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Grapevine trunk diseases, caused by many different fungal pathogens, are among the most economically important diseases affecting the grapevine industry worldwide. Pruning wounds are the main point of entry for these fungal pathogens and, thus, disease control is focused on preventative pruning wound protection by chemical products or biological control agents (BCAs). In this study, we evaluated a broad variety of chemical products and BCAs already registered or at the experimental stage in greenhouse and in field trials for the protection of table- and wine-grape vines against infection by Eutypa lata and Neofusicoccum parvum, major pathogens responsible for Eutypa and Botryosphaeria dieback, respectively. Our study showed that Trichoderma asperellum and T. gamsii consistently provided pruning wound protection in greenhouse and field trials, with a mean percent disease control (MPDC) of 88 and 100% for E. lata and N. parvum, respectively, when compared with the water-treated inoculated positive control (P < 0.05). The chemical protectants thiophanate-methyl + myclobutanil, fluopyram, and trifloxystrobin were also able to effectively protect wounds, with a MPDC of up to 86% when compared with the water-treated inoculated positive control (P < 0.05). When biological treatments were evaluated for recovery from treated canes at the end of the growing season, Trichoderma-based treatments had a rate of recovery between 0 and 100%, Aureobasidium-based treatments had a recovery rate between 25 and 100%, and Bacillus-based treatments had a recovery rate between 0 and 25%.

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.

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.

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.

International audience; Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. Previously (Geiser et al. 2013; Phytopathology 103:400-408. 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 by one research group (Lombard et al. 2015 Studies in Mycology 80: 189-245) who proposed dividing Fusarium into seven genera, including the FSSC as the genus Neocosmospora, with subsequent justification based on claims that the Geiser et al. (2013) concept of Fusarium is polyphyletic (Sandoval-Denis et al. 2018; Persoonia 41:109-129). 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 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 recently described as Neocosmospora were recombined in 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.

The specificity of ambrosia beetle-fungus relationships is unknown for the vast majority of ambrosia symbioses, yet has important implications for the success of these beetles and fungi in their surrounding environments. The polyphagous shot hole borer and the Kuroshio shot hole borer (Euwallacea spp.) are invasive ambrosia beetles that exist sympatrically in California, and together with their fungi cause Fusarium Dieback in a variety of hosts. The potential for the two beetle species to exchange symbionts is unknown, but has important ecological consequences. Here we perform symbiont switching experiments, to determine if the polyphagous and Kuroshio shot hole borers can survive and reproduce on each other’s symbiotic fungi in their invasive range in California. Aposymbiotic foundresses were assessed on their ability to reproduce on each of the beetles’ primary (Fusarium spp.) and auxiliary (Graphium spp., Paracremonium pembeum) fungal symbionts, grown on sawdust-based artificial media. Offspring were collected to confirm which fungi were present in the different sexes and life stages. Additionally, the ability of aposymbiotic and non-aposymbiotic foundresses to survive and reproduce on non-ambrosial Fusarium species was tested. We found that reproduction is maximized on the beetles’ primary Fusarium symbionts, regardless of whether a beetle was paired with its own Fusarium symbiont or its congener’s, indicating that there is potential for symbiont switching in natural populations of these beetles. The addition of auxiliary fungi to treatments containing Fusarium spp. had no effect on overall offspring production, and females that were fed solely on auxiliary fungi produced significantly fewer offspring. Females that were fed non-ambrosial Fusarium species produced significantly fewer offspring than when reared on symbiotic Fusarium, and this was especially pronounced in aposymbiotic females. From our experiments, the putative roles of the primary versus auxiliary fungal symbionts, as well as the implications of these roles for beetle reproduction and development in their surrounding environment, are discussed.

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 β-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.

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.

The rhizosphere microbiome is critical for plant growth and protection against plant pathogens. However, rhizosphere microbial communities are likely to be restructured upon plant infection by fungal pathogens. Our objective was to determine the shifts in rhizosphere bacterial communities of avocado trees (Persea americana Mill.) after Fusarium dieback (FD), a disease triggered by the symbiotic fungi of invasive ambrosia beetles (Euwallacea kuroshio and Euwallacea sp. nr. fornicatus), using 16S rDNA gene amplicon sequencing and a culture-dependent approach. Rhizosphere soil samples were collected from five asymptomatic and five FD-symptomatic avocado trees in a Californian orchard. Sequence analysis showed that diversity metrics of the rhizosphere bacterial communities associated with asymptomatic avocado trees were larger than those of communities from FD-symptomatic trees. Moreover, FD produced significant shifts in rhizobacterial community structure, which were mainly caused by rare OTUs. Bacterial taxa such as Armatimonadetes, Sporocytophaga or Cellvibrio were exclusively associated with the rhizosphere of asymptomatic trees and may act as an insurance mechanism against fungal invasions. Conversely, genera such as Myxococcus or Lysobacter, which have been described as effective biocontrol agents against Fusarium oxysporum, Colletotrichum gloeosporioides or Rhizoctonia spp., among other phytopathogens, were only found in the rhizosphere of FD-symptomatic trees. The culturable bacterial communities in the rhizosphere of both FD-symptomatic and asymptomatic trees were dominated by isolates from the Bacillus and Pseudomonas genera, indicating that potential biocontrol agents against FD may be isolated from healthy and diseased avocado trees. Altogether, our results showed that FD elicited shifts in the avocado rhizosphere microbiome, which could potentially affect soil microbial processes, and provide a basis for the selection of biocontrol agents that could be used for FD prevention., This research was funded by a 2015 UC MEXUS −CONACYT collaborative research grant and by a CONACYT postgraduate fellowship awarded to the first author.

