Grapevine trunk diseases (GTDs) are caused by multiple unrelated fungal pathogens, and their management remains difficult worldwide. Biocontrol is an attractive and sustainable strategy given the current need for a cleaner viticulture. In this study, twenty commercial vineyards were sampled across California to isolate endophytic and rhizospheric bacteria from different grapevine cultivars with the presence and absence of GTD symptoms. A collection of 1344 bacterial isolates were challenged in vitro against Neofusicoccum parvum and Diplodia seriata, from which a subset of 172 isolates exerted inhibition levels of mycelial growth over 40%. Bacterial isolates were identified as Bacillus velezensis (n = 154), Pseudomonas spp. (n = 12), Serratia plymuthica (n = 2) and others that were later excluded (n = 4). Representative isolates of B. velezensis, P. chlororaphis, and S. plymuthica were challenged against six other fungal pathogens responsible for GTDs. Mycelial inhibition levels were consistent across bacterial species, being slightly higher against slow-growing fungi than against Botryosphaeriaceae. Moreover, agar-diffusible metabolites of B. velezensis strongly inhibited the growth of N. parvum and Eutypa lata, at 1, 15, and 30% v/v. The agar-diffusible metabolites of P. chlororaphis and S. plymuthica, however, caused lower inhibition levels against both pathogens, but their volatile organic compounds showed antifungal activity against both pathogens. These results suggest that B. velezensis, P. chlororaphis and S. plymuthica constitute potential biocontrol agents (BCAs) against GTDs and their application in field conditions should be further evaluated.

Grapevine trunk diseases are caused by a broad diversity of fungal taxa that have serious impacts on the worldwide viticulture industry due to significant reductions in vineyards yield and lifespan. Field surveys carried out from 2018 to 2022 in California nurseries and young vineyards revealed a high incidence of Fusarium. Since Fusarium species are important pathogens of other perennial crops, the present study aimed to identify and determine the pathogenicity of the Fusarium species on grapevines. Morphology of the fungal colonies coupled with multilocus phylogenetic analyses using nucleotide sequences of the translation elongation factor 1-alpha (tef1) and the RNA polymerase II second largest subunit (rpb2) genes revealed the occurrence of 10 species clustering in six species complexes, namely F. fujikuroi (FFSC), F. oxysporum (FOSC), F. solani (FSSC), F. sambucinum (FSAMSC), F. incarnatum-equiseti (FIESC), and F. tricinctum (FTSC) species complexes. The species F. annulatum (FFSC) was the most prevalent in samples from both symptomatic young vineyards (73.5% incidence) and nursery propagation material (62.5% incidence). Pathogenicity of the 10 most frequent species was confirmed by fulfilling Koch’s postulates on living woody tissue of 1103 Paulsen rootstocks. Our results suggest that Fusarium spp. are involved in the development of young vine decline, probably as opportunistic pathogens when grapevines are under stress conditions.

Anthracnose caused by Colletotrichum species is one of the most frequent and damaging fungal diseases affecting avocado fruits (Persea americana Mill.) worldwide. In Chile, the disease incidence has increased over the last decades due to the establishment of commercial groves in more humid areas. Since 2018, unusual symptoms of anthracnose have been observed on Hass avocado fruits, with lesions developing a white to gray sporulation. Morphological features and multi-locus phylogenetic analyses using six DNA barcodes (act, chs-1, gapdh, his3, ITS, and tub2) allowed the identification of the causal agent as Colletotrichum anthrisci, a member of the dematium species complex. Pathogenicity was confirmed by inoculating healthy Hass avocado fruits with representative isolates, reproducing the same symptoms initially observed, and successfully reisolating the same isolates from the margin of the necrotic pulp. Previously, several Colletotrichum species belonging to other species complexes have been associated with avocado anthracnose in other countries. To our knowledge, this is the first record of C. anthrisci and of a species of the dematium species complex causing anthracnose on avocado fruits in Chile and worldwide.

