Epidemiology of Colletotrichum acutatum in apple orchards using realtime PCR

Abstract

In the past, bitter rots of apples in New Zealand have been caused most commonly by Colletotrichum gloeosporioides (teleomorph Glomerella cingulata). However, due to a change in fungicide use, Colletotrichum acutatum is now the most common fungus causing bitter rots in apples. Inoculum has previously been found on symptomless leaves, buds, petals, fruits and twigs using conventional isolations. This study developed a protocol to use a SYBR Green I real-time polymerase chain reaction (PCR) assay to investigate the pattern of distribution, spread of inoculum within the apple canopy, relationship between canopy density and inoculum level and to quantify the amount of C. acutatum in each inoculum source. Apple buds, petals, leaves, bark and fruit peel were collected from bud break to harvest time in two orchards in the Waikato region. An initial screening using the CaITS F701/R699 and R815 primers was conducted following the method described by Debode et al. (2009). A low amount of C. acutatum was found in all plant materials except bud samples which have the highest amount of inoculum (30 spores/mg). Bud samples were further tested in real-time PCR with the plant cytochrome oxidase (COX F1/R1) primer set following the method of Garrido et al. (2009). Normalization to the COX reference gene did not improve results. However, because DNA extraction efficiency was high (83%) and there was low variations (standard deviation of 2.6%) between samples, inferences could be made by comparing Ct values of DNA from different extractions. Our results showed that the distribution of C. acutatum at the lower, middle and upper canopy was not significantly different. In addition, the distribution was not affected by exposure to the sun. A relationship between canopy density and the amount of C. acutatum was not found because trees included in the study were of similar height and canopy densities. Results of the realtime PCR for leaves and fruit peel samples agreed with field disease assessment at harvest both showing low levels of C. acutatum in apple fruits. These initial results suggest that it may be possible to use the techniques developed during this study to provide useful tools to the growers for predicting the level of rots in the fruit at harvest by sampling leaf tissue earlier in the season.