Influence of organic fertilization on the number of culturable diazotrophic endophytic bacteria isolated from sugarcane

The numbers of culturable diazotrophic endophytic bacteria (CDEB) from roots, stems and leaves of sugarcane submitted to organic, inorganic or no fertilization were compared. In order to determine the size of the N₂ fixing populations, the Most Probable Number technique (MPN) was used. The quantification of diazotrophic bacteria by using the acetylene reduction assay (ARA) was more accurate than observing the bacterial growth in the vials; to confirm N₂ fixing capability, the detection of gene nifH was performed on a sample of 105 isolated bacteria. The production of extracellular enzymes involved in the penetration of the plants by the bacteria was also studied. The results showed that organic fertilization enhances the number of CDEB when compared with conventional fertilization used throughout the growing season. The maximum number of bacteria was detected in the roots. Roots and stems presented the greatest number of CDEB in the middle of the cropping season and in leaves numbers varied according to the treatment. Using two pairs of primers and two different methods, the nifH gene was found in 104 of the 105 tested isolates. Larger amounts of pectinase were released by isolates from sugarcane treated with conventional fertilizers (66%), whereas larger amounts of cellulase were released by strains isolated from sugarcane treated with organic fertilizers (80%).     

Genetic diversity among Elaeagnus compatible Frankia strains and sympatric-related nitrogen-fixing actinobacteria revealed by nifH sequence analysis

Elaeagnus compatible Frankia isolates from Tunisian soil have been previously clustered with Frankia, colonizing Elaeagnaceae and Rhamnaceae in two different phylogenetic subgroups, while strain BMG5.6 was described as a new lineage closely related to Frankia and Micromonospora genera. In this study we further assess the diversity of captured Frankia and the relationship with BMG5.6-like actinobacteria, by using nifH gene sequences. Using PCR-RFLP screening on DNA extracted from lobe nodules, additional microsymbionts sharing BMG5.6 features have been detected proving a widespread occurrence of these actinobacteria in Elaeagnus root nodules. Neighbour-Joining trees of Frankia nifH sequences were consistent with previously published 16S rRNA and GlnII phylogenetic trees. Although four main clades could be discerned, actinobacterial strain BMG5.6 was clustered with Frankia strains isolated from Elaeagnus. The present study underscored the emanation of new diazotrophic taxon isolated from actinorhizal nodules occupying intermediate taxonomic position between Frankia and Micromonospora. Moreover, its aberrant position in nifH phylogeny should open network investigations on the natural history of nitrogen-fixing gene among actinobacteria.     

Diversity of 16S-rRNA and nifH genes derived from rhizosphere soil and roots of an endemic drought tolerant grass,Lasiurus sindicus

Lasiurus sindicus is a highly nutritive, drought tolerant, perennial grass, endemic to the Thar Desert of Rajasthan, India. In order to characterize the diversity of bacteria associated with roots of this grass that had survived severe drought stress, 16S-rRNA gene clone libraries were established from RT-PCR amplified products of the total RNA extracted from the washed roots and rhizosphere soil samples. Eight major bacterial taxa were identified in a total of 121 16S-rRNA gene clones. The majority of sequences belonged to Gram-positive bacteria, Actinobacteria being the most predominant ones, closely followed by Firmicutes. Most of the sequences showed similarity with sequences from cultivated bacteria or uncultivated environmental clones associated with arid, semi-arid environments, cold deserts and contaminated soils. PCR amplification of nifH genes using total DNA as template produced a total of 48 nifH clones from the rhizosphere soil and root samples and revealed a predominance of nifH sequences closely affiliated to Pseudomonas pseudoalcaligenes, isolated in a previous study from root samples of Lasiurus sindicus. Some nifH sequences showed close similarity to cultivated diazotrophs like Azospirillum brasilense, Rhizobium sp., and a variety of uncultured nitrogen fixing bacteria. Thus, this study provides us with evidence that L. sindicus harbors a diversity of bacteria with potential for nitrogen fixation.     

