Comparison of g23 gene sequence diversity between Novosphingobium and Sphingomonas phages and phage communities in the floodwater of a Japanese paddy field

Our previous study indicated that the diversity of the major capsid gene (g23) of T4-type bacteriophages (phages) of Novosphingobium and Sphingomonas strains isolated from the floodwater of a Japanese paddy field is comparable to those of the clones obtained from other Japanese paddy fields. For more strict comparison of the diversity, this study examined g23 sequences between Novosphingobium and Sphingomonas phages and phage communities in the identical floodwater of a Japanese paddy field. The clones were obtained by applying g23-specific primers to DNA extracted from the floodwaters. Many 23 clones in the floodwater were grouped into the same clusters of Paddy Groups I-VI with g23 genes of Novosphingobium/Sphingomonas phages with some clones belonging to an additional cluster. In addition, the remaining clones belonged to the clusters of marine clones and T4-type enterophages. These findings indicate that the g23 genes in the floodwater are more diversified than those of Novosphingobium/Sphingomonas phages including g23 genes closely related to the genes of enterophages and marine origins.     

Morphological analysis of viral communities in the floodwater of a Japanese paddy field

Capsid size distributions of viral communities in the floodwater of a Japanese paddy field under a long-term fertilizer trial were surveyed during the rice cultivation period by using transmission electron microscopy. The capsid size distributions were monophasic, and the median values fell within the range of 50-70 nm. The quartile intervals were narrow from either 40-50 nm or 50-60 nm to either 60-70 nm or 70-80 nm for most samples. There was no clear seasonal variation in the capsid size distribution of viral communities. The difference in size distributions among different fertilizer plots was also not found. Viral communities in the floodwater were predominated by isometric icosahedral forms. Viruses with octahedral capsids and elongated ones were rare and sporadic in the floodwater.     

The distribution characteristics of the major capsid gene (g23) of T4-type phages in paddy floodwater in Northeast China

Our previous study revealed the high diversity of the major capsid gene (g23) of T4-type phages that existed in the paddy field soils in Northeast China. In this study, the phylogeny and genetic diversity of the g23 gene in the paddy floodwater samples collected from five sampling sites at three sampling times during the rice (Oryza sativa L.) growth season in Northeast China are reported. In total, 104 different g23 clones were isolated, among which 50% of the clones exhibited the highest identities with the clones retrieved in paddy soils and upland black soils. The remaining clones had the highest identities with lake origins. Phylogenetic analysis revealed that 43% of the g23 clones grouped into three novel subgroups which included the clones unique to paddy floodwater, and no g23 sequences obtained in paddy floodwater fell into the paddy soil groups II, III, IV, V, VI, VII and NPC-A. UniFrac analysis of g23 clone assemblages demonstrated that T4-type phage communities in paddy floodwater were changed spatially and temporally, and the communities were different from those in paddy soils. Further comparison of the g23 clone assemblages from different environments demonstrated that T4-type phages were biogeographically distributed, and the distribution was both affected by geographical separation and ecological processes across the biomes.     

Isolation, enumeration, and characterization of diazotrophic bacteria from paddy soil sample under long-term fertilizer management experiment

A study was undertaken to determine the free-living culturable diazotrophic bacteria of paddy soils from a long-term fertilizer management experiment. Long-term application of different fertilizers significantly affected the population of free-living diazotrophs. Out of 165 distinct bacterial morphotypes observed during the isolation process, only 32 were positive for both acetylene reduction assay (ARA), and nifH gene screening. The ARA activity of the isolates ranged from 1.8 to 2,844.7 nmol ethylene h^?1 mg protein^?1. The 16S rRNA analysis identified the isolates to be members of 13 different genera viz. Bacillus, Pseudomonas, Paenibacillus, Serratia, Ochrobactrum, Lysinibacillus, Burkholderia, Brevundimonas, Herbaspirillum, Novosphingobium, Sphingomonas, Xanthomonas, and Azorhizobium. Though partial nifH gene sequencing of diazotrophic isolates showed good consistency with that of 16S rRNA-based identification, some nifH sequences were similar to a variety of uncultured nitrogen-fixing bacteria. The diversity of free-living diazotrophic bacteria and the wide distribution of nifH sequences indicate the potential contribution of these microorganisms to nitrogen input to paddy fields.     

