3种水稻土中7株固氮蓝细菌的分离与特征

【背景】蓝细菌是水生和陆地生态系统中生物固氮的主要贡献者。【目的】增加对稻田土壤固氮蓝细菌的了解,获得用于进一步研究的可培养固氮蓝细菌菌株。【方法】选择3种具有不同固氮能力的水稻土,采用BG11-N培养基分离培养固氮蓝细菌菌株,对新分离菌株进行形态特征观察,通过基因组DNA的nifH基因扩增明确其固氮潜力,进一步采用乙炔还原法和~(15)N_2示踪法定量测定其固氮能力,通过基因组DNA的16SrRNA基因序列比对进行鉴定。【结果】在光照培养条件下,采用BG11-N培养基共分离纯化得到自养菌株7株,细胞呈圆形或椭圆形、单列、无分枝、丝状和念珠状,在固体培养基上形成团垫状菌落。新分离菌株在BG11-N培养基中生长状况良好,以基因组DNA为模板可扩增出nifH基因,乙炔还原法和~(15)N_2示踪法测定结果显示具有较高固氮能力,同时具有铁载体生成能力。结合16S rRNA基因序列比对和形态特征,7株菌被初步鉴定隶属于念珠藻科(Nostocaceae)。【结论】从水稻土中分离到在稻田生物固氮中发挥重要作用的蓝细菌(念珠藻科)菌株,可培养固氮蓝细菌菌株固氮能力较高,兼具铁载体生成能力,可作为进一步深入研究的微生物资源,具有潜在的研究应用价值。     

轻型栽培技术对稻田潜杂草群落多样性的影响

为明确轻型栽培技术对稻田潜杂草群落多样性的影响,研究了旱直播、水直播、抛秧、机插秧、麦套稻5种轻型栽培稻作模式及传统手插秧模式下稻田杂草种子库的数量特征.结果表明：旱直播稻田种子库密度显著高于其他模式,达228416粒·m^-2,麦套稻模式其次.另外,旱直播稻田莎草类杂草与麦套稻稻田阔叶类杂草种子密度显著高于其他模式的各类杂草种子.传统手插秧稻田具有最高的物种丰富度,Margalef指数达1.86; 麦套稻与水直播模式中,Shannon-Wiener指数、Gini指数、Pielou群落均匀度指数明显高于旱直播和机插秧等模式.与传统手插秧相比,各轻型栽培稻作模式具有不同的潜杂草群落优势种.为防治稻田杂草的持续危害,应轮流实施不同轻型栽培稻作模式.     

稻草还田对水稻土固氮基因(nifH)组成结构和多样性的影响

以中国科学院桃源农业生态试验站长期定位施肥试验为平台,选取稻草还田(C)、氮磷钾(NPK)、氮磷钾加稻草还田(NPK+C)和不施肥对照(CK)4个处理,在晚稻的分蘖期、孕穗期和成熟期分别采集土样,利用实时定量PCR (Q-PCR)和末端限制性片段多态性(TRFLP)等分子生物学方法研究长期稻草还田对水稻土含nifH基因固氮微生物群落丰度、组成和多样性的影响.结果表明:与对照相比,稻草还田和单施化肥处理均显著增加nifH基因的丰度(分蘖期除外),NPK+C处理中含nifH基因的微生物数量最高;nifH基因组成也受到长期施肥的影响,其中CK处理nifH基因组成与各施肥处理明显不同,C与NPK处理间nifH基因组成存在一定差异,而NPK与NPK+C处理间无显著差异.长期施肥不会引起含nifH基因微生物群落多样性的显著改变.可见,稻草还田不仅引起nifH基因群落的组成发生变化,而且导致其数量显著增加,因而可增加土壤的固氮能力.     

