First Report of White Leaf Spot Caused by Hypomontagnella monticulosa on Pachira glabra in China.

Abstract

Pachira glabra Pasq. is an ornamental tree widely distributed in tropical and subtropical regions of China. In August 2021, an unknown leaf spot was observed on P. glabra in Xiangtan County, Hunan, China (27.976°N, 113.041°E). Over 1,200 plants were evaluated, and up to 20% of the plants were diseased. In moderately diseased plants, approximately one third of leaves had symptoms with the disease severity estimated to be 31.6 ± 9.4% (n=100). The symptoms first appeared as pale yellow to yellow small dots often confined between leaf veins. These dots gradually enlarged, and coalesced into large pale or white spots with brown borders and yellow halo. In severe infections, early leaf death and defoliation occurred. Thirty lesions (2 × 2 mm) collected from ten trees were sterilized in 75% ethanol for 15 s, 5% sodium hypochlorite for 15 s, rinsed in sterile water three times, placed on oatmeal agar medium (OA) plate with lactic acid (3 ml/liter), and incubated at 28°C for 15 days. After incubation, five isolates with a similar morphology were obtained by single-spore culture. Colonies on OA were white and then turned pale grey. Pigments on the reverse side were pale brown. Conidiophores were hyaline, smooth to finely roughened, usually with virgariella-like branching patterns. Conidiogenouscells were hyaline, smooth, and measured 13.9 to 53.8 long and 1.5 to 2.3 µm wide (average 30.8 × 2.0, n=50). Conidia were single-celled, transparent, smooth, ellipsoid to obovoid, 2.3 to 4.6 × 1.7 to 3.1 µm (average 3.1 × 2.3, n=100) in measurement. For further molecular identification, internal transcribed spacer (ITS), β-tubulin (TUB2), RNA polymerase II (RPB2), and large ribosomal subunit (LSU) genes of a representative isolate TT422 were amplified from genomic DNA, using primers ITS1/ITS4 (Mills et al. 1992), T1/T22 (O'Donnell et al. 1997), RPB2-5F/7cR (Liu et al. 2000), and LROR/LR7 (Rehner et al. 1994), respectively. Sequences of ITS (accession no. OM070368), TUB2 (OM201746), LSU (OM070369), and RPB2 (OM141478) from the isolate TT422 showed >98% identity where sequences overlapped to the reference strain of Hypomontagnella monticulosa MUCL 54604 (KY610404, KX271273, KY624305, and KY610487). Concatenated sequences were used for a phylogenetic analysis based on Maximum Likelihood using MEGA7. Based on morphological and molecular data, the isolate TT422 was identified as H. monticulosa (Ju & Rogers 1995; Lambert et al. 2019). Pathogenicity tests were performed three times on healthy leaves using the isolate TT422. Three leaves on one-year-old plants were slightly wounded by a sterile needle, and sprayed with conidial suspension (1×106 conidia/ml, containing 0.05% Tween 20) . Control leaves were sprayed with sterile water containing 0.05% Tween 20. All plants were kept in a greenhouse for 24 h at 28°C and 80% relative humidity, with a 16-h photoperiod and then transferred to natural conditions. All inoculated leaves developed white leaf spot symptoms after 7 days similar to those observed in the field, whereas no visible symptoms appeared on the control leaves. H. monticulosa strains were reisolated from all symptomatic leaves, fulfilling Koch's postulates. H. monticulosa isolated from marine or endophytic origin has been reported to produce bioactive metabolites with anticancer and microbial activities (Leman-Loubière et al. 2017; Lutfia et al. 2021; Zhang et al. 2021), but not as a phytopathogen. To our knowledge, this is the first report of H. monticulosa causing white leaf spot on P. glabra in China and worldwide.