First Report of Calosphaeria pulchella causing Canker and Twig Dieback of Peach (Prunus persica) in California, U.S.A.

California leads the United States in peach (Prunus persica L.) production, with approximately 505,000 tons produced in 2021 and valued at $378.3 million (California Agriculture Statistics Review, 2021-2022). During the spring and summer of 2023, twig and branch dieback were observed in three peach orchards (cvs. Late Ross and Starn) in San Joaquin County, California. Wood cankers and discoloration also occurred in branches, generally initiating at pruning wounds. Approximately 8 symptomatic twigs or branches per orchard were collected to proceed with the isolation of necrotic tissues on acidified potato dextrose agar (APDA). Isolations consistently yielded colonies of the fungal pathogen Calosphaeria pulchella (Pers. : Fr.) J. Schröt. (Réblová et al. 2004; Trouillas et al. 2012). Pure cultures were obtained by transferring single hyphal tips onto new APDA Petri plates. Colonies on APDA grew dark pink to red or purple in their center, with a white margin. Conidiogenesis was phialidic, producing round conidial masses at the tip of phialides. Conidia were produced abundantly on APDA, and were hyaline, allantoid to oblong-ellipsoidal, 4 to 5.5 (7) × 1.2 to 2.3 µm (n = 60). Two representative isolates (SJC-62 and SJC-64) were selected for genomic DNA extraction and sequencing of the internal transcribed spacer region (ITS) using ITS5/ITS4 universal primers and the beta-tubulin (TUB2) gene region using primers Bt2a and Bt2b. Consensus sequences of the two genes for the two isolates (ITS: PP063990, PP063991; TUB2: PP068303, PP068304) were compared to reference sequences (Réblová et al. 2015; Trouillas et al. 2012) using BLAST analysis. The ITS sequences of SJC-62 and SJC-64 were 99.8 and 99.5% identical to that of C. pulchella ex-type strain CBS 115999 (NR145357) and reference strain SS07 (HM237297); the TUB2 sequences were at least 98.5% identical to that of C. pulchella CBS 115999 (KT716476). Pathogenicity tests were conducted in 2- to 3-year-old healthy branches on 7-year-old peach trees, cvs. Loadel, Late Ross and Starn using the two fungal isolates and a control treatment (1 branch per treatment and 3 branches per tree) on each of 8-tree replicates. Branches were inoculated in June 2023 following wounding with a 5 mm cork borer to remove the bark and placing an agar plug from the margin of 10-day-old colonies on APDA directly into the fresh wound. Sterile agar plugs were used as controls. Inoculation sites were covered with petroleum jelly and wrapped with Parafilm to retain moisture. The experiment was completed twice. After four months, cankers and vascular discolorations developed around the inoculation sites. Length of vascular discoloration in inoculated branches averaged 72, 75, and 79 mm, for the Loadel, Starn, and Late Ross cvs., respectively. Calosphaeria pulchella was re-isolated from inoculated branches at 80 to 100% recovery rate, thus fulfilling Koch's postulates. The average length of vascular discoloration in the control was 13.5 mm and no fungi were recovered from control branches. Calosphaeria canker caused by C. pulchella is a global disease of sweet cherry. Recently, it was reported to cause cankers in peach trees in Chile (Grinbergs et al. 2023). To our knowledge, this is the first report of C. pulchella causing cankers and twig dieback of peach trees in the United States. These findings improve our knowledge of the etiology of canker diseases affecting peach trees and is critical for the development of effective disease management strategies.

Determining the Main Infection Courts in Sweet Cherry Trees of the Canker Pathogens Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata.

The major fungal canker pathogens causing branch dieback of sweet cherry trees in California include Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata. These pathogens have long been known to infect cherry trees mainly through pruning wounds. However, recent field observations revealed numerous shoots and fruiting spurs exhibiting dieback symptoms with no apparent pruning wounds or mechanical injuries. Accordingly, this study was conducted to assess the incidence of the three pathogens in symptomatic terminal shoots and dying fruiting spurs, in addition to the wood below pruning wounds in branches. Surveys were conducted in five sweet cherry orchards across three counties in California. We also investigated the possibility that leaf scars, bud scars, and wounds resulting from fruit picking could serve as infection courts for Cal. pulchella, Cyt. sorbicola, and E. lata by means of artificial inoculations in the field. Orchard surveys revealed that Cal. pulchella had the highest pathogen incidence below pruning wounds in branch samples, followed by Cyt. sorbicola and E. lata. Among terminal shoots with dieback symptoms and dying fruiting spurs, Cyt. sorbicola was the most prevalent, followed by Cal. pulchella. Results from field inoculations indicated that fruit-picking wounds could serve as important infection courts for Cal. pulchella, Cyt. sorbicola, and E. lata, with average pathogen recovery of 41.5, 63, and 36.2%, respectively. Results also indicated that leaf and bud scars could serve as an entry site for Cyt. sorbicola, although recovery was relatively low. The present study is the first to identify harvest-induced wounds on fruiting spurs of sweet cherry as an important infection court of Cal. pulchella, Cyt. sorbicola, and E. lata.

