| Fusarium head blight(FHB)is the most devastating wheat disease occurred in warm humid climate areas and semi-humid areas worldwide.Developing disease-resistant cultivars is the most efficient way to control the disease.Accurate evaluation of the wheat resistant phenotype is crucial for fine gene mapping and development of disease-resistant varieties.Several methods including the evaluation method under natural climate conditions,spore misting,infected kernels on soil surface,in vitro leaf tissue infection method and single floret inoculation method(SFI)have been used to evaluate the resistant phenotypes.However,all of these methods have limitations and deficiencies in some degree.SFI is the most frequently-used method for FHB inoculation and resistance evaluation.We developed a novel method of FHB inoculation and evaluation called BRDI,which is performed by injecting spore suspensions into the basal internodes of rachilla and then determine the phenotype according to both the time when the first infected spikelet appears and the percentage of symptomatic spikelets(PSS)in 22 to 25 days after inoculation.This research aimed to validate BRDI and to compare BRDI with the traditional SFI in the aspects of incidence of successful infection,the PSS,the toxin content as well as the infection pattern both in the greenhouse and in the field conditions,and also to understand if the optimized BRDI method can be diagnostic for Fhbl in NILs,the varieties with known resistance levels,recombinant inbred lines(RIL),and in backcross lines contrasting in FHB resistance.We found that the successful incidence of BRDI reached up to 88.2%to 100%while that of SFI was in the range of 30%to 77.8%by comparing the two methods in the greenhouse.In the resistant varieties,no symptoms were observed in the spikelets during the evaluation period between inoculation to scoring,but the pathogen spread within rachis and did not break through the rachis node.However,the symptoms appeared on one of the third to the fifth spikelet in 8 to 12 days,depending on genotypes,after inoculation.The BRDI method was generally superiorto the SFI method.However,the BRDI method produced a zero of PSS in the resistant genotypes and was unable to differentiate the different resistant genotypes,while SFI frequently produces pseudo resistance and underestimate the severity due to the failure of pathogen spread within spike.These two methods are complementary to each other and but are not replaceable,therefore,application of the two methods simultaneously can greatly improve the reliability of phenotyping.Based on the observed facts,we found that the BRDI method had high goodness of fit between the BRDI scores and the marker genotypes of Fhbl locus,which indicates that the BRDI method is more effective to evaluate if the varieties carry Fhb 1 or the functionally similar genes to Fhb1.Under the two methods,DON concentrations in grains were quantified and the correlations between DON concentration and PSS were calculated.Although the PSS under the BRDI method was lower or significantly lower than that of the SFI method,the grains under the BRDI method still accumulated similar DONconcentration to that of the SFI method.We speculated the rachis may be the crucial for inducing the generation of toxin,which evidenced that lower PSS may disagree with low DON.NP-77(BSYH)was used as a control(standard)due to the lowest DON content in grains and lowest PSS.Taking DON content and the PSS together,the Mahalanobis distance was applied to comprehensively evaluate the overall resistance level of a specific genotype and found that NP-154,NP-89,NP-90,NP-70 and NP-77 had superior FHB resistance levels that Sumai 3 had.We used the Fusarium spore with GFP proteins in the inoculation,combining with the BRDI method and laser confocal microscopy,we compared the temporal and spatial differences in the infection patterns in the R and S varieties respectively,and found that the cortical cell may play an important role in the defense response in wheat.The dominant advantage of the BRDI is that there is no need to have extra moisture measures after inoculation,either in the field or in the greenhouses.The humidity that is required for successful infection is provided by the water within plant,thus saves the cost that is essential for SFI method.SFI is usually performed inthe flowering time and inoculation is time-concentrated and thus is labor-intensive.However,BRDI can be conducted at flexible stages from heading,anthesis to the early stage of grain filling(in 10 days after flowering),therefore,the BRDI method has high flexibility in inoculating time,thus are labor-efficient.BRDI has higher efficiency and lower cost than SFI has.It also maximizes the differences in phenotyping scores between the resistant and susceptible genotypes,and is friendly to technicians and environments. |
