First Report of Causing Leaf Anthracnose on L in China.

Jatropha curcas L. (Euphorbiaceae), is a valuable multi-purpose crop, previously used for disease treatment and environmental restoration, recently is attention to use jatropha oil for produce biodiese. In June 2023, the leaf disease of approximately 60% of J. curcas was observed in Mazhang, Zhanjiang, Guangdong Province (E110°27'26.8'' N22°6'14.7''). Diseased leaves showed typical anthracnose symptoms of chlorotic regions with brownish sunken necrotic lesions (Figure 1). Sections from the junction of disease were surface disinfected in 75% ethanol and 3% hydrogen peroxide solution for 1 minute each. Four small pieces of infected tissue were removed from the lesion and placed on potato dextrose agar (PDA), and incubated at 25 to 28℃ in the dark. Hyphal tips from the inoculated tissues were subcultured on PDA and two isolates were purified by single spore method. The colonies on PDA were 7.8 cm diam after 10 d at 25 to 28 ℃, covered with dense, cottony, grayish-white aerial mycelium and small dark-based acervuli with orange ooze and dark brown straight setae. Conidia were hyaline, smooth-walled, aseptate, the apex and base rounded, slightly constricted near centre, 12.9 - 13.8 × 3.9 - 4.6 um (av.13.6 × 4.3 μm, n = 50). Appressoria were variable in shape, mostly simple, subglobose or irregular lobes, 5.8-9.6 × 5.7-11.2 um (Figure 2). Perithecia were not observed. These characteristics were consistent with Colletotrichum sp. (Weir, B. S., et al. 2012). Sequences of isolates ACCC 35630 and ACCC 35631 stored in Agricultural Culture Collection of China including internal transcribed spacer (ITS), actin (ACT), beta-tubulin (tub2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and chitin synthase (chs). were amplified (Weir et al, 2012), sequenced and submitted to GenBank (ITS: PP474979 and PP474984; ACT: PP505487 and PP505488; TUB: PP505493 and PP505494; GAPDH: PP505491 and PP505492; CHS: PP505489 and PP505490). The amplicon sizes of ITS, ACT, TUB, GAPDH, and CHS were 550, 652, 500, 264, and 301 bp, respectively. Phylogenetic analyses showed that Isolates 35630 & 35631 were clustered closely association with RHCOL1 and RHCOL3. Phylogenetic analysis with MEGA 7 using the combined ITS-ACT-CHS-GAPDH-TUB2 sequences showed that the two isolates clustered with C. queenslandicum (Figure 3). To test the pathogenicity, ten healthy leaves on plants in the field were wiped with 75% alcohol and sterile water, punctured with a sterile needle and inoculated by adding 10 uL of spore suspension (1 × 105 conidia/ml) to the wounded sites. And two other leaves were added sterile water as controls. Symptoms of anthracnose were observed on leaves similar to the disease described above after 7 days of inoculation, whereas the leaves from the controls remained asymptomatic. C. queenslandicum was reisolated from the inoculated leaves. C. queenslandicum has been reported as a pathogen causing leaf and fruit anthracnose on papaya, coffee, rambutan, avocado and Persian lime etc. in tropical and temperate regions (Kunta, M., et al. 2018), and this is the first report on J. curcas in China as well as worldwide. This disease may have a significant negative impact on J. curcas cultivation.