【作者】 李玫；

【导师】 白嘉雨；

【作者基本信息】 中国林业科学研究院 ， 森林培育， 2009， 博士

【摘要】 红树林具有重要的生态、经济和社会效益。由于长期人为干扰,我国红树林出现面积减少、林分质量下降、防护功能衰退等问题。对红树林主要造林树种的PGPR开展研究,不仅有助于开发红树林微生物资源,而且可利用筛选出的优良PGPR菌株研制促生菌剂,接种于红树林幼苗根际以促进其生长、增强抗逆能力,提高红树林的造林存活率、保障沿海防护林体系建设顺利实施。本研究利用常规分离鉴定方法、乙炔还原法、钼锑抗比色法、Salkowski比色法、Biolog微生物鉴定系统和16S rDNA序列分析等试验技术和分析方法,对我国红树林主要造林树种根际固氮菌和溶磷菌,进行了菌株分离筛选及促生特性分析、红树林幼苗的接种效应研究,以及PGPR微胶囊菌剂的研制。结果表明:1.红树林主要造林树种的根际存在种类较丰富的PGPR菌。分别从广东湛江、珠海及海南东寨港等三地的木榄Bruguiera gymnorrhiza、秋茄Kandelia candel、无瓣海桑Sonneratia apetala、红海榄Rhizophora stylosa、海莲Bruguiera sexangula和海桑S. caseolaris等6种红树植物的根际,分离出固氮菌20株和溶磷菌35株。筛选出的7株优良固氮菌分属Azotobacter(1个)、Pseudomonas (1个)、Bacillus(4个)和Ochrobactrum(1个),7株优良溶磷菌分属Pseudomonas (1个)和Bacillus(6个)。2.利用改良OAB培养基分离的20株菌株大部分具有固氮能力,固氮酶活性在50.72~ 385.6 nmol C2H4·h-1 mL-1之间。固氮酶活性较高的菌株偏少,大于200nmol C2H4·h-1 mL-1的固氮菌株仅有NGWB4-wy1和NGHHL2-r14。利用改良SRMS1培养基分离得到的溶磷菌株数(35株)较多,其解磷酸盐的能力差异较大。溶磷强度较大的菌株有PGHHL -y7(180.2 mg·L-1),PGWB-y2(158.9 mg·L-1)和PGWB-wp5(153.2 mg·L-1)。采用Salkowski比色法定量测定部分菌株分泌IAA能力,所测试的9株固氮菌株和9株溶磷菌株所分泌的IAA浓度在11.40~14.10μg·mL-1之间。3.根据固氮酶活性、分泌IAA能力等初筛出15株固氮菌,用于固氮菌盆栽单接种试验。结果表明,接种固氮菌可显著促进秋茄苗和木榄苗的生长和氮磷吸收。综合比较各项生长指标和氮磷水平,NGWB3-w14、NGWB4-wy1、NLQQ2-w14、NGHHL2-r14、NLQQ2-r14、NGWB2-y1和NLQQ3-wy4等7株固氮菌株促生效果较好。接种这7株固氮菌后,秋茄苗高、地下生物量、地上生物量比对照组增长30.09%~65.19%, 61.64%~79.13%和55.97%~75.21%,木榄的苗高、地下生物量、地上生物量增长11.98%~23.81%,37.93%~51.90%和38.00%~63.78%。4.根据溶磷能力、分泌IAA能力等初筛出溶磷菌株16株,用于溶磷菌盆栽单接种试验。结果表明,接种溶磷菌能显著促进秋茄和木榄苗的生长和氮磷吸收。PGWB-wp5、PGHHL-y7、PGWB-y8、PLQQ-y12、PGWB-y2、PGML-y1和PGWB-y6等7株溶磷菌株促生效果较好;接种后,秋茄苗高、地下生物量、地上生物量比对照组增长31.92% ~45.61%,49.28%~70.56%和45.80%~67.71%,木榄的对应值分别增长20.17%~ 26.79%,34.87%~51.20%和55.92%~67.53%。5.通过室外盆栽接种试验,研究了PGPR接种对白骨壤Avicennia germina幼苗抗寒性的影响。结果表明PGPR的接种在促进白骨壤苗生长的同时增强了其抗寒能力,表现在对照组死亡率为60%,而各接菌处理组幼苗的死亡率为10% ~ 50%。6.采用固氮菌和溶磷菌交互组合方法,进行红树植物苗木的盆栽混合接种试验。结果表明,混合接种可显著促进秋茄苗和木榄苗的生长和氮磷吸收。综合比较各项生长指标和氮磷水平,以组15(NGHHL2-r14+ PGWB-y8+ NGWB3-w14+ PGML-y1)、组6(NGWB2-y1+ PGHHL-y7)和组9(NLQQ2-w14+ PGWB-wp5)的促生效果最佳。接种组15、组6和组9后,秋茄苗高、地径、根干重、总干重和叶面积分别比对照组增长35.13%~69.93%,10.03% ~13.27%,85.90%~99.33%,116.8%~131.8%和93.66% ~105.2%,木榄的苗高、地径、根干重、总干重和叶面积分别增长22.60%~24.38%,9.85%~10.92%,101.0%~106.6%,97.17% ~116.6%和74.53% ~85.47%。7.苗圃接种试验表明,混合接种可明显促进秋茄苗和木榄苗的生长。经方差分析及多重比较,混合接种处理组秋茄和木榄苗的各生长指标与对照比差异极显著。促生效果最佳的是组15(NGHHL2-r14+ PGWB-y8+ NGWB3-w14+ PGML-y1),秋茄苗高、根干重和总干重分别比对照组增长25.76%、42.22%和47.70%,木榄的分别增长15.15%、35.50%和42.20%。8.采用锐孔-凝固浴法,对PGPR微胶囊菌剂的研制进行了探讨。加工制造出微胶囊菌剂的专用生产设备,并形成较完整的PGPR微胶囊菌剂生产工艺流程。采用单因子试验确定微胶囊制作的适宜条件,即海藻酸钠质量分数为2.5%,CaC12质量分数为2.0%,菌体与海藻酸钠的体积比为1: 2,固化时间为1 h。经干燥处理后的PGPR微胶囊菌剂其有效期至少6个月,可在秋茄苗根际定殖并缓慢释放菌体,经盆栽接种试验表明其接种效果优于液体菌剂。更多还原

【Abstract】 Mangrove has important ecological, economic and social benefits. Because of long-term human damage, the mangrove area reduced, stand quality decreased, and protective effects declined. Study on PGPR of mangrove afforestation species is helpful to utilize mangrove microbial resources, and develop effective bacteria inoculants based on excellent strains. Inoculation of PGPR promotes the growth of mangrove plants and enhances their resistance. This utilization of inoculated seedlings should increase the survival rate of mangrove reforestation and promote the construction of coastal shelter forest system. The present study tries to screen and identify PGPR strains from the rhizosphere of mangrove plants, evaluate