First Report of causing wilt disease of guava in Guangdong, China.

Guava (Psidium guajava L.) is a tropical fruit with great economic value. Guangdong is one of the most important guava production areas. In November 2019, guava wilt disease (GWD) was observed in a 10.6 HA commercial orchard in NanSha district, Guangzhou, Guangdong (22°37'37.626" N, 113°35'56.089" E). Disease incidence was up to 35%. Initially, leaves on the top of some branches became purple or yellow interveinal chlorosis, later dry. Infection severely became systemic developing vascular discoloration of stem, black root rot, eventually entire trees wilted and died. The root tissues were cut into 5-mm2 pieces and surface disinfected with 70% ethanol for 30 sec, 3 % sodium hypochlorite for 4 min, rinsed by the sterile water, then plated onto potato dextrose agar and incubated for 5 days at 25°C. A total of 8 monoconidial isolates with identical colony morphology were obtained. All formed cottony, whitish to pale yellow colonies. Conidiophores were dimorphic, penicillate and acremonium-like. Penicillate conidiophores gave rise to ovoidal, one-celled conidia (4.15 to 6.55×2.28 to 4.61 μm) (n=100) with truncated ends. Cylindrical or fusiform conidia (7.02 to 15.57×2.01 to 5.30 μm) (n=100) arose in long chains on acremonium-like conidiophores. Morphological characteristics of the isolates were consistent with those of Nalanthamala psidii (syn. Myxosporium psidii) reported by Schroers (2005). The rDNA internal transcribed spacer (ITS) and partial nuclear large-subunit ribosomal DNA (LSU) of two representative isolates (GDNS02 and GDNS08) were amplified using the primers pairs ITS4/ITS5 (White et al. 1990) and V9G/LR5 (de Hoog and Gerrits van den. 1998), respectively. The obtained sequences were deposited in GenBank under the accession nos. OM278372 to 73 (ITS) and OM278377 to 78 (LSU). BLASTn analysis showed 99.81% and 100% identities with the reported sequences of N. psidii CBS 116952 (AY864836) and CBS 110507 (AY554243). Maximum likelihood analyses of combined ITS and LSU sequences indicated that these two isolates being clustered with N. psidii strains. Pathogenicity tests were performed twice using healthy seedlings (60-70 cm height, cv. pearl). Each stem of five seedlings was wounded using a 5-mm sterile cork borer, and 5-day-old mycelium plugs of isolate GDNS08 were inoculated into the holes (25-cm above the soil line) and covered with Parafilm, sterile PDA plugs were placed into the wounds of additional 5 control seedlings. All plants were kept in a greenhouse (25℃, 80% relative humidity, 16/8-h day/night). After 3 months, all inoculated plants developed purple leaf, defoliation and wilt symptoms resembling those observed in the orchards, while the controls remained asymptomatic. Nalanthamala psidii was reisolated from the roots tissue of the inoculated plants, identity was confirmed by morphological characteristics and ITS sequence analyses as described above, but not from the controls, fulfilling Koch's postulates. Nalanthamala psidii has been previously reported as the causal agent of guava wilt in Taiwan, Philippines, South Africa and Bangladesh (Hsieh et al. 1976; Opina 1995; Schoeman et al. 1997; Alam et al. 2019). To our knowledge, this is the first report of N. psidii causing guava wilt in Guangdong, China. The outbreak of GWD in South Africa in the 1980s resulted in devastating losses to guava industry (Schoeman et al. 1997). Further research is needed to develop the integrated management to constrain this disease from spreading.