Responses of Lentil to Co-Inoculation with Phosphate-Solubilizing Rhizobial Strains and Arbuscular Mycorrhizal Fungi

The purpose of this study was to evaluate the responses of lentil (Lens culinariscv. ‘Ziba’) to co-inoculation with arbuscular mycorrhizal (AM) fungi and some indigenous rhizobial strains varying in phosphorus (P)-solubilizing ability in a calcareous soil with high pH and low amounts of available P and nitrogen (N). A factorial experiment with completely randomized block design was conducted under controlled greenhouse conditions. The treatments consisted of (1) three inoculants of Rhizobium leguminosarum bv. viciae strains and a mixed rhizobial inoculant with an effective P-solubilizer strain of Mesorhizobium ciceri, (2) two AM fungal species, Glomus mosseae and Glomus intraradices, (3) two P sources, superphosphate and phosphate rock. Four replications were prepared for each treatment and a related control. After the growth period of three months, the dry matter of shoots plus seeds, their P and N contents, and percent of root colonized by AM fungus were measured. The results showed that the effects of AM fungi, rhizobial strains, and P fertilizers were highly significant (p < 0.01) for all the characteristics studied. The rhizobial strain with P-solubilizing ability showed a more beneficial effect on plant growth and nutrient uptake than the strain without this ability, although both strains had similar effectiveness for N₂-fixation in symbiosis with lentil. Synergistic relationships were observed between AM fungi and some rhizobial strains that related to the compatible pairing of these two microsymbionts. The P-uptake efficiency was increased when P fertilizers were applied along with AM fungi and/or P-solubilizer rhizobial strains.     

Benefits of inoculation of the common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) crop with efficient and competitive<Emphasis Type="Italic"> Rhizobium tropici</Emphasis> strains

Cropping in low fertility soils, especially those poor in N, contributes greatly to the low common bean (Phaseolus vulgaris L.) yield, and therefore the benefits of biological nitrogen fixation must be intensively explored to increase yields at a low cost. Six field experiments were performed in oxisols of Paraná State, southern Brazil, with a high population of indigenous common bean rhizobia, estimated at a minimum of 10³ cells g^–1 soil. Despite the high population, inoculation allowed an increase in rhizobial population and in nodule occupancy, and further increases were obtained with reinoculation in the following seasons. Thus, considering the treatments inoculated with the most effective strains (H 12, H 20, PRF 81 and CIAT 899), nodule occupancy increased from an average of 28% in the first experiment to 56% after four inoculation procedures. The establishment of the selected strains increased nodulation, N₂ fixation rates (evaluated by total N and N-ureide) and on average for the six experiments the strains H 12 and H 20 showed increases of 437 and 465 kg ha^–1, respectively,in relation to the indigenous rhizobial population. A synergistic effect between low levels of N fertilizer and inoculation with superior strains was also observed, resulting in yield increases in two other experiments. The soil rhizobial population decreased 1 year after the last cropping, but remained high in the plots that had been inoculated. DGGE analysis of soil extracts showed that the massive inoculation apparently did not affect the composition of the bacterial community.     

Influence of arbuscular mycorrhizae and phosphorus fertilization on growth, nodulation and N2 fixation (15N) inMedicago sativa at four salinity levels

