Assessing the Reaction of American Wildrice to Inoculated Pathogens. Raymond Porter*, Robert Nyvall, and Laura Carey University of Minnesota, NCROC, 1861 E. Hwy 169, Grand Rapids, MN Assessing the Reaction of American Wildrice to Inoculated Pathogens. Raymond Porter*, Robert Nyvall, and Laura Carey University of Minnesota, NCROC, 1861 E. Hwy 169, Grand Rapids, MN INTRODUCTION American wildrice (Zizania palustris— Fig. 1) has several known stem rot diseases, caused by the fungal pathogens Nakataea sigmoidea (Fig. 2), Bipolaris oryzae, and B. sorokiniana. The Bipolaris species also cause the foliar diseases fungal brown spot and spot blotch (Fig. 3). Since these diseases have previously been the target of selection to improve varietal resistance, methods have been developed to inoculate leaves with conidia suspensions to develop resistant varieties. Selection for resistance to stem rots in wildrice has not been done. In this study, methods were developed for growing inoculum of N. sigmoidea and for delivering known quantities of propagules of all three species to the stems of wildrice varieties. These methods were applied in a multilocation trial, where plots of a single variety were inoculated with all three species to test several fungicides for efficacy in reducing the effects of these diseases. This experiment was used as an opportunity to develop methods to quantify differences in disease severity for future assessment of varietal resistance in a breeding program. Specific objectives included: 1) Develop and assess the effectiveness of a method of production and inoculation of N. sigmoidea, and 2) Assess the effectiveness of leaf and stem disease rating methods in detecting statistical differences produced by different treatments (fungicides). INTRODUCTION American wildrice (Zizania palustris— Fig. 1) has several known stem rot diseases, caused by the fungal pathogens Nakataea sigmoidea (Fig. 2), Bipolaris oryzae, and B. sorokiniana. The Bipolaris species also cause the foliar diseases fungal brown spot and spot blotch (Fig. 3). Since these diseases have previously been the target of selection to improve varietal resistance, methods have been developed to inoculate leaves with conidia suspensions to develop resistant varieties. Selection for resistance to stem rots in wildrice has not been done. In this study, methods were developed for growing inoculum of N. sigmoidea and for delivering known quantities of propagules of all three species to the stems of wildrice varieties. These methods were applied in a multilocation trial, where plots of a single variety were inoculated with all three species to test several fungicides for efficacy in reducing the effects of these diseases. This experiment was used as an opportunity to develop methods to quantify differences in disease severity for future assessment of varietal resistance in a breeding program. Specific objectives included: 1) Develop and assess the effectiveness of a method of production and inoculation of N. sigmoidea, and 2) Assess the effectiveness of leaf and stem disease rating methods in detecting statistical differences produced by different treatments (fungicides). MATERIALS AND METHODS Variety: Itasca (a high-yielding, shattering resistant variety selected for resistance to fungal brown spot. Design: RCBD, 1.5 x 3.0 m plots, 8 fungicide treatments, 6 reps at each of 3 locations. Inoculation and treatments: Eighteen isolates of B. sorokiniana were cultured on a medium consisting of course perlite, wildrice flour, and 1% PDA (1:2:4). Separately, 18 isolates of B. oryzae were cultured on same medium. Conidia were filtered into suspension with water after 15 (B. sorokiniana) or 18 (B. oryzae) days of growth. Inoculum of B. sorokiniana was applied at a rate of 4.5 million conidia per plot with a CO 2 sprayer at mid-tillering. B. oryzae was applied 5 days later at 1.8 million conidia per plot. Six N. sigmoidea isolates were cultured on the same medium. Mature sclerotia were produced in 14 days, at which time medium was dried, crushed, and filtered through a #10 seive (Fig. 4) to produce the dried inoculum (750 sclerotia/ml). At late tillering, inoculum was spread with a Spred-Rite granular applicator on the water surface just inside the side borders of each plot, at 16,000 sclerotia per plot (Fig. 5). Five fungicides—Tilt (propaconizole), Headline (pyraclostrobin), Quadris (azoxystrobin), Quilt (azoxystrobin+propaconizole), and Stratego (trifloxystrobin+propaconizole) were applied at boot stage, and two of these were also applied at heading, making up seven fungicide-timing treatments plus a control. MATERIALS AND METHODS Variety: Itasca (a high-yielding, shattering resistant variety selected for resistance to fungal brown spot. Design: RCBD, 1.5 x 3.0 m plots, 8 fungicide treatments, 6 reps at each of 3 locations. Inoculation and treatments: Eighteen isolates of B. sorokiniana were cultured on a medium consisting of course perlite, wildrice flour, and 1% PDA (1:2:4). Separately, 18 isolates of B. oryzae were cultured on same medium. Conidia were filtered into suspension with water after 15 (B. sorokiniana) or 18 (B. oryzae) days of growth. Inoculum of B. sorokiniana was applied at a rate of 4.5 million conidia per plot with a CO 2 sprayer at mid-tillering. B. oryzae was applied 5 days later at 1.8 million conidia per plot. Six N. sigmoidea isolates were cultured on the same medium. Mature sclerotia were produced in 14 days, at which time medium was dried, crushed, and filtered through a #10 seive (Fig. 4) to produce the dried inoculum (750 sclerotia/ml). At late tillering, inoculum was spread with a Spred-Rite granular applicator on the water surface just inside the side borders of each plot, at 16,000 sclerotia per plot (Fig. 5). Five fungicides—Tilt (propaconizole), Headline (pyraclostrobin), Quadris (azoxystrobin), Quilt (azoxystrobin+propaconizole), and Stratego (trifloxystrobin+propaconizole) were