First Report on Neopestalotiopsis aotearoa of Rubber Tree in China

Natural rubber is an important industrial raw material and an economically important perennial in China. In recent years, A new leaf fall disease, caused by Neopestalotiopsis aotearoa Maharachch., K.D. Hyde & Crous, has occurred in Indonesia, Malaysia, Thailand, Sri Lanka, and other major rubber planting countries. In May and July of 2020, this disease was first found on 2-year-old rubber seedlings in two plantations located in Ledong and Baisha counties in Hainan Province, China. In the two plantations of approximately 32 ha, 15% of the rubber seedlings had the disease and the defoliation was more than 20%. The infected leaves turned yellow and watery, and dark brown and nearly round lesions of 1-2 mm in diameter were formed on the leaves. When the humidity was high, the center of the lesion was grey-white, and the lesions had many small black dots, black margins and surrounded by yellow halos. When the disease was severe, leaves fell off. To identify the pathogen, leaf tissues were collected from lesion margins after leaf samples were surface-sterilized in 75% ethanol, rinsed with sterile water for three times, and air dried. The leaf tissues were plated on potato dextrose agar (PDA) and incubated at 28°C for seven days. Fungal cultures with similar morphology were isolated from 90% of tested samples and two isolates (HNPeHNLD2001 and HNPeHNLD2002) were used in pathogenicity and molecular tests. Rubber leaves (clone PR107) were inoculated with conidial suspension (106 conidia/ml), and inoculated with PDA were used as the control, Each treatment had 3 leaves, and each leaf was inoculated with 3 spots and incubated at 28oC under high moisture conditions. Five days later, leaves inoculated with conidial suspension showed black leaf spots resembling the disease in the field, whereas the control leaves remained symptomless. The fungal cultures isolated from the inoculated tissues, had identical morphology compared with the initial isolates. Colonies on PDA were 55-60 mm in diameter after seven days at 28°C, with undulate edges, pale brown, thick mycelia on the surface with black, gregarious conidiomata; and the reverse side was similar in color. Black conidia were produced after eight days of culture on PDA. Conidia were fusoid, ellipsoid, straight to slightly curved, 4-septate, ranged from 18.35 to 27.12 μm (mean 22.34 μm) × 4.11 to 7.03 μm (mean 5.41 μm). The basal cells were conic with a truncate base, hyaline, rugose and thin-walled, 4.35 to 6.33 μm long (mean 4.72 μm). Three median cells were doliform, 12.53 to 18.97 μm long (mean 15.26 μm), hyaline, cylindrical to subcylindrical, thin- and smooth-walled, with 2-3 tubular apical appendages, arising from the apical crest, unbranched, filiform, 14.7 to 25.3 μm long (mean 19.94 μm). The basal appendages were singlar, tubular, unbranched, centric, 3.13 to 7.13 μm long (mean 5.48 μm). Morphological characteristics of the isolates were similar to the descriptions of N. aotearoa (Maharachchikumbura et al. 2014). The rDNA internal transcribed spacer (ITS) region, translation elongation factor 1-αgenes (TEF), and beta-tubulin (TUB2) gene were amplified using the primer pairs ITS1/ITS4, EF1-728F/EF1-986R and T1/Bt-2b (Pornsuriya et al. 2020), respectively. The sequences of these genes were deposited in GenBank (ITS Accession Nos.: MT764947 and MT764948; TUB2: MT796262 and MT796263; TEF: MT800516 and MT800517). According to the latest classification of Neoprostalotiopsis spp. (Maharachchikumbura et al. 2014) and multilocus phylogeny, isolates HNPeHNLD2001 and HNPeHNLD2002 were clustered in the same branch with N. aotearoa. Thus, the pathogen was identified as N. aotearoa, which is different from N. cubana and N. formicarum reported in Thailand (Pornsuriya et al. 2020; Thaochan et al. 2020). The Neopestalotiopsis leaf spotdisease of rubber tree (H. brasiliensis) was one of the most serious and destructive leaf diseases in major rubber planting countries in Asia. ( Tajuddin et al. 2020) The present study of leaf fall disease on rubber tree caused byN. aotearoa is the first report in China. The finding provides the basic pathogen information for further monitoring the disease and its control.