| Early blight, caused by Alternaria solani, is a foliar disease of cultivated tomatoes, affecting both tomato yield and quality. Boscalid was first introduced to China in 2008 to control tomato early blight. However, knowledge on the sensitivity/resistance of A. solani to boscalid is lacking after five years of field application. Thus, Alternaria solani isolates, which were collected from different districts in Shanxi province, were tested the sensitivity to boscalid and determined the current situation for boscalid resistance. And the fitness, the biological characteristic, the variation of cell membrane permeability, the physiological-biochemical characteristic of resistant strains, and the resistant molecular mechanism were studied.1. In this study,100 A. solani isolates were collected from five distantly separated localitions in Shanxi Province in China. The EC50 values to boscalid of these isolates ranged from 0.31 to 164.20 μg·mL-1 (mean=12.94μg·mL-1, SD=25.01). Field isolates of A. solani in Shanxi Province varied in their sensitivities to boscalid. Among these isolates,54% showed resistance to boscalid, and 76% of the resistant isolates had moderate resistance (RM) against boscalid. Boscalid-resistant isolates were most frequently observed in the Xinzhou and Yuncheng districts, and the only two highly-resistant isolates in this study were detected in Xinzhou. The resistance indexes (R1) to boscalid of these isolates ranged from 0.28 to 0.49 (mean=0.38, SD=0.08), but the total resistance situation to boscalid was no significant differences among the five districts(X2=0.78<X20.05,20,P>0.05).2. In this study, the fitness was tested, and evaluated the control efficacy of commonly-used fungicides against boscalid-resistant A. solani strains on leaf discs. The results showed that resistance was stable after the 20th generation. Compared with sensitive strains, the mycelium growth of resistant strains were significantly declined, the production of spores was significantly increased, and the germination rate of spores was not significantly changed (P>0.05); the toxin productions were increased, so the pathogenicity was also increased. Therefore, it was concluded that the survival probability of resistant strains was big in the nature, and they become the dominant population easily. No cross-resistance was detected between boscalid and the commonly-used fungicides, pyrisoxazole, difenoconazole, polyoxin, and sandofan.There was no significantly defference to the sensitive and resistant strains for polyoxin, including the RM strains (P>0.05). Thus, polyoxin could play a key role in early blight management for boscalid resistance.3. The mycelial morphologies of both A. solani boscalid sensitive and resistant strains were compared, and the optimum temperature, optimum pH value and the best carbon-nitrogen source of them were tested. The result showed that R strains showed more diaphragms, deeper color, smaller diameter (RM and RVH) than S strains. The R strains grew lower than S ones prominently in 25℃ (P<0.05), and the growth rate were no difference between them in 30℃ (P>0.05). The optimum growth temperature for the S and RM strains was 25℃, for the RH and RVH strains was 25～30℃. The optimum growth pH value were 6-8 for S strains,5～9 for RM strains,5～8 for RH strains and 5～8 for RVH strains, respectively. The optimum growth carbon source for S, RM, RH and RVH were lactose and starch, lactose and starch, lactose, starch, respectively. The optimum growth nitrogen source for S, RM, RH and RVH were peptone, peptone, soybean meal, corn meal and peptone, respectively. Therefore, when A. solani was resistant to boscalid, its mycelial morphology, range of optimum growth pH value and requirement for nutrition would varied, its optimum growth temperature would increased.4. The sensitivities to osmotic pressure of boscalid sensitive and resistant strains were compared, the conductivities of their menbrane with boscalid stress were observed, and associated substances in the cell were studied. The result showed that the correlation was highly significant between the boscalid resistance and the sensitivity to NaCl osmotic pressure of A. solani (P<0.01). The adaptive capacities to boscalid of RM and RVH strains were significant higher than of S ones, and the injuried membrane of them was lessThe adaptive capacities to boscalid of RH strains were significant lower than of S ones, and the injuried membrane of them was more severe.lt was indicated that RH strains probably speeded the drug excretion to keep growth, and RM and RVH strains probably prevented the drug by its lower membrane permeability to keep growth. The soluble sugar content of R strains was droped significantly, and soluble protein content increased significantly. For the soluble protein, there were two belt (Rf=0.06 and Rf=0.45) belonged to R strains only, which were related to the boscalid resistant. Meanwhile, the expressions of soluble protein were increased in R strains. With the increase of resistant degree, the membrane glycerol content was decreased gradually.5. The activities of succinate dehydrogenase (SDH) and protective enzymes in sensitive and resistant strains were studied and specific differences on the esterase isozymes between them were compared. The activities CAT, SOD and SDH of R strains were significant higher than of S ones (P<0.05). The activities CAT and SOD of S strains with boscalid stress were significant higher than with non-boscalid stress (P< 0.05), and those of R strains with boscalid stress were declined than with non-boscalid stress. But with boscalid stress, the activities SOD of R strains were always higher than of R strains. The activities SDH of S and R strains were significant inhibited with the increase of boscalid concentrations (P<0.05). And the higher bocalid resistance, the lower activities of SDH. There were no the two specific esterase isoenzymes (Rf=0.60 and Rf=0.74) in RH and RVH strains, and these enzymes probably varied because of the higher boscalid resistant.6. To investigate the molecular basis of A. solani boscalid resistance, Sdh gene of S and R strains were cloned and translated to the protein sequence, and then the protein sequence were put into sequence alignment. The mutation of SDH protein would aroused to cellular excessive ROS, thus the variation of ROS content between S and R strains were determined. The results showed that there were H278R,163T and K261M mutation in SdhB subunit of RM strain, T47A mutation in SdhD subunit of RM strain, V245G and E263D mutation in SdhB subunit of RH strain, S61L mutation in SdhB subunit of RVH strain. For SdhC subunit, no observation of gene mutation had been presented in the tested, and the same to SdhD subunit of RH and RVH strains. The phenomenon may be due to the difference of boscalid application in the sample sites between in this paper and in the mentioned reference, or Sdh protein had mutated because of other non-boscalid fungicide resistance, or the difference of strain sources. R strain performed higher ROS than S strain (P<0.05). Under boscalid gradient concentration stress, R strain performed higher ROS than S strain, and ROS of all declined. It indicated that the metabolism and ROS were influenced when the SDH complex were inhibited by boscalid. In addition, non-sense mutation were appeared in SdhB, SdhC and SdhD, whether these mutations would influence on the ROS should been deeper studied in the future. |
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