T4-type bacteriophage communities estimated from the major capsid genes (g23) in manganese nodules in Japanese paddy fields

The present study compared the capsid gene ( g23 ) of T4-type bacteriophages (phages) in Mn nodules with those in the plow layer soil and subsoils of two Japanese paddy fields by applying the primers MZIA1bis and MZIA6 to DNA extracts from the nodules and soils. The deduced amino acid sequences of the g23 genes in the Mn nodules were similar to those in the plow layer soil and in the subsoils. This result indicated that similar T4-type phage communities developed at these sites and that the diversity of T4-type phage communities was wide enough to cover those in the plow layer soil and in the subsoils. The majority of g23 clones formed several clusters with the clones and phages obtained from far-apart paddy fields, and the sequences of two clones were completely identical to a phage and a clone from other paddy fields at the nucleotide or amino acid level, indicating horizontal transfer of g23 genes between those paddy fields. A clone with a long nucleotide residue (686 bp) and a distribution remote from the other clones in the phylogenetic tree indicated that there were many uncharacterized, novel g23 genes in the paddy fields.     

Frequency of phage-infected bacterial cells in the floodwater of a Japanese paddy field

In aquatic environments, viruses play an important role in the microbial food web through microbial mortality from viral lysis. Bacteriophages (phages) compose the majority of viral communities in the floodwater of paddy fields. The present study evaluated bacterial mortality from phage lysis in the floodwater of a Japanese paddy field based on the frequency of phage-infected bacterial cells by transmission electron microscopy. Floodwater was sampled five times during the rice cultivation period from two plots of NPK plus lime (no-compost plot) and NPK plus lime and compost (compost plot) under a long-term and ongoing fertilizer trial that began in 1925. The frequency of visibly infected cells in the compost plot was larger, ranging from 2.4 to 3.6% (average 3.0%), than that in the no-compost plot, which ranged from 1.6 to 2.9% (average 2.0%). The fractions of bacterial mortality from phage lysis in the floodwater samples were estimated to range from 12.8 to 27.3% (average 17.2%) and from 21.7 to 35.0% (average 27.9%) for the samples collected from the no-compost plot and the compost plot, respectively. This is the first study to estimate bacterial mortality from phage lysis in the paddy field ecosystem, and the frequency of phage lysis in floodwater was found to be within the frequency ranges observed in other aquatic environments.     

Survey of major capsid genes (g23) of T4-type bacteriophages in Japanese paddy field soils

Many studies have shown the ecological importance of viruses as the greatest genomic reservoirs on the planet. As bacteriophages (phages) comprise the majority of viruses in the environment, we surveyed the capsid genes (g23) of T4-type phages, Myoviridae, from DNA extracts of three paddy field soils located in northern, central and southern Japan using the degenerate primers MZIA1bis and MZIA6. Denaturing gradient gel electrophoresis (DGGE) was performed to separate PCR-amplified g23 products, and 56 DGGE bands were identified as g23 fragments. Only nine clones were grouped into T-evens, PseudoT-evens and ExoT-evens, and most of the other clones were classified into Paddy Groups I-VI. No significantly different distribution of g23 clones was observed among the paddy fields at the group level, indicating that phage communities estimated from the g23 composition were common on the nationwide level. Comparison of g23 sequences showed that g23 genes in paddy fields were different from those in marine environments, and more divergence of g23 genes was estimated in the paddy fields compared to the marine environment. Two novel g23 clones with very short amino acid residues were detected, suggesting the existence of uncharacterized, novel groups of g23 genes in paddy field soils.     

