Using the spermosphere model technique to describe the dominant nitrogen-fixing microflora associated with wetland rice in two Egyptian soils

Summary This study is an attempt to describe the dominant N₂-fixing microflora associated with the roots of wetland rice. Rice cultivar Giza 171 was grown in a phytotron on two alluvial Egyptian soils for 8 days, a stage when the nitrogenase activity of undisturbed plants reached a level of 245 × 10^–6 mol C₂H₄ h^–1 g^–1 dry weight of leaf. The roots and rhizosphere soils were then used for counting and isolating dominant diazotrophs. Counts and initial enrichment steps were carried out on a selective medium made of an axenic rice plantlet, the spermosphere model, incubated under 1 % acetylene. The counts were very high, exceeding 10⁸ bacteria g^–1 dry weight of rhizosphere soil. Enterobacteriaceae were dominant; most isolates were Enterobacter cloacae belonging to different biotypes in the two soils. Enterobacter agglomerans, Citrobacter freundii and Klebsiella planticola were also present as members of the dominant microflora. Azospirillum brasilense and Azospirillum lipoferum were present as well, but less abundant.     

Infectivity and acetylene reduction of diazotrophic rhizosphere bacteria in wheat (Triticum aestivum) seedlings under gnotobiotic conditions

Summary Wheat seedlings were inoculated with rhizosphere nitrogen-fixing bacteria and grown gnotobiotically for 15 days. The growth medium consisted of semisolid agar with or without plant nutrients. The bacteria, isolated from roots of field-grown wheat, were three unidentified Gram-negative rods (A1, A2, E1), one Enterobacter agglomerans (C1) and two Bacillus polymyxa (B1, B2). A strain of Azospirillum brasilense (USA 10) was included for comparison.Nitrogenase activity (acetylene reduction activity, ARA) was tested on intact plants after 8 and 15 days of growth. In semisolid agar without plant nutrients, five isolates showed ARA of 0.01–0.9 nmol C₂H₄ plant^–1 h^–1, while the two strains of B. polymyxa had higher ARA of 3.3–10.6 nmol C₂H₄ plant^–1 h^–1.Plant development was not affected by inoculation with bacteria, except that inoculation with B. polymyxa resulted in shorter shoots and lower root weight.Transmission electronmicroscopy of roots revealed different degrees of infection. A. brasilense, A1 and A2, occurred mainly in the mucilage on the root surface and between outer epidermal cells (low infectivity). B. polymyxa strains and E1 were found in and between epidermal cells (intermediate infectivity) while E. agglomerans invaded the cortex and was occasionally found within the stele (high infectivity).     

Trace gas emissions from soil of the central highlands of Mexico as affected by natural vegetation: a laboratory study

In the central highlands of Mexico, mesquite (Prosopis laevigata) and huisache (Acacia schaffneri), N₂-fixing trees or shrubs, dominate the vegetation and are currently used in a reforestation program to prevent erosion. We investigated how natural vegetation or cultivation of soil affected oxidation of CH₄, and production of N₂O. Soil was sampled under the canopy of mesquite (MES treatment) and huisache trees (HUI treatment), outside their canopy (OUT treatment) and from fields cultivated with maize (ARA treatment) at three different sites while production of CO₂, and dynamics of CH₄, N₂O and inorganic N (NH₄⁺, and NO₃^–) were monitored in an aerobic incubation. The production of CO₂ was 2.3 times higher and significantly greater in the OUT treatment, 3.0 times higher in the MES treatment and 4.0 times higher in the HUI treatment compared to the ARA treatment. There was no significant difference in oxidation of CH₄ between the treatments, which ranged from 0.019 g CH₄–C kg^–1 day^–1 for the HUI treatment to 0.033 CH₄–C kg^–1 day^–1 for the MES treatment. The production of N₂O was 30 g N₂O–N kg^–1 day^–1 in the MES treatment and >8 times higher compared to the other treatments. The average concentration of NO₃^– was 2 times higher and significantly greater in the MES treatment than in the HUI treatment, 3 times greater than in the OUT treatment and 10 times greater than in the ARA treatment. It was found that cultivation of soil decreased soil organic matter content, C and N mineralization, but not oxidation of CH₄ or production of N₂O.     

