Impacts of 2,4-D application on soil microbial community structure and on populations associated with 2,4-D degradation

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) application rate on microbial community structure and on the diversity of dominant 2,4-D degrading bacteria in an agricultural soil was examined using cultivation-independent molecular techniques coupled with traditional isolation and enumeration methods. Fingerprints of microbial communities established under increasing concentrations of 2,4-D (0-500 mg kg-1) in batch soil microcosms were obtained using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments. While a 2,4-D concentration of at least 100 mg kg-1 was required to obtain an apparent change in the community structure as visualized by DGGE, the greatest impact of 2,4-D concentration occurred in the 500 mg kg-1 treatment, resulting in significantly reduced diversity of the dominant populations and enrichment by Burkholderia-like populations. The greatest diversity of 2,4-D degrading isolates was cultivated from the 10 mg kg-1 treatment, indicating that under these conditions, cultivation was more sensitive than DGGE for detecting changes in community structure. Most of these isolates harbored homologs of Ralstonia eutrophus JMP134 and Burkholderia cepacia tfdA catabolic genes. Results from this study revealed that agriculturally relevant application rates of 2,4-D may provide a temporary selective advantage for organisms capable of utilizing 2,4-D as a carbon and energy source.     

Growth and survival of Pseudomonas cepacia DBO1(pRO101) in soil amended with 2,4-dichlorophenoxyacetic acid

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading pseudomonad, Pseudomonas cepacia DBO1(pRO101), was inoculated at approximately 10⁷ CFU/g into sterile and non-sterile soil amended with 0, 5 or 500 ppm 2,4-D and the survival of the strain was studied for a period of 44 days. In general, the strain survived best in sterile soil. When the sterile soil was amended with 2,4-D, the strain survived at a significantly higher level than in non-amended sterile soil. In non-sterile soil either non-amended or amended with 5 ppm 2,4-D the strain died out, whereas with 500 ppm 2,4-D the strain only declined one order of magnitude through the 44 days.The influence of 0,0.06, 12 and 600 ppm 2,4-D on short-term (48 h) survival of P. cepacia DBO1(pRO101) inoculated to a level of 6×10⁴, 6×10⁶ or 1×10⁸ CFU/g soil was studied in non-sterile soil. Both inoculum level and 2,4-D concentration were found to have a positive influence on numbers of P. cepacia DBO1(pRO101). At 600 ppm 2,4-D growth was significant irrespective of the inoculation level, and at 12 ppm growth was stimulated at the two lowest inocula levels. P. cepacia DBO1(pRO101) was able to survive for 15 months in sterile buffers kept at room temperature. During this starvation, cells shrunk to about one third the volume of exponentially growing cells.Abbreviations AODC acridine orange direct count - CFU colony forming units - PTYG-Agar peptone, tryptone, yeast & glucose agar - TET tetracycline - LB Luria Bertani medium     

Effects of temperature and soil moisture on survival of eggs of the mosquito Psorophora columbiae (Diptera: Culicidae)

The results of laboratory tests indicated the average survival rates for Psorophora columbiae eggs remained quite high for all of the egg populations exposed to a temperature of 27°C (range 83.0–100.0% survival) after 96 days of exposure, except for the non‐diapausing eggs on dry soil (66.3%). In regard to the exposure of egg populations to moderately cold temperatures (i.e. 8°C, 4°C and ?2°C) for periods of up to 16 days, survival rates for egg populations exposed to 8°C continued to remain relatively high (average >85%) for the remainder of the experimental exposure period (i.e. 96 days). Diapausing Ps. columbiae eggs were more tolerant (82.0% survival) to low temperatures (?2°C) than non‐diapausing eggs (2.4% survival) for 64 days, particularly at temperatures of and below 4°C. Diapausing and non‐diapausing eggs were similar in their ability to survive under high temperatures (34°C and 38°C). High soil moisture (30–40%) or substrate moisture (95% relative humidity) content appeared to enhance the ability of the mosquito eggs to survive both low and high temperature extremes.     