Long-term agronomic practices alter the composition of asymbiotic diazotrophic bacterial community and their nitrogen fixation genes in an acidic red soil

The asymbiotic diazotrophic bacteria are important for nitrogen (N) input to soil. Here, we investigated asymbiotic diazotrophic bacteria in an acidic red soil from functional, phylogenetic, and ecological perspectives. We firstly confirmed that phosphorus (P) availability determines the overall asymbiotic N fixation potential in the red soil. Then, we analyzed the soil bacterial community and N fixing (nifH) gene composition. Long-term different fertilizations significantly affected the composition of soil bacterial community. In addition, long-term organic cultivations increased most of the asymbiotic diazotrophic bacteria and the corresponding nifH gene abundances. Few asymbiotic diazotrophic bacteria, belonging to Chloroflexaceae, Methylocystaceae, Enterobacteriaceae, and Pseudomonadaceae, and their corresponding nifH genes were more abundant in N and P co-limited than in not co-limited soils, suggesting that some bacterial taxa from these families might be activated under nutrient limited conditions. Our findings provided new information for the distribution of asymbiotic diazotrophic bacteria in red soil and gave insights into the ecology of diazotrophic bacteria.     

Paddy System with a Hybrid Rice Enhances Cyanobacteria Nostoc and Increases N2 Fixation

Biological nitrogen(N) fixation(BNF) plays a significant role in maintaining soil fertility in paddy field ecosystems. Rice variety influences BNF, but how different rice varieties regulate BNF and associated diazotroph communities has not been quantified. Airtight,field-based ~(15)N_2-labelling growth chamber experiments were used to assess the BNF capacity of different rice varieties. In addition,both the 16 S rRNA and nifH genes were sequenced to assess the influence of different rice varieties on bacterial and diazotrophic communities in paddy soils. After subjecting a rice-soil system to 74 d of continuous airtight, field-based ~(15)N_2 labelling in pots in a growth chamber, the amounts of fixed N were 22.3 and 38.9 kg ha~(-1) in inbred japonica(W23) and hybrid indica(IIY) rice cultivars planted in the rice-soil systems, respectively, and only 1%–2.5% of the fixed N was allocated to the rice plants and weeds. A greater abundance of diazotrophs was found in the surface soil(0–1 cm) under IIY than under W23. Sequencing of the 16 S rRNA gene showed significantly greater abundances of the cyanobacterial genera Nostoc, Anabaena, and Cylindrospermum under IIY than under W23.Sequencing of the nifH gene also showed a significantly greater abundance of Nostoc under IIY than under W23. These results indicate that the hybrid rice cultivar(IIY) promoted BNF to a greater extent than the inbred rice cultivar(W23) and that the increase in BNF might have been due to the enhanced heterocystous cyanobacteria Nostoc.     

Isolation and Characterization of Cadmium- and Arsenic-Absorbing Bacteria for Bioremediation

The contamination of hazardous metal(loid) is one of the serious environmental and human health risks. This study isolated a total of 40 cadmium (Cd)- and arsenic (As)-resistant bacterial isolates from coastal sediments by pour plate technique using tryptic soy agar supplemented with Cd or As (50 mg l^?1) for use as metal(loid) bioremediation agents. Out of 40, 4 isolates, RCd3, RCd6, RAs7, and RAs10, showed a relatively higher growth rate in Cd- or As-supplemented culture media which were selected for further study. The selected isolates showed a high minimum inhibitory concentration (60–400 mg l^?1 for Cd and 400–2200 mg l^?1 for As), which demonstrated their remarkable Cd and As resistance capabilities. The metal(loid) removal efficiencies (0.032–0.268 μg Cd h^?1 mg^?1 and 0.0003–0.0172 μg As h^?1 mg^?1 [wet weight cell]) of selected isolates indicated their greater magnitude in absorbing Cd compared to As from water. Phylogenetic analysis of the 16S rDNA sequences revealed that isolates RCd3, RCd6, RAs7, and RAs10 were closely related to Acinetobacter brisouii, Pseudomonas abietaniphila, Exiguobacterium aestuarii, and Planococcus rifietoensis, respectively. Because of high Cd and As resistance and removal efficiency, the selected isolates can survive in a high metal(loid)-contaminated environment and could be a potential tool for bioremediation of high metal(loid)-contaminated effluents to protect the aquatic environment.     

