Genomic diversity of vibrios associated with the Brazilian coral Mussismilia hispida and its sympatric zoanthids (Palythoa caribaeorum, Palythoa variabilis and Zoanthus solanderi)

Aims: A taxonomic survey of the vibrios associated with the Brazilian endemic coral Mussismilia hispida and the sympatric zoanthids (i.e. Palythoa caribaeorum, Palythoa variabilis and Zoanthus solanderi). Methods and Results: Mucus of 54 cnidarian specimens collected in three different places at São Sebastião in two consecutive years (i.e. 2005 and 2006) was used for taxonomic characterization of the cnidarian microbiota. Ninety‐eight of the 151 vibrio isolates fell within the vibrio core group according to partial 16S rDNA sequences. We performed the sequencing of recA and pyrH genes of all vibrio isolates. The most abundant taxa belonged to the vibrio core group (Vibrio harveyi, Vibrio rotiferianus, Vibrio campbellii and Vibrio alginolyticus), Vibrio mediterranei (=Vibrio shillonii) and Vibrio chagasii. With the exception of V. chagasii which was found only in the mucus of M. hispida, the other species appeared in different hosts with no evidence for the presence of host‐specific clones or species. Using rep‐PCR analysis, we observed a high genomic heterogeneity within the vibrios. Each vibrio isolate generated a different rep‐PCR fingerprint pattern. There was a complete agreement between the grouping based on rep‐PCR and concatenated sequences of pyrH, recA and 16S rDNA, but the pyrH gene has the highest discriminatory power for vibrio species identification. Conclusion: The vibrio core group is dominant in the mucus of these cnidarians. There is a tremendous diversity of vibrio lineages within the coral mucus. pyrH gene sequences permit a clear‐cut identification of vibrios. Significance and Impact of the Study: The taxonomic resolution provided by pyrH (but not recA) appears to be enough for identifying species of vibrios and for disclosing putative new taxa. The vibrio core group appears to be dominant in the mucus of the Brazilian cnidarians. The overrepresentation of these vibrios may reflect as yet unknown ecological functions in the coral holobiont.     

Detoxification of mercury and organomercurials by nitrogen-fixing soil bacteria

Minimal inhibitory concentration values of HgCl₂ and 5 organomercurials were determined against 24 mercury-resistant N₂-fixing soil bacteria previously isolated from soil and identified in our laboratory. These bacterial strains also displayed multiple antibiotic resistant properties. Typical growth pattern of a highly mercury-resistantBeijerinckia sp (KDr₂) was studied in liquid broth supplemented with toxic levels of mercury compounds. Four bacterial strains were selected for determining their ability to volatilize mercury and their Hg-volatilizing capacity was different. Cell-free extracts prepared from overnight mercury-induced cells catalyzed Hg²⁺-induced NADPH oxidation. Specific activities of Hg²⁺-reductase which is capable of catalyzing conversion of Hg²⁺ →Hg(o) of 10 Hg-resistant bacterial strains are also reported.     

Distribution and Abundance of Fauna on Living Tissues of Two Brazilian Hermatypic Corals (Mussismilia hispida (Verril 1902) and Siderastrea stellata Verril, 1868)

This study describes the distribution and abundance patterns of the associate fauna on the living surface of the corals Siderastrea stellata Verril, 1868 and Mussismilia hispida (Verril 1902) using a non-destructive method, on the northern coast of Rio de Janeiro State. For each coral species, infestation density and proportions of infested colonies, colonies attached and unattached to the substrate were estimated. A total of 474 colonies of S. stellata and 452 colonies of M. hispida were examined. The barnacle Ceratoconcha floridana (Pilsbry, 1931) was the dominant coral associate found, followed by gall-crabs of the family Cryptochiridae Paulson, 1875 and the bivalve Lithophaga bisulcata (d’Orbigny, 1842). Both coral species presented similar patterns of infestation dominance. S. stellata colonies were more commonly infested and showed a greater mean infestation density of 0.62 ind/cm² at Armação dos Búzios, whereas M. hispida colonies had infestation densities of only 0.20 ind/cm². Infestation density does not appear to impact negatively on corals of Armação dos Búzios. A clear negative relationship between the number of associates in the coral colony and coral size was found. Evidently abundance and frequency of occurrence of associated fauna is highly related to coral community structure and composition and the results highlight the importance of local scale studies.     