their inoculation effects, develop PGPR microbeads inoculants by using advanced experimental techniques and analysis methods, such as conventional bacteria selection and identification methods, acetylene reduction method, molybdenum-antimony anti-colorimetry, Salkowski colourimetry, Biolog automated microbial identification system, 16s rDNA sequence analysis. The results obtained were as follows:1. There were abundant PGPR strains colonized on rhizosphere of mangrove afforestation species. Plant samples were collected for PGPR screen from Zhanjiang, Zhu hai and Dongzhai Harbor. Total 20 nitrogen fixing strains and 35 phosphorus solubilizing strains were isolated from 6 mangrove species, including Bruguiera gymnorrhiza,Kandelia candel,Sonneratia apetala,Rhizophora stylosa,Bruguiera sexangula and Sonneratia caseolaris. According to identification, seven excellent nitrogen fixing strains belonged to Azotobacter (1 strain),Pseudomonas (1strain),Bacillus(4 strains),Ochrobactrum(1 strain). And seven excellent phosphorus solubilizing strains belonged to Pseudomonas (1 strain) and Bacillus (6 strains).2. Using modified OAB medium, 20 nitrogen fixing strains were screened from the the rhizosphere of mangrove plants. Most of these strains showed positive nitrogenase activity ranged from 50.72 to 385.6 nmol C2H4·h-1·mL-1. Only two strains of NGWB4-wy1 and NGHHL2-r14 had their C2H4 production higher than 200 nmol C2H4·h-1mL-1. A batch of (35 strains) phosphorus solubilizing strains with different phosphorus solubilizing abilities, were isolated by using modified SRMS1 medium. There were three strains with marked phosphorus solubilizing intensities, including PGHHL-y7 180.2 mg·L-1 ) , PGWB-y2(158.9 mg·L-1), PGWB-wp5(153.2 mg·L-1). Results of Salkowski colourimetry showed that all tested strains ( i.e. 9 nitrogen fixing strains and 9 phosphorus solubilizing strains ) could secrete IAA, which ranged from 11.40 to 14.10μg·mL-1.3. On the basis of nitrogenase activity and IAA secretion, 15 strains of nitrogen fixing bacteria were selected for single-inoculation pot test. The results showed that inoculation of nitrogen fixing strains significantly increased the height, biomass, nitrogen and phosphorus levels of K. candel and B. gymnorrhiza seedlings. According to different performances on growth indicators, nitrogen and phosphorus levels, 7 strains were screened with marked effects on growth promotion, including NGWB3-w14, NGWB4-wy1, NLQQ2-w14, NGHHL2-r14, NLQQ2-r14, NGWB2-y1, NLQQ3-wy4. As to the K. candel seedlings, height increased 30.09%~65.19%, below-ground biomass increased 61.64%~79.13%, above-ground biomass increased 55.97%~75.21% compared to the control. As to the B. gymnorrhiza seedlings, the corresponding values were 11.98%~23.81%, 37.93%~51.90%, 38.00%~63.78% respectively.4. On the basis of phosphorus solubilizing activity and IAA secretion, 16 strains of phosphorus solubilizing bacteria were selected for single-inoculation pot test. The results showed that inoculation of phosphorus solubilizing strains significantly increased the seedling growth, nitrogen and phosphorus levels of K. candel and B. gymnorrhiza. According to different performances on growth indicators, nitrogen and phosphorus