Arthrocladiella mougeotii causing powdery mildew on goji berry plants (Lycium barbarum and L. chinense) and mixed infections with Phyllactinia chubutiana in California.

Goji berries (Lycium barbarum and L. chinense) have a rich historical significance in traditional Chinese medicine and have gained popularity as a superfood in Western cultures. From 2021 to 2023, powdery mildew was observed on goji plants of both species in community and residential gardens in Yolo County, California (USA). Disease severity varied from 20 to 100% of infected leaves per plant. Powdery mildew was characterized by the presence of white fungal colonies on both sides of leaves and fruit sepals. Additionally, a brownish discoloration was observed in infected mature leaves, resulting in further defoliation. Morphologically, the fungus matched the description of Arthrocladiella mougeotii. The pathogen identity was confirmed by phylogenetic analyses of the rDNA internal transcribed spacer and the 28S rDNA gene sequences. Pathogenicity was confirmed by inoculating healthy L. barbarum plants using infected leaves and successfully reproducing powdery mildew symptoms after 28 days (22°C, 60% RH), with A. mougeotii colonies confirmed by morphology. Control leaves remained symptomless. Co-infection with Phyllactinia chubutiana was detected on plants from two separate gardens, with A. mougeotii observed first in late spring (May to June) and P. chubutiana later in the summer (July to August). These results revealed that both A. mougeotii and P. chubutiana constitute causal agents of powdery mildew on goji berry plants, often infecting the same plant tissues simultaneously. To our knowledge, this is the first report of A. mougeotii causing powdery mildew on L. barbarum and L. chinense in California, which provides a better understanding of the etiology of powdery mildew of goji plants in California.

Identification and Pathogenicity of Fungal Pathogens Associated with Branch Canker and Shoot Blight on Persimmons (Diospyros kaki) in California.

Persimmon is a relatively new crop to California agriculture. Asian persimmons (Diospyros kaki) are the dominant species commercially cultivated in the United States, primarily grown in California, covering approximately 1,153 ha of bearing trees. In the growing seasons of 2020 and 2021, unusual shoot blight and branch cankers were observed in several persimmon orchards in San Joaquin and Solano counties in California. The most prevalent symptoms were well-defined black discoloration in the cambium and streaking in the vascular tissues of green shoots. On woody branches and old pruning wounds, symptoms manifested as black wedge-shaped cankers. Isolations from affected tissues revealed the occurrence of Diaporthe species, including D. chamaeropis, D. foeniculina, and an undescribed Diaporthe sp., as the most frequent isolated pathogens, followed by Eutypella citricola, and Phaeoacremonium iranianum. The isolates were identified through multi-locus phylogenetic analyses using nucleotide sequences of the rDNA internal transcribed spacer, ß-tubulin, and translation elongation factor 1-alpha genes. To fulfill Koch's postulates, mycelium plugs of the various fungal species identified were introduced in 2-year-old branches of mature persimmon trees after making wounds using a corkborer in the field conditions. Results showed that, Diaporthe spp., E. citricola, and P. iranianum are the main causal agents of branch canker and shoot dieback of persimmon trees in California, with Diaporthe spp. being the most frequently isolated pathogen.

Identification and Pathogenicity of Fusarium Species Associated with Young Vine Decline in California.

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.

Reassessing the Etiology of Aspergillus Vine Canker and Summer Bunch Rot of Table Grapes in California.

Fungal taxonomy is in constant flux, and the advent of reliable DNA barcodes has enabled the enhancement of plant pathogen identification accuracy. In California, Aspergillus vine canker (AVC) and summer bunch rot (SBR) are economically important diseases that affect the wood and fruit of grapevines, respectively, and their causal agents are primarily species of black aspergilli (Aspergillus section Nigri). During the last decade, the taxonomy of this fungal group has been rearranged several times using morphological, physiological, and genetic analyses, which resulted in the incorporation of multiple cryptic species that are difficult to distinguish. Therefore, in this study, we aimed to reassess the etiology of AVC and SBR using a combination of morphological observations with phylogenetic reconstructions based on nucleotide sequences of the calmodulin (CaM) gene. Results revealed that the isolates causing AVC from recent isolations corresponded to A. tubingensis, whereas the isolates obtained from initial surveys when the disease was discovered were confirmed as A. niger and A. carbonarius. Similarly, the isolates obtained from table grapes with SBR symptoms and from spore traps placed in those vineyards were identified primarily as A. tubingensis, followed by A. niger and A. carbonarius. Notably, the A. niger isolates formed a subclade with strains previously known as A. welwitschiae, which is a species that was recently synonymized with A. niger. Overall, the most prevalent species was A. tubingensis, which was associated with both AVC and SBR, and representative isolates recovered from AVC-symptomatic wood, berries SBR symptoms, and spore traps were equally pathogenic in healthy wood and berries of 'Red Globe' grapevines. This study also constitutes the first report of A. tubingensis causing AVC and SBR of grapes in California and in the United States.