Tree fallows: A comparison between five tropical tree species

The natural abundance of ¹⁵N and ¹³C, conventional soil analyses, and biomass production by maize were used to study the influence of five tropical tree species on soils and their fertility. The experiment was conducted in Morogoro, Tanzania, to compare Cassia (Senna) siamea, Eucalyptus camaldulensis, E. tereticornis (all non-N₂-fixing), Leucaena leucocephala, Prosopis chilensis (both N₂-fixing), and a grass fallow. Maize biomass production, which was correlated with N uptake (P=0.001), was higher on soils from plots with 5-year-old Leucaena and Prosopis spp. compared to the grass fallow, while other tree species had less favourable effects on maize growth. The per cent N was higher in soil and ¹⁵N of soil total N was lower under Prosopis sp. compared to soil under other tree species, which suggests an input from N₂ fixation by Prosopis sp. A transfer of fixed N to maize or to understorey grass species was, however, not indicated by the ¹⁵N natural abundance. Prosopis sp. contributed more C to the soil than the other four tree species; the difference in ¹³C between soils from Prosopis sp. plots and from grass fallow plots showed that the tree contributed 11% to the total C of the soil over a period of 8 years. The leaves of the N₂-fixing species had a low ratio of lignin+phenols to N, and maize growth was negatively correlated with this parameter. The Eucalyptus spp. had leaves with a high lignin+phenols to N ratio, contributed very little C to the soil, and lowered the soil pH.     

N2-fixing bacteria in the rhizosphere: Quantification and hormonal effects on root development

The paper summarizes the results of a series of experiments on enumeration of N₂-fixing bacteria (diazotrophs) and hormonal effects of Azospirillum on root development. Numbers of N₂-fixing and N-heterotrophic bacteria were determined on the root (rhizoplane plus “inner” root surface) and in the rhizosphere soil (0–3 mm from the root surface) of Arrhenatherum elatius, other forage grasses and some herbaceous plant species. Pot experiments involved freshly collected soil from an unfertilized grassland area containing its natural population of N₂-fixing bacteria. The MPN (most probable number) of diazotrophs in relation to the MPN of the total bacterial population was always lower on the root than in the rhizosphere soil, suggesting that diazotrophs were not selectively advantaged at the root surface. Supply of mineral nitrogen (NH₄NO₃) decreased the proportion of N₂-fixing bacteria at the rhizoplane as well as in the rhizosphere soil. Similar results were obtained when N was supplied via the leaves. The data suggest that N₂-fixing bacteria in the rhizosphere are poor competitors once they loose their competitive advantage of binding dinitrogen. Correspondingly, the increase in the MPN of the diazotrophs found during plant development was interpreted as a result of decreased available combined N in the rhizosphere. The proportion of N₂-fixing bacteria relative to the total number of bacteria was generally below 1%. Considering the potential amount of substrate released from the roots and the substrate requirement of the bacterial population, N₂-fixation was considered insignificant for plant growth under the given conditions. For the investigations on possible beneficial effects on plant development by bacterial hormones, Azospirillum brasilense was chosen because evidence suggests that amongst the soil bacteria releasing hormones, especially IAA, certain strains of this species are more important than other bacteria. Application of A. brasilense Cd (ATCC 29710) onto the roots of young wheat plants grown in soil increased the number of lateral roots, the total root length and the number of root hairs. Similar results were obtained after application of IAA. This suggests that IAA is an important factor responsible for the effects observed after inoculation with A. brasilense. The increase in root surface may improve acquisition of nutrients and enhance growth of plants. Another hormonal effect of A. brasilense was an increase in nodulation of Medicago sativa grown on agar. Again pure IAA resulted in a similar increase in nodule number. Increases in nodule number were only in part associated with a change in root morphology. Therefore an effect of IAA on the plant immanent regulation system for nodulation is likely.     

