The two RIL populations were phenotyped for the severity of PRR development in inoculated field experiments in order to select RILs with high and low disease phenotypes.
Field experiments: The BC*susceptible RIL (n = 181) and the BC*tolerant RIL (n = 165) population were sown on 18 and 19 June 2014 in separate experiments at Hermitage Research Facility, Queensland (−28.204908 S, 152.102689 E) in 2014. The methods used for the RIL field experiments are described by Amalraj et al. (2019) (link). Briefly, the plots were sown with a four-row seeder with separate in-furrow delivery of in-solution Mesorhizobium ciceri rhizobia inoculant and the 10 isolate mixture of P. medicaginis at sowing at a concentration of ~1,500 oospores/seed. Each plot had 20 seeds per single 1.2-m row plot. The experiments had a randomised block design with four replicates. Check varieties covering a resistance spectrum were supra-replicated on block and sub-block basis. The soil type at the Hermitage site was a deep, self-mulching, black vertosol (Thomson et al., 2007 (link)). No in-crop irrigation was applied, and 97 mm of in-crop rainfall was received during the field experiments.
Establishment and disease assessments: The number of seedlings in each plot was counted 48 days after sowing (DAS) to determine establishment. A minimum of three disease assessments were then made; the first assessment was performed when early disease symptoms were evident in susceptible check varieties (85 DAS, pre-flowering 12–14 nodes), the second assessment was made mid-season (118 DAS, immature pods present), and the final assessment (135 DAS) occurred at the beginning of pod maturity. At each disease assessment, separate counts of the number of chlorotic, dead, and total number of plants were made. Late-season assessments were carried out before widespread plant senescence had occurred. At the final assessment, dead plants were categorised into development categories as having produced no pods (died as seedlings prior to flowering) or as podded, and counts of each category were made. At this assessment, counts were also made of the number of chlorotic, senescent, and healthy non-senesced plants.
Selected RIL disease phenotype groups: To select RILs with high and low disease phenotypes, the proportion of plants that had died at the 135 DAS assessment timing was used as the criterion. From each RIL population, six lines were randomly selected as low disease lines using a random number function in Excel (Microsoft Office Standard, 2016 ) on the basis of having no plant death. Six high-disease RILs were randomly selected from each RIL population based on more than 30% plant death for the BC*susceptible RIL and greater than 10% plant death for the BC*tolerant RIL.
Field experiments: The BC*susceptible RIL (n = 181) and the BC*tolerant RIL (n = 165) population were sown on 18 and 19 June 2014 in separate experiments at Hermitage Research Facility, Queensland (−28.204908 S, 152.102689 E) in 2014. The methods used for the RIL field experiments are described by Amalraj et al. (2019) (link). Briefly, the plots were sown with a four-row seeder with separate in-furrow delivery of in-solution Mesorhizobium ciceri rhizobia inoculant and the 10 isolate mixture of P. medicaginis at sowing at a concentration of ~1,500 oospores/seed. Each plot had 20 seeds per single 1.2-m row plot. The experiments had a randomised block design with four replicates. Check varieties covering a resistance spectrum were supra-replicated on block and sub-block basis. The soil type at the Hermitage site was a deep, self-mulching, black vertosol (Thomson et al., 2007 (link)). No in-crop irrigation was applied, and 97 mm of in-crop rainfall was received during the field experiments.
Establishment and disease assessments: The number of seedlings in each plot was counted 48 days after sowing (DAS) to determine establishment. A minimum of three disease assessments were then made; the first assessment was performed when early disease symptoms were evident in susceptible check varieties (85 DAS, pre-flowering 12–14 nodes), the second assessment was made mid-season (118 DAS, immature pods present), and the final assessment (135 DAS) occurred at the beginning of pod maturity. At each disease assessment, separate counts of the number of chlorotic, dead, and total number of plants were made. Late-season assessments were carried out before widespread plant senescence had occurred. At the final assessment, dead plants were categorised into development categories as having produced no pods (died as seedlings prior to flowering) or as podded, and counts of each category were made. At this assessment, counts were also made of the number of chlorotic, senescent, and healthy non-senesced plants.
Selected RIL disease phenotype groups: To select RILs with high and low disease phenotypes, the proportion of plants that had died at the 135 DAS assessment timing was used as the criterion. From each RIL population, six lines were randomly selected as low disease lines using a random number function in Excel (Microsoft Office Standard, 2016 ) on the basis of having no plant death. Six high-disease RILs were randomly selected from each RIL population based on more than 30% plant death for the BC*susceptible RIL and greater than 10% plant death for the BC*tolerant RIL.
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