连栽桉树根际微生物分析及固氮菌的研究
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摘要
桉树(eucalyptus)是速生用材树种,具有很高的经济价值,对土壤的适应性强,但对氮肥的需求量较大。因而种植桉树需要大面积施加氮肥。然而,大量施用化肥,不仅增加营林成本,而且导致水土污染,影响环境和破坏生态平衡。因此寻找新的生物固氮途径,增加氮素供应水平,促进桉树的生长,是当前桉树生产中亟需解决的课题,但目前对桉树根际固氮菌研究工作的相关报道还很少。
本研究主要针对广西七坡林场的不同世代连栽桉树人工林地的土壤微生物数量和酶的活性以及根际固氮菌的筛选、培养、回接等方面进行了初步的研究。本试验对不同栽植世代连栽桉树人工林中一代林(G_1)、二代林(G_2)、三代林(G_3)以及对照林(栽种桉树林前的皆伐马尾松林)林地(G_0)的0～20cm土层中根际土壤进行细菌、真菌、放线菌数量测定,并对土壤中果聚糖蔗糖酶和磷酸酶的活性进行了分析。研究表明,连栽桉树人工林土壤中真菌数量表现为G_3＞G_2＞G_1＞G_0,放线菌数量为G_3＞G_0＞G_2＞G_1,细菌数量为G_2＞G_0＞G_3＞G_1;其中二代林中微生物总量最多;各代林地之间土壤中果聚糖蔗糖酶活性差异显著,且表现出随着连栽代数的增加其活性逐渐降低的趋势;土壤中磷酸酶活性不同样地之间差异不显著。
在对不同世代连栽桉树林地根际固氮菌的筛选、培养、回接的研究中,经用阿须贝无氮培养基的培养和筛选,共筛选出8株生长势较好的菌株:G_(1143)、G_(3321)、G_(1163)、G_(2252)、G_(1151)、G_(1161)、G_(1152)、G_(2221),经对其进行固氮酶活性测定后,从中筛选到两株活性较高且表现稳定的菌株G_(3321)和G_(1143)。
为了检验试验中所得8株生长势较好的固氮菌菌株是否对桉树苗木的生长有促进作用,将经液体培养后制成的菌液对桉树组培苗进行回接试验,本试验采用同一浓度的菌液以100 ml/m~2喷洒和蘸根半小时两种方式对桉树苗进行接种。3个月后对接种苗木的苗高、地径测定,结果表明,两种接种方式接种的各固氮菌对桉树苗生长均有不同程度的促进作用,其中蘸根方式处理比喷洒效果明显。效果最显著的G_(3321)菌株蘸根处理后的平均苗高是19.64 cm,比喷洒处理的苗高高出15.9%,比对照高出32.2%蘸根处理桉树苗的地径分别比喷洒处理和对照处理高出8.6%和19.2%。显然,本试验中蘸根比喷洒处理对桉树苗的生长促进作用更为明显。
测定了对苗高、地径生长的促进效果更为明显的蘸根苗木的鲜重、干重和叶绿素含量以及土壤有机质含量、速效氮、速效磷、速效钾含量等指标。结果表明,与CK相比苗木的鲜重、干重和叶绿素的平均含量提高范围分别为10.3%～32.3%、10.8%～30.3%、2.1%～24.1%;与CK相比土壤有机质、速效N、速效P、速效K含量分别提高1.2%～16.1%、1.0%～21.9%、0.7%～12.2%、1.2%～12.5%。可见,各根际固氮菌肥对桉树三个月的组培幼苗的生长和土壤肥力的提高均有不同程度的促进作用,其中菌株G_(3321)和G_(1143)促进效果更为明显。上述结果显示,根际固氮菌发挥了其固氮作用,促进了桉树苗木生长和土壤的改良,但其机理尚不十分清楚,还需进一步研究。
Eucalyptus is a fast-growing timber tree species which has very high economic value and ecological benefit.Eucalyptus can resist drought and has strong adaptability to soil,but it has a larger demand for nitrogen fertilizer.So nitrogen fertilizer needs to be applied in large area in planting eucalyptus. However,the application of a large quantity of fertilizer not only increases the cost of forest culture and management but also results in the pollution of water and soil,affects the environment and destroys the ecological balance.Hence,it is a problem which needs to be solved urgently to seek a new approach to biological nitrogen fixation,increase the supply level of nitrogen,and promote the growth of eucalyptus in the production of eucalyptus.However,currently there are still few relevant reports on the research work of rhizosphere azotobacter of eucalyptus.