First report of Diplodia bulgarica causing black canker on 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.

First report of Phyllactinia chubutiana causing powdery mildew on Goji berry plants (Lycium barbarum and L. chinense) in the United States.

Goji berries, both Lycium barbarum, and L. chinense, are native to Asia and have been highly valued for food and medicinal purposes for more than 2,000 years (Wetters et al. 2018). These species are difficult to distinguish due to the extensive cultivar development of the former and the plasticity of the latter's phenotypes. During the summers (from July to September) of 2021 and 2022, powdery mildew was observed in Goji berry plants (L. barbarum and L. chinense) in both community and residential gardens, in Yolo Co., California. Disease severity varied between 30 and 100% of infected leaves per plant. Host identity was confirmed by phylogenetic analysis using sequences of the psbA-trnH intergenic region (Wetters et al. 2018). Powdery mildew was characterized by the presence of white fungal colonies on both sides of the leaves and the fruit sepals. Colorless adhesive tape mounts of the fungal structures were examined in drops of 3% KOH. Epidermal strips of infected leaves were peeled off for analysis of the mycelia. Hyphae were both external and internal, hyaline, septate, branched, smooth, and 2.5 to 5.8 (4.3) µm wide (n = 50). Appressoria were nipple-shaped to irregularly branched and solitary or opposite in pairs. Conidiophores were hyaline, erect, and simple. Foot cells were cylindrical, straight, 13.1 to 48.9 (29.8) × 5.0 to 8.2 (6.8) µm (n = 20), followed by 0 to 2 cells. Conidia lacked fibrosin bodies, were borne singly, unicellular, hyaline, and ellipsoid when young. Mature conidia were either cylindrical or slightly centrally constricted to dumb-bell-like, and 36.2 to 51.8 (44.9) × 15.1 to 22.0 (18.9) µm (n = 50), with conspicuous subterminal protuberances. Germ tubes were subterminal, either short with multilobate apex or moderately long with a simple end. Chasmothecia were not observed. Morphologically the fungus matched the description of Phyllactinia chubutiana Havryl., S. Takam. & U. Braun (Braun and Cook, 2012). The pathogen identity was further confirmed by amplifying and sequencing the rDNA internal transcribed spacer (ITS) and the 28S rDNA gene using the primer pairs ITS1/ITS4 (White et al. 1990) and PM3/TW14 (Takamatsu and Kano 2001, Mori et al. 2000). The resulting sequences (GenBank OP434568 to OP434569; and OP410969 to OP410970) were compared with the NCBI database using BLAST, showing 99% similarity to the ex-type isolate of P. chubutiana (BCRU 4634, GenBank AB243690). Maximum parsimony phylogenetic analysis clustered our isolates with reference sequences of P. chubutiana from various hosts deposited in GenBank. Pathogenicity was confirmed by inoculating two two-year-old L. barbarum potted plants. Four leaves per plant were surface disinfected (75% ethanol, 30 s) before gently rubbing powdery mildew infected leaves onto healthy leaves. Healthy leaves were used for mock inoculations. All plants were maintained in a growth chamber at 22°C and 80% relative humidity (RH) for five days and then 60% RH thereafter. Inoculated leaves developed powdery mildew symptoms after 28 days, and P. chubutiana colonies were confirmed by morphology, hence fulfilling Koch's postulates. Control leaves remained symptomless. Phyllactinia chubutiana (= Oidium insolitum, Ovulariopsis insolita) was first described on L. chilense in Argentina (Braun et al. 2000, Havrylenko et al. 2006), and later reported on L. chinense in China (Wang Yan et al. 2016). To our knowledge, this is the first report of P. chubutiana causing powdery mildew on L. barbarum and L. chinense in the United States, which provides crucial information for developing effective strategies to monitor and control this newly described disease.

