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Establishment of inoculated biofilms on root surfaces and their effect on plant enzyme activities and nutrient availability in the rhizosphere are less investigated. Two beneficial inoculants- Trichoderma spp. and Azotobacter spp., and the biofilm developed using them as partners, were evaluated in chickpea crop. The hypothesis tested was that the efficacy of the fungal-based bacterial biofilm in colonizing the root and rhizosphere may be better, as compared to their individual inoculation. Scanning electron microscopy and root staining illustrated the colonisation of developed T. viride (Tv) – A. chroococcum (Az) biofilm and their partners. Inoculation effects were analysed on selected plant growth parameters, soil nutrients, plant enzyme activities and organic acid exudations in the rhizosphere soil. Tv-Az biofilm was observed to exhibit better colonization in the rhizosphere and rhizoplane of chickpea, as compared to single inoculation. Significant enhancement of 30–45% in root volume, root proteins, root-shoot ratio, exudation of succinic and fumaric acids, soil available nutrients, along with two fold enhancement in the activity of plant enzymes was recorded, as compared to recommended doses of NPK fertilizers. Tv-Az biofilm is a promising biofertilizing option for enhancing plant growth parameters, available soil nutrients, along with providing a savings of 25% nitrogenous fertilizers in chickpea.

Transgenic insect-resistant cotton expressing the Cry1Ac and/or CpTI protein has been released into the environment for commercial cultivation for over a decade in China. In this consecutive 3-year field study (2007–2009), we characterized the microbial populations in two different transgenic cotton fields and one non-transgenic cotton field to assess the environmental impact of long-term cultivation of transgenic insect-resistant plants in credible field conditions. We obtained samples at four different growth stages of cotton (seedling, budding, boll forming and boll opening). The results showed significant seasonal variation in the number of bacteria, fungi, azotobacter, denitrifying bacteria and ammonia-oxidizing bacteria and in diversity indices of microorganisms, but no significant difference in the number of each microbial population or diversity indices attributable to long-term cultivation of transgenic cotton.

Azospirillum spp. represents one of the most studied plant growth promoting bacteria. A meta-analysis was conducted on 59 available articles to evaluate the extent to which Azospirillum may contribute to wheat growth properties. A mean increase of 8.9% in seed yield and 17.8% in aboveground dry weight was found to result from inoculation of wheat with Azospirillum. However, key determinants for the plant growth promoting effect were found to be the amount of N fertilization applied–maximum plant growth promoting effect was reported in the absence of N fertilization- and identities of wheat cultivar—Triticum aestivum was superior to Triticum durum-and Azospirillum isolate—Azospirillum lipoferum was more effective than Azospirillum brasilense. Co-inoculation with Azotobacter tended to further increase the growth promoting effect of Azospirillum on seed yield. A weak relationship between plant growth promoting effect on seed yield and aboveground biomass was detected. Inoculation with Azospirillum decreased the shoot:root ratio of wheat in field trials; but for pot trials, a key determinant of the shoot:root effect was the size of the pot used. The authors, thus, raise the scepticism of whether the growth parameters recorded in pot trials are representative of those under field conditions. Results highlight the efficiency of Azospirillum as an inoculant of wheat.

The establishment and productivity of a Manchurian walnut (Juglans mandshurica Maxim.) plantation can be improved by inter-planting with larch (Larix gmelini Rupr.) in Northeast China, but the potential mechanism remains obscure. We carried out a series of experiments in a 20-year-old mixed-species plantation, as well as in Manchurian walnut and larch plantations. Manchurian walnut seedlings had difficulty surviving in the Manchurian walnut plantation because their growth was inhibited by their own soil and root exudates. In sharp contrast, Manchurian walnut seedlings grew well in larch and mixed-species plantations. Larch soil and root exudates greatly stimulated the growth of Manchurian walnut seedlings in controlled conditions. In particular, larch root exudates can increase the soil microbial populations, including bacteria, actinomycetes, azotobacter and cellulose-decomposing microorganisms; larch root exudates can also increase the enzyme activities of saccharase, urease, proteinase and polyphenol oxidase. Significant results led to a rapid degradation of the root-exuded phytotoxic juglone from Manchurian walnut. Manchurian walnut root exudates contained juglone at a high concentration of 121.3 ± 6.6 mg g−1, while juglone concentrations in the soil beneath Manchurian walnut trees ranged from 2.9–6.2 µg g−1 soil. It appears from the results that juglone may be released from Manchurian walnut roots into the soil in a sufficient quantity but rapidly degrades due to interactions with soil factors. Furthermore, juglone was more resistant toward degradation in the Manchurian walnut soil (t 1/2 = 7.36 ± 0.63 h) when compared to the larch soil (t 1/2 = 4.66 ± 0.82 h). The results suggest that larch may improve the establishment and productivity of Manchurian walnut in a mixed-species plantation through the release of root exudates.

