Viticola Isolates Research Articles

Diversity and fitness of Plasmopara viticola isolates resistant to QoI fungicides

The effectiveness of Quinone outside Inhibitor (QoI) fungicides against grape downy mildew in European vineyards has significantly decreased in the last decade. One nucleotide polymorphism, G143A in the cytochrome b gene of Plasmopara viticola, is involved in resistance to QoIs. Previous genetic examination on the mitochondrial genomes showed four major haplotypes (IR, IS, IIR, IIS) coexisting in European vineyards. A resistant allele (G143A) was present in IR and IIR haplotypes. The purpose of the present study was to estimate the diversity of the different mitochondrial haplotypes and their distribution in QoI-resistant populations before evaluating the potential cost of the resistant mutation G143A in P. viticola population. From 2000 to 2004, the frequencies of resistant isolates ranged from 0% to 23.25% with an average of 4.64 % among the populations examined. To evaluate the fitness of sensitive and resistant isolates, a comparison of different biological parameters including latent period, spore production and infection frequency was performed, enabling a fitness index (FI) to be determined. Resistant isolates exhibited greater infection frequency than sensitive isolates, whereas no significant difference was found in sporulation ability and latent period between sensitive and resistant isolates. To further investigate competitiveness among isolates, an assay including two resistant isolates in different proportion with a sensitive isolate was conducted on eight asexual growing cycles in the absence of a QoI fungicide. The competitiveness of resistant isolates varied according to their fitness parameters, suggesting that there is no noticeable cost of QoI resistance in controlled conditions in Plasmopara viticola.

A six locus phylogeny reveals high species diversity in Botryosphaeriaceae from California almond

Botryosphaeriaceae are important pathogens on a variety of woody hosts, including almond, a major crop in California. Almond is susceptible to Botryosphaeria dothidea that forms band cankers on almond trunks, and the same fungus was also isolated from cankers of the canopy. To study the diversity and host range of B. dothidea and allied species from almond we used 132 isolates from 36 plant hosts from five continents, including 45 strains from almond in California. Species were identified by comparison to 13 ex-type strains with phylogenetic analyses based on six loci, including the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene repeat and portions of the coding genes elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, heat shock protein, histone-3 and beta-tubulin. Seven species were found from almond: Botryosphaeria dothidea, Neofusicoccum parvum, Neof. mediterraneum, Neof. nonquaesitum, Diplodia seriata and Macrophomina phaseolina were identified from band cankers, and B. dothidea, Neof. mediterraneum, Neof. parvum and Dothiorella sarmentorum from canopy cankers. All were capable of inducing cankers on inoculated almond branches in the field. All species found on almond also occurred on other hosts, suggesting that infected vegetation adjacent to almond orchards could serve as source of inoculum of virulent almond strains. Of the 19 monophyletic groups obtained at the species level, 13 contained ex-type strains, five were morphologically similar to established species and one was morphologically distinct from its closest relatives, Neof. andinum and Neof. arbuti, as well as from the more than 190 described species of Fusicoccum and Neofusicoccum, and thus was described as the new species, Neof. nonquaesitum. Evidence for cryptic speciation was found in B. dothidea, Neof. ribis and Spencermartinsia viticola. Botryosphaeria dothidea and Neof. ribis comprised lineages that formed the morphologically distinct Dichomera anamorph not found in any other isolates recognized as B. dothidea and Neof. ribis. An S. viticola isolate from California was phylogenetically divergent and had conidia that differed morphologically from the type. Neofusicoccum parvum was diverse but lacked any morphological features correlating with molecular diversity. Phylogenetic analyses of combinations of datasets showed that pooled analyses of all six datasets resulted in the highest number of supported branches, suggesting that addition of more data might yet improve phylogenetic resolution.

Breakdown of resistance to grapevine downy mildew upon limited deployment of a resistant variety.

