The Effects And Mechanisms Of Putative Trichoderma Harzianum Mutant T-E5 And Its Bio-organic Fertilizer On Growth Of Cucumber

Trichoderma species,as well-known plant growth-promoting fungi?PGPF?and widelydistributed in soils,have ability to promote plant growth and crop productivity.They alsohave been used as potent biocontrol agents to prevent plant diseases caused by numeroussoil-borne pathogens.However,few studies have been conducted to improve their abilities.T-E5 was chosen as the most excellent putative mutant by ultraviolet?UV?irradiation aftera series of screening procedures.Based on laboratory and greenhouse experiments,weexplored the effects and mechanisms of putative mutant Trichoderma harzianum T-E5 andits bio-organic fertilizer on growth of cucumber?growth-promotion effects and biocontrolof fusarium wilt disease?.The results were obtained as follows:1.High-performance liquid chromatography?HPLC?analysis in vitro showed thatindole acetic acid?IAA?producted by T-E5 was enhanced by 30.2%as compared with wildtype T.harzianum SQR-T037.In soil and hydroponic experiments,T-E5 treatmentsignificantly?P?0.05?increased cucumber plant height,shoot dry weight,root dry weightand root vigor by 8.01%,9.56%,13.8%and 16.1%,respectively,when compared withSQR-T037.Based on TaqMan Real-time PCR,the population of T-E5 was nearly ten timeshigher than SQR-T037 in soil samples at 30 days.The two strains?T-E5 and SQR-T037?had the same dynamic tendency both in endophytic colonization of root and stem,however,the population of T-E5 was significantly?P?0.05?higher than SQR-T037 at any samplingtime.2.In the split-root hydroponic system,when compared with CK?non-inoculation withT-E5?,inoculation with T-E5?T?significantly?P?0.05?increased cucumber plants height,shoot and root dry weight by 63.7%,55.1%and 36.1%,respectively.The T-E5 hyphaedensely covering the cucumber root surface were observed by scanning electronmicroscopy?SEM?.Three organic acids?oxalic acid,malic acid and citric acid?were identified from both CK and T-E5 treatment by HPLC and LC/ESI-MS procedures.The amounts of oxalic acid and malic acid in T were significantly higher than those in CK.All the organic acids exhibited different and significant stimulation effects on mycelial growth and conidial germination of T-E5 in vitro.An additional hydroponic experiment demonstrated the positive effects of organic acids on T-E5 colonization of cucumber roots.3.In greenhouse experiment,fusarium wilt disease incidence of control?CK,no Trichoderma T-E5 added?was 3.9 and 5.1 times of T1 and T2?bio-organic fertilizers enriched with T-E5?,respectively.Based on Real-time PCR,the population of F.oxysporum in CK increased from 10³ to 10⁴ ITS copies g^-1 soil,whereas the population decreased from 10³ to 10² ITS copies g^-1 soil in T1 and T2 treatments.T1 and T2 treatments significantly?P?0.05?altered rhizosphere fungal diversity and community.Based on HPLC analysis,6 phenolic compounds were detected from CK:gallic acid,p-hydroxybenzoic acid,vanillic acid,ferulic acid,benzoic acid and cinnamic acid.Meanwhile,we identified 4 and 5 phenolic compounds from T1 and T2,respectively.The detected levels of phenolic compounds in CK were significantly higher than the levels in the samples subjected to T1 and T2 treatments.The root exudates of CK significantly?P<0.05?increased the numbers of germinating spores of the pathogen compared with those from the samples treated with T1 and T2.4.A series of laboratory experiments were designed to characterize the importance of mycoparasitism,exoenzymes and VOCs by T.harzianum T-E5 for the antagonism of F.oxysporum f.sp.cucumerinum?FOC?.We further tested whether these mechanisms were inducible and upregulated in presence of FOC.The results were as follows:T-E5 heavily parasitized FOC by coiling and twisting the entire mycelium of the pathogen in dual cultures.T-E5 growing medium conditioned with deactivated FOC?T2?significantly increased proteins content by 31.65%,as compared with T1,in which T-E5 grew on glucose medium.Meanwhile,the cell wall-degrading enzyme activities(chitinase,?^-1,3-glucanase and protease)were significantly?P<0.05?higher than T1.The presence of deactivated FOC?T2'?also resulted in the upregulation of VOCs whereas 5 and 8 different types T-E5-derived VOCs were identified from T1' and T2',respectively.Further,the excreted VOCs in T2' showed significantly?P<0.05?higher antifungal activities against FOC than Tl'.5.Algae mud and other agricultural organic solid wastes were used to maximize T-E5 population through a design based on response surface methodology?RSM?.A composting test showed that the optimal ratios of organic materials,that is,73.2%pig manure organicfertilizer,7.3%algae mud,9.9%rapeseed meal and 9.6%feather meal at 28 ?,couldmaximize T-E5 population for 12 d fermentation.FTIR spectroscopy analysis showed thatthe possible degradation speed of these biopolymers in fermentation was protein>cellulose>polysaccharides,and the degradation sequence followed the order:polysaccharides,protein,cellulose.6.Hydrolytic amino acids and other agricultural organic solid wastes were used tomaximize T-E5 population based on RSM.Another composting experiment showed that65.2%cattle manure compost,19.3%maggot manure,15.5%hydrolytic amino acids and4.7%inoculum contents at 28 ? predicted a maximum T-E5 population(3.72 × 10⁸ ITScopies g^-1)after 14 d composting.3D-EEM fluorescence spectroscopy combined with FRIanalysis demonstrated that composting was a process of degradation of protein-likesubstances and formation of fulvic-like and humic-like substances.FRI parameters?P?,n,P?,n,P?,n and P?,n?could be used to characterize the degree of compost maturity.In addition,the application of the new T-E5-contained BIO significantly increased chlorophyll content,shoot length and plant dry weight in cucumber,tomato and pepper pot experiments.