Endophytic microbial assemblage in grapevine.

The plant vascular system has remained an underexplored niche despite its potential for hosting beneficial microbes. The aim of this work was to determine the origin of the microbial endophytes inhabiting grapevine. We focused on a single commercial vineyard in California over a two-year period and used an amplicon metagenomics approach to profile the bacterial (16S-V4) and fungal (ITS) communities of the microbiome across a continuum of six grapevine compartments: bulk soil, rhizosphere, root, cordon, cane and sap. Our data supported that roots are a bottleneck to microbial richness and that they are mostly colonized with soilborne microbes, including plant growth-promoting bacteria recruited by the host, but also saprophytic and pathogenic fungal invaders. A core group of taxa was identified throughout the vine; however, there was clear partitioning of the microbiome with niche adaptation of distinct taxonomic groups. Above- and belowground plant tissues displayed distinct microbial fingerprints and were intermixed in a limited capacity mostly by way of the plant sap. We discuss how cultural practices and human contact may shape the endosphere microbiome and identify potential channels for transmission of its residents.

First Report of Wood Canker Caused by Neoscytalidium dimidiatum on Grapevine in California.

In May 2012 in the Coachella valley, Riverside County, California, the decline of vines in table grape (Vitis vinifera) vineyards was observed. Foliar symptoms consisted of shoot blight with wilting and necrosis of leaves and drying and shriveling of berries. In some cases, the entire vine collapsed in the middle of the growing season (apoplexia). Wood cankers in the spurs, cordons, and trunks of affected vines were also present. The nine isolates recovered from the cankers were identified as Neoscytalidium dimidiatum (Penz.) Crous & Slippers based on morphological characteristics and DNA sequence comparisons. Two isolates were grown on potato dextrose agar (PDA) medium and a total of 50 conidia were measured per isolate. Conidia were ellipsoid to ovoid, with a truncate base and an acutely rounded apex, initially aseptate, becoming brown and two-celled at maturity, 7.2 ± 1.2 µm × 3.8 ± 0.4 µm. The rDNA internal transcribed spacer (ITS), and ß-tubulin (BT) loci were amplified using primer pairs and methods previously described (4). A total of five isolates were sequenced. The DNA sequences of one N. dimidiatum grapevine isolate (UCR-Neo1) were deposited in the GenBank database (ITS, KC937066; BT, KC937067). Pathogenicity tests were performed by inoculating 12 grape cuttings cv. Thompson Seedless with isolate UCR-Neo1 and 12 control cuttings with sterile medium using a technique previously described (1). The experiment was repeated twice. After 20 weeks of incubation period in the greenhouse, the lesions length produced by N. dimidiatum averaged 13.5 mm and was significantly longer (P < 0.05) from the control (average 3 mm). N. dimidiatum was reisolated from all the inoculated plants and identified by colony morphology. The incidence of N. dimidiatum in table grape vineyards of the Coachella valley has been estimated at 15%, with nine vines infected out of 60 vines total. This pathogen has been identified in California in walnut nursery causing the death of trees due to the development of canker at the graft union (2). N. dimidiatum has also been identified as the causal agent of shoot blight, canker, and gummosis on citrus in Italy (3). The crop is also being grown in the Coachella valley and these findings warrant further investigation in order to determine the host range, distribution, and incidence of this pathogen in the area. References: (1) K. Baumgartner et al. Plant Dis. 97:912, 2013. (2) S. F. Chen et al. Plant Dis. 97:993, 2013. (3) G. Polizzi et al. Plant Dis 93:1215, 2009. (4) J. R. Urbez-Torres et al. Plant Dis. 92:519, 2008.

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.

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.

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.

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.

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.