基于RFLP技术的鹿角杯形珊瑚共附生固氮菌多样性分析

对三亚湾鹿回头与西沙石岛的常见鹿角杯形珊瑚(Pocillopora damicornis)的固氮酶nif H基因多样性进行了分析,并对其共附生固氮微生物多样性进行了比较。利用Taq I和HaeⅢ两种限制性内切酶分别对三亚湾珊瑚的粘液(SYN)、组织(SYZ)和西沙珊瑚组织(XSZ)的阳性克隆子进行聚合酶链反应-限制性片段长度多态性(PCR-RFLP)分析,得到了23,25和13个OTUs,文库的覆盖率分别为90.3%,87.5%和94.1%,多样性指数Shannon-Wiener指数(H)分别为2.28,3.02和2.08,Evenness指数(E)分别为0.5,0.65和0.56。研究结果表明:1)3个文库重要组成部分都是绿菌门(Chlorobia)和变形菌门(Proteobacteria)α-亚门、γ-亚门、δ-亚门细菌;2)3个文库绝大部分OTUs的最相似序列来自珊瑚礁、海绵、水体、沉积物等环境中的不可培养细菌,反映鹿角杯形珊瑚组织和粘液可能蕴含着丰富的尚未被培养的固氮菌资源;3)发现了两大重要的类群——分别与绿菌门和脱硫弧菌属(Desulfovibrio)细菌序列最相似的类群,它们通过为珊瑚共生体提供有机氮源参与到珊瑚白化的调节或恢复过程中,在珊瑚共生体营养物质的循环中充当重要角色;4)从多样性指数分析可以看出,SYZ的固氮微生物多样性高于SYN,SYZ的固氮微生物多样性高于XSZ,反映了鹿角杯形珊瑚生活的地理位置不同,固氮菌共附生的生态位不同(如珊瑚粘液、组织等不同部位),都将导致珊瑚共附生固氮菌多样性的差异。     

A comparison of the thermal bleaching responses of the zoanthid Palythoa caribaeorum from three geographically different regions in south Florida

Coral bleaching involves the loss of symbiotic algae (zooxanthellae) from reef corals and other cnidarians during periods of environmental stress, particularly elevated temperature. In this study we compared the thermal bleaching responses of the zoanthid Palythoa caribaeorum from three populations along the southeast coast of Florida. Winter (2002-2003) and summer (2003) samples from three geographically separate sites were experimentally exposed to increased temperatures and the loss of zooxanthellae was measured. Population densities of zooxanthellae were analyzed and their genetic identity determined using PCR-DGGE analysis of the internal transcribed spacer region 2. The results showed that samples of P. caribaeorum from reefs that experienced the smallest range in annual seawater temperature released the most zooxanthellae. Seasonal comparisons revealed that winter samples lost more zooxanthellae than summer samples. P. caribaeorum harbored two genetic types of zooxanthellae, C1 and D1a. Individual colonies contained populations of only C1 or D1a, or combinations of C1 and D1a. However, these genotypic patterns did not relate latitudinal distribution nor to differences in experimental thermal tolerance.     

Diversity of nitrogen-fixing cyanobacteria under various ecosystems of Thailand: population dynamics as affected by environmental factors

Investigation of N₂-fixing cyanobacteria from Thai soil was carried out at 2-month intervals between July 1997 and November 1999 to determine the population number, population dynamics and favourable habitats. Sites were selected in three parts of Thailand; North, Central and Northeast. In each part, various soil ecosystems were used as sampling sites; at highest elevation as on the top of the mountain, in the middle and at the foot of the mountain, as well as in flat areas of agricultural practice and uncultivated areas. Generally, a high population of N₂-fixing cyanobacteria was found in agricultural areas where rice cultivation was practised, rather than in other sites. The population dynamics in the mountain and uncultivated areas were less fluctuating than in agricultural areas. The population densities in agricultural areas increased in the rainy season and decreased during the dry season. Other environmental factors such as temperature, moisture and pH also affected the population densities in different habitats. Cyanobacterial diversity was notably influenced by the type of ecosystem in both dry and rainy seasons. The cultivation area containing rice in rotation with other crops contained the most genetically diverse range of species.     

