Identification and Pathogenicity of Fungal Species 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 multilocus 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 inserted in 2-year-old branches of mature persimmon trees after making wounds using a corkborer in 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.

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 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.

Dothiorella sarmentorum Causing Branch Dieback of English Walnut in Maule Region, Chile.

English walnut (Juglans regia), cv. Chandler is the most cultivated tree nut in Chile, with 43,734 ha. In Maule Region, central Chile, English walnut plantings have expanded over an additional 7,000 ha in the last five years. During a routine orchard survey in 2019, branch and twig dieback symptoms were observed in two commercial orchards located in San Rafael (10 years old) and Longaví (12 years old) in the Maule Region, with an incidence of 45% to 65% of affected trees, respectively. Symptomatic branch samples (n = 15) were collected from the two commercial orchards and transported to the laboratory in a cooler and then surface sterilized in 96% ethanol for 3 s and briefly flamed. Cross-section of symptomatic branches revealed brown to dark-brown wedge-shaped wood cankers. Small (5 mm) pieces of wood from the edge of cankered tissues were placed on Potato Dextrose Agar (PDA, 2%) amended with 0.005% tetracycline, 0.01% streptomycin, and 0.1% Igepal CO-630 (PDAm) (Díaz and Latorre 2014) and incubated at 25°C for five days in the dark. Pure cultures were obtained by transferring a hyphal tip from growing colonies to fresh PDA media. Each fungal isolate was recovered from a single diseased branch (47%). Seven isolates (Dsar-1 to Dsar-7) developed dark to olive-brown fast-growing colonies with scarce aerial mycelium after seven days at 25°C on PDA. These isolates showed a dark-olive color on the reverse side of Petri dishes and developed abundant, aggregated, and dark-brown pycnidia after 15 days at 25°C. Conidia were hyaline and aseptate, dark brown, 1-septate, with a brown wall, ovoid with a broadly rounded apex and truncated base, (17.5-) 19.5 ±1.2 (-22.0) x (7.6-) 8.9 ± 0.6 (-10.1) µm (n = 30). These isolates were tentatively identified morphologically as Dothiorella sp. (Phillips et al. 2005). Molecular identification was performed using ITS1/ITS4 and EF1-728F/EF1-986R primers (White et al. 1990; Dissanayake et al. 2015) of the internal transcribed spacer (ITS1-5.8S-ITS2) region and part of the translation elongation factor (EF1-) genes, respectively. A MegaBlast search in GenBank showed a 100% similarity to isolate CBS 115038, the ex-type of Dothiorella sarmentorum. The sequences were added to GenBank (OM161950 to OM161956 for ITS; OM177188 to OM177194 for EF1-). Pathogenicity of two isolates (Dsar-2 and Dsar-7) was tested in the orchard on freshly made pruning wounds on attached branches of 2-year-old-pruned English walnut trees cv. Chandler. A second pathogenicity test was done on freshly made pruning wounds in 1-year-old rooted cuttings (n=15) (40 cm of long) of English walnut cv. Chandler. Each pruning wound was inoculated with 40 µL conidial suspension (105 conidia/mL). Sterile distilled water was used as a control treatment. Both pathogenicity tests were repeated once. After seven months for attached branches and four months for rooted plants, necrotic streaks with a mean length of 81.3 and 44.5 mm were observed below the inoculated pruning wounds, respectively. No necrotic streaks were observed in any of the control wounds. Dothiorella sarmentorum was 100% reisolated from symptomatic tissues of inoculated branches and molecularly identified (EF1-), thus fulfilling Koch's postulates. Recently, D. sarmentorum has been reported causing English walnut dieback in Spain (López-Moral et al. 2020). To our knowledge, this is the first report of D. sarmentorum causing branch dieback of English walnut in Chile. Further studies are needed to know the impact and extent of canker and branch dieback of walnut in commercial orchards in the Maule Region, central Chile.

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.

