First Report ofPyricularia oryzae Causing Blast onWild Rice (Oryza rufipogon) in China.

Abstract

Wild rice (Oryza rufipogon) is an excellent genetic resource for rice breeding programs. In June 2019, typical symptoms of blast on the leaves of wild rice cv. 'Haihong-12' were observed in a 3.3-ha field in Zhanjiang (20.93° N, 109.79° E), China. The symptoms included fusiform lesions with yellowish halo at the age of lesion, grayish-white color at the center, brown and elongated central veins at both ends of lesion, and grayish-white mold layer formed on the back of lesion under humid weather conditions. Disease incidence was more than 10%. Thirty diseased leaves were collected, and infected tissues were cut into 2 × 2 mm pieces, surface disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s and rinsed three times with sterile water. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 °C in darkness for 3 days. Three single-spore isolates (Pos-1, Pos-2, and Pos-3) were obtained using the method described by Jia (Jia 2009) and were subjected to further morphological and molecular identification. Colonies on PDA were light grey, with cottony mycelium. Conidiophores were solitary, erect, straight or curved, septate, and pale brown and measured 68 to 125×3 to 4 µm. Conidiogenous cells were sympodial and denticulate. Conidia were pale brown, pyriform, and 18.2 to 42.4×5.1 to 8.5 µm (n=30) in size, with two septa. Appressoria were spherical and had the size ranging 4.3 to 6.5×4.7 to 6.5 µm (n = 20). These morphological features agreed with the previous description of Pyricularia oryzae Cavara (Klaubauf et al. 2014). For molecular identification, the colony PCR method with MightyAmp DNA Polymerase (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS), calmodulin (CAL), actin (ACT), -tubulin (TUB) loci of the isolates using primer pairs ITS1/ITS4, CL1C/CL2C, ACT-512F/ACT-783R, and T1/βt2b, respectively (O'Donnell et al. 1997; Weir et al. 2012; White et al. 1990). Analysis of ITS (acc. nos. MW042176 to MW042178), ACT (MW091444 to MW091446), CAL (MW091447 to MW091449), and TUB (MW091441 to MW091443) sequences revealed 100% identity with the corresponding ITS (MH859782), ACT (MH589787), CAL (MH589663), and TUB (MH589547) sequences of P. oryzae in GenBank. A phylogenetic tree was generated based on the ITS sequences using maximum likelihood method that clustered Isolates Pos-1, Pos-2, and Pos-3 with known P. oryzae. Thus, the isolates were identified asP. oryzae. Pathogenicity tests were performed using Isolate Pos-1 in a greenhouse at 24to 30°C with 80% relative humidity. Individual rice plants (cv. 'Haihong-12') with three leaves were grown in 10 pots, with 50 plants per pot (40 × 60 cm). Five pots were spray inoculated with a spore suspension (105 spores/ml) until runoff from leaves, and the remaining five pots were sprayed with sterile water to serve as the controls. The test was conducted three times. Disease symptoms were observed on 10% of leaves at 10 days after inoculation, but the control plants remained healthy. The fungus was re-isolated from the diseased plants and morphologically identified as P. oryzae. Thus, this is the first report of P. oryzae causing blast on O. rufipogon in China. The results provide the information that can be used by rice breeders and fungal geneticists for further studies.