Defense Mechanism Of Response To Thrips Damage In Alfalfa (Medicago Sativa L.)

Thrips is one the main biotic stress factors affecting the yield and nutritive value of alfalfa in northwest of China.It was imperative to study the physiological and molecular changes of different thrips-resistant alfalfa cultivars in response to thrips,and to further understand the defense mechanism of alfalfa against thrips,which could lay a foundation for the germplasm innovation of thrips-resistant alfalfa.In this study,Medicago sativa L.including thripsresistant cultivar Gan-nong NO.9(G9),thrips-susceptible cultivar Gan-nong NO.3(G3)and highly thrips-susceptible cultivar WL363HQ(W3)as test materials,the effects of nutrients,secondary metabolites,defensive enzymes and endogenous hormone on the three alfalfa cultivars at seedling stage were investigated under the different thrips numbers(0,3,5,7 and 9-thrips per branch)and damage time(0,48 h,72 h,96 h,120 h).Then,the transcriptomics,and metabolomics analysis were used to clarify the molecular mechanisms in the leaves of the thrips-resistant cultivar G9 and highly thrips-sensitive cultivar W3,and to identify several key stress-responsive candidate genes,and metabolites.The defense mechanisms of alfalfa against thrips were revealed from the physiological,molecular,and metabolic levels,so as to provide a theoretical basis for the germplasm innovation of insect-resistant alfalfa.The main results showed that:1.With the increasing of thrips numbers,compared with CK,the damage indices,soluble sugar,free proline,flavonoids,tannin,and H2O2 of G9,G3,and W3 were significantly increased,but the activities of PPO and SOD were significantly reduced(P<0.05).In G9,the contents of tannin(except for 3-thrips per branch)and lignin under the same numbers were significantly greater than that of W3,but H2O2 contents were significantly less than that of W3(P<0.05).According to the correlation and principal component analysis,the damage index,aboveground biomass dry weight,chlorophyll(a,b,a+b),soluble sugar,free proline,flavonoids,tannin,PPO,SOD and H2O2 could be used as effective evaluation indexes for different thrips-resistant alfalfa cultivars to resist thrips damage.In G9,G3,and W3,5-thrips adults per branch through the principal component comprehensive evaluation was used as the inoculation threshold to continue the follow-up test.2.Under 5-thrips per branch treatment,with time extension of thrips damage,the different thrips-resistant alfalfa cultivars could resist thrips stress by significantly increasing the contents of soluble sugar,free proline,flavonoids,tannin,lignin,cinnamic acid,pcoumaric acid,apigenin,luteolin and MDA,CAT activities and the relative gene expressions of FLS,DFR,ANR,C3H and CCoAOMT.However,the PPO activities of the three cultivars were obviously decreased.In addition,G9 was significantly improved the contents of naringin,rutin and quercetin,the activities of POD and PAL,and the relative gene expressions of C4H,4CL,LAR,F5H,COMT1,and CCR in response to thrips damage.In G3,the contents of soluble protein,rutin,quercetin,and H2O2,POD activities,and the relative gene expressions of C4H,CHS2,and F5H were significantly improved by the thrips stress.However,the contents of naringin and catechin in G3 were significantly reduced after thrips feeding.W3 was significantly increased the contents of soluble protein,epicatechin and H2O2,and the relative gene expressions of CHS2,as well as significantly reduced the catechin contents under thrips damage.3.Under 5-thrips per branch treatment,the contents of JA,SA,and ZR in tested alfalfa cultivars showed the change trend of first increasing and then decreasing with time extension of thrips damage.The thrips damage had a significant effect on the SA contents of the three alfalfa cultivars,and the SA contents of G9 was significantly higher than W3 at the same time of thrips damage.Furthermore,the contents of JA,GA3 and ZR in G9 were significantly accumulated after thrips damage.G3 was significantly increased the contents of JA and IAA in response to thrips damage.In W3,the contents of GA3,IAA and ZR were significantly improved under thrips stress.4.Compared with CK,under 5-thrips per branch treatment at 120 h,the differentially expressed genes involved in response to thrips damage were screened between G9 and W3 through RNA sequencing-based transcriptomics analysis.The 4390(2616 up-regulated and 1774 down-regulated)differentially expressed genes were identified in G9,as well as 3052(1811 up-regulated and 1241 down-regulated)differentially expressed genes were present in W3,while both alfalfa cultivars shared 185 up-regulated and 307 downregulated differentially expressed genes.Compared with W3,the differentially expressed genes of G9 involved in hormones(JA,SA,ABA,ET),secondary metabolic biosynthesis,and carbon fixation in photosynthetic organisms were significantly improved,which could promote the accumulation of primary metabolites(sugars,amino acids,etc.),and secondary metabolites(flavonoids,dihydroflavonols,anthocyanins,lignin,alkaloids,terpenoids,etc.).5.Under 5-thrips per branch treatment at 120 h,based on the quasi-targeted metabolomics analysis,the differential metabolites of G9 and W3 were identified,including 311(128 up-regulated and 183 down-regulated)metabolites in G9,357(212 up-regulated and 145 down-regulated)metabolites in W3,as well as 92 up-regulated and 102 down-regulated simultaneously expressed in both cultivars.Thrips damage induced significant changes in the contents of sugars and their derivatives,amino acids and their derivatives,flavonoids and other metabolites of G9 and W3.The main metabolic pathways involved were starch and sucrose metabolism,galactose metabolism,valine,leucine and isoleucine degradation,etc.G9 also enhanced its resistance to thrips by glucosinolate biosynthesis,indole alkaloid biosynthesis,and aminoacyl-tRNAbiosynthesis,compared with W3.6.Based on combining analysis of transcriptomics and metabolomics,the galactose metabolism and glucosinolate biosynthesis were the key metabolic pathways of alfalfa in response to thrips damage.