce's disease susceptibility in Vitis vinifera cultivars with different pedigrees

Pierce's disease (PD) of grapevine is caused by the bacterium Xylella fastidiosa. In this study, an integrated approach was applied to assess PD susceptibility among different Vitis vinifera cultivars that incorporated disease severity, bacterial pathogen abundance and loss of stem xylem hydraulic conductivity. It was hypothesized that levels of PD susceptibility in V. vinifera can be attributed in part to the host anatomical features that are shaped by its pedigree background. Two popular wine grape cultivars were initially selected from the occidentalis group, Merlot and Cabernet Sauvignon, and one from the orientalis group, Thompson Seedless. The more recently bred table grape cultivar Scarlet Royal, that has mixed pedigree parentage, was also included. PD susceptibility was compared to the known PD resistant b43-17 V. arizonica/candicans wild grape species from North America. The data indicated that Thompson Seedless was ranked as the most susceptible to PD because it significantly exhibited the most severe disease symptoms at 12 weeks post-inoculation and hosted the highest X. fastidiosa titre of the cultivars, and lost over 90% of its stem hydraulic conductivity. In contrast, the other three cultivars displayed less susceptibility to PD. The way in which the xylem anatomy could impact PD susceptibility in V. vinifera cultivars is discussed, together with how grape pedigrees and their cognate centre of domestication may have influenced xylem anatomical features. This work provides a reference framework to further test the hypothesis that V. vinifera cultivars with wide xylem vessels may be more prone to PD decline.     

Asian grapevine leaf rust caused by Phakopsora euvitis: an important disease in Brazil

Anthropogenic influences such as tourism and climate change affect ecosystems. One consequence is that invasive species can spread to and colonize novel areas. A recent example of this scenario is the appearance, in the early 2000s in Brazil and Australia, of the plant pathogenic fungus Phakopsora euvitis, which causes Asian grapevine leaf rust. It is speculated that the disease was introduced either by long distance transport of spores in the atmosphere or the import of contaminated plant material. While the disease could be eradicated from the Australian continent it is still present in Brazil. The pathogen has spread successfully throughout most Brazilian grapevine‐growing regions, most probably because of the lack of resistant plant cultivars. Today, the disease is endemic in Brazil. This review was written to highlight the increasing impact of the disease in Brazil and summarizes the current knowledge on the disease monocycle under different environmental conditions and the consequences for grapevine management. Gaps in the present state of knowledge are highlighted with the purpose of stimulating further research aimed at identifying efficient strategies to combat the disease.     

Aetiology of anthracnose on grapevine shoots in Brazil

Anthracnose is an important disease in vineyards in south and southeast Brazil, the main grape‐producing regions in the country. This study aimed to identify the causal agents of grapevine anthracnose in Brazil through multilocus phylogenetic analyses, morphological characterization and pathogenicity tests. Thirty‐nine Elsinoë ampelina and 13 Colletotrichum spp. isolates were obtained from leaves, stems and berries with anthracnose symptoms collected in 38 vineyards in southern and southeastern Brazil. For E. ampelina isolates, the internal transcribed spacer (ITS), histone H3 (HIS3) and elongation factor 1‐α (TEF) sequences were analysed. HIS3 was the most informative region with 55 polymorphic sites including deletions and substitutions of bases, enabling the grouping of isolates into five haplotypes. Colonies of E. ampelina showed slow growth, variable colouration and a wrinkled texture on potato dextrose agar. Conidia were cylindrical to oblong with rounded ends, hyaline, aseptate, (3.57–) 5.64 (?6.95) μm long and (2.03–) 2.65 (?3.40) μm wide. Seven species of Colletotrichum were identified: C. siamense, C. gloeosporioides, C. fructicola, C. viniferum, C. nymphaeae, C. truncatum and C. cliviae, with a wide variation in colony and conidium morphology. Only E. ampelina caused anthracnose symptoms on leaves, tendrils and stems of Vitis vinifera and V. labrusca. High disease severity and a negative correlation between disease severity and shoot dry weight were observed only when relative humidity was above 95%. In this study, only E. ampelina caused anthracnose symptoms on grapevine shoots in Brazil.     