Evaluation of Biological Control Agents for the Protection of Almond Pruning Wounds Against Infection by Fungal Canker Pathogens.

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 SC1 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 SC1 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.

Seasonal Susceptibility of Sweet Cherry Pruning Wounds to Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata.

Fungal canker pathogens commonly infect trees at pruning wounds leading to branch dieback and loss of productivity in sweet cherry orchards. However, the seasonal susceptibility of sweet cherry pruning wounds to Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata is not well understood. This study compared the susceptibility of sweet cherry pruning wounds made during the dormant season (January) and the postharvest season (late May to June) to infection by main canker pathogens in California. Field trials were conducted in three cherry orchards and trees were pruned at the different periods over 2 years. Fresh pruning wounds were inoculated with spores of each pathogen, and pathogen recovery was assessed through microbiological isolations at 3 to 4 months after inoculations. Pruning wounds made in late May and June resulted in significantly higher infection by Cal. pulchella compared to pruning wounds made in January. Pruning wounds made during both seasons were generally equally susceptible to Cyt. sorbicola and E. lata infections. However, there was one orchard where dormant pruning wounds were more susceptible to infection by E. lata and there was one particularly cold winter where Cyt. sorbicola did not infect pruning wounds. Overall, our findings suggest that Cal. pulchella infections of cherry pruning wounds are more likely to occur during periods of warm temperatures such as late spring and early summer. However, infections by Cyt. sorbicola and E. lata can occur year-round if inoculum is present and if winter temperatures are not abnormally low for California. Finally, our results suggest that the emergence of Cal. pulchella as a major canker pathogen of sweet cherry in California may be the result of a shift from dormant to after-harvest pruning of sweet cherry trees.

Effects of Temperature on Spore Germination and Mycelial Growth of Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata Isolates Associated with Sweet Cherry Canker Diseases.

Although fungal canker diseases constitute a limiting factor to orchard productivity and longevity, little is known about the effects of temperature on spore germination and mycelial growth of the fungal causal agents. Accordingly, the germination of spores and colony growth of Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata were evaluated after incubation on 2% water agar and 4% potato dextrose agar, respectively, at 5, 10, 15, 20, 25, 30, 35, and 40°C. Temperature optima for spore germination and mycelial growth were derived from nonlinear models fitted to germination rates and colony diameter data. The optimal temperatures for spore germination of Cal. pulchella were 28.5°C for ascospores and 29.2°C for conidia. The optimal temperatures for Cyt. sorbicola conidia and E. lata ascospore germination were 25.8 and 23.1°C, respectively. The germination of ascospores and conidia of Cal. pulchella at temperatures below 15°C required an incubation time of at least 72 h. Ascospores of E. lata and conidia of Cyt. sorbicola germinated at 10°C after 36 h. The optimal temperature for colony growth of Cal. pulchella was 24.6°C, whereas it was 21.7°C for both Cyt. sorbicola and E. lata. Our study indicates that temperature requirements for basic biological functions are higher for Cal. pulchella than for Cyt. sorbicola and E. lata. The overall higher temperatures of California relative to other cherry-producing regions in the United States or worldwide could explain the prevalence of Calosphaeria canker in the state. Conversely, Cyt. sorbicola and E. lata appear better adapted to cooler temperatures.

Field Evaluation of Fungicides for the Management of Neofabraea Leaf Lesion of Olive in California.