The rose of an isolate of the arbuscular mycorrhizal (AM) fungusGlomus mosseae in the protection ofMedicago sativa (+Rhizobium meliloti) against salt stress induced by the addition of increasing levels of soluble salts was studied. The interactions between soluble P in soil (four levels), mycorrhizal inoculum and degree of salinity in relation to plant growth, nutrition and infective parameters were evaluated. Salt stress was induced by sequential irrigation with saline water having four concentrations of three salts (NaCl, CaCl₂, and MgCl₂).¹⁵N-labelled ammonium sulphate was added to provide a quantitative estimate of N₂ fixation under moderate to high salinity levels. N and P concentration and nodule formation increased with the amount of plant-available P or mycorrhizal inoculum in the soil and generally declined as the salinity in the solution culture increased from a moderate to a high level. The mycorrhizal inoculation protected the plants from salt stress more efficiently than any amount of plant-available P in soil, particularly at the highest salinity level applied (43.5 dS m^–1). Mycorrhizal inoculation matched the effect on dry matter and nutrition of the addition in the soil of 150 mg P kg^–1. Nevertheless the highest saline solution assayed (43.5 dS m^–1) affected more severely plants supplemented with phosphorus than those with the addition of mycorrhizal inoculum. Such a saline-depressing effect was 1.5 (biomass), 1.4 (N) and 1.5 (P) times higher in plants supplied with soluble phosphate than with AM inoculum. Mechanisms beyond those mediated by P must be involved in the AM-protectioe effect against salinity. The¹⁵N methodology used allowed the determination of N₂ fixation as influenced by different P applications compared to mycorrhizal inoculation. A lack of correlation between nodule formation and function (N₂ fixation) was evidenced in mycorrhizal-inoculated plants. In spite of the reduced activity per nodule in mycorrhizal-inoculated In spite of the reduced activity per nodule in mycorrhizal-inoculated plants, the N contents determined indicated the highest acquisition of N occurred in plants with the symbiotic status. Moreover, N and P uptake increased while Ca and Mg decreased in AM-inoculated plants. Thus P/Ca ratios and cation/anion balance in general were altered in mycorrhizal treatments. This study therefore confirms previous findings that AM-colonized plants have optional and alternative mechanisms available to satisfy their nutritive requirements and to maintain their physiological status in stress situations and in disturbed ecosystems.     

Competition between inoculated and indigenous Rhizobium/Bradyrhizobium spp. strains for nodulation of grain and fodder legumes in Pakistan

Summary The competitive ability of inoculated and indigenous Rhizobium/Bradyrhizobium spp. to nodulate and fix N₂ in grain legumes (Glycine max, Vigna unguiculata, Phaseolus vulgaris) and fodder legumes (Vicia sativa, Medicago sativa, and Trifolium subterraneum) was studied in pots with two local soils collected from two different fields on the basis of cropping history. The native population was estimated by a most-probable-number plant infectivity test in growth pouches and culture tubes. The indigenous rhizobial/bradyrhizobial population ranged from 3 to 2×10⁴ and 0 to 4.4×10³ cells g^-1 in the two soils (the first with, the second without a history of legume cropping). Inoculated G. max, P. vulgaris, and T. subterraneum plants had significantly more nodules with a greater nodule mass than uninoculated plants, but N₂ fixation was increased only in G. max and P. vulgaris. A significant response to inoculation was observed in the grain legume P. vulgaris in the soil not previously used to grow legumes, even in the presence of higher indigenous population (>10³ cells g^-1 soil of Rhizobium leguminosarum bv phaseoli). No difference in yield was observed with the fodder legumes in response to inoculation, even with the indigenous Rhizobium sp. as low as <14 cells g^-1 soil and although the number and weight of nodules were significantly increased by the inoculation in T. subterraneum. Overall recovery of the inoculated strains was 38–100%, as determined by a fluorescent antibody technique. In general, the inoculation increased N₂ fixation only in 3 out of 12 legume species-soil combinations in the presence of an indigenous population of rhizobial/bradyrhizobial strains.     

Response of Mucuna pruriens to symbiotic nitrogen fixation by rhizobia following inoculation in farmers' fields in the derived savanna of Benin

