Effect of Fungicides and Insecticides on Growth and Enzyme Activity of Four Cyanobacteria

Cyanobacterial populations introduced into crop fields as biofertilizer become non-target organisms for the pesticides and fungicides applied in the field. Effect of four commonly used pesticides viz. Bagalol, Mancozeb (fungicides), Thiodan and Phorate (insecticides) was studied on growth and different enzymes of four cyanobacterial species viz. Nostoc ellipsosporum, Scytonema simplex, Tolypothrix tenuis, and Westiellopsis prolifica. EC 50 concentration of each pesticide was determined for all cyanobacteria. Bagalol and Thiodan were found to be the most toxic. Both the fungicides and insecticides inhibited the activity of nitrogenase and glutamine synthetase (GS) at EC 50 concentration in all the four species studied. Bagalol incurred maximum inhibition of nitrogenase and GS activity on N. ellipsosporum and S. simplex while Thiodan and Phorate had maximum effect on T. tenuis, and W. prolifica. Mancozeb had lesser effect on all the above enzymes. One catabolic enzyme of carbohydrate metabolism, isocitrate dehydrogenase (ICDH) and one anabolic enzyme isocitrate lyase (ICL), which is related to glyoxylate pathway as well as gluconeogenesis, were also assayed. Cell free extracts of cyanobacteria treated with pesticides for 7 days show a drastic reduction of ICDH activity. ICL activity was induced in the organisms when treated with pesticides.     

Impacts of Organic and Conventional Crop Management on Diversity and Activity of Free-Living Nitrogen Fixing Bacteria and Total Bacteria Are Subsidiary to Temporal Effects

A three year field study (2007–2009) of the diversity and numbers of the total and metabolically active free-living diazotophic bacteria and total bacterial communities in organic and conventionally managed agricultural soil was conducted using the Nafferton Factorial Systems Comparison (NFSC) study, in northeast England. Fertility management appeared to have little impact on both diazotrophic and total bacterial communities. However, copy numbers of the nifH gene did appear to be negatively impacted by conventional crop protection measures across all years suggesting diazotrophs may be particularly sensitive to pesticides. Impacts of crop management were greatly overshadowed by the influence of temporal effects with diazotrophic communities changing on a year by year basis and from season to season. Quantitative analyses using qPCR of each community indicated that metabolically active diazotrophs were highest in year 1 but the population significantly declined in year 2 before recovering somewhat in the final year. The total bacterial population in contrast increased significantly each year. It appeared that the dominant drivers of qualitative and quantitative changes in both communities were annual and seasonal effects. Moreover, regression analyses showed activity of both communities was significantly affected by soil temperature and climatic conditions.     

Biological nitrogen fixation by alternative nitrogenases in terrestrial ecosystems: a review

Biological nitrogen fixation (BNF), a key reaction of the nitrogen cycle, is catalyzed by the enzyme nitrogenase. The best studied isoform of this metalloenzyme requires molybdenum (Mo) at its active center to reduce atmospheric dinitrogen (N₂) into bioavailable ammonium. The Mo-dependent nitrogenase is found in all diazotrophs and is the only nitrogenase reported in diazotrophs that form N₂-fixing symbioses with higher plants. In addition to the canonical Mo nitrogenase, two alternative nitrogenases, which use either vanadium (V) or iron (Fe) instead of Mo are known to fix nitrogen. They have been identified in ecologically important groups including free-living bacteria in soils and freshwaters and as symbionts of certain cryptogamic covers. Despite the discovery of these alternative isoforms more than 40 years ago, BNF is still believed to primarily rely on Mo. Here, we review existing studies on alternative nitrogenases in terrestrial settings, spanning inland forests to coastal ecosystems. These studies show frequent Mo limitation of BNF, ubiquitous distribution of alternative nitrogenase genes and significant contributions of alternative nitrogenases to N₂ fixation in ecosystems ranging from the tropics to the subarctic. The effect of temperature on nitrogenase isoform activity and regulation is also discussed. We present recently developed methods for measuring alternative nitrogenase activity in the field and discuss the associated analytical challenges. Finally, we discuss how the enzymatic diversity of nitrogenase forces a re-examination of existing knowledge gaps and our understanding of BNF in nature.

     

Nitrogenase Switch-Off by Ammonium Ions in Azospirillum brasilense Requires the GlnB Nitrogen Signal-Transducing Protein

Nitrogenase activity in several diazotrophs is switched off by ammonium and reactivated after consumption. The signaling pathway to this system in Azospirillum brasilense is not understood. We show that ammonium-dependent switch-off through ADP-ribosylation of Fe protein was partial in a glnB mutant of A. brasilense but absent in a glnB glnZ double mutant. Triggering of inactivation by anaerobic conditions was not affected in either mutant. The results suggest that glnB is necessary for full ammonium-dependent nitrogenase switch-off in A. brasilense.     