亚热带小流域土壤氮磷分布及其环境效应

土壤氮磷积累是引起农业面源污染的主要原因。为探讨土壤氮磷含量的分布状况及其对环境的影响,选取位于长沙县金井镇的小流域为研究区域,以表层土壤(0-20cm)为采样对象,按不同的土地利用方式共采集样品1 118个。以土地利用类型为分析单元,分析说明了土壤全氮、全磷的分布特征及其环境效应。结果表明:土壤全氮和全磷含量的平均值分别为1.66,0.54g/kg,变异系数分别为34.9%和46.3%,均属于中等变异。菜地土壤全氮含量主要分布在1.8～2.4g/kg区间,旱地和水田为1.2～1.8g/kg,林地和茶园为0.6～1.2g/kg;菜地土壤全磷含量主要分布在大于1.0g/kg区间,旱地为0.6～0.8g/kg,水田为0.4～0.6g/kg,林地和茶园为0.2～0.4g/kg。结合地下水氮磷含量分析表明,土壤氮素含量与地下水铵态氮含量增加有关,对地下水水质产生影响,其中水田的影响尤为突出;土壤磷素的淋失可能具有季节性差异,还有待进一步验证。     

Seasonal variations in the abundance of virus-like particles and bacteria in the floodwater of a Japanese paddy field

Abstract

Viruses are the most abundant biological entities in marine and freshwater environments. Many studies have shown the ecological importance of viruses in the primary production and microbial food web in aquatic environments. However, no studies have examined viral abundance in the floodwater of paddy fields. The present study surveyed the abundance of virus-like particles (VLPs) and bacteria in the floodwater of a Japanese paddy field under a long-term fertilizer trial since 1925 during the rice cultivation period. Virus-like particles and bacterial abundances in the floodwater ranged from 5.6 × 10⁶ to 1.2 × 10⁹ VLPs mL^?1 and from 9.2 × 10⁵ to 4.3 × 10⁸ cells mL^?1 with mean abundances of 1.5 × 10⁸ VLPs mL^?1 and 5.1 × 10⁷ cells mL^?1, respectively, and increased with an increase in the turbidity of the floodwater with suspended particles. The magnitude of seasonal variation was more than 50-fold for VLP abundance and 100-fold for bacterial abundance. The virus-to-bacterium ratios fluctuated over the rice cultivation period, ranging from 0.11 to 72 and their increase correlated with the decrease in bacterial abundance. Our results suggest that viral abundance in the floodwater of paddy fields is larger than in natural marine and freshwater environments.     

Ammonia Volatilization from Urea Applied to Acid Paddy Soil in Southern China and Its Control

Results showed that ammonia loss from urea broadcast into floodwater and incorporated into soil at transplanting was as high as 40% of applied N,and the corresponding total nitrogen (N) loss was 56%.Ammonia loss was measured with simplified micrometeorological method (ammonia sampler),and total N loss was concurrently measured using ^15N balance technique.The experiment was conducted under strong sunshine conditions on acid paddy soil derived from Quaternary red clay.The ammonia loss in this particular condition was much greater than those obtained from previous studies when urea was also applied to acid paddy soil but under cloudy conditions.It is concluded that the strong sunshine conditions with high temperature and shallow floodwater during the period of present experiment favoured ammonia volatilization.Application of stearyl alcohol on the surface of the floodwater reduced ammonia loss to 23% of applied N.However,the effect of stearyl alcohol was short-lived,probably due to the microbiological decomposition.     

间歇灌溉对稻田毒死蜱迁移转化特征的影响

间歇灌溉作为丘陵区稻田常见的灌溉方式之一,其强烈的干湿交替过程会影响稻田中污染物的环境行为。在室内批量平衡吸附试验的基础上,通过农药野外喷施试验与动态观测,研究了间歇淹水和持续淹水条件下石灰性紫色土发育的稻田中毒死蜱的迁移转化特征。结果表明,土壤对毒死蜱的吸附能力远远强于其对毒死蜱主要降解产物3,5,6-三氯-2-吡啶醇(3,5,6-TCP)的吸附能力,毒死蜱的吸附容量常数范围为34~170,TCP的吸附容量常数范围为0.62~0.67,且对毒死蜱和TCP的吸附容量常数及分配系数均以耕作层土壤高于非耕作层土壤;施药后田面水中毒死蜱及TCP的浓度均随时间迅速下降,两者均可通过土壤大孔隙优先流快速迁移至50cm深处;间歇灌溉处理稻田土壤孔隙水中两者的浓度总体低于持续淹水处理;降雨和灌溉事件会导致两者由土壤固相迅速向水相发生短时间、高浓度释放与淋失。     

