Estimation of nodulation tendency among Rj-genotype soybeans using the bradyrhizobial community isolated from an Andosol

The nodulation tendency of indigenous soybean bradyrhizobia on Rj -genotype soybean cultivars was investigated using approximately 260 bradyrhizobia isolated from an Andosol with 13 soybean cultivars of five Rj -genotypes (non- Rj , Rj ₂ Rj ₃, Rj ₃, Rj ₄ and Rj ₂ Rj ₃ Rj ₄). A dendrogram was constructed based on restriction fragment length polymorphism analysis of the polymerase chain reaction products (PCR-RFLP) of the 16S–23S rDNA internal transcribed spacer (ITS) region. Bradyrhizobium USDA strains were used as a reference. The dendrogram indicated nine clusters based on similarities among the reference strains. The ratio of beta diversity to gamma diversity ( H' _β/ H' _γ), which represents differences in the bradyrhizobial communities by pair-wise comparison between each cultivar, was obtained from Shannon–Wiener diversity indices. The results showed that bradyrhizobial communities among the same Rj -genotype cultivars were similar to each other, whereas bradyrhizobial communities between the Rj ₂-genotype and non- Rj , Rj ₃ or Rj ₄-genotype cultivars were significantly different. These results suggest that the Rj ₂-gene might not only affect the nodulation compatibility between Rj -genotype soybeans and bradyrhizobia, but also the nodulation tendency of the bradyrhizobia.     

Diversity and geographical distribution of indigenous soybean-nodulating bradyrhizobia in Japan

Abstract

Genetic diversity and distribution of indigenous soybean-nodulating bradyrhizobia in Japan were investigated based on restriction fragment length polymorphism analysis of PCR product (PCR-RFLP) analysis of the 16S?23S rDNA internal transcribed spacer (ITS) region using Bradyrhizobium USDA strains as reference strains. Soil samples were collected from five field sites in Hokkaido, Fukushima, Kyoto, Miyazaki and Okinawa in Japan. A total of 300 isolates were derived from three Rj-genotype soybean cultivars, Akishirome (non-Rj), CNS (Rj ₂ Rj ₃) and Fukuyutaka (Rj ₄), and five field site combinations. The PCR products of the ITS region were digested with HaeIII, HhaI, MspI and XspI. Electrophoresed patterns were analyzed for phylogenetic relationship using Bradyrhizobium reference strains. Results revealed 22 RFLP patterns and 11 clusters. The RFLP patterns of the seven clusters were similar or identical to Bradyrhizobium japonicum USDA 6, 38, 110, 115, 123 and Bradyrhizobium elkanii USDA 76 and 94. Four minor clusters were independent from the clusters of the reference strains. The isolation ratio revealed the major clusters at each field site. These results suggested that major clusters of indigenous bradyrhizobia might be in the order Bj123, Bj38, Bj110, Bj6 and Be76 from the northern to southern regions in Japan.     

Serological properties and intrinsic antibiotic resistance of soybean bradyrhizobia isolated from Thailand

A group of Bradyrhizobium strains isolated from soybean plants in Thailand did not correspond to any known DNA homology groups of Bradyrhizobium japonicum and Bradyrhizobium elkanii reported by Hollis et al. (J. Gen. Microbiol., 123, 215–222, 1981). To clarify the phenotypic characteristics of the group, serological properties and intrinsic antibiotic resistance (IAR) profile of 94 Thai strains were compared with those of USDA and Japanese strains. Indirect ELISA tests for each Thai strain were performed agaiIl.st polyclonal antisera prepared against 15 USDA standard serotype strains of B. japonicum and B. elkanii. Among the 94 Thai strains tested, 36 which were previously identified as B. elkanii, with the exception of one strain, were strongly responsive to an antiserum prepared against USDA 31. The remaining 58 strains, with the exception of two strains, showed multiple cross reactions which were peculiar to the Thai strains. These serological reaction patterns did not correspond to any known serogroups labeled as B. japonicum and B. elkanii. In the IAR test, the taxonomically unknown Thai soybean bradyrhizobia exhibited a high level of resistance to neomycin (50 µg/mL), polymyxin (50 µg/mL), nalidixic acid (15 µg/mL), and kanamycin (15 µg/mL). Kanamycin could thus be useful in combination with neomycine and nalidixic acid for distinguishing between the unknown Thai strains and strains of B. japonicum and B. elkanii. Our results demonstrated that the unknown Thai strains were serologically and IAR-phenotypically remote from both B. japonicum and B. elkanii.     

