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Fungal canker pathogens of almond initiate infection in trees primarily through pruning wounds. Biological control agents (BCAs) have the potential to provide long-term protection of pruning wounds by colonizing the wound surfaces and underlying tissues. Laboratory and field tests were performed to assess the efficacy of various commercial and experimental BCAs as wound protectants against almond canker pathogens. Four Trichoderma-based BCAs were evaluated using detached almond stems in the laboratory against the canker pathogens Cytospora plurivora, Eutypa lata, Neofusicoccum parvum, and Neoscytalidium dimidiatum. Results indicated that Trichoderma atroviride SC1 and T. paratroviride RTFT014 significantly reduced infections by all four pathogens. The abilities of these four BCAs to protect almond pruning wounds against E. lata and N. parvum were further evaluated in field trials using two almond cultivars and during two consecutive years. Both T. atroviride SC 1 and T. paratroviride RTFT014 protected almond pruning wounds against E. lata and N. parvum as efficiently as thiophanate-methyl, the recommended fungicide for treatment of almond pruning wounds. Comparisons of different application timings of BCA in relation to pathogen inoculation revealed a significant improvement in wound protection when inoculations were conducted 7 days versus 24 h post-BCA application for N. parvum, but not for E. lata. T. atroviride SC 1 and T. paratroviride RTFT014 are promising candidates for the preventive protection of almond pruning wounds and for inclusion in integrated pest management programs and organic almond production systems. [ABSTRACT FROM AUTHOR]

Seimatosporium spp. and closely related "pestalotioid fungi" have been isolated from vineyards worldwide, but their ecological status in grapevine wood is unclear. To determine their involvement in the grapevine trunkdisease complex, we tested the pathogenicity of Californian isolates obtained from vines with general symptoms of Botryosphaeria, Eutypa, and Phomopsis diebacks. Multilocus phylogenetic analyses revealed three species: Seimatosporium vitis and two newly described and typified species, S. luteosporum sp. nov. and S. vitifusiforme sp. nov. Inoculations to woody stems of potted grapevines of both isolates of S. vitis and one isolate of S. vitifusiforme, but not S. luteosporum, were associated with significantly larger lesions than those of noninoculated controls. Coinoculations with trunk pathogens (Cryptovalsa ampelina, Diaporthe ambigua, Diatrypella verruciformis, Dipladia seriata, and Eutypa lata), coisolated from the same wood cankers in the field, brought about increased lesion lengths for S. vitifusiforme paired with D. seriata, and S. luteosporum paired with Diaporthe ambigua. In contrast, there were no differences in lesion lengths of 5. vitis and Diatrypella verruciformis or 5. vitis and E. lata, inoculated alone or together. Our findings suggest that Seimatosporium spp. are involved in the grapevine trunk-disease complex, and their virulence may depend on or affect that of trank pathogens. [ABSTRACT FROM AUTHOR]

Pathogen adaptation to different hosts can lead to specialization and, when coupled with reproductive isolation, genome-wide differentiation and ecological speciation. We tested the hypothesis of host specialization among California populations of Eutypa lata (causal fungus of Eutypa dieback of grapevine and apricot), which is reported from >90 species. Genetic analyses of nine microsatellite loci in 182 isolates from three hosts (grapevine, apricot, and willow) at three locations were complemented by cross-inoculations on cultivated hosts grapevine and apricot to reveal patterns of host specialization. The cultivated hosts are likely more important sources of inoculum than the wild host willow, based on our findings of higher pathogen prevalence and allelic richness in grapevine and apricot. High levels of gene flow among all three hosts and locations, and no grouping by clustering analyses, suggest neither host nor geographic differentiation. Cross-inoculations revealed diversified phenotypes harboring various performance levels in grapevine and apricot, with no apparent correlation with their host of origin. Such phenotypic diversity may enable this pathogen to persist and reproduce as a generalist. Regular genetic reshuffling through sexual recombination, frequent immigration among hosts, and the lack of habitat choice in this passively dispersed fungus may prevent fixation of alleles controlling host specialization. [ABSTRACT FROM AUTHOR]

