| Fusarium graminearum, an important agricultural pathogen in China and throughout the world, is the major causal agent of Fusarium head blight (FHB) of wheat, barley, maize and other cereal grain crops. This pathogen can produce different kinds of trichothecene mycotoxins during infecting plants. Fusarium mycotoxins can destroy the host cells directly promoting the disease expanding and thus leading to huge losses of yield. More importantly, Fusarium mycotoxins pose a serious health threat to humans and animals. These mycotoxins are diffcult to be detected and controlled thus representing a potential threat to food and feed safety. Under the agricultural ecological condition in China, wheat, barley and maize are the top three crops jeopardized by FHB pathogens. Systemic analysis of the pathogenicity and mycotoxin productivity of F. graminearum pathogens on these three crops is of great importance to the research of the interaction between plant and their pathogens as well as the development of new methods for detecting and controlling F. graminearum and trichothecene mycotoxins. Meanwhile, based on the genome sequence of F. graminearum and the pathways of trichothecene mycotoxin synthesis and their regulations, methods for genetic chemotypeing of trichothecene mycotoxins in FHB pathogens can be developed, which will provide theory and technical supports to the establishment of a rapid and accurate mycotoxin detection and analysis system. In this study, we studied the relationship between the pathogenicity and mycotoxin productivity of representative F. graminearum strains in China, and further developed molecular identification techniques for chemotyping FHB pathogens. The major results are listed following:1. Fifteen Fusarium strains, isolated from different hosts, with different chemotypes were selected to inoculate wheat (Annong 8455, Sumai 3), barley (E 32380, E 9) and maize (Zhengdan 958). GC/MS method was applied to analyze the relationship between the pathogenicity and mycotoxin productivity of F. graminearum. The results showed that pathogenicity and the mycotoxin productivity were positively correlated and they both dramatically varied among different strains. Furthermore, the PCR detected chemotypes of different strains were confirmed by the chemical analysis. In addition, the chemotypes and amounts of mycotoxins of strains were not dependent on the hosts investigated. The mycotoxin amounts produced by strains mixture was in the average of the amounts produced by every single strain. Therefore, we conclude that the mycotoxin productivity and pathogenicity are peculiar characters of every individual strain and are not affected by the origins of hosts or locations.2. For the purpose of differentiating the three different chemotypes of Fusarium by only one PCR reaction, the specific primer pair Tri13-P1/2 was designed based on the sequence of Tri13, an essential gene in the trichothecene mycotoxin synthesis pathway. Fifty-four F. graminearum strains from different hosts and countries were assayed by the PCR with these two primers, the result of which showed a coherency with results obtained by PCR with other reported primers and chemical assays. Therefore, these primers are able to identify the genetic chemotypes of F. graminearum. Among the 54 strains of F. graminearum, fifteen strains were selected for verifying by GC/MS analysis after the inoculation on wheat, barley and maize. The chemical identification results further confirmed the validity of primers Tri13-P1/2.3. A set of multiple PCR primers were designed according to the result of the multiple alignment analysis of the sequences of Tri11genes from different F. graminearum strains with different chemotypes. With this set of primers we designed, a single specific fragment of different sizes can be amplified for 3-AcDON-,15-AcDON-and NIV-chemotype strains, respectively. Firstly, the chemotypes of 65 strains from different hosts and countries were identified with this multiple PCR strategy, the result of which was confirmed by chemical identification and PCR amplification with other primers. Subsequently, fifteen strains were inoculated on wheat, barley and maize followed by a GC/MS analysis, which proved that this multiple PCR strategy was a rapid and efficient method for chemotype identification.4. Based on the comparative analysis of sequences of Trill gene and Tri13 gene and the results of molecular chemotyping, we found a new subspecies of 3-AcDON producing strain, which may exist in Europe exclusively. These new 3-AcDON type strains showed the same deletion type of Tril3 gene as that observed for 15-AcDON type strains.5. The analysis of the population structure of F. graminearum strains isolated from maize in Hubei province was performed. The results of SCAR analysis of 100 strains isolated from maize in Hubei province with F. graminearum specific primers Fg16F/R showed a coexistence of SCAR type I (F. graminearum) (27 strains,27%) and SCAR type V (F asiaticum) (73 strains,73%) in the province. The chemotypes of these 100 strains were identified with the multiple PCR identification system we developed and confirmed by a number of specific primers. Seventy strains (70%) were identified as NIV-type and 30 strains (30%) were identified as 15-AcDON-type, while no 3-AcDON-type was detected. Based on the results of SCAR analysis and molecular chemotype identification described above, all the F. graminearum strains produce 15-AcDON and 70 strains in F. asiaticum produces NIV while other three strains, isolated from Wuchang, produce 15-AcDON mycotoxin.6. A specific primer pair of T2-P1/2 was designed for the identification of type A trichothecenes producers through multiple sequence alignments. One hundred and forty-one strains belonging to 16 species of Fusarim genus were assayed with the primer pair. The specific fragment of 470 bp was successfully amplified only in two strains, F. poae and F. langsethiae from France. Sixty-two strains among 141 strains were identified by GC/MS. Type A trichothecene mycotoxins were only detected in F. poae and F. langsethiae, which was in accordance with the PCR results. These results provided strong evidence that T2-P1/2 was reliable primer pair that could be used for detection of type A trichothecene mycotoxins.7. Two pairs of primers were designed based on two essential genes PKS4, PKS13 in ZEN biosynthesis pathway, respectively, to develop a molecular detecting system for ZEN. Corresponding fragments were successfully amplified from the genomes of 54 F. graminearum strains with both two pair of primers. Five randomly selected strains were inoculated on rice media for mycotoxin extraction and identification. ZEN was detected in all the five strains by LC/MS, the results indicated that these two primer pairs could be used to identify ZEN-producing strains in F. graminearum clade. However, whether this method can be use to detect ZEN from other Fusarium fungi other than F. graminearum should be further investigated. |
PDF Full Text Request