薇甘菊根际可培养固氮菌和氨化细菌的分离鉴定与促生作用

【目的】固氮菌和氨化细菌是氮循环产生生物有效氮的关键起始环节,直接影响了外来入侵植物的生长速度和扩散进程。然而,关于典型入侵植物薇甘菊根际可培养固氮菌和氨化细菌的研究尚未见报道,这在很大程度上制约了我们对薇甘菊根际高效的氮素转化机制的深刻理解。【方法】采用传统平板涂布培养法对野外采集的薇甘菊根际土壤中的可培养固氮菌和氨化细菌进行了分离鉴定,并进行了接种验证实验。【结果】结果表明,入侵植物薇甘菊根际土壤中的固氮菌和氨化细菌的菌群密度显著高于两个本地伴生植物(火炭母和鸡屎藤),其固氮效率及有机氮矿化效率也优于2个本地种;系统发育分析表明：薇甘菊根际的固氮菌菌株归类于5个属,分别为伯克霍尔德氏菌属(Burkholderia)、肠杆菌属(Enterobacter)、植物杆菌属(Phytobacter)、新肠杆菌属(Kosakonia)和根瘤菌属(Rhizobium);氨化细菌归类于7个属,分别为沙雷氏菌属(Serratia)、不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)、博德特氏菌属(Bordetella)、寡养单胞菌属(Stenotrophomonas)、苍...     

茅苍术叶片可培养内生细菌多样性及其促生潜力

对江苏省道地药材茅苍术叶片可培养内生细菌的多样性及其固氮、解磷、解钾、产生长素的能力进行研究。依据菌落形态的不同,共分离得到52株内生细菌。能正常传代培养的45株内生细菌经ARDRA分析后归入14个聚类簇,簇内菌株的BOX-PCR指纹图谱相似度不高,在属水平上显示出茅苍术内生细菌丰富的多样性。各聚类簇代表菌株16S rDNA的序列分析表明分离得到的内生细菌与泛菌属、微杆菌属、短杆菌属、农杆菌属、假单胞菌属、芽孢杆菌属细菌亲缘关系相近,优势内生细菌与假单胞菌属细菌亲缘关系相近。45株能正常传代培养的内生细菌中,有10株能够在无氮培养基上正常生长,具固氮潜力。使用nifH基因通用引物对其基因组进行扩增后,除ALEB 33外,其它9株内生细菌均可获得与nifH基因片段大小相近的条带。分别使用NBRIP培养基和蒙金娜有机磷培养基筛选后获得19株和15株能够溶解磷酸钙和卵磷脂的内生细菌,其中ALEB 43溶解无机磷的能力最强,达(251.43±6.55)mg/L;ALEB 4A溶解有机磷的能力最强,达(23.63±1.46)mg/L。部分内生细菌溶解无机磷的能力与其产酸能力呈正相关,而菌株溶解有机磷的能力却无此相关性。通过硅酸盐培养基的筛选,获得具有解钾潜力的菌株24株。43株内生细菌能够将色氨酸转化为生长素,其中ALEB 44产生长素的能力最强,达(268.44±10.12)μg/mL。本研究首次揭示了江苏省道地药材茅苍术体内丰富的内生细菌资源及其促生长潜力,对进一步阐述茅苍术与内生菌之间的相互关系具有重要意义。     

Ecological factors and adaptive processes in N2-fixing bacterial populations of the plant environment

Summary Physiology and genetics of non-symbiotic N₂-fixing bacteria have made much progress in recent years, especially in the case of a few reference strains. Nevertheless, understanding the ecology of diazotrophs cannot be achieved by studying only laboratory microorganisms. It is necessary to study naturally-occurring populations, to characterize their densities, size, composition, variability and variations in order to understand how a plant can select a rhizosphere population from a soil population. Very few comparisons of phenotypic diversity and dominant phenotypes in these two habitats have been made up to now. More studies of this type would allow a better knowledge of the selective pressures which actually drive the shift of population and they would permit investigation of the underlying mechanisms. These can vary from mere metabolic adaptation to selection of pre-adapted genotypes. A third mechanism is possible in which pre-adapted genes are maintained in soil populations at very low frequencies and energy costs, and whose transfer is triggered by the selective factor to which they constitute an adaptation.     