转 Bt 基因水稻对三化螟自然种群的影响

为了了解水稻重要害虫三化螟 Scirpophaga incertulas（Walker）对转 Bt 基因水稻产生抗性的潜在风险,本文利用自然种群生命表,研究了第 3 代三化螟在转Bt基因杂交稻汕优 63 及非转 Bt 基因杂交稻汕优 63 上的种群参数。结果表明：在 Bt 稻上1～2 龄幼虫的存活率为 4.03%,极显著低于非转基因 SY63（55.76%）;Bt 稻上幼虫为害率为1.75%,极显著低 于非转基因 SY63（5.98%）;Bt 稻上存活的幼虫发育历期明显长于非转 Bt SY63。     

稻草还田对水稻土固氮基因(nifH)组成结构和多样性的影响

以中国科学院桃源农业生态试验站长期定位施肥试验为平台,选取稻草还田(C)、氮磷钾(NPK)、氮磷钾加稻草还田(NPK+C)和不施肥对照(CK)4个处理,在晚稻的分蘖期、孕穗期和成熟期分别采集土样,利用实时定量PCR(Q-PCR)和末端限制性片段多态性(T-RFLP)等分子生物学方法研究长期稻草还田对水稻土含nifH基因固氮微生物群落丰度、组成和多样性的影响.结果表明：与对照相比,稻草还田和单施化肥处理均显著增加nifH基因的丰度(分蘖期除外),NPK+C处理中含nifH基因的微生物数量最高;nifH基因组成也受到长期施肥的影响,其中CK处理nifH基因组成与各施肥处理明显不同,C与NPK处理间nifH基因组成存在一定差异,而NPK与NPK+C处理间无显著差异.长期施肥不会引起含nifH基因微生物群落多样性的显著改变.可见,稻草还田不仅引起nifH基因群落的组成发生变化,而且导致其数量显著增加,因而可增加土壤的固氮能力.     

Sodium requirement and metabolism in nitrogen-fixing cyanobacteria

Sodium affects the metabolism of eukaryotes and prokaryotes in several ways. This review collates information on the effects of Na⁺ on the metabolism of cyanobacteria with emphasis on the N₂,fixing filamentous species. Na⁺ is required for nitrogenase activity inAnabaena torulosa, Anabaena L-31 andPlectonema boryanum. The features of this requirement have been mainly studied inAnabaena torulosa. The need for Na⁺ is specific and cannot be replaced by K⁺, Li⁺, Ca 2 + or Mg²⁺. Processes crucial for expression of nitrogenase such as molybdenum uptake, protection of the enzyme from oxygen inactivation and conformational activation of the enzyme are not affected by Na⁺. Mo-Fe protein and Fe protein, the two components of nitrogenase are synthesized in the absence of Na⁺ but the enzyme complex is catalytically inactive. Photoevolution of O₂ and CO₂ fixation, which are severely inhibited in the absence of Na⁺, are quickly restored by glutamine or glutamate indicating that Na⁺ deprivation affects photosynthesis indirectly due to deficiency in the products of N₂ fixation. Na⁺ deprivation decreases phosphate uptake, nucleoside phosphate pool and nitrogenase activity. These effects are reversed by the addition of Na⁺ suggesting that a limitation of available ATP caused by reduced phosphate uptake results in loss of nitrogenase activity during Na⁺ starvation. Na⁺ influx inAnabaena torulosa andAnabaena L-31 is unaffected by low K⁺ concentration, is carrier mediated, follows Michaelis-Menten kinetics and is modulated mainly by membrane potential. Treatments which cause membrane depolarisation and hyperpolarisation inhibit and enhance Na⁺ influx respectively. These cyanobacteria exhibit rapid active efflux of Na⁺, in a manner different from the Na⁺/H⁺ antiporter mechanism found inAnacystis nidulans. Na⁺ requirement in nitrogen metabolism including nitrate assimilation, synthesis of amino acids and proteins, in respiration and oxidative phosphorylation, in transport of sugars and amino acids, cellular distribution of absorbed sodium, physiological basis of salt tolerance and prospects of reclamation of saline soils by cyanobacteria are the other aspects discussed in this review.     

