A Lateral Flow-Recombinase Polymerase Amplification Method for Colletotrichum gloeosporioides Detection

Abstract

The greater yam (Dioscorea alata), a widely cultivated and nutritious food crop, suffers from widespread yield reduction due to anthracnose caused by Colletotrichum gloeosporioides. Latent infection often occurs before anthracnose phenotypes can be detected, making early prevention difficult and causing significant harm to agricultural production. Through comparative genomic analysis of 60 genomes of 38 species from the Colletotrichum genus, this study identified 17 orthologous gene groups (orthogroups) that were shared by all investigated C. gloeosporioides strains but absent from all other Colletotrichum species. Four of the 17 C. gloeosporioides-specific orthogroups were used as molecular markers for PCR primer designation and C. gloeosporioides detection. All of them can specifically detect C. gloeosporioides out of microbes within and beyond the Colletotrichum genus with different sensitivities. To establish a rapid, portable, and operable anthracnose diagnostic method suitable for field use, specific recombinase polymerase amplification (RPA) primer probe combinations were designed, and a lateral flow (LF)-RPA detection kit for C. gloeosporioides was developed, with the sensitivity reaching the picogram (pg) level. In conclusion, this study identified C. gloeosporioides-specific molecular markers and developed an efficient method for C. gloeosporioides detection, which can be applied to the prevention and control of yam anthracnose as well as anthracnose caused by C. gloeosporioides in other crops. The strategy adopted by this study also serves as a reference for the identification of molecular markers and diagnosis of other plant pathogens.

Keywords: Colletotrichum gloeosporioides; Dioscorea alata; disease diagnosis; pathogen identification; primer development.

Figure 1

Statistics of orthologous gene clustering analyses. (A) Number of orthogroups occupied by genes of different C. gloeosporioides strains, Venn diagram shows the number of orthogroups which were strain-specific (1) or shared by 2, 3, or 4 strains. (B) Comparison of orthogroups shared by all C. gloeosporioides strains with all non-redundant orthogroups of the other 56 Colletotrichum genomes. (C) Details of C. gloeosporioides-specific orthogroups, including gene IDs and length of each genome. The four colored orthogroups were eventually the molecular markers used in this study.

Figure 2

Determination of the primers targeting four C. gloeosporioides-specific orthogroups by PCR. M = 2000 bp DNA Ladder; 1 = C. gloeosporioides strain CgDa01; 2 = C. gloeosporioides strain CgDaM3; 3 = Fusarium graminearum; 4 = F. oxysporum; 5 = Sclerotinia sclerotiorum; 6 = Botrytis cinerea; 7 = Blumeria graminis; and 8 = C. fructicola. (A–D) PCR results of different primer pairs.

Figure 3

Sensitivity testing of four pairs of specific primers. M = 2000 bp DNA Ladder, and 1~8 = different DNA concentrations: 1 ng/μL, 100 pg/μL, 10 pg/μL, 1 pg/μL, 100 fg/μL, 10 fg/μL, 1 fg/μL, and NTC (no-template control), respectively. (A–D) PCR results of different primer pairs.

Figure 4

Evaluation of specific primers for C. gloeosporioides detection of infected plant tissues. (A) Phenotypes of leaves post inoculation with C. gloeosporioide on different days. Dpi = days post infection. (B–E) PCR results of DNA extracted from plant leaves at 0, 1, 3, and 5 days post inoculation (dpi) with C. gloeosporioides, respectively. M = 2000 bp DNA Ladder; CK = control check, namely greater yam leaves that were not inoculated with C. gloeosporioides; Lanes 1~3 = independent samples infected by C. gloeosporioide at the same time point.

Figure 5

Sensitivity test of LF-RPA C. gloeosporioides detection methods. NTC = no template control; Dpi = days post infection. (A) Sensitivity of LF-RPA at different concentrations of C. gloeosporioides genomic DNA. (B) Results of LF-RPA conducted on DNA samples extracted from greater yam leaves infected by C. gloeosporioides from 0 to 5 dpi.