Effect of incorporation of crop residues on development of diazotrophs and patterns of acetylene-reducing activity in Nile Valley soils

Summary Acetylene-reducing activity and populations of diazotrophs were estimated simultaneously in Giza soils after harvest of wheat and maize crops. Amendment of soil with residues of either crop together with flood irrigation enhanced the development of diazotrophs and nitrogenase activities in the soil. Bacterial numbers and acetylene-reducing activity decreased as soils dried following flood irrigation. Activities decreased progressively with each cycle of irrigation following the original incorporation of organic matter. Nitrogenase activity in the soil was greater in the cooler winter than in summer.     

Acetylene Reduction by Soil Cores of Maize and Sorghum in Brazil

Nitrogenase activity was measured by the C₂H₂ reduction method in large soil cores (29 cm in diameter by 20 cm in depth) of maize (Zea mays) and sorghum (Sorghum vulgare). The activity was compared to that obtained by a method in which the roots were removed from the soil and assayed for nitrogenase activity after an overnight preincubation in 1% O₂. In a total of six experiments and 28 soil cores, the nitrogenase activity of the cores was an average of 14 times less than the activity of roots removed from the same cores and preincubated. Nitrogenase activity in the cores was very low and extrapolated to an average nitrogen fixation rate of 2.8 g of N/hectare per day. It was shown that inadequate gas exchange was not a reason for the lower activity in the soil cores, and the core method gave satisfactory results for nitrogenase activity of soybeans (Glycine max) and Paspalum notatum.     

Diversity of diazotrophs in the sediments of saline and soda lakes analyzed with the use of the nifH gene as a molecular marker

Phylogenetic analysis of the nifH genes, encoding the Fe protein of the nitrogenase enzymatic complex, was carried out for pure cultures of anoxygenic phototrophic bacteria of diverse origin, as well as for heterotrophic alkaliphilic sulfate reducers isolated from saline and soda lakes. Topology of the nitrogenase tree correlated with that of the 16S rRNA gene tree to a considerable degree, which made it possible to use the nifH gene as a molecular marker for investigation of diazotrophic bacterial communities in sediments of hyper saline and soda lakes. Although diazotrophs were revealed in all environmental samples, their phylogenetic diversity was relatively low. Sulfate-reducing deltaproteobacteria and photo- and chemotrophic gammaproteobacteria were predominant in integrated samples. Analysis of the upper sediment layers revealed predominance of phototrophic diazotrophs of various phyla, including purple sulfur and nonsulfur proteobacteria, green nonsulfur bacteria, heliobacteria, and cyanobacteria. Some phylotypes could not be identified, probably indicating the presence of bacterial groups which have not yet been studied by conventional microbiological techniques.     

Nitrogenase switch-off by ammonium ions in Azospirillum brasilense requires the GlnB nitrogen signal-transducing protein

Nitrogenase activity in several diazotrophs is switched off by ammonium and reactivated after consumption. The signaling pathway to this system in Azospirillum brasilense is not understood. We show that ammonium-dependent switch-off through ADP-ribosylation of Fe protein was partial in a glnB mutant of A. brasilense but absent in a glnB glnZ double mutant. Triggering of inactivation by anaerobic conditions was not affected in either mutant. The results suggest that glnB is necessary for full ammonium-dependent nitrogenase switch-off in A. brasilense.     

Nitrogen fixation with the soybean crop in Brazil: Compatibility between seed treatment with fungicides and bradyrhizobial inoculants

Biological nitrogen fixation with the soybean crop can be improved by seed inoculation with superiorBradyrhizobium strains, but factors that reducethe population of inoculated bradyrhizobiaon the seedwill directly affect the efficiency of the process. Seed treatment with fungicides has been broadly practiced as cheap insurance against seed-and soil-borne pathogens, but toxicity of most fungicides to bradyrhizobia has often been underestimated. The compatibility between seed treatment with fungicides in single or mixed applications (including Benomyl, Captan, Carbendazin, Carboxin, Difenoconazole, Thiabendazole, Thiram, Tolylfluanid) and bradyrhizobial inoculants was examined in laboratory, greenhouse and field experiments during five crop seasons in Brazil. Bacterial survivalon the seeds was severely affected by all fungicides, resulting in mortalities of up to 62% after only 2 h and of 95% after 24 h. Fungicides also reduced nodule number, total N in grains and decreased yield by up to 17%. The toxic effects of fungicides were more drastic in sandy soils without soybean inoculation and cropping history, reducing nodulation by up to 87%, but were also important in areas with established populations of soybean bradyrhizobia. Therefore, fungicides should be used only when the seeds or soil are contaminated with pathogens, otherwise biological N₂ fixation may be severely affected.     