Severe Outbreak of Dry Core Rot in Apple Fruits cv. Fuji Caused by Kalmusia variispora During Pre-harvest in Maule Region, Chile.

Apple (Malus × domestica) is an important fruit crop in Chile, with a cultivated area of 32,313 ha concentrated (63%) in Maule Region (35°25' S). Unusual core rot on 'Fuji' apples was observed at harvest in a commercial orchard in Curicó, Maule Region, with an incidence ranged between 22 to 35% in 2018 and 2019. Previously, in 2017, an incidence of 30% was estimated on 'Fuji' fruits destined to the Asian market. Internal decay symptoms consisted of dry, corky light to dark-brown tissue, within the seed locules initially. In moderate to severe cases, the necrotic lesion progresses deeper into the mesoderm. External symptoms were quite subtle, and typically, the disease goes unnoticed. However, infected fruit ripen earlier. Small pieces (2-3 mm) from the internal lesion margin of symptomatic apples (n = 50) were placed on Potato Dextrose Agar (PDA) (2%) and incubated at 20°C for 10 days. Pure cultures (n = 41) were obtained and transferred to Malt Extract Agar (MEA) (2%). Colonies on MEA produced an even to slight undulating buff margin with white woolly aerial mycelium, and immerse ochreous in the center, changing gray to olivaceous aerial mycelium with age. On the underside, colonies were umber and buff in the center and margin, respectively. After 10 days, numerous densely aggregate dark-brown mature pycnidia were observed. Aseptate conidia were subglobose to cylindrical, straight, and sometimes curved with rounded at both ends, that was initially hyaline to pale olive, thin, smooth wall with mean dimensions of (2.9-) 3.4 (-4.4) x (1.5-) 1.8 (2.2) µm (n=50). Based on morphology, the fungus was identified as Kalmusia variispora (Verkley et al. 2014). The internal transcribed space (ITS), portion of ß-tubulin (TUB), and large subunits of the nuclear ribosomal RNA (LSU) loci were used for molecular identification, using primers ITS4/ITS5, Bt2a/Bt2b, and LR0R/LR5 (Ariyawansa et al., 2014). BLAST searches indicated 100% identity with K. variispora (ex-type CBS 121517). The maximum parsimony phylogenetic analysis placed Chilean isolates in the K. variispora clade. Sequences were deposited in GenBank (OL711706 to OL711709, OL739499 to OL739502 and OL711710 to OL711713 for ITS, TUB and LSU, respectively). Pathogenicity tests were conducted using four K. variispora isolates. 'Fuji' apples (n = 20) were surface disinfested (75% ethanol, 30 s) and then wounded and inoculated with conidial suspension (50 L of 106 conidia/mL) deposited in the middle and into the core region using a sterile fine-tipped micropipette. Additionally, 20 one-year dormant rooted cuttings 'Fuji' and 'Cripps Pink' were pruned and immediately inoculated on the pruning wound. An equal number of apples and rooted cuttings treated with sterile water were used as controls. The experiments were repeated once. All inoculated fruits developed lateral lesions (22 to 37 mm) and dry core rot (18 to 36 mm) symptoms identical to those described in the original outbreak, after 20 days at 20°C in a commercial packing box. The inoculated cuttings produced canker lesions of 10 to 21 mm in length, and dieback symptoms were observed after 3 months. No symptoms were observed on the negative controls. Koch's postulates were fulfilled by 100% reisolating K. variispora. Previously, Alternaria spp. have been reported as the primary pathogen associated with moldy core and dry core rot of apples worldwide (McLeod et al., 2014) and in Chile (Elfar et al., 2018). However, Kalmusia spp. have been associated with dry core rot in apples (McLeod et al., 2014) and have been isolated from canker symptoms on apples in Chile (Díaz et al. 2021). To our knowledge, this is the first report of a severe outbreak of K. variispora causing dry core rot in apples in Chile and worldwide.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Probe-Based Multiplex Real-Time PCR as a Diagnostic Tool to Distinguish Distinct Fungal Symbionts Associated With Euwallacea kuroshio and Euwallacea whitfordiodendrus in California.