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

Specimens of an ambrosia beetle, Xyleborus monographus (Fabricius), were found infesting oak trees in California. This is the first record of this species established in North America. Based on collection information, this species most likely has been established in the Napa County area for several years. A modified key to Xyleborus in North America, and diagnosis of the species is provided.

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 EC50values 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.

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.

The cryptic species that make up the Euwallacea fornicatus species complex can be readily distinguished via their DNA sequences. Until recently, it was believed that the Hawaiian Islands had been invaded by only one of these cryptic species, E. perbrevis (tea shot hole borer; TSHB). However, following the 2016 deposition of a DNA sequence in the public repository GenBank, it became evident that another species, E. fornicatus (polyphagous shot hole borer; PSHB), had been detected in macadamia orchards on Hawaiʻi Island (the Big Island). We surveyed the two most-populous islands of Hawaiʻi, Big Island and Oʻahu, and herein confirm that populations of TSHB and PSHB are established on both. Beetles were collected using a variety of techniques in macadamia orchards and natural areas. Individual specimens were identified to species using a high-resolution melt assay, described herein and validated by subsequent sequencing of specimens. It remains unclear how long each species has been present in the state, and while neither is currently recognized as causing serious economic or ecological damage in Hawaiʻi, the similarity of the newly-confirmed PSHB population to other damaging invasive PSHB populations around the world is discussed. Although the invasive PSHB populations in Hawaiʻi and California likely have different geographic origins within the beetle’s native range, they share identical Fusarium and Graphium fungal symbionts, neither of which have been isolated from PSHB in that native range.

California has been invaded by two distinct

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.

Several species of the ambrosia beetle Euwallacea (Coleoptera: Curculionidae: Scolytinae) cultivate Ambrosia Fusarium Clade (AFC) species in their galleries as a source of food. Like all other scolytine beetles in the tribe Xyleborini, Euwallacea are thought to be obligate mutualists with their fungal symbionts. Published diversification-time estimates suggest that the Euwallacea – Fusarium symbiosis evolved once approximately 21 million years ago. Female Euwallacea possess paired oral mycangia within which foundresses transport their Fusarium symbiont vertically from their natal gallery to new woody hosts. During the past two decades, exotic Asian Euwallacea – Fusarium mutualists have been introduced into the United States, Israel and Australia. Because these invasive pests attack and can reproduce on living woody hosts, they pose a serious threat to native forests, urban landscapes and the avocado industry.

The polyphagous shot hole borer (PSHB) and the Kuroshio shot hole borer (KSHB) are newly invasive ambrosia beetles in California. They are vectors of the plant pathogen Fusarium euwallaceae (S. Freeman, Z. Mendel, T. Aoki, K. O’ Donnell), the causal agent of Fusarium dieback in a broad host range that includes commercial avocados, landscape trees, and native tree species in urban and wildland environments. Management of these beetles using contact insecticides is challenging because the beetles spend little time outside their hosts. Trunk injection of systemic insecticides has been proposed as an alternative to contact treatments because insecticides can more effectively target the vascular tissues where the beetles establish their colonies. In this study, several field trials were conducted to evaluate the efficacy of trunk injections of the systemic insecticide emamectin benzoate in avocado trees. The uptake and persistence of emamectin benzoate were determined by quantifying residues in wood cores sampled at various heights within the trees where beetles would likely target. In conjunction with the field trials, a series of bioassays was conducted with a KSHB colony using an avocado-based artificial diet infused with the insecticide. The bioassays showed a dose-dependent effect of emamectin benzoate on the survival and development of the beetle in diet. We derived a tentative working threshold of 300 ng/g insecticide from the bioassay data that we subsequently used as a guide in evaluating the efficacy of the trunk injections. Emamectin benzoate established quickly within trees at the threshold concentration in the areas most vulnerable to attack and colonization by KSHB. Injection of the insecticide in a more dilute form promoted both faster uptake and more rapid establishment of effective concentrations than the undiluted form, thereby providing potential options in how the material is injected based on the levels of infestation of groves.