Field experiments were conducted during the fall-winter seasons of 2017 to 2018 and 2018 to 2019 to evaluate the efficacy of various fungicides to control Neofabraea leaf lesion of olive. Field trials were conducted in the highly susceptible cultivar Arbosana in a commercial, super-high-density orchard in San Joaquin County, California. Up to eight fungicidal products were applied using an air blast backpack sprayer, and their efficacy was compared with different application strategies. Results showed that most products were effective in reducing infection by the pathogens and limiting disease severity. Overall, best disease control was achieved by thiophanate-methyl, cyprodinil, difenoconazole + cyprodinil, and chlorothalonil, providing up to 75% reduction in disease severity. Copper hydroxide did not control the disease. In 2018 to 2019, the fungicides difenoconazole + cyprodinil and ziram were evaluated in additional field trials using different application strategies (single, dual, and combined applications) suitable for pathogen resistance management. Results showed that both products provided significant reduction in disease severity (∼50%), although no differences in efficacy were found between the two products nor between the different application strategies. Both products performed equally using one or two applications at 2-week intervals following harvest.

First Report of Cytospora azerbaijanica Causing Cytospora Canker and Shoot Dieback on Peach (Prunus persica) in California, U.S.A.

Peaches (Prunus persica L.) are an important crop in the United States with California leading the nation in peach production, with approximately 505,000 tons valued at $378.3 million (USDA National Agricultural Statistics Service, 2021, https://www.nass.usda.gov/). From April to July 2022, symptoms of branch and scaffold canker as well as shoot dieback were observed in three peach (cvs. Loadel, Late Ross and Starn) orchards located in San Joaquin County, California. Samples were collected from about 12 trees for each cultivar. Fast-growing, white, flat colonies were consistently isolated from active cankers on acidified potato dextrose agar (APDA) following the method described by (Lawrence et al. 2017). Pure fungal cultures were obtained by transferring single hyphal tips onto new APDA Petri plates. A total of 22 isolates were obtained. Each fungal isolate was recovered from a single diseased branch (40 to 55% recovery). All isolates in this study shared similar morphological characteristics. Fungal colonies were fast-growing with relatively even but slightly dentate margin, flat with white to off-white mycelium that turned vinaceous buff to pale greyish sepia (Rayner 1970) with age. Black, globose, ostiolated pycnidia, 0.8-(1.3)-2.2 mm diameter, with brownish surface hyphae formed on peach wood embedded in PDA after approximately three weeks and exudated buff-colored mucilage. Pycnidia were both solitary and aggregated and had multiple internal locules sharing invaginated walls. Conidiogenous cells were hyaline, smooth-walled, septate, tapering towards the apex, 13-(18.2)-25.1 × 0.8-(1.3)-1.9 µm (n = 40). Conidia were hyaline, allantoid, smooth, aseptate, 5.5-(6.3)-7.1 × 1.4-(1.9)-2.3 µm (n = 40). Genomic DNA was extracted and sequences of the internal transcribed spacer region (ITS) using ITS5/ITS4 universal primers, translation elongation factor 1α gene (TEF) using primers EF1-728F/EF1-986R, second largest subunit of RNA polymerase II (RPB2) using primers RPB2-5F2/fRPB2-7cR, and actin gene region (ACT) using primers ACT-512F/ACT-783R were obtained and compared with sequences available in GenBank (Lawrence et al. 2018; Hanifeh et al. 2022). Isolates were identified as Cytospora azerbaijanica following DNA sequencing and morphological identification. Consensus sequences of the four genes of two representative isolates (SJC-66 and SJC-69) were deposited into GenBank database (ITS: OQ060581 and OQ060582; ACT: OQ082292, OQ082295; TEF: OQ082290 and OQ082293; RPB2: OQ082291 and OQ082294). The Basic Local Alignment Search Tool (BLAST) indicated that the sequenced RPB2 genes of isolates (SJC-66 and SJC-69) were at least 99% identical to that of Cytospora sp. strain shd47 (Accession: MW824360) covering at least 85% of the sequences. The actin genes from our isolates were at least 97.85% identical to that of Cytospora sp. strain shd47 (Accession: MZ014513), covering 100% of the sequences. The translation elongation factor gene from isolates (SJC-66 and SJC-69) was at least 96.4% identical to that of Cytospora sp. strain shd166 (Accession: OM372512), covering 100% of the query. Those top hit strains belong to C. azerbaijanica, recently reported by Hanifeh et al. (2022). Pathogenicity tests were performed by inoculating eight wounded, 2- to 3-year-old healthy branches on each of eight 7-year-old peach trees, cvs. Loadel, Late Ross and Starn, using 5-mm-diameter mycelium plugs collected from the margin of an actively growing fungal colony on APDA. Controls were mock-inoculated with sterile agar plugs. Inoculation sites were covered with petroleum jelly and wrapped with Parafilm to keep moisture. The experiment was performed twice. After four months, inoculation tests resulted in vascular discoloration (canker) above and below the inoculation sites (average necrosis length of 114.1 mm). Cytospora azerbaijanica was re-isolated from all infected branches (70 to 100% recovery) completing Koch's postulates. Controls remained symptomless and no fungi were isolated from the slightly discolored tissue. Cytospora species are destructive canker and dieback pathogens of numerous woody hosts worldwide. Recently, C. azerbaijanica was reported in causing canker disease of apple trees in Iran (Hanifeh et al. 2022). To our knowledge, this is the first report of C. azerbaijanica causing canker and shoot dieback of peach trees in the United States and worldwide. These findings will aid towards a better understanding of genetic diversity and host range of C. azerbaijanica.

