Function Of Introns In β-Tubulin Gene In Fusarium Graminearum And Contribution To Carbendazim Resistance

Fusarium graminearum, which is the overriding pathogen of Fusarium head blight (FHB) on wheat, is a leading cause of economic loss in these crops. In addition to reducing seed mass and quality, the fungus contaminates grain with toxic metabolites such as DON and NIV that are a threat to human and other mammals’ health.Carbendazim and other benzimidazole fungicides have been used to control FHB for more than40years since Shenyang Research Institute of Chemical Industry industrialized manufacture in1970led by Zhang Shaoming. Benzimidazole fungicides combine with β-tubulin in plant pathogenic fungi’s cell, preventing the formation of spindle fiber, thus inhibiting mitosis. Plant pathogenic fungi retain the tubulin gene’s random mutation in evolutionary process to adapt to infaust medium. Therefore, most resistance of plant pathogenic fungi to benzimidazole fungicides owes to some definite amino acid mutation of β-tubulin, which causes decreased affinity between benzimidazole fungicides and the target.Unlike other fungi, there are two β-tubulin genes in Fusarium graminearum named β1-tubulin gene and β2-tubulin gene, respectively. The amino acid sequence homology they encode reaches77%. There are1341bp exons in β1-tubulin gene with three introns while1344bp exons in β2-tubulin gene with six introns, which means β2-tubulin gene has three more introns than β1-tubulin gene. To better understand the relationship between carbendazim resistance mechanism and β-tubulin genes to provide the theoretical foundation to control Fusarium head blight of wheat and manage carbendazim-resistance in G. zeae, we did the following research.We constructed the cassettes of β1-tubulin gene inserted with one, two and three introns from β2-tubulin gene and cassettes of β2-tubulin gene deleting three introns or all the introns by double-joint PCR. Then we transferred them into into wild type strains containing2021which was sensitive to carbendazim and JT04which was resistant to carbendazim by PEG-mediated protoplast transformation.Our study of the mutants showed that inserting introns into β1-tubulin gene did not change the strain’s mycelial growth, conidiation, germination and infection significantly. The insertion improved the mutant’s EC50to carbendazim, however, JT04’s mutants were more obvious. RT-PCR indicated that the insertion improved the expression of β1-tubulin gene to about1.5folds which led to a1.5-fold up-regulation of expression of p2-tubulin gene.On the other hand, the absence of introns in β2-tubulin gene did not affect the mycelial growth of the strain significantly. However, on the other hand, the mutants showed deficiency in conidiation and germination. Besides, the mutants could barely infect plants and became more sensitive to carbendazim compared with2021or JT04. The results of RT-PCR indicated that the loss of introns in β2-tubulin gene led to a lower expression of P2-tubulin gene while a higher expression of β1-tubulin gene.Besides, The incidence of FHB was monitored in2010-2012to evaluate the risk of FHB resistance to MBC, the effect of different fungicides on wheat production, the population distribution and epidemic dynamics of resistance of FHB to MBC in China. The result showed that the resistant population of Fusarium graminearum was mainly located in Jiangsu province, and MBC enhanced the resistance frequency of FHB. The use of JS-399or MBC twice during the flowering stage can control FHB effectively. The occurrence of FHB not only reduces crop production, but also contaminates grain with toxic metabolites that are a big threat to human and livestock. FHB broke out in the middle and lower reaches of Yangtze River in2010and2012, especially in Jiangsu Province, resulted in huge losses. In this paper, we investigated the occurrence of FHB in Jiangsu Province based on the allocation of wheat production to research the harm of the disease and the population distribution and epidemic dynamics of resistance of FHB to carbendazim and proposed control strategies in the future. The result showed that the average disease incidence of FHB were14.71%and41.86%, disease index37.11and20.48, yield losses0.44million ton and0.77million ton, and resistance frequency10.51%and25.64%in2010and2012, respectively.