BackgroundNatural disease resistance is a cost-effective and environmentally friendly way of controlling plant disease. Breeding programmes need to make sure that the resistance deployed is effective and durable. Grapevine downy mildew, caused by the Oomycete Plasmopara viticola, affects viticulture and it is controlled with pesticides. Downy mildew resistant grapevine varieties are a promising strategy to control the disease, but their use is currently restricted to very limited acreages. The arising of resistance-breaking isolates under such restricted deployment of resistant varieties would provide valuable information to design breeding strategies for the deployment of resistance genes over large acreages whilst reducing the risks of the resistance being defeated. The observation of heavy downy mildew symptoms on a plant of the resistant variety Bianca, whose resistance is conferred by a major gene, provided us with a putative example of emergence of a resistance-breaking isolate in the interaction between grapevine and P. viticola.ResultsIn this paper we describe the emergence of a P. viticola isolate (isolate SL) that specifically overcomes Rpv3, the major resistance gene carried by Bianca at chromosome 18. We show that isolate SL has the same behaviour as two P. viticola isolates avirulent on Bianca (isolates SC and SU) when inoculated on susceptible plants or on resistant plants carrying resistances derived from other sources, suggesting there is no fitness cost associated to the virulence. Molecular analysis shows that all three isolates are genetically closely related.ConclusionsOur results are the first description of a resistance-breaking isolate in the grapevine/P. viticola interaction, and show that, despite the reduced genetic variability of P. viticola in Europe compared to its basin of origin and the restricted use of natural resistance in European viticulture, resistance-breaking isolates overcoming monogenic resistances may arise even in cases where deployment of the resistant varieties is limited to small acreages. Our findings represent a warning call for the use of resistant varieties and an incentive to design breeding programmes aiming to optimize durability of the resistances.

QoI Resistance of Plasmopara viticola and Erysiphe necator in the Mid-Atlantic United States

Of 20 Plasmopara viticola isolates collected in four locations in Virginia and northwest North Carolina in 2005, 16 were resistant to QoI fungicides. The resistance factor was over 100, and label rates of formulated azoxystrobin and pyraclostrobin provided little or no control of these isolates. Additional sampling in 2006 revealed at least 15 additional vineyards with QoI-resistant P. viticola in Virginia, Maryland, and Pennsylvania. Of 22 isolates of Erysiphe (Uncinula) necator collected in 2005 from five Virginia locations, 20 isolates from 4 locations showed resistance to QoI fungicides. The G143A mutation for resistance was detected in several isolates of both pathogens. This is the first detection of this type of resistance in P. viticola in North America, and the second North American report of QoI resistance in E. necator. Accepted for publication 26 November 2007. Published 11 February 2008.

At least two origins of fungicide resistance in grapevine downy mildew populations.

Quinone outside inhibiting (QoI) fungicides represent one of the most widely used groups of fungicides used to control agriculturally important fungal pathogens. They inhibit the cytochrome bc1 complex of mitochondrial respiration. Soon after their introduction onto the market in 1996, QoI fungicide-resistant isolates were detected in field plant pathogen populations of a large range of species. However, there is still little understanding of the processes driving the development of QoI fungicide resistance in plant pathogens. In particular, it is unknown whether fungicide resistance occurs independently in isolated populations or if it appears once and then spreads globally by migration. Here, we provide the first case study of the evolutionary processes that lead to the emergence of QoI fungicide resistance in the plant pathogen Plasmopara viticola. Sequence analysis of the complete cytochrome b gene showed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Phylogenetic analysis of a large mitochondrial DNA fragment including the cytochrome b gene (2,281 bp) across a wide range of European P. viticola isolates allowed the detection of four major haplotypes belonging to two distinct clades, each of which contains a different QoI fungicide resistance allele. This is the first demonstration that a selected substitution conferring resistance to a fungicide has occurred several times in a plant-pathogen system. Finally, a high population structure was found when the frequency of QoI fungicide resistance haplotypes was assessed in 17 French vineyards, indicating that pathogen populations might be under strong directional selection for local adaptation to fungicide pressure.

First Report of Botryosphaeria iberica and B. viticola Associated with Grapevine Decline in California.