Development of PCR-Based Assays for Rapid and Reliable Detection and Identification of Canker-Causing Pathogens from Symptomatic Almond Trees.

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.

Evaluation of Pruning Wound Protection Products for the Management of Almond Canker Diseases in California.

Almond trunk and branch canker diseases constitute a major cause of tree mortality in California. Numerous fungal pathogens have been associated with these canker diseases and pruning wounds act as major infection courts. Before this study, there were no products registered in California for the management of these diseases. In this study, fungicidal products including synthetic chemistries, biocontrols, paint, and a sealant were evaluated for preventing fungal pathogen infection via pruning wounds. In four field trials conducted over two dormant seasons, 16 pruning wound treatments were tested using handheld spray applications against five almond canker pathogens, namely Botryosphaeria dothidea, Neofusicoccum parvum, Cytospora sorbicola, Ceratocystis destructans, and Eutypa lata. The fungicide thiophanate-methyl (Topsin M; United Phosphorus, Bandra West, Mumbai, India) provided 82% overall disease prevention against four fungal pathogens. The biological control agent, Trichoderma atroviride SC1 (Vintec; Bi-PA, Londerzeel, Belgium), tested at three application rates, resulted in 90 to 93% protection of pruning wounds in field trials, and for individual pathogens ranged from 81 to 100% protection for the three rates. At the time of this publication, Vintec is being considered for registration as a biological control product for the prevention of almond canker diseases, while Topsin M is recommended to growers for the prevention of almond canker diseases. This research indicates that effective protection of pruning wounds from infection by almond canker pathogens can be achieved with a one-time spray application of thiophanate-methyl or the biocontrol T. atroviride SC1 (recommended 2 g/liter) after pruning.

Drought Exacerbates Botryosphaeria Dieback Symptoms in Grapevines and Confounds Host-based Molecular Markers of Infection by Neofusicoccum parvum.

Neofusicoccum parvum, causal fungus of the grapevine trunk disease Botryosphaeria dieback, attacks the wood of Vitis vinifera. Because lesions are internal, using putative host-based markers of infection from leaves for diagnosis is a nondestructive option. However, their specificity under drought stress is unknown. Potted 'Cabernet-Sauvignon' were inoculated with N. parvum in the greenhouse after wounding (IW), and with wounded and nonwounded noninoculated controls. At 2 weeks postinoculation (WPI), half of the plants were severely stressed (SS), receiving 30% water volume of the well-watered (WW) plants. Larger lesions at 12 WPI among IW-SS plants, compared with all other treatments, revealed an interactive effect of inoculation and drought on lesion length. Expression of eight putative marker genes was analyzed in leaves by qPCR at the onset of drought stress, and at 8 and 12 WPI. One marker showed consistent over-expression at 8 WPI in IW plants, regardless of water treatment, suggesting specificity to infection. By 12 WPI, higher expression of seven genes in all SS plants (across inoculation treatments) revealed specificity to drought. Cross-reactivity of markers to drought, therefore, limits their utility for disease diagnosis in the field, where drought induced by climate and deficit irrigation is common.

Identification and Characterization of Neofabraea kienholzii and Phlyctema vagabunda Causing Leaf and Shoot Lesions of Olive in California.