 Leguminous cover crops such as Mucuna pruriens (mucuna) have the potential to contribute to soil N and increase the yields of subsequent or associated cereal crops through symbiotic N fixation. It has often been assumed that mucuna will freely nodulate, fix N₂ and therefore contribute to soil N. However, results of recent work have indicated mucuna's failure to nodulate in some farmers' fields in the derived savanna in Benin. One of the management practices that can help to improve mucuna establishment and growth is the use of rhizobial inocula to ensure compatibility between the symbiotic partners. Experiments were conducted in 1995 and 1996 on 15 farmers' fields located in three different villages (Eglimé, Zouzouvou and Tchi) in the derived savanna in Benin. The aim was to determine the response of mucuna to inoculation and examine the factors affecting it when grown in relay cropping with maize. The actual amount of N₂ fixed by mucuna in the farmers' fields at 20 weeks after planting (WAP) averaged 60 kg N ha^–1 (range: 41–76 kg N ha^–1) representing 55% (range: 49–58%) of the plant total N. The result suggested that mucuna in these farmers' fields could not meet its total N demand for growth and seed production only by N₂ fixation. It was estimated that after grain removal mucuna led to a net N contribution ranging from –37 to 30 kg N ha^–1. Shoot dry weight at 20 WAP varied between 1.5 and 8.7 t ha^–1 and N accumulation ranged from 22 to 193 kg N ha^–1. Inoculation increased shoot dry matter by an average of 28% above the uninoculated treatments, but the increase depended on the field, location and year. For the combinations of inoculated treatments and farmers' fields, the response frequency was higher in Eglimé and Tchi than in Zouzouvou. The response to inoculated treatments was dependent on the field and inversely related to the numbers of rhizobia in the soil. Soil rhizobial populations ranged from 0 to >188 cells g^–1 soil, and response to inoculation often occurred when numbers of indigenous rhizobia were <5 cells g^–1 soil. In two farmers' fields at Zouzouvou where extractable P was below 10 μg g^–1 soil, mucuna did not respond to rhizobial inoculation despite a higher population of rhizobia. Significant relationships between mycorrhizal colonization, growth and nodulation of mucuna were observed, and inoculated plants with rhizobia had a higher rate of colonization by arbuscular mycorrhizal fungi (%AMF) than uninoculated ones. Therefore, it was shown that mucuna will establish and fix N₂ effectively in those fields where farmer's management practices such as good crop rotation and rhizobial inoculation allow a build up of AMF spores that might lead to a high degree of AMF infection and alleviate P deficiency. Received: 14 June 1999     

Arbuscular mycorrhizal fungi reduce ammonia emissions under different land-use types in agro-pastoral areas

Ammonia (NH₃) emissions, the most important nitrogen (N) loss form, always induce a series of environmental problems such as increased frequency of regional haze pollution, accelerated N deposition, and N eutrophication. Arbuscular mycorrhizal (AM) fungi play key roles in N cycling. However, it is still unclear whether AM fungi can alleviate N losses by reducing NH₃ emissions. The potential mechanisms by which AM fungi reduce NH₃ emissions in five land-use types (grazed grassland, mowed grassland, fenced grassland, artificial alfalfa grassland, and cropland) were explored in this study. Results showed that AM fungal inoculation significantly reduced NH₃ emissions, and the mycorrhizal responses of NH₃ emissions were determined by land-use type. Structural equation modeling (SEM) showed that AM fungi and land-use type directly affected NH₃ emissions. In addition, the reduction in NH₃ emissions was largely driven by the decline in soil NH⁺₄-N and pH and the increases in abundances of ammonia-oxidizing archaea (AOA) amoA and bacteria (AOB) amoB genes, urease activity, and plant N uptake induced by AM fungal inoculation and land-use type. The present results highlight that reducing the negative influence of agricultural intensification caused by land-use type changes on AM fungi should be considered to reduce N losses in agriculture and grassland ecosystems.     

Effect of nitrogen levels and Rhizobium strains on symbiotic N2 fixation and grain yield of Phaseolus vulgaris L. genotypes in normal and saline-sodic soils

Summary Following screening, selection, characterization, and symbiotic N₂ fixation with 12,5, 25.0, and 40.0 mg N kg^–1 in normal and saline-sodic soils, only two Phaseolus vulgaris genotypes (HUR 137 and VL 63) and two Rhizobium spp. strains (ND 1 and ND 2) produced maximum nodulation, nitrogenase activity, plant N contents, and grain yields in saline-sodic soil, with 12.5 mg N kg^–1, compared with the other strains. However, interactions between strains (USDA 2689, USDA 2674, and ND 5) and genotypes (PDR 14, HUR 15, and HUR 138) were significant and resulted in more nodulation, and greater plant N contents, nitrogenase activity, and grain yields in normal soils with 12.5 mg N kg^–1 compared with salt-tolerant strains. Higher levels of N inhibited nodulation and nitrogenase activity without affecting grain yields. To achieve high crop yields from saline-sodic and normal soils in the plains area, simultaneous selection of favourably interacting symbionts is necessary for N economy, so that bean yields can be increased by the application of an active symbiotic system.     