Grapevine decline symptoms in California include dead spurs and cordon and trunk dieback due to canker formation in the vascular tissue. Seven Botryosphaeria spp. are known to be associated with grapevine cankers in California, viz. Botryosphaeria australis, B. dothidea, B. lutea, B. obtusa, B. parva, B. rhodina, and B. stevensii (3). Recently, B. iberica and B. viticola also were isolated from grapevine cankers in a field survey that was conducted throughout California. Identification was based on morphological comparisons along with DNA analyses with previously identified isolates from Spain (1,2): B. iberica (CBS115035, ex-type) and B. viticola (CBS117006 and CBS117009, ex-type). DNA sequences of the rDNA internal transcribed spacer region (ITSI-5.8S-ITS2), part of the β-tubulin gene (BT2), and part of the translation elongation factor 1-α gene (EF1-α) from B. iberica and B. viticola isolates from California were amplified using primers ITS4/ITS5, Bt2a/Bt2b, and EF-728F/EF-986R, respectively. All DNA sequences of B. iberica and B. viticola from California showed 99 to 100% homology with those previously identified and deposited in GenBank. B. iberica, isolated from grapevine cankers from San Luis Obispo County (central coast), formed colonies on potato dextrose agar (PDA) that were dark green with aerial mycelium, optimum growth at 20 to 25°C, and formed pycnidia after 15 days of incubation at 25°C. Conidia were brown, one-septate, oblong to ovoid with a rounded apex, and measured (20.1-) 22.5 to 23.5 (-27.1) × (8.1) 9.3 to 9.8 (-11.2) μm, length/width ratio = 2.4 (n = 60). B. viticola, isolated from grapevine cankers in Sonoma (north coast), San Luis Obispo, Santa Barbara (south coast), Riverside (southern California), and Yolo (Sacramento Valley) counties, formed colonies on PDA that were dark green to grayish with aerial mycelium, optimum growth at 25°C, and formed pycnidia after 2 weeks. Conidia were brown, one-septate, oval to oblong, and measured (16.6-) 19.3 to 20.3 (-23.5) × (8.1) 9.3 to 9.6 (-11.1) μm, length/width ratio = 2.1 (n = 60). Two isolates of each species were used to complete pathogenicity tests (B. iberica: ATCC MYA-4110, ATCC MYA-4111; B. viticola: ATCC MYA-4115, ATCC MYA-4116). Ten fresh pruning wounds on 15-year-old cv. Zinfandel vines were inoculated per isolate using 50 μl of a 5 × 106 conidia per ml suspension. Twenty control pruning wounds were inoculated with the same amount of sterile water. Twelve months after inoculation, all wood inoculated with B. iberica and B. viticola showed internal necrosis extending 35 to 50 and 30 to 35 mm from the point of inoculation, respectively. Necrosis and extent of vascular discoloration in infected wounds was significantly greater (P < 0.05) than in control inoculations (6.5 mm). B. iberica and B. viticola were reisolated from the necrotic region surrounding all inoculation sites. Representative isolates of B. iberica and B. viticola from California were deposited at the American Type Culture Collection (B. iberica: MYA-4110, MYA-4111; B. viticola: MYA-4112 to MYA-4116). Sequences from the studied DNA regions of all isolates were deposited at GenBank. To our knowledge, this is the first report implicating either species as a cause of grapevine decline in California and B. iberica as a pathogen of Vitis vinifera anywhere in the world.

Differences in zoospore germination and host penetration in response to temperature among Western Australian isolates of Plasmopara viticola

Plasmopara viticola, causal agent of grape downy mildew, was first detected in Western Australia (WA) in widespread commercial viticulture in the Swan Valley (–31.85 S, 116.02 E) in 1998. It has since been found in all viticultural areas in WA, which extend from the far north (–15.75 S, 128.74 E) to the far south (–35.02 S, 117.80 E) of the state across a diverse range of climate zones. Not all of these zones are considered conducive for the development of grape downy mildew. The early infection behaviour of P. viticola isolates, obtained from climatically different grape-growing locations, was examined under different temperatures of incubation (10, 20, and 30°C). Variation in early infection behaviour was used to discern ecotypes of the pathogen. Ten isolates were obtained from WA and 8 of these responded similarly. Three isolates obtained from the east of Australia and used as comparisons in the study behaved similarly to most of the WA isolates. Zoospore germination and host penetration occurred at 10 and 20°C for all isolates. None of the isolates penetrated the host at 30°C. Only 2 isolates, from WA, germinated at 30°C. The early infection behaviour of most of the P. viticola isolates examined in this study appears to be reasonably analogous, despite the influence of varied local environmental conditions. The capacity of certain isolates to germinate under a wider spectrum of temperatures could be indicative of ecotypic specialisation. Such behaviour could confer advantage for the pathogen in viticultural regions that experience high temperatures throughout the growing season.

