First report of Colletotrichum plurivorum causing anthracnose on pecan (Carya illinoinensis) in China.

Abstract

Pecan (Carya illinoinensis) is an economically important nut crop worldwide (Xiao et al 2021). Anthracnose symptoms were found on pecan fruits and leaves in plantations in Anhui and Jiangsu provinces, China in August 2019. Irregular, dark brown or black spotted lesions firstly appeared on the surface and inside of fruits, and spread to all leaves. The symptoms resulted in 30% to 50% leaf drop and nearly a half of fruit decay in almost all trees of the susceptible cv. Wichita. The causal agent were isolated from fruits with target symptoms following the steps: surface disinfected with 75% ethanol (2×, 30 s), rinsed with sterile deionized water (3×), ~ 0.5 cm small fragments of the fruits excised and plated on potato dextrose agar (PDA) medium and incubated at 28 °C in dark for 3-d. Mycelium of each colony was picked and incubated on fresh PDA at 25 °C with a 12-hour light/dark cycle for 6-d to induce conidia formation. One 5-mm hyphal plug produced from each single spore isolate was transferred onto fresh PDA to obtain the pure cultures. Koch's postulates was employed for pathogenicity determination of the isolates. Non-wounded healthy leaves from seedlings of the disease susceptible cv. Pawnee were disinfected with 1% NaClO and inoculated with 5-mm 5-d hyphal of each isolate at 25 ℃. Tiny lesion spots were visible on the leaves after 2 days post inoculation (DPI) with isolate W-6 (the only pathogenic one among all isolates), and expanded over time until to the leaves withered, while the control leaves and leaves inoculated with other isolates remained asymptomatic. The pathogenicity of W-6 were confirmed using leaves and fruits of living Pawnee trees growing in Linglong Mountain Plantation, Lin'an, Hangzhou, China (119⁰38'51″E, 30⁰12'39″N, elevation: 119m). Three experimental replicates were conducted separately with three bio-replicates for all pathogenetic testing. The same symptoms were observed on both detached leaves and leaves and fruits of living trees.. The colony of W-6 have round cottony mycelium with complete edges and showed the fastest growth rate 3 - 4 DPI. After 7 DPI, white aerial mycelium turned yellowish brown and formed Acervulus in the mycelium. Conidia (n=50) one-celled, 12.0 - 20.0 μm × 3.5 to 6.0 μm width. Hyaline cylindrical with slightly rounded ends and two or three large guttulate at the centre. Most Acervulus dark brown and slightly irregular in shape, 12.70 × 18.79 μm (n=10). Setae were dark brown in color with average length around 34.10 μm (n=10). These characteristics matched previous descriptions of Colletotrichum orchidearum species complex, including C. plurivorum (Damm et al 2019). The identity of W-6 was confirmed by multi-locus phylogenetic analysis using the internal transcribed spacer (ITS) rDNA region and partial sequences of the conserved genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), beta-tubulin 2 (TUB2), and chitin synthase (CHS). The sequences of W-6 were used for Basic Local Alignment Search Tool (BLAST) against NCBI GenBank and the sequences with 100% identity to that of W-6 were achieved, respectively. The concatenated sequences of the ACT-CHS-GAPDH-ITS-TUB2 was used for building a phylogenetic tree. The molecular analyses allowed the identification of the pathogen as C. plurivorum. It was known that 9 of the 11 Colletotrichum species causing pecan anthrax worldwide were reported in southern China (Brenneman 1989; Oh et al 2021). This is the first report of C. plurivorum as causal agent of pecan in China.