1. GOAL: Characterize the aggressiveness of sclerotinia isolates for use in future pathogen biology and resistance screening assays
I aim to collect a panel of S. sclerotium isolates from soybean-growing fields throughout Minnesota that can be characterized and used in pathogen biology assays and to screen breeding and commercial lines for resistance to SSR. Diverse isolate populations have been documented across US soybean fields (Aldrich-Wolfe et al. 2015 and Willbur et al. 2017).
Aggressiveness, as indicated by the severity of infections, is often different between isolates and soybean genotypes resistance rankings can also shift, depending on the isolate used in screenings (Willbur et al. 2016). Due to these differences, Willbur et al. (2017) concluded that multiple isolates with a range of characterized responses are recommended to screen for resistance to SSR. Additionally, biological studies will benefit from a representative isolate collection, as isolates are not uniform. For example, the number of sclerotia that can produce apothecia and the rate of apothecial production differ by isolate (Lui and Paul 2007). Additionally, the reference isolate for which we have sequenced genome (isolate 1980) is not as aggressive as several other isolates, and oddly, does not have mycelial compatibility with isolates that it was assayed with (Willbur et al. 2017). Due to these differential characteristics, this study aims to characterize Minnesota isolates in soybean to establish a range of native, biologically relevant isolates for germplasm screening and fungal biology assays in Minnesota.
Obj.1) Collect a panel of S. sclerotiorum isolates from Minnesota soybean fields and researchers
In the summer of 2021, isolates from 10 or more soybean fields will be collected from throughout the state. I will collect isolates through visits to research sites and through communications with growers. Sclerotia from diseased plants will be plated on PDA and sclerotia will be collected after approximately 14 days. New sclerotia will be collected and stored at 4° C, in the dark with desiccator beads, as described in Pottinger et al. (2008).
Obj. 2) Characterize their growth and relative aggressiveness on soybean lines known to have differential resistance
New isolates will be characterized alongside isolates currently used by researchers at UMN, including isolates in the Malvick Lab, and with the isolate for which we have sequenced genome, isolate 1980 (Amselem et al. 2007). Isolate growth will be characterized by measuring four replicated fungal cultures of each isolate over time. Area under the growth curves will be assessed for 3-4 days, until the edge of plates are reached. To evaluate isolate aggressiveness, susceptible cultivar Dwight will be challenged with all isolates in the greenhouse using a cut petiole technique (Peltier and Grau 2008), where an agar core is collecting from the leading edge of mycelia and introduced to the petiole. These inoculations will be repeated three times and lesions will be measured 7-14 days post-inoculation, at three intervals, to assess disease progress (McCaghey et al. 2017 and Willbur et al. 2017). The study will be organized in a randomized complete block design.
Once we identify a panel of isolates with different levels of aggressiveness, we can test whether isolates will distinguish the resistance ranking of cultivars. Using three representative isolates, we will inoculate soybean check lines (Webster et al. 2021, Figure 2) developed by Dr. Damon Smith’s Lab at University of Wisconsin-Madison with known low, moderate, and high levels of resistance (compared to susceptible controls) to see if resistance rankings are similar when challenged with the new, UMN isolates.
2. GOAL : Define relationship between canopy architecture and SSR development
In addition to physiological resistance, plant architecture may be an important component of avoiding infection by S. sclerotiorum in the field. The development of SSR and the production of apothecia that cause disease in soybean are highly dependent on environmental conditions. Two of these particularly important conditions include moisture and light wavelength penetration to the surface of the soil, where apothecia develop. Developing a better understanding
of plant architecture in relation to disease development may lend to future possibilities of screening for architecture traits, combining genetic and architecture traits while breeding. Additionally, wide rows and high seeding rates are associated with decreased SSR development, but yields are reduced in this planting scenario (Webster et al., 2021). However, it may be possible to identify architecture traits that allow planting densities to remain high without enhancing disease or harming yields. In soybean, branch angle is the primary trait associated with canopy architecture. This trait impacts canopy closure and light penetration.
Obj. 1) Characterize the branching angle of lines in the field
To quantify branch angle, the angles between two branches growing in opposite directions near the base of 20 fully mature individuals from each line in this study will be measured in the field with a protractor; average branch angles will be calculated and compared for the lines. Lines used in this study will be from Dr. Aaron Lorenz’s research program and include 1) a branch angle diversity panel of 10 mutant lines, 2) near isogenic lines (lines with nearly identical genetic makeups except for branch angle loci), and 3) an “era panel” of historical soybean accessions. Evaluations will occur both in the greenhouse, in a randomized complete block design, and in the field. Evaluations in the field will occur within existing trials of Dr. Aaron Lorenz, and postdoctoral researcher Dr. Suma Sreekanta. The lines will be grown in two locations per group of lines, for a total of 6 trial locations. Research farm locations include St. Paul, Rosemont, Waseca, and Lamberton, Minnesota.