类芦和狗牙根内生固氮菌初步研究

为探索水土保持禾草的固氮能力,利用乙炔还原法(ARA)对类芦(Neyraudia reynaudiana)和狗牙根(Cynodon dactylon)植株内生固氮菌的固氮酶活性进行研究.结果表明:在类芦茎部和狗牙根叶片中存在高固氮酶活性的内生固氮菌群;这些菌群在无氮培养基上经划线分离纯化得到40个菌株,其中26株有微弱固氮酶活性,14株检测不到固氮酶活性;从类芦茎部和狗牙根叶片中筛选到的固氮菌群在兼性厌氧的环境中有较高的固氮酶活性,且在石蜡油密封条件下,两者的固氮酶活性均最高;在不改变自然界微生物之间的协同关系情况下,混合菌群均具有较高的固氮酶活性,而人为组配混合得到的菌群固氮酶活性较低. reynaudiana)和狗牙根(Cynodondxtylon)植株内生固氮菌的固氮酶活性进行研究.结果表明:在类芦茎部和狗牙根叶片中存在高固氮酶活性的内生固氮菌群;这些菌群在无氮培养基上经划线分离纯化得到40个菌株,其中26株有微弱固氮酶活性,14株检测不到固氮酶活性;从类芦茎部和狗牙根叶片中筛选到的固氮菌群在兼性厌氧的环境中有较高的固氮酶活性,且在石蜡油密封条件下,两者的固氮酶活性均最高;在不改变自然界微生物之间的协同关系 况下,混合菌群均具有较高的固氮酶     

尾矿区不同植被恢复模式下高效固氮菌的筛选及Biolog鉴定

为了从铁尾矿中获得高效土著固氮菌,采用选择性无氮培养基从不同恢复模式下铁尾矿土样中筛选高效固氮菌,并且通过乙炔还原法对分离出的48株固氮菌进行固氮酶活性测定。结果表明:筛选出两株具有较高固氮活性菌株Db1与Ec1,Biolog鉴定为固氮菌属(Azotobacter),固氮比活力分别为203.20 nmol·mg-1·h-1和307.23 nmol·mg-1·h-1。不同植被恢复模式、物种多样性、尾矿坡向、工程措施、恢复年限均对尾矿固氮菌株数量有影响。样地处于尾矿阳坡,或属于人造混交林,或植被恢复年限长,又或者实施六孔砖、坡岩造地等工程措施的尾矿植被恢复模式下筛得的固氮菌菌株数较多或固氮活力较高。     

Biological nitrogen fixation in a post-volcanic chronosequence from south-central Chile

Biological nitrogen fixation is a key ecosystem function incorporating new nitrogen (N) during primary successions. Increasing evidence from tropical and northern temperate forests shows that phosphorus (P) and molybdenum (Mo) either alone or in combination limit the activity of free-living diazotrophs. In this study, we evaluated the effects of Mo, P, and carbon (C) addition, either singly or in combination, and moisture, on diazotrophic activity in a post-volcanic forest chronosequence in south-fentral Chile. Diazotrophic activity, both free-living (associated with fine litter) and symbiotic (associated with the moss Racomitrium lanuginosum and the cyanolichens Pseudocyphellaria berberina and P. coriifolia), was evaluated by incubation of samples and subsequent acetylene reduction assays conducted in the field and laboratory, in winter, spring and autumn of two consecutive years. Results showed that diazotrophic activity varied with the season of the year (lowest during the drier spring season), successional stage (highest in the maximal stage), and N-fixer community type (highest in symbiotic diazotrophs). In general, C+P+Mo limitation was documented for heterotrophic diazotrophs and P+Mo limitation for symbiotic diazotrophs. Limitation of diazotrophic activity was not associated with soil nutrient and C status in the chronosequence. Strong inhibition of diazotrophic activity by high N addition and by low moisture suggests that reductions in precipitation expected for south-central Chile under climate change, as well as increasing inputs of reactive N from atmospheric deposition due to increasing use of N fertilizers, may drastically alter the composition and functional role of cryptogamic assemblages in native forests.     

Diazotrophic diversity in the rhizosphere of two exotic weed plants, Prosopis juliflora and Parthenium hysterophorus

This study is aimed at assessing culturable diazotrophic bacterial diversity in the rhizosphere of Prosopis juliflora and Parthenium hysterophorus, which grow profusely in nutritionally-poor soils and environmentally-stress conditions so as to identify some novel strains for bioinoculant technology. Diazotrophic isolates from Prosopis and Parthenium rhizosphere were characterized for nitrogenase activity by Acetylene Reduction Assay (ARA) and 16S rRNA gene sequencing. Further, the culture-independent quantitative PCR (qPCR) was performed to compare the abundance of diazotrophs in rhizosphere with bulk soils. The proportion of diazotrophs in total heterotrophs was higher in rhizosphere than bulk soils and 32 putative diazotrophs from rhizosphere of two plants were identified by nifH gene amplification. The ARA activity of the isolates ranged from 40 to 95 nmol ethylene h⁻¹ mg protein⁻¹. The 16S rRNA gene analysis identified the isolates to be members of alpha, beta and gamma Proteobacteria and firmicutes. The qPCR assay also confirmed that abundance of nif gene in rhizosphere of these two plants was 10-fold higher than bulk soil.     