Dual inoculation of pretransplant stage Oryza sativa L. plants with indigenous vesicular-arbuscular mycorrhizal fungi and fluorescent Pseudomonas spp.

Summary This study examined the response of rice (Oryza sativa L.) plants at the pretransplant/nursery stage to inoculation with vesicular-arbuscular mycorrhizal (VAM) fungi and fluorescent Pseudomonas spp., singly or in combination. The VAM fungi and fluorescent Pseudomonas spp. were isolated from the rhizosphere of rice plants. In the plants grown in soil inoculated with fluorescent Pseudomonas spp. alone, I found increases in shoot growth, and in root length and fine roots, and decreases in root growth, and P and N concentrations. In contrast, in the plants colonized by VAM fungi alone, the results were the reverse of those of the pseudomonad treatment. Dual inoculation of soil with VAM fungi and fluorescent Pseudomonas spp. yielded plants with the highest biomass and nutrient acquisition. In contrast, the plants of the control treatment had the lowest biomass and nutrient levels. The dual-inoculated plants had intermediate root and specific root lengths. The precentages of mycorrhizal colonization and colonized root lengths were significantly lower in the dual-inoculated treatment than the VAM fungal treatment. Inoculation of plants with fluorescent Pseudomonas spp. suppressed VAM fungal colonization and apparently reduced photosynthate loss to the mycorrhizal associates, which led to greater biomass and nutrient levels in dual-inoculated plants compared with plants inoculated with VAM fungi alone. Dual inoculation of seedlings with fluorescent Pseudomonas spp. and VAM fungi may be preferable to inoculation with VAM alone and may contribute to the successful establishment of these plants in the field.     

Influence of arbuscular mycorrhizae and phosphorus fertilization on growth, nodulation and N2 fixation (15N) inMedicago sativa at four salinity levels

The rose of an isolate of the arbuscular mycorrhizal (AM) fungusGlomus mosseae in the protection ofMedicago sativa (+Rhizobium meliloti) against salt stress induced by the addition of increasing levels of soluble salts was studied. The interactions between soluble P in soil (four levels), mycorrhizal inoculum and degree of salinity in relation to plant growth, nutrition and infective parameters were evaluated. Salt stress was induced by sequential irrigation with saline water having four concentrations of three salts (NaCl, CaCl₂, and MgCl₂).¹⁵N-labelled ammonium sulphate was added to provide a quantitative estimate of N₂ fixation under moderate to high salinity levels. N and P concentration and nodule formation increased with the amount of plant-available P or mycorrhizal inoculum in the soil and generally declined as the salinity in the solution culture increased from a moderate to a high level. The mycorrhizal inoculation protected the plants from salt stress more efficiently than any amount of plant-available P in soil, particularly at the highest salinity level applied (43.5 dS m^?1). Mycorrhizal inoculation matched the effect on dry matter and nutrition of the addition in the soil of 150 mg P kg^?1. Nevertheless the highest saline solution assayed (43.5 dS m^?1) affected more severely plants supplemented with phosphorus than those with the addition of mycorrhizal inoculum. Such a saline-depressing effect was 1.5 (biomass), 1.4 (N) and 1.5 (P) times higher in plants supplied with soluble phosphate than with AM inoculum. Mechanisms beyond those mediated by P must be involved in the AM-protectioe effect against salinity. The¹⁵N methodology used allowed the determination of N₂ fixation as influenced by different P applications compared to mycorrhizal inoculation. A lack of correlation between nodule formation and function (N₂ fixation) was evidenced in mycorrhizal-inoculated plants. In spite of the reduced activity per nodule in mycorrhizal-inoculated In spite of the reduced activity per nodule in mycorrhizal-inoculated plants, the N contents determined indicated the highest acquisition of N occurred in plants with the symbiotic status. Moreover, N and P uptake increased while Ca and Mg decreased in AM-inoculated plants. Thus P/Ca ratios and cation/anion balance in general were altered in mycorrhizal treatments. This study therefore confirms previous findings that AM-colonized plants have optional and alternative mechanisms available to satisfy their nutritive requirements and to maintain their physiological status in stress situations and in disturbed ecosystems.     