Effects of thiamine and nitrogen fertilizer form on the number of N2−fixing and total bacteria in the rhizosphere of maize plants

The effects of thiamine (vitamin B1) application as seed dressing and of N form supplied (NH₄⁺ versus NO₃^?) on rhizosphere pH and on rhizosphere microorganisms were evaluated in two different soils. Imbibition of maize (Zea mays L.) seeds with thiamine (1 g kg^?1) increased seed thiamine content by a factor of 370. Maize plants from untreated and treated seeds were cultivated in a growth chamber under controlled conditions for 10 d in a sandy loam soil, pH 7.1 (Mascherode soil) or in a sandy soil, pH 4.8 (Niger soil) fertilized with two different N sources (NO₃?N or NH₄?N with dicyandiamide, 100 and 250 mg N kg^?1 soil). The rhizosphere pH was not affected by thiamine, only slightly affected by N source in the Mascherode soil, but markedly affected in the Niger soil. Thiamine application and N source affected the most probable number (MPN) of diazotrophs and total bacteria isolated from the rhizosphere soil of 10 d old maize plants. In the Mascherode soil, thiamine application increased MPN of diazotrophs 4-fold and total bacteria 2-fold when the soil was fertilized with 100 mg NO₃?N compared to untreated seedlings. Compared to Mascherode soil, in the Niger soil, MPN of diazotrophs was extremely low, especially after NH₄?N treatment which significantly decreased pH of the rhizosphere. Thiamine application had only marginal effects on the MPN of diazotrophs and total bacteria. Total bacteria isolated from Niger soil fertilized with NH₄?N were about 10-fold lower compared to the soil from Mascherode. However, in the other two treatments, total bacteria were higher in the Niger soil compared to the Mascherode soil. In the Niger soil, apparently some of the heterotrophs (the Actinomycetes dominated in this soil) might have suppressed the diazotrophs. The results of the present study demonstrate that in many cases seed treatment with thiamine enhances MPN of diazotrophs and total bacteria in the rhizosphere of maize seedlings.     

GREENHOUSE GAS EMISSIONS FROM AN ALKALINE SALINE SOIL CULTIVATED WITH MAIZE (ZEA MAYS L.) AND AMENDED WITH ANAEROBICALLY DIGESTED COW MANURE: A GREENHOUSE EXPERIMENT

Sludge derived from cow manure anaerobically digested to produce biogas (methane; CH₄) was applied to maize (Zea mays L.) cultivated in a nutrient-low, alkaline, saline soil with electrolytic conductivity 9.4 dS m^?1 and pH 9.3. Carbon dioxide (CO₂) emission increased 3.1 times when sludge was applied to soil, 1.6 times when cultivated with maize and 3.5 times in sludge-amended maize cultivated soil compared to the unamended uncultivated soil (1.51 mg C kg^?1 soil day^?1). Nitrous oxide (N₂O) emission from unamended soil was -0.0004 μg nitrogen (N) kg^?1 soil day^?1 and similar from soil cultivated with maize (0.27 μg N kg^?1 soil day^?1). Application of sludge increased the N₂O emission to 4.59 μg N kg^?1 soil day^?1, but cultivating this soil reduced it to 2.42 μg N kg^?1 soil day^?1. It was found that application of anaerobic digested cow manure stimulated maize development in an alkaline saline soil and increased emissions of CO₂ and N₂O.     

Nitrogen fixation by Azospirillium brasilense in soil and the rhizosphere under controlled environmental conditions

Summary Acetylene reduction activity by Azospirillum brasilense, either free-living in soils or associated with wheat roots, was determined in a sterilised root environment at controlled levels of O₂ tension and with different concentrations of mineral N. In an unplanted, inoculated soil nitrogenase activity remained low, at approximately 40 nmol C₂H₄ h^-1 per 2kg fresh soil, increasing to 300 nmol C₂H₄ h^-1 when malic acid was added as a C source via a dialyse tubing system. The N₂ fixation by A. brasilense in the rhizosphere of an actively growing plant was much less sensitive to the repressing influence of free O₂ than the free-living bacteria were. An optimum nitrogenase activity was observed at 10 kPa O₂, with a relatively high level of activity remaining even at an O₂ concentration of 20 kPa. Both NO _inf3 ^sup- and NH _inf4 ^sup+ repressed nitrogenase activity, which was less pronounced in the presence than in the absence of plants. The highest survival rates of inoculated A. brasilense and the highest rates of acetylene reduction were found in plants treated with azospirilli immediately after seedling emergence. Plants inoculated at a later stage of growth showed a lower bacterial density in the rhizosphere and, as a consequence, a lower N₂-fixing potential. Subsequent inoculations with A. brasilense during plant development did not increase root colonisation and did not stimulate the associated acetylene reduction. By using the ¹⁵N dilution method, the affect of inoculation with A. brasilense in terms of plant N was calculated as 0.067 mg N₂ fixed per plant, i.e., 3.3% of the N in the root and 1.6% in the plant shoot were of atmospheric origin. This ¹⁵N dilution was comparable to that seen in plants inoculated with non-N₂-fixing Psudomonas fluorescens.     