Recent studies suggest a high diversity of diazotrophic bacteria in maize. However, none of these works have been based on a sufficient number of samples to provide reasonable quantitative estimates of diazotrophic bacterial diversity. Here we present the use of molecular tools and statistical inference to assess diazotrophic bacterial diversity within rhizosphere soils, roots and stems of field grown maize. DNA was isolated from the latter collected from six maize growing regions within the southern most state in Brazil, Rio Grande do Sul. Using conserved primers, nifH Cluster I gene fragments were amplified from each of the three zones, and the products cloned and sequenced. The majority of the sequences were classified within the Proteobacteria with the α-proteobacteria and β-proteobacteria being the most abundant in the rhizosphere soil and stem samples. The γ-proteobacteria were most abundant in rhizosphere soils, less so in roots, and least in the stem samples. According to three different diversity measures, the rhizosphere soil samples possessed greater diazotrophic bacterial diversity than the roots and stems of the maize plants. Only two genera, Azospirillum and Azotobacter, were found in virtually all samples at an abundance of over 1% of the total nifH sequences obtained. Other genera were largely restricted to soil (Methylocystis, Beijerinckia, Geobacter, Rhodovulum, Methylobacterium, Gluconacetobacter, Methylocella, and Delftia), roots (Dechloromonas), or stems (Methylosinus, Raoultella, and Rhizobium). Three genera, Herbaspirillum, Ideonella, and Klebsiella, appeared to dominate in the interior of the plant but were much rarer in soil.

The abandoned “Monte-Fresco” rock phosphate mine in Tachira, Venezuela, was sampled to study the biodiversity of phosphate-solubilizing microorganisms (PSM). Rhizosphere and bulk soils were sampled from colonizer plant species growing at a mined site where pH and soluble P were higher than the values found at a near by unmined and shrubby soil. Counting and isolating of PSM choosing strains showing high solubilization halos in a solid minimal medium with hydroxyapatite as phosphate source were evaluated using ammonia or nitrate as nitrogen sources and dextrose, sucrose, and mannitol as carbohydrate sources. A larger number of PSM were found in the rhizospheric than in the bulk soil. Six fungal strains belonging to the genus Penicillium and with high hydroxyapatite dissolution capacities were isolated from bulk soil of colonizer plants. Five of these strains had similar phenotypes to Penicillium rugulosum IR94MF1 but they solubilized hydroxyapatite at different degrees with both nitrogen sources. From 15 strains of Gram-negative bacteria isolated from the rhizosphere of colonizer plants, 5 were identified as diazotrophic free-living encapsulated Azotobacter species able to use ammonium and/or nitrate to dissolve hydroxyapatite with glucose, sucrose and/or mannitol. Different nitrogen and carbohydrate sources are parameters to be considered to further characterize the diversity of PSM.

Inoculation of sugar mill by-products compost with N2-fixing bacteria may improve its quality by increasing total N and available P. Compost was inoculated with Azotobacter vinelandii(ATCC 478), Beijerinckia derxii (ATCC 49361), and Azospirillumsp. TS8, each alone and all three together. Numbers of all N2-fixing bacteria in compost declined from an initial population of 5 × 105 cells g−1 during incubation. The population of Azotobacter declined to approximately 2 × 102 cells g−1 and the population of Beijerinckia and Azospirillum declined to approximately 9 × 103 and 3.5 × 104 cells g−1 respectively, at day 50. Inoculation with N2-fixing bacteria increased acetylene reduction, total N by 6–16 and available P by 25–30% in comparison to the uninoculated control. Increasing the N content and P availability of compost increases its value and there may be additional benefit from providing N2 fixing bacteria.

Bacteria with the ability to grow on nitrogen-free media and with nitrogenase activity under aerobic or microaerobic conditions were isolated from sugarcane roots collected from four different agricultural locations in Granada (Spain). Isolates were Gram negative rods and were identified as Azotobacter chroococcum and Azospirillum brasilense. Our results suggest that Azotobacter isolates do not have a particular affinity for sugarcane rhizospheres and that, on the contrary, Azospirillum isolates show specific association and perhaps endophytic colonization of sugarcane. However, obligate endophytes (Gluconacetobacter diazotrophicus) were not found in the apoplastic fluid of the stems and macerates extracts of sugarcane tissues with the procedure applied. Population of this microorganism might be in low number in the Spanish sugarcane varieties studied which is also discussed.

The side effects of sulfonylurea and imidazolinone herbicides on plant-associated bacteria were investigated under pure culture conditions. Eighteen isolates, belonging to the genera Azotobacter, Azospirillum, Bacillus, Enterobacter Pseudomonas and Serratia, were exposed to four active compounds at concentration ranges similar to those in field soil. The sulfonylureas chlorsulfuron and rimsulfuron inhibited the growth of one of two Azospirillum and one of four Pseudomonas strains, while the imidazolinones imazapyr and imazethapyr were effective on two out of five Bacillus isolates. Surfactants in commercial formulation significantly enhanced rimsulfuron toxicity. With the exception of one Azospirillum strain, the differential tolerance of rhizobacteria to these herbicides was related to a differential sensitivity of their target, the activity of the first enzyme in branched-chain amino acid biosynthesis, acetohydroxyacid synthase (AHAS).