Obj. 2) Define the genetic, SSR resistance of architecturally diverse lines
In addition to examining branch angle, the genetic resistance of lines will be assessed, independent of branch angle in the greenhouse. This will be helpful to observe whether infection rates in the field are different due to genetic/physiological resistance or because of disease escape from canopy architecture variation. To examine the genetic resistance of lines, they will be directly challenged with the pathogen, S. sclerotium, as described in Goal 1, Obj. 2. Greenhouse screenings will compare the resistance reactions of the lines measured in the field, and lines will be evaluated for resistance alongside defined check lines (Webster et al. 2021) that are susceptible, moderately resistant, and highly resistant lines (compared to the susceptible and moderate controls). Dr. Ashish Ranjan is developing check lines from Dr. Aaron Lorenz’s research program, and these may be used in comparisons as well.
Obj. 3) Measure canopy closure and light penetration along with apothecia and SSR development
Within each of the evaluated lines, four plots will be monitored for canopy closure, wavelength at the ground, disease, and apothecia production. As the canopy closes, a favorable microclimate promotes sclerotial germination and SSR development by maintaining high humidity, prolonging leaf wetness, and lowering temperature (Blad et al. 1978 and Kurle et al. 2001). Light, temperature, and moisture are considered to be the three most important factors for the germination of sclerotia of S. sclerotiorum (Abawi and Grogan 1979; Letham 1975; Sun and Yang 2000). Measurements of canopy closure will start before beginning bloom growth stage (R1) until full pod (R4) and will occur in by pictures in three fixed spots at a height of 1.5m in each plot, as described in (Mamadou et al. 2018). For scale reference, every picture included a plastic ruler. The percentage of canopy closure will be calculated for each sampling day as: Canopy closure=1-(daily measured distance/row spacing distance).
Additionally, the wavelengths of light penetrating to the ground where apothecia germinate and the intensity of light will be measured using a portable, fibre optic spectrometer. The spectrometer will be placed at three positions relative to the soybean stem base, into the row, and at 3-5 locations in each sampling plots. Measurements will be performed on sunny days, within a three-hour window each sampling period. Disease severity will be monitored in each naturally infested plot (if disease pressure is present). Thirty arbitrarily selected plants in each plot of the field nursery at the R6 soybean growth stage will be evaluated. Plants were scored either 0 (no infection), 1 (infection on branches), 2 (infection on, but not girdling, the main stem), or 3 (infection on the main stem resulting in death or poor pod fill) (McCaghey et al 2017). The number of apothecia will be monitored during each growth stage. Apothecia counts will be collected from 3-5 squares placed randomly throughout plots so that the middle of each square was aligned with a row.
Importantly, Dr. Lorenz’s group will evaluate the lines for yield, which would be a priority consideration in line selection for breeding and further trialing.
GOAL: Develop reliable S. sclerotiorum nurseries for future SSR field trials
Currently, researchers do not have field sites with reliable and uniform inoculum where we can conduct SSR experiments (personal communication). High disease pressure across plots is often required to observe the impact of experimental treatments (such as variety resistance differences or fungicide efficacy).
To produce apothecia and simulate natural ascospore infections, sclerotia need to undergo a period of cold conditioning for about eight weeks (Pethybridge et al. 2008). This summer, I am interested in trialing three methods to develop disease pressure for trials in 2023. These include:
1) growing sunflowers, which are very susceptible to white mold, inoculating them the back of the head with a slurry of S. sclerotiorum, and then incorporating residue into the soil in the fall of 2022.
2) sprinkle sclerotia inoculum generated in the lab on sorghum/millet seed into the field during the fall before 2023 trials. Cold conditioning over the winter should allow the inoculum to produce apothecia in the following field season.
3) I will grow sclerotia in the lab, cold condition them in the fridge, and then spring-apply the sclerotia to the field.
Untreated, naturally infested plots will be left as controls to compare with plots treated with the described infestation methods. This trial will be conducted in strips, replicated three times per inoculation method at each site. SSR trials will be conducted in the disease nursery locations in 2023, and apothecia and disease incidence will be monitored in the untreated plots of next year’s trials.
The goals and objectives described in this proposal will set the stage for my soilborne fungi pathology lab to conduct biologically relevant SSR research in soybean and will open new, creative avenues to improve resistance to this challenging fungal disease.