Phenotyping protocol for soybeans of maturity groups 000 and 00: screening for tolerance to Sclerotinia sclerotiorum

As soybeans are grown in higher latitudes in the northern regions, double (00) and triple zero (000) maturity group (MG) varieties will be bred. White mold (WM) is a concern in Northern growing environments, and appropriate screening protocols for reliable and reproducible phenotyping will be required. The available data on pathogen resistance screening is currently minimal for MG 0 to 000. Investigation of field and greenhouse inoculation methods were conducted to make preliminary observations and assess their suitability to early maturity soybeans. The first objective of this research was to determine if soybean inoculation with Sclerotinia sclerotiorum (Lib.) de Bary (Ss), the fungus that causes WM and Sclerotinia stem rot (SSR) in soybeans, with spray inoculum can cause infection that can be differentiated for disease ratings. The second objective was to establish a protocol for applying spray inoculum in a greenhouse, which can be used to phenotype plants and build datasets for the future genotyping of resistant breeding lines for soybean cultivar development.\nDuring 2017 and 2018, research was conducted in Saskatoon, Saskatchewan, Canada, on fields with a history of SSR occurrence in canola (Brassica napus L.). Greenhouse protocols were conducted in the L. F. Kristjanson Biotechnology Complex in Saskatoon. Soybean varieties of early maturing groups 00 and 000 with known reactions to WM were sprayed with Ss inoculum of ascospores and/or mycelium fragments to screen for WM tolerance. In both environments, inoculum treatments differed in their composition and application frequencies. Data was intended to be collected for percentage stand, canopy width, disease presence, and disease severity. The high ambient temperatures and low precipitation resulted in unfavorable conditions for disease establishment. The greenhouse conditions were better suited for disease expression; however, unknown contamination of the control precluded data collection. An important observation of this preliminary research indicated spray inoculum is unreliable for field and greenhouse use.\nAfter a series of unsuccessful spray inoculation trials, the cut stem and mycelium plug method was executed in the L. F. Kristjanson Biotechnology Complex in Saskatoon in 2019. The study was a continuation of the preliminary research, which intended to identify a reliable and reproducible screening protocol for phenotyping genetic resistance to Sclerotinia sclerotiorum (Lib.) de Bary (Ss) in soybeans (Glycine max L. Merr.). Soybeans from the 00 and 000 maturity group (MG) were inoculated with mycelium plugs made of agar from two different Ss isolates in a greenhouse environment over two experimental periods. Plants were inoculated and then moved to the humidity chamber for the 48-hour incubation period. Once incubation was complete, plants were removed from the humidity chamber, and qualitative scores on overall plant health were taken at seven, ten, and fourteen days post-inoculation. On the 14th-day post-inoculation, measurements of the lesion length and width were also recorded.\nANOVA with a Tukey Test and correlations were used to determine the significance of experimental factors. Isolate was found to have a significant effect on lesion length in one of two experiments, while lesion width was not significantly affected by isolate in either experiment, with the ARC isolate having a greater ability to produce differentiated results for phenotyping. Plant health scores taken at fourteen days post-inoculation were found to be significant. Correlation coefficients were calculated between the two experiments to indicate repeatability. These values increased over the three days of data collected (7, 10, and 14 days post-inoculation). When comparing the two experiment replications, lesion length had a notably higher correlation compared to lesion width. Lesion width was found to have no significance.\nThe greenhouse screening with agar plugs was successful in its ability to generate a dataset that differentiated between genotypes. One of the two Ss isolate produced larger lesions in plants, but either could be used for screening in future studies as plant health was not significantly different between the two. The data also found that collecting data on the overall health of plants is a better indication of plant tolerance to the pathogen than measurements of lesion size. Health scores can be taken at multiple dates to track the progress of the disease, but scores 14 days post-inoculation was the most differentiated.

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