Multiple occupancy of nodules by nodulating rhizobia on field-grown soybeans with attendance of Sinorhizobium spp.

Abstract

This study analyzed the phenotypic and genotypic characters of nodulating rhizobia isolated from two soybean cultivars, Kyushu 151 and Sachiyutaka, in the same field of the Yamaguchi Prefectural Technology Center of Agriculture and Forestry in Japan. The isolates were classified into groups using phenotypic characteristics, such as growth rate, color change on Bromothymol blue-containing yeast extract-mannitol agar (YMA) plates and colony morphology on YMA plates, and by genotypic characteristics, such as polymerase chain reaction–restriction fragment length polymorphism patterns of the 16S ribosomal RNA genes (16S rDNA) and the internal transcribed sequence (ITS) regions. In Kyushu 151, single phenotypic and genotypic groups were isolated from every nodule examined. In Sachiyutaka, plural strains belonging to distinct groups were obtained frequently from single nodules, indicating that multiple occupancy was established at high frequency. No fixed combination of the groups was found in the composition of multiple occupancy. An increase in the relative abundance of isolates belonging to Sinorhizobium fredii (Ensifer fredii) occurred concomitantly with the increase in the proportion of nodules with multiple occupancy. Nearly 60% of the isolates from Sachiyutaka belonged to S. fredii; 75% of them were obtained from nodules with multiple occupancy.     

半干旱地区湿地-干草原生态系统植物群落多样性分析

利用极点排序法对宁夏盐池县四儿滩湿地交错带的边界进行确定,并在此基础上研究其植物的生物多样性.结果表明：1）沿湿生带-交错带-旱生带的生境梯度,交错带划分结果为北样线交错带样方为N8 ～ N13,南样线为S15～ S24,东样线为E13 ～ E22,西样线为W23～W30.2）4条样线多样性指数与丰富度指数均表现为交错带＞旱生带＞湿生带,均匀度指数均表现为旱生带＞交错带＞湿生带,优势集中性指数均表现为湿生带＞交错带＞旱生带.3）受人为干扰和地形差异的影响,不同方向植物群落差异较大,北样线和东样线丰富度指数、多样性指数、均匀性指数较高,优势集中性指数较低.     

三种不同种植模式对土壤细菌群落多样性的影响

采用变性梯度凝胶电泳(DGGE )技术,研究了不同种植模式对土壤细菌群落多样性的影响。结果表明,不同种植模式下DGGE图谱条带的数量及亮度有较大区别,且有几条特征性条带发生了明显的变化。0—15 cm土层细菌群落的丰富度、多样性指数及均匀度指数均表现为:“菜稻菜模式”（RVCs） “休闲轮作模式”（FRCs） “蔬菜连作模式”（VCCs）;15—30 cm土层细菌群落的丰富度、多样性指数及均匀度指数均表现为:FRCs RVCs VCCs。UPGMC聚类分析表明,RVCs 处理的细菌群落结构相似性较低。主成分分析表明,对各主成分起分异作用的主要是 RVCs和FRCs;与主成分因子1 正相关程度较高的种植模式为:RVCs和VCCs,与主成分因子2 正相关程度较高的是 FRCs。可见,水稻土细菌群落多样性与种植模式密切相关,这些变化对土壤细菌群落有重要的调节作用。     

Evaluation of effective Myanmar Bradyrhizobium strains isolated from Myanmar soybean and effects of coinoculation with Streptomyces griseoflavus P4 for nitrogen fixation