Elevated CO<Subscript>2</Subscript> alters the abundance but not the structure of diazotrophic community in the rhizosphere of soybean grown in a Mollisol

The effect of elevated CO₂ (eCO₂) on rhizospheric diazotrophic community in cropland has little been studied, although eCO₂ facilitates nodulation and N₂ fixation in legumes. In this study, four soybean cultivars (Xiaohuangjin, Suinong 8, Suinong 14, and Heinong 45) were grown in Mollisols for 65 days under ambient CO₂ (aCO₂) (390 ppm) or eCO₂ (550 ppm). Quantitative PCR and Illumina MiSeq sequencing targeting the nifH gene that reflects the composition of diazotrophic community were determined. Elevated CO₂ significantly increased the abundance of nifH gene copies in the rhizospheres of the Suinong 8 and Heinong 45 cultivars, but not in the Suinong 14 and Xiaohuangjin cultivars. The nifH abundance correlated negatively with nodule density (p?≤?0.01) but positively with nodule size (p?≤?0.01). Elevated CO₂ did not significantly alter the composition of diazotrophic community, nor shift dominant bacterial operational taxonomic units (OTUs). These results indicated that eCO₂ stimulated the growth but did not alter the community composition of diazotrophs in the rhizosphere of soybean, which depended on cultivar and might contribute to nodulation responses to eCO₂.     

Straw amendment to paddy soil stimulates denitrification but biochar amendment promotes anaerobic ammonia oxidation

Purpose

Organic matter amendment is usually used to improve soil physicochemical properties and to sequester carbon for counteracting climate change. There is no doubt that such amendment will change microbial activity and soil nitrogen transformation processes. However, the effects of straw and biochar amendment on anammox and denitrification activity and on community structure in paddy soil are unclear.

Materials and methods

We conducted a 30-day pot experiment using rice straw and rice straw biochar to deepen our understanding about the activity, microbial abundance, and community structure associated with soil nitrogen cycling during rice growth.

Results and discussion

Regarding activity, anammox contributed 3.1–8.1% of N₂ production and denitrification contributed 91.9–96.9% of N₂ production; straw amendment resulted in the highest denitrification rate (38.9 nmol N g^?1 h^?1), while biochar amendment resulted in the highest anammox rate (1.60 nmol N g^?1 h^?1). Both straw and biochar amendments significantly increased the hzsB and nosZ gene abundance (p < 0.05). Straw amendment showed the highest nosZ gene abundance, while biochar amendment showed the highest hzsB gene abundance. Phylogenetic analysis of the anammox bacteria 16S rRNA genes indicated that Candidatus Brocadia and Kuenenia were the dominant genera detected in all treatments.

Conclusions

Straw and biochar amendments have different influences on anaerobic ammonia oxidation and denitrification within paddy soil. Our results suggested that the changes in denitrification and anammox rates in the biochar and straw treatments were mainly linked to functional gene abundance rather than microbial community structure and that denitrification played the more major role in N₂ production in paddy soil.

