Utility of some nitrogen-fixing microorganisms in the phyllosphere of crop plants

Summary The utility of spraying some known N₂-fixing microorganisms on rice leaves grown both in N-less sand culture and under field conditions was examined. The effect was compared with that of spraying a phyllosphere N₂-fixing isolate of Klebsiella, KUPBR₂, and application of nitrogenous fertilizers. All the growth parameters studied including dry weight and N-content were enhanced. Under field conditions number of tillers was increased by 26% withKlebsiella pneumoniae M5al and by 65% with Aphanothece. The dry weight of the plants was enhanced by 61–119%. The yield per 10 m² was almost doubled with Aphanothece, Beijerinckia 8007,Mycobacterium flavum, K. pneumoniae M5al and KUPBR₂. The increases observed withStreptomyces sp. G12 though less spectacular was significant at 1% level with respect to several growth parameters.K. pneumoniae M5al,M. flavum andStreptomyces sp. G12 exhibited nitrogenase activity both in laboratory culture and in association with rice plants.     

植物叶际固氮菌研究进展

固氮菌广泛存在于植物叶际,能固定空气中的氮气,满足自身或植物的部分氮素需求。基于纯种分离和培养的传统生物学方法已经研究了部分叶际固氮菌的特性,但对叶际固氮菌的物种组成、群落结构及生态功能等方面的认识还非常有限。随着分子生物学技术的发展、微生物分子生态学研究方法的逐步成熟,人们对叶际微生物的多样性和生态功能的研究越来越深入。叶际固氮菌具有丰富的多样性,温度、湿度、光照等环境因素及植物种类和微生物互作均会影响叶际固氮菌的组成。不同于根际固氮菌,叶际固氮菌具有非专一性,且不易受化肥的影响,其在农业生产上已经表现出潜在的应用价值。为此,本文综述了农作物、森林、海域生态系统中叶际固氮菌的群落结构组成及生态功能,以及外界因素对叶际固氮系统的影响。     

Effect of spraying nitrogen-fixing phyllospheric bacterial isolates on wheat plants

Summary Culture of two nitrogen-fixing bacteria (REN₂ and JN₁) isolated from rice and jute phyllospheres respectively, were sprayed on wheat plants as substitute for nitrogenous fertilisers. There was a marked improvement in yield and growth of the plants. An average increase in yield by 70% was obtained which was very near to that obtained by fertilizer treatment.     

Microclimatic parameters influencing nitrogen fixation in the phyllosphere in a Costa Rican premontane rain forest

The acetylene reduction method was used to measure nitrogen fixation in the phyllosphere of attached leaves of different phorophytes under natural conditions in a premontane rain forest in Costa Rica. Maximum rates of nitrogen fixation (26 ng N · cm⁻² leaf area · h⁻¹) – mainly due to the activity of two species of Scytonema (Cyanobacteria) – were measured in the rainy season in bright sunlight. Rates of nitrogen fixation were correlated with the leaf area covered by Scytonema. In periods without precipitation the fixation activity decreased to zero within 2–3 days. As long as the epiphylls were sufficiently supplied with water, other microclimatic factors like temperature and light intensity also influenced nitrogen fixation rates, but to a lesser extent. Relative humidity and species of phorophyte showed no direct influence. It was concluded that the most important factor for nitrogen fixation in the phyllosphere was the availability of liquid water. Linking these results to meteorological data, the input of nitrogen by biological nitrogen fixation in the phyllosphere in the investigation area was estimated to be as much as 1.6 ± 0.8 kg N · ha⁻¹ · year⁻¹ per unit of leaf area index (LAI). For an LAI of 2 for the understory the nitrogen input would vary between 2 and 5 kg N · ha⁻¹ · year⁻¹. This work also demonstrates the importance of detailed knowledge of variation in microclimate throughout the year as a basis for extrapolation of the annual nitrogen input. Received: 21 December 1997 / Accepted: 14 June 1998     

Utility of phyllosphere N2-fixing micro-organisms in the improvement of crop growth