Relative importance of the effect of 2,4-D,glyphosate, and environmental variables on the soil microbial biomass

Two post-emergence herbicides (glyphosate and 2,4-D) were applied at field application levels to tilled field plots in a mixed cropping area in south-central Alberta. The effects of these chemicals on certain variables associated with microbial biomass and activity were monitored in these plots (as well as corresponding control plots) for 45 days. Glyphosate did not influence any of the microbial variables tested. Addition of 2,4-D significantly influenced all microbial variables investigated but these effects were transient, being detectable only within the first 1–5 days of herbicide addition. The effects of 2,4-D addition on the microbial variables tested, even when significant, were typically small and probably of little ecological consequence especially when spatial and temporal variation in these variables is taken into account.     

Plant protection and rhizosphere colonization of barley by seed inoculated herbicide degrading Burkholderia (Pseudomonas) cepacia DBO1(pRO101) in 2,4-D contaminated soil

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacterium, Burkholderia cepacia (formerly Pseudomonas cepacia) DBO1(pRO101) was coated on non-sterile barley (Hordeum vulgare) seeds, which were planted in two non-sterile soils amended with varying amounts of 2,4-D herbicide. In the presence of 10 or 100 mg 2,4-D per kg soil B. cepacia DBO1(pRO101) readily colonized the root at densities up to 10⁷ CFU per cm root. In soil without 2,4-D the bacterium showed weak root colonization. The seeds coated with B. cepacia DBO1(pRO101) were able to germinate and grow in soils containing 10 or 100 mg kg^–1 2,4-D, while non-coated seeds either did not germinate or quickly withered after germination. The results suggest that colonization of the plant roots by the herbicide-degrading B. cepacia DBO1(pRO101) can protect the plant by degradation of the herbicide in the rhizosphere soil. The study shows that the ability to degrade certain pesticides should be considered, when searching for potential plant growth-promoting rhizobacteria. The role of root colonization by xenobiotic degrading bacteria is further discussed in relation to bioremediation of contaminated soils.     

2,4-D impact on bacterial communities, and the activity and genetic potential of 2,4-D degrading communities in soil

The key role of telluric microorganisms in pesticide degradation is well recognized but the possible relationships between the biodiversity of soil microbial communities and their functions still remain poorly documented. If microorganisms influence the fate of pesticides, pesticide application may reciprocally affect soil microorganisms. The objective of our work was to estimate the impact of 2,4-D application on the genetic structure of bacterial communities and the 2,4-D-degrading genetic potential in relation to 2,4-D mineralization. Experiments combined isotope measurements with molecular analyses. The impact of 2,4-D on soil bacterial populations was followed with ribosomal intergenic spacer analysis. The 2,4-D degrading genetic potential was estimated by real-time PCR targeted on tfdA sequences coding an enzyme specifically involved in 2,4-D mineralization. The genetic structure of bacterial communities was significantly modified in response to 2,4-D application, but only during the intense phase of 2,4-D biodegradation. This effect disappeared 7 days after the treatment. The 2,4-D degrading genetic potential increased rapidly following 2,4-D application. There was a concomitant increase between the tfdA copy number and the 14C microbial biomass. The maximum of tfdA sequences corresponded to the maximum rate of 2,4-D mineralization. In this soil, 2,4-D degrading microbial communities seem preferentially to use the tfd pathway to degrade 2,4-D.     

The influence of soil moisture and flooding on formation of VA-endo- and ectomycorrhizae in Populus and Salix

Native mixtures of extomycorrhizal fungi were found to infect Populus and Salix roots primarily in very moist but well drained soils in both the field and in controlled experiments (0 to –0.2 MPa), whereas native mixtures of VA-endomycorrhizal fungi infected roots over a much wider range of soil moisture (flooded to –3.4 MPa). Although a moisture gradient experiment showed endomycorrhizal formation was greater in moist soil than in very dry or flooded soils, this pattern was reversed in field transects along drainage gradients. Infection by VA-endomycorrhizal fungi in the field was the lowest where infection by ectomycorrhizal fungi was high, which suggests possible antagonism among the fungal symbionts. The narrow moisture range for ectomycorrhizal formation, and antagonism among endo- and ectomycorrhizal fungi, apparently combine to produce the mycorrhizal distributions found in nature.     