     

Anaerobic ammonium oxidation and denitrification in a paddy soil as affected by temperature,pH, organic carbon,and substrates

Anaerobic ammonium oxidation (anammox process) widely occurs in paddy soil and may substantially contribute to permanent N removal; however, little is known about the factors controlling this process. Here, effects of temperature, pH, organic C, and substrates on potential rate of anammox and the relative contribution of anammox to total N₂ production in a paddy soil were investigated via slurry incubation combined with ¹⁵N tracer technique. Anammox occurred over a temperature range from 5 to 35 °C with an optimum rate at 25 °C (1.7 nmol N g^?1 h^?1) and a pH range from 4.8 to 10.1 with an optimum rate at pH 7.3 (1.7 nmol N g^?1 h^?1). The presence of glucose and acetate (5–100 mg C L^?1) significantly inhibited anammox activities and the ratio of anammox to total N₂ production. The response of potential rates of anammox to ammonium concentrations fitted well with Michaelis-Menten relationship showing a maximum rate (V_max) of 4.4 nmol N g^?1 h^?1 and an affinity constant (K_m) of 6.3 mg NH₄⁺-N L^?1. Whereas, nitrate addition (5–15 mg ¹⁵NO₃^?-N L^?1) significantly inhibited anammox activities and the ratio of anammox to total N₂ production. Our results provide useful information on factors controlling anammox process and its contribution to N loss in the paddy soil.     

Isolation of an endophytic diazotroph,Klebsiella oxytoca,from sweet potato stems in Japan

Abstract

The population density of endophytic bacteria in the stem of field-grown sweet potato cultivars (Beniotome [BO], Koganesengan [KS], and Shiroyutaka [SYD in Miyakonojo, Miyazaki, Japan, ranged from 10² to 10⁴ cells g^?1 fresh weight sample using a semi-solid nitrogen-free medium. Eleven strains were isolated from the stems and two isolates, BO-1 and BO-5, showed a positive reaction in the acetylene reduction activity (ARA) test. BO-1 and BO-5 were isolated from cv. Beniotome in September 1999 and 2000, respectively. Morpho-physiological characterization of these isolates revealed that BO-1 and BO-5 showed a similar colony color in potato sucrose agar slants, produced bubbles in a modified semi-solid medium, acidified the medium, and displayed similar characteristics using the API 20NE rapid diagnostic kit. Partial sequence analysis of 16S rRNA from BO-1 revealed a 100% similarity (491 bp) to that of Klebsiella oxytoca. The other 9 isolates showed a negative reaction in the ARA test, slightly acidified or did not acidify the medium. Partial sequence analysis of the 16S rRNA revealed that the isolate SY-2 corresponded to Methylobacterium sp. (99.3% similarity for 1,241 bp), BO-3 to Pantoea agglomerans (99.1% similarity for 469 bp), and BO-8 to Sphingomonas sanguinis (98.8% similarity for 419 bp).     

Isolation,partial identification and application of diazotrophic rhizobacteria from traditional Indian rice cultivars

A diversity of N₂-fixing (diazotrophic) bacteria was isolated from two traditional rice cultivars, Sataria and Kartiki, from the rice growing area of Mithila region of North Bihar, India, where low levels of nitrogen fertilizers are applied. Nitrogen-free semisolid media NFb, JMV and LGI with different carbon sources and pH-values were used for enrichment and isolation of root-associated diazotrophs. The colonization density of roots by diazotrophs, as estimated from positive pellicle formation at highest dilution in nitrogen-free enrichment media, was 10⁶–10⁸ diazotrophic bacteria per g fresh root weight. Roots of the cultivar Kartiki were found to be more densely colonized endophytically by diazotrophs as detected after chloramine T (1%) surface disinfection. To ascertain the phylogenetic affiliation of the isolates, phylogenetic oligonucleotide probes and the Fluorescent in situ Hybridization (FISH) technique were applied. Using group-specific rRNA directed oligonucleotide probes, the majority of the isolates could be identified as alpha-, beta-, or gamma-proteobacteria. Using 16S and 23S rRNA-directed genus- or species-specific probes, Herbaspirillum seropedicae, Azospirillum amazonense, Burkholderia cepacia/vietnamiensis, Rhizobia and Pseudomonas spp. were found to be the most prominent root associated culturable diazotrophs. Diazotrophic Gluconacetobacter spp. were also demonstrated as colonizers of rice roots. Burkholderia cenocepacia, Pseudomonas sp. and three diazotrophic PGPR reference strains were used for the inoculation of axenically grown rice seedlings to determine the plant growth promoting potential. Significant increases in the shoot length (up to 60%), shoot dry weight (up to 33%) and the grain yield (up to 26%) per plant were observed in non-axenic pot and field trials. Using semisolid enrichment media after surface sterilization of field grown inoculated rice roots and oligonucleotide probing of the diazotrophic enrichment cultures, a sustainable colonization with the inoculated bacteria could be demonstrated.     