applied at boot stage, and two of these were also applied at heading, making up seven fungicide-timing treatments plus a control. Disease assessment and analysis: All plots at a location were harvested on the same day. Ten stems and 20 flag leaves per plot were collected at harvest and frozen to be later rated for area affected by disease. Diseased leaf area was estimated by comparison with the Clive James key for Septoria leaf blotch (Key 1.6.1). Diseased stem area was estimated without the aid of a key. Two pieces were cultured from each stem and one piece from each leaf (a total of 20 each of stem and leaf pieces). Five to seven days later, fungal species were identified for each lesion to estimate the incidence of each pathogen. Indexes were calculated for leaf and stem diseased area due to a particular pathogen by multiplying the visual estimate by the frequency of the pathogen incidence on cultured leaf pieces. Each location was analyzed separately using SAS Proc Mixed, adjusting for spatial variability with the function sp(powa). Significance of pairwise comparisons at P<0.05 was used as the criterion for declaring treatments statistically different. Disease assessment and analysis: All plots at a location were harvested on the same day. Ten stems and 20 flag leaves per plot were collected at harvest and frozen to be later rated for area affected by disease. Diseased leaf area was estimated by comparison with the Clive James key for Septoria leaf blotch (Key 1.6.1). Diseased stem area was estimated without the aid of a key. Two pieces were cultured from each stem and one piece from each leaf (a total of 20 each of stem and leaf pieces). Five to seven days later, fungal species were identified for each lesion to estimate the incidence of each pathogen. Indexes were calculated for leaf and stem diseased area due to a particular pathogen by multiplying the visual estimate by the frequency of the pathogen incidence on cultured leaf pieces. Each location was analyzed separately using SAS Proc Mixed, adjusting for spatial variability with the function sp(powa). Significance of pairwise comparisons at P<0.05 was used as the criterion for declaring treatments statistically different. Fig. 1Cultivated American wildrice, Zizania palustris cv. 'Petrowske Purple' Fig. 2Sclerotia inside of a wildrice stem infected with Nakataea sigmoidea Fig. 3Fungal brown spot (left), caused by B. oryzae, and Spot blotch caused by B. sorokiniana. Fig. 4Granular inoculum of N. sigmoidea in perlite medium to be filtered (left). Sclerotia amid perlite granules in inoculum (right). Fig. 5Granular applicator used to apply dry inoculum of N. sigmoidea (left). Granules on water surface adhering to wildrice stem (circled in photo on right). Fig. 6Estimated diseased leaf area of flag leaves for treatments at Aitkin. Treatments having a letter in common are not significantly different at P=0.05. RESULTS Significant treatment differences were found for diseased leaf area at all three locations, but the Aitkin location was especially severe, with many significant comparisons (Fig. 6). Incidence of B. oryzae was far greater than B. sorokiniana in leaf lesions and also showed significant differences (not shown). When multiplied by diseased leaf area, the index magnified treatment differences (Fig. 7). RESULTS Significant treatment differences were found for diseased leaf area at all three locations, but the Aitkin location was especially severe, with many significant comparisons (Fig. 6). Incidence of B. oryzae was far greater than B. sorokiniana in leaf lesions and also showed significant differences (not shown). When multiplied by diseased leaf area, the index magnified treatment differences (Fig. 7). Fig. 7Index estimating percentage of leaf area diseased due to B. oryzae at Aitkin. photo by Dave Hansen, MAES Stem lesion area was difficult to estimate, and analysis showed no significant treatment differences. B. oryzae was predominant among the stem lesions, and N. sigmoidea did not have as high an incidence as expected (1-3% vs 10-30% for B. oryzae). However, when stem lesion area was multiplied by incidence of all three stem pathogens combined, significant treatment differences were seen (Fig. 8). Fig. 8Index estimating percentage of stem area diseased due to Bipolaris spp. and N. sigmoidea at Aitkin. CONCLUSIONS 1.Because incidence of Nakatae in stems was low, this method of inoculation needs to be improved. Earlier application of inoculum and development of a method to apply sclerotia directly to stems are priorities for future research. 2.Indexing of both diseased leaf and stem area with pathogen incidence in cultured lesions improves statistical discrimination between treatments. This may indicate that a significant number of lesions (both stem and leaf) are of non-fungal origin. CONCLUSIONS 1.Because incidence of Nakatae in stems was low, this method of inoculation needs to be improved. Earlier application of inoculum and development of a method to apply sclerotia directly to stems are priorities for future research. 2.Indexing of both diseased leaf and stem area with pathogen incidence in cultured lesions improves statistical discrimination between treatments. This may indicate that a significant number of lesions (both stem and leaf) are of non-fungal origin. ACKNOWLEDGMENTS Funded through USDA-ARS Cooperative Agreement No Wildrice growers Tom Godward, Rod Skoe, and Ed Mohs graciously provided space for experiments. Dan Braaten and Henry Schumer provided technical assistance. Representatives of BASF, Bayer, and Syngenta provided fungicides for the treatments. ACKNOWLEDGMENTS Funded through USDA-ARS Cooperative Agreement No Wildrice growers Tom Godward, Rod Skoe, and Ed Mohs graciously provided space for experiments. Dan Braaten and Henry Schumer provided technical assistance. Representatives of BASF, Bayer, and Syngenta provided fungicides for the treatments. photos by James Percich, U of MN