California produces over 95% of the olives grown in the United States. In 2017, California's total bearing acreage for olives was 14,570 hectares producing 192,000 tons of olives valued at $186.6 million. During the early spring of 2016, unusual leaf and shoot lesions were detected in olive trees from superhigh-density orchards in the Northern San Joaquin and Sacramento valleys of California. Affected trees displayed numerous leaf and shoot lesions developing at wounds created by mechanical harvesters. The 'Arbosana' cultivar was highly affected by the disease, whereas the disease was sporadic in 'Arbequina' and not found in 'Koroneiki' cultivar. Two fungal species, Neofabraea kienholzii and Phlyctema vagabunda, were found to be consistently associated with the disease, and Koch's postulates were completed. Species identity was confirmed by morphology and molecular data of the partial large subunit rDNA, the internal transcribed spacer region, and partial beta-tubulin region. The disease signs and symptoms are described and illustrated.

Two dominant loci determine resistance to Phomopsis cane lesions in F1 families of hybrid grapevines.

KEY MESSAGE: Rapid characterization of novel NB-LRR-associated resistance to Phomopsis cane spot on grapevine using high-throughput sampling and low-coverage sequencing for genotyping, locus mapping and transcriptome analysis provides insights into genetic resistance to a hemibiotrophic fungus. Phomopsis cane and leaf spot, caused by the hemibiotrophic fungus Diaporthe ampelina (syn = Phomopsis viticola), reduces the productivity in grapevines. Host resistance was studied on three F1 families derived from crosses involving resistant genotypes 'Horizon', Illinois 547-1, Vitis cinerea B9 and V. vinifera 'Chardonnay'. All families had progeny with extremely susceptible phenotypes, developing lesions on both dormant canes and maturing fruit clusters. Segregation of symptoms was observed under natural levels of inoculum in the field, while phenotypes on green shoots were confirmed under controlled inoculations in greenhouse. High-density genetic maps were used to localize novel qualitative resistance loci named Rda1 and Rda2 from V. cinerea B9 and 'Horizon', respectively. Co-linearity between reference genetic and physical maps allowed localization of Rda2 locus between 1.5 and 2.4 Mbp on chromosome 7, and Rda1 locus between 19.3 and 19.6 Mbp of chromosome 15, which spans a cluster of five NB-LRR genes. Further dissection of this locus was obtained by QTL mapping of gene expression values 14 h after inoculation across a subset of the 'Chardonnay' × V. cinerea B9 progeny. This provided evidence for the association between transcript levels of two of these NB-LRR genes with Rda1, with increased NB-LRR expression among susceptible progeny. In resistant parent V. cinerea B9, inoculation with D. ampelina was characterized by up-regulation of SA-associated genes and down-regulation of ethylene pathways, suggesting an R-gene-mediated response. With dominant effects associated with disease-free berries and minimal symptoms on canes, Rda1 and Rda2 are promising loci for grapevine genetic improvement.

Novel Seimatosporium Species from Grapevine in Northern California and Their Interactions with Fungal Pathogens Involved in the Trunk-Disease Complex.

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 trunk-disease 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, Diplodia 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 S. vitis and Diatrypella verruciformis or S. 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 trunk pathogens.

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 ß-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 ß-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.

Distinctive expansion of gene families associated with plant cell wall degradation, secondary metabolism, and nutrient uptake in the genomes of grapevine trunk pathogens.

BACKGROUND: Trunk diseases threaten the longevity and productivity of grapevines in all viticulture production systems. They are caused by distantly-related fungi that form chronic wood infections. Variation in wood-decay abilities and production of phytotoxic compounds are thought to contribute to their unique disease symptoms. We recently released the draft sequences of Eutypa lata, Neofusicoccum parvum and Togninia minima, causal agents of Eutypa dieback, Botryosphaeria dieback and Esca, respectively. In this work, we first expanded genomic resources to three important trunk pathogens, Diaporthe ampelina, Diplodia seriata, and Phaeomoniella chlamydospora, causal agents of Phomopsis dieback, Botryosphaeria dieback, and Esca, respectively. Then we integrated all currently-available information into a genome-wide comparative study to identify gene families potentially associated with host colonization and disease development. RESULTS: The integration of RNA-seq, comparative and ab initio approaches improved the protein-coding gene prediction in T. minima, whereas shotgun sequencing yielded nearly complete genome drafts of Dia. ampelina, Dip. seriata, and P. chlamydospora. The predicted proteomes of all sequenced trunk pathogens were annotated with a focus on functions likely associated with pathogenesis and virulence, namely (i) wood degradation, (ii) nutrient uptake, and (iii) toxin production. Specific patterns of gene family expansion were described using Computational Analysis of gene Family Evolution, which revealed lineage-specific evolution of distinct mechanisms of virulence, such as specific cell wall oxidative functions and secondary metabolic pathways in N. parvum, Dia. ampelina, and E. lata. Phylogenetically-informed principal component analysis revealed more similar repertoires of expanded functions among species that cause similar symptoms, which in some cases did not reflect phylogenetic relationships, thereby suggesting patterns of convergent evolution. CONCLUSIONS: This study describes the repertoires of putative virulence functions in the genomes of ubiquitous grapevine trunk pathogens. Gene families with significantly faster rates of gene gain can now provide a basis for further studies of in planta gene expression, diversity by genome re-sequencing, and targeted reverse genetic approaches. The functional validation of potential virulence factors will lead to a more comprehensive understanding of the mechanisms of pathogenesis and virulence, which ultimately will enable the development of accurate diagnostic tools and effective disease management.

