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.

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.

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.

Evaluations of Insecticides and Fungicides for Reducing Attack Rates of a new invasive ambrosia beetle (Euwallacea Sp., Coleoptera: Curculionidae: Scolytinae) in Infested Landscape Trees in California.

A recently discovered ambrosia beetle with the proposed common name of polyphagous shot hole borer (Euwallacea sp., Coleoptera: Curculionidae: Scolytinae), is reported to attack >200 host tree species in southern California, including many important native and urban landscape trees. This invasive beetle, along with its associated fungi, causes branch dieback and tree mortality in a large variety of tree species including sycamore (Platanus racemosa Nutt.). Due to the severity of the impact of this Euwallacea sp., short-term management tools must include chemical control options for the arboriculture industry and private landowners to protect trees. We examined the effectiveness of insecticides, fungicides, and insecticide-fungicide combinations for controlling continued Euwallacea sp. attacks on previously infested sycamore trees which were monitored for 6 mo after treatment. Pesticide combinations were generally more effective than single pesticide treatments. The combination of a systemic insecticide (emamectin benzoate), a contact insecticide (bifenthrin), and a fungicide (metconazole) provided some level of control when applied on moderate and heavily infested trees. The biological fungicide Bacillus subtilis provided short-term control. There was no difference in the performance of the three triazole fungicides (propiconazole, tebuconazole, and metconazole) included in this study. Although no pesticide combination provided substantial control over time, pesticide treatments may be more effective when trees are treated during early stages of attack by this ambrosia beetle.

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.

Identification and pathogenicity of Botryosphaeriaceae species associated with coast live oak (Quercus agrifolia) decline in southern California.

Symptoms of decline have been observed on dying coast live oak (Quercus agrifolia) trees in areas throughout southern California that are both infested and uninfested by the gold-spotted oak borer (GSOB). The purpose of this study was to identify and assess the pathogenicity of several anamorph species of the Botryosphaeriaceae, including Diplodia corticola, Dothiorella iberica and Diplodia agrifolia sp. nov., that were recovered consistently from symptomatic tissues. Species were identified morphologically and by phylogenetic analyses of the complete sequence of the internal transcribed spacer (ITS) of the rDNA and partial sequences of ß-tubulin and elongation factor (EF1-α) genes. Results from morphological assessments and phylogenetic analyses support the erection of a new species closely related to D. mutila, described herein as Diplodia agrifolia sp. nov. Pathogenicity of all species was verified by wound inoculation of 1 y old coast live oak seedlings under controlled conditions. Isolates of D. corticola were the most aggressive tested, and isolates of D. agrifolia were the second most aggressive. Both species caused bleeding symptoms on inoculated seedlings. Seedlings inoculated with D. corticola died within 4 wk, with the pathogen progressing up and down through the xylem in advance of living phloem and moving throughout the taproot in 70% of inoculated seedlings. Colonization and re-isolation was successful for all species. All three fungal species represent newly recorded fungal pathogens of coast live oak in California. Results from the pathogenicity test suggest that these fungi play a role in the decline of coast live oaks throughout southern California.