Agricultural factors affecting methane oxidation in arable soil

CH₄ oxidation activity in a sandy soil (Ardoyen) and agricultural practices affecting this oxidation were studied under laboratory conditions. CH₄ oxidation in the soil proved to be a biological process. The instantaneous rate of CH₄ consumption was in the order of 800 mol CH₄ kg^–1 day^–1 (13 mg CH₄ kg^–1 day^–1) provided the soil was treated with ca. 4.0 mmol CH₄ kg^–1 soil. Upon repeated supplies of a higher dose of CH₄, the oxidation was accelerated to a rate of at least 198 mg CH₄ kg^–1 day^–1. Addition of the plant-growth promoting rhizopseudomonad strains Pseudomonas aeruginosa 7NSK2 and Pseudomonas fluorescens ANP15 significantly decreased the CH₄ oxidation by 20 to 30% during a 5-day incubation. However, with further incubation this suppression was no longer detectable. Growing maize plants prevented the suppression of CH₄ oxidation. The numbers of methanotrophic bacteria and fungi increased significantly after the addition of CH₄, but there were no significant shifts in the population of total bacteria and fluorescent pseudomonads. Drying and rewetting of soil for at least 1 day significantly reduced the activity of the indigenous methanotrophs. Upon rewetting, their activity was regained after a lag phase of about 3 days. The herbicide dichlorophenoxy acetic acid (2,4-D) had a strong negative effect on CH₄ oxidation. The application of 5 ppm increased the time for CH₄ removal; at concentrations above 25 ppm 2,4-D CH₄–oxidizing activity was completely hampered. After 3 days of delay, only the treatments with below 25 ppm 2,4-D showed recovery of CH₄–oxidizing activity. This finding suggests that it can be important to include a CH₄–removal bioassay in ecotoxicology studies of the side effects of pesticides. Changes in the native soil pH also affected the CH₄–oxidizing capacity. Permanent inhibition occurred when the soil pH was altered by 2 pH units, and partial inhibition by 1 pH unit change. A rather narrow pH range (5.9–7.7) appeared to allow CH₄ oxidation. Soils pre-incubated with NH ₄ ⁺ had a lower CH₄–removal capacity. Moreover, the nitrification inhibitor 2-chloro-6-trichloromethyl pyridine (nitrapyrin) strongly inhibited CH₄ oxidation. Probably methanotrophs rather than nitrifying microorganisms are mainly responsible for CH₄ removal in the soil studied. It appears that the causal methanotrophs are remarkably sensitive to soil environmental disturbances.     

Biological nitrogen fixation in Azospirillum strain-maize genotype associations as evaluated by the 15N isotope dilution technique

Few studies of the inoculation of cereal crops with N₂-fixing bacteria have included more than one or two plant genotypes. In a recent study performed in Argentina using 12 different maize genotypes, it was found in 2 consecutive field experiments that several of them responded consistently, either negatively or positively, to inoculation with a mixture of strains of Azospirillum spp. The present study in post was performed to investigate the effect of inoculation of individual strains (and a mixture) of Azospirillum spp., and their nitrate reductase negative (NR-) mutants, on the growth of four of these maize genotypes. Two of these genotypes were grown in ¹⁵N-labelled soil with the aim of quantifying any contributions of biological N₂ fixation. Two genotypes (Morgan 318 and Dekalb 4D-70) produced similar increases in grain yield when they were inoculated with a mixture of Azospirillum spp. strains or fertilized with the equivalent of 100 kg N ha^-1. The other genotypes (Dekalb 2F-11 and CMS 22) showed little response to inoculation or N fertilization. The Morgan 318 and Dekalb 4D-70 genotypes showed a large increase in total N accumulation, suggesting that the response was due to increased N acquisition, but not due to bacterial nitrate reductase as the NR- mutants generally caused plant responses similar to those of the parent strains. Despite problems with the stabilization of the ¹⁵N enrichment in the soil, the ¹⁵N isotope dilution results indicated that there were very significant biological nitrogen fixation (BNF) contributions to the Dekalb 4D-70 and CMS 22 maize genotypes.Dedicated to Professor J.C.G. Ottow on the occasion of his 60th birthday     

