Philippe E. Rolshausen

Use of Boron for the Control of Eutypa Dieback of Grapevines

Eutypa dieback is a perennial canker disease of grapevine (Vitis vinifera) caused by Eutypa lata. The fungus produces ascospores, which infect grapevines through pruning wounds during the dormant season. Management of the disease has been achieved with fungicide applications during the dormant period. However, no effective fungicide was available for this purpose after Benlate was withdrawn from the market. Boric acid (17.5% a.i. boron), a potential alternative to Benlate, was tested in the present study against E. lata. The EC50 values for inhibition of mycelial growth and ascospore germination were 125 and 475 μg of boric acid per ml (22 and 83 μg a.i./ml), respectively. Two boron-based treatments were developed and tested in vitro and in four field trials during 2001 to 2003. One product, biopaste, contained 5% boric acid (8.75 mg a.i./ml) in a commercial paste. The second product, bioshield, contained 5% boric acid in a spore suspension of Cladosporium herbarum. Both products significantly reduced disease in vitro and in field trials in comparison with a water control treatment. Boron was not found to accumulate in leaves and shoots, but bud failure at the first node below the treated wound occurred at a higher rate than in untreated vines.

Pathogenesis of Eutypa lata in grapevine: identification of virulence factors and biochemical characterization of cordon dieback.

Eutypa lata is a vascular pathogen of woody plants. In the present study we (i) determined which component(s) of the cell wall polymers were degraded in naturally infected grapevines and in artificially inoculated grape wood blocks; (ii) compared the pattern of wood decay in the tolerant grape cv. Merlot versus the susceptible cv. Cabernet Sauvignon; and (iii) identified secondary metabolites and hydrolytic enzymes expressed by E. lata during wood degradation. Biochemical analyses and a cytochemical study indicated that glucose-rich polymers were primary targets of E. lata. Structural glucose and xylose of the hemicellulose fraction of the plant cell wall and starch were depleted in infected woods identically in both cultivars. Moreover, the more tolerant cv. Merlot always had more lignin in the wood than the susceptible cv. Cabernet Sauvignon, indicating that this polymer may play a role in disease resistance. In vitro assays demonstrated the production by E. lata of oxidases, glycosidases and starch degrading enzymes. Phytotoxic secondary metabolites were also produced but our data suggest that they may bind to the wood. Finally, we demonstrated that free glucose in liquid cultures repressed primary but not secondary metabolism.

Identification of Eutypa lata by PCR-RFLP

Eutypa lata is a vascular canker pathogen of woody plants commonly diagnosed by isolating the pathogen from infected tissue. Related fungi from the same family, the Diatrypaceae, also have been found in association with grapevine in Californian vineyards. An in situ polymerase chain reaction (PCR) method has been developed for detection of E. lata in infected wood tissue. However, our results indicate that this method also would amplify other Diatrypaceous fungi, which could potentially lead to an incorrect diagnosis. Therefore, we developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay. The internal transcribed spacer (ITS)1/5.8S/ITS2 ribosomal DNA region was amplified by PCR using universal primers, and RFLP patterns were determined after digestion with AluI. The restriction profiles obtained served to distinguish E. lata from wood trunk pathogens of grapevine (Phomopsis viticola, Botryodiplodia sp., Phaeoacremonium aleophilum, and Phaeomoniella chlamydospora), Diatrypaceous fungi (Diatrype sp., Diatrypella sp., Eutypella vitis, and Eutypa leptoplaca), and Cryptovalsa sp. found on dead wood of grapevine, and other Eutypa spp. (E.petrakii var. hederae, E. astroidea, E. crustata, and E. lejoplaca), with the exception of E. armeniacae, which we regard as a synonym for E. lata, and E. laevata, which has not been found on grapevine.

A Reassessment of the Species Concept in Eutypa lata, the Causal Agent of Eutypa Dieback of Grapevine

ABSTRACT Eutypa dieback is a vascular disease of several cultivated crops and trees worldwide. The attribution of the name to the agent responsible for branch dieback is ambiguous. Pathogenicity of Eutypa sp. first was reported on apricot and the causal agent was named E. armeniacae. However, no morphological differences were reported with the previously described E. lata, and some authors considered both species synonymous. Others regarded them as distinct species on the basis of pathogenesis and molecular analysis. We further investigated the relatedness of both species by phylogenetic analyses of the internal transcribed spacer region and beta-tubulin gene. These analyses included several other taxa placed in the same family (Diatrypaceae), and yielded three groups. The isolates referred to as E. lata in previous work clustered with Diatrype stigma in one group. Isolates of E. armeniacae and E. lata clustered in a second group, supporting the synonymy of these species. The third group included other Eutypa spp. supporting the polyphyletic origin of this genus. Measurements of conidia length and secondary metabolite production of isolates supported the phylogenetic analyses. Secondary metabolites appeared to be a synapomorphic character shared by several taxa including E. lata, E. armeniacae, E. laevata, and E. petrakii var. petrakii.

