First report of Neopestalotiopsis clavispora causing leaf blight on Aegicerascorniculatum in China.

Abstract

Aegicerascorniculatum is an important mangrove plant that mainly grows in tropical and subtropical regions. Ithasimportantecological,economicandsocialbenefits (Bandaranayake 1998). In April 2021, a leaf disease on A.corniculatum was observed in Zhanjiang (21.21° N, 110.41° E), Guangdong province, China. Diseaseincidenceandseverity were 15%and 20% (n = 100 investigated plants), respectively. The disease symptoms on leaves primarily appeared as small brown spots, then enlarged and coalesced into regular or irregular gray necrotic lesions with dark margins. At the late stage of symptom development, black acervuli appeared on the necrotic lesions. Ten symptomatic leaves from 10 plants were collected. Small pieces of tissue (4 × 4 mm) were cut from lesion borders and were surfaced disinfected in 75% ethanol for 30 s, followed by 1 min in 1% NaClO, rinsed three times with sterile water, plated on potato dextrose agar (PDA), and incubated at 28°C. After 7 days, a total of 10 fungal isolates with 100% isolation frequency were obtained and three representative strains (THS-1, THS-2, and THS-3) were used for morphological and molecular characterization.Colonies were white with cottony aerial mycelium and irregular margins. Black viscous acervuli were scattered on the colony surface 10 days after incubation. Conidia were spindle shaped, five cells, four septa, 18.77-28.70 × 4.53-6.80μm (mean 23.13 × 5.14μm) in size (n = 50). Basal and apical cells were colorless while the three medium cells were dark brown and lightly versicolor. All conidia had one basal appendage of 3.51-7.27μm (mean 5.49 μm; n = 50) and two to three apical appendages of 15.80-33.64μm (mean 25.87 μm; n = 50). These morphological characteristics are consistent with those of Neopestalotiopsis clavispora(Maharachchikumbura et al. 2012). The ITS (OM698813-15), tub2 (OM810165-67) andtef1α(OM810186-88) sequences were 99.38%, 99.09%, and 99.17% identical to the type N.clavispora strain MFLUCC12-0281 (accession nos. JX398979, JX399014, and JX399045) through BLAST analysis, respectively. A phylogenetic tree was generated using the concatenated sequences of ITS, tub2, and tef1α. The result showed that these three isolates were clustered with N.clavispora strains including the type MFLUCC12-0281. To perform pathogenicity tests, 20 healthy potted seedlings of A.corniculatum (2-year-old) were selected. Ten surface-sterilized leavesof 10 seedlings were wounded and inoculated by spraying conidial suspension ( 105conidia/ml). The same number wounded leaves of the other 10 plants treated with sterile water served as controls. All plants were wrapped in polyethylene bags for 24 h and incubated at 28°C in a growth chamber (at 90% relative humidity). After 10 days, all the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic. N. clavispora was reisolated from the lesions in terms of morphology and molecular characterization, whereas no fungus was isolated from the control leaves. The pathogenicity test was repeated three times under the same conditions. Thus, Koch's postulates were fulfilled. This pathogen has been reported on a wide host range worldwide, such as leaf spot on strawberry in China (Zhao et al. 2016) and twig blight on blueberry in Spain (Borrero et al. 2018). This is the first report of N. clavispora causing leaf blight on A.corniculatum in China.This study provides valuable information for the identification and control of the disease.