Co-selection of compatible rhizobia and vesicular-arbuscular mycorrhizal fungi for cowpea in sterilized and non-sterilized soils

Summary We selected two isolates of Rhizobium for cowpea (Vigna unguiculata) with sterilized soil tests and two different isolates by non-sterilized soil testing. The four rhizobia were then paired individually with either Glomus pallidum, Glomus aggregatum, or Sclerocystis microcarpa in separate, sterilized, or non-sterilized soil experiments. The purpose of the experiments was to determine the effect of soil sterilization on the selection of effective cowpea rhizobia, and to see whether these rhizobia differed in their effects on cowpea growth when paired with various vesicular-arbuscular mycorrhizal (VAM) fungi. Our experiments showed that the rhizobia selected in sterilized soil tests produced few growth responses in the cowpea compared to the other introduced rhizobia, irrespective of pairing with VAM fungi in sterilized or non-sterilized soil. In contrast, the two rhizobia initially selected by non-sterilized soil testing significantly improved cowpea growth in non-sterilized soil, especially when paired with G. pallidum. Our results suggest that it is important to select for effective rhizobia in non-sterilized soil, and that pairing these rhizobia with specific, coselected VAM fungi can significantly improve the legume growth response.     

Influence of a vesicular-arbuscular mycorrhizal fungus on the competitive ability of Bradyrhizobium spp. for nodulation of cowpea Vigna unguiculata (L.) Walp in non-sterilized soil

Summary We examined the influence of a vesicular-arbuscular (VAM) fungus (Glomus pallidum Hall) on the competitive ability of introduced and native Bradyrhizobium strains to nodulate cowpeas [Vigna unguiculata (L) Walp]. Our experiments in non-sterilized soil revealed that in the presence of VAM fungus, introduced Bradyrhizobium spp. strains become more competitive than native rhizobia. For example, strain JRC29 occupied 59.2% of the total nodules when inoculated alone, but this figure increased to 71.2% when JRC29 was used in dual inoculations with VAM fungus. A similar pattern of enhanced competitiveness for nodule formation was observed with the two other strains in the presence of the VAM fungus. Our results suggest that the competitiveness of rhizobia can be enhanced by co-inoculating with a selected strain of a VAM fungus.     

Growth response of field-grown Siratro (Macroptilium atropurpureum Urb.) and Aeschynomene american L. to inoculation with selected vesicular-arbuscular mycorrhizal fungi

Summary The effect of inoculation with a selected isolate of Glomus etunicatum Becker and Gerdemann and one of G. intraradices Schenck and Smith on the growth and nutrient content of Macroptilium atropurpureum Urb. cv. Siratro and Aeschynomene americana L., at applied P levels of 10, 30, 60, and 120 kg ha^-1, was studied under field conditions. At all P levels and for all harvests, the shoot dry mass of Siratro and A. americana were greater for the plants inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungi than the control plants. Differences between the VAM fungus-inoculated and the control plants were most marked between 30 and 90 kg ha^-1 of applied P and diminished at 120 kg ha^-1. At the first harvest of Siratro, the plants inoculated with G. etunicatum had a greater shoot dry mass than those inoculated with G. intraradices, for all levels of applied P. However, for subsequent harvest of Siratro and for the one harvest of A. americana the response of shoot dry mass to the two VAM fungi was equivocal. Fungal inoculation gave at least a 30% saving in the amount of P fertilizer required (40 kg ha^-1) for the maximum yield. The plants inoculated with VAM fungi had a greater tissue concentration and total content of P and N than the control plants at low and intermediate levels of applied P. The percentage of root colonized by VAM fungi for the inoculated plants of the two legumes increased linearly with P additions up to 60 kg ha^-1. The conclusion is that under amended (limed and fertilized) soil conditions, inoculation with selected VAM fungi can improve the establishement and growth of forage legumes in fields that contain ineffective populations of native VAM fungi.     

Selection of efficient vesicular-arbuscular mycorrhizal fungi for wetland rice (Oryza sativa L.)

Summary We tested the response of the wetland rice cultivar Prakash to inoculation with ten vescular-arbucular mycorrhizal (VAM) fungi (three selected from the first screening and seven isolated from local paddy fields) in a pot experiment under flooded conditions in order to select the most efficient mycorrhizal fungi to inoculate the rice nursery. A sandy clay loam soil was used as the substrate, fertilized with the recommended N and K levels (100 kg N ha^–1 as ammonium sulphate and 50 kg K ha^–1 as muriate of potash) and half the recommended level of P (25 kg ha^–1 as super phosphate). The inoculation was made into dry nursery beds and the beds were flooded when the seedlings were about 25 cm high, in 15 days. Twenty-eight-day old seedlings were transferred to pots filled with well puddled soil flooded with 5 cm of standing water. Based on the increase in grain yield and total biomass, Glomus intraradices and Acaulospora sp. were considered efficient and suitable for inoculation into rice nurseries.     