levels, 7 strains were screened with marked effects on plant growth promotion, including PGWB-wp5, PGHHL-y7, PGWB-y8, PLQQ-y12, PGWB-y2, PGML-y1, PGWB-y6. As to the K. candel seedlings, the height increased 31.92%~45.61%, below-ground biomass increased 49.28%~70.56%, above-ground biomass increased 45.80%~67.71% compared to the control. As to the B. gymnorrhiza seedlings, the corresponding values were 20.17%~26.79%, 34.87%~51.20%, 55.92%~67.53%.5. Inoculation effects of PGPR strains on cold resistance of Avicennia germina seedlings were studied through the outdoor pot experiment. Inoculation not only promoted seedling growth but also enhanced the cold resistance of A. germina. The mortality rate of the control group was 60%, while those treatment groups 10% ~ 50%.6. Using the interactive combinations of nitrogen-fixing strain and phosphorus solubilizing strain, mixed inoculation pot experiments were conducted in greenhouse. Results showed that mixed inoculation could promote the growth of K. candel and B. gymnorrhiza seedlings significantly. According to different performances on growth indicators, nitrogen and phosphorus levels, there were 3 strain combinations having marked growth promoting effects, i.e.Group15 ( NGHHL2-r14+ PGWB-y8+ NGWB3-w14+ PGML-y1),Group 6(NGWB2-y1+ PGHHL-y7),Group 9(NLQQ2-w14+ PGWB-wp5. As to the K. candel seedlings, height increased 30.09%~65.19%, basal diameter increased 10.03%~13.27%, below-ground biomass increased 85.90%~99.33%, total biomass increased 116.8%~131.8%, leaf area increased 93.66%~105.2% compared to the control. As to the B. gymnorrhiza seedlings, the corresponding values were 22.60%~24.38%, 9.85%~10.92%, 101.0%~106.6%, 97.17%~116.6%, 74.53%~85.47%.. 7. Mixed inoculation experiments were carried out in mangrove nursery. Results showed that mixed inoculation promoted the seedling growth of K. candel and B. gymnorrhiza significantly. By analysis of variance and multiple comparisons, the growth indicators, nitrogen and phosphorus levels of these inoculated seedlings had significant differences compared to the control. The growth-promoting effect was the best when treated with Group15. As to the K. candel seedlings, height increased 25.76%, below-ground biomass increased 42.22%, total biomass increased 47.70% compared to the control. As to the B. gymnorrhiza seedlings, the corresponding values were 15.15%, 35.50%, 42.20%. 8. Microbead inoculants were explored according to the piercing-solidifying method. A special equipment for the production of microbeads was processed and manufactured, and a complete technological process was formed. Several single-factor tests were conducted to determine suitable conditions for microbead production. The results were as follows, 2.5% (mass fraction) sodium alginate, 2.0% (mass fraction) CaC12, with the volume ratio of bacterium solution and Sodium Alginate at 1:2, with the curing time of 1h. The validity of PGPR microbead inoculants reached at least 6 months after drying process. Microbead inoculants could colonize in the rhizosphere of K.candel, and release bacteria slowly. According to the pot experiments, effects of microbead inoculants on the plant growth promoting were better than those of liquid inoculants.更多还原