Molecular and phenotypic characterization of strains nodulating Anthyllis vulneraria in mine tailings, and proposal of Aminobacter anthyllidis sp. nov., the first definition of Aminobacter as legume-nodulating bacteria

Bacterial strains from Zn-Pb mine tailings were isolated by trapping with Anthyllis vulneraria, a legume-host suitable for mine substratum phytostabilisation. Sequence analysis of the 16S rRNA gene and three housekeeping genes (atpD, dnaK and recA) showed that they were related to those of the genus Aminobacter. DNA-DNA relatedness of representative isolates supported the placement of novel strains in Aminobacter as a new species. Phenotypic data emphasize their differentiation from the other related species of Aminobacter and Mesorhizobium. Aminobacter isolates exhibited nodA sequences tightly related with M. loti as the closest nodA relative. By contrast, their nodA sequences were highly divergent from those of M. metallidurans, another species associated with A. vulneraria that carries two complete copies of nodA. Therefore, the novel bacterial strains efficient on A. vulneraria represented the first occurrence of legume symbionts in the genus Aminobacter. They represent a new species for which the name Aminobacter anthyllidis sp. nov. is proposed (type strain STM4645(T)=LMG26462(T)=CFBP7437(T)).     

Effects of the fish anesthetic, clove oil (eugenol), on coral health and growth

Ecological research within coral reefs often requires the use of anesthetics to immobilize organisms. It is therefore important to consider the effect of these chemicals on the surrounding flora and fauna, particularly to the corals themselves. We quantified the effects of clove oil, a commonly used fish anesthetic, on the growth and occurrence of bleaching in three species of corals: Acropora striata, Pocillopora verrucosa, and Porites australiensis. We compared coral responses to five treatments: a gradient of four clove oil concentrations (0-28%) in seawater, and one concentration of clove oil (14%) in ethanol. Each week, we assessed the presence of bleaching, and then applied the treatment. We measured growth over the duration of the 6-week experiment using the buoyant weight technique. Growth and bleaching showed a dose response to clove oil exposure, and the use of ethanol as a solvent had an additional deleterious effect, as also suggested by observed changes in concentrations of eugenol following field application. Overall, growth was reduced by 37.6% at the highest concentration (28% clove oil in seawater) relative to the control (0% clove oil). The reduction in growth was nearly as great (35.3% of the control) at half the concentration of clove oil (14%) when dissolved in ethanol. These results suggest the repeated use of clove oil (even without a solvent) can deleteriously affect corals.     

The role of epibotic bacteria from the surface of the soft coral Dendronephthya sp. in the inhibition of larval settlement

It has been suggested that bacteria associated with soft-bodied organisms are suggested to produce bioactive compounds against the attachment of invertebrate larvae and bacteria onto the surface of these organisms. Our recent study has demonstrated that epibiotic bacteria from the surface of the soft coral Dendronephthya sp. (Coelenterata: Octocoralia, Alcyonacea) inhibit the growth of bacteria commonly found in marine natural biofilms. In the present study, the effect of 11 epibiotic bacteria isolated from the surface of Dendronephthya sp. on larval settlement of the tubeworms Hydroides elegans was examined using laboratory bioassay. Among 11 bacterial isolates, 2 strains (18%) inhibited the larval settlement of H. elegans (Haswell), 4 strains (36%) were “inductive” to larvae and the remaining 5 strains (46%) were “non-inductive”. There was no correlation between the antifouling activities of bacterial isolates and their phylogenetic origin, i.e. closely related bacterial strains showed different effects on larval settlement of H. elegans. When all “inductive”, “non-inductive” and “inhibitive” bacterial isolates were mixed in a 1:1:1 ratio, the effect of the resultant multispecies film on larval settlement became “inhibitive”. Waterborne compounds of Vibrio sp. and an unidentified α-Proteobacterium, which suppressed the settlement of H. elegans and Bugula neritina (L.) larvae, were further investigated using size fractionation and bioassay-guided enzymatic analysis. It was found that antilarval settlement compounds from these bacteria were heat-stable polysaccharides with a molecular weight >100 kDa. The results indicate that the bacteria associated with the soft coral Dendronephthya sp. may contribute to the antifouling mechanisms of the soft-bodied organisms by producing compounds that are against bacterial growth and settlement of macrofoulers on the surface of their host.     