Diversity and activity of free-living nitrogen-fixing bacteria and total bacteria in organic and conventionally managed soils

Agricultural soils are heterogeneous environments in which conditions affecting microbial growth and diversity fluctuate widely in space and time. In this study, the molecular ecology of the total bacterial and free-living nitrogen-fixing communities in soils from the Nafferton Factorial Systems Comparison (NFSC) study in northeast England were examined. The field experiment was factorial in design, with organic versus conventional crop rotation, crop protection, and fertility management factors. Soils were sampled on three dates (March, June, and September) in 2007. Total RNA was extracted from all soil samples and reverse transcribed. Denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were used to analyze nifH and 16S rRNA genes in order to study free-living diazotrophs and the total bacterial community, respectively. Crop rotation was shown to have a significant effect on total bacterial diversity (and that of free-living N fixers) (P ≤ 0.001). On all three dates, nifH activity was higher in the conventional crop rotation. In contrast, qPCR analysis of free-living N fixers indicated significantly higher levels of activity in conventionally fertilized plots in June (P = 0.0324) and in plots with organic crop protection in September (P = 0.0143). To our knowledge, the effects of organic and conventional farming systems on free-living diazotrophs have never been studied. An increased understanding of the impacts of management practices on free-living N fixers could allow modifications in soil management practices to optimize the activity of these organisms.     

Environmental conditions influence the plant functional diversity effect on potential denitrification

Global biodiversity loss has prompted research on the relationship between species diversity and ecosystem functioning. Few studies have examined how plant diversity impacts belowground processes; even fewer have examined how varying resource levels can influence the effect of plant diversity on microbial activity. In a field experiment in a restored wetland, we examined the role of plant trait diversity (or functional diversity, (FD)) and its interactions with natural levels of variability of soil properties, on a microbial process, denitrification potential (DNP). We demonstrated that FD significantly affected microbial DNP through its interactions with soil conditions; increasing FD led to increased DNP but mainly at higher levels of soil resources. Our results suggest that the effect of species diversity on ecosystem functioning may depend on environmental factors such as resource availability. Future biodiversity experiments should examine how natural levels of environmental variability impact the importance of biodiversity to ecosystem functioning.     

Cyanobacteria in Uruguayan rice fields: diversity, nitrogen fixing ability and tolerance to herbicides and combined nitrogen

It has been established that cyanobacteria play a vital role in the maintenance of flooded rice field fertility. To evaluate the potential use of nitrogen-fixing cyanobacteria as a natural biofertilizer for rice in Uruguay, the diversity, abundance and nitrogen fixing ability of these microorganisms were studied in the field and in the laboratory. The effect of urea fertilization on population density and diversity of heterocystous cyanobacteria was determined on a 3-year assay. The highest number of cyanobacteria, 1.6x10(4) CFU x m(-2), was found at the control 8 weeks after flooding. About 90% of the heterocystous cyanobacteria found in both treatments belong to the genera Nostoc and Anabaena. Maximal nitrogenase activity was reached after 12 weeks of flooding in both treatments, with an average of about 20 micromol C2H4 x m(-2) x h(-1). To improve the understanding of the environmental factors that can limit nitrogenase activity in rice fields, two of the most abundant cyanobacteria isolates were tested for tolerance to combined nitrogen and two herbicides. In both isolates 0.2 mM ammonium inhibited nitrogenase activity after 24 h of culture. The addition of field-recommended doses of quinclorac and propanil affected oxygen photoevolution but nitrogenase activity was only inhibited by propanil.     

Discovery and evidence of nitrogen fixation by thermophilic heterotrophs in hot springs

A previously unknown phenomenon, nitrogenase activity at high temperatures by nonphotosynthetic prokaryotes in thermal springs, is reported. This activity was revealed by dark-bottle incubations during cyanobacterial nitrogenase studies in hot springs of Yellowstone National Park. Benthic mat cores from alkaline thermal streams reduced acetylene in the dark from 30° to 60°C. the bacterial activity was distinguished from that of sympatric cyanobacteria by its sensitivity to oxygen (light bottles), heterotrophic response to organic amendments, and distinctive spatial distribution. Field tests for other biotic or abiotic explanations of the activity were negative. Anaerobic enrichment cultures supported the field evidence. Fourteen morphological types of presumptive diazotrophs, including sulfate-reducing and spore-forming rods. have been observed in these cultures.