In order to substitute the use of chemical fertilizers in legume production, there is a need for the production of rhizobial inoculants which are capable of being used as biofertilizers. To achieve this, an effective symbiotic nitrogen (N) fixation between legumes and root nodule bacteria will be essential. Evaluation of effective Myanmar Bradyrhizobium (Jordan 1982) strains isolated from Myanmar soybean (Glycine max L. Merr.) and effects of coinoculation with Streptomyces griseoflavus Krainsky 1914 P4 for N fixation were studied in pot experiments using sterilized vermiculite and Hoagland solution in the Phytotron (25°C and 70% relative humidity) with completely randomized design and three replicates. N fixation ability of soybean was evaluated by acetylene reduction activity (ARA) by gas chromatography. It was found that MAS23 showed a relatively high degree of stability and a high level of ARA per plant on both Yezin-3 and Yezin-6 soybean varieties. In the symbiotic relationship between Bradyrhizobium strains and P4 experiments, the treatments consisted of six Bradyrhizobium strains (MAS23, MAS33, MAS34, MAS43, MAS48 and USDA110) and Streptomyces griseoflavus P4 were evaluated with four Myanmar soybean varieties (Yezin-3, Yezin-6, Hinthada and Shan Sein). In the Yezin-3 soybean variety, the best treatment for ARA per plant was found in the dual inoculation of P4 and MAS34. In the Yezin-6 soybean variety, the highest nodule dry weight was found in dual inoculation of P4 with MAS34 but the highest ARA per plant was observed in the dual inoculation of P4 and MAS23. On the other hand, single inoculation of MAS43 and coinoculation of P4 with MAS48 were significantly higher in N fixation of Hinthada, and coinoculation of P4 with MAS33 was significant improvement of ARA per plant (P < 0.05) in Shan Sein soybean.     

毒死蜱对棉花根际土细菌群落多样性和结构的影响

通过室内盆栽试验模拟自然环境条件,采用高效液相色谱(HPLC)和末端限制性片段长度多态性(T-RFLP)技术,研究了土壤使用推荐剂量(5 mg·kg~(-1))及推荐剂量的2倍、3倍和4倍(10 mg·kg~(-1)、15 mg·kg~(-1)、20 mg·kg~(-1))毒死蜱对棉花根际土壤细菌群落多样性和结构的影响,以不施用毒死蜱的土壤为对照。结果表明,5 mg·kg~(-1)、10 mg·kg~(-1)、15 mg·kg~(-1)和20 mg·kg~(-1)毒死蜱在土壤中的半衰期分别为10.04 d、11.36 d、11.55 d和12.16 d,60 d时基本完全降解。毒死蜱处理60 d后,棉花生物量显著降低;毒死蜱浓度越高,棉花生物量越低。无毒死蜱条件下不同取样时间根际细菌多样性无显著差异,毒死蜱处理组前30 d细菌多样性均显著降低,60 d时毒死蜱处理组细菌多样性恢复到正常水平。研究发现毒死蜱浓度越高对细菌多样性抑制作用越显著,恢复越缓慢。主成分分析结果发现,第10 d、30 d和60 d毒死蜱处理组与对照组细菌群落结构差异显著,其中60 d时20 mg·kg~(-1)毒死蜱处理组差异最显著,即使土壤中毒死蜱完全降解,根际细菌群落结构仍不会恢复到正常水平。60 d时,被毒死蜱抑制的细菌有硝化刺菌属(Nitrospina sp.)和Cellulophaga sp.等,被激活的有芽孢杆菌属(Bacillus sp.)和链霉菌属(Streptomyces sp.)等。可见,毒死蜱的引入,重新构建了土壤细菌群落结构,显著影响棉花生长,对棉花根际土壤微生态环境冲击较大,应对其生态安全性予以重视。     