Esca (black measles) and Petri disease (young esca) are two of the most destructive diseases of grapevines in California and other grape-producing countries. This disease is now known to be caused by multiple species of Phaeoacremonium including P. aleophilum, P. angustius, P. parasiticum, P. rubrigenum, and P. mortoniae. The teleomorph of P. aleophilum was confirmed recently as Togninia minima (Tul. & C. Tul.) Berl. (2), but the teleomorph for the other Phaeoacremonium species are not known. During the summer of 2004, plant specimens were collected from declining ash trees surrounding vineyards as well as from grapevines that were showing typical symptoms of esca including the presence of purple-to-brown spots on berries and necrosis between the veins and on margins of leaves. The specimens were cut into small pieces (15 cm), placed in plastic bags, and thoroughly sprayed with 50 ml of sterile distilled water. The specimens were soaked in water for 10 min at room temperature (25 ± 2°C). Plant materials were then removed from the water and air dried under a laminar flow hood for future examination. Each wash solution was passed through 5.0- and 0.45-μm filters attached to a sterile 35-ml syringe. The 5-μm filters removed large fragments of plant tissues and larger spores, while the 0.45-μm filters trapped spores of Phaeoacremonium spp. The 0.45-μm filters were inverted onto 1.5-ml Eppendorf tubes, and 1 ml of sterile distilled water was passed through to wash out the trapped spores. Aliquots of 200 μl were spread onto potato dextrose agar amended with 0.01% tetracycline hydrochloride (PDA-tet). Colonies of P. mortoniae were identified on the plates after 10 days. Previously wetted wood pieces (grapevine and ash) were examined for fruiting bodies with a stereomicroscope. In this study, perithecia of T. fraxinopennsylvanica were discovered for the first time on diseased grapevines in California. Perithecia were embedded in decayed vascular tissue of ash branches as well as pruning wounds, cordons, and trunks of grapevine cv. Riesling from Mendocino County and cv. Thompson Seedless from Madera County. Perithecia were black, globose to subglobose, and ranged in diameter from 183 to 276 μm. They were embedded and superficial and the lengths of the perithecial necks ranged in size from 200 to 400 μm. The asci were hyaline, clavate, and ranged in size from 16.7 to 23.2 × 4.7 to 5.6 μm. Ascospores were hyaline, ellipsoid to allantoid, and ranged in size from 3.9 to 5.5 × 1.4 to 2.0 μm. When plated onto PDA-tet media, ascospores formed colonies typical of P. mortoniae. Perithecia, asci, and ascospore dimensions as well as phylogenetic analysis of sequences from the internal transcribed spacer region confirmed that the perithecia are in fact T. fraxinopennsylvanica (Hinds) Hausner et al. (1) and that this fungus is the teleomorph of P. mortoniae. Perithecia of T. fraxinopennsylvanica were also produced in vitro by crossing compatible isolates of P. mortoniae. Furthermore, spore trapping studies show that propagules of this fungus are present as airborne inoculum in infected vineyards during rainfall events throughout the growing season. References: (1) G. Hausner et al. Can. J. Bot. 70:724, 1992. (2) L. Mostert et al. Mycologia 95:646, 2003.

Togninia minima is an important pathogen causing esca and grapevine declines worldwide. Although perithecia of T. minima have been produced in the laboratory, their presence in diseased vineyards has not been shown. In our study, perithecia of T. minima were found on grapevines in the field in five California counties. Perithecia were clustered on dead vascular tissue in deep cracks along trunks and cordons or on the surfaces of decayed pruning wounds. Field-collected perithecia were characteristic of T. minima perithecia previously produced in vitro and molecular sequence analysis of the internal transcribed spacer region of the nuclear ribosomal DNA additionally confirmed their identity. Ascospores from perithecia germinated on agar medium and formed colonies typical of T. minima. This is the first report of T. minima perithecia in diseased vineyards and suggests ascospores as an additional source of inoculum for new grapevine infections.

Fresh to 10-month-old pruning wounds on grapevine cvs. Thompson Seedless and Cabernet Sauvignon were inoculated with Phaeoacremonium aleophilum or Phaeomoniella chlamydospora at approximately 1 × 106 spores/ml. Successful infection was determined by isolation of the pathogen from necrotic margins of cankers or from vascular discoloration assessed 4 months after each inoculation date. Disease incidence decreased as the length of time between pruning and inoculation increased; however, wounds remained susceptible for up to 4 months. Vascular discoloration was significantly less extensive in the noninoculated control than in inoculated spurs. Reduction of shoot length in both cultivars varied depending on pruning wound age at the time of inoculation. In a separate study, inoculation of fresh pruning wounds of Cabernet Sauvignon resulted in successful infection regardless of the time of pruning from February to December.

During the 2006 growing season, grape berries in several Red Globe vineyards in Kern County, California were found exhibiting black fungal sporulation and typical sour rot symptoms. Symptoms included berry cracking and leakage along with a pungent vinegar smell. In California, sour rot (also known as summer bunch rot) has been attributed to a complex of microorganisms that invade ripe berries following injury. Fungi typically associated with sour rot include Aspergillus niger, Alternaria tenuis, Cladosporium herbarum, Rhizopus arrhizus, and Penicillium spp. Various yeasts and bacteria have also been associated with the disease, especially Acetobacter bacteria, which give the grapes their pungent acetic acid smell (2). In June 2006, the two fungi most commonly isolated from affected berries (postveraison) were A. niger and A. carbonarius. Identification of the two species was confirmed by colony and spore morphology (1) and PCR analysis of the internal transcribed spacer (ITS) region of rDNA. Although A. niger was more common, A. carbonarius has not previously been reported as a pathogen of grape in California. Conidia of A. carbonarius were globose, dark with very rough walls, and could be distinguished from A. niger by their wall structure and larger size ([5.1] 6.0 to 7.6 [8.8] μm in diameter). Sequence analysis of the ITS region of isolates morphologically identified as A. carbonarius showed 100% similarity to known isolates of this species. To confirm pathogenicity, postveraison Red Globe berries on standing grapevines were wounded with a needle and inoculated by dipping entire clusters into A. carbonarius spore suspensions (106 conidia/ml) for 30 s. Sterile water was used as a control. Twenty berries on each cluster were wounded and 10 clusters were used for each treatment. The experiment was repeated in two different vineyards in Kern County in 2006 and 2007. After 48 h, water-soaked lesions could be seen on the wounding site of grapes inoculated with A. carbonarius. After 1 week, 100% of the inoculated grapes exhibited dark, black sporulation, and after 3 weeks, the clusters were almost completely rotted. Koch's postulates were completed by isolating A. carbonarius from the affected berries. A few (less than 5%) of the wounded control berries also exhibited black sporulation and rot after 3 weeks. These infections were probably the result of natural inoculum at the sites because spore traps placed in the vineyards also contained A. carbonarius and A. niger propagules. Furthermore, soil surveys in the same vineyards showed that both A. niger and A. carbonarius were present on plant debris on the vineyard floor and in the soil. To our knowledge, this is the first report of A. carbonarius causing sour rot of grapes in California. References: (1) M. A. Klich. Identification of Common Aspergillus Species. Centraalbureau voor Schimmelcultures, Urecht, The Netherlands, 2002. (2) J. J. Marois et al. Bunch rots: Miscellaneous secondary invaders and sour rot. Page 69 in: Grape Pest Management. 2nd ed. The Regents of the University of California, Division of Agriculture and Natural Resources, Oakland, 1992.