First Report of Neofusicoccum mediterraneum and Neofusicoccum parvum Causing Pine Ghost Canker on Pinus spp. in Southern California.

Pinus eldarica, P. halepensis and P. radiata are important conifer species native to Mediterranean regions that are cultivated in the southwestern United States for landscaping (Phillips and Gladfelter, 1991; Chambel et al., 2013). Among them, Monterey pine (P. radiata) is native to restricted areas of California and Mexico, but it is extensively grown for timber production in other countries, especially in the Southern Hemisphere (Rogers, 2004). From 2018 to 2022, severe dieback and cankers have been detected on more than 30 mature pines of the three species within a 40-ha urban forest in Orange County, Southern California. Symptoms initiate on the lower portion of the canopy and advance into the crown, leading to quick dieback and, in some cases, to tree death. Cross sections of affected branches revealed wedged cankers with irregular, indistinct margins, and cryptic discoloration (i.e., "ghost cankers"). Pycnidia were observed on the surface of each bark scale of branches with advanced infections. Two morphotypes of Botryosphaeriaceae colonies (n = 34 isolates) were recovered consistently from more than 90% of the symptomatic pines. Two isolates per morphotype were grown on pistachio leaf agar (Chen et al., 2014) for 14 days to induce pycnidia formation. Conidia (n = 50) were hyaline, thin-walled and fusoid to ellipsoidal in shape, ranging from 16.1 to 27.9 (22.6) × 5.4 to 8.2 (6.8) µm for the first morphotype and 11.5 to 20.4 (16.3) × 4.8 to 8.6 (6.3) µm for the second morphotype. The rDNA internal transcribed spacer (ITS), beta-tubulin (tub2), and translation elongation factor 1-alpha (tef1-α) partial gene regions were amplified and sequenced using the primers ITS5/ITS4 (White et al., 1990), Bt2a/Bt2b (Glass and Donaldson, 1995), and EF1-728F/EF1-986R (Carbone and Kohn, 1999), respectively. A multi-locus phylogenetic analysis revealed that isolates UCD9433 and UCD10439 clustered with the ex-type strain of Neofusicoccum mediterraneum (CBS:113083), and isolates UCD9161 and UCD9434 grouped with N. parvum (CMW:9081). Sequences were submitted to GenBank (nos. OP535391 to OP535394 for ITS, OP561946 to OP561949 for tef1-α, and OP561950 to OP561953 for tub2). Pathogenicity tests were performed with above-mentioned isolates on 20-mm-diameter healthy branches of mature Monterey pines (n = 10, 14 years old) located in a research field at UC Davis. Isolates were grown for 7 days on potato dextrose agar and inoculated in the internode area by removing a 5-mm-diameter disk of the bark with a sterile cork borer and placing a 5-mm-diameter mycelial plug. Controls were mock-inoculated with sterile agar plugs, and the experiment was performed twice. After three months, inoculations resulted in vascular lesions that ranged from 20.6 to 49.7 (32.7) mm with N. mediterraneum and from 13.5 to 71.0 (33.6) mm with N. parvum, and the same pathogens were reisolated (70 to 100% recovery). Controls remained symptomless and no botryosphaeriaceous colonies were recovered. Both N. mediterraneum and N. parvum are polyphagous pathogens associated with multiple woody plant hosts (Phillips et al., 2013). Previously, only N. parvum has been associated with pine cankers in Iran, however, the pine species was not indicated (Abdollahzadeh et al., 2013). The detection of these pathogens in urban forests raises concerns of potential spillover events to other forest and agricultural hosts in Southern California. To our knowledge, this is the first report of N. mediterraneum and N. parvum causing Pine Ghost Canker on P. eldarica, P. halepensis and P. radiata.

Evaluation of the Antifungal Activity of Endophytic and Rhizospheric Bacteria against Grapevine Trunk Pathogens.

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.

First Record of Colletotrichum anthrisci Causing Anthracnose on Avocado Fruits in Chile.

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.

First Report of Fusarium annulatum Associated with Young Vine Decline in California.

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.