The economic feasibility of using cultures of nitrogen fixing microorganisms in programmes to increase crop production, as a selfgenerating source of nitrogen, has been proved beyond doubtviz. Legume-Rhizobium symbiosis and blue green algal ‘fertilizer’ for rice. The extent to which the free living, N-fixing, aerobic, heterotropicAzotobacter chroococcum could replace the application of nitrogenous fertilizer to maize was investigatedin vivo. Total nitrogen uptake (kg ha−1) by maize after inoculation with Azotobacter combined with moderate applications of nitrogen fertilizer and farmyard manure was influenced significantly and resulted in a higher nitrogen concentration in grain and stover along with a higher yield.

Azotobacter bacteriophages were studied using liquid as well as solid enrichment cultures. The study included 16 samples representing the soils of Ruzyně, Prague. The effect of long-term application of mineral fertilizers (N, P, K) and organic manure (straw enriched by inorganic nitrogen) to the soil on Azotobacter and their phages was investigated. Liquid enrichments yielded phages in fairly high titers, and phages were found in most of the soils while solid ones only gave low titers in occasional soils. On the basis of plaque morphology 4 different phage types were obtained. Electron micrographs showed that the phages belong to the groups with long non-contractile, contractile, and short contractile tails. Host specificity assay showed that the majority of the strains of A. chroococcum, A. vinelandii, and one strain of A. beijerinckii were lysed by phage, while none of A. macrocytogenes, A. agilis and A. insignis strains were lysed. Phage inactivation and neutralization was carried out with homologous antisera. re]19741128

The influence of application of hexachlorocyclohexane (HCH=gamma BHC), to a submerged tropical field soil at rates equivalent to recommended field rates (1–2.5kg a.i./ha) and twice this level, upon the rhizosphere soil nitrogenase, nitrogen fixers, and soil redox potential (Eh) was investigated. The rhizosphere soil from HCH-treated field exhibited significantly higher nitrogenase activity than that from untreated fields. HCH retarded the drop in redox potential of the field soil upto 80 days after transplantation under submerged conditions. Populations of nitrogen-fixingAzospirillum sp. and Azotobacter, to a greater extent, and anaerobic organisms, to a lesser extent, were stimulated in HCH-treated soils. Results indicate the stimulation of heterotrophic nitrogen-fixing bacteria by HCH in submerged paddy soils.

Field trials were conducted during theKharif seasons of 1979 and 1980 to examine the effect of the nitrogen-fixing bacteriumAzotobacter chroococcum (isolate M4) on yields of maize and N-economy. Different levels of nitrogen and farmyard manure (FYM) were supplied to assess their interaction with Azotobacter inoculation. Seed inoculation without fertilization raised grain and stover yields significantly. FYM coupled with inoculation gave higher yields than either could singly. The financial gain due to an increase in grain yield upon applying per ha 80 kg N, 10×103 kg FYM and Azotobacter over the treatment 40N + FYM + Azotobacter was offset by the cost of the additional N; thus, the higher N-application was not economical. Azotobacter inoculation was economically most efficient at lower doses of fertilizer nitrogen which not only increased yields but resulted in a saving of fertilizer N when applied in combination with FYM.

Fungal propagules, in general, were drastically reduced in vapam amended soils. The toxicant from 250 to 1000 ppm was lethal toFusarium spp. Only in the early part of the experiment, higher concentrations of vapam appreciably reduced the population of bacteria and actinomycetes, though later on, their population gradually increased. The numbers of Azotobacter in soil amended with 125, 250 and 500 ppm did not alter appreciably, but their population in treated soils increased over the check on the 45th day. All concentrations of vapam lowered the population of Rhizobia.

Acetylene-reducing activities (ARA) of strains ofEnterobacter agglomerans, Azospirillum brasilense, Azotobacter chroococcum, and Bacillus, isolated from temperate or tropical soils, were compared at different temperatures to study temperature adaptability. All Enterobacter strains and Bacillus strain C-11-25 reduced C2H2 at temperatures as low as 5°C. ARA by Enterobacter strains declined sharply above 30°C but ARA by Bacillus strain C-11-25 continued to increase with an increase in temperature.A. brasilense strain sp 245, isolated from wheat roots in Brazil, reduced more C2H2 at lower temperatures than strain Cd, isolated from a Californian soil. Similarly, the temperate strain ofA. chroococcum was a better N2 fixer than the tropicalA. chroococcum strain at lower temperatures. Tropical strains ofA. brasilense andA. chroococcum reduced more C2H2 than temperate strains at higher temperatures. Therefore, it appears that temperate and tropical N2-fixing organisms adapt themselves to their particular environment and should have more potential to benefit crops grown at the particular temperatures favorable to them.