Involvement of autoregulation in the interaction between rhizobial nodulation and AM fungal colonization in soybean roots

Soybean plants autoregulate to suppress excessive nodulation. It has been revealed recently that the autoregulation of various legumes controls both nodulation and arbuscular mycorrhizal (AM) fungal colonization. We investigated the involvement of autoregulation in the interaction between rhizobial nodulation and AM fungal colonization. We used a wild-type soybean cv. Enrei and its hypernodulating mutant Kanto100, defective in the autoregulation. We included four different treatments: an uninoculated control, inoculation with rhizobium Bradyrhizobium japonicum alone, inoculation with AM fungus Gigaspora rosea alone, and dual inoculation with rhizobium and AM fungus. In both Enrei and Kanto100, AM fungal colonization enhanced the weight and N₂ fixation of nodules, suggesting that autoregulation of host plant is not involved in the stimulatory effect of AM fungal colonization on rhizobial nodulation. In plants with the AM fungus alone, the AM fungal colonization of Enrei was comparable to that of Kanto100. In plants with dual inoculation, however, this was significantly (P?<?0.05) lower than in Kanto100. To confirm the control of AM fungal colonization by the autoregulation of host plant, a reciprocal grafting experiment was performed between Enrei and Kanto100. In plants with the AM fungus alone, AM fungal colonization was comparable among Enrei (shoot)/Enrei (root), Enrei/Kanto100, Kanto100/Enrei, and Kanto100/Kanto100 grafts. In plants with dual inoculation, however, AM fungal colonization of Enrei/Enrei and Enrei/Kanto100 grafts was significantly (P?<?0.05) lower than that of Kanto100/Enrei and Kanto100/Kanto100. These results indicate that rhizobial nodulation suppresses AM fungal colonization, and the autoregulation of host plant, initiated by nodulation, is involved in this phenomenon.     

Single and Multistrain Rhizobial Inocula for Pinto and Black Bean Cultivars

ABSTRACT

Common bean (Phaseolus vulgaris L.) is relatively poor in dinitrogen (N₂) fixation, so selecting compatible host cultivar and Rhizobium strain combinations may offer an improvement. The effectiveness of six rhizobial strains was evaluated using five bean cultivars of bean (three pinto and two black bean) in a growth-room experiment. We subsequently selected the three best strains to assess whether multi-strain inoculation had advantages over single-strain inoculation in growth-room and field experiments. In the first-growth-room experiment, Rhizobium strains UMR 1899, RCR 3618, and USDA 2676 were selected for high nodulation, plant dry weight, shoot nitrogen (N), and N₂ fixation. In a second growth-room experiment, the individual strains and a mixture of the three strains generally did not differ in the parameters evaluated. Total shoot N accumulated ranged from 172.9 to 162.8 mg plant^?1, of which 32.1% to 33.6% (equivalent to 54.0 to 59.2 mg plant^{? 1}) was fixed. In field experiments, plant biomass and seed N₂ fixed did not differ among the inoculants at any site. These results suggest that the three strains were equally effective and that the multi-strain inoculant offered no consistent advantage over the single-strain inoculants.     