Protein synthesis in enuleated fertilized and unfertilized mouse eggs

Summary Fertilized and unfertilized C57BL/6J eggs were microsurgically enucleated and then analyzed for their capacity to synthesize proteins using 2-dimensional polyacrylamide gel electrophoresis. In both types of enucleated eggs (cytoplasts), protein synthesis continued and was still detected up to three days in culture. Shortly after enucleation, the pattern of polypeptides remained similar to the respective non-operated control eggs but it later became gradually reduced in intensity and complexity. After two days of culture the appearance of some new proteins typical for 2-cell embryos was observed in enucleated fertilized eggs only. Our findings suggest that maternal mRNA stored during oogenesis is utilized during the preimplantation period.     

Development of a biofertilizer based on filamentous nitrogen-fixing cyanobacteria for rice crops in Chile

The purpose of this study was to develop a biofertilizer based on filamentous nitrogen-fixing cyanobacteria selected from rice fields and to generate a technological package compatible with its use for the rice crop in Chile. Thirty-four Chilean rice fields, located between Maule and BioBío regions, were sampled during the 1998/1999 and 1999/2000 growing seasons. A total of 9 species and 3 varieties of cyanobacteria were found, and the nitrogen fixation rate under laboratory conditions was determined for 6 of them. Only 4 were used for the small-scale production of a biofertilizer, which was assayed in field trials. To check the efficiency of the biofertilizer during the rice crop, the nitrogen fixation rates in soil samples were estimated. Additionally, the biofertilizer application efficiency was tested in combination with nitrogen synthetic fertilizer, in rates that were previously established in field trials. Biofertilization allowed a decrease of up to 50% in the use of nitrogen synthetic fertilizer (50 kg N ha⁻¹), resulting in the same grain yield (7.4 t ha⁻¹) and quality in relation to the fertilized control. The use of biofertilizers based on local strains of cyanobacteria shows promise to increase nitrogen use efficiency in rice.     

Characterization of water in unfertilized and fertilized sea urchin eggs

The water in unfertilized and fertilized sea urchin eggs was characterized with a proton nuclear magnetic resonance (NMR) titration method assuming fast proton diffusion (FPD) between water compartments. This method involves stepwise dehydration with sequential T1 relaxation time and water content determinations. The results analyzed by the FPD model give evidence of intracellular water compartments with three different correlation times: 6 X 10(-12) sec (bulk water), 1 X 10(-10) sec (structured water) and about 2 X 10(-9) sec (bound water). Fertilization is accompanied by a substantial increase in bulk water (from 111 to 414 g H2O per 100 g dry mass) and by a decrease in the water of hydration (from 128 g to 56 g per 100 g dry mass). This study shows that 54% of the water in the unfertilized sea urchin egg has motional properties different from bulk water and that this percentage decreases dramatically shortly after fertilization. Most of the change in T1 relaxation rate observed at fertilization can be accounted for by uptake of bulk water associated with elevation of the fertilization membrane.     

Enhancement of phycobiliprotein production in nitrogen-fixing cyanobacteria

The influence of several environmental factors on the phycobiliprotein content of two phycoerythrin-rich nitrogen-fixing cyanobacteria has been studied in order to maximize pigment production. Total phycobiliprotein content was enhanced when either temperature within the optimum range for growth or cell density of the culture was increased. The phycobiliprotein level increased also in response to a decrease in irradiance. In all cases the effect was more marked for C-phycoerythrin than for C-phycocyanin and allophycocyanin. The cellular content in C-phycoerythrin was also preferentially enhanced when cultures were irradiated with green light. On the other hand, red light induced an increase in the C-phycocyanin content, but the C-phycoerythrin level decreased considerably.     

