Study On Microflora And Characteristic Of Plant Growth Promoting Rhizobacteria(PGPR) In Rhizosphere Of Kentucky Bluegrass

Kentucky bluegrass is a cold-season turf grass with stronger resilience capacity. It can be planted in most northern part of China and is widely used in urban area for turf establishment in parks, football pitch, golf courses, schools, residential area and so on. Based on the growth characteristics of Kentucky bluegrass turf, the study utilizes the selective culture medium to analyze dynamic changes of both rhizosphere and non-rhizosphere bacteria, actinomyces and fungi under different light conditions, in different soil fertilities and before and after turning green. Then it investigates the relations between the rhizosphere of Kentucky bluegrass and external environment conditions. Finally it probes into the optimum growth conditions of Kentucky bluegrass by separating, identifying and selecting the PGPR strains, which aims to lay down both practical and theoretical foundations for adjusting the ecosystems of Kentucky bluegrass rhizosphere and developing turfgrass PGPR biological manure. The following is the main results and conclusions.(1)With the illumination goes down, the amount of different rhizosphere micro-organisms change correspondingly as follows, the amount of both rhizosphere and non-rhizosphere bacteria in Kentucky bluegrass falls; the amount of rhizosphere actinomyces falls at the beginning and goes up in the end; the amount of non-rhizosphere actinomyces declines at a slow pace; the amount of fungi increased gradually; the rhizosphere soil of Kentucky bluegrass transforms from bacterial type into fungic type. In one word, various bacterial groups of Kentucky bluegrass demonstrate a strong rhizosphere effect.(2)As the soil fertility declines, the amount of different rhizosphere micro-organisms change correspondingly as follows: the amount of both rhizosphere and non-rhizosphere bacteria in Kentucky bluegrass falls; the amount of both rhizosphere and non-rhizosphere actinomyces falls at the beginning and goes up in the end; the amount of fungi goes up; the rhizosphere soil of Kentucky bluegrass transforms from bacterial type into fungic type.(3)As the Kentucky bluegrass turf turns green, the amount of different rhizosphere micro- organisms change correspondingly as follows, the amount of bacteria and fungi, both rhizosphere and non-rhizosphere, increases dramatically; the amount of both rhizosphere and non-rhizosphere actinomyces falls; various bacterial groups of Kentucky bluegrass demonstrate a strong rhizosphere effect after the Kentucky bluegrass turns green; the amount of both rhizosphere or non-rhizosphere bacteria accounts for the majority of the soil microbial biomass(SMB) both before and after the Kentucky bluegrass turns green. In conclusion, the numerical changes of bacteria represent changing tendency of the whole microorganism groups.(4) The NFM culture medium was employed for separating and purifying the nitrogen-fixing bacteria strains from soil samples of Kentucky bluegrass rhizosphere, and 21 pure cultures of nitrogen-fixing bacteria strains were obtained. The result indicates that the strains ranking is Azoobacter, Pseudomonas, Bacillus, and Enterobacter.(5)The mengjinna organic culture medium and PKO inorganic culture medium were introduced to separate and purify the phosphate solubilizing bacteria(PSB)from rhizosphere soil samples of Kentucky bluegrass, and pure cultures of 8 organic phosphate solubilizing bacteria strains and 7 organic phosphate solubilizing bacteria strains were obtained. It is identified that the organic phosphorus bacteria are as Bacillus sp. And strains PO1, PO3, PO4 and PO5 are Bacillus cereus; strain PO2, PO6 and PO7 are Bacillus megaterium; strain PO8 is Bacillus badius; all inorganic phosphorus bacterial strains are Pseudomonas sp.(6)The silicate culture medium was introduced for separation and the purification of Potassium solubilizing bacteria strains from Kentucky bluegrass rhizosphere, and 7 pure cultures were obtained. All Potassium solubilizing bacteria strains are identified as Bacillus mucilaginosus.