Response of Vigna Unguiculata Grown Under Different Soil Moisture Regimes to the Dual Inoculation with Nitrogen-Fixing Bacteria and Arbuscular Mycorrhizal Fungi

The interaction between legumes, rhizobial and arbuscular mycorrhizal (AM) partners benefits plant nutrition and improves plant tolerance to water stress. The present research evaluated the effectiveness of symbioses between cowpea plants (Vigna unguiculata (L.) Walp.), AM fungi (Glomus intraradices) and two strains of Bradyrhizobium japonicum on the mycorrhization, acid phosphatase activity (APase), enzymes related to nitrogen fixation and assimilation, and biomass accumulation at three soil moisture levels. The results revealed that the soil moisture optimal for the formation of active symbiotrophic associations in cowpea cultivation was about 60% water-holding capacity (WHC), where both Bradyrhizobium strains and AM fungi function well with respect to mycorrhization, nitrogen and phosphorus uptake, nitrogen fixation and plant biomass production. Under conditions of reduced water supply, the symbiotic association between Br. japonicum-273 and Gl. intraradices was better for cowpea cultivation, while in elevated soil moisture association between Br. japonicum-269 and Gl. intraradices was more appropriate.     

Evaluation of N2‐fixation measured by the 15N‐dilution and N‐difference methods in Nicaraguan and Ecuadorian Phaseolus vulgaris L. plants inoculated with Rhizobium leguminosarum biovar

Pot and field experiments were performed to assess N₂ fixation in Nicaraguan (R79 and R84) and Ecuadorian (Imba) common bean (Phaseolus vulgaris L.) cultivars, with the aim of improving their productivity by inoculating them with commercially produced Rhizobium phaseoli. With maize (Zea mays L.) as the non‐N₂‐fixing control, the percentage of N₂ fixed predicted by the ¹⁵N‐dilution method was significantly (P ≤ 0.05) higher than that predicted by the N‐difference method. However, the N₂ amounts predicted by the two methods were not significantly different. The correlation between the two methods was significant and positive (P ≤ 0.0001, n = 36). Compared with the native rhizobial strain, symbiotic associations of the bean cultivars with UMR1073, UMR1077 and UMR1899 rhizobial inoculants did not significantly (P ≤ 0.05) influence plant dry matter (DM) and N yields, the extent of N₂ fixation and uptake of soil and fertilizer N. Nevertheless, the UMR1077 and UMR1899 strains markedly increased the uptake of soil N by R84 plants, while decreasing N2 fixation. In contrast, the Imba‐UMR1899 association enhanced positive effects on all variables. About 60–70% of the total N taken up by the Imba plants was fixed N_2. The R79 and R84 plants fixed about 50% of their total N uptake. N₂ fixation rates were positively correlated with DM and total N yields, while being negatively correlated with soil N uptake (P ≤ 0.001, n = 36). Future research in Nicaragua should focus on selecting rhizobial strains suitable for indigenous common bean cultivars.     

The potential for rhizobial inoculation to increase soybean grain yields on acid soils in Ethiopia

In Ethiopia, inoculation of soybean with rhizobial inoculants is not common practice, but could provide an option to increase grain yields in low nitrogen (N) acidic soils. In these acid soils, the selection of acid tolerant rhizobia is one strategy that may increase the performance of soybean. In this study, rhizobial strains isolated from Ethiopian soils were evaluated for their acid tolerance and symbiotic N fixation efficiency with soybean, in controlled environments. Following this, four isolated rhizobial strains were evaluated in six field experiments in major soybean growing areas of Ethiopia. Inoculation with the commercial strain or with one of two locally sourced isolates, that were developed as inoculants, improved soybean yield. The yield increase due to inoculation with the commercial strain was consistent and greater than other treatments, while the increase due to the two locally sourced strains was comparable to, or greater than, application of 46 kg N/ha in soils, where the resident rhizobial population was ≤1.4 × 10³ cfu/g soil. For soils with high background rhizobial populations, there was no response to inoculation. In one of the experimental sites (Bako), the percentage of N fixed (%Ndfa) was 55 for the commercial strain and 35 for the local strain, ES3. This study demonstrated that field validation is a necessary step in the selection of acid-tolerant strains of rhizobia to increase soybean production for Ethiopia.     

