Optimization and Utilization of Immature Spike Culture System to Identify and Characterize Fusarium Head Blight Resistant Wheat Genotypes
Date
2019-03-04
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Doctoral
Abstract
Fusarium head blight (FHB) is a potentially devastating fungal disease-causing yield loss and grain quality reduction in wheat, barley, oat and other small grain cereals. In wheat (Triticum aestivum L.) FHB is predominantly caused by Fusarium graminearum Schwabe [telomorph: Gibberella zeae (Schaw) Petch), although a few other species also cause FHB. Fusarium infects wheat immature spikes during flowering and produces a trichotehecene group of mycotoxins that are toxic to humans and animals, but also act as virulence factors responsible for the spread of the disease in the plant tissue. To mitigate the effect of FHB, there is a need to develop durable FHB resistance in wheat. To select for durable FHB resistance, an immature spike culture-based method was developed to distinguish FHB resistant and susceptible genotypes. Screening of an ethyl methyl sulfonate (EMS) treated spike culture derived variant (SCDV) population (134 lines) of wheat cultivar AC Nanda identified 15 FHB-resistant and 9 FHB-susceptible lines.
A LC-MS/MS method was optimized to quantify type B trichothecenes in infected wheat spikes. The optimized method separated four mycotoxins: DON, 15-ADON, 3-ADON and Nivelanol; and DON-3-Glycoside (D3G) within 25 min with intermediate precision and repeatability. The coefficient of determination was >0.99 for all the mycotoxins whereas, the level of detection and quantification ranged from 0.30 to 0.75 and from 0.5 to 2.5 ng/mL, respectively. The FHB resistant genotypes had reduced concentrations of all mycotoxins compared to the FHB-suscpeptible genotypes, suggesting the utility of the method to determine grain mycotoxin concentration and to identify the chemotype of a F. graminearum isolate.
UDP-glucosyl transferases (UGT) catalyze the conversion of DON to non-toxic D3G by transferring a glucoside moiety to the 3’- carbon on DON which contributes to FHB resistance, thus preventing the spread of the fungus. Nucleotide sequence comparisons revealed one nucleotide change in TaUGT-2B and TaUGT-3B between AC Nanda and Sumai-3. Statistical analysis determined that the nucleotide difference present at positions 450 and 1558 bp from the translation initiation site between AC Nanda and Sumai-3 in the TaUGT-2B and TaUGT-3B genes respectively correlated significantly (P<0.01) with the disease severity of 134 SCDV lines.
FHB- resistant and susceptible lines were also screened in FHB nurseries at Carman Manitoba (University of Manitoba) (2017) and University of Saskatchewan (2017, 2018). FHB disease development and severity, observed during in vitro spike culture screening method, showed a positive correlation with mycotoxin accumulation in the spikes as determined using a LC-MS/MS method, and FHB disease development and severity in the field experiments. Theses results supported the utilization of in vitro spike culture to identify FHB resistant SCDV lines that can potentially be used in breeding programs to develop FHB resistant wheat germplasm.
The immature spike cultures from 55 SCDV lines were separately inoculated with five F. graminearum isolates, Carman-NIV (NIV), Carman-705-2 (3-ADON), M9-07-1 (3-ADON), M1-07-2 (15-ADON), and China-Fg809 (15-ADON) that produced different mycotoxins (in parenthesis). Five genotypes with stable FHB resistance were identified based on their FHB disease development and severity at 5, 7, 9, and 11 days after inoculation (DAI). Compared with the susceptible control AC Nanda, all the resistant genotypes had significantly reduced FHB susceptibility. LC-MS/MS analysis identified five DON chemotypes. The SCDV lines FHB213.4, FHB244.1, FHB245.6, FHB250.2, and FHB252.3 were identified as having resistance to multiple F. graminearum chemotypes.
The SCDV lines FHB202, FHB244, FHB250 and FHB256 were also used as resistant parent to cross with an elite wheat cultivar PT588 (CDC Hughes) to generate FHB resistant wheat breeding populations. The genotypes with the same SNP as Sumai-3 in crossing combinations FHB202/PT588, FHB244/PT588, FHB250/PT588 and FHB256/PT588 accounted for 5.88%, 9.09%, 34.62% and 10.53% within each BC2 progeny population, with an average rate of 15.03%. The rates of FHB resistance in crossing combinations of PT588*2/FHB202, PT588*2/FHB244, PT588*2/FHB250 and PT588*2/FHB256 were 0.0%, 0.0%, 17.39% and 27.27%, with an average of 11.17%. The ANOVA results showed significant differences of phenotypic FHB severity among different genotypic groups (P<0.01), indicating a strong correlation between phenotypic and genotypic data.
In conclusion, the study demonstrated the use of immature spike cultures as a method to screen for FHB disease development and resistance. A LC-MS/MS method was optimized to characterize four mycotoxins and D3G commonly present during FHB infection. The SCDV population yielded FHB resistant lines, which were used to cross with CDC Hughes to develop FHB resistant wheat genotypes.
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Keywords
Wheat, Fusarium Head Blight (FHB), Immature Spike Culture, Mycotoxin, Deoxynivalenol (DON), Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), High Resolution Melt (HRM), Single Nucleotide Polymorphism (SNP), Backcross
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Plant Sciences
Program
Plant Sciences