The influence of phosphorus application on soil nitrogenase and N2-fixers in tropical paddy soils differing in their properties was investigated in a laboratory incubation study. Application of P stimulated the soil N2-ase in an alluvial soil and in a P-deficient soil under both flooded and nonflooded conditions. The stimulation of N2-ase by P was more pronounced under nonflooded conditions. A corresponding increase in N2-ase occurred with an increase in the P level at least up to 80 ppm level. A depressive effect of P on N2-ase occurred after 16 days under nonflooded conditions when the level of P was increased to 100 ppm. But under flooded conditions the stimulation was almost continuous. Results indicate that the effect of P on N2-ase depended on the water regime, level of P and soil type. Addition of P had a little effect on the population of N2-fixing micro-organisms in alluvial soil. On the contrary, addition of P stimulated the population of Azospirillum and Azotobacter in a P-deficient soil. Data suggested that the alteration in the N2-fixing microbial populations and the levels of available P might be responsible for changes in the N2-ase activity in these soils. Application of superphosphate and dicalcium phosphate stimulated N2-ase activity; while the rock phosphate exhibited an innocuous effect in alluvial and P deficient soils. In Sukinda soil, however, super phosphate slightly stimulated N2-ase at early stages, while other P sources had innocuous effect. Results indicate that the level and source of applied P exhibited differential influence on N2-ase and N2-fixers in tropical paddy soils.

The influence of clay minerals, humic acid and capsular polysaccharide on the survival of Klebsiella aerogenes exposed to drying and heating in soils was investigated. The study of three types of soils showed that the survival of the bacteria was higher in montmorillonitic than in kaolinitic soils. When a sandy soil was amended with 10 per cent montmorillonite, 10 per cent kaolinite or 0.5 per cent humic acid, the survival of K. aerogenes was increased in the presence of montmorillonite but was not affected by kaolinite or humic acid. It was suggested that montmorillinite affected the pattern of water retention by the cells during the drying process. The influence of capsular polysaccharides was also investigated and no significant difference was abserved between the survival of the encapsulated and non-capsulated strains of K. aerogenes exposed to drying and heating in soils. re]19750321

Microbial populations of 11 Nigerian soils are reported. Four soils from the dry forest zone of Nsukka, East Central State, are characterized by few algae, nitrifiers, denitrifiers, and aerobic cellulose decomposers. Four soils from four northern States in the Guinea savanna have abundant bacteria and algae but in none of these soils do Azotobacter or Beijerinckia exceed 100 bacteria/g soil. Three soils from the dry forest zone of Ibadan, Western State, contain normal densities of the microbes examined. Populations of fungi and actinomycetes in the 11 soils vary little. Acidity has a great influence on the densities of the microbes examined. No conclusions can be drawn on the relation of temperature, soil type, or vegetation to the densities of the microbes examined.

A field experiment was conducted with ADT-31 rice variety. Foliar spray ofAzotobacter chroococcum was applied on 15th, 30th and 45th day after transplanting of rice crop. The foliar spray of Azotobacter culture significantly increased the grain and straw yield of rice crop.

The factors that influence inocula establishmentviz. soil fertility, organic manuring and interactions between Azotobacter and phosphobacteria were examined in unsterilised and sterilised soil with and without farm yard manure in green house conditions. During the experiments, the number of nitrogen-fixing and phosphate-solubilising bacteria were enumerated. At the end of experiment, dry weight of grain and its nitrogen and phosphate uptake were determined. Increased number of both types of bacteria in rhizosphere soil and greater yield and nutrients uptake were observed, when seeds were bacterised with single or mixed cultures as compared to uninoculated treatment. The number of organisms remained quite high in mixed cultures and was so for longer periods of plant growth. Addition of 2% farmyard manure further enhanced the bacterial population and crop productivity. This effect was more pronounced in sterilised soil.

Out of ten isolates ofAzospirillum spp. isolated from the root and rhizosphere of wheat plant, seven belonged toA. brasilense and three belonged toA. lipoferum. All eight isolates ofAzotobacter spp. belonged toAzotobacter chroococcum. Two strains, one fromA. lipoferum and another fromA. chroococcum having high nitrogen fixing capacity with negative test for denitrification were used as inoculant to supplement the nitrogen need of wheat crop. Significant increases in the yield of wheat grain and uptake of nitrogen by the crop over the control were found in pot tests when the seeds were inoculated either withAzospirillum spp. orAzotobacter spp. or the combination of both the inoculants.

Organic components leaked fromSorghum bicolor seedlings (‘root exudates’) were examined by recovering14C labelled compounds from root solutions of seedlings inoculated withAzospirillum brasilense, Azotobacter vinelandii orKlebsiella pneumoniae nif-. Up to 3.5% of the total14C recovered from shoots, roots, and nutrient solutions was found in the root solutions. Inoculation with Azospirillum and Azotobacter increased the amounts of14C and decreased the amounts of carbohydrates in the root solutions. When sucrose was added as a carbon source for the bacteria, the increase of14C in the solutions did not occur. Quantities of14C found in the root solutions were proportional to amounts of mineral nitrogen supplied to the plants. Bacterial growth also was proportional to nitrogen levels. When sorghum plants were grown in soil and labelled with14CO2, about 15% of the total14C recovered within 48 hours exposure was found in soil leachates.

