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Identification of Two Novel Fungal Species Associated with Kuroshio Shot Hole Borer (Euwallacea sp.) and Evaluation of Novel Biological Control Method to Inhibit the Fungal Associates of the Invasive Ambrosia Beetle Species in California

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The invasive ambrosia beetle species polyphagous shot hole borer (Euwallacea sp., PSHB) and its ambrosial fungal associates have been studied extensively since the beetle’s discovery in the Los Angeles County in 2012. Another closely related ambrosia beetle species has been discovered in San Diego County (Kuroshio shot hole borer, KSHB), and the ambrosial fungal associates of this beetle species have been found to be distinct from the related fungal associates of PSHB, Fusarium euwallaceae and Graphium euwallaceae. The KSHB-associated Fusarium kuroshium sp. nov. and Graphium kuroshium sp. nov. have been found to be genetically distinct from F. euwallaceae and G. euwallaceae in multilocus phylogenetic analyses using ITS, EF1α, RPB1 and RPB2 sequences. F. kuroshium was also found to differ in morphology and symptom severity compared to F. euwallaceae, while G. kuroshium was not found to differ from G. euwallaceae in morphology or symptom severity. The results of this study suggest that the KSHB-associated F. kuroshium and G. kuroshium are novel fungal associates of KSHB. A method to inhibit the Fusarium ambrosial fungi of PSHB and KSHB using plant-endophytic bacteria was evaluated in vitro and in vivo. Endophytic bacterial isolates were obtained from the wood tissues of avocado (Persea americana) and California sycamore (Platanus racemosa), and were identified using molecular identification methods. These bacteria were simultaneously cultured with F. euwallaceae in order to screen for endophytic bacteria with the ability to inhibit the Fusarium ambrosial fungi. 15 bacterial isolates were found to inhibit F. euwallaceae during the screen. Inhibition efficacies of the bacterial endophytes were calculated for both Fusarium associates of PSHB and KSHB as percent inhibition, and 4 Bacterial isolates that showed the highest percent inhibition were chosen to be used as injection treatments in the greenhouse in vivo study. In greenhouse, stems of avocado and California sycamore were bacterized using the endophytic bacteria then challenged with F. euwallaceae or F. kuroshium. The sizes of fungal lesions resulting from the Fusarium infection were measured then compared. The results showed that the endophytic bacteria had significant effects in the sizes of lesions, and bacteria were only recovered from bacterized plants.

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