In this experiment,the preliminary research was conducted in terms of the amount of soil microbes,the activity of enzyme,and the screening,culture and baCK inoculation of rhizosphere azotobacter of the successive planting eucalyptus in Qipo Forest Farm of Guangxi.The amount of bacteria,epiphytes and actinomycetes of rhizosphere soil in 0-20 cm depth soil layer of different planting generations including the first generation forest(G_1)land,the second generation forest(G_2)land,the third generation forest(G_3)land and the comparison forest(G_0)(clearcutting Pinus massoniana forest before planting eucalyptus)land of the successive planting eucalyptus was tested,and the activities of levansucrase and phosphatase in soil were analysed.The research indicated that the amount of epiphytes in the soil of the eucalyptus plantation in successive rotations was presented as G_3>G_2>G_1>G_0,the amount of actinomycetes was G_3>G_0>G_2>G_1 and the amount of bacteria was G_2>G_0>G_3> G_1.The total amount of microbes in the second generation forest land was the highest.The activity discrepancy of levansucrase in soil between each generation forest land was significant and represented the trend of gradual decrease of the enzyme activity with the increase of successive planting generations.The activity discrepancy of phosphatase in soil between different plots was not significant.
In the research on the screening,culture and baCK inoculation of rhizosphere azotobacter of the successive planting eucalyptus,eight strains with better growth vigor were screened out totally by the culture and screening of Ashy nitrogen-free culture medium,which were represented as G_(1143)、G_(3321)、G_(1163)、G_(2252)、G_(1151)、G_(1161)、G_(1152)、G_(2221)in this paper.After the nitrogenase activities of them were tested,two strains among them,G_(3321)and G_(1143),which had higher activities and a stable expression,were screened out.
In order to test whether the eight azotobacter strains gained in the experiment,which grew better,had promotion functions for the growth of the eucalyptus seedlings,the baCK inoculation experiment was conducted on the tissue culture seedlings of eucalyptus by the bacteria solution after liquid culture. In this experiment two methods of spraying and dipping roots in the same concentration(100ml/m~2)for half an hour were adopted to conduct inoculation on the eucalyptus seedlings.After three months the height and the ground diameter of the inoculation seedlings were determined.The results showed that each azotobacter inoculated by using two inoculation methods had the promotion function for the growth of eucalyptus seedlings to different extent. The effect of dipping roots is more significant than that of spraying.The average seedling height of strain G_(3321)with the most significant effect after the treatment of dipping roots is 19.64cm,15.9%higher than the height of seedling after the treatment of spraying,32.0%higher than the comparison seedlings.The ground diameter of eucalyptus seedlings of dipping roots treatment is respectively higher than that of spraying treatment and comparison treatment by 8.6%and 16.2%.Apparently,in this experiment the treatment of dipping roots has more significant promotion function for the growth of eucalyptus seedlings than that of spraying.
The fresh weight,dry weight and the content of chlorophyll of dipping roots seedlings which had significant promotion function for the growth of the height of seedlings and ground diameter were tested.So were the indicators including the content of organic matter in soil,available N,available P,available K and so on.The results indicated that compared with CK,the range of improvement of the fresh weight,dry weight and the content of chlorophyll was respectively 10.3%～32.3%、10.8%～30.3%and 2.1%～24.1%,and compared with CK,the range of improvement of the content of organic matter in soil, available N,available P and available K,was respectively 1.2%～16.1%、1.0%～21.9%、0.7%～12.2%and 1.2%～12.5%.It can be seen that every bacterial fertilizer of nitrogen fixation in rhizosphere had promotion function for the growth of tissue culture seedlings of three-month eucalyptus and the improvement of soil fertility to different extent.The strain G_(3321)and G_(1143)with better growth vigor and activity had more significant promotion effect.The results above show that rhizosphere azotobacter gives to play its function of nitrogen fixation and promotes the growth of the eucalyptus seedlings and the improvement of soil,but its mechanism is still not very clear and needs further study.