浮萍对稻田田面水中氮素转化与可溶性氮的影响

浮萍是稻田田面水中大量存在的典型水生植物，本试验采用室内培养的方法，以采自浙江省嘉兴市双桥农场的青紫泥水稻土为例，探讨了不同初始密度的浮萍对稻田施尿素后田面水尿素水解及可溶性氮浓度的影响。结果表明，浮萍可明显加快田面水尿素态氮的水解过程，对照、低浓度浮萍（D1）和高浓度浮萍（D2）处理中尿素水解速率常数k分别为0．02，0．03，0、04／h；试验前期，浮萍将大量的铵态氮（NH4^＋-N）吸收同化后储存于体内．而从第12天（D2处理）和15天（D1处理）开始，由于浮萍的释氮作用导致田面水中NH4^＋-N浓度逐步回升，同时硝态氮（NO3^--N）浓度也明显增加，说明浮萍在田面水氮索浓度较高时可大量积累氮索而浓度较低时可以向田面水中释放氮素，这有利于降低施肥初期田面水氮素流失潜能和保证施肥后期作物的氮营养供应。     

Mapping pathways of possible phage-mediated genetic interchange among soil bacilli

Bacillus pumilus (Strain W43) has been shown to sustain the growth of an unusually large number of different phages. From 22 isolates 16 distinguishable phages have been obtained. Phage BPPX which is similar to the defective particle PBSX of B. subtilis is induced by four of the phages. When tested against a variety of Bacillus spp 12 of the non-defective phages had host ranges crossing at least one species line: Phage K13 infected 14 of 25 strains, distributed among six species some of which are considered to be taxonomically distant. It is suggested that the relatively restricted host ranges noted for most Bacillus phages may result from the use. as test organisms, of bacteria isolated from soils ecologically distinct from the source(s) of phage. A genetic “circuit diagram” constructed from the host range table, maps possible genetic connexions between various soil bacilli made possible by the phages. These data are set in the context of recent theories which postulate that viruses are agents of accelerated cell evolution.     

东北典型县域稻田土壤肥力评价及其空间变异

  【目的】  明确东北典型县域稻田土壤肥力空间变异特征，为该区域稻田土壤合理培肥管理提供科学依据。  【方法】  以黑龙江省方正县为研究区域，2017年在该研究区域采取114个代表性点位的稻田土壤，选取容重、pH、全氮含量、有效磷含量、速效钾含量、有机质含量和阳离子交换量作为土壤综合肥力评价指标，采用相关系数法确定各个指标的权重，根据东北稻田土壤特征，选择隶属度函数曲线，并确定隶属度函数转折点，依据模糊数学法的加乘原理，利用各土壤肥力指标的权重值和隶属度值计算土壤综合肥力指数；采用GIS和地统计学相结合的方法，确定各项肥力指标和综合肥力指数的空间变异特征和分布格局；通过主成分分析探究土壤肥力差异的主控因子。  【结果】  描述性统计分析表明，方正县稻田土壤综合肥力指数在0.18～0.99，平均值为0.60。土壤容重和pH的变异系数分别为9.15%和5.69%，属于弱变异强度，其他肥力指标的变异系数在20.01%～36.18%，属于中等变异强度。地统计学研究表明，土壤容重、全氮含量、有机质含量和阳离子交换量的块金系数在39%～50%，它们具有中等强度的空间自相关性，土壤pH、有效磷含量、速效钾含量和综合肥力指数的块金系数均在25%以下，它们具有强烈的空间自相关性。方正县土壤综合肥力指数值在0.70以上的稻田占16%，在0.60～0.70的稻田占45%。各个肥力指标中，土壤有效磷含量和速效钾含量的分布特征与综合肥力指数相似，呈南高北低的分布格局；土壤pH和阳离子交换量由稻区中部向南北方向逐渐降低；土壤容重由西北向东南呈逐渐降低的趋势；土壤有机质和全氮含量均表现为由东南向西北逐渐降低的趋势，土壤有机质含量在30 g/kg以上的稻田占比为97%，土壤全氮含量在1.5 g/kg以上的稻田占比为84%；其中北部蚂蚁河沿岸土壤全氮、有效磷、速效钾和有机质含量相对较低。利用主成分分析得到各项肥力指标的综合得分值由大到小依次为:土壤有效磷含量、速效钾含量、有机质含量、阳离子交换量、全氮含量、pH和容重。  【结论】  方正县61%的稻田土壤肥力处于中等及以上水平，稻田土壤肥力整体呈现出南高北低的分布格局。土壤有效磷和速效钾含量是造成土壤肥力差异的主要因子。     