Cultivars of European grapevine, Vitis vinifera, show varying levels of susceptibility to Eutypa dieback and Esca, in terms of foliar symptoms. However, little is known regarding cultivar susceptibility of their woody tissues to canker formation. Accordingly, we evaluated the relative susceptibility of V. vinifera cultivars ('Cabernet Franc', 'Caber-net Sauvignon', 'Chardonnay', 'Merlot', 'Riesling', 'Petite Syrah', and 'Thompson Seedless') and species or interspecific hybrids of North American Vitis (Vitis hybrid 'Concord', V. arizonica 'b42-26', V. rupestris × V. cinerea '111547-1', and Fennell 6 [V. aestivalis] × Malaga [V. vinifera] 'DVIT0166') to canker formation by seven trunk patho-gens (Neofusicoccum pan'um, Lasiodiplodia theobromae, Phaeomoni-ella chlamydospora, Togninia minima, Phomopsis viticola, Eutypa lata, and an undescribed Eutypa sp.). Susceptibility was based on the length of wood discoloration (LWD) in the woody stems of rooted plants in duplicate greenhouse experiments. Cultivars of V. vinifera and Concord did not vary significantly in susceptibility to N. pan'um or L. theobromae (LWD of 21 to 88 mm at 14 weeks post inoculation; P > 0.16), suggesting that they are similarly susceptible to Botryosphaeria dieback. The table-grape Thompson Seedless was most susceptible to P. viticola (mean LWD of 61 mm at 11 months post inoculation; P < 0.0001). V. vinifera cultivars and Concord showed similar susceptibility to the Esca pathogens, Phaeomoniella chlamydospora and T. minima. Susceptibility to E. lata was greatest in V. arizonica b42-26 (mean LWD of 96 mm at 11 months post inoculation; P < 0.03). In fact, all four American Vitis spp. were more susceptible to Eutypa dieback than the V. vinifera cultivars. Our findings suggest that no one cultivar is likely to provide resistance to the range of trunk pathogens but that certain cultivars may be promising candidates for commercially rele-vant host resistance in grape-production systems where the dominant cultivars are very susceptible. [ABSTRACT FROM AUTHOR]

Armillaria mellea infects hundreds of plant species in natural and managed ecosystems throughout the Northern hemisphere. Previously reported nuclear genetic divergence between eastern and western U.S. isolates is consistent with the disjunct range of A. mellea in North America, which is restricted mainly to both coasts of the United States. We investigated patterns of population structure and genetic diversity of the eastern (northern and southern Appalachians, Ozarks, and western Great Lakes) and western (Berkeley, Los Angeles, St. Helena, and San Jose, CA) regions of the United States. In total, 156 diploid isolates were genotyped using 12 microsatellite loci. Absence of genetic differentiation within either eastern subpopulations (θST = -0.002, P = 0.5) or western subpopulations (θST = 0.004, P = 0.3) suggests that spore dispersal within each region is sufficient to prevent geographic differentiation. In contrast to the western United States, our finding of more than one genetic cluster of isolates within the eastern United States (K = 3), revealed by Bayesian assignment of multilocus genotypes in STRUCTURE and confirmed by genetic multivariate analyses, suggests that eastern subpopulations are derived from multiple founder sources. The existence of amplifiable and nonamplifiable loci and contrasting patterns of genetic diversity between the two regions demonstrate that there are two geographically isolated, divergent genetic pools of A. mellea in the United States. [ABSTRACT FROM AUTHOR]

