First report of Causing Fusarium Dieback on in China.

(Jack) Benth. (common name: Monkey-pod) is a perennial arbor tree belonging to the family Fabaceae. It is used to produce a traditional medicine to treat a variety of heat toxicity symptoms in China. The distribution of mainly include Guangdong, Fujian, and Zhejiang provinces in China as well as other countries in Southeast Asia. In 2018, about 30 Monkey-pod trees were observed showing typical dieback symptoms in a commercial plantation (114°36'09.401″E, 22°58'38.553″N) of Huizhou, Guangdong Province, China. In addition, white cylindrical sawdust exudates from exit holes of ambrosia beetles were observed on the trunks and main branches of the diseased trees. Discoloration was seen around the exit holes of the beetles by splitting the wood. Samples of symptomatic and asymptomatic wood tissues of branches, trunks, and roots were collected from 11 trees. To identify the pathogen, the samples were disinfected with a sodium hypochlorite solution containing 50 g/l chlorine for 60 s, rinsed three times with sterile distilled water, and placed on potato dextrose agar (PDA) for culture at 25℃ for 14 days. Beetles from exit holes of the pathogen-infected wood tissues were also collected and identified as by their cytochrome c oxidase I (COI) sequences (GenBank accession numbers: MT921005 and MT921006) as well as morphological features. After two weeks incubation, 37 single-spore isolates were obtained (31 from galleries of branches and trunks, and 6 from mycangia and the body surface of beetles). Colonies on PDA were fusarium-like, pale orange, floccose with abundant aerial mycelia, and growing at the rate of 3.0 to 4.5 mm/d at 25℃. Conidiophores from the aerial mycelia often had single phialides. Microconidia were hyaline, 0-1 septate, (4-)6-12(-16) × 2-4 μm (n=100), Macroconidia were hyaline, ellipsoidal to falcate, 2-4 septate, (10-)25-45(-50) × 3-6 μm (n=100), with basal foot cells shaped to pointed and apical cells tapered and curved. Two representative isolates (CYML 367, 368) were used for molecular identification and pathogenicity tests. The internal transcriptional spacer region (ITS), β-tubulin (TUB), and translation elongation factor-1α (TEF 1-α) regions were amplified with primers ITS1F/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and EF1-728F (Carbone and Kohn 1999) /EF-2 (O'Donnell et al.1998), respectively. DNA extraction and PCR conditions were followed the methods described by Yin et al. (2019). The amplified fragments were sequenced. The results of BLAST demonstrated that the fragment sequences of ITS (468 bp), TEF 1-α (616 bp), and TUB (295 bp) of isolates CYML367 and CYML368 had 100% similarity to correspondence sequences from the Schlecht. emend. Snyder & Hansen species complex (GenBank accession numbers MT032694, JF740777, and MN451171, respectively). Sequences generated from this study were deposited into GenBank under accession numbers MN826824 and MN826825 (ITS), MN839680 and MN839681 (TUB), and MN839682 and MN839683 (TEF 1-α). Pathogenicity tests were conducted on 2-year-old seedlings (60-70 cm height, 1.0-1.2 cm in diameter) of with the representative isolates. Ten seedlings were inoculated with each isolate. The stem surface was disinfected with 75% ethanol for 30 s, and rinsed with sterilized water, wounded by removing part of the phloem and xylem, and placed with a mycelial plug (5 mm diameter) from the margin of a 7-day-old PDA plate with mycelia facing the cambium. The inoculated wounds were wrapped with a medical sterile gauze to prevent desiccation and contamination. Control plants were inoculated with non-colonized PDA plugs. These inoculated and control plants were incubated in a chamber at 25℃, 50% humidity, 12 h light/12 h dark cycle. After 3 weeks, all inoculated plants displayed Fusarium dieback symptoms similar to those observed on the original diseased trees in the plantation. The average lesion lengths (1.2/1.7 cm) caused by two isolates were all significantly longer than the wounds in the negative controls (P<0.05). The same fungus was re-isolated from the symptomatic plants, thus fulfilling the Koch's postulates. Further studies should be conducted on the potential vector association, transmission route, and management strategies of this pathogen on in growing regions of China. To our best knowledge, this is the first report of causing dieback on in China.