Characterization of Fungicide Resistance in Venturia inaequalis Populations in Virginia

Apple scab (causal organism: Venturia inaequalis) is an economically devastating disease of apples that is predominantly controlled with fungicides. Of the chemical classes currently available, the sterol-inhibiting (SI) and strobilurin (QoI) fungicides are the most commonly used. Recent observations indicate that V. inaequalis populations in Virginia have developed resistance to myclobutanil and other SIs. However, little is known about the frequency and distribution of SI and QoI resistance in Virginia's scab populations. The first objective of this research was to evaluate V. inaequalis populations in Virginia for SI and QoI resistance. Fungal isolates were collected from experimental orchards at the Alson H. Smith Jr., Agricultural Research and Extension Center (AHS AREC) and from commercial orchards in Virginia and Maryland. Sensitivities were determined by assessing colony growth at 19°C on potato dextrose agar (PDA) amended with 0 or 1.0 µg ml-1 of myclobutanil (SI) (N=87) or trifloxystrobin (QoI) (N=25) at 28 days. A range of fungicide sensitivity was observed for both chemical classes. The second objective of this research was to monitor the temporal dynamics of SI resistance over five sequential field seasons. To monitor shoot growth, neon rubber bands were placed over actively growing shoot tips following myclobutanil application or sample collection. Fungal isolates were collected from the same trees from 2007 through 2010 (N=176) and compared with isolates collected from wild apple seedlings (N=3). A continuum of SI resistance was observed for each year, and the V. inaequalis population exhibited a baseline shifted toward reduced sensitivity. The third objective of this research was to examine the spatial distribution of SI fungicide resistance within the tree canopy in a lower-density orchard (less than 150 trees A-1). Leaves collected from larger trees (>8m) in a lower-density orchard at the AHS AREC were analyzed for manganese deposition, pre- and post-mancozeb application. Fungal isolates (N=105) were collected from several locations within the canopy in replicated trees in the same orchard. Weather sensors also monitored the microclimates within those tree canopies. Spray deposition, microclimate and SI resistance were influenced by canopy location. The fourth objective of this research was to investigate potential SI resistance mechanisms. Previously classified isolates were screened for point mutations within the CYP51A1 gene (Appendix C), differences in polymorphic bands (alleles) (Appendix D), and differences in metabolism of myclobutanil (Appendix E). The consensus sequences for the CYP51A1 gene were identical for all isolates tested (N=9), and results from amplified fragment length polymorphism experiment (N=82) were inconclusive. There were, however, significant differences among incubation time and myclobutanil concentration in the bioassay (N=11). Our results indicate that myclobutanil is still an effective compound for control of apple scab in many areas of Virginia.