Bilogical Activities Of A Novel Fungicide JS399-19 To Fusarium Graminearum And Its Resistance Risk Assessment

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. In order to develop a sound recommendation for the use and exploitation of a new crop fungicide, sufficient biological properties, antifugal spectrum, risk assessment and baseline sensitivity of a fungicide should be studied.The toxicity of a new compound JS399-19 (2-cyano-3-amino-3-phenylacrylic acetate) was studied against 13 species of important agricultural pathogens of different taxonical position. The results showed that JS399-19 inhibited strongly mycelia growth of F. graminearum and F. moniliforme with EC₅₀ values of 0.141μg/mL and 0.459μg/mL, respectively. Compared with carbendazime, the EC₅₀ values of JS399-19 inhibiting growth of F. graminearum and F. moniliforme were 4.06 and 1.43 times lower, respectively. At the same time, JS399-19 is highly efficient against F. oxysporum, too. Its EC₅₀ inhibiting mycelium growth was 3.565μg/mL. However, it appeared much lower activity against other tested nine pathogenic fungi and bacterium. The result showed that JS399-19 was highly inhibitory to mycelial growth of either carbendazim-sensitive (CS) or carbendazim-resistant (CR) isolates of F. graminearum, and EC₅₀ values of inhibiting mycelia growth of CS and CR isolates were 0.117±0.036μg/mL and 0.107±0.020μg/mL, respectively. The fungicide could not inhibit the conidia germination, but caused abnormal morphology of germ-tube of the CS and CR isloates.51 isolates of F. graminearum were isolated from wheat samples from Nantong, Yancheng Jiangsu Province and Dafeng farm, Shanghai where JS399-19 had never been used before. The sensitivities to JS399-19 were determined by the method of mycelial growth test on PDA medium. The results that the sensitivity of wild-type population in the nature against JS399-19 exhibit a certain extend diversity and the the EC₅₀ values of the compound inhibiting growth of F. graminearum was ranged from 0.075～0.245μg/mL and with the mean value of 0.153±0.050μg/mL. The mean values of EC₉₀ and MIC were 0.153±0.050μg/mL and 0.712±0.255μg/mL, repectively. It showed that JS399-19 inhibited strongly mycelia growth of F. graminearum.In laboratory, we obtained total fourteen resistant mutants derived from wild-type isolate 2021 through ultra-violet (UV) irradiating at the dosage of LD₉₀ or fungicide training methods with a frequency of 1.67×10^-7 for UV. These mutants could be divided into three resistance phenotypes of low level resistance (LR), moderate level resistance (MR) and high level resistance (HR), of which determined by the EC₅₀ values 1.5-15.0μg/mL, 15.1-75.0μg/mL and more than 75.0μg/mL, respectively. Resistance of F. graminearum to JS399-19 was stable during the period of 8 subcultures. As compared with their parent isolate 2021, mutants increased or decreased their mycelia growth rate on PDA plate, mycelia dry weight and sexual reproduction ability. However, the amount of conidia production obviously reduced and their pathogenicity did not appear changeable. The growth of mutants were more quickly than their parent, except UV-2021-4 grew slowly in the media with low osmotic pressure. While, all mutants were much more favourable in growth at moderate and high osmotic pressure. The result also indicated that there was no cross-resistance between JS399-19 and well-known fungicides, such as benzimidazoles, ergosterol biosynthesis inhibitors, strobilurins, dithiodicarbamates and aromatic hydrocarbons.According to the bioassay of translocation results, JS399-19 could be absorbed by the roots and leaves of the wheat and show xylem-systemic activity in the acropetal direction, but failed to translocate basipetally and be absorbed by the stems of wheat. JS399-19, applied at the concentration of 400μg/mL, showed the excellently protective and curative activity against F. graminearum at various intervals, the control efficacy values of JS399-19 applied 24, 48 and 72h before inoculation, were 88%, 78% and 73%, repectivity. JS399-19 provided 95% disease control as applied 24h post-inoculation, but only 75% and 62% when applied 48h and 72h after inoculation. In comparison, the standard fungicide, carbendazim, provided only marginal reduction in disease development when applied at 400μg/ml at various intervals. Meanwhile, the compound exhibited stable residual activity and excellent translaminar activity.The activity of the dual mixture of JS399-19 with tebuconazole and the ternary mixure of JS399-19, carbendazim with tebuconazole to three Fusarium graminearum strains 2021, HR52-7 and UV-2021-8 was evaluated by inhibiting mycelium growth on PDA medium. The dual mixture of tebuconazole with carbendazim, tebuconazole with thiram and the ternary mixture of tebuconazole, thiram with carbendazim were used as comparison. The results showed that the activity of different mixtures and different ratio of the same mixture were difference. The ratio of all the mixtures revealed additive biological response to F. graminearum, expect for the ratio of the ternary mixture of JS399-19, carbendazim with tebuconazole and dual mixture of JS399-19 with tebuconazole at 1:1:0.3, 1:1:0.4 and 1:0.3. The ratio of the dual mixture of tebuconazole with carbendazim, tebuconazole with thiram at 1:10, 1:15, 1:20, 1:25 and 1:5, 1:10, 1:20, 1:40, 1:80 revealed synergistic effect to HR52-7 and the ternary mixtures of tebuconazole, carbendazim with thiram at the ratio of 1:10:15, 1:10:10, 1:5:10 and 1:10:15 revealed the same effect. The mixures of tebuconazole, cabendazim and thiaosan have excellent activity against F. graminearum using different ratio and ingredient, which can filtrate appropriate mixtures to reduce the resistance risk of F. graminearum to JS399-19 by alternating with JS399-19 in the field.The control effect of JS399-19 against the wheat scab in the field and effect of various fungicides pressure on development of the effect of controlling the wheat scab was 72.2% and 86.1%, respectively, when JS399-19 was applied as 375 g a.i./ha one and two times. JS399-19 applied 562.5 g a.i. and 375 g a.i./ha showed the same effect as the carbendazim applied 562.5 g a.i./ha and the frequency of resistance to the carbendazim increased from 2.91% to 14.81% in the plots treated with carbendazim, however, the frequency of resistance was only 0-1.39% in the plots treated carbendazim-resistant populations of F. graminearum on wheat were studied. The result showed that with JS399-19. JS399-19 seems to be a good candidate for controlling Fusarium disease in the field, especially in the areas where carbendazim-resistant fungal populations have been developed, which indicated that JS399-19 had not selective effect on development of carbendazim resistance population and the compound seems to be a good candidate for controlling Fusarium disease in the field,...