In 1953, branch cankers on California avocado (Persea americana Mill.) trees were attributed to a Botryosphaeria anamorph, Dothiorella gregaria (teleomorph B. ribis) (2), and the disease was known as Dothiorella canker. Since this time, it has been suggested that this fungus should probably be classified as Fusicoccum aesculi Corda (teleomorph B. dothidea) (3). To our knowledge, B. dothidea is the only reported Botryosphaeriaceae species causing Dothiorella canker on avocado in California. Between the summer of 2008 and the winter of 2009, five trees from each of eight avocado orchards in five counties (San Diego, Riverside, Ventura, Santa Barbara, and San Luis Obispo) were surveyed for Dothiorella canker symptoms to verify the associated Botryosphaeriaceae species. Typical Dothiorella canker symptoms observed included darkened and friable bark with a dried, white, powdery exudate. Underneath the bark, cankers were variable in shape and some penetrated into the heartwood. Small sections of tissue (0.5 cm2) were excised from two to four separate cankers per tree and placed onto potato dextrose agar amended with tetracycline (0.01%) (PDA-tet). The most frequently isolated fungi, based on general growth pattern, speed, and colony color, were in the Botryosphaeriaceae with the following percent recovery by county: Riverside–40 and 100% (site 1 and 2, respectively); San Diego–60% (site 3); Ventura–42 and 53% (site 4 and 5, respectively); Santa Barbara–33% (site 6); and San Luis Obispo–32 and 60% (site 7 and 8, respectively). Pycnidia of Botryosphaeriaceae species were also observed on old diseased avocado tree branches. Sequenced rDNA fragments (ITS1, 5.8S rDNA, ITS2, amplified with ITS4 and ITS5 primers) were compared with sequences deposited in GenBank. Four different Botryosphaeriaceae species were identified and included Neofusicoccum australe, B. dothidea, N. luteum, and N. parvum, with species nomenclature based on the work of Crous et al. (1). Pathogenicity tests were conducted in the greenhouse on 1-year-old avocado seedlings, cv. Hass, with one randomly chosen isolate from each of the Botryosphaeriaceae species noted above. Four replicate seedlings were stem-wound inoculated with a mycelial plug and covered with Parafilm. Sterile PDA plugs were applied to four seedlings as a control. Over a period of 3 to 6 months, seedlings were assessed for disease symptoms that included browning of leaf edges and shoot dieback. Mean vascular lesion lengths on stems were 64, 66, 64, and 18 mm for B. dothidea, N. parvum, N. luteum, and N. australe, respectively. Each fungal isolate was consistently reisolated from inoculated seedlings, thus fulfilling Koch's postulates. To our knowledge, this is the first report of N. australe, N. luteum, and N. parvum recovered from branch cankers on avocado in California. These results are significant because Botryosphaeriaceae canker pathogens are known to enter the host plant through fresh wounds (pruning, frost, and mechanical). With high-density planting becoming more common, which requires intensive pruning, the transmission rate of these pathogens could increase in California avocado groves. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) F. F. Halma and G. A. Zentmyer. Calif. Avocado Soc. Yearb. 38:156, 1953. (3) W. F. T. Hartill and K. R. Everett. N. Z. J. Crop Hortic. Sci. 30:249, 2002.

