| In recent years, the problem of fungicide resistance causes a decline effect in kinds of diseases. Besides, some kinds of diseases, which can be easily managed before, show a tendency of more and more severe. Along with the advance of agricultural modernization and chemical control of plant diseases, the resistant problem becomes more serious than before. B. cinerea, which has the characteristic of wide hosts, fastly genetic mutation and severe damage, is of a pathogen with serious resistance problem. Propamidine, a kind of aromatic diamidine compound with Chinese intellectual property rights, is developed by Research & Development Center of Biorational Pesticide in Northwest A&F University independently and is used for controlling grey mould caused by B. cinerea. Until now, few articles about resistance of propamidine had been reported. So, this research is based on the target pathogen B. cinerea and in terms of baseline-sensitivity assay, assess of resistance risk and the mechanism of development of resistance on resistant mutants. The main results and conclusions were outlined as follows:1. The sensitivity of 41 strains of B. cinerea which was isolated from 5 districts of Xi'an, Xianyang, Yangling, Weinan and Baoji was tested by the method of mycelial growth rate. According to the result, all the tested strains were inhibited seriously by propamidine. Besides, low differential of EC50 values was tested among the different strains. The EC50 values were 0.182 to 1.460μg/mL. The mean EC50 was 0.791±0.269μg/mL, representing a range-of-variation factor of 8.0. The frequency distribution of sensitivities of different strains to propamidine was representing a curve of continuous single peak which was approaching a unimodal curve. No low sensitivity of resistant population was detected. So the mean EC50 of these strains could be used as a baseline for observing the sensitivity in B. cinerea populations to propamidine.2. 10 resistance mutants with high genetic stability were obtained by UV irradiation and selection for resistance to fungicide. One resistant mutant was induced by UV irradiation and nine resistant mutants were obtained by selection for resistance to fungicide with the mutation frequency 1.98×10-9 and 0.025 respectively. Based on the standard of resistance created by FAO and the baseline sensitivity and the resistance level of resistant mutants, the resistant mutants were divided into low (L), moderate (M) and high (H) resistant level, determined by the EC50 values of 5.0-15.0μg/mL, 15.1-75.0μg/mL and more than 75.0μg/mL, respectively. So, 8 LR mutants, 2 MR mutants and no HR mutant were obtained. Low mutation frequency and resistant level showed the fact that it is hard for B. cinerea developing resistance to propamidine. According to the result of fitness parameters of the mutants, the hyphal growth, sporulation, spore germination and pathogenicity, showed significantly lower than the sensitive strains. Besides, with the result of cross resistance test, propamidine has no cross resistance with the botryticides in common use like pyrimethanil, carbendazim, iprodione and procymidone. Above all, B. cinerea has low resistance risk to propamidine.3. According to the result of activity of mitochondrial complexⅢ, propamidine had no effect on both sensitive strain and resistance-strains during the test of post-treatment on mitochondrial. However, during the pre-treatment activity test, propamidine possessed obvious inhibition on sensitive strains and had no significant effect on resistance-strains. The inhibition of propamidine on mitochondrial complexⅢrelative genes expression is a key factor, according to previous research.4. During the osmolarity sensitivity test, sensitive strain was sensitive against low and high osmolarity. And, LR (low resistance) strain was sensitive against low osmolarity only. Besides, MR (moderate resistance) strain possessed no sensitivity against both low and high osmolarity. Basing on the result of cell membrane permeability, both sensitive strains and LR strains showed high cell membrane permeability after treated by different concentration of propamidine, especially treated by high concentration of propamidine (25μg/mL and 50μg/mL). Although the final relative leakage rate (240min) of sensitive strains and LR strains were close, the membrane injury speed of sensitive strains showed more fast than LR strains during the treatment of propamidine, which indicates that the cell membrane permeability and the adaptation to propamidine between sensitive strains and LR strains is different. Further more, the final relative leakage rate (240min) of MR strains showed more lower than sensitive strains and LR strains, which indicates that MR strains with much higher ability of adapting to propamidine than sensitive strains and LR strains. The lower cell membrane injury degree makes the lower cell membrane permeability of MR strains comparing with sensitive strains and LR strains, and the ability of adapting to propamidine become lower with the concentration increasing, which also can be indicated by the result of osmolarity test. So, the decrease of osmotic sensitivity and the change of cell membrane permeability may be one of the physiological and biochemical mechanisms on resistance of B. cinerea to propamidine. 5. The difference of esterase isoenzyme and soluble proteins were compared by the PAGE between resistance-strains and sensitive strains . The result showed obvious difference of the band amount and content between resistance-strains and sensitive strains, which indicates that the resistance of B. cinerea to propamidine is relative to the formation or content change of esterase isoenzyme and soluble proteins.6. Gene expression of resistance strain (091) and MR strain (F091-16) was analyzed by the cDNA-AFLP. About 38 differentially expressed TDFs were isolated and 6 of them were cloned and sequenced. The result showed that 4 of 6 differentially expressed TDFs were found with homological sequences. Besides, TDF2, TDF4 and TDF4, which belongs to the same gene fragment, possess high homology with the gene sequence of cytochrome P450 alkane hydroxylase. And, another differential TDF named TDF1 possesses high homology with gene sequence of C-1-tetrahydrofolate synthase and the others two TDFs, TDF3 and TDF6, possesses no homology with existing sequences. Up-regulated expression of C-1-tetrahydrofolate synthase gene can promote the synthesis speed of nucleic acid which lead to the decrease of target gene expression which caused by retrieving of medical molecular effect, and the activity of mitochondrial complexⅢcan be recovered. Along with the result of mechanism on agriculture fungal inhibition of propamidine, the conclusion is speculated that the up-regulated expression of C-1-tetrahydrofolate synthase gene may be the main fact of resistance development on B. cinerea to propamidine. Further more, the up-regulated expression of cytochrome P450 alkane hydroxylase gene is also relation to the resistance development.Above all, the resistance risk is low on B. cinerea to propamidine. The resistance of B. cinerea to propamidine is leaded by lots of factors. Among the factors, the up-regulated expression of C-1-tetrahydrofolate synthase gene may be the main factor: The decrease of osmolarity and cell membrane permeability makes propamidine hard to pass through the cell membrane into plasma. Part of some medical molecular in plasma can be degraded by esterase isoenzyme or cytochrome P450 alkane hydroxylase. C-1-tetrahydrofolate synthase gene can promote the synthesis speed of nucleic acid which leads to the decrease of target gene expression caused by retrieving of medical molecular effect and resistance is possessed finally on resistance-strains. Besides, the gene expression which inhibition of propamidine on mitochondrial complexⅢis a key factor, according to previous research.
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