First Report of Pestalotiopsis clavispora Causing Leaf Spot on Pandanus amaryllifolius in China

Abstract

Pandanus amaryllifolius, also known as pandan, is a perennial herb, growing in Indonesia, China and the Maluku Islands (Wakte et al. 2009). It is the only plant with aromatic leaves in the Pandanaceae. It is widely used in food, medicine, cosmetics and other industries, and is also known as "Oriental Vanilla." Pandan is planted in Hainan province over 1,300 ha and is the main plant intercropped among the forest trees. From 2020, the leaf spot was surveyed for three years. Diseased leaves occurred on 30 to 80% of the surveyed plants, with an incidence of 70% and yield losses of 40%. The disease occured from mid-November to April and was most severe at low temperatures and humidity. Initial symptoms were pale green spots, that formed dark brown, nearly circular lesions. As the lesions expanded, their centers became greyish white, with yellow halos at the junction of the diseased and healthy tissue. When the humidity was high, there were small black spots scattered in the center of the lesion. Symptomatic leaf samples were collected from four different sites. The leaf surface was disinfested with 75% ethyl alcohol for 30 s and washed with sterile distilled water three times. Samples from the junction of diseased and healthy tissue (0.5 × 0.5 cm) were removed and placed on potato dextrose agar (PDA) medium containing 100 µg/mL of cefotaxime sodium and cultivated in a dark incubator at 28°C. After two days, hyphal tips from the edges of growing colonies were transferred to fresh PDA plates for further purification. Following Koch's postulates, colonies from strains were used as inoculum in pathogenicity tests. Colonies with 5 mm diameter were inoculated upside onto fresh and healthy pandan leaves via wounding method (pinpricked by sterilized needles) and non-wounding method. Sterilized PDA was used as control. All plants were setted three replicates and were incubated at 28℃ for 3 to 5 days. When symptoms on leaves similar to those in the field appeared, the fungus were reisolated The colonies formed on PDA were also consistent with the original isolate (Scandiani et al, 2003). After seven days, the colony covered the whole petri dish with white, petal-shaped growth with a slight concentric, annular bulge in the center, irregular edges, with black acervuli emerging at a later stage of colony growth. Conidia were fusiform, 18.1±1.6 × 6.4±0.3 μm, showing four septations and five cells, the middle three cells were brownish black to olivaceous, and the apical cell colorless with two to three filaments, 21.8±3.5 μm long. The caudate cell was colorless with one stalk 5.9±1.8 μm long (Zhang et al. 2021; Shu et al. 2020). According to the colony and conidia characteristics, the pathogen was initially identified as Pestalotiopsis spp. (Benjamin et al. 1961). To confirm the pathogen identity, we used the universal primers ITS1/ITS4, targeting primers EF1-728F/EF1-986R and Bt2a/Bt2b sequences (Tian et al. 2018). The sequences of the PCR products were deposited in NCBI GenBank with accession numbers OQ165166 (ITS), OQ352149 (TEF1-α) and OQ352150 (TUB2). BLAST results showed that the sequences of the ITS, TEF1-α and TUB2 genes shared 100% homology with the sequences of Pestalotiopsis clavispora. The maximum likelihood method was used in the phylogenetic analysis. The result showed that LSS112 was clustered with Pestalotiopsis clavispora with a support rate of 99%. Based on morphological and molecular characteristics, the pathogen was confirmed as Pestalotiopsis clavispora. To our knowledge, this is the first report of leaf spot of pandan caused by Pestalotiopsis clavispora in China. This research will be immediately helpful for the diagnosis and control the disease on pandan.