Azotobacter vinelandii strains caused the formation of increased numbers of root nodules onGlycine max, Vigna unguiculata andTrifolium repens by their respective rhizobial symbionts. Increased nodulation due to inoculation withA. vinelandii also occurred in field grownG. max. Mutant strains ofA. vinelandii unable to fix nitrogen caused nodulation increases comparable to those caused by nitrogen-fixing strains. This indicates that nitrogen fixation byA. vinelandii was not responsible for the enhanced nodulation. The effect ofA. vinelandii on nodulation was greatest when cells from the mid-exponential phase of growth were applied as inoculants. Non viable cell preparations ofAzotobacter vinelandii were also found to cause an increase in the number of root nodules formed onGlycine max Rhizobium japonicum under greenhouse conditions. The nodulation enhancement activity was influenced by the method chosen to kill theA. vinelandii cells. Heat treatment and treatment with lethal levels of streptomycin destroyed the activity, whereas the activity was unaffected by ultraviolet-light treatment of the cells. Cell-free extracts ofA. vinelandii were found to enhance nodulation. On the other hand, culture supernatants ofA. vinelandii had no effect on nodulation. A split-root experiment suggested that the agent(s) responsible for the increased nodulation was not translocatable throughout the plant. The results suggest a non-excretable protein, produced byA. vinelandii, as a possible mechanism for nodulation enhancement.

Respiration and N2-fixation (acetylene reduction) ofAzotobacter vinelandii have been studied at a variety of soil water potentials. Both processes were strictly linked and strongly reduced at water potentials between −0.6 and −1.3 MPa. Complete inhibition occurred below −2.1MPa. Osmotic potentials in soil compared to matric potentials of the same value were less inhibitory to respiration and acetylene reduction by Azotobacter. The N2-fixing efficiency (mg N/g glucose) was not influenced by water potentials ranging from −0.1 to −2.1 MPa.

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%.

It has been observed that in the case ofVigna radiata andGlycine max incorporation of suitable strain of Azotobacter gave higher yield than obtained by the use of Rhizobium as inoculant. In the case ofVigna radiata even a strain of Azotobacter isolated from the rhizosphere of berseem gave similar yields as Rhizobium. In the case ofPisum sativum association of Rhizobium with a strain ofAzotobacter chroococcum isolated from the rhizosphere of pea gave numerically higher yield than Rhizobium alone. It may be possible that statistically higher yield may be obtained when a suitable strain of Azotobacter is used after screening a large number of strains of Azotobacter from the rhizosphere of pea.

Captan was applied to laboratory-incubated agricultural soil and to bacterial cultures to determine its effects on total counts of soil microorganisms, nitrification, ammonification of urea and asymbiotic dinitrogen fixation.

The pattern of N2-ase activity in clay-loam soil of Nile Delta was determined. However, unamended soil showed somewhat low activity: an amount of 18–95 mg N2 fixed/kg soil/year was calculated. Addition of glucose greatly enhanced such activity and efficiencies of N2-fixation increased with decreasing carbon source concentration. Highest activities (800 n moles C2H4/gh−1) and efficiencies (18.06 mg N2/g glucose added) were reported in soil amended with 1% glucose, adjusted to 50% W.H.C. and incubated at 30°C. Enrichment of the soil with straw lead to a significant nitrogen gain particularly under water-logged conditions. During a short period of 16 days 5.8–9.3 mg N2 were fixed/g straw added at the latter conditions.

A field experiment was conducted to study the effect ofAzotobacter chroococcum andAzospirillum brasilense inoculation under graded levels of fertilizeer nitrogen (0, 30, 60, 90, 120 kg/ha) on wheat (Triticum durum Desf. var. HD2189). The results revealed that both the inolculants sigificnatly increased the growth parametersviz. tilles, dry-matter accumulation, grain yield and protein content of wheat gains. Fertilizer—N @ 90 kg/ha with both inoculations was found to be the most responsive, however 120 kg/ha with Azospirillum inoculation recorded maximum number of tillers (3.5/plant), dry-matter accumulation (13.5 g/plant), grain yield (41.9 g/ha) and protein content in grains (13.3%). Maximum percent increase in grain yeidl with Azospirillum (25.8%) and Azotobacteer (19.2%) over uninoculated control was observed at 90 kg N/ha level. Azospirillum inoculation, Azotobacter inoculation and uninoculated control significnatly differed between each other.

