Per capita consumption of avocado in the United States has nearly doubled between 2000 and 2010. The California avocado industry supplies almost 40% of U.S. demand and the remaining 60% is supplied by imports from Latin America and New Zealand. The Tea Shot Hole Borer (TSHB) is an ambrosia beetle from Asia that forms a symbiosis with a new, yet undescribed Fusarium sp. and is a serious problem for the Israeli avocado industry (3). The beetle also causes severe damage on the branches of tea (Camelia sinensis) in Sri Lanka and India (1). In California, TSHB was first reported on black locust (Robinia pseudoacacia) in 2003, but there are no records of fungal damage (4). In 2012, nine backyard avocado trees (cvs. Hass, Bacon, Fuerte, and Nabal) exhibiting branch dieback were observed throughout the residential neighborhoods of South Gate, Downey, and Pico Rivera in Los Angeles County. Upon inspection, symptoms of white powdery exudate, either dry or surrounded by wet discoloration of the outer bark in association with a single beetle exit hole, were found on the trunk and main branches of the tree. Examination of the cortex and wood under the exit hole revealed brown discolored necrosis. The TSHB was also found within galleries that were 1 to 4 cm long going against the grain. Symptomatic cortex and sapwood tissues were plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet). The TSHB was dissected and plated onto PDA-tet after surface disinfestation following methods described by Kajimura and Hijii (2). After 5 days of incubation at room temperature, regular fungal colonies with aerial mycelia and reddish brown margins were produced. Single spore isolations were used to establish pure culture of the fungus. Fifty conidia were hyaline, clavate with a rounded apex, and initially aseptate (4.1 to 12.0 × 2.4 to 4.1 μm) becoming one- to three-septate (7.6 to 15.1 × 2.8 to 4.5 μm, 9.2 to 17.2 × 3.4 to 4.8 μm, and 13.5 to 17.6 × 4.3 to 4.7 μm, respectively). Identity of the fungal isolates was determined by amplification of the rDNA genes with primers ITS4/5 and EF1/2, respectively. Sequences were deposited into GenBank under Accession Nos. JQ723753, JQ723760, JQ723756, and JQ723763. A BLASTn search revealed 100% similarity to Fusarium sp. (Accession Nos. JQ038020 and JQ038013). Detached green shoots of healthy 1-year-old avocado were wounded to a depth of 1 to 2 mm and 5-mm mycelial plugs from 5-day-old cultures (UCR 1781 and UCR 1837) were placed mycelial side down onto the freshly wounded surfaces and then wrapped with Parafilm. Control shoots were inoculated with sterile agar plugs and five replicates per treatment were used. Shoots were incubated at 25 ± 1°C in moist chambers for 3 weeks. Lesions were observed on all inoculated shoots except for the control. Mean lesion lengths were 10.7 and 12.8 cm for UCR1781 and UCR1837, respectively, and were significantly different (P ≤ 0.05) from the control. Both isolates were reisolated from 100% of symptomatic tissues of inoculated shoots to complete Koch's postulates. This experiment was conducted twice and similar results were obtained. To our knowledge, this is the first report of Fusarium sp. and its vector E. fornicatus causing Fusarium dieback on Avocado in California. References: (1) W. Danthanarayana. Tea Quarterly 39:61, 1968. (2) H. Kajimura and N. Hijii. Ecol. Res. 7:107, 1992; (3) Mendel et al., Phytoparasitica, DOI 10.1007/s12600-012-0223-7, 2012. (4) R. J. Rabaglia. Annals Entomol. Soc. Amer. 99:1034, 2006.

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), β-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.

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.

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.

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.