Occurrence of effective nitrogen-scavenging bacteria in the rhizosphere of kallar grass

Bacteria occurring in high numbers on the rhizoplane of kallar grass grown at a natural site in Pakistan were effective scavengers of traces of combined nitrogen from the atmosphere. Bacteria grew under appropriate conditions in nitrogen-free semi-solid malate medium in the form of a typical subsurface pellicle which resulted in a significant nitrogen gain in the medium within 3 to 4 days of incubation; this could be also measured by¹⁵N-dilution. Bacteria grew and incorporated nitrogen under an atmosphere containing NH₃ and N₂O. A rapid and strong binding of strain W1 to roots of kallar grass grown in hydroponic culture was found by using a³²P-tracer technique. We obtained no evidence for diazotrophy of our strains because they failed to grow on nitrogen-free media when gases of high purity were used. No¹⁵N₂ was incorporated when bacteria were grown on¹⁵N₂ although a nitrogen gain was found, no acetylene reduction was observed and no homology with DNA containing sequences ofnifHDK structural genes for the nitrogenase components fromKlebsiella pneumoniae were detected. Owing to close contact of these bacteria with roots of kallar grass, utilization of scavenged nitrogen by the plant may have to be taken into account.     

High zooxanthella density shortens the survival time of coral cell aggregates under thermal stress

To investigate bleaching mechanisms in coral-zooxanthella symbiotic systems, it is important to study the cellular- or tissue-level responses of corals to stress. We established an experimental system to study the stress responses of coral cells using coral cell aggregates. Dissociated coral cells aggregate to form spherical bodies, which rotate by ciliary movement. These spherical bodies (tissue balls) stop rotating and disintegrate when exposed to a thermal stress. Tissue balls prepared from dissociated cells of Fungia sp. and Pavona divaricata were exposed to either elevated temperature (31 °C, with 25 °C as the control) or elevated temperature in the presence of exogenous antioxidants (ascorbic acid and catalase, or mannitol). The survival curves of tissue balls were markedly different between 31 and 25 °C. At 31 °C, most tissue balls disintegrated within 24 h, whereas at 25 °C, most tissue balls survived for more than 24 h. There was a negative correlation between survival time and the zooxanthella density of tissue balls at 31 °C, but no significant relationship was found at 25 °C. Antioxidants extended the survival time of tissue balls at high temperature, suggesting that zooxanthellae produce reactive oxygen species under stress. These results indicate that zooxanthellae produce harmful substances and damage coral cells under high-temperature stress. Tissue balls provide a good experimental system with which to study the effects of stress and various chemical reagents on corals cells.     

Two groups of entomopathogenic bacteria, Photorhabdus and Xenorhabdus, share an inhibitory action against phospholipase A2 to induce host immunodepression

Photorhabdus and Xenorhabdus are two genera of entomopathogenic bacteria having a mutualistic relationship with their respective nematode hosts, Heterorhabditis and Steinernema. One of the pathogenic mechanisms of these bacteria includes host immunodepression, which leads to lethal septicemia. It has been known that X. nematophila inhibits phospholipase A2 (PLA2) to induce host immunodepression. Here, we tested the hypothesis of PLA2 inhibition using another bacterial species involved in other genera. P. temperata subsp. temperata is the intestinal symbiont of an entomopathogenic nematode, H. megidis. The bacteria caused potent pathogenicity in a dose-dependent manner against the fifth instar larvae of a test target insect, Spodoptera exigua, as early as 24 h after the intra-hemocoelic injection. In response to the live bacterial injection, hemocyte nodulation (a cellular immune response) and prophenoloxidase (pPO) activation were inhibited, while the injection of heat-killed bacteria significantly induced both immune reactions. The immunodepression induced by the live bacteria was reversed by the addition of arachidonic acid, the catalytic product of phospholipase A2. In contrast, the addition of dexamethasone, a specific PLA2 inhibitor to the heat-killed bacterial treatment, inhibited both immune capacities. In addition to a previously known PLA2 inhibitory action of X. nematophila, the inhibition of P. temperata temperata on PLA2 suggests that bacteria symbiotic to entomopathogenic nematodes share a common pathogenic target to result in an immunodepressive state of the infected insects. To prove this generalized hypothesis, we used other bacterial species (X. bovienni, X. poinarii, and P. luminescens) involved in these two genera. All our experiments clearly showed that these other bacteria also share their inhibitory action against PLA2 to induce host immunodepression.     