(7)Acetylene reducing method was employed to measure nitrogenase activity of 21 nitrogen-fixing bacteria strains isolated from Kentucky bluegrass rhizosphere soil samples. And the result shows that 14 strains have an activity of more than 100nmol/mL·h. In addition, the solubilizing phosphorus intensity of nitrogen-fixing bacteria is between 13.38μg/mL to 58.21μg/mL and among these strains N4, N16 and N20 demonstrates a stronger solubility of phosphorus. At the same time a colorimetric assay method was employed for measuring the strong ability of strains N2, N4, N5, N10, N14, N16, N17and N20 in excreting plant growth hormone(8)The organic phosphate bacteria demonstrate a solubilizing P capability between 17.05μg/mL to 62.37μg/mL. Specifically, strains PO1, PO4, PO5 and PO6 have a stronger performance;and the inorganic phosphate solubilizes P between 17.05μg/mL to 62.37μg/mL; strains PM6,PM1 and PM5 have a strong solubility.(9)The capacity of solubilizing K is between 5.7mg/L to 14.5 mg/L for Potassium bacteria strains isolated from Kentucky bluegrass rhizosphere. And strain K5 ranks the first in solubilizing K, strain K3 the second and strain K7 the third.(10)After being tested of the biological effectiveness and various index being considered, 18 high-performing PGPR strains are chosen, i.e., N2, N4, N5, N10, N14, N16, N17 and N20 nitrogen-fixing bacteria strains, PO1, PO4, PO5 and PO6 organic phosphate bacteria strains, PM1, PM5 and PM6 inorganic phosphate bacteria strains, and K3, K5 and K7 potassium bacteria strains.(11)The optimum growth temperature of various PGPR strains was measured by gradient method. The result shows that the nitrogen-fixing bacteria, phosphate solubilizing bacteria and potassium solubilizing bacteria can grow well between 10～45°C. It indicates that the temperature adaptation scope of PGPR strains is wide, but the optimum growth temperature of most PGPR strains is 20~35°C. (12)The measuring of various strains optimum growth initial pH value shows that the nitrogen fixation strain is 7.0～7.5; the inorganic and organic phosphate solubilizing bacteria is 7.5～8.5 and 5.5～6.5 respectively; the optimum growth initial pH value of Potassium solubilizing bacteria is 7.5～8.0. These data indicates that PGPR strains can grow well in a neutral or slightly alkaline pH condition, except the organic phosphate solubilizing bacteria strain, which requires a medium acidity or slight acidity condition.(13)According to the measuring of optimum ventilation volume conditions, strain N4, N5, N10, N16, N17, N20, PM5, PO6 and K7 are aerobes. They have achieved the peak value of growth in 220 mL of ventilation volume. Facultative anaerobes strains including N2,PM1 and K5 have achieved the peak value in 150～200 mL of ventilation volume. And strain N14, PM6 and K3 are microaerobes which have achieved the peak value in 100 mL of ventilation volume; In addition, the strain PO1, PO4 and PO5 are obligate anaerobes, which have achieved the peak value in 70 mL.(14)The different light intensity grads were set for measuring of optimum growth light condition of various PGPR strains. It indicates that the optimum growth light condition of most PGPR strains including N5, N14, N20, PM1, PO6, K3, K5 and K7 is 3500LX; however, various PGPR strains do not present growth peak value when the light intensity is 0LX and 7500 LX. These data indicates that PGPR strains isolated from Kentucky bluegrass rhizosphere require certain illumination to achieve the highest growth efficiency, but strong illumination condition will suppress growth and reproduction of PGPR strains.(15)Under the optimum growth factors such as proper temperature, pH value, ventilation volume condition and illumination condition, the typical growth curve of each PGPR strain is obtained by making the y-coordinate by the cell number value of various strain, and making the x-coordinate by the time of germ culture. According to the typical growth curve above, the lag phase, the logarithmic phase, the stationary phase and the declining phase can be obtained for each PGPR strain. These data is of vital significance for understanding and mastering growth rhythm of PGPR strains, as well as promoting the strain's growth effectively.