Geographic distance and amorphous iron affect the abundance and distribution of <Emphasis Type="Italic">Geobacteraceae</Emphasis> in paddy soils in China

Purpose

Geobacteraceae are important dissimilatory Fe (III)-reducing microorganisms, influencing the cycling of metals, nutrients as well as the degradation of organic contaminants. However, little is known about their distribution, diversity, and abundance of Geobacteraceae and the effects of environment factors and geographic distance on the distribution and diversity of Geobacteraceae in paddy soils remain unclear. Therefore, the objectives of this study were to investigate the distribution, diversity, and abundance of Geobacteraceae in paddy soils and to determine key factors in shaping the Geobacteraceae distribution, environmental factors, geographic distance, or both and to quantify their contribution to Geobacteraceae variation.

Materials and methods

Illumina sequencing and quantitative real-time PCR using a primer set targeting 16S rRNA genes of bacteria affiliated with the family Geobacteraceae were employed to measure the community composition, diversity, and abundance patterns of 16S rRNA genes of Geobacteraceae in 16 samples collected from north to south of China. MRT, Mantel test, and VPA were used to analyze the relationship between communities of Geobacteraceae and environmental factors and geographic distance.

Results and discussion

Quantitative PCR showed that the abundance of 16S rRNA genes of Geobacteraceae ranged from (1.20?±?0.18)?×?10⁸ to 1.13?×?10⁹?±?2.25?×?10⁸ copies per gram of soil (dry weight) across different types of soils. Illumina sequencing results showed Geobacter was the dominant genus within the family of Geobacteraceae. Multivariate regression tree (MRT) analysis showed that soil amorphous iron contributed more (22.46 %) to the variation of dominant species of Geobacteraceae than other examined soil chemical factors such as pH (14.52 %), ammonium (5.12 %), and dissolved organic carbon (4.74 %). Additionally, more geographically distant sites harbored less similar communities. Variance partitioning analysis (VPA) showed that geographic distance contributed more to the variation of Geobacteraceae than any other factor, although the environmental factors explained more variation when combined. So, we detected the uneven distribution of Geobacteraceae in paddy soils of China and demonstrated that Geobacteraceae community composition was strongly associated with geographic distance and soil chemical factors including aFe, pH, Fe, DOC, C:N, and NO₃ ^?-N. These results greatly expand the knowledge of the distribution of Geobacteraceae in environments, particularly in terrestrial ecosystems.

Conclusions

Our results showed that geographic distance and amorphous iron played important roles in shaping Geobacteraceae community composition and revealed that both geographic distance and soil properties governed Geobacteraceae biogeography in paddy soils. Our findings will be critical in facilitating the prediction of element cycling by incorporating information on functional microbial communities into current biogeochemical models.

     

Manure as a Source of Antibiotic-Resistant Escherichia coli and Enterococci: a Case Study of a Wisconsin, USA Family Dairy Farm