引种植物江南桤木改变了土著芦苇根际细菌群落的结构和多样性

Alnus trabeculosa, a rhizobia-nodulating tree, was introduced into the Chongxi tidal wetland in the Yangtze River estuary of China to increase the biodiversity of plants and restore tidal wetland functions. However, the effect of the introduced plant on soil bacterial communities and restoration outcomes remains unknown. In this study, the rhizosphere bacterial community structure and diversity were compared between Phragmites australis monospecific community and A. trabeculosa-P. australis mixed communities,aiming to assess whether A. trabeculosa influenced the rhizosphere bacterial communities of P. australis and to investigate whether different taxonomic groups within a soil community may respond similarly to the presence of an introduced exotic plant. Among the14 phylogenetic phyla detected, Proteobacteria and Acidobacteria were the dominant bacterial taxa in the rhizosphere. Phylogenetic analysis of the predominant Proteobacteria showed that the clones from the rhizosphere soils of A. trabeculosa and P. australis in A. trabeculosa-P. australis mixed communities were more diverse than those in the rhizosphere soil of P. australis in P. australis monospecific community. The rhizosphere community in the wetland potentially included active microbial community related to carbon, nitrogen, and sulfur cycling in the Yangtze River estuary. The rhizosphere soil of P. australis in A. trabeculosa-P. australis mixed communities exhibited the highest Shannon diversity index(H′) and Simpson diversity index(1/D)(H = 4.52, 1/D =253). Correspondence analyses revealed that the bacterial community structures were altered after A. trabeculosa was introduced.     

玉米秸秆生物炭对褐土微生物功能多样性及 细菌群落的影响

生物炭施入土壤被认为是一种有效的固碳减排措施,可增加土壤有机碳及矿质养分含量,提高土壤的持水能力及保肥能力。为探明其施入土壤后对土壤微生物活性及多样性的影响,本文在盆栽试验条件下,采用Biolog与高通量测序相结合的方法,研究了CK(不施生物炭)和施用5 g·kg~(-1)、10 g·kg~(-1)、30 g·kg~(-1)、60 g·kg~(-1)玉米秸秆生物炭对土壤微生物碳源利用能力(AWCD)、功能多样性指数以及土壤细菌的丰度和多样性的影响。结果表明,随着生物炭施用量的增加,表征土壤微生物活性的AWCD值呈下降趋势,表现为:5 g·kg~(-1)处理≈CK10 g·kg~(-1)处理30 g·kg~(-1)处理60 g·kg~(-1)处理,其中CK和5 g·kg~(-1)处理间差异不显著(P0.05),而10 g·kg~(-1)、30 g·kg~(-1)和60 g·kg~(-1)处理在整个培养期间的AWCD值显著低于CK处理(P0.05);土壤微生物群落代谢功能多样性指数(H′)、碳源利用丰富度指数(S)均随生物炭施用量的增加而呈下降趋势,但均匀度指数(E)表现出相反趋势,5g·kg~(-1)、10 g·kg~(-1)、30 g·kg~(-1)、60 g·kg~(-1)各处理的H′较CK处理分别增加0.16%、-0.88%、-3.14%、-11.09%,S分别增加-2.82%、-11.27%、-18.31%、-47.89%,E分别增加1.14%、3.00%、3.73%和13.76%。主成分分析表明,与CK处理比较,5 g·kg~(-1)处理对土壤微生物群落碳源利用方式没有显著影响(P0.05),而10 g·kg~(-1)、30 g·kg~(-1)和60g·kg~(-1)处理对土壤微生物群落碳源利用方式影响显著(P0.05)。随着生物炭施用量的增加,土壤细菌OTU数目及丰富度指数(Chao1)呈增加趋势,5 g·kg~(-1)处理与CK处理差异不显著,而10 g·kg~(-1)、30 g·kg~(-1)、60 g·kg~(-1)处理的OTU数目较CK处理分别增加1.09%、5.26%、24.42%,Chao1分别增加5.73%、10.21%、37.68%。土壤中施用生物炭后土壤细菌变形菌门(Proteobacteria)的丰度在CK处理和5 g·kg~(-1)处理间差异不显著(P0.05),而10g·kg~(-1)、30 g·kg~(-1)、60 g·kg~(-1)处理较CK处理分别增加32.3%、21.1%、16.7%,拟杆菌门(Bacteroidetes)的丰度随着生物炭施用量的增加各处理较CK处理分别减少22.1%、55.3%、66.8%、50.5%。生物炭施入土壤后降低了土壤可培养微生物的活性,减少或改变了土壤微生物碳源利用的种类,使土壤原有微生物群落组分发生改变,生物炭也影响了土壤细菌各菌群在土壤中的丰度,使其分布的均匀性降低。为了不影响微生物群落结构和功能,石灰性褐土上生物炭一次还田量不能超过5 g·kg~(-1)(干土)。     

