Research On The Resistance Mechanism Of Fusarium Graminearum To JS399-19

JS399-19,2-cyano-3-amino-3-phenylancryic acetate is a novel cyanoacrylate fungicide introduced by Jiangsu Branch of National Pesticide Research & Department South Center. This fungicide was demonstrated to have activity against Fusarium. spp, especially for Fusarium graminearum and had a good control of Fusaruim head blight（FHB） or scab in the field. But In vitro, JS399-19-resistant mutants were obtained easily by ultra-violet （UV） irradiating and fungicide training and most of the resistant mutants belong to MR or HR and the fitness of JS399-19-resistant mutants was not decreased significantly. Indoor resistance risk of Fusarium graminearum to JS399-19 can be divided into the level of moderate-high. In order to develop a sound recommendation for the use of a new crop fungicide and detection technique for resistance, the resistance mechanism of Fusarium graminearum to JS399-19 should be studied before the resistance appear in the field.The efficient genetic transformation system of protoplast for Fusarium graminearum was established. F.graminearum isolate Y2021A high-resistant to JS399-19（JS399-19HR） was used for optimizing enzyme system for digesting cell wall and hph transformation system. The optimum condition for preparing protoplasts was:one milliliter enzyme mixture of 2% Driselase and 2% Snailase digested the 0.12 g germLings at 30℃for 1.5 hours. The efficiency of hph transformation was 40-50 transformants per microgram of DNA.Based on efficient genetic transformation system of protoplast for Fusarium graminearum, hph and hph-hsv were transformed to JS399-19HR isolate Y2021A respectively. Two mutation libraries were established and the mutations sensitive to JS399-19 were selected.10163 mutations inserted by hph were totally obtained,10125 of which considered as positive mutations could grow normally on PSA plate containing 100μg/mL hygromycin B. All of the positive mutations could grow normally on PSA plates contraining 10μg/mL JS399-19 and were resistant to JS399-19 as Y2021A.4745 mutations inserted by hph-hsv were totally obtained,4336 of which considered as positive mutations could grow normally on PSA plate containing 100μg/mL hygromycin B and could not grow on PSA plate containing 0.2M F2dU.328 mutations were sensitive to JS399-19. These sensitive mutations were important materials for studying resistance-related genes of F. graminearum to JS399-19.Target DNA segments sequences flanking hph-hsv sequences of sensitive mutation were amplified by Tail-PCR and blast in Genbank of Fusarium graminearum. The result indicated that hph-hsv was inserted into tubulin beta chain（FGSG₀6611.3）（β2-tub） of F.graminearum. However, the results of PCR, sequencing, Southern blot and RT-PCR illuminated that hph-hsv was not inserted intoβ2-tub but replaced the open reading frame （ORF） ofβ2-tub. Compared toβ2-tub of wild-type strain 2021, amino acids at codon 12, 17,21,22,41 and 74 changed differently in the strains with different resistance level.Theβ2-tub deletion and complemented mutations of wild-type strain 2021,JS399-19HR strain JT04-4 and sensitive mutations were studied. Theβ2-tub deletion mutant of wild type strain 2021 and the sensitive mutations had the similar EC50 values to JS399-19 with wild type strain 2021, but MIC value decreased 4 times. Theβ2-tub deletion mutants of JS399-19HR strain JT04-4 was moderately-resistant to JS399-19. The fitness of theseβ2-tub deletion mutants and sensitive mutations were significantly decreased as well. However, the finess of the sensitive mutations were decreased less than theseβ2-tub deletion mutants. The complementation mutants possessingβ2-tub origined from wild-type or JS399-19HR strain exhibited a JS399-19-sensitivity corresponding to the their parental strains, and had a comparative fitness to their original strains as well. These results indicated thatβ2-tub deletion or missing inserted by hph-hsv were important mechanism of decreased resistance or losing resistance.Two-dimensional Electrophoresis Profile of total protein of Fusarium graminearum was analysed. The effect of JS399-19 to proteomics of sensitive-strain 2021 and resistant-strain Y2021A was studied respectively. The sensitive-strain 2021 and resistant-strain Y2021A were treated with EC90 of JS399-19（0.5μg/mL） respectively and the controls were the sensitive-strain 2021 and resistant-strain Y2021A treated with nothing. Thirty-eight protein spots whose expression changed significantly in the sensitive-strain 2021 treated with EC90 of JS399-19 while did not change significantly in the resistant-strain Y2021A treated with EC90 of JS399-19 were identified by MALDI-TOF-MS/MS. Thirty-seven protein spots were identified successfully and the success rate reached more than 95%. But sixteen of thirty-seven identified were hypothetical protein and the function of them was not clear. The amino acid sequences of those hypothetical protein were blast in the database of Broad and the complete amino acid sequences of them were obtained. Then the complete amino acid sequences of those hypothetical protein were blast in the database of NCBI. According to the E-value, the highest homology protein were obtained. Based on the function of the highest homology protein, the function of those hypothetical protein was speculated. Those protein related to resistance of F.graminearum to JS399-19 were divided into metabolism-related protein, motility-related protein, control protein, signal transduction-related protein, and other protein. The metabolism-related protein contain pyruvate decarboxylase, pyruvate kinase, glutamate-1-semialdehyde 2,1-aminomutase, 1-aminocyclopropane-l-carboxylate deaminase, DUF455 domain protein, phenylalanyl-tRNA synthetase beta chain, glutamine synthetase and so on; The motility-related protein contain ARP2/3 complex 34 kDa subunit and diatom spindle kinesin 1; The control protein contain eukaryotic translation initiation factor 3 subunit 6, fungal specific transcription factor domain-containing protein, mitochondrial dicarboxylate carrier,30 kDa heat shock protein, heat shock protein SSC1, ATP-binding cassette sub-family F member 2, LAGLIDADG endonuclease and so on; The signal transduction-related protein contain GTP-binding nuclear protein GSP1/Ran and adenylyl cyclase-associated protein. JS399-19 had less effect on the energy metabolism system and more effect on the amino acid metabolism system, control system and signal transduction system. As there was no cross-resistance between JS399-19 and well-known fungicides, such as benzimidazoles, ergosterol biosynthesis inhibitors, strobilurins, dithiodicarbamates and aromatic hydrocarbons and JS399-19-resistant mutants were obtained easily by ultra-violet （UV） irradiating and fungicide training and most of the resistant mutants belong to MR or HR, it was speculated that the resistance of Fusarium graminearum to JS399-19 may be caused by the expression change of control-related protein or signal transduction-related protein.