First report of brown leaf spot on Juglans sigillata caused by Ophiognomonia leptostyla in Sichuan, China.

Abstract

Juglans sigillata Dode (Iron walnut) is mostly distributed in southwestern China, and valued for wood and nuts (Feng et al. 2018). In April 2020, we surveyed a walnut garden located in Baisha Town, Wanyuan City, (Sichuan, China), where brown spot symptoms were observed on leaves of ten trees among of 100 plants, and this disease can result in a reduced growth potential when trees are severely infected. Necrotic and subcircular lesions with conidiamata were observed on diseased leaves. Symptomatic leaves were collected and taken back to the laboratory forfurther analysis. Using the single spore isolation technique developed by Chomnunti et al. (2014), five isolates were grown from the infected leaves on Potato Dextrose Agar medium (PDA). The five isolates had similar colony morphology, which was initially white, suborbicular, gradually turning yellowish with black spots, developing fluffy aerial mycelium. Morphological characteristics were examined using light microscopy on the PDA. Conidiogenous cells were subcylindrical to cylindrical, or ampulliform, hyaline, rarely branched. Macroconidia were lunate, reniform, hyaline, 1-3-septate, mostly 1-septate, distinctly constricted at the septum, the basal cell was bluntly rounded, the apical cell had an acute end, and the basal cell was equal to or larger than the apical cell, measuring 22 to 40.5 × 2.5 to 8.3 μm (mean = 32 × 6.2 μm, n = 50). Microconidia were botuliform, or subfusiform, hyaline, both ends were rounded, straight or curved, aseptate, and measured 10 to 28.5 × 1.9 to 3.7 μm (mean= 17.2 × 2.7 μm, n = 20). A multilocus approach was conducted for precise identification of a representative isolate SICAUCC 20-0012. The internal transcribed spacer regions (ITS), guanine nucleotide-binding protein subunit beta gene (MS204), and translation elongation factor 1-alpha (tef1-α) of isolate SICAUCC 20-0012 were amplified and sequenced as described by Sogonov et al. (2008) and Walker et al. (2012a). GenBank Accession Nos. for ITS, MS204, and tef1-α are MW250303, MW246773, and MW246775, respectively. Phylogenetic analyses showed 100% support with Ophiognomonia leptostyla (Fr.) Sogonov, and the morphology was consistent with the asexual stage of O. leptostyla documented by Walker et al. (2012b). To test Koch s postulates, five healthy plants of J. sigillata (2- to 3-year-old) with 5-8 leaves per plant were inoculated with conidial suspensions (104 conidia/mL) after wounded with a small pin as described by Desai et al. (2019), and the same number of healthy plants were wounded and sprayed with sterile distilled water as controls. Plants were sprayed regularly with distilled water every day and placed in a growth chamber at 25℃ with a 12-h fluorescent light/dark regimen. After 15 days, typical brown spot symptoms developed on inoculated leaves, but not on the controls. The fungus O. leptostyla was reisolated from the lesion as described above but not from non-inoculated leaves. O. leptostyla has been reported on some walnut trees; for example: J. ailantifolia, J. californica, J. cinerea, J. hindsii, J. major, J. mandshurica, J. nigra, and J. regia (Farr & Rossman 2020). However, to our knowledge, this is the first report of O. leptostyla causing brown leaf spot on J. sigillata. J. sigillata is an economically important tree in southwest China, and fungicide treatments should be considered to prevent the spread of this fungus before it becomes more widespread. Chunlin Yang, Yu Deng, and Feihu Wang contributed equally to this work. This research was supported by the Key Research and Development Project of Sichuan Province (2021YFYZ0032).