Development And Mechanisms Of Synergetic Combinations Of Bioloigical And Chemical Fungicides For Rice Disease Control

Rice is one of the most important crops in China,every year huge losses are caused by kinds of rice diseases,such as rice blast,rice sheath blight,and rice false smut.Although chemical control is the most important and reliable measure in rice disease management,it confronts with increasing problems,such as environment pollution,resistance,and residues.With the increasing of the environmental awareness,re-consideration on chemicals and development of organic agriculture,biological control showed brilliant potentials in management of rice diseases because of its advantages,such as environmental friendliness,safety,and low cost for developing new biological control agents?BCAs?.In this study,we assessed effects of two strains,Bacillus H158 and Streptomyces HSA312 on rice disease control,and studied the related mechanisms.Considering advantage complementary between fungicides and BCAs,synergetic effects were evaluated for combinations of these strains and commonly used fungicides.Results are as follows:1. H158 was identified as Bacillus subtilis by morphology,physiological and chemical characteristics,fatty acid composition of cell wall,sequences of 16S r DNA and gyr B.H158 showed obvious antagonism effects?AE?against many rice fungal pathogens,especially against pathogens of rice blast and shealth blight with AE of83.3 and 75.6%.H158 can form mature and stable biofilm on different media,such as MSGG,PDA?B?,and PSA?B?.The stain showed cellulase and chitinase activity indicated its fungal lysis ability.It proven that H158 could regulate the rice defense related enzymes and genes to enhance the disease resistance level by induced systemic resistance?ISR?.RNA-seq analysis showed that confront culture with H158made many genes differently expressed in Magnaporthe oryzae,especially genes involving the lipid metabolism related pathways.In the field,H158 showed obvious control effect?CE?on rice blast,rice sheath blight,and rice false smut,CE among38.4-50.1%.H158 also showed excellent compatibility with two fungicides,azoxystrobin and tebuconazole,respectively.The two combinations showed synergetic effects on control of rice sheath blight and false smut,and coefficient of synergism?CS?were 1.9 and 0.6.Fermentation broth of H158 showed no obvious adverse effect on rice quality and mill properties,even showed better effects for some characters like chalkiness,content of protein and amylose.2. Combined application of H158 with three Qo Is,azoxystrobin,pyraclostrobin,and trifloxystrobin showed strong synergistic effects on control of rice sheath blight under conditions of both artificial inoculation and natural infection.Combination of H158with azoxystrobin showed the highest control effect?88.8%?and with trifloxystrobin showed the strongest synergistic effect?3.7?.H158 could tolerate high concentrations of Qo Is,and its growth was stimulated even at concentrations below 200 mg L^-1.The survival of H158 on rice leaves was not obviously inhibited by Qo Is and was slightly stimulated at 10 days after application,especially when applied with trifloxystrobin.In early and middle culture stages of H158?0-48h?,trifloxystrobin could accelerate the growth and formation of biofilm.Trifloxystrobin did not show obvious influence on ISR induced by H158 on rice,thus the synergistic effects probably from the stimulation of the rice growth,enhancement of stress resistance and colonization of H158 on rice by Qo Is.3. Combination of H158 and tebuconazole showed synergistic effect only when the application rate of the fungicide was as low as 64.5 g a.i.ha^-1 with a CS of 2.5,and showed antagonistic effect at higher application rates.Tebuconazole showed toxic to H158 and the concentration of 50 mg L^-1 was almost enough to stop its growth.Biofilm formation of H158 was also susceptible to tebuconazole and completely inhibited when concentration was above 25 mg L^-1.This combination showed obvious induction effect on rice defense related enzymes and genes,which means that enhancement of ISR probably was the mechanism of synergistic effect of this combination.4. HSA312,a strain isolated from Naqu,Tibet Plateau,was finally identified as Streptomyces araujoniae by a series of studies including morphology observation,physiological and chemical analysis,measurement of cell wall fatty acid composition,and phylogenetic analysis of 16S r DNA.This strain showed excellent antagonistic effects against the pathogens of rice blast?51.1%?and rice false smut?56.7%?,but only slight inhibition?<22%?on the other pathogens.HSA312 showed fungal lysis ability and strong UV tolerance and colonization ability on rice plant,ability to enhance ISR of rice by regulating the defense related enzymes and genes.RNA-seq analysis for confront cultured M.oryzae showed many down regulated genes.In paddy field,HSA312 showed excellent control effect on rice blast as high as 52.2%,but only slight on rice sheath blight and rice false smut.Fermatation broth displayed significant antagonistic effect against mycelial growth,conidial germination,and especially appressoria formation of M.oryzae.The concentration of 10⁷ cfu m L^-1 was enough to completely prevent formation of appressoria.HSA312 showed excellent CE on rice blast especially in leaf blast in conditions of both artificial inoulation and nature ocurrence,and the highest CE reach 83.9%.HSA312 did not show good compatibility with general fungicides as well as H158,and only showed synergetic effects with CS of 1.5 when combined with tricyclazole,which is one of the most effective fungicides for rice blast.Fermentation broth of HSA312 did not show adverse effect on quality and mill properties,even showed enhancement on some characters like chalkiness,adhesive strength,and refined rice rate.5. Combination of HSA312 and tricyclazole showed obvious but unstable synergistic effects on control of leaf blast,and stable synergistic effects on control of panicle blast.The CS of this combitnation was 1.0 and 1.2 in consecutive two years'experiments to control rice neck blast.Tricyclazole showed certain inhibition effect on the spore germination and colony growth of HSA312;however,the inhibition may be disappeared over time.The colony growth can't be inhibited when the concentration of tricyclazole was below 160 mg L^-1.The addition of tricyclazole did not show influence on antagonistic effect of HSA312 against M.oryzae as well as transcriptome in confront culture condition.Defense related enzymes and genes were up-regulated by combination of HSA312 and tricyclazole,therefore ISR probably was the mechanism of synergistic effect of this combination.This work laid the foundation for application of H158 and HSA312 in management of rice diseases and explored the related mechanisms of the synergistic effects of the combinations between BCAs and fungicides,which can be used as references for further research of synergistic effects between fungicides and BCAs.