Physiological and symbiotic characteristics of Rhizobium fredii isolated from subtropical-tropical soils

Summary Physiological and symbiotic characteristics were identified in Rhizobium fredii isolated from subtropical-tropical soils. The generation times of R. fredii Taiwan isolated-SB 357 and -SB 682 were 1.7 and 2.5 h, respectively. These strains were associated with acid production in yeast-extract mannitol medium. They were able to use hexoses, pentose, sucrose, trehalose and raffinose. Strain SB 357 can resist a high concentration of kanamycin (100 g ml^–1 and penicillin (400 g ml^–1). It can tolerate up to 2.34% NaCl and 1031.3 mosmol kg^–1 (23.4 bars). The growth rate of R. fredii SB 357 under the concentration of approximately 450 mosmol kg^–1 (10.2 bars) was not affected by salinity, but responded to osmotic pressure. Both strains (SB 357 and SB 682) isolated from subtropical-tropical soils were able to form an effective N₂-fixing symbiosis with the US soybean cv Clark lanceolate leaflet.     

Short-term measurements of uptake of nitrogen fixed in the rhizospheres of sorghum (Sorghum bicolor) and millet (Pennisetum americanum)

Summary In a series of short-term experiments root systems of young sorghum and millet plants inoculated with N₂-fixing bacteria were exposed to ¹⁵N₂-enriched atmospheres for 72 h. The plants were grown in a normal atmosphere for up to 22 days after the end of the exposure to allow them to take up the fixed N₂. Environmental conditions and genotypes of sorghum and millet were selected to maximise N₂-fixation in the rhizosphere. Detectable amounts of fixed N (> 16 g/plant) were rapidly incorporated into sorghum plants grown in a sand/farmyard manure medium, but measurable fixation was found on only one occasion in plants grown in soil. N₂ fixation was detectable in some experiments with soil-grown millet plants but the amounts were small (2–4 g/plant) and represented less than 1 % of plant N accumulated over the same period. In many cases there was no detectable ¹⁵N₂ incorporation despite measurable increases in ethylene concentration found during an acetylene reduction assay.Published as ICRISAT Journal Article No. JA 740     

Acetylene reduction,hydrogen evolution and nodule respiration in Phaseolus vulgaris

Summary In an experiment performed under greenhouse conditions, four cultivars of Phaseolus vulgaris L. (Venezuela-350; Aroana; Moruna; Carioca) were inoculated with three Rhizobium leguminosarum biovar phaseoli strains (C-05; C-40 = CIAT 255; C-89 = CIAT 55) and were fertilized with an N-free mineral nutrient solution. The plants were harvested 25, 40, and 55 days after emergence and the following paramenters were evaluated: Nitrogenase activity of nodulated roots, H₂ evolution by the nodules; relative efficiency of nitrogenase; respiration rates of nodulated roots and detached nodules; dry weight and total N of stems, leaves, pods, roots, and nodules. Generally the bean cultivar, Rhizobium strain, had an effect and there was an interaction effect with both symbiotic partners, on all parameters. On average, nodules represented 23% of total root respiration but the best symbiotic combinations showed lower ratios of C respired to N fixed. The maximum N-assimilation rate (between 40 and 55 days after emergence) of 11.93 mg N plant^–1 day^–1 occurred with the symbiotic combination of Carioca × C-05, while the poorest rate of 0.55 mg N plant^–1 day^–1 was recorded with Venezuela-350 × C-89. The best symbiotic combinations always showed the highest relative nitrogenase efficiency, but the differences in N₂-fixation rates cannot be explained solely in terms of conservation of energy by recycling of H₂. This requires further investigation.     

Influence of methyl isocyanate on the growth,ATP levels,and nitrogen fixation of Azotobacter chroococcum

Summary Growth, ATP levels, and N₂-fixing activity of Azotobacter chroococcum culture exposed to 500 g ml^-1 of methyl isocyanate were investigated. In methyl isocyanate-exposed cells, the lag phase was extended up to 16 h but the control cells started to multiply immediately. ATP levels and N₂-fixing activity as determined by acetylene reduction assay were markedly reduced in the treated cells.     

