First report of Neopestalotiopsis clavispora Causing guava scab in China.

Abstract

Psidium guajava L. is widely cultivated in southern China. In May 2021, guava scab on cv. Zhenzhu was observed in Zhanjiang (21.18° N, 110.21° E), Guangdong province, China. Guavascab was corky with ovoid or round lesions on the surfaces of green fruits. Gradually the lesions sunk. Disease incidence was estimated as 85% in 500 investigated plants in about 50 ha. Twenty diseased fruits were collected from twenty trees in the field. From each fruit 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, successively; and rinsed thrice with sterile water. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. Thirty-four isolates were obtained. Single-spore isolation method (Liu et al. 2021) was used to recover pure cultures of three isolates (PGNC-1, PGNC-2, and PGNC-3) . The colonies were initially white with cottony aerial mycelium at 7 days on PDA. Then, these colonies form black acervular conidiomata at 10 days. Conidia were clavate to fusiform, four-septate, straight or slightly curved, and measured 15.8 to 21.2 µm × 4.5 to 6.5 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 were consistent with the description of Neopestalotiopsisclavispora (Maharachchikumbura et al. 2012). Molecular identification was performed using PCR 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 (Maharachchikumbura et al. 2012). The sequences of the isolates were submitted to GenBank (ITS, OQ996557 to OQ996559; TEF, OR101037 to OR101039; β-tubulin, OR100971 to OR100973). 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). N. clavispora and Pestalotiopsis clavispora are synonyms. The pathogenicity was tested in vivo. Plants (cv. Zhenzhu) were grown ( 3 years old) in a quarantine orchard at 25℃ to 32℃ with 60 to 80% relative humidity in May 2022. Disease-free green fruits were inoculated. 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 wounded fruits with transparent tape. Five fruits on one plant per isolate were inoculated. Five fruits on one plant severed as control. The test was performed thrice. Disease symptoms were found on the inoculated fruits after 20 days, whereas the controls remained healthy. The pathogen was re-isolated from infected fruits and was phenotypically identical to the original isolates thus fulfilling Koch's postulates. Neopestalotiopsis or Pestalotiopsis spp. were reported to be the causal agents of guava scab in Colombia and in Hawaii (Keith et al. 2006; Solarte et al. 2018). N. clavispora has been reported to cause disease in a broad range of hosts (Ge et al. 2009; Chen et al. 2018), but not in guava. This is the first report of N. clavispora causing guava scab in China. There would be no harvest if this disease is left unmanaged.