菌肥对青稞根际土壤理化性质以及微生物群落的影响

应用化学分析、聚合酶链反应-变性梯度凝胶电泳(PCR-DGGE)技术和DNA测序技术,研究了西藏棕色砂壤土中微生物肥料不同施用量和施用期对青稞根际土壤理化性质和细菌群落多样性的影响。结果表明,施用谷特菌肥能显著提高土壤全氮、全磷、全钾、有机质、碱解氮、有效磷和速效钾的水平,如播前施用菌肥浓度750 ml hm-2的处理较不施用菌肥的处理上述指标分别提高13.32%、28.42%、16.20%、9.81%、21.36%、39.35%和30.48%,拔节期施用菌肥浓度2 250 ml hm-2的处理较不施用菌肥的处理分别提高7.25%、29.35%、18.04%、12.86%、15.90%、43.27%和53.99%。DGGE分析表明,相同施用方式中不同施用量土样中微生物的DGGE图谱相似。非加权组平均法(UPGMA)聚类分析将DGGE图谱分为2大类群。Shannon-Wiener指数表明,施用菌肥的土壤细菌多样性先增加后逐渐降低,播前以喷施谷特菌浓度750 ml hm-2时的细菌多样性最高;拔节期则以喷施谷特菌浓度2 250 ml hm-2处理的细菌多样性最高,且两种施用方式土壤养分的释放与Shannon指数的变化规律均为播前﹥拔节期。测序结果表明,不同施肥浓度土样微生物种群分布较为广泛,其中Actinobacteria纲细菌种类略多,少数菌种为未经培养菌种(Uncultured bacterium)。典型对应分析(CCA)表明,DGGE图谱条带分布与土壤理化性质密切相关,碱解氮、全磷和全氮是影响微生物群落的主要环境因子。研究结果表明,施用谷特菌肥可明显改善青稞根际土壤理化性状,提高土壤细菌多样性。     

大豆施用肥料增效剂的应用效果

以春大豆为试验材料，研究利用沸石、腐殖酸钠等为主要无机、有机原料配方所研制的肥料增效剂的效果。结果表明：与不施肥对照相比，施用所研制的肥料增效剂能极显著提高大豆产量。在施用尿素、磷酸二铵、过磷酸钙300kg／hm^2商品最3种条件下，采用25％、50％与75％等量比例肥料增效剂与75％、50％和25％等量比例尿素、磷酸二铵、过磷酸钙结合各处理的大豆产量，以75％等量比例增效剂与25％等量比例各肥料结合处理增产效果最好，分别比纯施尿素、磷酸二铵、过磷酸钙增产20．3％、5．7％和1．2％，表现出良好的节肥与增加肥效功能。     

Genetic diversity of fast-and slow-growing soybean rhizobia determined by random amplified polymorphic DNA analysis

The genetic relationships among six strains of rhizobia, including three strains of Rhizobium fredii and three strains of Bradyrhizobium japonicum, was determined using random amplified polymorphic DNA (RAPD) technique. In this study, 46 arbitrary 10mer primers were employed for RAPD, generating a total of 251 informative fragments. A dendrogram of phylogenetic relationships among the six strains was constructed. The results indicated that geographical distribution may affect phylogeny, as there were closer relationships among the four Taiwanese strains, SB138, SB562, SB368 and SB651, than between these strains and USDA192, which originated from mainland China. The strain USDA110, obtained from the United States, was used in the parsimony analysis. The greatest similarity (55.6%), existed between two strains of B. japonicum, SB562 and SB138, which both, and the lowest R. fredii (44.4%) between two strains of R. fredii, SB368 and USDA192. We also found a RAPD marker specific to the four Taiwanese SB strains used in the study. The RAPD technique is a potential tool for the identification of the genetics and systematics of different populations. Received: 23 January 1997     

Low pH and aluminum tolerance of Bradyrhizobium strains isolated from acid soils in Indonesia

