Studies On The Pathway Of Ethylene Biosynthesis And Its Relationship With Casing Soil Triggering Sporophore Formation Of Agaricus Bisporus

Agaricus bisporus is the most widely and the largest cultivated in the world. However,it must be casing in the process of cultivation, the mechanism is unclear. This study of Agaricus bisporus ethylene biosynthesis revealed the mechanism of casing. The main results are as follows:1, The pathway of ethylene biosynthesis in Agaricus bisporus may be the same with the higher plants, high concentration of ACC and ethylene exist in the culture material of Agaricus bisporus and casing. Ehylene can inhibite mycelium growth. Were used gas chromatography and ion chromatography detected the content of ethylene and ACC in the process of mushroom cultivation. The results revealed that Agaricus bisporus can produce ACC and ethylene in the growth process. Agaricus bisporus is cultured in the liquid PDA medium and the second fermentation for 15 days ,then sealed 12 hours .we found that the average concentration of ethylene reached 9.26μl / L.h and 5.92μl / L.h; Agaricus bisporus is cultured in the liquid PDA medium that the ACC reached 13.76μg/g. we study the effects of ethylene on mycelium growth of Agaricus bisporus that different concentrations of ethylene exist in environment.The results showed that: the average growth rate of the control was 4.0mm / d in the process of 6 days, while concentration of ethylene in the environment was 100nmol / L, 200nmol / L, 400 nmol / L, the average growth rate was 3.0mm / d, 2.9mm / d, 1.6mm / d. Obviously, ethylene inhibite the mycelium growth of Agaricus bisporus.2, We can detecte ACC synthase and oxidase activity from the mushroom mycelium. Agaricus bisporus cultured in PDA liquid medium for 15 days, and then adding L-Met and ACC solution to the medium.The final concentration was 5mmol / L. After 12h cultivation, We extracted crude enzyme solution and determinated enzyme activity. Activity test showed that: ACC synthase activity (in nmol / mg.h) of were 9.81 and 1.94, respectively. The control was 0.84. The L-Met and ACC solution treated samples were 2.31and 11.68 times of the control samples. There was a significant difference (p <0.05) in the three treated; The vivo ACC oxidase activity of L-Met, ACC solution treatment and control were 0.72,3.14,0.89. ACC solution treatment and the control samples was significant difference (p <0.05) , ACC solution treatment and L-Met solution treatment samples was the same as it. But the control and L-Met the solution treatment was no significant difference; L-Met, ACC solution treatment and control of ACC oxidase activity in vitro were 0.23,0.96,0.27, consistent with the laws of determination results in vivo. However, due to the determination of in vitro activity may lead to the loss of some activity, so control、L-Met and ACC treatment samples, the ACC oxidase enzyme activity in vivo than in vitro activity to be much higher, the former were 3.30 times, 3.13 times and 3.27 times than the latter.Agaricus bisporus cultured in PDA liquid medium for 15 days and extracted crude enzyme solution, then added the AOA and CoCl2 solution to the medium. The final concentration was 5mmol / L, and incubated it for 30min and determinated enzyme activity. Activity test showed that: ACC synthase activity (in nmol / mg.h) of AOA and CoCl2 treatment samples were 0.888 and 1.728, respectively. They were 2 / 5 and 4 / 5 of the control what is 2.184. There were significant differences (p <0.05). AOA inhibited the ACC synthase; ACC oxidase activity (in nmol / mg.h) of AOA and CoCl2 treatment samples were 0.835 and 0.192, respectively. The control was 2.225. It was 2.7 and 11.6 times than the AOA and CoCl2 treatment samples.Both AOA and CoCl2 inhibited ACC oxidase, but the latter inhibition is more intense.3, ACS and ACO genes were cloned from Agaricus bisporus. According to ACS and ACO genes conserved sequences of the fungi and higher plants GeneBank , primers were designed. We cloned ACS and ACO genes by PCR, RT-PCR and other methods. We compared the sequences of experimental strain with ACS and ACO genes conserved sequences of GeneBank ,and then we confirmed that we Successed .So a molecular level evidence was provided that ACC ethylene metabolic pathway is in Agaricus bisporus. 4, ACC deaminase producing strains can promote the growth of Agaricus bisporus mycelium and fruiting body formation which is the dominant bacteria in casing.The dominant groups in the casing were Pseudomonas, Serratia rubidaea spp, Klebsiella pneumoniae and Bacillus cereus spp. These bacteria have the ACC deaminase genes and we can detecte their ACC deaminase activity. Their activity reached118% of the control strain (131 was the strain of Pseudomonas), so they can take the ACC in casing.The same trend is between the total number of culturable bacteria and the total number of culturable ACC degrading bacteria in the entire cultivation process. whether the total number of culturable bacteria increase or decrease, the total number of culturable ACC degrading bacteria can be rapidly becoming the dominant flora that the highest ratio reached 28.5%. This may be mycelium produced ACC and diffusion by capillary action then move to the casing .A large number of these bacteria can be maintained in the period of primordia formation by using ACC in casing. The content of ACC can make ACC degrading bacteria growth and reproduction, the disappearance of ( or increase) rate ACC degrading bacteria was significantly less (or greater than) other culturable bacteria (or increase) the speed, quickly became the dominant flora. Mycelium of Agaricus bisporus produced ACC rule can be knowed from the trend of ACC deaminase bacterial number. We found that primordia of Agaricus bisporus number were 20.7 times between the casing with ACC degrading bacteria and without degrading bacteria, it indicates that ACC deaminase bacterial in the casing is necessary.5,For study the effects of ACC deaminase bacterial on Agaricus bisporus primordia formation , we sterilized casing, and then added ACC degrading bacteria. 131 strain (Pseudomonas) is ACC degrading bacteria (with the ACC deaminase), and the 459 strain is the strain of 131 mutant. The first time of fruiting primordia number of shows that: the fruiting primordia number of 131 strain casing is 248 / m2、the fruiting primordia number of no sterilization casing is 184 / m2; the fruiting primordia number of 459 strain casing is 12 / m2; the fruiting primordia number of sterilization casing is 24 / m2. the before two are 7.7 ~ 20.7 times than the latter two.It indicats that ACC deaminase bacterial in the casing is necessary. The second time of fruiting primordia number of shows that: the fruiting primordia number of 131 strain casing is 84 / m2、the fruiting primordia number of no sterilization casing is 88 / m2; the fruiting primordia number of 459 strain casing is 16 / m2; the fruiting primordia number of sterilization casing is 40 / m2. the before two are 2.1 ~ 5.5 times then the latter two. So ACC deaminase bacterial in the casing have an impact on the second time primordia formation.In summary, the pathway of ethylene biosynthesis in Agaricus bisporus is from ACC to ethylene, and ethylene inhibit the growth of its mycelium and primordia formation. The dominant bacteria of casing is the ACC deaminase bacteria. The ACC deaminase bacteria in casing can promote the formation of fruiting bodies, but the ACC deaminase mutant in casing is no effects. So, the mechanism of casing is :after the ACC produced mycelium and diffusion by capillary action to move to the casing, the ACC deaminase bacteria use it .This action reduced the Agaricus bisporus ethylene production and removed the inhibition of ethyleneon the growth of mycelium and primordia formation in Agaricus bisporus.