Summary The beneficial effect of spraying some highly active phyllosphere N₂-fixing microorganisms on high and low yielding cultivars of rice plants as compared with that of urea applied at different doses are described. The dry weight, N-content, 1000 grain weight, and yield were remarkably increased in all cases with the application of phyllosphere microorganisms. The performance of two isolates KUP₄ and KUPBR₂ with IR-8 and IR-26 rice, was better than that of 52 kg urea-N per hectare. IR-579 rice leaves in association with some phyllosphere bacteria reduced acetylene at the rate of 664–816 nmoles/g leaf/h. In IR-26 rice the effect of application of bacterial suspension at three phases of plant growth corresponded very well with that of urea application in three split doses under identical conditions. Recommended fertilizer rates produced the same yield as the half dose plus bacterial spray in the cultivars Pankaj and Rupsail. Fertilizer application in Pankaj and Rupsail rice reduced nitrogenase activity and the beneficial effects of phyllosphere N₂-fixation was reduced by 40–55%.     

一株高效甘蔗内生固氮细菌GXS16的鉴定及其对甘蔗的促生长作用

【背景】我国甘蔗生产中氮肥过量施用严重,导致生产成本居高不下,充分发挥甘蔗与内生固氮菌的联合固氮作用,减少氮肥施用量,对促进我国甘蔗产业可持续发展具有重要意义。【目的】筛选优势甘蔗内生固氮菌,对其基本特性、联合固氮效率及促生长功能进行评价。【方法】从甘蔗根系分离到一株内生固氮菌GXS16,利用乙炔还原法测定固氮酶活性,通过PCR扩增nifH基因确定菌株为固氮菌;通过形态观察、Biolog检测和16S rRNA基因序列分析等对菌株进行分类;通过接种盆栽甘蔗检测菌株的促生长作用,采用¹⁵N同位素稀释法检测菌株相对固氮效率。【结果】菌株GXS16固氮酶活性为2.42μmol-C₂H₄/(h·mL),根据菌株培养性状和菌体形态观察、Biolog检测、16S rRNA、nifH、acdS基因序列分析结果,菌株GXS16属于伯克氏菌属(Burkholderia);菌株GXS16还具有1-氨基环丙烷-1-羧酸脱氨酶(1-Aminocyclopropane-1-Carboxylate Deaminase,ACC)活性及合成生长素吲哚乙酸...     

Seasonal and spatial distribution pattern of nitrogen fixing bacteria in fish ponds under different management systems

Enumeration of nitrogen fixing bacterial population in water and sediment samples of six fish farming ponds (polyculture, monoculture and traditional systems) over a period of two and a half years revealed significant differences between the culture systems with maximal and lowest counts in the monoculture and traditional systems, respectively. Polynomial equations of the 2nd to 4th degree were used to represent the observed seasonal data in these ponds. The bacterial populations peaked in summer, while the lowest count was observed in winter. The environmental factors such as pH, different species of nitrogen, dissolved organic carbon were responsible for the seasonal changes in nitrogen fixing bacteria.     

Study of rhizosphere and phyllosphere bacterial community and resistance to bacterial canker in genetically engineered phytochrome A cherry plants