Sharp decline and mortality of coast live oak (Quercus agrifolia) has been observed in San Diego County, CA since 2002. Much of this decline has been attributed to a new pest in California, the goldspotted oak borer (GSOB, Agrilus coxalis) (1). Symptoms include crown thinning, bark cracking and/or peeling, patches of stain (1 to 10 cm in diameter), bleeding on the bole, and tree death and are most often observed on trees with a diameter at breast height (DBH) >30 cm. In 2008, a Botryosphaeria sp. was recovered from necrotic tissue of bleeding bole cankers from GSOB-affected trees in Jamul, CA. Zone lines separated dead and live tissue in affected phloem and xylem. Pycnidia were observed on the bark surface of the infected host. Fifty conidia averaging 32 × 18 μm, one-septate with age, and morphologically similar to conidia described by Úrbez-Torres et al. were observed (4). Oak stands with tree mortality were surveyed in GSOB-infested and -uninfested sites over eight locations throughout San Diego and Riverside counties in 2009 and 2010. Symptomatic tissue or conidia from pycnidia of affected trees, plated onto potato dextrose agar amended with 0.01% tetracycline and incubated at 25°C for 1 week, consistently produced cultures with dense, wooly, olive-green mycelium. Mycelia fit the description of Botryosphaeria corticola A.J.L. Phillips, Alves et Luque (anamorph Diplodia corticola) (2). The resulting amplified ITS4/5 region of two sequences matched 100% to published D. corticola sequences (GU799472 and GU799460) (4). These sequences were deposited with NCBI GenBank (HM104176 and HM104177). Koch's postulates were conducted by inoculating 2-mm-diameter holes on five coast live oak trees with D. corticola. Holes were drilled to the cambium at 2 to 4 locations per tree within 1 to 2 m up the bole using a 0.157-cm portable electric drill. Trees ranged from 3.7- to 32.4-cm DBH. Either single agar plugs from two isolates each of a 7-day-old culture (UCR454 and UCR793) or noncolonized agar plugs as uninoculated controls were inserted into the holes and then covered with petroleum jelly and Parafilm. Average temperature was 10°C, relative humidity of 64%, and no precipitation during inoculation. Inoculations were conducted at a location in San Diego County uninfested by GSOB and repeated twice. After 3.5 months, bark was removed from inoculation sites. Average lesion length was not significantly different between inoculations, thus data were combined (one way analysis of variance [ANOVA]; P = 0.05). Lesions averaged 13.9 × 2.3 cm and were significantly different (n = 30; one way ANOVA; P = 0.05) from controls that measured 0.31 × 0.3 cm. Staining was observed around the inoculation points on all trees and three trees exhibited bleeding. Necrotic tissue was observed in the phloem and 3 mm into the xylem tissue, where the lesion had extended up and down the grain. D. corticola was consistently reisolated from necrotic tissue but not from control treatments. B. corticola was originally described as a canker pathogen on Quercus spp. in the western Mediterranean (2), and is known to contribute to the decline of cork oak (Q. suber) in the region (3). To our knowledge, this is the first report of D. corticola causing bot canker on coast live oak in California. References: (1) T. W. Coleman and S. J. Seybold. U. S. For. Serv. R5-PR-08, 2008. (2) A. Correia et al. Mycologia 96:598, 2004. (3) J. Luque et al. For. Pathol. 38:147, 2008. (4) J. R. Úrbez-Torres et al. Plant Dis. 94:785, 2010.

McDonald, V., and Eskalen, A. 2011. Botryosphaeriaceae species associated with avocado branch cankers in California. Plant Dis. 95:1465-1473. Members of the family Botryosphaeriaceae cause branch cankers and dieback on California avocado trees. More intensive pruning, a practice associated with high-density planting that is becoming more common in the California avocado industry, may increase the occurrence of branch canker. This study was undertaken to identify and characterize the Botryosphaeriaceae spp. involved in the branch canker disease complex in order to develop future management strategies. From 2008 to 2009, branch cankers were sampled from four or five trees from each of eight avocado groves in five California counties. Six Botryosphaeriaceae spp. were identified based on morphology as well as phylogenetic analysis of the internal transcribed spacer region (ITS15.8S-ITS2) and a partial sequence of the -tubulin gene. These six species included Neofusicoccum australe, N. luteum, N. parvum, an unknown Neofusicoccum sp., Fusicoccum aesculi, and Dothiorella iberica. Members of the Botryosphaeriaceae were isolated from all avocado-growing regions sampled in California; however, incidence and distribution of species varied. This report is the first description of the isolation of D. iberica from avocado branch cankers in California.

Eutypella is one of the few genera in the Diatrypaceae considered plant pathogens (1). In California, E. vitis and other members of the Diatrypaceae cause branch and trunk canker on grapevine (3,4). Eutypella spp. have not previously been documented as pathogens of citrus. In a 2010 survey on citrus branch canker and dieback in six citrus-growing counties of California, four isolates of Eutypella species were detected in Riverside and San Diego counties. Canker symptoms included dieback and bark cracking, and cuts made through symptomatic trees showed that the cankers were expanding through the center of the tree. Branch samples were collected from 10 trees per orchard and 5 to 10 orchards per county (102 trees for two counties). Pieces of symptomatic tissue (1 to 2 mm2) were plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet) and incubated at 25°C for 4 days. All isolates were identified by morphological and molecular characteristics. PCR of isolates was performed in a thermal cycler using two primer pairs, ITS4/5 and Bt2a/2b for amplifying the internal transcribed spacer (ITS1), 5.8S, and ITS2 region and the β-tubulin gene, respectively (2,3). PCR products were sequenced at the University of California, Riverside Genomics Core and the sequences compared in a BLAST search. Four isolates identified as Eutypella spp. included two (UCR1088 and UCR1101) from San Diego County and two (UCR1148 and UCR1149) from the Riverside County samples. The sequences were deposited in GenBank (HQ880579, JF758610, HQ880581, and HQ880582 and HQ880583, JF758611, HQ880585, and HQ880586 for the ITS regions and β-tubulin gene, respectively. ITS sequences for UCR1088 and UCR1101 had 98 and 100% match, respectively, to Eutypella spp. ITS sequences in GenBank (GQ293959 to GQ293961), while UCR1148 and UCR1149 matched 99% (GQ293956 to GQ293958). On the basis of morphological characteristics, UCR1088 and UCR1101 were similar to Eutypella spp. group 1, while UCR1148 and UCR1149 were similar to Eutypella spp. group 3 (4). Pathogenicity tests were conducted with all four isolates on detached shoots from healthy citrus trees of the same cultivar/rootstock from which each isolate was obtained. One wound per shoot was made on 1-year-old, green, detached shoots using a 3-mm-diameter cork borer and the wounded surfaces were inoculated with 3-mm-diameter mycelial plugs of 5-day-old cultures of each isolate growing on PDA-tet. Inoculated wounds and shoot ends were covered with petroleum jelly and wrapped with Parafilm (3). Control shoots were inoculated with sterile agar plugs. There were 10 inoculated shoots per isolate and noninoculated control treatment. Shoots were incubated at 25°C in moist chambers for 6 weeks. Lesions similar to those on the original infected shoots were observed on all inoculated shoots except the control treatment. Reisolation and identification of fungi from inoculated and control shoots were done using methods described above. Inoculated isolates were recovered from 100% of inoculated shoots but none was recovered from noninoculated shoots, indicating association of Eutypella spp. with citrus branch canker. To our knowledge, this is the first report of Eutypella spp. associated with cankers on citrus in California. References: (1) B. Piskur et al. Plant Dis. 91:1579, 2007. (2) B. Slippers et al. Mycologia 96:83, 2004. (3) F. P. Trouillas and W. D. Gubler. Plant Dis. 94:867, 2010. (4) F. P. Trouillas et al. Mycologia 102:319, 2010.