Pre-inoculation with arbuscular mycorrhizal fungi suppresses root knot nematode (<Emphasis Type="Italic">Meloidogyne incognita</Emphasis>) on cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>)

A pot experiment was conducted to evaluate the influence of pre-inoculation of cucumber plants with each of the three arbuscular mycorrhizal (AM) fungi Glomus intraradices, Glomus mosseae, and Glomus versiforme on reproduction of the root knot nematode Meloidogyne incognita. All three AM fungi tested significantly reduced the root galling index, which is the percentage of total roots forming galls. Numbers of galls per root system were significantly reduced only in the G. intraradices + M. incognita treatment. The number of eggs per root system was significantly decreased by AM fungus inoculation, no significant difference among the three AM fungal isolates. AM inoculation substantially decreased the number of females, the number of eggs g⁻¹ root and of the number of eggs per egg mass. The number of egg masses g⁻¹ root was greatly reduced by inoculation with G. mosseae or G. versiforme. By considering plant growth, nutrient uptake, and the suppression of M. incognita together, G. mosseae and G. versiforme were more effective than G. intraradices.     

Uptake and metabolism of nitrate in mycorrhizal plants as affected by water availability and N concentration in soil

We compare the effect of arbuscular mycorrhizal (AM) colonization and PO₄^?3 fertilization on nitrate assimilation, plant growth and proline content in lettuce plants growing under well‐watered (?0.04 MPa) or drought (?0.17 MPa) conditions. We also tested how AM‐colonization and PO₄^?3 fertilization influenced N uptake (¹⁵N) and the percentage of N derived from the fertilizer (% NdfF) by plants under a concentration gradient of N in soil. Growth of mycorrhizal plants was comparable with that of P‐fertilized plants only under well‐watered conditions. Shoot nitrogen content, proline and nitrate reductase activity were greater in AM than in P‐fertilized plants under drought. The addition of 100 μg g^?1 P to the soil did not replace the AM effect under drought. Under well‐watered conditions, AM plants showed similar (at 3 mmol N), greater (at 6 mmol N) or lesser (at 9 mmol N) %NdfF than P‐fertilized plants. Comparing a control (without AM inoculation) to AM plants, differences in % NdfF ranged from 138% (3 mmol N) to 22.6% (6 mmol N) whereas no differences were found at 9 mmol N. In comparison with P fertilization, mycorrhizal effects on %NdfF were only evident at the lowest N levels, which indicated a regulatory mechanism for N uptake in AM plants affected by N availability in the soil. At the highest N level, P‐fertilized plants showed the greatest %NdfF. In conclusion, AM symbiosis is important for N acquisition and N fertilizer utilization but this beneficial mycorrhizal effect on N nutrition is reduced under large quantities of N fertilizer.     

Inoculation of rice plants with the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp.

 Four experiments were performed under gnotobiotic conditions to select strains of the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp. as inocula of rice plants. Eighty strains of H. seropedicae originally isolated from rice, sorghum and maize plants, were tested in test tube cultures with N-free agar as the substrate. Rice plants showed medium and high increases in their fresh weight in response to inoculation with nineteen strains. These strains were tested again, and six strains were then selected to evaluate their contribution to the N of the plant via biological N₂ fixation (BNF) using an agar growth medium containing 5 mg N l^–1of ¹⁵N-labelled (NH₄)₂SO₄. The contribution of the strains to plant N via BNF varied from 54% when rice plants were inoculated with strain ZAE94, to 31% when strain ZAE67 was used. These results were confirmed in the fourth gnotobiotic experiment, which also included strains of the new N-fixing bacteria belonging to the genus Burkholderia, isolated from rice, as well as a strain of Burkholderia vietnamiensis, isolated from rice rhizosphere. Burkholderia spp. strains showed similar effects to those observed for H. seropedicae strains, while B. vietnamiensis fixed only 19% of plant total N. The best four strains were tested in a pot experiment where pre-germinated, inoculated rice seedlings were grown in soil labelled with ¹⁵N. The results confirmed the gnotobiotic experiments, although the levels of N in the rice plants derived from BNF of the selected H. seropedicae and Burkholderia spp. strains were lower. Nevertheless, there was an increase in N content in grains of inoculated plants, and the results showed that the method used for strain selection is very useful and can be applied to other strains of N₂-fixing bacteria and plants. Received: 4 May 1999     

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     

Impact of arbuscular mycorrhizal fungi on the growth and expression of gene encoding stress protein – metallothionein BnMT2 in the non‐host crop Brassica napus L.