本研究主要针对广西七坡林场的不同世代连栽桉树人工林地的土壤微生物数量和酶的活性以及根际固氮菌的筛选、培养、回接等方面进行了初步的研究。本试验对不同栽植世代连栽桉树人工林中一代林(G_1)、二代林(G_2)、三代林(G_3)以及对照林(栽种桉树林前的皆伐马尾松林)林地(G_0)的0～20cm土层中根际土壤进行细菌、真菌、放线菌数量测定,并对土壤中果聚糖蔗糖酶和磷酸酶的活性进行了分析。研究表明,连栽桉树人工林土壤中真菌数量表现为G_3＞G_2＞G_1＞G_0,放线菌数量为G_3＞G_0＞G_2＞G_1,细菌数量为G_2＞G_0＞G_3＞G_1;其中二代林中微生物总量最多;各代林地之间土壤中果聚糖蔗糖酶活性差异显著,且表现出随着连栽代数的增加其活性逐渐降低的趋势;土壤中磷酸酶活性不同样地之间差异不显著。
在对不同世代连栽桉树林地根际固氮菌的筛选、培养、回接的研究中,经用阿须贝无氮培养基的培养和筛选,共筛选出8株生长势较好的菌株:G_(1143)、G_(3321)、G_(1163)、G_(2252)、G_(1151)、G_(1161)、G_(1152)、G_(2221),经对其进行固氮酶活性测定后,从中筛选到两株活性较高且表现稳定的菌株G_(3321)和G_(1143)。
为了检验试验中所得8株生长势较好的固氮菌菌株是否对桉树苗木的生长有促进作用,将经液体培养后制成的菌液对桉树组培苗进行回接试验,本试验采用同一浓度的菌液以100 ml/m~2喷洒和蘸根半小时两种方式对桉树苗进行接种。3个月后对接种苗木的苗高、地径测定,结果表明,两种接种方式接种的各固氮菌对桉树苗生长均有不同程度的促进作用,其中蘸根方式处理比喷洒效果明显。效果最显著的G_(3321)菌株蘸根处理后的平均苗高是19.64 cm,比喷洒处理的苗高高出15.9%,比对照高出32.2%蘸根处理桉树苗的地径分别比喷洒处理和对照处理高出8.6%和19.2%。显然,本试验中蘸根比喷洒处理对桉树苗的生长促进作用更为明显。
测定了对苗高、地径生长的促进效果更为明显的蘸根苗木的鲜重、干重和叶绿素含量以及土壤有机质含量、速效氮、速效磷、速效钾含量等指标。结果表明,与CK相比苗木的鲜重、干重和叶绿素的平均含量提高范围分别为10.3%～32.3%、10.8%～30.3%、2.1%～24.1%;与CK相比土壤有机质、速效N、速效P、速效K含量分别提高1.2%～16.1%、1.0%～21.9%、0.7%～12.2%、1.2%～12.5%。可见,各根际固氮菌肥对桉树三个月的组培幼苗的生长和土壤肥力的提高均有不同程度的促进作用,其中菌株G_(3321)和G_(1143)促进效果更为明显。上述结果显示,根际固氮菌发挥了其固氮作用,促进了桉树苗木生长和土壤的改良,但其机理尚不十分清楚,还需进一步研究。
Eucalyptus is a fast-growing timber tree species which has very high economic value and ecological benefit.Eucalyptus can resist drought and has strong adaptability to soil,but it has a larger demand for nitrogen fertilizer.So nitrogen fertilizer needs to be applied in large area in planting eucalyptus. However,the application of a large quantity of fertilizer not only increases the cost of forest culture and management but also results in the pollution of water and soil,affects the environment and destroys the ecological balance.Hence,it is a problem which needs to be solved urgently to seek a new approach to biological nitrogen fixation,increase the supply level of nitrogen,and promote the growth of eucalyptus in the production of eucalyptus.However,currently there are still few relevant reports on the research work of rhizosphere azotobacter of eucalyptus.