Spectral dependence of energy transfer in wild-type peripheral light-harvesting complexes of photosynthetic bacteria

The precise position of the upper exciton component and relevant vibronic transitions of the B850 ring in peripheral light-harvesting complexes from purple photosynthetic bacteria are important values for determining the exciton bandwidth and electronic structure of the B850 ring. To determine the presence of these components in wild-type LH2 complexes the pump-probe femtosecond transient spectra obtained with excitation into the 730-840 nm spectral range are analyzed. We show that at excitation wavelengths less than 780 nm B850 absorption bands are present and that, in accordance with exciton theory, these bands peak further in the blue when the lowest optically allowed transition is more red-shifted.     

Isolation and distribution of iridescent Cellulophaga and other iridescent marine bacteria from the Charente-Maritime coast,French Atlantic

An intense colored marine bacterium, identified as Cellulophaga lytica, was isolated previously from a sea anemone surface on the Charente-Maritime rocky shore (Atlantic Coast, France), and iridescence of its colonies under direct light was recently described. In addition, iridescence intensities were found to differ strongly between C. lytica strains from different culture collections. However, importantly, the occurrence and distribution of iridescent bacteria in the marine environment were still unknown. Therefore, in this study, a search was undertaken for marine iridescent bacterial strains in different biotopes of the Charente-Maritime coast. Various marine samples (water, sediment, macroalgae, other macroorganisms and detritus) were collected from seven biotopes using a direct plate inoculation method. As a result, 34 iridescent strains related to the genus Cellulophaga, as well as 63 iridescent strains affiliated to the genera Tenacibaculum and Aquimarina, were isolated. Iridescent colors were different according to the genera but iridescent marine bacteria were widely distributed. However, a majority of strains were isolated from rocky shores and, in particular, red seaweed surfaces and mollusks. The data from the study suggested that isolates with iridescent properties were well conserved in stressful environments such as the coastal shoreline. This origin may provide an insight into the ecological and biological functions of iridescence.     

Plant-bacteria interactions with special emphasis on the kallar grass association

Kallar grass is a highly salt-tolerant grass grown as a pioneer plant on alkaline, salt-affected soils in Pakistan. Nitrogen-fixing bacteria and kallar grass were found to be in close association, which was even root-zone specific: rhizoplane and endorhizosphere were colonized by two different populations. Among theAzospirillum isolates originating from the root surface, some were of a new species, now namedA. halopraeferens. To study plant-bacterium interactions, this natural kallar grass association was chosen. The possible role of bacterial chemotaxis and oxygen tolerance are discussed.     

cDNA cloning and characterization of human and mouse Ca-independent phosphatidylethanolamine N-acyltransferases

The formation of N-acylphosphatidylethanolamine by N-acylation of phosphatidylethanolamine (PE) is the initial step in the biosynthetic pathway of bioactive N-acylethanolamines, including the endocannabinoid anandamide and the anti-inflammatory substance N-palmitoylethanolamine. We recently cloned a rat enzyme capable of catalyzing this reaction, and referred to the enzyme as Ca²⁺-independent N-acyltransferase (iNAT). Here we report cDNA cloning and characterization of human and mouse iNATs. We cloned iNAT-homologous cDNAs from human and mouse testes, and overexpressed them in COS-7 cells. The purified recombinant proteins abstracted an acyl group from both sn-1 and sn-2 positions of phosphatidylcholine, and catalyzed N-acylation of PE as well as phospholipase A₁/A₂-like hydrolysis. The iNAT activity was mainly detected in soluble rather than particulate fractions, and was only slightly increased by Ca²⁺. These results demonstrated that the human and mouse homologues function as iNAT. As for the organ distribution of iNAT, human testis and pancreas and mouse testis exhibited by far the highest expression level, suggesting its physiological importance in the specific organs. Moreover, mutagenesis studies showed crucial roles of His-154 and Cys-241 of rat iNAT in the catalysis and a possible role of the N-terminal domain in membrane association or protein–protein interaction.     

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.