First report of Colletotrichum siamense causing leaf spot on Manglietia decidua in the world.

Abstract

Manglietia decidua Q. Y. Zheng is a deciduous broad-leaved plant and native to Jiangxi province, China. It is cultivated for its timber and urban landscaping (Xiong et al., 2014). In September 2019, a new foliar disease was observed on approx. 25% of 121 M. decidua trees in Jiangxi Agricultural University (N28°45'56″, E115°50'21″), Nanchang city, Jiangxi Province, China. The disease site belongs to the subtropical monsoon humid climate, with rainfall (1,600-1,700 mm) and red soil region. Initially, infection appeared on the leaf margins or tips as water-soaked, irregular lesions, then expanded to the center, developed into large black-brown, irregular necrotic lesions. Finally, the lesions fall off the leaves. To identify the pathogen, 15 diseased leaves were collected from 5 trees (3 leaves per tree) randomly. Small pieces (5 × 5 mm) cut from the lesion margins were surfaced sterilized (70% ethanol for 30 s, 3% NaOCl for 1 min, rinsed 3 times with sterile water), and placed on potato dextrose agar (PDA) at 25 °C. Among the isolated fungi, Colletotrichum-like colonies were about 91%, and 18 monoconidial isolates were obtained. Isolates HML-1, HML-4, and HML-7 were selected and preserved for further studies. Colonies on PDA were white, cottony, and grayish-white on the reverse side. Setae absent. Acervuli were brown, circular. Conidiophores were clear, septate, non-branching or branching at the base, conidiogenous cells were enteroblastic, phialidic, colorless, cylindrical, ampulliform. Conidia were elliptical, single-celled, straight, hyaline, and measured 13.3-17.9 × 4.3-5.7 µm (14.8 ± 1.2 × 4.8 ± 0.4 µm, n = 100). Appressoria were oval to irregular, dark brown, and ranged from 5.3-9.1 × 4.4-6.3 µm (7.2 ± 0.3 × 5.1 ± 0.2 µm, n=100). Morphological characteristics matched the description of Colletotrichum gloeosporioides sensu lato (Weir et al. 2012). The internal transcribed spacer regions (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), chitin synthase (CHS-1), calmodulin (CAL), and beta-tubulin 2 (TUB2) were sequenced (Weir et al., 2012), and deposited in GenBank (ITS: OL757565-OL757567; ACT: OL627398-OL627400; CHS-1: OL757358-OL757360; GAPDH: OL757361-OL757363; CAL: OL757355-OL757357; TUB2: OL757364-OL757366). Six loci were concatenated, and the aligned sequences (2056 bp) were 99.9%, 99.8% homologous to C. siamense ICMP 18574 and ex-type ICMP18578, respectively. In the maximum-likelihood phylogenetic tree, the highest log likehihood was -9259.74, and 3 isolates were in the C. siamense clade. Based on the phylogeny and morphology, 3 isolates were identified as C. siamense. The pathogenicity of 3 isolates was tested on 12 M. decidua plants (variety: Yi lin ke) grown in the field. Healthy leaves were wounded slightly with a needle (Φ=0.5 mm) and inoculated with 10 µL of spore suspension (106 conidia/mL). Controls were treated with ddH2O (Si et al. 2021). All the treated leaves were covered with plastic bags to keep a high-humidity environment for 2 days. The experiments were repeated twice. Within 9 days, all the inoculated points showed similar symptoms to those observed in the field, whereas controls were asymptomatic. The same isolate was re-isolated from the lesions, whereas no fungus was isolated from control leaves. Manglietia decidua is an ancient and endangered plant, threatened with southern blight (Sclerotium rolfsii) (Yi et al. 2021a), root rot (Calonectria ilicicola) (Yi et al. 2021b). This is the first report of the newly emerging disease caused by C. siamense in the world. The potential threat should be evaluated for conservation in the future. This study provided crucial information for epidemiological studies and appropriate control strategies.