Molecular identification of fungal organisms associated with cool-season turfgrass
Creators: name: Groben, Glen
Publisher: name: No Publisher Supplied
Year: 2023

description: Recent advances in molecular biology have made it possible to investigate complex plant-fungal interactions. In this dissertation two molecular methods, qPCR and metabarcoding with two next generation sequencing platforms, were utilized to better understand the interaction between fungi and two cool-season turfgrasses. The main objectives of these studies were: 1) to develop and use a qPCR assay specific to the Clarireedia genus to conduct molecular epidemiological studies with dollar spot and creeping bentgrass (Agrostis stolonifera) managed as fairway turf, and 2) adapt the Oxford Nanopore Technologies MinION platform to sequence near full-length fungal ribosomes, compared to the Illumina MiSeq short-read sequences, for analysis of the mycobiome associated with tall fescue (Festuca arundinacea) grown under drought stress in the field. A qPCR assay specific to the Clarireedia genus was developed and validated. The assay was able to discern differences in Clarireedia concentration in dollar spot susceptible and tolerant creeping bentgrass cultivars, but did not cross react with closely related taxa, other turfgrass pathogens, or fungal species commonly isolated from turfgrass. The assay detected as little as 38.0 fg (3.8 x10-14 g) of Clarireedia genomic DNA in three hours and identified Clarireedia in both symptomatic and asymptomatic turf. The assay was used to assess the spatial (horizontal) distribution of Clarireedia in asymptomatic and symptomatic creeping bentgrass from foliage and crown tissue in cores extracted from the field. Clarireedia was found to be randomly distributed in both asymptomatic and symptomatic turfgrass. Positive detections ranged from 37% to 69% in asymptomatic turfgrass and 77% to 95% in symptomatic turfgrass. Similarly, the vertical distribution of Clarireedia was measured in creeping bentgrass foliage, crowns, and thatch over two years. Clarireedia was most abundant in foliage, followed by crown tissue, and thatch (lowest abundance). qPCR identified significant differences in Clarireedia concentration between dollar spot tolerant (‘Declaration’) and susceptible (‘Independence’) creeping bentgrass cultivars in both asymptomatic and symptomatic turf. Using linear model analysis, the susceptible cultivar always had a higher Clarireedia concentration compared to the tolerant cultivar. Different sampling methods (using individual cores vs composites of cores vs tissue from verticutting) were evaluated to determine the most effective method for quantifying Clarireedia spp. in foliage of two dollar spot susceptible creeping bentgrass cultivars, ‘Crenshaw’ and ‘Independence’. Overall, verticutting was found to be the most accurate sampling method for quantifying the concentration of Clarireedia in foliage of both cultivars. Verticutting sampled more of the plot area (2.62 %) than individual cores or composites (0.23% each), had the lowest variability in average cycle threshold (Ct) values, and consistently identified Clarireedia in asymptomatic and symptomatic tissue. Moreover, verticutting is a sampling method that could be easily adopted by golf course superintendents to monitor the population of the dollar spot pathogens in the field to better time fungicide applications and potentially reduce fungicide inputs. The qPCR assay was also used to evaluate the impact of autumn fungicide timing (seven application timings of fluazinam + propiconazole and one application timing of chlorothalonil) on the concentration of Clarireedia at the onset of dollar spot symptoms and as disease progressed the following growing season in ‘007’ creeping bentgrass. Treatments that included September applications of fluazinam + propiconazole had the greatest impact on reducing the Clarireedia concentration the following spring, while chlorothalonil was much less effective at reducing the concentration of the pathogen. Additional research is needed to determine if other fungicide chemistries applied in autumn can also affect the pathogen population in the field the following spring. From the different qPCR studies performed in the field, it is apparent that Clarireedia lives throughout the year in creeping bentgrass foliar and crown tissue as an endophytic and that dollar spot symptoms do not appear until a concentration threshold is reached. After pooling results from all the qPCR studies, symptom expression occurred between a Ct of 23.1 and 25.6. More observations in this Ct range will be required to determine the exact Clarireedia concentration required for symptom development in ‘Independence’ creeping bentgrass, and additional research with will be needed to determine if this range applies to other creeping bentgrass cultivars and turfgrass species. Objective 2 was completed by evaluating the fungal mycobiome associated with tall fescue exposed to prolonged periods of drought stress in a rainout shelter over two years. Six sets of half-sib progenies, one exhibiting a drought tolerant phenotype and the other a susceptible phenotype, were selected for analysis. The mycobiome associated with shoots, roots, and rhizosphere soil was evaluated for each tall fescue half-sib pair using both short (Illumina MiSeq) and long-reads (Oxford Nanopore Technology (ONT) MinION). Both platforms sequenced portions of the fungal nuclear ribosomal RNA genes. The Illumina MiSeq sequenced the internal transcribed spacer region (ITS, 600 bp), while the ONT MinION covered the small subunit, ITS, and partial large subunit (4,600 bp). Comparative analysis of the fungal mycobiome associated with the roots, shoots, and soil showed that the ONT MinION platform identified more diverse fungal lineages and had higher taxonomic resolution compared to the Illumina MiSeq. Furthermore, MinION was able to identify statistical differences in microbial populations between the two tall fescue phenotypes which the MiSeq could not detect. The only taxon that was consistently different in both years and both sequencing platforms was the Magnaporthales which contains pathogens known to decay turfgrass roots. It is possible that root infections caused by the Magnaporthales may increase the plant’s susceptibility to drought, further research would be needed to test this hypothesis.