Juglans sigllata Dode, known as the iron walnut, is widely planted in Liangshan prefecture of southwest China for its nuts and wood. Liangshan prefecture is a major traditional growing area of J. sigllata and has unique advantages for walnut industrial development because of its good soil, climate, and availability of water. Currently there are 2.7 million hectares of walnut, contributing important incomes for farmers. In April 2013, numerous J. sigllata were found infected with root rot in the Muli county of Liangshan prefecture. Symptoms included dried leaves, dead branchcs, and even death. Rotted roots were collected and surface-sterilized in 2% NaOCl and 70% ethanol. The junction (1 cm) between infected and healthy regions was removed, plated on rose bengal-glycerin-urea medium, and incubated at 20°C for 12 h. A fungus was found and purified successively by transferring hyphal tips from the margin of a thinly growing colony on 2% water agar (3). Morphological characteristics were identified both on potato dextrose agar (PDA) and carnation leaf-piece agar. Evaluation of pigmentation and colony growth rate were also measured using PDA. Ovoid microconidia (average dimensions 10.6 × 9.1 μm) were observed after 2 to 3 days, and most of them had no septa or only one septum. Macroconidia (average dimensions 47.4 × 5.3 μm), with one to three septate sickle shapes, were found after 3 to 6 days. Single or paired chlamydospores (average dimensions 10.3 × 9.2 μm), which were circular to ovate, smooth or not smooth, were observed after 7 days of incubation in clean water. According to the cultural characteristics, the fungus was primarily identified as Fusarium solani (1). To better determine the species, universal primers ITS1/ITS4 for the ribosomal internal transcribed spacer (ITS) coupled with translation elongation factor (EF-1α) primers EF1/EF2 were used for PCR-based molecular identification. Against GenBank and the FUSARIUM-ID databases, our sequences shared 99 and 98% identities with ITS (FJ459973.1) and EF-1α (JX677562.1) of F. solani, respectively. Both sequences produced in this study have been deposited in GenBank under accession numbers KJ528277 for ITS and KJ528278 for EF-1α. Pathogenicity tests were conducted by drop inoculating 20 ml of microconidia suspension (106 spores/ml) on the roots of 1-year-old healthy potted J.sigllata, Mianyang walnut, and Xinjiang walnut. Controls were not treated with F. solani. Fifteen plants were in each group. All materials, including pots and soil, were disinfected. After 12 days, all J. sigllata inoculated with F. solani exhibited dried leaves, and after 17 days, Mianyang walnut and Xinjiang walnut infected with F. solani also developed the same symptoms. After 24 days, the inoculated J. sigllata died. However, control plants remained asymptomatic. The fungus re-isolated from infected roots showed the same characteristics as described above and was totally identical in appearance to the isolates used to inoculate the plants. No colonies of F. solani were isolated from untreated plants. At present, F. solani has been reported in stem cankers on English walnut in South Africa (2). To our knowledge, this is the first report of root rot caused by F. solani in J. sigllata in China.
References: (1) C. Booth. Fusarium Laboratory Guide to the Identification of the Major Species. CMI, Kew, England, 1977. (2) W. Chen and W. J. Swart. Plant Dis. 84:592, 2000. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.