In this experiment,the preliminary research was conducted in terms of the amount of soil microbes,the activity of enzyme,and the screening,culture and baCK inoculation of rhizosphere azotobacter of the successive planting eucalyptus in Qipo Forest Farm of Guangxi.The amount of bacteria,epiphytes and actinomycetes of rhizosphere soil in 0-20 cm depth soil layer of different planting generations including the first generation forest(G_1)land,the second generation forest(G_2)land,the third generation forest(G_3)land and the comparison forest(G_0)(clearcutting Pinus massoniana forest before planting eucalyptus)land of the successive planting eucalyptus was tested,and the activities of levansucrase and phosphatase in soil were analysed.The research indicated that the amount of epiphytes in the soil of the eucalyptus plantation in successive rotations was presented as G_3>G_2>G_1>G_0,the amount of actinomycetes was G_3>G_0>G_2>G_1 and the amount of bacteria was G_2>G_0>G_3> G_1.The total amount of microbes in the second generation forest land was the highest.The activity discrepancy of levansucrase in soil between each generation forest land was significant and represented the trend of gradual decrease of the enzyme activity with the increase of successive planting generations.The activity discrepancy of phosphatase in soil between different plots was not significant.
In the research on the screening,culture and baCK inoculation of rhizosphere azotobacter of the successive planting eucalyptus,eight strains with better growth vigor were screened out totally by the culture and screening of Ashy nitrogen-free culture medium,which were represented as G_(1143)、G_(3321)、G_(1163)、G_(2252)、G_(1151)、G_(1161)、G_(1152)、G_(2221)in this paper.After the nitrogenase activities of them were tested,two strains among them,G_(3321)and G_(1143),which had higher activities and a stable expression,were screened out.
In order to test whether the eight azotobacter strains gained in the experiment,which grew better,had promotion functions for the growth of the eucalyptus seedlings,the baCK inoculation experiment was conducted on the tissue culture seedlings of eucalyptus by the bacteria solution after liquid culture. In this experiment two methods of spraying and dipping roots in the same concentration(100ml/m~2)for half an hour were adopted to conduct inoculation on the eucalyptus seedlings.After three months the height and the ground diameter of the inoculation seedlings were determined.The results showed that each azotobacter inoculated by using two inoculation methods had the promotion function for the growth of eucalyptus seedlings to different extent. The effect of dipping roots is more significant than that of spraying.The average seedling height of strain G_(3321)with the most significant effect after the treatment of dipping roots is 19.64cm,15.9%higher than the height of seedling after the treatment of spraying,32.0%higher than the comparison seedlings.The ground diameter of eucalyptus seedlings of dipping roots treatment is respectively higher than that of spraying treatment and comparison treatment by 8.6%and 16.2%.Apparently,in this experiment the treatment of dipping roots has more significant promotion function for the growth of eucalyptus seedlings than that of spraying.
The fresh weight,dry weight and the content of chlorophyll of dipping roots seedlings which had significant promotion function for the growth of the height of seedlings and ground diameter were tested.So were the indicators including the content of organic matter in soil,available N,available P,available K and so on.The results indicated that compared with CK,the range of improvement of the fresh weight,dry weight and the content of chlorophyll was respectively 10.3%～32.3%、10.8%～30.3%and 2.1%～24.1%,and compared with CK,the range of improvement of the content of organic matter in soil, available N,available P and available K,was respectively 1.2%～16.1%、1.0%～21.9%、0.7%～12.2%and 1.2%～12.5%.It can be seen that every bacterial fertilizer of nitrogen fixation in rhizosphere had promotion function for the growth of tissue culture seedlings of three-month eucalyptus and the improvement of soil fertility to different extent.The strain G_(3321)and G_(1143)with better growth vigor and activity had more significant promotion effect.The results above show that rhizosphere azotobacter gives to play its function of nitrogen fixation and promotes the growth of the eucalyptus seedlings and the improvement of soil,but its mechanism is still not very clear and needs further study.
引文
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