Beneficial effects on yield and N-assimilation in wheat cv. HD2329 due to inoculation with nine strains belonging to six species and cowpea miscellany of genus rhizobium and Azotobacter were observed. Stimulation of soil Azotobacter and Azospirillum in the root region of wheat occurred due to seed inoculation with Rhizobium.

Azotobacter associated withEichhornia crassipes were found to be concentrated in the slime formed around petiole bases. ‘Massive’ colonies formed where raw slime was applied to nitrogen-free medium inoculated with Azotobacter. Slime in which Azotobacter had been killed did not stimulate the formation of massive colonies unless live Azotobacter cells were mixed into it. Acetylene reduction tests indicated that Azotobacter associated with Eichhornia did not fix nitrogen unless the plant was suffering extreme nitrogen deficiency, and that the maximum rate of fixation was inadequate for rapid plant growth.

Under controlled conditions in pots filled with sand, vermiculite and field soil, inoculation withA. brasilense-Cd ATCC 29729 or withAzotobacter chroococcum caused increases above controls in the weight and N content of panicles ofSetaria italica. In no case, however, did N increases in test plants exceed the initial total N content. High acetylene reduction activities (1,000–2,000 nmole/h/pot) could be found only in plants inoculated withAzospirillum. Inoculation withAzospirillum (strain-Cd) in the field caused a significant increase above noninoculated controls of 18.5% in shoot dry weight, ofSetaria italica. Azotobacter caused a non significant increase of 8%. No significant differences were found between yields ofSetaria italica grown in soil inoculated withA. chroococcum, and those of plants grown in the presence ofA. vinelandii. A. brasilense-Cd was more effective in the field thanA. brasilense Sp-7 ATCC 29145. The results suggest that Azospirillum may increase yields ofS. italica more efficiently than Azotobacter under local field conditions.

A new medium has been evolved for counting Azotobacter by the plating method. The advantages of this medium are: 1) contaminants are greatly suppressed 2) the colonies of Azotobacter are bigger in size than those of the contaminants and being black in colour are easily identified.

Seed of maize, tomato, and wheat was inoculated with cultures of Azotobacter, Clostridium, and a nitrogen-fixing facultative Bacillus and grown in a nutrient-deficient sand and a highly fertile silt loam. In sand, wheat showed a significant positive response to inoculation with Azotobacter and Clostridium but maize and tomato were unaffected by inoculation. When inoculated seed was planted in Lima silt loam there were significant increases in the growth of maize, tomato, and wheat to treatment with Clostridium, inoculated maize and wheat responded to Azotobacter inoculation while only wheat responded to inoculation with the facultative Bacillus. In pure-culture studies of the ability of these cultures to establish upon plant roots it was shown that Azotobacter did not colonize the roots of lucerne, maize, tomato, or wheat to any great extent. Bacillus and Clostridium were moderate colonizers of plant roots reaching from 1 to 20 per cent the levels reached byPseudomonas fluorescens on the same plants.

A series of investigations were conducted with the objective of elucidating natural pathways of electron transport from respiratory processes to the site of N2 fixation in nodule bacteroids. A survey of dehydrogenase activities in a crude extract of soybean nodule bacteroids revealed relatively high activities of NAD-specific β-hydroxybutyrate and glyceraldehyde-3-phosphate dehydrogenases. Moderate activities of NADP-specific isocitrate and glucose-6-phosphate dehydrogenases were observed. By use of the ATP-dependent acetylene reduction reaction catalyzed by soybean bacteroid nitrogenase, and enzymes and cofactors from bacteroids and other sources, the following sequences of electron transport to bacteroid nitrogenase were demonstrated: (1) H2 to bacteroid nitrogenase in presence of a nitrogenase-free extract ofC. pasteurianum; (2) β-hydroxybutyrate to bacteroid nitrogenase in a reaction containing β-hydroxybutyrate dehydrogenase, NADH dehydrogenase, NAD and benzyl viologen; (3) β-hydroxybutyrate dehydrogenase, to nitrogenase in reaction containing NADH dehydrogenase, NAD and either FMN or FAD; (4) light-dependent transfer of electrons from ascorbate to bacteroid nitrogenase in a reaction containing photosystem I from spinach chloroplasts, 2,6-dichlorophenolindophenol, and either azotoflavin from Azotobacter or non-heme iron protein from bacteroids; (5) glucose-6-phosphate to bacteroid nitrogenase in a system that included glucose-6-phosphate dehydrogenase, NADP, NADP-ferredoxin reductase from spinach, azotoflavin from Azotobacter and bacteroid non-heme iron protein. The electron transport factors, azotoflavin and bacteroid non-heme iron protein, failed to function in the transfer of electrons from an NADH-generating system to bacteroid nitrogenase. When FMN or FAD were added to systems containing azotoflavin and bacteroid non-heme iron protein, electrons apparently were transferred to the flavin-nucleotides and then nitrogenase without involvement of azotoflavin and bacteroid non-heme iron protein.