The spread of antibiotic-resistant bacteria in the environment is raising serious public health concerns, and manure is being increasingly recognized as a major source of antibiotic-resistant bacteria. In this research, we isolated Escherichia coli and enterococci from manure produced in a Wisconsin, USA family dairy farm to determine their resistance to six representative antibiotics. The average densities for E. coli and enterococci were 6.37(±4.38)?×?10⁷ colony formation units (CFU)?g^?1 and 1.60(±1.57)?×?10⁴ CFU g^?1, respectively. The E. coli isolates were found to be resistant to cephalothin, ampicillin, tetracycline, and erythromycin. In addition to these four antibiotics, the Enterococcus isolates were also resistant to gentamicin and ciprofloxacin. Additionally, we examined the survival and growth of E. coli and enterococci in dairy manure over a period of ~3 days. While the densities of enterococci remained stable over the study period, the concentrations of E. coli on average increased by 1.5 log10 units. Further tests of the bacterial antibiotic resistance over time showed no significant changes in the prevalence of antibiotic resistance. This result indicated that slightly aged manure could represent a larger source of antibiotic-resistant E. coli than fresh manure and the accumulation of antibiotic-resistant E. coli and enterococci in the agricultural fields must be accounted for in the modeling of the spread of antibiotic-resistant bacteria in the environment.     

Synergistic effect of root-associated bacteria on plant growth and certain physiological parameters of banana plant (Musa acuminata)

Fifty strains of bacteria isolated from banana roots were studied for their plant growth promoting (PGP) activities. Indole -3- acetic acid (IAA) production by root-associated bacteria ranged from 20 to 302 µg ml^?1. Seventeen isolates (34%) were positive for siderophore production and 18 isolates (36%) showed phosphate solubilization. None of the isolates showed potassium solubilization. All the isolates showed growth on nitrogen free Jensen medium. Identification of the bacteria based on 16S rRNA gene sequencing revealed that the isolates belonged to genus Bacillus sp, Klebsiella sp, Microbacterium sp and Enterobacter sp. A pot experiment in a greenhouse was conducted to investigate the effect PGP bacteria on banana plant growth and enzyme activities. The results demonstrated a significant (P < 0.05) increase in plant growth, chlorophyll, total phenolics, proline, catalase and ascorbic acid oxidase in banana plants treated with PGP bacteria as compared to control. However, the plant-growth response was variable and dependent on the bacterial strains, enzyme activity, and growth parameter observed. The present study revealed that bacteria showing multiple PGP activity could be used as biostimulants in enhancing banana production.     

不同施肥处理稻田系统磷素输移特征研究

磷是水体富营养化限制性元素,近年来由于磷肥的过量施用,农田迁移的磷素已成为水体磷素的主要来源。本研究通过野外测坑定位试验,研究有机肥处理(OT)、混施肥处理(MT)和化肥处理(CT)3种施肥处理下,稻田中磷素的迁移流失特征及这3种处理对水稻产量和磷素利用率的影响,以探求稻田系统的最佳施磷方式。结果表明,CT、MT和OT 3种施肥方式的磷径流流失负荷分别为0.56 kg(P)·hm-2、1.13 kg(P)·hm-2和4.20 kg(P)·hm-2,渗漏流失负荷分别为0.42 kg(P)·hm-2、0.44 kg(P)·hm-2和0.45 kg(P)·hm-2;磷的径流流失占流失总量的56.86%~90.38%,是水稻田磷素流失的主要途径。磷的径流流失主要受施肥和降雨的影响,50%左右磷的流失发生在第1次径流过程;磷素渗漏流失特征不受施磷处理的影响,80%以上的流失发生在施肥后的前30 d。在磷素流失形态上,坑面水、渗漏水和径流水中磷素的主要形态均为可溶性磷;在土壤方面,MT处理和OT处理能保证土壤磷营养,CT处理的土壤有效磷和有机质含量则显著下降。3种施肥处理的水稻产量显著高于空白对照,且MT最高,为6 728.84 kg·hm-2;磷肥利用率CT和MT处理显著高于OT,CT和MT间差异不显著。综合比较,混施肥处理在磷素流失、土壤养分利用和水稻产量等方面更符合我国生态农业发展的要求。     

Effect of 7-year application of a nitrification inhibitor, dicyandiamide (DCD), on soil microbial biomass, protease and deaminase activities, and the abundance of bacteria and archaea in pasture soils

Purpose

The nitrification inhibitor dicyandiamide (DCD) has been shown to be highly effective in reducing nitrate (NO₃ ^?) leaching and nitrous oxide (N₂O) emissions when used to treat grazed pasture soils. However, there have been few studies on the possible effects of long-term DCD use on other soil enzyme activities or the abundance of the general soil microbial communities. The objective of this study was to determine possible effects of long-term DCD use on key soil enzyme activities involved in the nitrogen (N) cycle and the abundance of bacteria and archaea in grazed pasture soils.