The cherry rootstock 'Colt' line was transformed with a phytochrome A rice gene with the aim of altering light perception. Three transgenic events were chosen because of a modified developmental behavior. When red enriched light was supplied horizontally to stems, the PD3 transgenic line showed an increased rate of phytomer formation associated to a superior rate of plant growth compared to wild type (WT). Under the same light conditions, the PO1 and PA lines were less altered in morphology and development. When far-red enriched light was supplied, all transgenic lines had a reduced rate of growth, with the PD3 line being the most similar to the WT. The influence of the alien gene on root and leaf-associated bacteria was studied for a duration of 1 year. Significantly more culturable bacteria were recovered from PA lines than from PO1, PD3 and WT lines. On average, significantly more fluorescent pseudomonads were recovered from the rhizosphere of PA and PO1 lines than from PD3 and WT. No significant differences were detected in the number of bacteria recovered from the phyllosphere of transgenic and WT plant lines. A total of 143 Pseudomonas fluorescens strains isolated from rhizosphere of transgenic and WT lines were tested for their antagonistic activity against Phytophthora nicotianae and differences between bacteria derived from transgenic and WT were not detected. Fluorescent pseudomonads strains isolated from phyllosphere of PA and PO1 lines showed antagonistic activity against P. syringae pv. syringae, whereas no difference among the transgenic and WT lines was detected when fluorescent Pseudomonas strains were tested against P. syringae pv. mors-prunorum. Pathogenicity tests were conducted on rooted and micropropagated plants with P. s. pv. syringae and P. s. pv. mors-prunorum: in all assays, the PO1 lines were the most tolerant to P. s. pv. Syringae, and the PO1 and PD3 were tolerant to P. s. pv. mors-prunorum.     

Enrichment and isolation of nitrogen fixing hydrogen bacteria

An enrichment method for nitrogen fixing hydrogen bacteria is described. The procedure invariably resulted in the isolation of yellow-pigmented coryneform bacterial strains assigned to Corynebacterium autotrophicum. The procedure included a serial transfer in an ammonium-free mineral liquid medium under an atmosphere of 10% hydrogen, 5% oxygen, 10% carbon dioxide and 75% nitrogen, followed by a short alkali treatment and by streaking on nutrient broth-succinate agar. The ability to fix nitrogen was confirmed by the acetylene reduction test and by ¹⁵N₂ incorporation.     

Aphid honeydew and its effect on the phyllosphere microflora of Picea abies (L.) Karst

Aphids of the genus Cinara, feeding on Norway spruce, excrete copious amounts of honeydew, a carbon-rich waste product, which accumulates locally on needles and twigs. We investigated the role of honeydew as a potential source of energy which might promote the growth of micro-organisms in the phyllosphere of conifer trees. To approach this question, we followed the population dynamics of Cinara spp. in a natural forest stand over two seasons. We also studied the amounts of honeydew produced by individual aphids and identified potential parameters which might influence honeydew production. Finally, we determined the growth of micro-organisms on infested and uninfested needles of Norway spruce during the growing season. Confined to Picea abies, the investigated Cinara species only became abundant in midsummer, when needles and shoots were expanding. The populations showed only a single peak in abundance, the timing and magnitude of which may vary from year to year due to weather conditions, changes in plant quality in a yearly cycle or the impact of natural enemies. The amount of honeydew produced by individual aphids was dependent on the developmental stage of the aphid, the nutritional supply of its host plant and on the developmental state of the Norway spruce (e.g. bud burst, end of shoot extension). The presence of honeydew significantly increased the growth of bacteria, yeast and filamentous fungi on the surface of needles and there was a pronounced seasonal trend, with the highest abundance in midsummer correlating with the period of peak aphid abundance. Taken together, these findings indicate that aphids have an influence on microbial ecology in the phyllosphere of trees. The implication of our study, from interactions at the population level to effects and potential consequences for C and N fluxes at the level of forest ecosystems, is discussed.     

Fungal biomass associated with the phyllosphere of bryophytes and vascular plants

Little is known about the amount of fungal biomass in the phyllosphere of bryophytes compared to higher plants. In this study, fungal biomass associated with the phyllosphere of three bryophytes (Hylocomium splendens, Pleurozium schreberi, Polytrichum commune) and three vascular plants (Avenella flexuosa, Gymnocarpium dryopteris, Vaccinium myrtillus) was investigated using ergosterol content as a proxy for fungal biomass. Phyllosphere fungi accounted for 0.2-4.0 % of the dry mass of moss gametophytes, representing the first estimation of fungal biomass associated with bryophytes. Significantly more fungal biomass was associated with the phyllosphere of bryophytes than co-occurring vascular plants. The ergosterol present in moss gametophytic tissues differed significantly between species, while the ergosterol present in vascular plant leaf tissues did not. The photosynthetic tissues of mosses had less associated fungal biomass than their senescent tissues, and the magnitude of this difference varied in a species-specific manner. The fungal biomass associated with the vascular plants studied varied significantly between localities, while that of mosses did not. The observed differences in phyllosphere community biomass suggest their size could be affected by host anatomical and physiological attributes, including micro-niche availability and chemical host defenses, in addition to abiotic factors like moisture and nutrient availability.     