Arbuscular mycorrhizal (AM) fungi are an important component of the soil biota in most agroecosystems, and their association can directly or indirectly affect the diversity of soil microorganisms, nutrient cycling, and growth of host plants. Since not all crops are symbiotic, we hypothesized that the presence of AM fungi can: (1) inhibit the growth of non‐host plants by resulting in biotic stress, or (2) promote their growth indirectly by increased nutrient mobilization. These hypotheses were tested in the present study on the non‐mycorrhizal crop canola (Brassica napus L.) in the presence and absence of other autochthonous soil microorganisms. The soil was inoculated with a mixture of AM fungi (Acaulospora longula, Glomus geosporum, G. mosseae, Scutellospora calospora) and as a control, a non‐inoculated soil was used. The impact of inoculation on plant growth (biomass production, nutrient concentrations) and expression of the stress protein metallothionein gene BnMT2 was investigated in the shoots. B. napus L. did not form mycorrhizal associations on its roots, but its growth was promoted after inoculation with AM fungi. In the soil with autochthonic microorganisms, growth inhibition after inoculation was observed compared to the control. The concentrations of N, P, K, and S in the shoot were always significantly increased after inoculation with AM fungi. However, this was partly combined with reduced growth and thereby decreased total uptake of nutrients. Expression of BnMT2 in the leaves was increased after inoculation with AM spores at the soil devoid of indigenous microorganisms, but decreased in their presence. The expression of stress proteins (BnMT2) significantly increased with increasing length and biomass of shoots. In conclusion, the inhibition of the non‐host plant B. napus L. following inoculation with AM fungi was confirmed, however, only in combination with autochthonous microorganisms. Growth promotion of B. napus L. in the presence of AM fungi in the absence of autochthonous soil microorganisms suggest that plant growth depression in the presence of AM fungi was based on interactive effects of AM fungi with the autochthonous microorganisms in the soil rather than on a direct impact of the AM fungi.     

Wheat plants invest more in mycorrhizae and receive more benefits from them under adverse than favorable soil conditions

Soil chemistry and biota heavily influence crop plant growth and mineral nutrition. The stress-severity and optimal resource allocation hypotheses predict mutualistic symbiotic benefits to increase with the degree of metabolic imbalance and environmental stress. Using two cross-factorial pot experiments with the same biologically active calcareous soil, one time highly saline and nutrient-deficient, and the other time partially desalinated and amended with mineral soil fertilizer, we explored whether these general predictions hold true for zinc (Zn) nutrition of bread wheat in mycorrhizal symbiosis. Increased arbuscular mycorrhizal (AM) fungal root colonization positively correlated with plant Zn nutrition, but only when plants were impaired in growth due to salinity and nutrient-deficiency; this was particularly so in a cultivar-responsive to application of mineral Zn fertilizer. Evidence for direct involvement of AM fungi were positive correlations between Zn uptake from soil and frequency of fungal symbiotic nutrient exchange organelles, as well as the quantitative abundance of AM fungi of the genera Funneliformis and Rhizophagus, but not Claroideoglomus. Combined partial soil desalination and fertilization swapped the dominance ranking from Claroideoglomus spp. to Funneliformis spp. Positive growth, nitrogen, and Zn uptake responses to mycorrhization were contingent on moderate soil fertilization with ZnSO₄. In agreement with the predictions of the stress-severity and optimal resource allocation hypotheses, plants limited in growth due to chemically adverse soil conditions invested relatively more into AM fungi, as evident from heavier root colonization, and took up relatively more Zn and nitrogen in response to mycorrhization, than better growing and less mycorrhized plants. It thus appears that crop plant cultivar-dependent mycorrhization and Zn fertilizer-responsiveness may reinforce each other, provided that there is bioavailable Zn in soil and plant growth is impaired by suboptimal chemical soil conditions.