From 2018 to 2021 a decline was detected in young vineyards of both wine and table grape (Vitis vinifera L.) in seven counties across California (Kern, Monterey, Napa, Sonoma, Tulare, Yolo, and Yuba). Affected vines showed poor or no growth throughout the season, dieback, sap exudation and internal cankers around the graft union. Lack of feeder roots was detected, indicating weak root development. In some cases, graft failure was associated with the symptomatology in recently established vineyards (<3 years old). A prevalence from 5 to 50% was estimated in 10 vineyards. Affected vines (n=34) were collected by farm advisors and submitted to the laboratory. Symptomatic vines were surface disinfected with 70% ethanol for 1 minute and air dried under sterile conditions. Vascular discoloration around the graft union was observed and inspected by removing the bark using a sterile knife. Isolations were performed from the margin of lesions by placing five wood sections (1×1 mm) per vine onto potato dextrose agar acidified with 0.5 mL/L of 85% lactic acid (APDA) and incubated for 7 days at 25°C in the dark. Even though other fungi associated with young vine decline were isolated and identified as Phaeoacremonium, Ilyonectria, and Botryosphaeriaceae species, Fusarium colonies (Leslie and Summerell, 2006) were the most prevalent among all the symptomatic vines. Pure cultures were obtained by transferring single hyphal tips onto fresh PDA. After 5 days of incubation, colonies formed white aerial mycelium with orange to purple colors on the bottom. Colonies in Spezieller Nährstoffarmer agar (SNA) produced abundant microconidia that were hyaline and ovoid to elliptical, ranging from 5.4 to 10.6 (7.4) × 1.4 to 3.3 (2.4) µm in size (n=50). Straight and slightly curved macroconidia varied from 15.5 to 42.3 (23.7) × 2.6 to 5.0 (3.6) µm in size (n=50). Upon DNA extraction, the translation elongation factor 1α (tef1) and the RNA polymerase II second largest subunit (rpb2) partial gene regions were amplified and sequenced using the EF1/EF2, 5F2/7cR and 7cF/11aR pair primers, respectively (O'Donnell et al. 1998, O'Donnell et al. 2007, Liu et al. 1999). Consensus sequences were compared to the NCBI database using BLAST, showing over 99% similarity with the ex-type sequence of F. annulatum CBS 258.54 (MT010994 and MT010983). A maximum likelihood multi-locus phylogenetic analysis confirmed that all the Californian isolates cluster with F. annulatum strains. Sequences were deposited in GenBank (nos. OK888534 to OK888537). Two representative isolates (UCD9188 and UCD9416) were used for pathogenicity tests. One-year-old 'Chardonnay' vines were inoculated between the second and third node by removing a 5-mm diameter disk of the bark using a sterile cork borer and placing a 5-mm agar plug with actively growing mycelium. Five replicates per isolate including controls with sterile agar plugs were incubated under greenhouse conditions for 2 months. The experiment was performed twice. Symptoms expressed as vascular linear necrotic lesions that ranged from 25.6 to 62.8 mm and the same pathogen was recovered, thus fulfilling Koch's postulates. Fusarium annulatum Bugnic. is a morphologically and genetically diverse species that has been widely known as F. proliferatum and known to be pathogenic in more than 200 plant hosts (Yilmaz et al. 2021). Fusarium spp. have been previously reported to cause young vine decline in Australia and British Columbia, Canada (Highet and Nair, 1995; Úrbez-Torres et al. 2017). To the best of our knowledge, this is the first report of F. annulatum associated with young vine decline complex in California.

