| The individual farmers are the main body in facility agriculture in our country.In recent years,the continuous cropping obstacle has become the restricting factor for the sustainable development of facility vegetables.In our previous studies,we found that continuous cropping obstacle of tomato(Solanum lycopersicum L.)could be alleviated by relay intercropping with garlic(Allium sativum L.),and the allelopathy of garlic root exudates may play a major role.Diallyl disulfide(DADS)is one of the main allelochemicals in garlic root exudates and garlic straw,however,the physiological and molecular mechanism of DADS on tomato are still unclear.The clarification of physiological and molecular mechanism of DADS-promoting effect on tomato plant and its alleviating effect on obstacles to continuous cropping of tomato have important significance in the sustainable production of greenhouse tomato.The objectives of this research were: to study the physiological and biochemical effects of DADS on tomato seeds germination and seedling growth;to investigate the effect of DADS on the expression of tomato root genes,analyse the sensitive metabolic pathways and functions of major genes which response to DADS;to test the allelopathic effect of DADS on tomato plant growth under continuous cropping conditions and analyze the impact of DADS on tomato root exudates changes.These researches will clarify the physiological and molecular mechanism of garlic allelochemical DADS in plant growth promoting effect and lay the theoretical basis for alleviating continuous cropping obstacle of tomato.The main results are as follows:1.DADS showed a biphasic,dose-dependent effect on tomato seed germination and root growth under different DADS concentrations.Lower concentrations(0.01-0.62 mM)of DADS significantly promoted root growth,whereas higher levels(6.20-20.67 mM)showed inhibitory effects.Cytological observations showed that the cell length of root meristem was increased and that the mitotic activity of meristematic cells in seedling root tips was enhanced at lower concentrations of DADS.In contrast,DADS at higher concentrations inhibited root growth by affecting both the length and division activity of meristematic cells.However,the cell width of the root meristem was not affected.Additionally,DADS increased the indole 3acetic acid(IAA)and zeatin riboside(ZR)contents of seedling roots in a dose-dependentmanner.The influence on IAA content may be mediated by the up-regulation of FZYs and PINs.Further investigation into the underlying mechanism revealed that the expression levels of tomato EXPs were significantly affected by DADS.The expression levels of EXPB2 and beta-expansin precursor were increased after 3 d,and those of EXP1,EXPB3 and EXLB1 were increased after 5 d of DADS treatment(0.41 mM).This result suggests that tomato root growth may be regulated by multiple expansin genes at different developmental stages.2.Based on the seedling growth test,DADS at low concentrations(0.14 and 0.21 mM)promoted tomato plant growth by increasing plant fresh weight,shoot height,total root length,root volume,root surface area,root activity and chlorophyll content.The effect of DADS on defense enzymes activity of tomato was also determined using a series concentrations of DADS.The results showed that DADS improved the activity of POD,CAT,PPO and PAL in tomato root and hypocotyl,and increased SOD activity in tomato root.The soluble protein content in tomato root and hypocotyl was increased by DADS treatment.However,DADS at low concentrations did not significantly affect MDA content in tomato root and hypocotyl compared with control.The expression of RBOHD was markedly decreased by DADS at lower levels(0.10-0.41 mM),but was increased by DADS at higher level(6.20 mM),suggests that DADS could regulate the generation of ROS by regulating the expression of RBOHD gene in tomato root.Moreover,DADS showed promoting or inhibiting effect on the expression of different defensive enzyme genes.This result suggests that DADS could regulate the activity of defense enzymes by affecting the expression of multiple defense enzyme genes,thus increase tomato resistance and affect its response to the external environment.3.According to RNA-seq,a total of more than 2500 DADS-responsive genes were identified,1828,1296 and 1190 differentially expressed genes(DEGs)were enriched in the 4,24 and 48 h samples,respectively.Most DEGs involved in assimilatory sulfate reduction and glutathione(GSH)metabolism were up-regulated after short-term(4 h)DADS treatment.The activity of Cysteine synthase,the key enzyme in GSH synthesis,was also promoted by DADS.In addition,increased activity of defensive enzymes,such as POD,CAT,SOD and PPO,and up-regulation of six peroxidase genes were observed,suggesting that DADS could induce tomato resistance by increasing the amount of nonenzymatic antioxidant and promoting the activities of defense enzymes.In plant-pathogen interactions,DEGs related to calcium signaling(CAM/CML and CDPKs)were primarily inhibited,while those encoding pathogenesis-related(PR)proteins were primarily up-regulated.This result suggests that DADS mainly activated the effector-triggered immunity(ETI)in tomato.Although planthormone synthesis and signal transduction were both significantly affected by DADS,the expression trends of the genes in these two pathways were conflicting,suggesting that DADS may influence tomato plant growth and resistance by regulating the synthesis of plant hormones and signal transduction.4.Under continuous cropping conditions,DADS increased tomato root activity,chlorophyll content,SOD,POD and PAL activity,and promoted the metabolism GSH and GSSG,thus increased tomato seedling growth and improved plant resistance,alleviated the stress of continuous cropping conditions.After DADS treatment,the contents of unsaturated fatty acids(e.g.,palmitic acid methyl ester,palm methyl oleate,methyl oleate)in tomato root exudates decreased from day 1 to day 3,but increased after 15 d.The germination test showed that palmitic acid methyl ester at 20 mM inhibited the growth of tomato hypocotyl,while methyl oleate at 20 mM to inhibited tomato root growth.Besides,the contents of root exudates which have root knot nematode resistance such as2,2’-Methylenebis(6-tert-butyl-p-cresol),dibutyl phthalate and 2,4-Di-tert-butylphenol were increased 1 d after DADS treatment,and gradually decreased in the following days.This result suggests that DADS may change the content of tomato root exudates to reduce autotoxicity and increase the tomato disease resistance.5.The coding region of SlDIR1 homologous gene was cloned from tomato cDNA by PCR,named SlDIR1-like.After sequence alignment,we found SlDIR1-like gene has the eight conservative cysteine residues like DIR1 genes in other species.DIR1 protein takes part in the formation and intercellular transfer of systemic acquired resistance(SAR)signal.The expression patterns of different DIR genes in SlDIR gene family were different,which means each SlDIR gene may has a special function in different tissues.The expression profiles of different SlDIR genes were also different after DADS treatment,suggesting the genes in Sl DIR gene family may have different role during SAR formation.In addition,the expression levels of NPR1(salicylic acid receptor protein gene)and PR1(SAR marker gene)were up-regulated,implying that DADS may increase salicylic acid level in different tomato tissues and induce SAR reaction. |
