Development of a Nested Polymerase Chain Reaction Assay for Detection of Colletotrichum acutatum on Symptomless...

A nested polymerase chain reaction (PCR) assay was developed for detection of Colletotrichum acutatum on symptomless strawberry leaves. In pure culture, the assay detected as little as 1.0 fg of DNA extracted from mycelium and as few as 1.5 conidia ml^-1 when conidial suspensions were sonicated. On detached inoculated leaves, three alternative protocols to dislodge the pathogen were assessed: (i) immersion of whole leaves in 0.05% Tween 20 and manual agitation in plastic bags for 1 min (A); (ii) immersion in Tween 20, sonication for 30 min, then agitation for 1 min (SA); and (iii) freezing for 3 h, incubation for 2 days at 27°C, immersion in Tween 20, then sonication for 30 min and agitation for 1 min (FISA). Each method removed significantly (P ≤ 0.05) more conidia from leaves than the nontreated control; however, removal of appressoria did not vary among assays. In composite samples of noninoculated and inoculated (1.5 ×10³ conidia ml^-1) strawberry leaves, the nested PCR assay using the FISA protocol detected C. acutatum in as few as 1 infested leaf in 50 noninfested leaves. In a strawberry field, the assay detected the presence of C. acutatum in samples of asymptomatic strawberry leaves, showing potential as a powerful tool for reliable diagnosis of the pathogen in the field.