Avocado branch canker in California is caused by a complex of fungal species in the families Botryosphaeriaceae and Diaporthaceae. As the popularity of avocado fruit increases, California growers are under pressure to increase their productivity in order to compete with imports. One way to increase production is through high-density planting, which entails intense canopy management, possibly leaving the trees vulnerable to infection through pruning wounds. A spore trap study was undertaken to determine the seasonal spore discharge of Botryosphaeriaceous and Diaporthaceous fungi. Based on colony counts, the highest population of Botryosphaeriaceous fungi (68%) occurred during or soon after precipitation events, which coincided with the winter months of December, January, and February. The spring and fall seasons had lower numbers of spores trapped (at 13 and 17%, respectively), with few spores trapped in the summer season. For members of the Diaporthaceae family, spores trapped were almost evenly split between winter and fall seasons (50 and 49%, respectively), with few to no spores trapped in the spring and summer seasons. A pathogenicity test of six fungi of Botryosphaeriaceae and one fungus of Diaporthaceae was conducted in the greenhouse. Internal plant vascular lesion lengths resulting from inoculation with any of the seven fungal species differed significantly from the noninoculated control. These studies suggest that pruning during the drier parts of the year would minimize infection by fungi in the Botryosphaeriaceae and Diaporthaceae families.

Esca and petri diseases are important grapevine trunk diseases in South Africa and most other grape-producing countries. The causal pathogens are Phaeomoniella chlamydospora and several species of Phaeoacremonium. In total, 25 species of Phaeoacremonium have been isolated from grapevines of which seven species have been linked to Togninia teleomorphs obtained through in vitro mating studies (3). Of these species, only perithecia of T. minima, T. fraxinopennsylvanica, and T. viticola have been found on grapevines in California (1,2,4). T. minima is heterothallic, and although both mating types are present in South African vineyards, perithecia have never been observed (3). In the current study, grapevine cordons and trunks were collected from vineyards and rootstock mother vines within Western Cape Province for examination in the laboratory under a dissecting microscope. The grapevines displayed general decline symptoms, including reduced vegetative growth, dead or dying shoots and cordons, as well as internal vascular streaking and/or a red/black/brown margin next to decayed wood typically associated with esca and petri disease. Rootstock mother vines were apparently healthy, although many old, cracked pruning wounds were visible. Togninia-like perithecia with distinctive long necks were found along the wood crevices, often on old pruning wounds. The perithecia were removed and placed on microscope slides with sterile water. Structures were measured and slides were washed with 500 μl of sterile water onto potato dextrose agar amended with chloramphenicol (250 mg/liter). Ascospores were allowed to germinate overnight to obtain single ascospore colonies. Perithecia were found on cultivars Muscat d' Alexandrie and Pinotage (Vitis vinifera) at Stellenbosch in May 2011 and on Ramsey (V. champinii) rootstock mother vines at Slanghoek in June 2012. Perithecia were globose to subglobose, black, and often embedded in the wood tissue but also present on the surface of the wood. The length of the necks was 250 to 300 × 47.5 to 55 μm. The asci were hyaline and ranged from 16 to 25 × 3.5 to 5 μm. Ascospores were hyaline, ellipsoid, and ranged from 5 to 6 × 1.5 to 2 μm. These measurements were similar to those reported by Mostert et al. (3) and Rooney et al. (4). Colony growth was typical of T. minima. DNA was extracted from the colonies and the partial betatubulin gene was amplified and sequenced using the primers T1 and Bt2b. Sequences were deposited into GenBank (JX962864 to 67). Based on a megablast search of the NCBI's GenBank nucleotide database, 100% similarity was found with other T. minima sequences (JQ691670.1, HQ605018.1, HQ605014.1; identities = 647/647 [100%], gaps = 0/647 [0%]). To our knowledge, this is the first report on the occurrence of T. minima perithecia on grapevines in Western Cape Province of South Africa. The removal of dead spurs and cordons will be instrumental in lowering the inoculum originating from perithecia, especially in rootstock mother blocks where no control strategies are applied for petri disease or esca. Spore trapping studies are currently in progress to study spore release patterns in order to determine whether pruning wounds are at risk during traditional pruning periods. References: (1) A. Eskalen et al. Plant Dis. 89:528, 2005. (2) A. Eskalen et al. Plant Dis. 89:686, 2005. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006. (4) S. Rooney-Latham et al. Plant Dis. 89:867, 2005.

