The Chemotaxis And Casing Soil Application Of Pseudomonas Putida In Agaricus Bisporus Cultivation

Agaricus bisporus is the most widely cultivated and consumed mushroom in the world.Our laboratory has found that the dominant bacterial population in the casing soil of Agaricus bisporus is the 1-aminocyclopropane-1-carboxylic acid(ACC)deaminase(Acd S)-producing bacteria,which degraded the ACC produced by Agaricus bisporus,reduced the subsequent ethylene production of the mushroom,and promoted the growth and development of the mushroom.The aim of this study is to explore the effect of casing soil with Acd S-producing bacteria Pseudomonas putida uw4 on the large-scale cultivation of Agaricus bisporus,and to confirm the bacterial strain chemotaxis to ACC at the gene level.The main results are as following:1.The effect of three different substrates on the Pseudomonas putida UW4 inoculant preparation was compared.The three substrates were decomposed coal,Dongbei peat soil,and Pindstrup peat soil from Danish.The results showed that the bacteria number was 8×108 cfu per gram in the inoculant prepared by Pindstrup peat soil,higher than the agricultural industry standard for the bacterial inoculants.While the bacteria numbers were only 5×104 and 2×106 cfu per gram in the inoculants prepared by the other two substrates.2.Six casing treatments were performed,the treatments were conventional casing soil,conventional casing soil added 2% microbial inoculum,sterilized conventional casing soil,sterilized conventional casing soil added 2% bacterial inoculant,vermiculite,vermiculite added 2% bacterial inoculant.Each treatment was replicated three plots;each plot was 2.4 m in length and 1.2 m in width.The mushroom yields of the treatments with bacterial inoculant were all higher than that without bacterial inoculant.The vermiculite added 2% bacterial inoculant treatment produced the highest yield,and consumed the least labour compared to the other treatments.The mushroom yield of the vermiculite added 2% bacterial inoculant treatment was higher 35% than that of conventional casing soil treatment,the mushroom yield of sterilized conventional casing soil added 2% bacterial inoculant treatment was higher 13% than that of conventional casing soil treatment.3.After harvesting the first and second crop of the mushroom,the number of Acd S-producing bacteria in different casing materials was detected.The number was 5.4,3.9,5.8,12.6,1.8,18×106 cfu per gram in the six casing materials,respectively,which accounted for 15.9%,8.6%,11.2%,18.9%,15.9%,18.8% of the number of total cultivable bacteria.The number of Acd S-producing bacteria and total cultivable bacteria in different casing mateiral was significantly positive related to the yields of Agaricus bisporus.4.In order to botain a chemotaxis mutant strain of Pseudomonas putida UW4,we try to knock out the Che R gene by using homologous recombination.The upstream and downstream flanking DNA fragments of the target gene and tetracycline resistance gene expression box were amplified by PCR,and then we fused the three fragments by using OE-PCR reaction.The DNA fragment was transformed into P.putida by electroporation,the transformants were been selecting.