A representative alkali soil sample from Tal-El-Kebir was treated in pots with different rates of gypsum or sulphur. These amounts were calculated according to the exchangeable sodium present in the soil. Chemical and microbiological properties were studied during reclamation process. Addition of gypsum in appropriate amounts to replace exchangeable sodium on the clay complex is much faster and more superior to the addition of the corresponding amounts of sulphur. Counts of total microbial flora, Azotobacter, nitrifiers, and aerobic cellulose-decomposers increased as the reclamation proceeded, on the other hand, counts of spores of aerobic sporeformers, streptomyces, and clostridia decreased. Cultivation of such reclaimed soils exerted beneficial effect on its microbial flora. Plant vigor which was taken as a resultant of the improvement of the physico-chemical and biological properties of the soil due to reclamation was found to be superior in case of addition of an amount of gypsum equivalent to the amount of adsorbed sodium.

Periodical nitrogen fixation experiments with two strains ofAzotobacter chroococcum for 16 months showed no definite correlation between gains of nitrogen and meteorological changes, as reported by Bortels. Variability in the gains of nitrogen seemed to be due largely to chance variation, together with certain abnormal depressions which did not clearly coincide with changes in atmospheric pressure. The conclusions of Bortels thus do not seem to be generally valid forAzotobacter. Certain long-term variations in nitrogen fixation seemed in one strain to be connected with the arising of a variant of lower nitrogen-fixing power.

The preceding observations showed that both Azotobacter and a mixed flora with Azotobacter are about equally capable of fixing atmospheric nitrogen. During subsequent transfer of the nitrogen there was a distinct differentiation between movement of the newly fixed nitrogen and the nitrogen that was already present in tissues introduced in the beginning of the experiment. Pathways and transfer coefficients of both N14 and N15 were different in the presence of the different microbial populations. Turnover was more complex and in general slower in the presence of a mixed population than with pure Azotobacter. The lower transfer coefficients in the mixed populations may reflect more recycling within these populations than in pure cultures of Azotobacter. Denitrification losses upon acidification were negligible for N14 but appreciable for N15. The effect of acidification tended to be greater in systems with pure cultures of Azotobacter than in systems with a mixed culture.

Four latosolic soils, varying in total nitrogen content from 0.050 to 0.098 per cent and in organic matter from 0.95 to 1.56 per cent, were collected from the farm of University College, Ibadan, Nigeria. No change in nitrogen content was observed when the soils were kept moist in the greenhouse for four months. Where a legume (Centrosema pubescens) was grown gains of nitrogen in the soil—plant system equivalent to from 100 to 200 pounds nitrogen per acre — 6 inches deep were obtained. Where a grass (Eleusine coracana) was grown gains of nitrogen in the soil—plant system equivalent to from 100 to 130 lb. nitrogen per acre — 6 inches deep occurred in the two soils with low initial nitrogen levels. The possible origin of this nitrogen accretion is discussed. The data support, but do not prove, the hypothesis that it is due to non-symbiotic fixation in the soil. In one of the soils showing a significant nitrogen gain under grass the presence of Azotobacter was not demonstrated and Beijerinckia occurred in only one of 24 samples examined. Thus, if non-symbiotic biological nitrogen fixation did occur, Clostridium and other micro-organisms not belonging to the Azotobacteriaceae must have been the agents of fixation involved. On the other hand, the presence of Azotobacter in the two soils in which no fixation occurred could easily be demonstrated. It is suggested that non-symbiotic nitrogen fixation by soil micro-organisms under grass cover in the humid tropics may be of greater significance than has been believed in the past.

Small amounts of indolyl-3-acetic acid (IAA) were detected in aerated cultures ofAzotobacter chroococcum grown with or withoutl-tryptophane in the medium, but IAA was detected in agar cultures only whenl-tryptophane was present. Most IAA was found in 7-day-old cultures and less in older cultures. Washed cells did not convert tryptophane enzymically to IAA. The time course of IAA formation byA. chroococcum strain A6 has been described and the effect of adding tryptophane to the medium has been studied. In contrast to results elsewhere strain A6 produced traces of IAA in aerated cultures with or without added tryptophane. IAA was detected only after the end of exponential growth when cells had begun to autolyse. The amount of IAA declined as cultures aged. The slight effect ofl- but not ofd-tryptophane in promoting IAA formation in ageing cultures suggests some kind of biological transformation but it seems unlikely that IAA formation is part of the normal metabolic processes of intact Azotobacter cells.

The mulberry leaves were shown to harbour substantial populations of bacteria, streptomycetes, yeasts, and moulds. Azotobacter and Beijerinckia were observed to contribute to nearly 5 to 10 per cent of the bacterial population. When grown in water culture under sterile conditions, Azotobacter inoculation on the leaf or root surface was found to increase plant growth, dry wt, and nitrogen content of the mulberry. The beneficial effect of Azotobacter was largely influenced by the presence of a carbon source in the plant nutrient solution. The root inoculation in comparison to leaf application was found to confer greater benefits to the growing plant. The presence of carbohydrates and amino acids in the leaf leachates of mulberry was shown. The mutual beneficial nature of the association of the plant and Azotobacter has been brought to light.