Materials and methods

Three field sites used for this study had been treated with DCD for 7 years in field plot experiments. The three pasture soils from three different regions across New Zealand were Pukemutu silt loam in Southland in the southern South Island, Horotiu silt loam in the Waikato in the central North Island and Templeton silt loam in Canterbury in the central South Island. Control and DCD-treated plots were sampled to analyse soil pH, microbial biomass C and N, protease and deaminase activity, and the abundance of bacteria and archaea.

Results and discussion

The three soils varied significantly in the microbial biomass C (858 to 542 μg C g^?1 soil) and biomass N (63 to 28 μg N g^?1), protease (361 to 694 μg tyrosine g^?1 soil h^?1) and deaminase (4.3 to 5.6 μg NH₄ ⁺ g^?1 soil h^?1) activity, and bacteria (bacterial 16S rRNA gene copy number: 1.64?×?10⁹ to 2.77?×?10⁹ g^?1 soil) and archaea (archaeal 16S rRNA gene copy number: 2.67?×?10⁷ to 3.01?×?10⁸ g^?1 soil) abundance. However, 7 years of DCD use did not significantly affect these microbial population abundance and enzymatic activities. Soil pH values were also not significantly affected by the long-term DCD use.

Conclusions

These results support the hypothesis that DCD is a specific enzyme inhibitor for ammonia oxidation and does not affect other non-target microbial and enzyme activities. The DCD nitrification inhibitor technology, therefore, appears to be an effective mitigation technology for nitrate leaching and nitrous oxide emissions in grazed pasture soils with no adverse impacts on the abundance of bacteria and archaea and key enzyme activities.     

稻田厌氧氨氧化菌群落结构对氮肥的响应

为探明稻田厌氧氨氧化菌多样性及其对氮肥用量的响应状况,利用厌氧氨氧化菌16S rRNA基因特异引物对定位试验稻田土壤DNA进行PCR-DGGE(聚合酶链反应变性梯度凝胶电泳)并结合DNA克隆测序,研究了氮肥供应量对厌氧氨氧化菌群落结构的影响。DGGE图谱及依据其条带位置和亮度数值计算的多样性指数均显示:高氮处理[N3:225 kg(N).hm 2]的厌氧氨氧化菌群落结构多样性在表层或根层土壤中均显著(P<0.05)高于中、低氮[N2:150 kg(N).hm 2;N1:75 kg(N).hm 2]处理和不施肥对照(CK);同时,高氮处理下表层土壤厌氧氨氧化菌群落多样性指数显著高于根层土壤(P<0.05)。冗余分析(RDA)结果表明,表层土壤中厌氧氨氧化菌群落结构组成与不同氮肥水平处理存在显著相关性(P=0.006)。此外,本试验获得厌氧氨氧化菌DGGE条带DNA序列18条,登录GenBank并获得登录号。研究表明稻田厌氧氨氧化菌群落结构对高氮水平具有较强的响应,尤其是在表层土壤中。     

Enhancement of Growth and Yield of Rice (Oryza Sativa) by Plant Probiotic Endophyte,Lysinibacillus sphaericus under Greenhouse Conditions

