| Rice bakanae,rice blast and false smut were occured in large areas with the large-scale cultivation of high-yielding varieties and the promotion of high-yield cultivation techniques,which seriously threatened the safe production of rice.It is is essential for disease management that identify the epidemic pattern of pathogens and their sensitivity to fungicides.This study was conducted to study the species of main pathogen and their resistance to fungicides and the early observing and monitoring technology of the main fungal pathogens of rice.The main results are as follows:(1)A total of 134 strains of Fusarium were obtained from Jinhua,Jiaxing and Shaoxing of Zhejiang Province,China,f which 101 strains were F.fujikuroi and 36,32 and 33 in Jinhua,Jiaxing and Shaoxing respectively,accounting of the population in the area for 75.0%,71.1%,and 80.48%,respectively.(2)The sensitivity of 134 strains of Fusarium to carbendazim,prochloraz,and carbendazim was determined.The results were as follows: 17 strains were sensitive to carbendazim strains and 117 strains were resistant to carbendazim,of which,92 strains and 9 strains were lowly and moderately resistant to carbendazim,respectively.All F.fujikuroi strains were resistant to prochloraz,of which 25 strains were low-resistance,44 strains were medium-resistance,and 32 strains were high-resistance.Other strains of Fusarium were prochloraz-sensitive strains.There were 121 Fusarium strains were resistant to phenamacril.The effective concentration of 50%(EC50)values ranged from 0.19 μg/m L to 0.85 μg/m L,with an average of 0.46 μg/m L.Among them,13 F.fujikuroi strains were phenamacril-resistant strain.The EC50 value was between 33.28μg/m L and 80.33 μg/m L with an average of 59.27 μg/m L,which was 129 times than the average EC50 value of sensitive strains.(3)The full-length nucleotide sequence of β-tubulin gene was cloned from different carbendazim sensitivity phenotypes strains.Sequence alignment showed that the amino acid at position 198 of β2-tubulin gene in highly resistant strains to carbendazim was changed from glutamic acid(GAG)to proline(GTG);the amino acid at position 200 of β2-tubulin gene from two lowly resistant strains to carbendazim was changed from phenylalanine(TTC)to tyrosine(TAC);the amino acid at position 167 of the β2-tubulin gene of 18 lowly resistant strains to carbendazim was mutated from phenylalanine(TTC)to tyrosine(TAC).The 235 codons of β2-tubulin gene from all resistant-strains were mutated from GGC to GGT and were nonsense mutations.(4)The CYP51 A gene,CYP51 B gene sequence,CYP51 A gene promoter sequence and the relative expression level of CYP51 A gene was analyzed from different prochloraz sensitivity phenotypes strains.Sequence alignment results indicated that there were no point mutations in CYP51 A and CYP51 C genes,and no sequence fragment t insertion in the promoter region of CYP51 A gene,but amino acid at position 312 of CYP51 B gene was changed from serine(TCT)to threonine(ACT);After treatment with 10 μg/m L prochloraz for 12 h,there was no significant difference in the relative expression of CYP51 A gene between the prochloraz low-resistant,high-resistant strains and sensitive strains;After treatment for 24 h,the relative expression of CYP51 A gene in the low-resistant,high-resistant strains was increased and was significantly different from the sensitive strains;after 48 h,the relative expression levels of CYP51 A gene in the low-resistant,high-resistant strains was decreased.The difference between high resistant strains and sensitive strains and low resistant strains was still significant,and the difference between low resistant strains and sensitive strains was not significant.(5)The full length of the myosin-1 gene was cloned from from different phenamacril sensitivity phenotypes strains.Sequence alignment showed that the amino acid at position 219 of myosin-1 gene in highly resistant strains to phenamacril was changed phenamacril.(6)Established LAMP detection system for F.fujikuroi in rice seeds.The detection system has good specificity,including DNA extraction and LAMP reaction process,it can effectively detect the genomic DNA of fungi from a concentration of 0.001 ng/μL within 70 minutes,and it can be used to rapidly detecte from seed and seedlings before seed germination and transplant rice seedlings.(7)A quantitative LAMP(q-LAMP)assay system was established for the spores of Pyricuiaria grisea.The system can theoretically detect from a concentration of 6.4 spores per m L,and can perform quantitative detection from complex samples.It can be used in conjunction with spore capture instruments to quantify airborne spores;this detection system provides technical support for the study of the relationship between airborne spore release patterns and disease occurrence of rice blast fungus.(8)A q-LAMP detection system for rice blast fungus was established.The system can theoretically detect from a concentration of 3.2 spores per m L.The detection system can quantitatively detect airborne spores,and can detect the rice blast fungus before symptoms. |
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