RESP-4753

Azotobacter and nitrogen-fixing clostridia are ubiquitous soil inhabitants in Egypt, Iraq and probably in all of the Near East. They occur in high numbers except where barrenness, NaCl accumulation or other depressing factors exist. The soil environment has proved favourable for their development since their response to supplementation with energy materials is quite marked. The organisms are resistant to drought, but optimal activity of Azotobacter is around 60% W.H.C. while that of clostridia is at 100%. Azotobacter as well as clostridia show optimal activity around 30°C, higher temperatures favour clostridia while lower ones favour Azotobacter. Gains of soil nitrogen are linked to the growth of Azotobacter rather than to that of Clostridium. The amounts of nitrogen gained and fixation efficiency are affected by the nature of the substrate, being greatest in clay, then in sand and calcareous soils and least in liquid media. Phosphate is essential, favouring nitrogen fixation firstly by satisfying the high phosphate requirement of Azotobacter and secondly by increasing the rate of decomposition of otherwise unavailable material. Gains of combined nitrogen and fixation efficiency are also affected by the type of organic matter added. A wide C/N ratio and susceptibility to decomposition are specially beneficial properties. Plant residues enrich the soil with nitrogen, partly by enhancing nitrogen fixation and partly by causing immobilization of mineral nitrogen which would otherwise be leached out of the soil by irrigation.

1. Rumen fluid from four sheep, one on a low nitrogen diet, showed slight acetylene reduction.Desulfotomaculum ruminis, a rumen anaerobe, fixes N2 but the effective organisms in rumen samples seem to resembleClostridium pasteurianum; this organism can persist in the sheep rumen. In domestic sheep the contribution of rumen fixation to the animal's N-nutrition is probably negligible; other ruminants on various diets require study.

The various types of blue-green algae known to fix nitrogen are considered. Particular attention has been paid to the effects of oxygen and other physiological parameters on nitrogenase activity and on the ecological distribution of the group. Data on nitrogen fixation by cell-free extracts of blue-green algae are presented.

The development ofAzotobacter chroococcum is promoted in the ivy (Hedera helix L.) rhizosphere when it is colonized by the pectinolytic moldMucor fragilis. The multiplication of Azotobacter cells in the mineral solution of ivy cultures induces a decrease of the amount of sugars excreted by the roots and a increase of the amount of nitrogen. Within the experimental period Azotobacter does not influence the growth of ivy roots although it seems to be promoted by Mucor. Filtrates of Mucor culture are, however, harmful to the plant.

Small samples of soil amended with 2% (w/w) of glucose were preincubated either aerobically or anaerobically and then assayed (N2 15 and C2H2-C2H4) either aerobically or anaerobically for different time periods. One-hour C2H2-C2H4 assays showed greatest activity when anaerobic assay followed anaerobic preincubation. During the anaerobic preincubation a lag of 12–24 h occurred before rapid increase in one-hour assay activity was observed. When aerobic assay followed aerobic preincubation a longer lag was observed and lower activities were obtained. When anaerobic assay followed aerobic preincubation (orvice versa) negligible activities were observed in short assays, and longer assays showed increasing activity related to changes in atmosphere and/or microbial population in the closed system. Preincubation of soil on a diffusion gradient at a series of different partial pressures of oxygen confirmed the above pattern and showed that as preincubation pO2 increased, the anaerobic assay activity rapidly decreased. As preincubation pO2 decreased from 0.2 atm the aerobic assay activity decreased but less rapidly. The activities observed were related to the sizes of the Azotobacter and Clostridium populations. There was no evidence of aerobic or anaerobic C2H2 reduction in any cultures of ‘oligonitrophiles’ isolated. Incorporation of N2 15 was related to C2H2 reduction activity in the soil system studied. However, observed C2H4/N2 molar ratios ranged from 10 to 22 and appeared to be highest in samples which were preincubated anaerobically.

The physico-chemical properties of the soil were gradually improved; a progressive increase in silt and clay, moisture equivalent, organic matter, total nitrogen, C : N ratio and cation exchange capacity was observed during reclamation. Microbiological analysis showed that bacteria, actinomycetes and fungi had increased progressively as a result of cultivation. The increase in the microbial counts showed a positive relation with the increase in the organic matter content of the soil. This indicates that one of the limiting factors for microbial proliferation is organic matter. It was also found that the aerobic cellulose-decomposers, nitrifiers, Azotobacter and Clostridia had increased gradually with the cultivation of these soils. The increase of Clostridia was more remarkable than that of Azotobacter.

Investigations were carried out to study the effect ofAzotobacter chroococcum VP-5 on the hatching of egg masses and eggs ofMeloidogyne incognita in vitro. Eggs were more susceptible and required lesser time for antagonistic action of Azotobacter than egg masses.