Ficus altissima, a new host of Colletotrichum fructicola Causing anthracnose in China

Ficus altissima Blume.，known as council tree or lofty fig, is a tall evergreen tree in the Moraceae family, widely planted as a roadside tree due to its exquisite tree posture in subtropical areas. During October 2023, an anthracnose disease was observed in approximately 90% of F. altissima plants in Mazhang, Zhanjiang, Guangdong Province ( 22°6'14.7''N,110°27'26.8''E ). Symptoms were small dark-brown or black spots with yellow halos on about 10% disease leaves, which downgraded its ornamental value (Figure 1). Twenty-four diseased tissues (5 × 5 mm) were cut and sterilized with 3% hydrogen peroxide for 2 min, rinsed three times with sterile water, and placed on potato dextrose agar (PDA) medium containing 50 mg/L of penicillin. The plates were incubated in the dark at 28°C. Nineteen isolates were obtained by the single-spore method (Choi et al. 1999). The colonies on PDA were cotton-like, gray-black with concentric ring on the mycelial mat, and orange conidiomata appeared after seven days (Figure 1). The conidia are unicellular, oblong in shape and 11.9-16.1 μm × 6.1-10.9 μm (av. 13.9 × 7.3 μm, n = 50) in size. Appressoria were irregularly, dark brown, some with lobes, elliptical, 10.9-12.4 × 6.4-9.5µm (av. 11.7 × 8.2 μm) (Figure 1). The morphological characteristics were consistent with those of Colletotrichum species (Weir et al. 2012). The internal transcribed spacer (ITS) region, actin (ACT), chitin synthase (CHS), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), and B-tubulin (TUB) were amplified and sequenced for two isolates (R3-2 and R3-4) with primer pairs of ITS1/ITS4 (White et al. 1990), ACT-512F/ACT-783R, CHS-79F/345R, GAPDH-F/R, and TUB-2Fd/4Rd, respectively (Weir et al. 2012). The sequences were deposited in GenBank (ITS: PQ013063 and PQ432515, TUB: PQ066260 and PQ433584, ACT: PQ066256 and PQ433581, GAPDH: PQ066259 and PQ433583, CHS: PQ066258 and PQ433582). BLAST research showed the sequences of R3-2 and R3-4 had above 99% identity with these of C. fructicola ex-type ICMP: 18581 (ITS: 481/481 (100%) and 481/482 (99%); GAPDH: 277/280 (99%) and 278/281 (99%); TUB: 406/406 (100%) and 379/379 (100%); CHS: 267/267 (100%) and 299/299 (100%); ACT: 239/241 (99%) and 269/270 (99%)). The concatenated data of ITS, TUB, CHS, ACT and GAPDH sequences was used to phylogenetic analysis using Maximum Likelihood method in MEGA-X. The isolates R3-2 and R3-4 clustered in the same clade with C. fructicola ICMP 18581 (Figure 2). To confirm pathogenicity, three healthy leaves of 8 to 10-year-old F. altissima were surface sterilized with 75% ethanol and sterile water, wounded on each leaf with sterile needles. The wounded sites were inoculated with 10 µl of spore suspension (1×105 spore/ml) and inoculated with sterile water as control. The experiment was repeated three times. After three days, symptoms of anthracnose were observed on the leaves, similar to those described above; no symptoms occurred in the controls. Colletotrichum fructicola was re-isolated from artificially inoculated leaves, all re-isolated fungal isolates similar to R3-2 and R3-4 morphology and re-confirmed by molecular analysis. In China, C. fructicola may infect 20 species of plants from 11 genera (Farr and Rossman 2024) and it is the first report causing F. altissima anthracnose, which provided a basic knowledge for diagnosing, preventing and controlling this disease.