天山北坡积雪消融对不同冻融阶段土壤温湿度的影响

积雪作为一种特殊的覆被,直接影响着土壤温度、土壤水分分布及其冻结深度、冻结速率等,影响当地的生态水文过程。利用2017年11月1日至2018年3月31日天山北坡伊犁阿热都拜流域的土壤含水率资料,划分土壤不同冻融阶段,结合积雪不同阶段,进而分析积雪消融对季节性冻土温湿度的影响。结果表明:在整个土壤冻融期间,土壤温湿度的变化取决于积雪深度、大气温度和雪面温度的高低,且与其稳定性有关。土壤冻结阶段,土壤温湿度持续下降,表层土壤温湿度受气温影响较大,且波动明显,而深层土壤的温湿度变化平缓;土壤完全冻结时,有稳定积雪覆盖,由于积雪的高反射性、低导热性,影响着地气之间的热量传递,因此土壤的温湿度变化较为平稳,积雪有一定的保温作用;冻土消融阶段,气温回升,积雪消融,地表出露,各层土壤温度随气温变化而变化,且越靠近地表,土壤温度越高,变幅越大,与冻结期完全相反。由于融雪水的下渗,土壤湿度快速增加。进一步分析积雪与土壤温湿度的相关性得出,积雪对土壤温湿度的影响分不同时期,对土壤温度的影响主要在积雪覆盖时,对土壤湿度的影响主要是在积雪消融时期,这对于研究该地生态水文循环及后续融雪性洪水的模拟与预报具有一定...     

土壤温度和水分对油松林土壤呼吸的影响

用LI-COR 6400-09土壤呼吸测定系统,在太原天龙山自然保护区对油松林的土壤呼吸进行了4a测定.结果表明,土壤呼吸具有明显的季节变化特点,最大值出现在8月份,在6～10 μmol m~(-2) s~(-1) 之间,最小值出现在12月份和3月份,在0.5 μmol m~(-2) s~(-1)左右.2005、2006、2007和2008年土壤呼吸CO_2的年平均值分别为(4.71±3.74)、(3.08±2.91)、(2.96±2.58) μmol m~(-2) s~(-1)和(2.12±1.54) μmol m~(-2) s~(-1);4a的CO_2总平均值为(3.27±2.95) μmol m~(-2) s~(-1).4个测定年土壤呼吸的平均值总体差异显著.4个测定年土壤CO_2释放C量分别为1103.5、882.8、918.4 g m~(-2)和666.3 g m~(-2),总C平均值为892.8 g m~(-2),具有明显的年际差异.指数方程可以很好的表达土壤呼吸与10 cm深度土壤温度的关系,R~2值4a分别为0.39,0.60,0.68和0.71,Q_(10)值分别为3.10,4.41、4.05和5.18,用4a全部数据计算的Q_(10)值为4.31.土壤水分对土壤呼吸的作用较弱,R~2值4a分别仅为0.31、0.25、0.13和0.02,但是夏季土壤干旱对土壤呼吸的抑制作用非常明显,可使土壤呼吸下降50%以上.夏季土壤干旱是导致土壤呼吸年际变化的主要原因.4个包括土壤温度和水分的双变量模型均可以很好地模拟土壤呼吸的季节变化, 拟合方程的R~2值从0.58到0.79.     