Nitrogenase activity associated with leaf,root, and rhizosphere soils in an Indian tropical forest

Summary N₂ fixation (acetylene reduction assay) by phylloplane microorganisms was measured in dominant and co-dominant plant species growing in a tropical rain forest. No significant acetylene reduction was recorded with intact leaf samples. Azotobacter sp., Beijerinckia sp., Derxia sp., and Klebsiella pneumoniae were isolated as phylloplane N₂-fixing bacteria. Azospirillum lipoferum was only isolated from soil samples containing the roots of Poaceae. Nitrogenase activity was recorded in culture derived from the roots and rhizosphere soil samples, although low acetylene reduction activity indicates that these associations did not provide large amounts of N to the systems studied.     

Biodiversity of diazotrophs associated to the plant cover of north sinai deserts: Biodiversität diazotropher assoziiert mit der pflanendecke der wüsten nordsinais

Efforts are made to record biodiversity of microflora and diazotrophs associated with the plant cover of the major agricultural development areas in north Sinai, around the El-Salam canal, a newly-constructed canal that brings Nile water westward across the Suez canal. Natural plant communities were collected from three major areas. Ectorhizosphere, endorhizosphere and phyllosphere samples were examined for total microbial population and diazotrophs. The vegetation of South Qantara (area I) is characterized by the dominance of Stipagrostis scoparia followed by Nitraria retusa, Convolvulus lanatus, Cornulaca monacantha and Filago desertorum. Rabaa-Bir El Abd (area II) is dominated by Artemisia monosperma, Panicum turgidum and Zygophyllum album. Euphorbia terracina, Oligomeris linifolia, Astragalus kahiricus, Hyoscyamus muticus and Thymelea hirsuta represent the major plants of El Ser and Al Quarir (area III). Microorganisms colonized root surfaces of all tested plants ranging from >?10⁵ to 10¹⁰ cfu g^???1. Diazotrophs were common residents (10¹⁰ cfu g^???1), invaded the root tissue and established endophytically (10²?–?10⁶ cfu g^???1). Fifty-one N₂-fixing isolates were obtained. Among the 32 bacilli isolates, Bacillus polymyxa and Bacillus circulans were more common compared to Bacillus macerans. BNF Gram-negative isolates belonged to Enterobacter agglomerans, Enterobacter gergoviae, Enterobacter amnigenus, Klebsiella pneumoniae, Pseudomonas luteola, Pseudomonas cepacia, Agrobacterium radiobacter and Azospirillum spp.     

Diversity of Paenibacillus durus strains isolated from soil and different plant rhizospheres evaluated by ARDRA and gyrB-RFLP analysis

Strains belonging to Paenibacillus durus isolated from the rhizosphere of various grasses and from bulk soil were previously divided into five phenotypic groups (A1–A5) based on the fermentation pattern of six carbohydrates (A1: sorbitol (+), A2: dulcitol and tagatose (+), A3: starch and glycogen (+), A4: starch, glycogen and d-arabitol (+) and A5: negative for these carbohydrates). This study aimed to assess whether plant types select for specific P. durus phenotypic groups. For that purpose, polymerase chain reaction-restriction fragment length polymorphism analysis of part of genes encoding 16S rRNA (ARDRA) and DNA gyrase subunit B (gyrB-RFLP) were used to produce genetic fingerprints. ARDRA and gyrB-RFLP data were clustered together to generate a dendrogram and two main clusters were observed. Cluster I showed a predominance of strains isolated from wheat, maize and sugarcane rhizospheres. Strains isolated from maize were distributed among the five patterns of carbohydrate metabolism, while strains isolated from sugarcane showed to be predominantly able to metabolize starch and glycogen. Neither sorbitol- nor arabitol-metabolizing strains were found in cluster II, which consisted of strains isolated from soil and from all plant species used. Our results suggest that the plants influenced the diversity of P. durus in their rhizospheres.     

