Dwiroopa punicae Causing Leaf Spot Disease on Pomegranate in Guizhou, China

Punica granatum 'Tunisia' is widely planted in several areas of Kaiyang County, Guizhou Province, China. Symptoms of leaf spot and fruit rot were observed in April 2020. Infected pomegranate leaves exhibited scattered, oval, yellow-to-brown spots (2-5 mm), and numerous melanized pycnidia were observed in the center of the lesions. All 7-year-old pomegranate trees in a private orchard (Shangyi orchard, E27°09'08", N107°10'58"), exhibited the same symptoms, and 25% of the diseased pomegranate trees had blighted leaves in the late stage. Eighty diseased P. granatum leaves were collected in the orchard. Abundant fruiting bodies were observed on the surface of diseased spots examined using a dissecting microscope. Portions of the symptomatic leaves were surface disinfected with 75% ethanol for 30 s and then in 2% NaClO for 1 min, rinsed three times with sterile distilled water, and transferred onto potato dextrose agar (PDA) at 28°C for 3 weeks. The morphological characteristics of potential pathogens were observed and photographed under a compound light microscope (Zeiss Scope 5) equipped with an AxioCam 208 color camera. Pycnidial conidiomata were erumpent, globose, black, with a central ostiole, and oozed a slimy black conidial mass. Conidiophores were reduced to conidiogenous cells, ampulliform to subcylindrical, hyaline to pale brown, and proliferated percurrently at the apex. Macroconidia were solitary, one-celled, obovoid, truncate at the base, rounded at the apex, dark brown, aseptate, granular, and thick-walled, (11.0-)12.8-18.2(-19.0) × (9.2-)10.0-15.1(-16.0) μm (av: 14.8 × 12.0 μm, n = 30). No meso- or microconidia were observed. In morphology, our fungi were very similar to Dwiroopa punicae K.V. Xavier, A.N. Kc, J.Z. Groenew, Vallad & Crous (Xavier et al., 2019). Primers ITS4 and ITS5 were used to amplify the ITS, LROR and LR5 were for the LSU (White et al. 1900), and fRPB2-5F and fRPB2-7cR for rpb2 (Liu et al. 1999). Phylogenetic analysis based on these three loci also suggested that our strain was D. punicae, since there were 100% match for ITS and LSU and only two base differences in rpb2 gene between our strain and the D. punicae (CBS 143163). The qualified sequences were submitted to GenBank (ITS: MZ816695, MZ816696, MZ816697; LSU: MZ816692, MZ816693, MZ816694; rpb2: MZ802953). To fulfill Koch's postulates, mycelial plugs (5 mm diameter) were used for an inoculation experiment, only because the fungal isolates failed to produce conidia on PDA, and conidia from diseased tissues were also fewer to make the required spore suspension. A total of 20 attached leaves were used from three pots of pomegranate seedlings in the artificial climate chamber (25°C). Each mycelial plugs cut from 1-week-old PDA cultures were placed on one healthy leaf wrapped with a plastic fresh-keeping film. As control, leaves were inoculated with sterile PDA plugs. After 24h, the plugs were removed. The inoculation experiment was performed three times. Symptoms similar to the original were observed at the 7th day after inoculation on all inoculated plant leaves and necrosis occurred in 30-50% of leaf tissue, but control plants remained healthy. Dwiroopa punicae was successfully re-isolated from the inoculated diseased tissues after morphological and phylogenetic analyses. To the best of our knowledge, this is the first report of D. punicae causing leaf spot disease on pomegranate trees in China. This first report of D. punicae in China provides a basis for the diagnosticians and researchers to identify the disease and formulate disease management strategies.