Members of the Botryosphaeriaceae family are known to cause Bot gummosis on many woody plants worldwide. To identify pathogens associated with Bot gummosis on citrus in California, scion and rootstock samples were collected in 2010 and 2011 from five citrus-growing counties in California. Symptoms observed on citrus included branch cankers, dieback, and gumming. Various fungal species were recovered from necrotic tissues of branch canker and rootstock samples. Species were identified morphologically and by phylogenetic comparison as ‘Eureka’ lemon, ‘Valencia’, ‘Washington Navel’, ‘Fukumoto’, grapefruit, ‘Satsuma’, and ‘Meyer’ lemon. Species were identified morphologically and by phylogenetic comparison of the complete sequence of the internal transcribed spacer regions, β-tubulin gene, and elongation factor α-1 genes with those of other species in GenBank. A consensus-unrooted most parsimonious tree resulting from multigene phylogenetic analysis showed the existence of three major clades in the Botryosphaeriaceae family. In total, 74 isolates were identified belonging to the Botryosphaeriaceae family, with Neofusicoccum spp., Dothiorella spp., Diplodia spp., (teleomorph Botryosphaeria), Lasiodiplodia spp., and Neoscytalidium dimidiatum (teleomorphs unknown) accounting for 39, 25, 23, 10, and 3% of the total, respectively. On inoculated Eureka lemon shoots, lesion length was significantly different (P < 0.05) among 14 isolates recovered from portions of cankered tissues of the original trees. Lesion lengths were significantly longer (P < 0.05) for shoots inoculated with isolates of Neofusicoccum luteum and shorter for shoots inoculated with isolates of Dothiorella viticola (P < 0.05) than those of other species. Identifying the distribution and occurrence of these fungal pathogens associated with Bot gummosis is useful for management applications during occasional outbreaks in California.

The Aegean region (western Turkey) is the center of table, raisin, and wine grape cultivation. During the 2012 growing season, wood canker symptoms were observed in vineyards in Manisa city. Symptoms adjacent to pruning wounds, including shoot dieback and wedge-shaped wood discolorations observed in cross section, were among the most prevalent symptoms of the vines. To identify the causal agents, symptomatic woody tissues were surface disinfested with 95% ethanol and flame-sterilized and the discolored outer bark was cut away. The internal tissues (0.5 cm2) were excised from cankers of vines and plated onto potato dextrose agar amended with tetracycline (0.01%) (PDA-tet). The most frequently isolated fungi, based on general growth pattern, speed of growth, and colony color, resembled species in the Botryosphaeriaceae family. According to morphological characteristics, four different groups have been identified based on visual discrimination. After DNA extraction, ribosomal DNA fragments (ITS1-5.8S-ITS2) (2) amplified with ITS4 and ITS5 primers were sequenced and sequences were compared with those deposited in NCBI GenBank database. Four different Botryosphaeriaceae isolates were identified, including Botryosphaeria dothidea (MBAi25AG), Diplodia seriata (MBAi23AG), Lasiodiplodia theobromae (MBAi28AG), and Neofusicoccum parvum (MBAi27AG) (Accession Nos. KF182329, KF182328, KF182331, and KF182330, respectively) with species nomenclature based on Crous et al. (1). Pathogenicity tests were conducted under greenhouse conditions (24°C, 16/8-h day/night, 70% RH) on 1-year-old own rooted grapevine (Vitis vinifera) cv. Sultana Seedless seedlings using one isolate from each of the Botryosphaeriaceae species specified above. Stems of grapevine seedlings were wounded by removing bark with 4-mm cork borer and fresh mycelial plugs were inoculated into the holes and covered with Parafilm. Sterile PDA plugs were placed into the wounds of control seedlings. Five vines were inoculated per isolate. The experiment was repeated twice. After 4 months of incubation, grapevine seedlings were examined for the extent of vascular discoloration and recovery of fungal isolates. Mean lesion lengths on wood tissues were 85.3, 17.2, 13.9, and 13.1 mm for N. parvum, B. dothidea, L. theobromae, and D. seriata, and 6.3 mm for control. Each fungal isolate was successfully re-isolated from inoculated seedlings to fulfill Koch's postulates. To our knowledge, this is the first report of multiple species in the Botryosphaeriaceae causing wood canker and dieback on grapevine in Turkey. These results are significant because Botryosphaeriaceae species are known causal agents of grapevine trunk disease worldwide (3). References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) B. Slippers et al. Mycologia 96:83, 2004. (3) J. R. Urbez-Torres. Phytopathol. Mediterr. 50:S5, 2011.