In late summer 2020, symptoms of leaf blotch and fruit spot were observed in two different commercial apple orchards (cultivars ‘Pink Lady’ and ‘Modi’) in San Joaquin County, California, USA. Ninety Alternaria isolates were obtained from symptomatic leaves and fruits collected from the orchards. Based on morphological characteristics of the colonies, sporulation patterns, and conidia, the isolates were preliminarily separated into three morphogroups, tentatively identified as A. alternata, A. tenuissima and A. arborescens. Multi-locus phylogenetic analyses, using nucleotide sequences of plasma membrane ATPase, calmodulin, and Alternaria major allergen genes, showed that the isolates initially identified as A. tenuissima clustered with strains of A. alternata, following the current taxonomical arrangement of the genus. Pathogenicity tests on detached wounded apple leaves and fruits, using representative isolates of the three morphogroups, fulfilled Koch’s postulates. This is the first report of A. alternata and A. arborescens as causal agents of leaf blotch and fruit spot of apple in California.

California is the sixth largest apple-producing state in the United States with a production that reached 4,654 ha in 2021. During the late winter of 2023, black canker symptoms were observed on branches of 'Gravenstein' apple (Malus domestica) in two commercial orchards in Sonoma County, California. The prevalence of symptomatic trees ranged from 10 to 30%. External symptoms included charcoal looking-cankers with the bark peeling off from the primary and secondary branches. Internally, cankers were dark brown in color with a hard consistency. Pycnidia were observed on the surface of older cankers. Fungal isolations were performed from disinfected (70% ethanol, 30 s) symptomatic branch samples (n = 15). Small wood pieces (5 mm length) were taken from the margin of diseased and healthy tissues, and placed on potato dextrose agar acidified with 92% lactic acid at 0.5 mL per liter (APDA). Plates were incubated at room temperature (20-22 °C) for 7 days. Colonies of Botryosphaeriaceae species (Phillips et al. 2013) (n = 12) were consistently recovered and pure cultures were obtained by transferring a single hyphal tip onto fresh APDA. Colonies were light gray with irregular margins. To induce pycnidia formation, two isolates (UCD11350 and UCD11351) were grown on pistachio leaf agar for 21 days. Conidia (n = 50) were thick-walled and ovoid in shape, initially hyaline, then turned pale brown and dark brown at maturity, and some of them became 1-septate, ranging from 18.9 to 24.0 (21.9) × 11.5 to 14.7 (13.4) µm. Isolates were identified by sequencing a partial region of the beta-tubulin (tub2) gene using the primers Bt2a/Bt2b (Glass and Donaldson 1995). BLAST searches on NCBI GenBank revealed 99.5 % identity with the Diplodia bulgarica ex-type (CBS 1245254). To confirm the identity, the rRNA internal transcribed spacer (ITS) and the translation elongation factor 1-alpha (tef1) were also sequenced using ITS5/ITS4 (White et al. 1990), and EF1-688F/EF1-1251R (Alves et al. 2008), respectively. A maximum parsimony multi-locus phylogenetic analysis clustered Californian isolates with reference strains of D. bulgarica. Sequences were deposited in GenBank (nos. OR631209 to OR631210, OR637361 to OR637362, OR637363 to OR637364 for ITS, tub2, and tef1, respectively). Pathogenicity tests were conducted on 2 to 3-year-old branches (n = 5) of over 20-year-old trees by inserting a 5-mm segment of a toothpick, completely colonized with each of the two isolates mentioned above, into a 1-mm-diameter hole made with a sterile drill bit. The same number of branches where mock inoculated with a non-colonized toothpick as negative control. The experiment was performed twice. After ten weeks, inoculations resulted in dark brown necrotic lesions that ranged from 54.0 to 59.8 mm in length. Negative controls remained asymptomatic. Koch's postulates were fulfilled by successfully recovering the isolates from the lesion margins, which were confirmed by morphology. Diplodia bulgarica was first described affecting M. sylvestris in Bulgaria (Phillips et al. 2012), and then detected on M. domestica causing cankers in Iran (Abdollahzadeh 2015), India (Nabi et al. 2020), Germany (Hinrichs-Berger al. 2021) and Türkiye (Eken 2021). The pathogen was also identified causing postharvest fruit rot (Eken 2022). To our knowledge, this is the first report of D. bulgarica causing branch canker on apple in California, which provides important information for developing detection and control strategies.