Spatial Patterns of Brown Rot Epidemics and Development of Microsatellite Markers for Analyzing Fine-Scale Genetic Structure of Monilinia fructicola Populations Within Peach Tree Canopies

Abstract

To better understand the fine-scale spatial dynamics of brown rot disease and corresponding fungal genotypes, we analyzed three-dimensional spatial patterns of pre-harvest fruit rot caused by Monilinia fructicola in individual peach tree canopies and developed microsatellite markers for canopy-level population genetics analyses. Using a magnetic digitizer, high-resolution maps of fruit rot development in five representative trees were generated, and M. fructicola was isolated from each affected fruit. To characterize disease aggregation, nearestneighbor distances among symptomatic fruit were calculated and compared with appropriate random simulations. Within-canopy disease aggregation correlated negatively with the number of diseased fruit per tree (r = −0.827, P = 0.0009), i.e., aggregation was greatest when the number of diseased fruit was lowest. Sixteen microsatellite primers consistently amplified polymorphic regions in a geographically diverse test population of 47 M. fructicola isolates. None of the test isolates produced identical multilocus genotypes, and the number of alleles per locus ranged from 2 to 16. We are applying these markers to determine fine-scale population structure of the pathogen within and among canopies. Accepted for publication 23 May 2012. Published 23 July 2012.

A comparison of UP‐PCR and RAPD markers to study genetic diversity of Fusicladium effusum (G. Winter), cause of pecan scab

“…Information on the genetic diversity of numerous other fungal pathogens of many crops is now available (McDonald et al 1999;McDonald and Linde 2002a,b;Bock et al 2005;Obanor et al 2010) using a range of different tools including restriction fragment length polymorphisms (RFLPs, McDonald et al 1999;Salamati et al 2000), simple sequence repeats (SSRs, Tenzer et al 1999;Everhart et al 2012), amplified fragment length polymorphisms (AFLPs, Majer et al 1998;Bock et al 2005), randomly amplified polymorphic DNAs (RAPDs, Arnau et al 1994; and universally primed PCR (UP-PCR, which are similar to RAPDs, Bulat et al 1998;Obanor et al 2010), as well as other methods (e.g. single-nucleotide polymorphisms).…”

Novel gene‐sequence markers for isolate tracking within Monilinia fructicola lesions

“…For SSR analysis, PCR was performed using primers and touchdown PCR described previously . SSR loci used in the present experiment were SED, SEF, SEI, SEL, SEN, SEP and SEQ, and to limit production of stutter peaks as a result of poly‐A tail formation during PCR, forward primers were pig‐tailed on the 5′ end with GTTTT (Table ).…”

“…In the present study, the fluorophore FAM was attached directly to primers instead of using the HEX‐CAG complementary primer, where direct FAM attachment allowed faster throughput of samples. Base pairs corresponding to the CAG primer used for indirect fluorescent labeling were added to the FAM‐labeled primer so that resulting fragment sizes would be similar to those described previously . Thermal cycler conditions used for amplification were: an initial denaturation cycle of 95 °C for 2 min and 30 s, followed by a touchdown cycle repeated 20 times of 95 °C for 20 s, 60 °C for 20 s, with temperature decreasing by 0.5 °C every cycle, and 72 °C for 30 s. The third cycle of 95 °C for 20 s, 50 °C for 20 s and 72 °C for 30 s was repeated 15 times.…”

“…Without availability of a reference genome, the most effective and sensitive method for fingerprinting M. fructicola isolates is microsatellite or simple‐sequence repeat (SSR) genotyping. Development of SSR markers has resulted in a number of studies on population dynamics and diversity of M. fructicola . Although previous studies have characterized fine‐scale population dynamics of M. fructicola over time within individual trees, none included resampling of isolates from the same infection lesions over a growing season.…”

“…Development of SSR markers has resulted in a number of studies on population dynamics and diversity of M. fructicola. 6,10,11 Although previous studies have characterized fine-scale population dynamics of M. fructicola over time within individual trees, 12 none included resampling of isolates from the same infection lesions over a growing season. This lack of resampling assumes that an infection lesion will possess the same genotype from beginning to end of the growing season, but that may not be a reliable assumption given the high polymorphism and mutability of these loci.…”