Cytoskeletal and contractile proteins in coelomic oocytes,unfertilized and fertilized eggs of discoglossus pictus (Anura)

The distribution of actin, myosin, and tubulin has been investigated in coelomic oocytes, unfertilized and fertilized eggs of Discoglossus pictus utilizing: (1) immunofluorescence; (2) electron microscopy; (3) incubation with heavy meromyosin (HMM), and (4) SDS-polyacrylamide gel electrophoresis (PAGE). In coelomic oocytes, the germinative area (GA) has long, irregular microvilli containing microfilaments. In the rest of the oocyte, the microvilli are shallow. During the transit of the oocyte in the oviduct, a dimple forms by the invagination of the GA. A palisade of microfialment bundles is present in the finger-shaped microvilli of the dimple and extends for about 10 μm in the cytoplasm. In the rest of the egg, microvilli are absent and only random filaments appear in the cortex. Following HMM incubation, the dimple microfilaments are decorated with arrowheads pointing toward the bulk of the cytoplasm. SDS-PAGE of egg extracts shows bands co-migrating with actin (43K), pyruvate kinase (57K), and phosphorylase (94K). As result fertilization, the pattern of microfilament bundles in the dimple disappers in parallel with the dimple invergination itself. Generally, the entire oocyte cortex is positive to immunofluorescent staining with anti-actin, antimyosin, and antitubulin antibodies. However, the pattern of distribution and intensity of immunofluorescent staining changes for each antiserum, during different stages. It is concluded that a contractile system is present in Discoglossus eggs, and it is particularly developed in the dimple. The dimple is probably a major compartment for the storage of unpolymerized tubulin.     

Studies on mixed mass cultivation of Anabaena spp. (nitrogen-fixing blue-green algae, cyanobacteria) on a large scale

Studies on mixed mass cultivation of Anabaena spp. on a large scale (5170m²) were conducted continuously for 3 years. Under the continental monsoon climate in northern subtropics (30°N, 115°E), 7–11 g dry weight m⁻² day⁻¹ of microalgal biomass on average was harvested in simple plastic greenhouses in the effective growth days during the warmer seasons. The maximum productivity was 22 g m⁻² day⁻¹ in the middle of summer. Observations on the productive properties of strains of Anabaena spp. indicated that they were different from and could compensate for each other in their productivities and adaptations to the seasonal changes. With different lining materials (PVC sheets, concrete, sand and soil) in the culture ponds, no significant variation of productivity was found, but bubbling with biogas in the middle of the day and the application of some growth regulating substances (2,4-D, NaHSO₃ and extracts of oyster mushroom spawn) was able to improve the production. The cost of microalgal biomass in this way was around 0·75–1·0 US dollar(s) per kilogram.     

Contribution of winter cover crop amendments on global warming potential in rice paddy soil during cultivation

Background and aims

Winter cover crop cultivation during the fallow season has been strongly recommended in mono-rice paddy soil to improve soil quality, but its impact in increasing the greenhouse gases (GHGs) emissions during rice cultivation when applied as green manure has not been extensively studied. In order to recommend a preferable cover crop which can increase soil productivity and suppress GHG emission impact in paddy soil, the effect of winter cover crop addition on rice yield and total global warming potential (GWP) was studied during rice cultivation.

Methods

Two cover crops (Chinese milk vetch, Astragalus sinicus L., hereafter vetch, and rye, Secale cerealis) having different carbon/nitrogen (C/N) ratios were cultivated during the rice fallow season. The fresh above-ground biomasses of vetch [25 Mg fresh weight (FW) ha^?1, moisture content (MC) 86.9 %, C/N ratio 14.8] and rye (29 Mg rye FW ha^?1, MC 78.0 %, C/N ratio 64.3) were incorporated as green manure 1 week before rice transplanting (NPK + vetch, and NPK + rye). The NPK treatment was installed for comparison as the control. During the rice cultivation, methane (CH₄) and nitrous oxide (N₂O) gases were collected simultaneously once a week using the closed-chamber method, and carbon dioxide (CO₂) flux was estimated using the soil C balance analysis. Total GWP impact was calculated as CO₂ equivalents by multiplying the seasonal CH₄, CO₂, and N₂O fluxes by 25, 1, and 298, respectively.

