Leaf Spot Disease of Syzygium cumini Caused by Neopestalotiopsis clavispora Newly Reported in India

Abstract

Syzygium cumini (L.) Skeels (Myrtaceae), commonly known as jambolan or Java plum, is a fast-growing, popular indigenous minor fruit of India. Its leaves, fruits, and seeds have immense medicinal value for treatments of diabetes, chronic diarrhea, enlargement of spleen, ringworm infection, and so on. During May 2018, severe outbreaks of leaf spots occurred on S. cumini plantations in the Bidhan Chandra Krishi Viswavidyalaya, West Bengal, India. Disease incidence ranged from 50 to 100% in some areas of the cultivated orchards. Leaf spot began as a black dot with a distinct yellow halo. These dots enlarged to a diameter of 3 to 5 mm and progressed to form deep brown, circular, elliptical or irregular lesions with a dark black halo around the margin. In a humid environment, black, sessile, and discoid conidiomata developed on the spots and exuded a pink spore mass that turned brown with age. Infected leaves were surface sterilized, plated onto potato dextrose agar (PDA), and incubated under 12-h/12-h cycles of light and darkness for 7 days at 22 to 24°C. The fungal colonies on PDA grew fast, covering the entire plate with a velvety white mycelium with numerous black acervuli. The colony reverse side showed light orange tonalities. Conidia were fusiform (average 26 × 8 µm) (n = 50) and five-celled. Apical and basal cells were hyaline, and the three median cells were dark brown. Conidia showed one short basal and two to four long apical appendages. The identification was confirmed by amplifying and sequencing the rDNA ITS1-5.8S-ITS2 region (GenBank accession no. MN367329) and the D1/D2 region in the 5′ end of the 28S rDNA gene (GenBank accession no. MN371275). BLAST analysis showed in both cases 100% identity with existing sequences of Neopestalotiopsis clavispora (GenBank accession nos. MH423984 and KC154000). The morphological (Maharachchikumbura et al. 2014) and molecular data corresponded to those of N. clavispora. Koch’s postulates were fulfilled for the fungus by spray inoculating two healthy young plants with 2 × 10⁶ conidia per milliliter of sterile distilled water. As a control, two similar plants were sprayed with sterile water. Plants were placed inside plastic bags to maintain humidity and incubated in a growth chamber at 25°C under fluorescent light for 14 h and at 20°C in darkness for 10 h. After 3 days, plastic bags were removed, and plants were maintained under the same conditions. More than 15 days after inoculation, the symptoms described above were observed on the leaves of the inoculated plants, whereas these symptoms did not develop on the control leaves. Cultures isolated from the leaf spots were similar to those isolated previously from plants in the orchard. Several species of Pestalotiopsis were recorded as one of the major problems of S. cumini, including postharvest fruit rot (Srivastava and Mehra 2004), foliar blight (Bhanumathi and Rai 2007), and bark infection (Abbas et al. 2013). Although a leaf blight disease caused by Pestalotiopsis sp. in India was reported in 2007 (Bhanumathi and Rai 2007), the pathogens and symptoms of these two diseases on S. cumini are different according to a comparison of the present study with that of Bhanumathi and Rai (2007). First, the disease symptom observed in our study was in matured plants, whereas in Bhanumathi and Rai (2007) it was S. cumini seedlings. Second, the characteristic symptoms observed in our study were typical 3- to 5-mm-sized leaf spots with a clear dark black halo around the margin of lesions, not previously reported (Bhanumathi and Rai 2007). Third, there were differences in the cultural and morphological characteristics of the mature conidia between the two Pestalotiopsis-like species, including color, conidiogenic cells number and size, and appendages observed under a compound microscope. Therefore, to the best of our knowledge, this is the first report of leaf spot disease on S. cumini caused by N. clavispora in India.