| Trichoderma harzianum(M3)is a biocontrol fungi of Fusarium oxysporum f.sp.niveum(FON).M3 can compete with pathogens for space and nutrition,and it can secrete antifungal enzymes,such as chitinase(CHIA)and β-1,3-glucanase(GA),to dissolve cell walls of pathogens,thereby preventing and controlling diseases effectively.Phenolic acids are main components of root exudates,so continuous cropping can lead to accumulation of them in the soil and affect growth of plants and survival and competition of microorganisms in the root zone.Therefore,in continuous cropping watermelon fields,the effect of phenolic acids on M3 antagonistic ability determines its disease prevention effect.This study aims to identify main phenolic acids in watermelon root exudates and to investigate their influence on M3 secretion of CHIA,GA and its colonization in the roots.The research results will provide a theoretical basis for biological control of watermelon wilt in continuous cropping environment.The main findings are as follows:1.Used high performance liquid chromatography-mass spectrometry(LC-MS)technology and gas chromatography-mass spectrometry(GC-MS)technology to analyze and identify main components of watermelon root exudates.The results showed that LC-MS detected 10 main phenolic acids in watermelon root exudates,and their mass fractions were cinnamic acid(RGS),ferulic acid(AWS),vanillic acid(XCS),syringic acid(DXS),benzoic acid(BJS),p-hydroxybenzoic acid(QBS),salicylic acid(SYS),phthalic acid(LBS),gallic acid(MZS)and p-coumaric acid(XDS).GC-MS detected 42 components in watermelon root exudates,including 6 organic acids,7 aldehydes,3 ketones,5 esters,4 amines,4 alcohols,5 hydrocarbons,3 phenols and 5 kinds of other chemical substances.Therefore,LC-MS technique is suitable for identification of phenolic acids in watermelon root exudates.2.On basis of clarifying types and concentrations of phenolic acids in watermelon root exudates,liquid culture method and ultraviolet photometer method were used to detect the effect of 0,1,12.5,25,and 50 μmol·L-1 of 10 phenolic acids and FON on M3 secretion of CHIA and GA.The results showed that:10 phenolic acid and FON had a significant promotion effect on 2 enzyme activities.With the increase of phenolic acid concentration,enzyme activities increased,but promotion effect of 10 phenolic acid was different,in order,QBS>BJS>LBS>AWS>XCS>DXS>RGS>SYS>MZS>XDS,of which 50μmol·L-1 QBS had the strongest promoting effect.Compared with CK1(M3 spore suspension+normal saline treatment),2 enzyme activities increased by 732.45%and 1185.09%.Compared FON+phenolic acid(FON+P)treatment with single phenolic acid treatment,tendency of the two to promote enzyme activity was similar but amplitude was different.Enzyme activities of FON+P treatment were higher.Among them,FON+50 μmol·L-1 QBS had the highest enzyme activities,which were 25.46 U·mL-1 and 21.38 U·mL-1.Compared with CK2(M3 and FON spore mixture+saline treatment),they increased 406.16%and 458.22%.Therefore,M3 can secrete CHIA and GA to inhibit FON,and accumulation of phenolic acid in the environment and the presence of pathogenic fungi can promote the production of M3 antifungal enzymes.3.Based on gene sequences,M3 and FON were constructed by real-time PCR technology.By optimizing reaction conditions,such as annealing temperature,primer sequence and primer concentration,a reaction system with better specificity and higher sensitivity were obtained.When fungi initial DNA concentration were in the range of 0.001～100 ng·μL-1,logarithmic value of M3 and FON DNA concentration and cycle number(Ct value)had a good linear relationship,which were suitable for research of M3 and FON colonization within plant roots.4.Used real-time PCR technology and pot experiment to study effects of 10 phenolic acids and FON on M3 colonization within roots of watermelon seedlings.The results showed that on the 10th day,M3 and FON were colonized in all roots.Single phenolic acid treatment,10 kinds of phenolic acid all inhibited M3 colonization,among which 25 μmol·L-1 MZS,BJS and 50 μmol·L-1 XDS had the lowest colonization and the strongest inhibitory effect on M3 strain;FON+P treatment,M3 colonization amount was significantly lower than other treatments.Among them,50 μmol·L-1 QBS and 12.5 μmol·L-1 LBS,BJS had the lowest colonization and the strongest inhibitory effect on M3 strain,and FON colonization amount in watermelon seedling roots increased over time.However,25 μmol·L-1 RGS and 12.5 μmol·L-11 XCS and QBS had the strongest inhibitory effect on FON.The pot experiment also showed that accumulation of phenolic acid and FON worked together to reduce control effect of M3.Therefore,accumulation of phenolic acid in the soil and FON significantly inhibit M3 colonization in the roots of watermelon seedlings,affecting the control effect of biocontrol Trichoderma harzianum on watermelon fusarium wilt. |