覆盖方式对旱地冬小麦土壤水分的影响

在黄土高原半干旱雨养条件下,研究了不同覆盖方式(夏季覆膜,T₁;秋季覆膜,T₂;小麦碎秆覆盖,T₃;小麦整秆覆盖,T₄;夏季覆膜+麦秆还田,T₅;旧膜二茬利用,T₆;无覆盖对照,CK)对旱地冬小麦土壤水分的影响.结果表明: T₆在各时期、各土层土壤含水量普遍高于CK,其他5个覆盖处理可明显改善开花前0～90 cm土壤墒情,但开花后0～90 cm土层以及全生育期90～200 cm土层含水量普遍低于CK.全生育期0～200 cm土层平均含水量T₆显著高于CK,两者差值为0.9%,其余处理均低于CK.0～200 cm土层平均含水量秸秆覆盖处理高于覆膜处理,旧膜二茬利用高于新覆膜.覆膜处理单位面积籽粒产量较CK提高20.3%～29.0%,秸秆覆盖处理较CK提高5.0%～16.7%,冬小麦产量与生育期耗水量呈显著正相关(r=0.77^*).     

水分对敦煌阳关湿地芦苇叶片与土壤C、N、P生态化学计量特征的影响

土壤水分是影响干旱区植物养分吸收和利用策略的关键因子之一。研究不同水分梯度叶片与土壤生态化学计量特征,有助于揭示植物对环境变化的响应特征及生态适应性。通过野外调查与实验分析,对敦煌阳关不同水分梯度芦苇叶片与土壤碳(C)、氮(N)、磷(P)生态化学计量特征及其关系进行了研究。结果表明:(1)随土壤含水率升高,叶片C、N、P含量降低,叶片C/N、C/P、N/P升高。(2)随土壤含水率升高,土壤有机碳(OC)、总氮(TN)、总磷(TP)含量及土壤N/P升高,土壤C/N降低,土壤C/P先升后降。(3)低水分梯度叶片N、C/N与土壤N、C/N显著负相关(P<0.05),叶片C、P、C/P、N/P与土壤C、P、C/P、N/P无显著相关性(P>0.05);高、中水分梯度叶片C、N、P与土壤C、N、P化学计量特征相关性均不显著(P>0.05)。低水分梯度叶片受干旱胁迫和土壤养分制约,且能够保持较高的叶养分含量,体现了干旱区湿地植物异质生境下独特的养分调节机制。     

水分和温度对若尔盖湿地和草甸土壤碳矿化的影响

土壤碳矿化及其温度和水分敏感性是研究生态系统碳循环的重要指标。本文以若尔盖高寒湿地和草甸为对象,在不同水分（70%,100%,130%饱和含水量（SSM））和温度（5,10,15,20,25℃）培养下定期测定土壤碳矿化速率（或土壤微生物呼吸速率）,探讨水分和温度对高寒湿地和草甸土壤碳矿化的影响,为揭示未来暖干化对若尔盖地区碳贮存及其碳汇功能的潜在影响提供科学依据。实验结果表明：增温显著促进了高寒湿地和草甸土壤碳矿化,而水分过高会抑制土壤碳矿化;此外,高寒湿地土壤碳矿化速率高于高寒草甸。土壤水分和草地类型对土壤碳矿化温度敏感性（Q10）的影响比较复杂。高寒草甸Q10随水分升高而显著升高,培养7天时的Q10变化趋势为70% SSM（1.21）< 100% SSM（1.76）< 130% SSM（2.80）,培养56天的Q10从1.17上升为4.53。高寒湿地的Q10在培养7天差异不显著,但整个56天培养期内Q10随水分升高而显著增加。在评估暖干化对若尔盖地区碳贮量和碳汇功能的影响时,应更加重视高寒草甸和高寒湿地Q10对水分和温度变化的不同响应。     

黄土半干旱区坡地土壤水分、养分及生产力空间变异

通过田问取样,分析黄土半干旱丘陵区陡坡面土壤水分、养分及其生产力空间变异性及其相关关系．结果表明,坡面0～20cm土层土壤各养分含量均不同程度地高于20～40cm土层,但变异程度却明显低于20～40cm土层,且除全磷外,土壤养分变异程度均明显高于水分．20～40cm土层土壤养分沿坡面向下逐渐增大,而0～20cm沿坡面纵向差异较小．坡面浅沟沟槽处的土壤水分、养分条件最优,但其地上生物量却低于坡顶．虽然纵向坡度(35°～45°)明显大于横向(5°～10°),但横向坡位对土壤养分的影响却明显大于纵向(除20～40cm土层速效磷含量除外),而对水分的影响纵向大于横向．相关分析表明,0～120cm土壤水分与20～40cm土壤养分以及土壤养分之间(0～20cm速效磷除外)均呈极显著相关．坡位对土壤水分、养分及地上生物量有很大影响,但土壤水分、养分对生物量影响不显著．     

