First Report of Ber (Zizyphus mauritiana) Leaf Spot Caused by Alternaria alternata in Pakistan

Ber (Zizyphus mauritiana Lamk.) is called the apple of the desert and is also known as the poor man’s fruit. In Pakistan, apart from direct consumption, the fruit is widely used in traditional medicines. It is cultivated on over 5,100 ha with a production of approximately 25,000 metric tons, but its production is continuously decreasing in the country (Government of Pakistan 2016). During February 2016, a survey was carried out in the ber growing areas of Lahore (31.5204°N, 74.3587°E) and Multan (30.1575°N, 71.5249°E). Leaves were observed with brown circular spots 3 to 6 mm in diameter with a gray to tan center and distinct brownish-yellow margins. Leaves with severe infection had lesions that gradually enlarged, coalesced, and covered the entire surface of the leaf, causing an early leaf drop. A total of 70 symptomatic leaves were surface sterilized with 1% sodium hypochlorite for 2 min and then rinsed thrice with sterile distilled water. The excised pieces of sterile tissues (4 mm²) were transferred to potato dextrose agar and incubated at 25 ± 2°C with a 12-h photoperiod. After 5 to 7 days, whitish brown fungal colonies were observed, which later turned dark-brown to black and produced abundant aerial hyphae. Conidiophores were short, septate, bent or sometimes branched, 25 to 55 μm long, and 2 to 3.5 μm wide. Conidia were borne singly or in short chains and were obpyriform to obclavate, 9.0 to 55.5 µm long × 6.2 to 24.60 µm wide with zero to three longitudinal and two to six transverse septa. Based on morphological characteristics, the fungus was tentatively identified as Alternaria alternata (Fr.) Keissl (Simmons 2007). To fulfill Koch’s postulates, pathogenicity tests were performed on 20 asymptomatic detached leaves by using two representative isolates, PAK45000_1 and PAK45000_2. The leaves were surface sterilized with sodium hypochlorite (1%) and then inoculated with a single-spore suspension (10⁶ conidia/ml) of each isolate. Leaves inoculated with sterile distilled water served as negative controls. The leaves were incubated at 25 ± 2°C in sterile glass chambers at 80% relative humidity, and spot symptoms were recorded on inoculated leaves 3 days after inoculation, whereas no symptoms were observed on the negative control leaves. The pathogen reisolated from the artificially inoculated leaves was morphologically indistinguishable from the pathogen used for inoculation. The experiment was conducted twice. For molecular identification, the internal transcribed spacer region (ITS1, 5.8S, and ITS2) of ribosomal DNA and the endopolygalacturonase gene (endoPG) of representative isolates (PAK45000_1 and PAK45000_2) were amplified with ITS1/ITS4 and PG3/PG2b primers, respectively (Andrew et al. 2009; White et al. 1990). Obtained sequences of ITS (MF972906 and MF972907) and endoPG (MF977271 and MF977272) were submitted to GenBank. BLAST analysis of sequences from both genes exhibited 99 to 100% genetic homology with previously reported sequences for ITS (HE579390 and HE579327) and endoPG gene region (EF504192 and KY969535) of A. alternata in NCBI, respectively. Previously, A. alternata has been reported to cause leaf spot in China (Bai et al. 2015), leaf spot on strawberry in Pakistan (Mehmood et al. 2018), and a postharvest fruit rot of ber in Pakistan (Alam et al. 2018). To our knowledge, this is the first report of A. alternata causing a leaf spot of ber in Pakistan. This finding will help to plan effective disease management strategies against Alternaria leaf spot because ber provides significant economic returns to small farmers in Pakistan.