Bacterial blight of rice and its control in Nepal

Research on Xanthomonas oryzae pv. oryzae, the bacterial blight of rice pathogen, was initiated at the Institute of Agriculture and Animal Science (IAAS) with the main objective of assessing the population structure of X. o. pv. oryzae through the use of both conventional and molecular markers in combination with virulence typing. A high DNA polymorphism was detected in the pathogen populations using different DNA probes and rep-PCR primers. Most strains were avirulent to cultivars containing the bacterial blight resistance gene Xa-21, which suggested the strategy that targets gene deployment is feasible in Nepal.     

Eradication of black rot (Guignardia bidwellii) from grapevines by drastic pruning

A drastic pruning strategy was developed to eradicate the fungal disease black rot (Guignardia bidwellii), which is exotic in Australia, from grapevines, while minimizing the economic cost of returning an affected vineyard to its previous quality and production levels. The protocol involved cutting off vines at the top of the trunk, removing debris from the ground beneath and between vines, mulching the vineyard floor, removing low watershoots during vine regrowth and applying a targeted fungicide programme. The protocol was initially evaluated and consequently modified in Australia using an endemic grapevine disease, black spot or anthracnose (Elsinoe ampelina), as an analogous model system. Then, it was validated in a black‐rot‐infested vineyard in New York, USA. Following two seasons of disease‐conducive weather conditions, no black rot was detected on treated vines, whereas leaf and fruit infections developed on the untreated control vines. These results confirmed the efficacy of the protocol for eradicating black rot from vineyards while allowing vines to return quickly to previous yield and quality levels without replanting. The protocol may have applicability to disease eradication protocols for other perennial crops as well. Evidence is also presented on the efficacy and potential pitfalls of burning infected grapevine material to eradicate E. ampelina.     

Protection of potatoes against pests in Ukraine1

Potato production in Ukraine is now mainly in the private sector, and the phytosanitary state of potato crops leaves much to be desired. Control of Leptinotarsa decemlineata is generally well managed, but potato late blight (Phytophthora infestons) is out of control. Potato wart disease continues to cause problems in areas where it occurs because of the unavailability or unacceptability of resistant cultivars. Potato viruses and the losses they cause are little studied or taken into account. Other fungal, bacterial and insect pests are causing increasing problems in the field and in store. Integrated pest management systems are proposed by scientific institutions, including adequate forecasting of potato blight and use of certified seed potatoes of cultivars resistant to wart and cyst nematodes, but these systems do not adequately reach private growers. The range of plant protection products authorized for sale to the general public is very limited, and this is a serious obstacle to adequate plant protection in potato.     

Identification of a genetic lineage within Xanthomonas arboricola pv. juglandis as the causal agent of vertical oozing canker of Persian (English) walnut in France

A new bacterial disease of Persian (English) walnut (Juglans regia) has been observed in France. This disease, called vertical oozing canker (VOC), is characterized by vertical cankers on trunks and branches of affected walnut trees with oozing exudates. To determine the aetiology of the disease, a study was carried out in 79 walnut orchards and nurseries located in southeastern and southwestern France. Bacterial analysis from diseased samples yielded 36 strains identified as Xanthomonas arboricola and 32 strains identified as Brenneria nigrifluens on the basis of biochemical tests. The causal agent of VOC was identified as X. arboricola by pathogenicity tests on walnut. Fluorescent amplified fragment length polymorphism (F‐AFLP) was carried out on 36 strains of X. arboricola collected in this study, 24 strains of X. arboricola pv. juglandis isolated from walnut blight symptoms and one strain of X. arboricola pv. corylina included as an outgroup. Based on cluster analysis of F‐AFLP data, most X. arboricola strains responsible for main VOC outbreaks showed a high degree of similarity, forming a cluster clearly separate from strains of X. arboricola pv. juglandis isolated from walnut blight symptoms. It is suggested that VOC is caused by a distinct genetic lineage within the pathovar juglandis of X. arboricola that is also able to cause classical bacterial blight symptoms on walnut leaves and fruits.     

