First Report of Neocosmospora pseudensiformis Causing Bark Cracking on Artocarpus heterophyllus L. in China.

Jackfruit (Artocarpus heterophyllus) is a tree of the Moraceae family and widely cultivated due to its fruit with diverse medicinal and nutritional properties in China. In June 2022, a serious bark cracking disease was observed on jackfruit in the 2400-acre orchards of Hekou, Yunnan (E103°52'39″ N22°34'14″). The disease mainly harms tree trunks and branches. The incidence rates were about 90% and the mortality rate of plants reached up to 50%. Symptoms initially appeared as small watered-spots with gumming. Subsequently the bark rotted and cracked, the xylem gradually turned brown (Figure 1). Ultimately, the upper branches of infected parts withered and died. Small 5×5 mm segments of infected tissue from 50 randomly selected trunk with typical symptoms were surface sterilized with 75% alcohol solution and 3% hydrogen peroxide solution respectively, rinsed three times with sterile water, and placed onto PDA medium with 50mg/L of penicillin. The plates were kept at 25 to 28 ℃ in the dark. Fusarium-like colonies were consistently isolated on potato dextrose agar (PDA) and 15 monoconidial isolates were obtained. On PDA, colonies exhibited white and fluffy aerial mycelia. Main hyphae are up to 5.5 µm wide. The microconidia were hyaline, falcate, measuring 4.50-9.00×2.30-4.00 µm (av.6.50×3.15 µm, n=50). Macroconidia with septation were 9.00-11.50×3.05-5.25 µm (av.10.50×4.15 µm, n=50) with foot-shaped basal cells, tapering to hooked apical cells (Figure 2). These morphological characterizations were similar to Fusarium sp. (Sun et al, 2018). For further identification, the internal transcribed spacer (ITS) region, RNA polymerase II subunit (RPB2) and translation elongation factor 1-alpha (tef1) genes sequence of isolate BLM1 and BLM2 were amplified and sequenced with primer pairs of ITS1/ITS4, RPB2-5F2/RPB2-7CR, EF-1/EF-728R respectively (Weir et al, 2012), The sequences were submitted to GenBank (ITS: PQ394640 and PQ394641, RPB2: PQ416997 and PQ416998, TEF: PQ416999 and PQ417000). Blast results showed the sequences of BLM1 and BLM2 had high identity to these of Neocosmospora pseudensiformis (Anamorp: Fusarium pseudensiformis) ex-type CBS 125729 (NRRL 46517) (ITS: 548/553(99%); TEF: 456/466(98%); RPB2: 823/823(100%)) (Sandoval-Denis, et al 2019). Polyphasic identification showed above 98% sequence similarity to N. pseudensiformis CBS 125729, CBS 130.78, NRRL22354, LC13838 (https://www.fusarium.org/Poly%20ID%20Fusarium). Phylogenetic analysis using MEGA 7 based on the combined ITS-TEF-RPB2 sequence data, employing the maximum likelihood (ML) method for the multi-locus dataset, showed that BLM1 and BLM2 clustered with F. pseudensiformis (Figure 3). Two five-year-old healthy Jackfruit trees and ten one-year-old seedlings were used for a pathogenicity test. Three plants were inoculated with PDA medium or with sterile water as an experimental control. The seedlings were cultured in a greenhouse (25℃, 70% relative humidity, 12 h light and dark cycle) and the trees cultured in the field, and the experiment was repeated three times. The inoculation sites on trunk were rinsed with sterile water, surface disinfected with alcohol, slightly wounded with sterilized needles, and inoculate fungal masses containing mycelium and spores, as well as 10 µL of spore suspension (1 × 105 conidia/ml). Seven days after inoculation, white slurry effluent as well as minor cracking and rotting of the trunks were observed on the trunks, whereas no symptoms were observed on the trunks of the controls. The pathogen was reisolated and identified by sequencing the ITS, TEF, and RPB2 genes as being consistent with the original isolates N. pseudensiformis has been reported as a pathogen of fruit rot on Passiflora edulis (Pereira et al, 2019), and this is the first report causing bark cracking on A. heterophyllus in China as well as worldwide. Bark cracking disease had seriously affected jackfruit cultivation, and the report provides an knowledge for the prevention and control of this disease.