淹水稻田氮的损失

A field microplot experiment was conducted during the tillering stage of paddy rice to investigate nitrogen(N) Iosses from flooded rice fields following fertilizer application. After application of ammonium bicarbonate, most of nitrogen in the flood water was present as NH₄-N and its concentration varied widely with time. Concentrations of both NO₃-N and NO₂-N in the floodwater were low due to the weakened nitrification. Under flooded anaerobic reducing conditions, soil solution concentrations of NO₃-N and NH₄-N were nothigh, ranging from 0.6 mg L^-1 to 4.8 mg L^-1, and decreased with soil depth. However, the ground water wasstill contaminated with NO₃-N and NH₄-N. Rainfall simulation tests showed that the N losses via runoff inrice fields were closely related to the time intervals between fertilizer applications and rainfall events. Whena large rain fell for a short period after fertilizer application, the N losses via runoff could be large, which could have a considerable effect on surface water quality. Both irrigation and N fertilizer application must be controlled and managed with great care to minimize N losses via runoff from agricultural land.     

太湖地区稻田氨挥发及影响因素的研究

应用微气象学方法研究太湖地区水稻三个不同施肥期施用尿素后的氨挥发损失 ,并对其影响因素 (气候、田面水中NH 4 N浓度和作物覆盖等 )的作用进行了分析研究。结果表明 ,水稻施用尿素后的氨挥发损失为各时期施氮量的 18 6 %～ 38 7% ,其中以分蘖肥时期损失最大 ,其次为基肥 ,穗肥氨挥发损失最小。氨挥发损失主要时期是在施肥后 7d内。在水稻不同生长期 ,各因素对氨挥发的影响能力大小并不一样 ,三个施肥期的氨挥发损失通量与施肥后田面水中铵态氮浓度呈显著正相关。     

土壤脲酶活性对稻田田面水氮素转化的影响

以浙江省嘉兴市双桥农场的青紫泥水稻田为研究对象，采用大田小区的研究方法，对不同供肥水平下水稻追施尿素后田面水中氮素主要形态的浓度变化进行了动态监测，并从土壤酶学的角度探讨了其氮素转化的深层机制。研究发现．土壤脲酶在稻田田面水氮素转化中起着关键作用，施尿素后3天内田面水中总氮、氨氮的浓度及氨氮／总氮比值达到峰值．且第二次追肥较第一次追肥后峰值低．随后急剧下降。5天后维持在较低的水平，说明施肥后前5天是氮素大量且快速流失的关键时期，其流失以氨氮为主；土壤脲酶活性在第一次追肥后3～5天也出现了明显的增长趋势，第二次追肥后脲酶活性增强幅度远小于第一次追肥．N270处理初期甚至出现了负增长；线性相关分析表明田面水中氨氮／总氮的比值与土壤脲酶活性之间存在显著的正相关关系．说明土壤脲酶在水土界面氮素迁移转化过程中发挥着关键作用，因此，抑制脲酶活性可能是降低稻田氮素流失的主要途径之一。     

Morphology and general characteristics of phages specific to Lens culinaris rhizobia

Four lytic phages, namely LRP-1, LRP-4, LRP-13, and LRP-15, active against indigenous rhizobial strains of Lens culinaris were isolated and characterized for their individual morphology, host range, plaque characteristics, lytic behavior, and restriction endonuclease profiling of phage DNA. All phages had a typical polyhedral head and long non-contractile tail, representing the bacteriophage family close to Siphoviridae. Phages produced distinct types of plaques on their indicator bacterial strains. The host range of the phage isolates was restricted to Rhizobium leguminosarum biovars and no cross infectivity among susceptible strains was observed. A study on the lytic cycle of the phages under identical conditions exhibited distinct latent period and burst size. Inactivation pattern of phages with temperature and UV light was quite distinct. Phage LRP-1 showed higher thermal resistance, though greater sensitivity to UV light, as compared to other phages. Genome sizes of the phages were estimated to vary between 50–72 kbp. The 16S rRNA sequence analysis of the phage indicator rhizobial strains revealed 81% to 100% similarity with R. leguminosarum bv. viciae. The phages could thus prove to be considerably useful in typing and investigating into the genetic variability which might exist amongst the soil rhizobia nodulating Lens culinaris.     