Role of stilbenes in the resistance of grapevine to powdery mildew

Stilbene phytoalexins are identified as defence response in pathogen–grapevine interactions, but little information is available on the role of stilbenes on Erysiphe necator, causal agent of grapevine powdery mildew. Analysis of stilbenes in artificially infected leaf discs from susceptible to highly resistant cultivars was performed and compared to the development of the pathogen. Results indicate that stilbene synthesis is confined in infected cells, penetrated by an appressorium–peg. Stilbene amounts expressed by infection site allow discriminating susceptible and resistant cultivars. Highest viniferins concentrations on resistant cultivars are in correspondence with the observed inhibition of the pathogen growth. The analysis of stilbenes at the infection site and viniferins accumulation in grapevine defence reaction is discussed.     

Genetic structure of the grapevine fungal pathogen Phaeomoniella chlamydospora in southeastern Australia and southern France

In this study, 18 microsatellite loci were used to examine the genetic structure and mode of reproduction of Phaeomoniella chlamydospora, the fungus that causes Petri disease of grapevine and is involved in another grapevine disease, esca. A total of 60 southeastern Australian isolates were tested and compared with 64 isolates from southern France. The French population possessed relatively high genotypic diversity (G = 29·2) whilst the Australian population showed low genotypic diversity (G = 11·1), consistent with a limited founder population. Haplotypes from four different grapevine cultivars were not significantly differentiated (Φpt values effectively zero). Likewise, genetic differentiation of haplotypes from different regions within each country was not significant, although small but significant genetic differentiation (9%) was identified between Australia and France. Based on bootstrapped cluster analysis, there did not appear to be any genetic groups within the overall sample of isolates. Significant linkage disequilibrium identified in both countries, together with overrepresented, widespread identical haplotypes, indicated limited genetic recombination and a largely clonal structure consistent with the absence of an observed sexual cycle in this species.     

Characterization of resistance mechanisms activated by Trichoderma harzianum T39 and benzothiadiazole to downy mildew in different grapevine cultivars

Downy mildew, caused by Plasmopara viticola, is one of the most destructive diseases of grapevine and is controlled with intense application of chemical fungicides. Treatment with Trichoderma harzianum T39 (T39) or benzothiadiazole‐7‐carbothioic acid S‐methyl ester (BTH) has been previously shown to activate grapevine resistance to downy mildew and reduce disease symptoms in the Pinot noir cultivar. However, enhancement of plant resistance can be affected by several factors, including plant genotype. In order to further extend the use of resistance inducers against downy mildew, the physiological and molecular properties of T39‐ and BTH‐activated resistance in different cultivars of table and wine grapes were characterized under greenhouse conditions. T39 treatment reduced downy mildew symptoms, but the degree of efficacy differed significantly among grapevine cultivars. However, efficacy of BTH‐activated resistance was consistently high in the different cultivars. Expression profiles of defence‐related genes differed among cultivars in response to resistance inducers and to pathogen inoculation. T39 treatment enhanced the expression of defence‐related genes in the responsive cultivars, before and after P. viticola inoculation. A positive correlation between the efficacy of T39 and the expression level of defence‐related genes was found in Primitivo and Pinot noir plants, while different genes or more complex processes were probably activated in Sugraone and Negroamaro. The data reported here suggest that the use of a responsive cultivar is particularly important to maximize the efficacy of resistance inducers and new natural inducers should be explored for the less responsive cultivars.     

First laboratory confirmation of Xylophilus ampelinus in Slovenia*

Bacterial blight of grapevine is caused by a slow‐growing bacterium Xylophilus ampelinus. It has been suspected to occur in Slovenia on the basis of visual observation of characteristic symptoms in the 1960s. In the present study, symptoms were recorded in an infected vineyard during three consecutive years (2002/2004). Samples from this vineyard were tested by nested‐PCR and isolation of bacteria on media was attempted. In the first year, angular lesions on leaves were highly expressed and an isolate morphologically similar to X. ampelinus was obtained from one sample. It was purified and identified as X. ampelinus using biochemical and nutritional tests, fatty acid analysis, immuno‐fluorescence, nested PCR and partial sequencing of the 16S rRNA gene. The 16S rDNA sequence showed 99–100% homology to known sequences of X. ampelinus strains, including the type strain. Pathogenicity of the isolate was confirmed in tissue‐cultured and potted grapevine plants. In the following two years, symptoms of bacterial blight were only faintly expressed. Using isolation on media and nested‐PCR, 23 and 17 extracts prepared from 10 and 8 grapevines, respectively, were analysed. In 2003, no positive sample was found, but X. ampelinus was again isolated and identified by colony morphology and nested‐PCR in 2004.     

