Pathogenicity Assay,Mycotoxin Determination And Molecular Biology Studies Of Fusarium Graminearum

Fusarium head blight or scab caused by species of Fusarium is an economically important disease on small grain cereal crops, prevalently in the areas with warm temperature and high humidity worldwide, which not only can cause yield loss, but also produce a variety of mycotoxins that can potentially pose serious threat on human health and animal production. Fusarium graminearum (sexual stage: Gibberella zeae) is the dominant pathogen responsible for wheat scab epidemics in China. Therefore, comprehensive analyses of widely distributed F. graminearum isolates in China would provide some references for further studies on the mechanisms of pathogenesis, more understanding on Fusarium-wheat interaction and development of new methods for mycotoxin analysis and food safety detection. In this study, 364 F. graminearum isolates from China and 39 foreign F. graminearum isolates from USA, Europe and Nepal were conducted as experimental materials. Main research aspects and results were concluded as follows:Pathogenicity of 58 F. graminearum isolates from different geographic area was assayed by field inoculation of florets of three wheat varieties over two years of 2001 and 2000. The results revealed that pathogenicity among 58 isolates were significantly different on all hosts of three wheat varieties, suggesting that pathogenicity of F. graminearum isolates were controlled by their own germ plasms. Also, pathogenicity of the same strain was highly correlated in the inoculation experiments on the highly resistant and highly susceptible wheat varieties, indicating that these two types of wheat varieties were more appropriate and stable for pathogenicity assessment in field trials.Through systematically comparative analyses of six inoculation experiments, ten representative F. graminearum isolates from China were obtained and subsequently applied for pathogenicity assay of foreign F. graminearum isolates and resistance assessment of wheat varieties. These isolates would be very valuable for pathogenicity-related studies of F. graminearum at the molecular level. And their indices could be a reference for population analysis of F. graminearum isolates in China.Six wheat varieties and three F. graminearum isolates with high, medium and low pathogenicity were used to develop a new method for inoculation of wheat coleoptiles, which consists of cutting off the coleoptile apex, covering the cut apex with a piece offilter paper soaked in conidial suspension, and measuring the lengths of brown lesions 7 days post inoculation. After coleoptile inoculation, distinct brown lesions in the diseased stems were observed, in which the presence of the fungus was verified by PCR amplifications with F. graminearum-specific primers. Analysis of the coleoptiles of Annong8455 inoculated with the same set of 58 different isolates of F. graminearum showed a significant difference in the lengths of the lesions, forming the basis by which pathogenicity of the isolates was assessed. These results indicate that the wheat coleoptile inoculation is a simple, rapid and reliable method for pathogenicity studies of F. graminearum in wheat. Comparative analyses of eight inoculation experiments of wheat with 58 F. graminearum isolates showed significantly direct linear correlations (P<0.001) between coleoptile and floret inoculations.In coleoptile inoculation, three resistant wheat varieties were observed to display more susceptible phenotypes, suggesting that the adult plant resistance (APR) mechanism might exist in the wheat-Fusarium scab interaction.HPLC method was used to determine deoxynivalenol (DON) concentration in mycotoxin samples of in vtiro cultures and scab-infected wheat grains. The results revealed that the DON detection peaks were obviously clear without disturbance and the retention time was about 9.5 min, indicating that the HPLC method could accurately detect DON in liquid and solid samples.Comparative analysis of the intergenic sequences of TRI5-TRI6 genes involved in the mycotoxin pathway of Fusarium resulted in the development of one generic PCR assay for the detection of both DON- and NIV-chemotypes of F. graminearum. This PCR assay produces a 300 bp fragment for DON-chemotypes and a 360 bp fragment for NIV-chemotypes that are easily differentiated on agarose gels. Mycotoxin chemotypes revealed by the PCR assay were confirmed by the chemical analyses of HPLC or GC/MS. Further analysis of 364 F. graminearum isolates collected from 12 provinces of China with this assay showed that 310 were DON-chemotypes and 54 were NIV-chemotypes, indicating that F. graminearum isolates in China were mainly DON-chemotypes. However, DON- and NIV-chemotypes coexisted in most areas throughout China.Nucleic acid sequence analyses of DON- and NIV-chemotypes fragments corroborate the differences between the two mycotoxin chemotypes in the regions. DON-chemotypes display more variations whereas NIV-chemotypes appear more conserved.Furthermore, this assay was used to detect mycotoxin chemotypes of DON and NIV in the wheat and maize grains with different disease symptoms, which respectivelycollected from the wheat grains inoculated by the equally mixed conidial suspension of DON- and NIV-producing F. graminearum isolates with florets and naturally Fusarium-'mftcttd maize grains in the field. There is no either DON- or NIV-specific amplification detected in healthy grains. Simultaneously, the mycotoxin chemotypes of DON and NIV were sensitively detected in all lowly, moderately and highly scab-infected grains of wheat and maize.This PCR assay is a rapid, reliable and cost-effective method for the identification of two mycotoxin chemotypes of DON and NIV in Fusarium fungi. Also, the method developed in this study could be used in the detection and control of mycotoxins contaminated in food and grains with some practical values and commercial prospect.