Cylindrocarpon species associated with black-foot of grapevine in Northeastern United States and Southeastern Canada.

Black-foot disease of grapevine is caused by a complex of soilborne fungi. The most common and virulent species, which are found across all major grapegrowing regions of the world, are Cylindrocarpon liriodendri (C. liriodendri) and C. macrodidymum (teleomorph = Neonectria). Other species with a more limited distribution and uncertainty regarding their pathogenicity include C. destructans, C. obtusisporum, C. pauciseptatum, Campylocarpon fasciculare (C. fasciculare), and C. pseudofasciculare. The goal was to identify the species associated with black-foot disease in vineyards of the northeastern United States (U.S.) and southeastern Canada as such regions have not previously been surveyed. Recent expansion of winegrape acreage in these regions necessitates a clear understanding of the disease risks. Eleven U.S. states and two Canadian provinces were surveyed. Genus-level identification was based preliminarily on colony morphology. Species-level identity was based on phylogenetic analysis of two nuclear loci, 5.8S rDNA and ß-tubulin, using voucher specimens and sequences with high sequence identity. We report for the first time from Canada recovery of C. liriodendri, C. macrodidymum, and C. destructans from symptomatic grapevines. Also reported are species not previously identified from black-foot symptomatic grapes anywhere in the world, including C. didymum and a Neonectria mammoidea-like species. Results suggest that local viticultural practices, primarily burying the vine underground during winter, may create injuries, and thus exacerbate infection by wound pathogens such as Cylindrocarpon. Overall this work improves the knowledge of black-foot disease in these nascent grapegrowing regions and will be helpful to growers in their decisions regarding viticultural practices, planting, and disease management.

Microsatellite markers for the grapevine pathogen, Eutypa lata

We isolated and characterized nine polymorphic microsatellite markers for Eutypa lata, a fungal pathogen responsible for Eutypa dieback of grapevine, in populations from two California vineyards (24 isolates per vineyard). Allele frequency ranged from two to 11 alleles per locus and haploid gene diversity ranged from 0.33 to 0.83. All samples comprised unique haplotypes. Our results suggest that there is sufficient allelic polymorphism to estimate fine‐scale spatial structure, and to identify possible sources of inoculum from habitats outside of vineyards.

A method to detect and quantify Eutypa lata and Diplodia seriata-complex DNA in grapevine pruning wounds

Trunk diseases are factors that limit sustainability of vineyards worldwide. Botryosphaeria and Eutypa diebacks are caused by several fungi belonging to the Botryosphaeriaceae and Diatrypaceae, respectively, with Diplodia seriata and Eutypa lata being two of the most common species. Previous information indicated that the traditional isolation method used to detect these pathogens from plant samples could underestimate their incidence levels. In the present study, we designed two sets of primers that target the β-tubulin gene and that are amenable for quantitative real-time PCR (qPCR) Sybr-Green assays for the detection and quantification of D. seriata-complex (DseCQF/R) and E. lata (ElQF/R) DNA. The design of a species-specific assay was achieved for E. lata. For D. seriata, a species-specific assay could not be designed. The low interspecific diversity across β-tubulin genes resulted in an assay that could not discriminate D. seriata from some closely related species either not yet reported or presenting a low prevalence on grapevine, such as D. intermedia. We validated our technique on grapevine spur samples naturally and artificially infected with D. seriata and E. lata during the dormant season. Experimental grapevines were located in two counties of northern California where the incidence of both pathogens was previously reported. The qPCR assays revealed that a high frequency of pruning wound infections (65%) was achieved naturally by E. lata, while low infection frequency (less than 5%) was observed using the reisolation method. For D. seriata-complex, low (5%) to no natural infection frequencies were observed by the qPCR and the reisolation method, respectively. These results also provided evidence that our qPCR detection methods were more sensitive to assess the incidence of E. lata and D. seriata-complex in plant samples, than traditional isolation techniques. Benefits of molecular methods for the detection of canker pathogens in the field under natural conditions are discussed.