Meio semi-seletivo para isolamento de Xanthomonas campestris pv. viticola

O cancro bacteriano causado por Xanthomonas campestris pv. viticola é a fitobacteriose mais importante da videira no Submédio São Francisco. O isolamento de X. campestris pv. viticola de tecidos vegetais infectados é dificultado pela presença de contaminantes bacterianos, entre os quais Microbacterium barkeri. Objetivando-se a formulação de meio de cultura semi-seletivo, 22 isolados de X. campestris pv. viticola foram testados com relação a 30 antibióticos. O meio semi-seletivo NYDAM (extrato de carne 3, peptona 5, glicose 10, extrato de levedura 5, ágar 18 e ampicilina 0,1 em g L-1) inibiu M. barkeri e bactérias fitopatogênicas podendo ser utilizado para isolar X. campestris pv. viticola de hospedeiros com infecção natural em campo.

Characterization of Phomopsis spp. Infecting Grapevines in the Great Lakes Region of North America

Seventy-five isolates of Phomopsis were obtained from grapes (Vitis spp.) with Phomopsis cane and leaf spot symptoms in Ohio, Michigan, New York, Pennsylvania, Maryland, and Ontario (Canada). Four isolates from California and one from Europe were also included in the study. Isolates were grouped on the basis of DNA sequences from intron regions in the translation elongation factor 1-α and calmodulin genes. According to DNA sequence comparisons with the type isolate, all isolates except two (OH-48 and CAL-5) were determined to be P. viticola, confirming the etiology of this disease in the Great Lakes region. Thirty representative isolates were evaluated for mycelial growth rate in vitro, conidial size, and pathogenicity on grapevine (Vitis interspecific hybrid 'Seyval') leaves and internodes. A subsample of 13 isolates was also evaluated for pathogenicity on fruit and rachises. All isolates of P. viticola caused disease on grape but differed in virulence. Among P. viticola isolates, virulence on leaves and internodes was positively correlated, and virulence on fruit and rachises was positively correlated, but there was no relationship between ratings on vegetative versus reproductive grape tissues. Some isolates that were not virulent on leaves or internodes were highly virulent on fruit and rachises and vice versa, indicating some specialization with respect to host tissues. However, differences were mostly of a quantitative nature, which makes it difficult if not impossible to assign biotypes. Among P. viticola isolates, virulence on fruit and rachises was positively correlated with mycelial growth rate in vitro. For the two isolates that were not P. viticola, the internal transcribed spacer regions of the nuclear ribosomal DNA were sequenced for identification purposes. Based on the best match available in GenBank, CAL-5 was determined to be close to Diaporthe phaseolorum, while the sequence of OH-48 matched that of Phomopsis sp. from Eucommia ulmoides in China. Both isolates had shorter alpha conidia and significantly higher mycelial growth rates than the P. viticola isolates, and were not or only slightly pathogenic to Vitis interspecific hybrid 'Seyval'.

Sensitivity of Australian isolates of Plasmopara viticola to acylanaline fungicides

A floating leaf disk technique was used to test the sensitivity of Australian isolates of Plasmopara viticola to 4 acylanaline fungicides, phosethyl-A1 and phosphorous acid. Sporulation of all 15 isolates was completely inhibited by 1 mg/L of metalaxyl. Benalaxyl, oxadixyl and ofurace at 10 mg/L and phosethyl-A1 and phosphorous acid at 100 mg/L also completely inhibited sporulation of each of the isolates tested for each fungicide. These results indicate that acylanaline insensitive isolates of P. viticola are either not present or are not widely distributed in grape growing areas of Australia. Cultures of selected P. viticola isolates have been established on tissue-cultured grapevines. Sporangia from these cultures can be used as standards for future fungicide sensitivity testing.