Sensitivity Of Magnaporthe Grisea To The Sterol Demethylation Inhibitor Fungicide Propiconazole And Agrobacterium Tumefaciensmediated Transformation Of Magnaporthe Grisea-Identification Of Pathogenicity Defective Mutants

The rice blast disease caused by the fungal pathogen Magnaporthe grisea is one of the most destructive diseases of rice. In this study, The baseline sensitivity to sterol demethylation inhibitor fungicide propiconazole of 53 isolate of Magnaporthe grisea which from different areas of Guangdong., Guizhou, Zhejiang, Anhui, Fujian, Jiangsu and Guangdong Province were measured by mycelium growth rate method. The baseline sensitivity can be used to develop and evaluate the effectiveness of resistance management programs. Cross-resistence between propiconazole, triadimefon, and carbendazim was determined using 53 isolates of Magnaporthe grisea. Two isolates were highly sensitive to propiconazole; the molecular mechanism of this highly sensitive to propiconazole was studied.Furthermore, we obtained one nonpathogenic mutants Fy-1230 by Agrobacterium tumefaciens-mediated transformation (ATMT) technique and identified a T-DNA tagged gene. We found this T-DNA tagged gene MGG₀9926 played an essential role on colony growth, conidiation, sexual reproduction and pathogenicity.â… . Sensitivity of M. grisea to the sterol demethylation inhibitor fungicide propiconazole and cross-resistance between propiconazole and other fungicides(1) Sensitivity of M. grisea to propiconazoleThe EC50 values of 53 wild-type isolates to propiconazole ranged from 0.145 to 1.446μg mL-1, with a mean of 0.901μg mL-1 . Among the 53 isolates, two were hypersensitive to propiconazole with a mean EC50 of 0.148μg mL-1, which was 6-fold lesser than that of other 51 isolates. The EC50s of these two isolates were significantly (P < 0.01) lower than those of the other 51 isolates. Thus, these two isolates, 07-82 and 2004-006 were designated as propiconazole highly sensitive (PHS) isolates, and the other 51 isolates were designated as propiconazole sensitive (PS) isolates.(2)Sensitivity of M. grisea to triadimefon and carbendazimThe EC50 values of 53 isolates to another DMI fungicide triadimefon ranged from 0.533 to 8.150μg mL-1. The mean EC50 value of these two PHS isolates to triadimefon was 7.5-time less than that of the other 51 PS isolates. The EC50s of these two isolates were significantly (P < 0.01) lower than those of the other 51 isolates.The two PHS isolates were also hypersensitive to DMI fungicide triadimefon. The EC50 values of 53 isolates to benzimidazole fungicide carbendazim ranged from 0.137 to 0.314μg mL-1. There was no significant difference between the two PHS isolates and other PS isolates. The EC50 values to carbendazim did not correlate with those to propiconazole, which indicated that there was no cross-resistance between carbendazim and propiconazole. â…¡. The molecular mechanism detection of high sensitivity to DMI fungicides in M. grisea(1)Genetic backgroundA genetic background difference between the PHS isolates 2004-006 and other 52 isolates was found by PCR fingerprinting with M13 primer.,but there was no obvious genetic background difference between the PHS isolate 07-82 and other 51 isolates .This indicated that high sensitivity to DMI fungicides in M. grisea was not decuded by genetic background difference.(2) Analysis of DNA sequence of CYP51 genesDMI fungicides specifically bind to the cytochrome P450 lanosterol 14α-demethyla encoded by the CYP51 gene. As compared to the deduced amino acid of CYP51 from PS isolate, the isolate 2004-006 had a substitution at amino acid position 450 of CYP51. The PHS isolate 07-82 also had a substitution at amino acid position 234. The results indicated that these substitutions in the CYP51gene might be related to high sensitivity to DMI fungicides in M. grisea.(3) Expression of CYP51 gene in the PHS and PS isolatesRNA from the PHS isolates 07-82, 2004-006 and the PS isolates 07-95, 07-110 were extracted for reverse transcription cDNA. The cDNA were samples for Real-time PCR. Real-time PCR assays showed that the levels of CYP51 expression in two PS isolates were not significantly (P < 0.01) different from those of two PHS isolates which indicated that the expression of CYP51 might not be related to sensitivity of M. grisea to DMI fungicide.â…¢. Obtainning non-pathogenicity mutant and identify T-DNA tagged gene of mutant(1) Obtainning non-pathogenicity mutant we isolated one mutants Fy-1230 of M. grisea, which was completely nonpathogenic to both susceptible barley and rice. Phenotypic analysis showed that the mutant was significantly reduced in vegetative growth, conidiation and unable to form appressoria on hydrophobic surfaces. The colony color of the mutant was lighter than that of the wild type strain Guy11. No perithecia were observed when the mutant was crossed with a standard tester strain TH3.(2) To identify T-DNA tagged gene of mutant Fy-1230The genomic DNA flanking T-DNA was amplified using hiTAIL-PCR. The tertiary 1000bp PCR product for the right border was purified and cloned into pGEM-T easy vector. The sequencing and BLAST results showed that T-DNA inserted in a gene of MGG₀9926. The T-DNA insert site was located at 751bp downstream from the start codon. This gene encodes a predicted protein of 507 amino acids. After NCBI BLAST search, we found high homogenous sequences to the predicted protein. It has respective similarity of 93%, 90% and 90% to the sequences of XP₀01908619 from Podospora anserina, XP₉58320from Neurospora crassa and XP₃81455 from Gibberella zeae.â…£. The test of T-DNA single copy insert into Fy-1230 genome(1) Isolation of single ascospore of mutant Fy-1230 and HPH+ resistance experiment30 single ascospores were obtained from mutant Fy-1230. The 30 single ascospore isolates were cultured in medium with hygromycin. 16 single ascospores had HPH+ resistance, 14 single ascospores hadn't HPH+ resistance, and the scale was nearly 1:1. This showed that T-DNA insertted into the Fy-1230 genome was single copy.(2) Pathogenicity test of single ascosporesConidia from 30 single ascospores were droped on barley leaves. 16 single ascospores with HPH+ resistance isolates were nonpathogenic, 14 single ascospores without HPH+ resistance isolatess were pathogenic, and the scale was nearly 1:1. Pathogenicity assay showed that T-DNA insertted into the Fy-1230 genome was single copy.â…¤. Construction of complementation vector of Fy-1230To determine whether the phenotypes of Fy-1230 were caused by a T-DNA insertion of MGG₀9926, we constructed a complementation vector Mg-GFP containing the MGG₀9926 and GFP fusion. The primer pair was designed to amplify full sequence of GFP gene and MGG₀9926 gene, the link recation was taken out on the two sequences. A complementation vector Mg-GFP was constructed.