Results

Methane mainly covered 79–81 % of the total GWP, followed by CO₂ (14–17 %), but the N₂O contribution was very small (2–5 %) regardless of the treatment. Seasonal CH₄ fluxes significantly increased to 61 and 122 % by vetch and rye additions, respectively, compared to that of the NPK treatment. Similarly, the estimated seasonal CO₂ fluxes increased at about 197 and 266 % in the vetch and rye treatments, respectively, compared with the NPK control plots. Based on these results, the total GWP increased to 163 and 221 % with vetch and rye applications, respectively, over the control treatment. Rice productivity was significantly increased with the application of green manure due to nutrient supply; however, vetch was more effective. Total GWP per grain yield was similar with the vetch (low C/N ratio) and NPK treatments, but significantly increased with the rye (high C/N ratio) application, mainly due to its higher CH₄ emission characteristic and lower rice productivity increase.

Conclusions

A low C/N ratio cover crop, such as vetch, may be a more desirable green manure to reduce total GWP per grain yield and to improve rice productivity.     

Epiphytic cyanobacteria on Chara vulgaris are the main contributors to N(2) fixation in rice fields

The distribution of nitrogenase activity in the rice-soil system and the possible contribution of epiphytic cyanobacteria on rice plants and other macrophytes to this activity were studied in two locations in the rice fields of Valencia, Spain, in two consecutive crop seasons. The largest proportion of photodependent N(2) fixation was associated with the macrophyte Chara vulgaris in both years and at both locations. The nitrogen fixation rate associated with Chara always represented more than 45% of the global nitrogenase activity measured in the rice field. The estimated average N(2) fixation rate associated with Chara was 27.53 kg of N ha(-1) crop(-1). The mean estimated N(2) fixation rates for the other parts of the system for all sampling periods were as follows: soil, 4.07 kg of N ha(-1) crop(-1); submerged parts of rice plants, 3.93 kg of N ha(-1) crop(-1); and roots, 0.28 kg of N ha(-1) crop(-1). Micrographic studies revealed the presence of epiphytic cyanobacteria on the surface of Chara. Three-dimensional reconstructions by confocal scanning laser microscopy revealed no cyanobacterial cells inside the Chara structures. Quantification of epiphytic cyanobacteria by image analysis revealed that cyanobacteria were more abundant in nodes than in internodes (on average, cyanobacteria covered 8.4% +/- 4.4% and 6.2% +/- 5.0% of the surface area in the nodes and internodes, respectively). Epiphytic cyanobacteria were also quantified by using a fluorometer. This made it possible to discriminate which algal groups were the source of chlorophyll a. Chlorophyll a measurements confirmed that cyanobacteria were more abundant in nodes than in internodes (on average, the chlorophyll a concentrations were 17.2 +/- 28.0 and 4.0 +/- 3.8 microg mg [dry weight] of Chara(-1) in the nodes and internodes, respectively). These results indicate that this macrophyte, which is usually considered a weed in the context of rice cultivation, may help maintain soil N fertility in the rice field ecosystem.     

Pattern of cyanophycin accumulation in nitrogen-fixing and non-nitrogen-fixing cyanobacteria

The temporal and spatial accumulation of cyanophycin was studied in two unicellular strains of cyanobacteria, the diazotrophic Cyanothece sp. strain ATCC 51142 and the non-diazotrophic Synechocystis sp. strain PCC 6803. Biochemistry and electron microscopy were used to monitor the dynamics of cyanophycin accumulation under nitrogen-sufficient and nitrogen-deficient conditions. In Cyanothece sp. ATCC 51142 grown under 12 h light/12 h dark nitrogen-fixing conditions, cyanophycin was temporally regulated relative to nitrogenase activity and accumulated in granules after nitrogenase activity commenced. Cyanophycin granules reached a maximum after the peak of nitrogenase activity and eventually were utilized completely. Knock-out mutants were constructed in Synechocystis sp. PCC 6803 cphA and cphB genes to analyze the function of these genes and cyanophycin accumulation under nitrogen-deficient growth conditions. The mutants grew under such conditions, but needed to degrade phycobilisomes as a nitrogen reserve. Granules could be seen in some wild-type cells after treatment with chloramphenicol, but were never found in Delta cphA and Delta cphB mutants. These results led to the conclusion that cyanophycin is temporally and spatially regulated in nitrogen-fixing strains such as Cyanothece sp. ATCC 51142 and represents a key nitrogen reserve in these organisms. However, cyanophycin appeared to play a less important role in the non-diazotrophic unicellular strains and phycobilisomes appeared to be the main nitrogen reserve.     