A standardised method for the quantitative analysis of resistance to grapevine powdery mildew

Powdery mildew caused by Erysiphe necator is one of the most important diseases affecting grapevine (Vitis vinifera, L.). Control of this pathogen is based on the use of fungicides, which cause environmental damage and increase production costs. A cost-effective and environmentally friendly alternative to control the disease relies on using resistant varieties. While most V. vinifera cultivars are susceptible to powdery mildew, several species belonging to the Vitaceae have been described as resistant. Several loci for resistance to grapevine powdery mildew have been identified through genetic analysis of segregating populations derived from different resistance sources. Identifying quantitative trait loci (QTL) with minor effects on the resistance may prove valuable in a strategy of pyramiding, which aims at increasing the durability of the resistance. However, current methods for evaluation of resistance either do not take into account quantitative variations, or, if they do, are not adapted to large sample sets. Here we develop a method for the analysis of quantitative resistance to grapevine powdery mildew in large populations. We devised a semi-quantitative resistance scale and confirmed the usefulness of a cell counter to quantify sporulation. We compared three inoculation methods and identified dry inoculation using a settling tower as the one giving the best infection. Finally, we confirmed the value of the method by applying it to a set of plants segregating for resistance to E. necator. Using the method described here for the quantitative analysis of the resistance to powdery mildew will prove valuable for breeding for durable resistance.     

Differential Control of Head Blight Pathogens of Wheat by Fungicides and Consequences for Mycotoxin Contamination of Grain

Fusarium head blight of wheat is caused by a disease complex comprised of toxigenic pathogens, predominantly Fusarium spp., and a non-toxigenic pathogen Microdochium nivale, which causes symptoms visually indistinguishable from Fusarium and is often included as a causal agent of Fusarium head blight. Four field trials are reported here, including both naturally and artificially inoculated trials in which the effect of fungicide treatments were noted on colonisation by Fusarium and Microdochium, and on the production of deoxynivalenol (DON) mycotoxin. The pathogen populations were analysed with quantitative PCR and samples were tested for the presence of the mycotoxin DON. Application of fungicides to reduce Fusarium head blight gave a differential control of these fungi. Tebuconazole selectively controlled F. culmorum and F. avenaceum and reduced levels of DON, but showed little control of M. nivale. Application of azoxystrobin, however, selectively controlled M. nivale and allowed greater colonisation by toxigenic Fusarium species. This treatment also lead to increased levels of DON detected. nobreak Azoxystrobin application two days post-inoculation increased the production of DON mycotoxin per unit of pathogen in an artificially inoculated field trial. This result indicates the potential risk of increased DON contamination of grain following treatment with azoxystrobin to control head blight in susceptible wheat cultivars. This is the first study to show differential fungicidal control of mixed natural pathogen populations and artificial inoculations in field trials.     

Diversity of populations of Phytophthora infestans in relation to patterns of potato crop management in Latvia and Lithuania

Potato crop losses can be substantial when conditions for late blight (Phytophthora infestans) development and spread are favourable. In this study, drivers of differences between the P. infestans population structures in Latvia and Lithuania, two neighbouring countries with similar potato-growing traditions, were investigated. Genotypes of P. infestans and population genetic diversity were analysed using a 12-plex simple sequence repeat (SSR) marker assay. High genetic diversity was demonstrated in both populations, with population diversity being higher in Latvia. It would appear that local populations established from soilborne oospores early in the season are well adapted to the conditions in the region. However, somewhat greater spread and survival of local clones was detected in Lithuania, suggesting that potato cropping there is more vulnerable to clonal invasion than in Latvia. For effective disease management, current strategies should be adjusted according to the specific pathogen populations in the region, considering the reproduction and survival of the pathogen. Potato growers should implement late blight preventive measures such as longer field rotation to prevent oospore infections, especially in Latvia, and should use more disease resistant cultivars and high-quality seed potatoes.     

