| Malus sieversii is the main constructed species of Xinjiang wild fruit forest distributed in Yili,Xinjiang.It is a rare natural gene bank of apple also quality gene bank in the world.However,in the past half century,the death rate of wild apple forest caused by disease has reached 80%.Trichoderma is a promising biological control fungus for plant diseases.,there are more than 100 Trichoderma-based commercial preparations in the world since Trichoderma was found to have antagonistic effect by Weindling in 1932,which have been widely used to control plant diseases in agriculture and forestry.Among them,wettable powders,granules and microcapsules are widely used.This study explored a solid-state fermentation process of Trichoderma harzianum T2,which produced.harzianum T2 spore powder effectively in the form of straw and wheat bran bags.Then,harzianum T2 conidial powder produced by this process as spore powder,the processing technology of.harzianum T2 wettable powder was preliminarily explored,and the pesticide effect of final formulation on leaf blight caused by Alternaria alternata on Malus sieversii was tested.By optimizing the solid-state fermentation technology of T.harzianum T2 in powder bag,the fermentation formula of 57%straw,40%wheat bran,1%soybean flour,1%corn flour,1%NH4NO3 and 75%water content was finally screened out.Each bag was filled with 200 g fermentation material.The optimum harvest time was 8 d after inoculation with spore suspension at 28?.The.harzianum spore powder was harvested by drying under natural condition,which is screened by vibration screening machine and then through 200 sifter screening.The final spore powder contains 2.63%water and 1.64×1012/g spores,and the spore germination rate reaches 99.25%.It provides a basis for further preparation processing.High-quality.harzianum T2 spore powder obtained by solid-state fermentation in the form of bags by straw mainly as raw powder,with kaolin,bentonite,attapulgite,diatomite as carrier,EFW,MF-5,SDS and pull-off powder as wetting agent,NNO,calcium lignosulfonate and sodium lignosulfonate as dispersant,sodium fluorescein,dextrin,VC and zinc oxide as ultraviolet protection,the above aids were screened.After preliminary screening and orthogonal experiments,the results showed that 1%MF-5,5%calcium lignosulfonate,0.3%dextrin and 10%.harzianum spore powder with diatomite as carrier as complement to 100%was the best.The quality test results of the.harzianum wettable powder showed that the wetting time was 5.89s and the mass suspension rate was 79.79%.Other indicators of the formulation were tested,including fineness(over 325 target quasi-sieves,average particle size of 28 microns)>98%,pH value of 6.5,and spore germination rate of 95.12%after 12 months at 4?,which accorded with the national standards for the formulation of fungal wettable powder.The efficacy of Trichoderma.harzianum T2 wettable powder on Malus sieversii seedlings against Alternaria alternata was tested.(1)Observation on the disease incidence of Malus sieversii after 7 d of inoculation showed that.harzianum T2 wettable powder could reduced the the disease incidence caused by A.alternate from 3 to 1.After 7 d of inoculation,the stomata on the back of leaves of Malus sieversii were observed by optical microscope.The results showed that.harzianum T2 wettable powder could inhibit the infection of A.alternata on guard cells of stomata and maintain its normal form.(2)The T.harzianum T2 wettable powder can also effectively improve catalase(CAT)activity in leaves of Malus sieversii and reduce the content of hydrogen peroxide and malondialdehyde(MDA).Compared with group which Malus sieversii treated with A.alternata alone,the CAT activity of Malus sieversii treated with.harzianum T2 wettable powder sprayed ahead of time was significantly increased,and reached the highest level at 2d with 212.44?mol/g,which was 2.61 times higher than the former,The content of hydrogen peroxide was 26.89?mol/g lower than it and the content of MDA was 60.724?mol/g lower at 1 d.Evans Blus and nitroblue tetrazolium(NBT)staining experiments have reached the same conclusion.Leaf necrosis area of group treated with T.harzianum T2 wettable powder sprayed ahead of time and then inoculated with A.alternata is less.This result also showed that.harzianum T2 wettable powder can improve defense enzyme activity and reduce the accumulation of reactive oxygen species in Malus sieversii.(3)RT-qPCR was used to detect the effects of T.harzianum T2 wettable powder and inoculation with A.alternata on the expression level of resistance-related genes in leaves of Malus sieversii.For salicylic acid pathway related genes PR1 and NPR1,the NPR1 gene in leaves of wild apples in Xinjiang showed a trend of negative regulation after spraying.harzianum T2 wettable powder alone,reaching the lowest level in 6h with-0.24 times,while PR1 maintained a higher expression level and reached the highest expression level of 2.38 times at 1d.For MYC2,COI and JAZ genes related to jasmonic acid pathway,after spraying T.harzianum T2 wettable powder and inoculating A.alternata,the expression level of MYC2 gene in Malus sieversii was higher at 6 h,1 d and 2 d,and reached the highest expression level of 2.43 times at 1 d.COI gene expression was up-regulated and reached the highest expression level of 4.05 times on 2 d and JAZ gene expression was up-regulated and reached the highest expression level of 3.30 times on 6 h,showing an overall up-regulated trend.For auxin-related gene LAX,spraying.harzianum T2 wettable powder before inoculation of A.alternata increased expression level of LAX gene and reached a maximum expression level of 6.42 times in 4 d,which also showed a positive regulation trend.Generally speaking,the solid-state fermentation of T.harzianum T2 in the form of straw and wheat bran bags provides a new idea for the solid-state fermentation technology of T.harzianum T2.The application of T.harzianum T2 wettable powder for Malus sieversii in Xinjiang is feasible for the special geographical environment and climate in the wild fruit forest area of Xinjiang.The study demonstrated that.harzianum T2 wettable powder could improved resistance of Malus sieversii of to A.alternate by aspects as physiological status of plants(stoma,area of disease spot,etc.),enzyme activity and expression level of resistance related genes,which provided a new basis for the control of disease of Malus sieversii in Tianshan wild fruit forest in Xinjiang. |
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