California has been invaded by two distinct Euwallacea spp. that vector unique plant pathogenic symbiotic fungi on multiple hosts and cause Fusarium dieback. The objective of this study was to develop multiplex real-time quantitative PCR assays using hydrolysis probes targeting the ß-tubulin gene to detect, distinguish, and quantify fungi associated with the polyphagous shot hole borer (PSHB; Euwallacea whitfordiodendrus, Fusarium euwallaceae, Graphium euwallaceae, and Paracremonium pembeum) as well as the Kuroshio shot hole borer (KSHB; Euwallacea kuroshio, Fusarium kuroshium, and Graphium kuroshium) from various sample types. Absolute quantification reaction efficiencies ranged from 88.2 to 104.3%, with a coefficient of determination >0.992 and a limit of detection of 100 copies µl-1 for all targets across both assays. Qualitative detection using the real-time assays on artificially inoculated avocado shoot extracts showed more sensitivity compared with conventional fungal isolation from wood. All symbiotic fungi, except P. pembeum, from PSHB and KSHB female heads were detectable and quantified. Field samples from symptomatic Platanus racemosa, Populus spp., and Salix spp. across 17 of 26 city parks were positively identified as PSHB and KSHB through detection of their symbiotic fungi, and both were found occurring together on five trees from three different park locations. The molecular assays presented here can be utilized to accurately identify fungi associated with these invasive pests in California.

Members of the Euwallacea fornicatus species complex exhibit promiscuous mutualism with ambrosia fungi in Taiwan.

Carrillo, J.D., Rugman-Jones, PF., Husein, D., Stajich, J.E., Kasson, M.T., Carrillo, D., Stouthamer, R., and Eskalen, A. 2019. Members of the Euwallacea fornicatus species complex exhibit promiscuous mutualism with ambrosia fungi in Taiwan. A number of ambrosia beetles have come to prominence in recent years because of the damage they inflict on a variety of trees within invaded habitats across the globe. Ambrosia beetles rely on symbiotic microorganisms, mainly fungi, as a dedicated food source and carry those microorganisms around with them within specialized organs termed mycangia. Investigation of members of the Euwallacea fornicatus species complex and their fungal symbionts in Taiwan revealed promiscuous symbioses with ambrosial Fusaria clade (AFC) members, Graphium spp., and Paracremonium spp. based on co-phylogenetic analyses. For AFC members, a novel diagnostic PCR assay targeting mating type genes MAT1-1-1 and MAT1-2-1 was developed and validated by amplicon size and sequencing. Mating type screening of AFC members revealed the isolates screened are all heterothallic (self-sterile), with both MAT types represented and recovered from fungi vectored by E. fornicatus (tea shot hole borer), E. kuroshio (Kuroshio shot hole borer), and E. whitfordiodendrus (polyphagous shot hole borer) in Taiwan. Members of the Euwallacea fornicatus species complex and the variety of ambrosia fungi they utilize further confirms that their relationship with these fungi are more likely promiscuous in native areas, as opposed to strictly obligate to a specific combination of fungi as observed in invaded areas.

Identification, Pathogenicity, and Spore Trapping of Colletotrichum karstii Associated with Twig and Shoot Dieback in California.

Colletotrichum Corda, 1831 species are well-documented pathogens of citrus that are associated with leaf and fruit anthracnose diseases. However, their role in twig and shoot dieback diseases of citrus has recently become more prominent. Recent surveys of orchards in the Central Valley of California have revealed C. gloeosporioides and a previously undocumented species, C. karstii, to be associated with twig and shoot dieback. Pathogenicity tests using clementine (cv. 4B) indicated that both C. karstii and C. gloeosporioides are capable of producing lesions following inoculation of citrus stems. Pathogenicity tests also revealed C. karstii to be the most aggressive fungal species producing the longest lesions after 15 months. The majority of spores trapped during this study were trapped during or closely following a precipitation event with the majority of spores being trapped from January through May. These findings confirm C. karstii as a new pathogen of citrus in California.