Amino acid transport in the unfertilized and fertilized mouse egg.

The transport of methionine into unfertilized and fertilized mouse eggs appears to involve active transport mechanisms with similar Vmax, Km, substrate specificity and independence from Na+. An exchange diffusion system with a similar amino acid specificity to the uptake system has also been found in both types of egg. An estimate of 6.5 fmol has been made for the size of the total internal pool of exchangeable amino acids.     

Effects of conservation tillage on soil organic matter in paddy rice cultivation

In this study, the effect of conservation tillage on soil organic matter (SOM) in paddy rice cultivation after 10 yr was investigated. Four treatments, disk till-fallow (DTF), disk till-wheat (DTW), conservation till-fallow (CTF) and conservation till-wheat (CTW) were used. The results indicated that the combinative application of no-tillage, ridge culture and wheat cultivation was a sound conservation practice in paddy rice cultivation. It not only significantly increased the concentration of SOM in the topsoil, but also affected optical and pyrolysis characteristics of humic acids (HA) through changing the composition and structure of SOM. At 0–10 cm, the greatest SOM content was in CTW, but declined sharply with depth, while in DTF, DTW and CTF the SOM content was not as high at the surface as in CTW, but did not decline as fast as in CTW. The oxidation stabilization of SOM was generally greater in no-tillage and ridge culture than that in disk till. The HA optical density in CTW at wavelength 665 nm and 465 nm was 0.122 and 0.705, while in DTF was 0.062 and 0.321, respectively. E₄/E₆ ratio in CTW was higher than that in the other treatments. The enthalpy capacity of the exothermal peak (360–365°C) for the HA DTA curve in no-tillage and ridge culture was lower than that in disk till, while the HA absorption peaks in 1000–1050 cm^?1 presented the reverse trend. The oxidation stabilization coefficient of HA in no-tillage and ridge culture was higher than that in disk till, indicating that the polycondensation degree and aromatization of HA were stronger. These findings suggest that it may be possible to manipulate paddy soil through conservational tillage and crop practices, and thereby maintain adequate SOM concentrations, and mitigate soil organic carbon loss from soil to atmosphere.     

Influence of rice cultivar on methane emission from paddy fields

Influence of rice cultivars on CH₄ emissions from a paddy field was studied using four Japonica types, two Indica types, and two Japonica/Indica F₁ hybrids. In addition, the suppression of CH₄ emission by interrupting irrigation at the flowering stage was investigated. Patterns of seasonal variation in CH₄ emission rates were similar among the eight cultivars. Two of the Japonica types showed the maximum and minimum CH₄ emissions among the cultivars investigated. Neither the number of tillers, shoot length, shoot weight, and root weight correlated with the CH₄ emission rates at the tillering and reproductive growth stages. Following temporary interruption of irrigation at the flowering stage, CH₄ emission rates decreased drastically and remained at very low levels until the harvesting stage, indicating its great effectiveness for the suppression of CH₄ emission from rice paddies.     

水稻植株对稻田甲烷排放的影响

稻田CH4排放是稻田土壤中CH4产生、氧化和传输不同过程的净效应,水稻植株强烈影响稻田CH4的产生、氧化和传输过程,是导致稻田CH4排放季节性变化规律的一个重要因素,本文综述了水稻植株对稻田CH4排放过程的不同影响,水稻植株根系分泌物和脱落物作为产甲烷前体促进稻田土壤中CH4的产生,在水稻生长后期,植株根系分泌物和脱落物被认为是稻田土壤甲烷产生的主要基质,是导致这一时期稻田CH4高排放通量的主要原因;水稻植株根系泌氧在根际环境形成一个微氧区域氧化稻田甲烷,整个水稻生长季稻田土壤中产生的CH4大约36％～90％在植株根际环境中被氧化;约80％甚至更多的稻田CH4通过水稻植株的通气组织进入大气圈,植株对稻田CH4的传输具有十分重要的意义。