Trunk and scaffold canker diseases (TSCDs) of almond cause significant yield and tree losses and reduce the lifespan of orchards. In California, several pathogens cause TSCDs, including Botryosphaeriaceae, Ceratocystis destructans, Eutypa lata, Collophorina hispanica, Pallidophorina paarla, Cytospora, Diaporthe, and Phytophthora spp. Field diagnosis of TSCDs is challenging because symptom delineation among the diseases is not clear. Accurate diagnosis of the causal species requires detailed examination of symptoms and subsequent isolation on medium and identification using morphological criteria and subsequent confirmation using molecular tools. The process is time-consuming and difficult, particularly as morphological characteristics are variable and overlap among species. To facilitate diagnosis of TSCD, we developed PCR assays using 23 species-specific primers designed by exploiting sequence differences in the translation elongation factor, ß-tubulin, or internal transcribed spacer gene. Using genomic DNA from pure cultures of each fungal and oomycete species, each primer pair successfully amplified a single DNA fragment from the target pathogen but not from selected nontarget pathogens or common endophytes. Although 10-fold serial dilution of fungal DNA extracted from either pure cultures or infected wood samples detected as little as 0.1 pg of DNA sample, consistent detection required 10 ng of pathogen DNA from mycelial samples or from wood chips or drill shavings from artificially or naturally infected almond wood samples with visible symptoms. The new PCR assay represents an improved tool for diagnostic laboratories and will be critical to implement effective disease surveillance and control measures. [ABSTRACT FROM AUTHOR]

Eutypa dieback of grapevine is caused by Eutypa lata in production areas with Mediterranean climates in California, Australasia, Europe, and South Africa. Eutypa dieback has also been described in the colder, eastern North American vineyards where cultivars adapted from native Vitis spp. (e.g., Vitis x labruscana 'Concord') are primarily grown. However, the causal agents associated with the diseases in this region have not been conclusively identified. Examination of 48 vine-yards showing symptoms of dieback in the northeastern United States (Connecticut, Massachusetts, Michigan, New York, Ohio, and Rhode Island) and Ontario, Canada revealed that vineyards were mainly in-fected by Eutypa spp. other than E. lata. Multigene phylogenies (inter- nal transcribed spacer ribosomal DNA, ß-tubulin, and RNA polymer-ase II) of isolates recovered from these vineyards indicated that Eutypa dieback is caused primarily by an undescribed Eutypa sp. and E. lae-vata. Eutypa sp. was recovered from 56% of the vineyards examined, whereas E. laevata and E. lata were less far common (17 and 6%, respectively). Fruiting body morphology and spore dimensions supported phylogenetic separation of the three taxa. Pathogenicity tests conducted on Vitis vinifera 'Chardonnay' in the greenhouse and in the field verified that all three species were able to cause wood canker and to infect pruning wounds, respectively. [ABSTRACT FROM AUTHOR]

In eastern North America, Phomopsis cane and leaf spot, caused by Phomopsis viticola, is a foliar disease of grape but, in the Mediterra-nean climate of western North America, P. viticola is primarily associ-ated with wood cankers, along with other Diaporthe spp. To determine the identity of wood-infecting Diaporthe spp. in eastern North Amer-ica, 65 isolates were cultured from 190 wood-canker samples from 23 vineyards with a history of Phomopsis cane and leaf spot. Identifica-tion of 29 representative isolates was based initially on morphology, followed by phylogenetic analyses of DNA sequences of the ribosomal DNA internal transcribed spacer region, elongation factor subunit 1-a, and actin in comparison with those of type specimens. Three species were identified: P. viticola, P. fukushii, and Diaporthe eres. Inoc-ulations onto woody stems of potted Vitis labruscana 'Concord' and V. vinifera 'Chardonnay' showed that D. eres and P. fukushii were pathogenic (mean lesion lengths of 7.4 and 7.1 mm, respectively, compared with 3.5 mm for noninoculated controls) but significantly less so than wood-canker and leaf-spot isolates of P. viticola (13.5 mm). All three species infected pruning wounds of Concord and Char-donnay in the field. Our finding of pathogenic, wood-infecting Di-aporthe spp. in all 23 vineyards suggests a frequent co-occurrence of the foliar symptoms of Phomopsis cane and leaf spot and wood can-kers, although the latter are not always due to P. viticola. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]