Synergistic effects of vesicular-arbuscular mycorrhizal fungi and diazotrophic bacteria on nutrition and growth of sweet potato (Ipomoea batatas)

Summary Sweet potatoes were micropropagated and then transplanted from axnic conditions to fumigated soil in pots in the greenhouse. Spores of Glomus clarum were obtained from Brachiaria decumbens or from sweet potatoes grown in soil infected with this fungus and with an enrichment culture of Acetobacter diazotrophicus. Three experiments were carried out to measure the beneficial effects of vesicular-arbuscular mycorrhizal (VAM) fungi-diazotroph interactions on growth, nutrition, and infection of sweet potato by A. diazotrophicus and other diazotrophs obtained from sweet potato roots. In two of these experiments the soils had been mixed with ¹⁵N-containing organic matter. The greatest effects of mycorrhizal inoculation were observed with co-inoculation of A. diazotrophicus and/or mixed cultures of diazotrophs containing A. diazotrophicus and Klebsiella sp. The tuber production was dependent on mycorrhization, and total N and P accumulation were increased when diazotrophs and G. clarum were applied together with VAM fungal spores. A. diazotrophicus infected aerial plant parts only when inoculated together with VAM fungi or when present within G. clarum spores. More pronounced effects on root colonization and intraradical sporulation of G. clarum were observed when A. diazotrophicus was co-inoculated. In non-fumigated soil, dual inoculation effects, however, were of lower magnitude. ¹⁵N analysis of the aerial parts and roots and tubers at the early growth stage (70 days) showed no statistical differences between treatments except for the VAM+Klebsiella sp. treatment. This indicates that the effects of A. diazotrophicus and other diazotrophs on sweet potato growth were caused by enhanced mycorrhization and, consequently, a more efficient assimilation of nutrients from the soil than by N₂ fixation. The possible interactions between these effects are discussed.     

Dual inoculation of Pisum sativum with Rhizobium leguminosarum and Penicillium bilaji

Summary To investigate the effect of single versus dual inoculation of peas (Pisum sativum L.) with Rhizobium leguminosarum biovar viceae and Penicillium bilaji (a soil fungi capable of solubilizing soil P) on N₂ fixation an experiment was carried out under controlled conditions. A sandy loam soil was selected which contained low levels of available N and P. P fertilizer [Ca(H₂PO₄)₂] and P. bilaji significantly increased dry matter production. Peas inoculated with R. leguminosarum showed only a small increase in dry matter, but the additional application of P significantly increased the yield. The total N accumulation was highly dependent on the presence of R. leguminosarum. Using the ¹⁵N method for estimating N₂ fixation, the highest level of N₂-fixing activity was observed in peas inoculated with R. leguminosarum and fertilized with inorganic P. Dual inoculation of peas with P. bilaji and R. leguminosarum significantly decreased the amount of N₂ fixed. Total P uptake was solely dependent on the P fertilizer.     

Effect of manganese-reducing rhizosphere bacteria on the growth of Gaeumannomyces graminis var. tritici and on manganese uptake by wheat (Triticum aestivum L.)

Summary Five bacterial strains capable of Mn reduction were isolated from the rhizosphere of plants growing in different South Australian soils. They differed in their Mn-reducing capacity. The antagonism of these strains compared to the imported strain 2–79 (from the United States) against Gaeumannomyces graminis var. tritici was tested in agar and in a soil sandwich experiment at different Mn²⁺ concentrations in the soil. In addition, wheat seeds were coated with the different strains and with MnSO₄ or with MnSO₄ only in order to investigate their effect on plant growth and Mn uptake. With one exception, all strains inhibited the growth of G. graminis in agar, but to different degrees. In contrast, only two strains significantly inhibited the growth of the fungus in the soil. The hyphal density was decreased more than the hyphal length. The Mn²⁺ concentration in the soil also had a marked effect on fungal growth; low Mn concentrations slightly increased while high Mn concentrations strongly decreased the fungal growth. Seed treatment with MnSO₄ only (+Mn) increased Mn uptake above that of the control (no seed treatment). Only the weakest Mn reducer on agar significantly increased plant growth and Mn uptake from soil in comparison with the Mn treatment. One strain was tested as seed coating without adding MnSO₄; it increased the plant growth to an extent similar to the Mn treatment. Increasing the Mn uptake by plants may be one of the growth-promoting effects exerted by rhizosphere bacteria.