First report of Neopestalotiopsis clavispora Causing Leaf Spots on Garcinia mangostana in China

Garcinia mangostana L. is a famous tropical fruit in Asia. In April 2021, a leaf disease on G. mangostana cv. Huazhu was observed in Zhanjiang (21.17° N, 110.18° E), Guangdong province, China. Symptoms was on new leaves of 2 year old plants. The spots were circular to irregular, gray in the center, and brown on the lesion margin. The disease incidence was estimated 25% (n = 500 investigated plants from about 50-ha). Twenty diseased leaves were collected from the orchard. The margin of the diseased tissues was cut into 2 mm × 2 mm pieces; surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively; and rinsed thrice with sterile water. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. Twenty-eight isolates were obtained (isolation frequency = 28/4×20 = 35%). Single-spore isolation method was used to recover pure cultures for three isolates (GMN-1, GMN-2, and GMN-3) (Liu et al. 2021). The colonies were initially white with cottony aerial mycelium at 7 days on PDA. Then, they developed black acervular conidiomata at 10 days. Conidia were clavate to fusiform, four-septate, straight or slightly curved, and measured 16.5 to 21.4 µm long (average 19.5 µm; n = 40) × 4.5 to 6.5 µm wide (average 5.2 µm; n = 40). The three median cells were versicolored, whereas the basal and apical cells were hyaline. Conidia had a single basal appendage (4.5 to 5.5 µm long; n = 40) and three apical appendages (19.2 to 24.5 µm long; n = 40). The morphological characteristics of the isolates are comparable with those of the genus Neopestalotiopsis (Sajeewa et al. 2012). Molecular identification was performed using the colony polymerase chain reaction method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012). Sequences were generated from the isolates using primers for the rDNA ITS (ITS1/ITS4), TEF1-α (EF1-728F/EF1-986R), and β-tubulin (T1/βt2b) loci (Sajeewa et al. 2012). The sequences of the isolates were submitted to GenBank (ITS, MZ026535-MZ026537; TEF, MZ032203-MZ032205; β-tubulin, MZ032206-MZ032208). The sequences of the isolates were 100% identical to the type strain MFLUCC12-0281 (accession nos. JX398979, JX399014, and JX399045) through BLAST analysis. The isolates clustered with N. clavispora (MFLUCC12-0280 and MFLUCC12-0281). The pathogenicity was tested in vivo. Individual plants (cv. Huazhu) were grown (n = 2, 1-1.5 year old) in a greenhouse at 24 ℃-30 ℃ with 80% relative humidity. Wounded leaflets were inoculated with 5-mm-diameter mycelial plugs or agar plugs (as control). Besides, sterile cotton balls were immersed in the spore suspension (1 × 105 per mL) and sterile distilled water (control) for about 15 s before they were fixed on the leaves for 3 days. One plant employed for each isolate with nine leaves. The test was performed thrice. Disease symptoms were found on the leaflets after 10 days, whereas the controls remained healthy. The pathogen was re-isolated from infected leaves and phenotypically identical to the original isolates to fulfill Koch's postulates. Neopestalotiopsis clavispora and Pestalotiopsis clavispora are synonyms. The fungus appeared to have a wide host range and distribution including in Thailand, Malaysia, North Queensland, and Australia (Sajeewa et al. 2012;Shahriar et al. 2022). Thus, this is the first report of N. clavispora causing leaf spot on G. mangostana in China. This finding will help improve management strategies against the leaf spots on G. mangostana in China.