Interaction of yeasts and some nitrogen fixing bacteria on nodulation of legumes

Summary Combined inoculation ofRhizobium trifolii withSaccharomyces cerevisiae and other yeasts generally enhanced the number of nodules, length of plants and dry weight of Egyptian clover (Trifolium alexandrinum) seedlings grown on agar slopes. Similar effects were observed when seedlings were inoculated withR. trifolii in the presence of dialyzed culture filtrate ofS. cerevisiae.     

植物叶际好氧反硝化细菌的筛选及其脱氮性能研究

【目的】探索叶际微生物协同植物削减大气氮氧化物的机制,了解叶际可培养好氧反硝化细菌的存在及多样性,获得高效的叶际好氧反硝化细菌资源。【方法】采用富集培养结合格里斯试剂检测、溴百里酚蓝(bromothymol blue, BTB)培养基筛选的方法从景观植物叶际分离筛选好氧反硝化细菌,对好氧反硝化细菌的16S rRNA基因序列进行系统发育分析,并选取其中一株高效好氧反硝化细菌进行脱氮性能研究。【结果】从6种景观植物石楠、女贞、木樨、樟树、卫矛冬青、荷花玉兰的叶际中分离到好氧反硝化细菌13株,经16S rRNA基因序列分析发现,13株细菌分别属于4门7科7属,其中4株为肠杆菌属(Enterobacter),3株为无色杆菌属(Achromobacter),2株为假单胞菌属(Pseudomonas),其余4株分别属于鞘氨醇杆菌属(Sphingobacterium)、不动杆菌属(Acinetobacter)、微杆菌属(Microbacterium)和假节杆菌属(Pseudarthrobacter)。定量分析发现菌株SF的反硝化效果较好。通过单因素试验和响应面设计试验,对菌株SF的脱氮性能进行了一系列研究,探究了碳源、温度、初始pH、碳氮比和转速等因素对菌株SF脱氮效果的影响。结果表明,菌株SF的最佳脱氮条件：碳源为葡萄糖,初始pH值为7.5,碳氮比为9.7,转速180 r/min,温度为33.5 ℃。在此条件下,当初始硝酸盐浓度为361 mg/L时,72 h总氮去除率可达到93.3%。【结论】景观植物叶际中存在较多种类的可培养好氧反硝化细菌,丰富了叶际氮循环相关微生物的类型,为探索叶际微生物协同削减大气氮氧化物的机制奠定了基础。通过高效脱氮菌株的筛选,为进一步应用微生物协同植物削减空气氮氧化物污染提供了候选菌株。     

Tolerance of pesticides and antibiotic resistance in bacteria isolated from wastewater-irrigated soil

A total of 64 bacterial isolates (40 Pseudomonas spp., 12 Azotobacter and 12 Rhizobium spp.) were characterized on the basis of morphological, cultural and biochemical characteristics. All the isolates were tested for their tolerance to the pesticides endosulfan, carbofuran, and malathion. 12.5% of the Pseudomonas isolates from soil tolerated concentrations of 1600 g malathion ml whereas 7.5% of isolates tolerated the same concentration of carbofuran. However, Pseudomonas isolates demonstrated a tolerance limit to endosulfan at a concentration of 800 g/ml. Asymbiotic N₂-fixers (Azotobacter) and symbiotic N₂-fixers (Rhizobium spp.) were also able to tolerate concentrations of pesticides up to 1600 g/ml. All the isolates were further tested for their antibiotic susceptibility against seven different antibiotics, nalidixic acid, cloxacillin, chloramphenicol, tetracycline, amoxycillin, methicillin, doxycycline. 100% of the Pseudomonas isolates were resistant to cloxacillin and 57.5% were resistant to methicillin. 7.5% of the isolates exhibited multiple resistance to five different antibiotics in three different combinations whereas 25% of the isolates showed multiple resistance to four different antibiotics in seven different combinations. Some of the resistant isolates were also screened for plasmid DNA and found to harbour a single plasmid.     

Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere

Extremophiles - Genome analyses are being used to characterize plant growth-promoting (PGP) bacteria living in different plant compartiments. In this context, we have recently isolated bacteria...     

秸秆覆盖免耕对土壤氮素转化细菌区系的影响

不同耕作方法影响土壤N素转化细菌的组成和数量.多年连续秸秆覆盖免耕试验结果表明,好氧固氮菌和厌氧固氮菌数量在铁茬处理土壤中较少,而在免耕与翻耕土壤中较多,免耕厌氧固氮菌数量明显地高于翻耕和铁茬,而铁茬最低,这种趋势特别在0～0.1m和0.1～0.2m土层中表现明显.土壤反硝化细菌的数量在表层土壤中较少,三种耕作方法相差较小,但在0.1～0.2m,0.2～0.3m土层中,翻耕反硝化细菌数量比免耕和铁茬多,而铁茬最少.不同耕作方法间氨氧化细菌数量相差不明显,免耕土壤中氨氧化细菌数量较高,动态变化趋势不规则.0～0.1m土层中免耕亚硝酸氧化细菌数量高于翻耕和铁茬,0.1～0.2m土层免耕与翻耕亚硝酸氧化菌数较多,而铁茬较少.     

Growth of lactic acid bacteria on oilseed crop pea- and chickpea-based media

Six strains of lactic acid bacteria isolated from different biotopes were cultivated on oilseed crop pea- or chickpea-based media, supplemented with the usual nitrogen supplements, yeast extract, meat extract or tryptic casein peptone. Growth was compared to that recorded on the widely used MRS and M17 media. All the media tested favoured growth of the selected strains, since the final c.f.u. gained at least 3 logs. Vegetal substrate (pea) appeared especially efficient for growth of a LAB species isolated from plants, Lactobacillus plantarum, illustrating the relationship between the supplementation used and the natural biotope of the LAB strain; during growth on oilseed crop pea- and chickpea-based media, the final c.f.u. was 0.58 and 1.28 log higher than the final cell numeration recorded on the reference medium MRS. The better efficiency of chickpea to stimulate LAB growth, if compared to oilseed crop pea, must be related to its higher protein content, 24.15 ± 0.1 and 28.2 ± 0.5% respectively. A vegetal substrate with high protein content can be therefore used to replace, at least partially, the usual nitrogen supplements.     

Cover crop effects on the fate of N following soil application of swine manure

Biological nitrogen fixation (BNF) in sugarcane is considered one of the principal reasons for the success of the Brazilian Ethanol Program (PRO-ALCOOL) for motor car fuel. The BNF influences positively the energy balance of sugarcane crops for alcohol production. Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium associated with sugarcane, and is particularly abundant and active in the early stages after germination. The objective of this work was to evaluate the effect of the addition of increasing amounts of two sources of mineral nitrogen (ammonium sulphate and calcium nitrate) on the population of G. diazotrophicus and also to evaluate its effect on nitrogenase (acetylene reduction) activity and accumulation of N by two sugarcane hybrids, SP 701143 and SP 792312. The results showed that both varieties differed in the form of nitrogen they prefer to uptake from the soil. The variety SP 701143 preferred ammonium sulphate, whilst the variety SP 792312 preferred N from calcium nitrate. In both varieties, the addition of increased doses of ammonium and nitrate inhibited the population of G. diazotrophicus but in the variety SP 701143 the inhibition was more pronounced in the presence of calcium nitrate. The acetylene reduction activity was inhibited in both varieties, especially in variety SP 792312 in the presence of either of the two nitrogen sources.