First Report of Heart Rot Disease on Pineapple Caused by <i>Phytophthora nicotianae</i> in Guangxi Province, China

Pineapple (Ananas comosus) is a perennial economically important tropical fruit in Guangxi Province, China. Between September to November 2022, the rot of heart leaves of pineapple was found on 6.7 hectares of pineapple, NaLang village Jiangxi Town, Jiangnan District, Nanning City and on 200 hectare Liutian Village, Wangmao Town, Bobai County, Yulin City. The disease incidence of the investigated 10500 plants was from 20% to 30%. The basal heart leaves near the soil surface showed symptoms of water-soaked lesions and soft rot. The infected heart leaves were easily pulled out. As the disease developed, 800 plants of the investigated diseased plants collapsed and died. The diseased leaves from each district were surface-disinfected for 3 min with 1.0% hypochlorite solution, then rinsed three times in sterile water, and plated to carrot agar medium (CA media) (Luo et al. 2012). Inoculated dishes were incubated at 28°C in the dark. After 5 days, Eight mycelium colonies were recovered from diseased tissue. Colonies developing on CA exhibited white cottony mycelium which was aseptate. Numerous sporangia and chlamydospores were visible after 10-15 d later. The chlamydospores were spherical in shape and measured an average of 25.8 to 37.5 μm. The shape of the sporangia was papillate, oval or spherical, and 34.5 to 58.2 μm. These characteristics were consistent with the description of Phytophthora (Stamps et al. 1990). For molecular identification, the direct colony PCR method (Lu et al. 2012) was used to amplify the ITS of rDNA, β-tubulin (Tu), and elongation factor (EF) locus of the 2 strains NNPN1 from Nanning city and YLPN1 from Yulin city using the primer pairs of ITS4/ITS5, T1/T2 (O'Donnell et al. 1997) and EF1/EF2 (O'Donnell et al. 1998). The resulting sequences were deposited in GenBank (OR612028 and OR612029 for ITS; OR620158 and OR620159 for elongation factor; OR620160 and OR620161 for β-tubulin). BLAST searches with the three gene sequences revealed the greatest similarity with the sequences of some Phytophthora sp. The complete genome sequences of highly similar Phytophthora sp. were downloaded and the sequences of the corresponding 3 genes of each genome was extracted by Bioedit software. Phylogenetic trees were constructed in MEGA 11 using the maximum likelihood (ML) method based on the concatenated sequences of ITS, EF1α, and Tubulin (Figure 1). The 2 isolates grouped with P. nicotianae in the phylogenetic tree. The morphology and multi-gene phylogenetic analysis indicated that the new isolate was P. nicotianae. Tests of pathogenicity were performed according to Koch's postulates using the P. nicotianae strains NNPN1 and YLPN1. Fresh wounds were made symmetrically with a sterile needle on 2 tender basal leaves of pineapple plants (Bali variety) at the 8- to 10-leaf stage and each wound was covered with a piece of mycelium disc from each isolate. Control seedlings were inoculated identically except CA medium disc was used to cover the wound. Plants were placed in pots in a greenhouse at 28°C and 90% relative humidity. After 12 days, soft rot was observed clearly on the base of heart leaves of 9 inoculated plants, while the 9 control plants appeared normal. The pathogenicity tests were conducted three times with similar results. The strains were then reisolated from the infected plants and found to be Phytophthora sp. as those of the inoculum by morphology. P. nicotianae was previously reported as the causal agent of heart rot of pineapple in Guangdong and Hainan Province of China (Luo et al. 2012; Shen et al. 2013). To our knowledge, this is the first report of P. nicotianae on pineapple in Guangxi province in China. Identification of the pathogen of heart rot disease on pineapple is vital to reduce yield losses of the disease using effective control method.