Molecular Polymorphism and Phenotypic Diversity in the Eutypa Dieback Pathogen Eutypa lata.

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.

Diversity of Diaporthe species associated with wood cankers of fruit and nut crops in northern California.

Diaporthe ampelina, causal agent of Phomopsis cane and leaf spot of grapevine (Vitis vinifera L.) is isolated frequently from grapevine wood cankers, causing Phomopsis dieback. The latter disease is associated with four other Diaporthe species, three of which also are reported from hosts other than grape. To better understand the role of this Diaporthe community in Phomopsis dieback of grapevine and the potential for infection routes among alternate hosts, 76 Diaporthe isolates were recovered from wood cankers of cultivated grape, pear, apricot, almond and the wild host willow in four California counties. Isolates were characterized morphologically and assigned to species based on multigene sequence analyses. This study identified eight Diaporthe species from grapevine and one novel taxon from willow, D. benedicti. We report the first findings of D. australafricana and D. novem in North America. Our findings also expand the host ranges of D. ambigua to apricot and willow, D. australafricana to almond and willow, D. chamaeropis to grapevine and willow, D. foeniculina to willow and D. novem to almond. The generalists D. ambigua and D. eres were the most genetically diverse species, based on high nucleotide and haplotypic diversity, followed by the grapevine specialist D. ampelina. Analyses based on multilocus linkage disequilibrium could not reject the hypothesis of random mating for D. ambigua, which is further supported by relatively high haplotypic diversity, reports of both mating types and reports of successful matings in vitro. Pathogenicity assays revealed that D. ampelina was the most pathogenic species to grapevine wood.

Identification of Eutypa spp. Causing Eutypa Dieback of Grapevine in Eastern North America.

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 × labruscana 'Concord') are primarily grown. However, the causal agents associated with the diseases in this region have not been conclusively identified. Examination of 48 vineyards 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 infected by Eutypa spp. other than E. lata. Multigene phylogenies (internal transcribed spacer ribosomal DNA, ß-tubulin, and RNA polymerase II) of isolates recovered from these vineyards indicated that Eutypa dieback is caused primarily by an undescribed Eutypa sp. and E. laevata. 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.

Susceptibility of Cultivated and Wild Vitis spp. to Wood Infection by Fungal Trunk Pathogens.

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', 'Cabernet 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 'Ill547-1', and Fennell 6 [V. aestivalis] × Malaga [V. vinifera] 'DVIT0166') to canker formation by seven trunk pathogens (Neofusicoccum parvum, Lasiodiplodia theobromae, Phaeomoniella 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. parvum 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 relevant host resistance in grape-production systems where the dominant cultivars are very susceptible.

Characterization of Species of Diaporthe from Wood Cankers of Grape in Eastern North American Vineyards.

In eastern North America, Phomopsis cane and leaf spot, caused by Phomopsis viticola, is a foliar disease of grape but, in the Mediterranean climate of western North America, P. viticola is primarily associated with wood cankers, along with other Diaporthe spp. To determine the identity of wood-infecting Diaporthe spp. in eastern North America, 65 isolates were cultured from 190 wood-canker samples from 23 vineyards with a history of Phomopsis cane and leaf spot. Identification 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-α, and actin in comparison with those of type specimens. Three species were identified: P. viticola, P. fukushii, and Diaporthe eres. Inoculations 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 Chardonnay in the field. Our finding of pathogenic, wood-infecting Diaporthe spp. in all 23 vineyards suggests a frequent co-occurrence of the foliar symptoms of Phomopsis cane and leaf spot and wood cankers, although the latter are not always due to P. viticola.