Antifungal activity of avocado rhizobacteria against Fusarium euwallaceae and Graphium spp., associated with Euwallacea spp. nr. fornicatus, and Phytophthora cinnamomi.

Plant rhizobacteria have been successfully used as biocontrol agents against fungal phytopathogens. However, their potential to control two important avocado diseases, namely Fusarium dieback (FD) and Phytophthora root rot (PRR), has been poorly studied. FD is an emerging disease triggered by fungi associated with two ambrosia beetle species (Euwallacea fornicatus species complex), while PRR is caused by Phytophthora cinnamomi, a soil-borne oomycete. In the present work, the antifungal activity of bacteria isolated from avocado rhizosphere was tested in dual culture assays against Fusarium euwallaceae, Graphium euwallaceae and Graphium sp., causal agents of FD, and against P. cinnamomi. In 2015, rhizosphere soil samples of FD infested and non-infested avocado trees were collected from a commercial avocado orchard in Escondido, California. In an initial screening, 72 of the 168 assessed bacterial isolates reduced mycelial growth of F. euwallaceae by up to 46%. Eight bacterial isolates showing inhibition percentages larger than 40% were then selected for further antagonism assays against the other fungal pathogens. Five bacterial isolates, determined by 16S rDNA sequencing to belong to the Bacillus subtilis/Bacillus amyloliquefaciens species complex, successfully inhibited the mycelial growth of both Graphium species by up to 30%. The same isolates and an additional isolate identified as Bacillus mycoides, inhibited the growth of P. cinnamomi by up to 25%. This is the first report of avocado rhizobacteria with antifungal activity against pathogens responsible for FD and PRR in avocado.

Two Novel Fungal Symbionts Fusarium kuroshium sp. nov. and Graphium kuroshium sp. nov. of Kuroshio Shot Hole Borer (Euwallacea sp. nr. fornicatus) Cause Fusarium Dieback on Woody Host Species in California.

Shot hole borer (SHB)-Fusarium dieback (FD) is a new pest-disease complex affecting numerous tree species in California and is vectored by two distinct, but related ambrosia beetles (Euwallacea sp. nr. fornicatus) called polyphagous shot hole borer (PSHB) and Kuroshio shot hole borer (KSHB). These pest-disease complexes cause branch dieback and tree mortality on numerous wildland and landscape tree species, as well as agricultural tree species, primarily avocado. The recent discovery of KSHB in California initiated an investigation of fungal symbionts associated with the KSHB vector. Ten isolates of Fusarium sp. and Graphium sp., respectively, were recovered from the mycangia of adult KSHB females captured in three different locations within San Diego County and compared with the known symbiotic fungi of PSHB. Multigene phylogenetic analyses of the internal transcribed spacer region (ITS), translation elongation factor-1 alpha (TEF1-α), and RNA polymerase II subunit (RPB1, RPB2) regions as well as morphological comparisons revealed that two novel fungal associates Fusarium kuroshium sp. nov. and Graphium kuroshium sp. nov. obtained from KSHB were related to, but distinct from the fungal symbionts F. euwallaceae and G. euwallaceae associated with PSHB in California. Pathogenicity tests on healthy, young avocado plants revealed F. kuroshium and G. kuroshium to be pathogenic. Lesion lengths from inoculation of F. kuroshium were found to be significantly shorter compared with those caused by F. euwallaceae, while no difference in symptom severity was detected between Graphium spp. associated with KSHB and PSHB. These findings highlight the pest disease complexes of KSHB-FD and PSHB-FD as distinct, but collective threats adversely impacting woody hosts throughout California.

