Development of novel species-specific primers for the specific identification of Colletotrichum nymphaeae based on conventional PCR and LAMP techniques

Colletotrichum nymphaeae is the primary causal agent of strawberry anthracnose in Iran resulting in epidemics in strawberry fields in recent years. Due to the hemibiotrophic nature of the pathogen, early detection in symptomless, infected plants, especially in nurseries could be advantageous for disease management. Assessing inter-species variation by rep-PCR fingerprinting of Colletotrichum spp. within the C. acutatum species complex causing strawberry anthracnose revealed a specific genomic segment amplified with only C. nymphaeae. The amplicon was extracted, purified and novel primer sets were developed based on conventional PCR and loop-mediated isothermal amplification (LAMP) techniques. The genus and species-specific PCR primers were able to discriminate the genus Colletotrichum and C. nymphaeae from other fungal species in pure culture and in assays of diseased, detached leaves. Similar results were observed for LAMP, but it was more sensitive compared to the PCR assay. Using both assays, asymptomatic strawberry plants infected with C. nymphaeae strain CCTUCch32 were readily detected. These results show that the primer sets developed in this study based on conventional PCR and LAMP techniques can be effective for early detection of C. nymphaeae, which can contribute to improved control strategies for strawberry anthracnose.