In May 2012 in the Coachella valley, Riverside County, California, the decline of vines in table grape (Vitis vinifera) vineyards was observed. Foliar symptoms consisted of shoot blight with wilting and necrosis of leaves and drying and shriveling of berries. In some cases, the entire vine collapsed in the middle of the growing season (apoplexia). Wood cankers in the spurs, cordons, and trunks of affected vines were also present. The nine isolates recovered from the cankers were identified as Neoscytalidium dimidiatum (Penz.) Crous & Slippers based on morphological characteristics and DNA sequence comparisons. Two isolates were grown on potato dextrose agar (PDA) medium and a total of 50 conidia were measured per isolate. Conidia were ellipsoid to ovoid, with a truncate base and an acutely rounded apex, initially aseptate, becoming brown and two-celled at maturity, 7.2 ± 1.2 μm × 3.8 ± 0.4 μm. The rDNA internal transcribed spacer (ITS), and β-tubulin (BT) loci were amplified using primer pairs and methods previously described (4). A total of five isolates were sequenced. The DNA sequences of one N. dimidiatum grapevine isolate (UCR-Neo1) were deposited in the GenBank database (ITS, KC937066; BT, KC937067). Pathogenicity tests were performed by inoculating 12 grape cuttings cv. Thompson Seedless with isolate UCR-Neo1 and 12 control cuttings with sterile medium using a technique previously described (1). The experiment was repeated twice. After 20 weeks of incubation period in the greenhouse, the lesions length produced by N. dimidiatum averaged 13.5 mm and was significantly longer (P < 0.05) from the control (average 3 mm). N. dimidiatum was reisolated from all the inoculated plants and identified by colony morphology. The incidence of N. dimidiatum in table grape vineyards of the Coachella valley has been estimated at 15%, with nine vines infected out of 60 vines total. This pathogen has been identified in California in walnut nursery causing the death of trees due to the development of canker at the graft union (2). N. dimidiatum has also been identified as the causal agent of shoot blight, canker, and gummosis on citrus in Italy (3). The crop is also being grown in the Coachella valley and these findings warrant further investigation in order to determine the host range, distribution, and incidence of this pathogen in the area. References: (1) K. Baumgartner et al. Plant Dis. 97:912, 2013. (2) S. F. Chen et al. Plant Dis. 97:993, 2013. (3) G. Polizzi et al. Plant Dis 93:1215, 2009. (4) J. R. Urbez-Torres et al. Plant Dis. 92:519, 2008.

Declining coast live oak (Quercus agrifolia) trees have been observed since 2012 throughout urban landscapes in Los Angeles, Orange, Riverside, Santa Barbara, Ventura, and Monterey counties in California. Symptoms causing branch dieback and tree death included a cinnamon-colored gum seeping through multiple 0.95-mm-diameter entry holes on the bole, followed by a prolific, cream-colored foamy liquid. Beneath the outer bark was phloem and xylem necrosis. Fifty 1- to 2.5-mm adult and larval beetles were collected. Adults fit the morphological description of Pseudopityophthorus pubipennis (western oak bark beetle) (R. Rabaglia, personal communication), and ~800 bp of the mitochondrial COI gene was amplified for three beetles using primer pairs and methods previously described (2,3). All three sequences were identical (GenBank Accession Nos. KJ831289 to 91) and a BLAST search confirmed the closest match (94%) as P. pubipennis. Necrotic wood tissues collected from two trees in each county were cultured on potato dextrose agar amended with 0.01% tetracycline (PDA-tet), and incubated at 25°C for 1 week. Ochre-colored cultures with plane or radially furrowed velutinous mycelium were consistently produced. Fifty conidia each measured from two isolates were 3.66 ± 0.04 μm × 1.77 ± 0.03 μm, and arranged in non-persistent conidial chains, at first roughly parallel, becoming tangled with age. These fungal colonies were observed within gallery walls. The rDNA internal transcribed spacer (ITS) was amplified using primer pairs and methods previously described (5). Three isolates were sequenced and matched 100% to known sequences of Geosmithia pallida in GenBank; sequences of two isolates (UCR2208 and UCR2210) were deposited in GenBank (KJ468687 and KJ468688). Pathogenicity tests were performed by inoculating twelve 27.0-cm detached coast live oak shoots for each isolate with a spore suspension of G. pallida (UCR2208 and UCR2210) and sterile distilled water for controls. A 2-mm-wide, 3-mm-deep hole was drilled into the center of each shoot, 20 μl of a 106 conidia/ml spore suspension was pipetted into the hole, and sealed with Vaseline and Parafilm. The experiment was repeated twice. After 4 weeks in a moist chamber at 25°C, lesions produced by G. pallida averaged 8.3 cm and was significantly longer (ANOVA; P < 0.0001) from the control (average 0.4 cm). G. pallida was re-isolated from all inoculated plants and identified by colony morphology. P. pubipennis is a native beetle, common as a secondary agent, and previously not associated with disease. However, cryptic species may be common among bark and ambrosia beetles (4). A larger sample (i.e., populations and loci) is needed to determine the precise taxonomic status of P. pubipennis. G. pallida was shown to inhibit root growth of Q. petraea by 25% in Europe (1), appears to have affinities with a range of subcorticolous insects, and is widely distributed (5), but there is no published record of the fungus occurring in the United States. This is the first report of G. pallida causing foamy bark canker in association with P. pubipennis on Q. agrifolia in California. Results suggest this new disease complex is causing decline of Q. agrifolia throughout the state. References: (1) D. Cizková et al. Folia Microbiol. 50:59, 2005. (2) A. I. Cognato and F. A. H. Sperling. Mol. Phylogenet. Evol. 14:445, 2000. (3) A. I. Cognato et al. Mol. Phylogenet. Evol. 36:494, 2006. (4) B. H. Jordal and M. Kambestad. Mol. Ecol. Res. 14:7, 2014. (5) M. Kolarík et al. Mycol. Res. 108:1053, 2004.

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), β-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 β-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.