Chemical Management of Invasive Shot Hole Borer and Fusarium Dieback in California Sycamore (Platanus racemosa) in Southern California.

Fusarium dieback (FD) is a new vascular disease of hardwood trees caused by Fusarium spp. and other associated fungal species which are vectored by two recently introduced and highly invasive species of ambrosia beetle (Euwallacea spp. nr. fornicatus). One of these ambrosia beetles is known as the polyphagous shot hole borer (PSHB) and the other as the Kuroshio shot hole borer (KSHB). Together with the fungi that they vector, this pest-disease complex is known as the shot hole borer-Fusarium dieback (SHB-FD) complex. Mitigation of this pest-disease complex currently relies on tree removal; however, this practice is expensive and impractical given the wide host range and rapid advancement of the beetles throughout hardwoods in southern California. This study reports on the assessment of various pesticides for use in the management of SHB-FD. In vitro screening of 13 fungicides revealed that pyraclostrobin, trifloxystrobin, and azoxystrobin generally have lower effective concentration that reduces 50% of mycelial growth (EC50) values across all fungal symbionts of PSHB and KSHB; metconazole was found to have lower EC50 values for Fusarium spp. and Paracremonium pembeum. Triadimefon and fluxapyroxad were not capable of inhibiting any fungal symbiont at the concentrations tested. A 1-year field study showed that two insecticides, emamectin benzoate alone and in combination with propiconazole, and bifenthrin, could significantly reduce SHB attacks. Two injected fungicides (tebuconazole and a combination of carbendazim and debacarb) and one spray fungicide (metconazole) could also significantly reduce SHB attacks. Bioassays designed to assess fungicide retention 1 year postapplication revealed that six of the seven fungicides exhibited some level of inhibition in vitro and all thiabendazole-treated trees sampled exhibiting inhibition. This study has identified several pesticides which can be implemented as part of an integrated pest management strategy to reduce SHB infestation in low to moderately infested landscape California sycamore trees and potentially other landscape trees currently affected by SHB-FD.

PCR Multiplexes Discriminate Fusarium Symbionts of Invasive Euwallacea Ambrosia Beetles that Inflict Damage on Numerous Tree Species Throughout the United States.

Asian Euwallacea ambrosia beetles vector Fusarium mutualists. The ambrosial fusaria are all members of the ambrosia Fusarium clade (AFC) within the Fusarium solani species complex (FSSC). Several Euwallacea-Fusarium mutualists have been introduced into nonnative regions and have caused varying degrees of damage to orchard, landscape, and forest trees. Knowledge of symbiont fidelity is limited by current identification methods, which typically requires analysis of DNA sequence data from beetles and the symbionts cultured from their oral mycangia. Here, polymerase chain reaction (PCR)-based diagnostic tools were developed to identify the six Fusarium symbionts of exotic Euwallacea spp. currently known within the United States. Whole-genome sequences were generated for representatives of six AFC species plus F. ambrosium and aligned to the annotated genome of F. euwallaceae. Taxon-specific primer-annealing sites were identified that rapidly distinguish the AFC species currently within the United States. PCR specificity, reliability, and sensitivity were validated using a panel of 72 Fusarium isolates, including 47 reference cultures. Culture-independent multiplex assays accurately identified two AFC fusaria using DNA isolated from heads of their respective beetle partners. The PCR assays were used to show that Euwallacea validus is exclusively associated with AF-4 throughout its sampled range within eastern North America. The rapid assay supports federal and state agency efforts to monitor spread of these invasive pests and mitigate further introductions.

Identification, pathogenicity and abundance of Paracremonium pembeum sp. nov. and Graphium euwallaceae sp. nov.--two newly discovered mycangial associates of the polyphagous shot hole borer (Euwallacea sp.) in California.