温度和湿度对我国内蒙古羊草草原土壤净氮矿化的影响

土壤氮素的矿化是反映土壤供氮能力的重要因素之一 ,也是目前国内外研究的热点。通过测定内蒙古典型羊草草原自由放牧地土壤净氮矿化量和净氮矿化速率 ,揭示影响草地生态系统土壤氮循环过程的有关机理 ,为草地生态系统建模提供理论依据。在实验室条件下 ,运用恒温恒湿培养箱控制土壤的温度与湿度 ,测定羊草草原长期自由放牧地土壤氮素矿化量的积累。将不同水分含量的土柱分别放在温度为 - 10℃、0℃、5℃、15℃、2 5℃和 35℃的恒温恒湿培养箱中培养 ,培养 1、2、3、5周后取出 ,分析培养前后的 NH 4- N和 NO- 3- N含量 ,以确定土壤净氮矿化 (NH 4- N NO- 3- N)的累积和不同时间段内的矿化速率。结果表明 :不同处理温度和水分之间的差异均达到显著水平 (p<0 .0 0 0 1)。温度和水分之间具有显著的交互作用 (p<0 .0 0 0 1)。随着培养时间的延长 ,矿化氮累积量增加 ,但是矿化速率下降     

Ecologically significant effects of Pseudomonas putida PPO301(pRO103), genetically engineered to degrade 2,4-dichlorophenoxyacetate, on microbial populations and processes in soil

Pseudomonas putida PPO301 (pRO103), genetically engineered to degrade 2,4-dichlorophenoxyacetate, affected microbial populations and processes in a nonsterile xeric soil. In soil amended with 2,4-dichlorophenoxyacetate (500 micrograms/g soil) and inoculated with PPO301 (pRO103), the rate of evolution of carbon dioxide was retarded for approximately 35 days; there was a transient increase in dehydrogenase activity; and the number of fungal propagules decreased below detection after 18 days. In unamended soil inoculated with PPO301(pRO103), the rate of evolution of carbon dioxide and the dehydrogenase activity were unaffected, and the numbers of fungal propagules were reduced by about two orders of magnitude. The numbers of total, spore-forming, and chitin-utilizing bacteria were reduced transiently in soil either amended or unamended with 2,4-dichlorophenoxyacetate and inoculated with PPO301(pRO103). The activities of arylsulfatases and phosphatases in soil were not affected by the presence of PPO301(pRO103), either in the presence or absence of 2,4-dichlorophenoxyacetate. In soil amended with 2,4-dichlorophenoxyacetate and inoculated with the parental strain (PPO301) or not inoculated, the evolution of carbon dioxide, the numbers of fungal propagules and of total, spore-forming, and chitin-utilizing bacteria, and the dehydrogenase activity were not affected as in soil inoculated with PPO301(pRO103). These results demonstrated that a genetically engineered microorganism, in the presence of the substrate on which its novel genes can function, is capable of inducing measurable ecological effects in soil.     

