First Report of Lasiodiplodia citricola and Neoscytalidium dimidiatum Causing Death of Graft Union of English Walnut in California

California produces 99% of the English walnuts (Juglans regia) in the USA. In August 2012 in Tulare County, about 5,000 out of 90,000 trees were killed in a walnut nursery by a distinct black canker that developed around the graft union. The cankers appeared to be initiated at the heading cut on the rootstock, and expanded down to the rootstock and through the budded union up to the scion, resulting in mortality of scion. The walnut nursery was located adjacent to a mature walnut orchard. The fungi isolated from the cankers were identified as Lasiodiplodia citricola and Neoscytalidium dimidiatum based on morphological characteristics and DNA sequence comparisons. L. citricola was isolated from one of the 10 graft unions, while N. dimidiatum from the other nine. L. citricola isolates were characterized by white, aerial mycelium on potato dextrose agar that turned gray after 4 days and produced ellipsoidal to ovoid hyaline one-celled conidia that became 2-celled and brown with thick walls and longitudinal striations in the wall (1). N. dimidiatum isolates were characterized by ellipsoid to ovoid, hyaline conidia with a truncate base and an acutely rounded apex, initially 1-celled, and some becoming brown and 2-celled at maturity; no muriform conidia were produced (3). These identifications were confirmed by analyses of the ITS, BT2, and TEF-1α gene regions. The three gene regions were amplified by using the primers and methods described in (4). For L. citricola (isolates 7E78 to 7E80), a DNA sequence BLASTn at GenBank showed 100% identity with accessions GU945354 (ITS) and GU945340 (TEF-1α) of the ex-type specimen (CBS124707, BT2 sequencing data was not available) (3). For N. dimidiatum (isolates 7E61 to 7E63), a BLASTn search showed a high identify (ITS, 100%; BT2, 99%; TEF-1α, 99%) with reference sequence of N. dimidiatum (ITS, GQ330903; BT2, GU251768; TEF-1α, GU251240). Sequences of the studied DNA regions were deposited to GenBank as KC357298 to KC357303 (ITS); KC357304 to KC357309 (BT2); and, KC357310 to KC357315 (TEF-1α). The pathogenicity of L. citricola in comparison with N. dimidiatum in J. regia cvs. Chandler, Tulare, and Vina was evaluated in an orchard at KARE, by using two isolates each of L. citricola (7E78, 7E80) and N. dimidiatum (7E61, 7E63). Pathogenicity tests were performed by inoculating ten 2-year-old branches per isolate in late September 2012 by the method described in (2). After 3 weeks, the average lesion lengths caused by L. citricola on Chandler, Tulare, and Vina were 152, 156, and 188 mm, respectively, and 32, 38, and 34 mm, respectively, caused by N. dimidiatum. The lesion length averages produced on the three cultivars by the four isolates were all significantly (P < 0.05) longer than their respective controls (average length 10 mm on all cultivars). L. citricola, but not N. dimidiatum, killed branches of Chandler, Tulare, and Vina in 10 days. Both L. citricola and N. dimidiatum were reisolated from the inoculated branches and no fungus was isolated from the control, confirming Koch's postulates. These results confirmed that the walnut graft union canker was caused by either L. citricola or N. dimidiatum. To our knowledge, this is the first report of death of newly grafted walnut trees caused by L. citricola and N. dimidiatum worldwide, and also the first report of L. citricola infecting walnut worldwide. References: (1) J. Abdollahzadeh et al. Persoonia. 25:1, 2010. (2) S. F. Chen et al. Plant Dis. 97:994, 2013. (3) D. Pavlic et al. Mycologia. 106:851, 2008. (4) B. Slippers et al. Mycologia. 96:83, 2004.