浮萍对福州平原稻田CH4和N2O排放的影响分析

浮萍是稻田中常见的漂浮在水面的水生植物,具有固氮作用,但是,浮萍对稻田温室气体排放的影响尚不明确.以位于湿润亚热带的福州平原稻田为研究对象,探讨浮萍对该区域稻田CH4和N2O排放的影响,为科学评价、准确编制我国水稻田温室气体排放清单提供基础数据.研究结果表明,观测期内,有萍小区和无萍小区CH4排放范围分别为0.19～26.50 mg·m-2·h-1和1.02～28.02 mg·m-2·h-1,平均值分别为9.28 mg·m-2·h-1和11.66 mg·m-2·h-1,有萍小区CH4排放低于无萍小区(P＜0.01),有萍小区CH4排放高峰比无萍小区约提前1周,高峰期后排放迅速降低;有萍小区和无萍小区N2O排放范围分别为-50.11～201.82 μg·m-2·h-1和-28.93～54.42μg·m-2·h-1,平均值分别为40.29 μg·m-2·h-1和11.93 μg·m-2·h-1,有萍小区N2O排放高于无萍小区(P＜0.05).稻田排干后,N2O排放迅速上升,2个小区N2O排放呈现出相似的规律.有萍小区和无萍小区的CH4与N2O排放的影响因子有所不同.综合考虑CH4和N2O两种温室气体,CH4仍是稻田温室效应产生的主要贡献者,浮萍可降低位于沿海区域的福州平原稻田综合温室效应的17.3％.     

Paddy management on different soil types does not promote lignin accumulation

Paddy soil management is generally thought to promote the accumulation of soil organic matter (SOM) and specifically lignin. Lignin is considered particularly susceptible to accumulation under these circumstances because of the recalcitrance of its aromatic structure to biodegradation under anaerobic conditions (i.e ., during inundation of paddy fields). The present study investigates the effect of paddy soil management on SOM composition in comparison to nearby agricultural soils that are not used for rice production (non‐paddy soils). Soil types typically used for rice cultivation were selected, including Alisol, Andosol and Vertisol sites in Indonesia (humid tropical climate of Java) and an Alisol site in China (humid subtropical climate, Jiangxi province). These soil types represent a range of soil properties to be expected in Asian paddy fields. All upper‐most A horizons were analysed for their SOM composition by solid‐state ¹³C nuclear magnetic resonance (NMR) spectroscopy and for lignin‐derived phenols by the CuO oxidation method. The SOM composition was similar for all of the above named parent soil types (non‐paddy soils) and was also not affected by paddy soil management. A substantial proportion (up to 23%) of the total aryl‐carbon in some paddy and non‐paddy soils was found to originate from condensed aromatic‐carbon (e.g ., charcoal). This may be attributed to the burning of crop residues. On average, the proportion of lignin was low and made up 20% of the total SOM, and showed no differences between straw, particulate organic matter (POM), and the bulk soil material. The results from CuO oxidation are consistent with the data obtained from solid‐state ¹³C NMR spectroscopy. The extraction of lignin‐derived phenols revealed low VSC (vanillyl, syringyl, cinnamyl) values for all investigated soils in a range (4 to 12 g kg⁻¹ OC) that was typical for agricultural soils. In comparison to adjacent non‐paddy soils, the data do not provide evidence for a substantial accumulation of phenolic lignin‐derived structures in the paddy soils, even for those characterized by higher organic carbon (OC) contents (e.g ., Andosol‐ and Alisol (China)‐derived paddy soils). We conclude that the properties of the parent soil types are more important for the lignin content of the soils than the effect of paddy management itself.