温室黄瓜根结线虫发生地土壤微生物宏基因组文库的构建及其一个杀线虫蛋白酶基因的筛选

【目的】本研究旨在通过非培养手段构建和筛选宏基因组文库,以求找到新型的杀线虫蛋白酶基因。【方法】采用密度梯度离心法提取和纯化温室土壤微生物总DNA,经平末端、连接、包装、转染后,构建宏基因组Fosmid文库,同时,以脱脂奶为底物,以根结线虫为靶标,对文库进行功能初筛。【结果】该文库库容31008个克隆,平均插入片段36.5kb,包含1.13Gbp的微生物基因组信息,适合大规模的微生物功能基因筛选,通过功能初筛,筛选到1个含杀线虫蛋白酶基因的Fosmid克隆(pro12)。进一步构建和筛选出亚克隆(espro124a5),通过对基因结构进行了初步分析发现:espro124a5是一种分泌型胞外蛋白酶,与来自于Maricaulis maris MCS10(accession no.YP_756822at NCBI)的丝氨酸蛋白酶S15仅有45%的同源性,是一种新型的丝氨酸蛋白酶,有其保守的催化三元组:Asp469、His541和Ser348。【结论】密度梯度离心法提取到的DNA纯度高、片段长,完全能满足构建宏基因组Fosmid文库的要求;同时,构建的宏基因组Fosmid文库库容大,有利于我们从中筛选其他的微生物基因资源。     

The influence of soil type and moisture on pupal survival of Bactrocera tryoni (Froggatt) (Diptera: Tephritidae)

Abstract  Larvae of the Queensland fruit fly, Bactrocera tryoni , pupate in the soil, but the influence of soil variables on B. tryoni pupal mortality is not known. For other tropical tephritid species, soil moisture has been identified as a major pupal mortality factor. In the laboratory, we tested the effects of soil moisture and soil type on pupal survival through a factorial experiment which used three soil types (loamy sand, loam, sandy clay) and seven soil moisture levels (0%, 10%, 25%, 50%, 75%, 90% and 100%). Minor, but significant, differences in pupal mortality were observed between the soil types, but the most significant factor affecting pupae was extremes of soil moisture. Eighty-five percent pupal mortality occurred at 0% soil moisture and 30% mortality at 100% soil moisture: very low levels of mortality occurred at all intermediate levels. We detected a significant interaction between soil type and moisture level but cannot explain it. In a follow-up experiment, we demonstrated that prepupal wandering larvae of B. tryoni could discriminate between different moisture levels, with significantly greater pupation in loam soil at 75% soil moisture than at either 0% or 100% soil moisture. Results are used to modify a pupal mortality/soil moisture equation used in a recently published DYMEX model of B. tryoni population dynamics .     

黄土丘陵区坡面整地和植被耦合下的土壤水分特征

水分是干旱半干旱地区植被恢复的主要环境制约因子。在黄土高原小流域,合理整地能够有效截留降雨补给土壤水,进而促进植被恢复。选择地处甘肃定西的半干旱黄土小流域为研究区,基于野外实测数据,分析不同植被和整地方式(柠条水平阶、山杏水平沟、侧柏反坡台,油松反坡台)综合影响下的土壤水分特征。采用最优分割法将不同整地方式土壤水分垂直层次划分为活跃层,次活跃层和相对稳定层。结果表明:生长季不同整地方式土壤水分的变化与降水量的变化密切相关,不同月份以及不同深度各整地方式土壤水分之间差异显著(P0.05)。根据土壤水分垂直变化特征,山杏水平沟水分活跃层与次活跃层为0—80cm,其深度范围均大于其他3种整地方式,而柠条水平阶土壤水分均在30 cm以下较为稳定,其深度范围均小于其他3种整地方式。不同整地方式土壤水分含量具体表现为:山杏水平沟侧柏反坡台柠条水平阶油松反坡台。     

间作经济作物对黄土丘陵区旱作红枣土壤水分的调控效应

研究行间种植经济作物饲料油菜和黄花菜对黄土丘陵区旱作红枣林地土壤水分的调控效应.结果表明： 饲料油菜和黄花菜处理0～180 cm土层土壤含水量较无作物对照分别提高6.2%和10.1%;枣树生育期内土壤水分变化主要集中在0～60 cm土层,饲料油菜和黄花菜处理均明显增加了0～60 cm土层土壤含水量,保证枣树生育期内正常生长;持续干旱条件下,各处理土壤水分消耗主要在0～60 cm土层,其中0～20 cm土层土壤含水量与次降雨后干旱天数存在显著指数负相关,雨后18 d干旱期饲料油菜和黄花菜处理0～60 cm土层土壤水分含量均高于对照.该间作系统显著改善了红枣林土壤水分环境,是黄土丘陵区克服季节性干旱的有效措施.