Screening?Evaluation And Application Technology Of Trichoderma Resources Collected From Tidal–Flat And Wetland

Trichoderma is a widely used microorganism in biological control.In this paper,A group of Trichoderma resources were collected from beach wetlands or tidal flat in selected five provinces in China,and also rapid screening methods and evaluation criteria for antagonistic and tolerant Trichodema strains was built-up.Meanwhile the combined use of hyperaccumulator-Trichoderma were demonstrated in two sites for heavy metal polluted soil remediation.The results suggested that a total of 462 strains of Trichoderma were isolated from 397 soil samples and 100 water samples mostly sourced from different areas in southern China.Based on the sequence of ITS-rDNA,sequence extension of translation elongation factor(Tef1-?)and morphological identification Thirteen Trichoderma were identified as T.asperellum,T.harzianum,T.guizhouense,T.velutinum,T.virens,T.tomentosum,T.orientale,T.yunnanense,T.viride,T.atroviride,Trichoderma longibrachiatum,Trichoderma viride,Trichoderma trixiae,T.aureoviride,and T.urophylla.T.asperellum and T.harzianum are the dominant species of tidal wetlands in China.Use glycerol tubeand liquid paraffin storage in two ways to save the strain.The diversity of distribution of these strains was analyzed.With antagonistic rate over 65% in dual culture of pathogen and candidate biocontrol strains as screening standard,we got 10 Trichoderma strains against Fusarium oxysporum and 77 Trichoderma strains antagonistic to Botrytis cinerea were obtained from 462 Trichoderma strains.Meanwhile 21 Trichoderma strains belong to uncommon species in Trichoderma genus were evaluated on their activity against five different pathogens.Furthermore,a multi-wells plate method ?hemi-living plant and test tube seedling screening method constructed for effectively screening biocontrol mircrobes,and the screening results with the both methods shared a good correlation with typical dual culture.A multi-wells plate for strains tolerance evaluation under three kinds of stress factors were construced and corresponding tolerance thresholds were also proposed.With the method we examined a total of 20 Trichoderma isolates from five different species to show their the tolerance performance to different stress conditions including high and/or low temperature,salt,and drought,then the evaluation thresholds were established.A significant correlation was found between Trichoderma strain mycelium dry weight and APX and CAT activity under each of stress factors.Results further showed that the thresholds for Trichoderma growth response to stress factors is 1 mol/L of NaCl(saltstress),36?(high temperature),14?(low temperature),and 400 g/L PEG-6000(drought stress).A linear regression analysis showed that a significant positive correlation emerged between results from colony diameter/area in 12-wells plate and in traditional confrontation culure in PDA plate with 9 cm in diameter under designed stress factors.Thus,the establishment of threshold of Trichoderma strains resistant to different stress factors provided the essential basis for future scientific evaluation and utilization of Trichoderma.The work is important for future scientific evaluation and utilization of tolerance of Trichoderma resources as biocontrol agents.This study also carried out the Trichoderma combined with plant removal of heavy metals in soil.Heavy metal-rich plants arepromoted to absorb heavy metals in soil by applying Trichoderma agents.In the remediation field test in Jinshan District,Shanghai,alfalfa +Trichoderma + organic fertilizer treatment was demonstrated as a more satisfactory bioremediation technique leading to significant reduction of soil heavy metals from polluted soil.In the experiment of remediation in Heqing Town,Pudong,the intercropping system of alfalfa and rapeseed in the presence of Trichodema resulted in better remediation of soil,the contents of heavy metals in soil were decreased significantly as compared with those in monoculture of each of both crops.Similarly under theintercropping model,the accumulations of heavy metals in plants were also higher.