Sixty-seven strains of Bradyrhizobium isolated from soybean plants growing on acid soils in West Java and Sumatra, Indonesia, were examined for the effect of the pH and aluminum concentration on their growth in nutrient media, compared with 61 strains of Bradyrhizobium from soils in Japan. The results in this study indicated that the indigenous population of Bradyrhizobium in the soils of Indonesia showed a large difference in acid- and Al-tolerance from that of Japan. Eighty-five and 48% of the isolates from Japanese soils and Indonesian soils, respectively, were unable to grow in YEM broth at pH below 4.5. The acid-tolerance was correlated with AI-tolerance of the isolates on YEM agar plates at pH 4.4. Seventy-five percent of the isolates that grew in YEM broth at pH 4.5 were also resistant to 400 µM Al on the YEM plates. Acetylene reduction assay of the root nodules revealed that 3 of the acid- and Al-tolerant isolates from Indonesian soils showed a significantly high nitrogen fixation activity.     

Long-term fertilization regimes affect bacterial community structure and diversity of an agricultural soil in northern China

Background, Aims, and Scope  Knowledge about shifts of microbial community structure and diversity following different agricultural management practices could improve our understanding of soil processes and thus help us to develop sound management strategies. A long-term fertilization experiment was established in 1989 at Fengqiu (35°00′N, 114°24′E) in northern China. The soil (sandy loam) is classified as aquic inceptisols and has received continuous fertilization treatments since then. The fertilization treatments included control (CK, no fertilizer), chemical fertilizers nitrogen (N) and potassium (K) (NK), phosphorous (P) and K (PK), NP, NPK, organic manure (OM), and half chemical fertilizers NPK plus half organic manure (1/2NPKOM). The objective of this study was to examine if the microbial community structure and diversity were affected by the long-term fertilization regimes. Materials and Methods  Soil samples were collected from the long-term experimental plots with seven treatments and four replications in April 2006. Microbial DNAs were extracted from the soil samples and the 16S rRNA genes were PCR amplified. The PCR products were analyzed by DGGE, cloning and sequencing. The bacterial community structures and diversity were assessed using the DGGE profiles and the clone libraries constructed from the excised DGGE bands. Results  The bacterial community structure of the OM and PK treatments were significantly different from those of all other treatments. The bacterial community structures of the four Ncontaining treatments (NK, NP, NPK and 1/2NPKOM), as well as CK, were more similar to each other. The changes in bacterial community structures of the OM and PK treatments showed higher richness and diversity. Phylogenetic analyses indicated that Proteobacteria (30.5%) was the dominant taxonomic group of the soil, followed by Acidobacteria (15.3%), Gemmatimonadetes (12.7%), etc. Discussion  Irrespective of the two fertilization treatments of OM and PK, the cluster analysis showed that bacterial communities of the remaining five treatments of CK, NK, NP, NPK and 1/2NPKOM seemed to be more similar to each other, which indicated the relatively weak effects of the four N-containing treatments on soil bacterial communities. N fertilizer may be considered as a key factor to counteract the effects of other fertilizers on microbial communities. Conclusions  Our results show that long-term fertilization regimes can affect bacterial community structure and diversity of the agricultural soil. The OM and PK treatments showed a trend towards distinct community structures, higher richness and diversity when compared to the other treatments. Contrasting to the positive effects of OM and PK treatments on the bacterial communities, N fertilizer could be considered as a key factor in the soil to counteract the effects of other fertilizers on soil microbial communities. Recommendations and Perspectives  Because of the extremely high abundance and diversity of microorganisms in soil and the high heterogeneity of the soil, it is necessary to further examine the effects of fertilization regimes on microbial community and diversity in different type soils for comprehensively understanding their effects through the appropriate combination of molecular approaches. ESS-Submission Editor: Chengrong Chen, PhD (c.chen@griffith.edu.au)     

银杏内生真菌刺盘孢遗传多样性的初步分析

为了研究银杏内生真菌的刺盘孢菌株的遗传分化,选用了分离自江苏5个县市的14个菌株,利用RAPD技术进行遗传多样性分析。从20个随机引物中筛选出5个引物,扩增出62条DNA带,在此基础上分析并建构了遗传相关聚类图。结果表明,不同地区的刺盘孢菌株其遗传差异性不同,14个菌株可聚为两大类,其中启东、邳州的6株聚为一类,南京、南通和泰兴的8株又聚为一类。所有的14个菌株的相似性系数范围在46%~89%。