First report of stem canker of <i>Mytilaria laosensis</i> saplings caused by <i>Lasiodiplodia theobromae</i> in southern China

Mytilaria laosensis is a fast–growing and high–yielding timber tree species widely grown in the southern subtropical region of China (Yu et al. 2023). In September 2024, a survey was conducted in Yingde City, Guangdong Province, southern China. Sapling stem canker symptoms were found on M. laosensis, and the incidence rate was 33.12% (investigated 1561 trees and infected 517 trees). The symptoms included dark brown necrotic spots that were sunken and shrunken, with cracked cortex and yellowing leaves. The epidermis was stripped from four stem pices (∼0.5 cm) between the healthy and infected tissues, the pieces were disinfected with 75% alcohol for two minutes, rinsed three times with sterile water, and dried for 10 minutes. They were then placed in Petri dishes on potato dextrose agar (PDA) medium and cultured at 28°C in the dark for 2–3 days to obtain pure cultures. Then, monosporic cultures were obtained (isolates MLP112, MLP414, MLP424, and MLP511). The fungal colonies were initially composed of white hyphae, after 5 days became gray and after 10 days turned black. Immature conidia were hyaline, aseptate, ellipsoid, thick–walled, and mature conidia elliptic or oblong, simple septate, dark brown in color, 19.3–31.3 μm in length and 9.2–15.7 μm in width, (mean length=23.9 μm, width average=11.9 μm, N=40), length–to–width ratio was 2.0. Based on morphological characteristics the fungus was temptatively identified as Lasiodiplodia theobromae (Alves et al. 2008). To confirm the indentity of the isolates, the internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1 (tef1) and beta tubulin genes were amplified with the universal ITS (White et al. 1990), EF1–α (Stielow et al. 2015) and β–tubulin (Glass &amp; Donaldson 1995) primers, respectively, and sequenced. The obtained sequences were deposited in Gen Bank (ITS: PV855669, PV855670, PV855671, PV855672; EF1–α: PV903164, PV903165, PV903166, PV903167; β–tubulin: PX564918, PX564919, PX564920, PX564921). BLAST results showed 100% similarity with reliable references of L. theobromae (ITS, OR116087.1; EF1–α, OR536247.1; β–tubulin, MT212400.1). To fulfill Koch’s postulates, 5 mm diameter mycelial plugs of PDA cultures were inoculated into eight M. laosensis stem segments, while four stem segments were inoculated with sterile PDA plugs and served as a control. Both ends of stem segments were sealed with paraffin, Segments were incubated at 28°C and 80% relative humidity. Within 5 to 7 days, inoculation sites exhibited obvious dark brown to black lesions and epidermal necrosis, consistent with symptoms of natural infections. The control stems remained asymptomatic. The fungus was isolated from the tissue and identified as L. theobromae based on morphology and DNA sequencing (ITS, tef1 and β–tubulin). The host range of L. theobromae include more than 500 plants species (Hattori Y. et al. 2023). To the best of our knowledge, this is the first report of L. theobromae causing sapling stem canker on M. laosensis in China. The results of this study will help to develop management strategies of M. laosensis sapling stem canker.