Fusarium euwallaceae is a well-characterized fungal symbiont of the exotic ambrosia beetle Euwallacea sp. (polyphagous shot hole borer [PSHB]), together inciting Fusarium dieback on many host plants in Israel and California. Recent discoveries of additional fungal symbionts within ambrosia beetle mycangia suggest these fungi occur as communities. Colony-forming units of Graphium euwallaceae sp. nov. and Paracremonium pembeum sp. nov., two novel fungal associates of PSHB from California, grew from 36 macerated female heads and 36 gallery walls collected from Platanus racemosa, Acer negundo, Persea americana and Ricinus communis. Fungi were identified based on micromorphology and phylogenetic analyses of the combined internal transcribed spacer region (nuc rDNA ITS1-5.8S-ITS2 [ITS barcode]), elongation factor (EF 1-α), small subunit (18S rDNA) sequences for Graphium spp., ITS, EF 1-α, calmodulin (cmdA), large subunit of the ATP citrate lyase (acl1), ß-tubulin (tub2), RNA polymerase II second largest subunit (rpb2) and large subunit (28S rDNA) sequences for Paracremonium spp. Other Graphium spp. recovered from PSHB in Vietnam, Euwallacea fornicatus in Thailand, E. validus in Pennsylvania and Paracremonium sp. recovered from PSHB in Vietnam were identified. F. euwallaceae was recovered from mycangia at higher frequencies and abundances in all hosts except R. communis, in which those of F. euwallaceae and P. pembeum were equal. P. pembeum was relatively more abundant within gallery walls of A. negundo and R. communis. In all hosts combined F. euwallaceae was relatively more abundant within PSHB heads than gallery walls. All three fungi grew at different rates and colonized inoculated excised stems of P. americana and A. negundo. P. pembeum produced longer lesions than F. euwallaceae and G. euwallaceae on inoculated avocado shoots. Results indicate PSHB is associated with a dynamic assemblage of mycangial fungal associates that pose additional risk to native and nonnative hosts in California.

Identification, Distribution, and Pathogenicity of Diatrypaceae and Botryosphaeriaceae Associated with Citrus Branch Canker in the Southern California Desert.

Several members of the families Botryosphaeriaceae and Diatrypaceae are known as canker and dieback pathogens of a number of woody hosts. Because desert citrus production in California can occur in proximity to table grape production, it was suspected that fungi associated with grapevine cankers might also be associated with citrus branch canker and dieback decline. To determine the fungi associated with branch canker and dieback disease of citrus in the southern California desert regions, surveys were conducted from 2011 to 2013 in the major citrus-growing regions of Riverside, Imperial, and San Diego Counties. Cankered tissues were collected from branches showing symptoms typical of branch canker and dieback. Various fungal species were recovered from necrotic tissues and species were identified morphologically and by phylogenetic comparison of partial sequences of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2), ß-tubulin gene, and elongation factor 1-α genes with those of other species in GenBank. Four fungi, including Neoscytalidium hyalinum, Eutypella citricola, E. microtheca, and an unnamed Eutypella sp., were associated with branch canker. N. hyalinum was the most frequently recovered fungus from symptomatic tissues (31%) followed by E. citricola (10%), E. microtheca (4%), and the Eutypella sp. (2%). In pathogenicity tests, all fungi caused lesions when inoculated on 'Lisbon' lemon (citrus) branches. Lesions caused by the Eutypella sp. were significantly longer than those of the other Eutypella spp.; however, they did not differ significantly from those produced by N. hyalinum. The most-parsimonious unrooted trees based on the combined data of ITS and partial ß-tubulin gene region sequences showed three distinct clades of Eutypella spp. (E. citricola, E. microtheca, and an unidentified Eutypella sp.). Similarly, ITS and partial translation elongation factor 1-α gene region sequences differentiated two species of Neoscytalidium, N. hyalinum and N. novaehollandiae. Identifying the diversity, distribution, and occurrence of these fungal pathogens is useful for the management of citrus branch canker and dieback disease in the desert citrus-growing regions of California.