First Report of Lasiodiplodia theobromae Causing Brown Leaf Spot on Bruguiera gymnorrhiza in China.

Abstract

Bruguiera gymnorrhiza (Linn.) Sav. is a dominant tree species of mangrove forests in tropical coastal areas of China. This species is commonly used for the greening of tidal flats and seawalls in tropical and subtropical regions (Allen, & Duke 2000). A survey that was conducted from August to September 2020, in the mangrove national nature reserve at Zhanjiang, Guangdong Province, South China. Brown leaf spot symptoms were observed on Bruguiera gymnorrhiza and disease incidence was over 10% (200 investigated trees). Symptomatic small spots initially appeared at the middle or edges of leaves, enlarged irregularly, and developed into brown necrotic spots with dying curly edges. The color of the lesion's center changed to dark brown or gray. To identify the causative agent, twenty diseased leaves were sampled for pathogen isolation. Affected foliar tissues cut into 5 × 5 mm pieces, disinfected in 75% ethanol for 2 mins, rinsed in sterile distilled water, and then air dried under a sterilized filter paper. Leaf pieces were plated onto potato dextrose agar (PDA) in Petri dishes and then incubated at 28°C in darkness for 3-5 days. Hyphal tips of fungal colonies growing from the tissue pieces were subcultured onto fresh PDA to obtain pure single hyphae cultures. The fungal colonies were initially composed of white aerial mycelia, but turned gray after 7 days. Immature conidia were hyaline, subovoid, and aseptate while mature conidia becoming dark brown, one-septate with longitudinal stripes, the length/width ratio is 19.98 to 29.50 μm (average 24.37 µm; n = 50) × 11.99 to 14.45 μm (average 13.09 µm; n = 50). On the basis of morphological features all isolates were identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl (Alves et al. 2008). For DNA-based identification, the internal transcribed spacer (ITS) region gene and fragment of elongation factor 1-alpha (EF1-α) gene of the three isolates were amplified and sequenced following the methods described in a previous study (White et al. 1990, Carbone & Kohn 1999). The obtained sequences of ITS and EF1-α were deposited in GenBank with accession numbers OK644200 and OL345571. The BLAST results showed at least 99.60% similarity with the sequences of Lasiodiplodia theobromae (ITS, MT644474.1 [99.79%]; EF1-a, MK961975.1 [99.60%]). To fulfill Koch's postulates, PDA plugs with actively growing mycelium of the isolates were inoculated on the leaves of Bruguiera gymnorrhiza plants that were wounded by using a sterilized needle or scalpel. Inoculated leaves were covered with sterilized wet cotton, and the plants were kept at 28°C and 80% relative humidity. The inoculated plants showed leaf spot symptoms that were similar to those previously observed in the field after 1-2 days, whereas control leaves remained healthy. Lasiodiplodia theobromae was consistently isolated from inoculated leaves again. Lasiodiplodia theobromae (Botryosphaeriaceae) is a plurivorous pathogen in a wide variety of hosts, mostly prevalent in tropical and subtropical climate regions. It has been previously reported to cause brown leaf spot on Broussonetia papyrifera (Luo et al. 2020), foliar diseases on Camellia oleifera (Zhu et al. 2014) and Kadsura longipedunculata (Fan et al. 2020). To our knowledge, this is the first report of Lasiodiplodia theobromae causing brown leaf spot on Bruguiera gymnorrhiza plants in China and worldwide. Our findings will help to make management strategies for control of this disease on Bruguiera gymnorrhiza.