The role of seedling infection in epiphytotics of ascochyta blight on chickpea

Didymella rabiei, the causal agent of ascochyta blight, survives on infected seeds and seedlings. Diseased seedlings originating from infected seeds occasionally serve as the source for primary infection in chickpea crops. Experiments carried out independently in Australia and in Israel provided quantitative information on the temporal and spatial distribution of ascochyta blight from initial infections and on the relationship between the amount of initial infection and the intensity of subsequent epiphytotics for cultivars differing in susceptibility to the pathogen. Disease spread over short distances (<10 m) from individual primary infections, was governed by rain and wind, and was up to five times greater down-wind than up-wind. Cultivar response to D. rabiei significantly affected the distance and area over which disease spread and the intensity of the disease on infected plants. At onset of the epiphytotic, the relationship between disease spread and time was exponential (P < 0.05; R ² > 0.95) and the area of the resulting foci was over 10 times greater in susceptible cultivars than in resistant cultivars. Regression equations showed the relationship between disease severity and the distance from the focus-plants was inverse-linear for all cultivars tested (P < 0.05). A simulation model based on the experimental data revealed that even if primary infection is infrequent (less than 1% of plants), the consequences are potentially devastating when susceptible cultivars are used. The epidemiological information and simulation model generated by this study provide an increased understanding of the development of an epiphytotic in which the primary foci of disease originate from infected chickpea seedlings.     

Clove-transmissibility of <Emphasis Type="Italic">Pseudomonas salomonii</Emphasis>, the causal agent of ‘Café au lait’ disease of garlic

Bacterial blight of garlic, caused by Pseudomonas salomonii, results in leaf and sheath necrosis and sometimes leads to soft rot and plant death. The epidemiology of this bacterial disease, known as ‘Café au lait’ disease, is poorly understood and no resistant cultivars are currently available. To develop control strategies for this disease, we investigated principal sources of inoculum. The pathogen was isolated from bulbs from plants with typical vegetative symptoms of bacterial blight. Subsequent development of typical foliar symptoms on plantlets originating from symptomatic bulbs demonstrated transmission of the pathogen in the planting material. In one of three field experiments the contamination rate of planting stock influenced the disease incidence in field-grown garlic. The importance of planting stock as a source of inoculum was demonstrated here and should be evaluated relative to other potential sources such as crop debris, soil or alternate hosts in order to develop successful control strategies.     

Sensitive detection of Xanthomonas axonopodis pv. dieffenbachiae on Anthurium andreanum by immunocapture-PCR (IC-PCR) using primers designed from sequence characterized amplified regions (SCAR) of the blight pathogen

One of the most devastating Xanthomonas diseases affecting the Anthurium cut flower industry worldwide is the bacterial blight caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad). The disease can be spread through latently infected tissue-cultured plants that are used for the propagation of Anthurium worldwide. Current disease diagnostic techniques involve the use of semi-selective media and serological tests. This study describes the development of a PCR tool combined with a genus-specific monoclonal antibody for the sensitive detection of the pathogen directly from plants. It was demonstrated that the immunocapture PCR (IC-PCR) was more sensitive than the conventional PCR and even more sensitive than indirect ELISA for the detection of the pathogen. Latently infected plants could be positively screened for the presence of the pathogen. Three sets of primers were designed from DNA probes that were reported to show some specificity to the pathovar dieffenbachiae. The use of all three sets of primers in a single reaction successfully amplified the three individual loci when bacterial DNA was used as a template. The multiplex PCR generated PCR profiles that could differentiate between the reference strains of X. axonopodis pv. dieffenbachiae from other control bacteria. The new primers could therefore be used both for the diagnosis of Anthurium blight in single PCR reactions and also for the profiling of Xanthomonas. pv. dieffenbachiae strains using the multiplex PCR technique.