ABSTRACT

Nitrogen (N) is the “prime” among the three major nutrients required for plant growth. The large potential demand of nitrogen for plant growth is mainly achieved through biological nitrogen fixation. We have attempted to study the growth enhancement of rice crop under greenhouse conditions using the endophytic diazotrophic bacteria Lysinibacillus sphaericus. The effect of inoculation of L. sphaericus on yield and nutrient uptake was studied. All the parameters studied showed significantly higher values than that obtained in uninoculated absolute control. The highest yield was given by treatment T7 (seed and foliar) followed by T9 (soil and foliar) and T5 (seed and soil) and were statistically at par. The highest dry weight of grains was found to be in treatment T2 (13.0 g^?1plant) which received soil treatment of L. sphaericus followed by T9 (12.2 g^?1plant) which received combined application of L. sphaericus as soil and foliar treatment. It can be deduced from the results, there is a high influence of endophytic bacteria L. sphaericus in plant growth despite the mode of inoculation. This establishes the potential of diazotrophic endophyte L. sphaericus to limit the use of industrial N fertilizers thereby enhancing the fertility of the soil.     

Diversity of endophytic actinomycetes in mandarin grown in northern Thailand,their phytohormone production potential and plant growth promoting activity

Abstract

The rapid expansion of mandarin (Citrus reticulata L.) production areas with high agrochemical input in the highland areas of northern Thailand has resulted in negative effects in terms of production, environment, soil quality, and public health. The use of microorganisms as plant growth promoters is an alternative method to reduce agrochemical input. Thus, we studied the diversity of endophytic actinomycetes in mandarin and their potential as plant growth promoters. A total of 252 endophytic actinomycete isolates were recovered from mandarin. Based on spore chain morphology, cell wall type, and 16S rRNA gene sequence, the isolates were classified into six genera: Streptomyces, Nocardia, Nocardiopsis, Spirillospora, Microbispora and Micromonospora. The most frequent isolates recovered were members of Streptomyces (85.3%). Selected isolates (64 isolates) from these genera were evaluated for their indole-3-acetic acid (IAA) production potential in a medium with 2 mg mL^?1 tryptophan, and all the selected isolates showed the potential to produce IAA, with average values of IAA production of 13.34, 3.36, 140.38, 12.55, 1.40, and 6.19 µg IAA mL^?1, respectively. Isolates of genus Nocardiopsis showed a very high ability to produce IAA that was the highest among all the genera, with values ranging from 62.23 to 222.75 µg mL^?1. Twelve isolates selected from these genera were inoculated onto mandarin seedlings, and the results indicated that the shoot height, fresh shoot weight and fresh root weight of the seedlings were promoted by the inoculation of endophytic actinomycetes, with values ranging from 20.2 to 49.1%, 14.9 to 53.6%, and 1.6 to 102% over the control, respectively.     

Effect of land use on the abundance and diversity of autotrophic bacteria as measured by ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO) large subunit gene abundance in soils

Elucidating the biodiversity of CO₂-assimilating bacterial communities under different land uses is critical for establishing an integrated view of the carbon sequestration in agricultural systems. We therefore determined the abundance and diversity of CO₂ assimilating bacteria using terminal restriction fragment length polymorphism and quantitative PCR of the cbbL gene (which encodes ribulose-1,5-biphosphate carboxylase/oxygenase). These analyses used agricultural soils collected from a long-term experiment (Pantang Agroecosystem) in subtropical China. Soils under three typical land uses, i.e., rice–rice (RR), upland crop (UC), and paddy rice–upland crop rotation (PU), were selected. The abundance of bacterial cbbL (0.04 to 1.25?×?10⁸ copies g^?1 soil) and 16S rDNA genes (0.05–3.00?×?10¹⁰ copies g^?1 soil) were determined in these soils. They generally followed the trend RR?>?PU?>?UC. The cbbL-containing bacterial communities were dominated by facultative autotrophic bacteria such as Mycobacterium sp., Rhodopseudomonas palustris, Bradyrhizobium japonicum, Ralstonia eutropha, and Alcaligenes eutrophus. Additionally, the cbbL-containing bacterial community composition in RR soil differed from that in upland crop and paddy rice–upland crop rotations soils. Soil organic matter was the most highly statistically significant factor which positively influenced the size of the cbbL-containing population. The RR management produced the greatest abundance and diversity of cbbL-containing bacteria. These results offer new insights into the importance of microbial autotrophic CO₂ fixation in soil C cycling.