Microbial&Ecological Analysis Of Soil Health-associated Microbe In Tobacco Continuous Cropping Fields And Its Application

The abundant biodiversity of soil microbe is an important foundationfor soil health, which is also important for plant healthy. But continuous cultivationmay lead to many problems, such as the rampancy of disease and insects, the fallingof soil available fertility and the reducing of sustainable production and so on, and ithas become a serious problem in tobacco production. Therefore, it is high time to findout the mechanism of soil micro-ecological balance affecting tobacco continuouscropping, and to develop a new way to control tobacco soil-borne disease instead ofcrop rotation and chemical prevention.PCR-DGGE program has been optimized in this study, and the best DGGEelectrophoresis conditions have been seeked out. Soil samples collected fromcontinuous cropping1～8year(except7year) tobacco fields in Luoyang andXuchang are analyzed, and the soil enzyme activity and soil microbe diversity weredetermined. Besides, the relationship between soil micro-ecological indicator andtobacco soil-borne disease has also been surveyed, and the soil health mechanism hasbeen explored Pot-trial proved that ecochar-based fertilizer can greatly repair soilhealth of tobacco continuous cropping. The main results are as follow:（1）PCR-DGGE program has been optimized on primer screening、DGGEelectrophoresis time and staining methods. The results show that the optimizednested-PCR primer pairs are10F/1507R and BV34F/BV56GC, the optimized samplevolume for DGGE is50ul, DGGE gel concentration is6％, concentration ofdenaturants varies from40％to60％, electrophoresis time is100V×12h, and EBstains for15min, the most ideal DGGE pattern can be obtained.（2）The soil enzyme activities have been analyzed, and the results indicate thatwith the increasing of continuous cropping year, soil amylase, sucrase activitiesconsistently increase in short-term continuous cropping year, showing a summit in thecontinuous cropping year of two, and then decrease significantly. The phenomenonreveals that short-term continuous cropping can temporarily increase the soil amylase,sucrase activities, but long-term continuous cropping decrease that greatly. The soilcellulase activities have the same change in Luoyang tobacco continuous croppingfields, while that in Xuchang tobacco continuous cropping fields decrease persistently,and, then keep in a lower level after3a.（3）The results also show that with the increasing of continuous cropping year,the soil microbial diversity declines. Compared with fungi and actinomycetes, soilbacterial diversity is more sensitive to continuous cropping, which reflects the essential situation of soil health.Traditional cultivation for soil microbe indicates that the soil microbe densityshow a decline curve with the increasing of continuous cropping year,. Similar withsoil actinomycetes, the bacterial density shows a summit within1a or2a continuouscropping year and then reduces year by year. Soil fungi are insensitive to continuouscropping because the fungi density in Luoyang and Xuchang fields shows irregularchanging.PCR-DGGE-based determination of soil bacterial diversity has been carried out.The number of detected bands is decreasing with the increasing of continuouscropping year, which reveals that the richness of soil bacteria is decreasing. However,the soil bacterial number has a temporarily increase in5a. Besides, the soil bacterialShannon index、Simpson index and Mclntosh index density also have significantdifference（p﹤0.01）among different continuous cropping years, and their changemode show high corresponding with the bacteria density.（4）The relation of soil micro-ecological indicator with continuous cropping andto tobacco black shank and root knot nematode disease are analyzed, the result showsthat tobacco disease index rise up with the increasing of continuous cropping year.The tobacco disease is not so serious in the continuous cropping year of1and2, butincreases rapidly after3a and keeps in a higher level. The results also show that thesoil amylase, sucrase, cellulase activities and soil bacterial number have a linearnegative correlation with the disease index.（5）The dose effect and additive effect of ecochar-based fertilizer have beenexplored, and the effects of ecochar-based fertilizer on tobacco maximum leaf area、plant height、root shoot ratio、root activities、soil enzyme activities and bacterialdiversity have also been analyzed. The results indicate that ecochar-based fertilizerhas a good effect on repairing soil health, and obviously, it exists dose effect andadditive effect, that is, the soil micro-ecological indicator in the soil treated byecochar-based fertilizer for two years is better than those treated for one year.