First Report of Lasiodiplodia theobromae Associated with Stem Canker of Almond in California.

Abstract

California is a major almond (Prunus dulcis) producer in the world. In September 2012, 2-year-old almond trees from an orchard in Fresno Co. with stem cankers were submitted for disease diagnosis. In a survey of the orchard, 12 ha (1,500 Nonpareil and 1,800 Monterey almond trees) of 48 ha trees had been killed apparently due to a stem canker. The cankers developed above the graft union, were covered with amber sap, and often girdled the trunk. Isolations made from tissues at the canker margins onto acidified potato dextrose agar (PDA) yielded two fungi, Macrophomina phaseolina (Tassi) Goid and Lasiodiplodia theobromae (Pat.) Griffon & Maubl (1). M. phaseolina and L. theobromae were isolated from eight and two of 10 cankered trees, respectively. No mixed infections were found. M. phaseolina isolates were characterized by gray hyphae that turned black with developing microsclerotia. L. theobromae isolates were characterized by white, aerial mycelium that turned mouse gray after 5 days. Young conidia were ellipsoidal, thick walled, initially hyaline, granular, and nonseptate; aged conidia were brown, 1-septate with longitudinal striations in the wall. Identity was confirmed by analyses of the internal transcribed spacer (ITS), β-tubulin 2 (BT2), and the translation elongation factor 1-alpha (TEF-1α) gene regions. BLAST searches at GenBank showed a high identity with reference sequences of type specimens both for M. phaseolina (isolates 7E64 to 7E69: ITS, 100%; BT2, 99%; TEF-1α, 99%) and L. theobromae (isolates 7E86 to 7E88: ITS, 99%; BT2, 99%; TEF-1α, 100%). Sequences of three gene regions were deposited as GenBank accessions KC357271 to KC357279 (ITS); KC357280 to KC357288 (BT2); and KC357289 to KC357297 (TEF-1α). The pathogenicity of M. phaseolina and L. theobromae to P. dulcis cultivars Butte, Carmel, Nonpareil, and Padre was investigated in an orchard at KARE using four isolates of M. phaseolina (7E64, 7E65, 7E66, and 7E69) and two isolates of L. theobromae (7E86 and 7E88). Ten 2-year-old branches per isolate from 7-year-old trees were inoculated with each isolate in late September 2012, after removing the bark with a 7-mm cork borer and placing a 7-day-old 7-mm-diameter agar plug bearing mycelium of each isolate directly into the fresh wound, mycelium side down. Ten additional branches of each of the four cultivars were inoculated with sterile PDA plugs and served as negative controls. Three weeks after inoculation, the average lesion produced by M. phaseolina on Butte, Carmel, Nonpareil, and Padre was 53, 52, 41, and 37 mm in length, respectively. Lesions produced by L. theobromae were 191, 206, 194, and 103 mm in length on the four cultivars, respectively. No disease lesion, only wounds, were produced on negative controls. Lesions produced by both pathogens were longer (P < 0.05) than wounds on the controls (average length 10 mm on all cultivars). Both L. theobromae isolates killed branches of cultivars Butte, Carmel, and Nonpareil in 2 weeks. M. phaseolina and L. theobromae were reisolated from the inoculated branches, and no fungus was reisolated from controls. Based on pathogenicity results, L. theobromae is more virulent to almond branches than M. phaseolina. To our knowledge, this is the second report of M. phaseolina (2) and the first report of L. theobromae as pathogens of P. dulcis trees in California.