Leucaena leucocephala seedling response to vesicular-arbuscular mycorrhizal inoculation in soils with varying levels of inherent mycorrhizal effectiveness

Summary The symbiotic effectiveness of vesicular-arbuscular mycorrhizal (VAM) fungi present in widely differring tropical soils was evaluated in a greenhouse experiment. Small volumes of field soil, a standard inoculum (Glomus aggregatum) or both were introduced into a fumigated sand-soil medium amended with nutrients for optimum VAM activity. Leucaena leucocephala (Lam.) de Wit var. K8 was grown in the medium as an indicator plant. VAM effectiveness was monitored as a function of time by determining the P status of pinnules. The soils differed from each other with respect to the time their endophytes required for the expression of initial and maximum effectiveness and in the level of maximum effectiveness they exhibited. The effect of mycorrhizal inoculation, calculated as the ratio of the areas enclosed by the effectiveness curve of G. aggregatum to that enclosed by the effectiveness curves of test soils, was found to be a good indicator of the response of L. leucocephala to inoculation of soils with G. aggregatum Hawaii Institute of Tropical Agriculture and Human Resources Journal series No. 3285     

Competitive interaction among strains of Rhizobium leguminosarum bv. phaseoli in the nodulation of kidney beans (Phaseolus vulgaris L.)

We examined the competitiveness of five effective Rhizobium leguminosarum biovar phaseoli strains in the nodulation of kidney beans (Phaseolus vulgaris L.), either alone or in pairwise combination, against the indigenous strains. The results showed that the introduced Rhizobium sp. strains (B2, B17, B36, T2, or CIAT 652) occupying 64–79% of the total nodules (as single inocula) were more competitive in nodulation than the native rhizobia. However, the competitiveness of the individual Rhizobium sp. strain either increased or decreased when used in a pairwise combination of double-strain inocula. For example, strain B17, although quite competitive against the indigenous population (68% nodule occupancy), became poorly competitive in the presence of strain B2 (reduced from 68 to 2.5%). A similar reduction in nodule occupancy by strain B17 was observed in the presence of B36 or CIAT 652, indicating that two competitive strains may not always be compatible. These results suggest that it is important to co-select competitive as well as compatible rhizobia for multistrain inoculant formulation.     

Phosphatase activity and cytokinin content in cowpeas (Vigna unguiculata) inoculated with a vesicular-arbuscular mycorrhizal fungus

We examined the effect of a vesicular-arbuscular mycorrhizal (VAM) fungus Glomus pallidum Hall on the phosphatase activity and cytokinin concentration in cowpea [Vigna unguiculata (L.) Walp] roots at successive stages of plant growth. Both acid and alkaline phosphatase activity were significantly (P=0.05) higher in mycorrhizal than in non-mycorrhizal roots 30 days after inoculation. Similarly, the cytokinin content was significantly increased in mycorrhizal roots compared to non-mycorrhizal roots. Our study suggests that these biochemical changes may improve the growth of mycorrhizal cowpea plants.     

Growth and iron nutrition of broccoli (Brassica oleracea L. var. italica Plenck), grown in a Typic Ustochrept, as influenced by vesicular-arbuscular mycorrhizal fungi in the presence of pyrite and farmyard manure

Greenhouse experiments were conducted using potted soil (Fe-deficient Typic Ustochrept) to study the influence of the vesicular-arbuscular mycorrhizal fungi (VAM), Glomus macrocarpum and G. fasciculatum, on the mobilisation of Fe in broccoli (Brassica oleracea L. var. italica Plenck) in the presence of pyrite and farmyard manure (FYM). Individual applications of either VAM or pyrite with NPK fertiliser significantly enhanced both the Fe²⁺ content in leaf tissue and total uptake of Fe and resulted in increased curd and straw yields of broccoli compared to those observed with NPK alone. Though the application of FYM decreased the Fe²⁺ content in leaf tissue relative to plants supplied NPK alone, this result was not statistically significant. The available Fe content in soil, after harvest of broccoli, was found to be lower in the presence of VAM than in the control. Received: 18 June 1997     

The tripartite symbiosis between legumes, rhizobia and indigenous mycorrhizal fungi is more efficient in undisturbed soil

We investigated how the rate of colonization by indigenous arbuscular mycorrhizal fungi (AMF) affects the interaction between AMF, Sinorrhizobium meliloti and Medicago truncatula Gaertn. To generate a differential inoculum potential of indigenous AMF, five cycles of wheat, each of 1 month, were grown in sieved or undisturbed soil before M. truncatula was sown. The early colonization of M. truncatula roots by indigenous AMF was faster in undisturbed soil compared with sieved soil, but by pod-fill the frequency of hyphae, arbuscules and vesicles was similar in both treatments. At this latter stage, M. truncatula grown in undisturbed soil had accumulated a greater biomass in aboveground tissues, had a greater P concentration and derived more N from the atmosphere than plants grown in disturbed soil, although soil compaction resulted in plants having a smaller root system than those from disturbed soil. The difference in plant P content could not be explained by modifications in hydrolytic soil enzymes related to the P cycle as the activity of acid phosphatase was greater in sieved than in undisturbed soil, and the activity of alkaline phosphatase was unaffected by the treatment. Thus, the results observed were a consequence of the different rates of AMF colonization caused by soil disturbance. Together with earlier results for soybean, this study confirms that soil disturbance modifies the interaction between indigenous AMF, rhizobia and legumes leading to a reduced efficacy of the bacterial symbiont.     

Effects of different phosphatase-producing fungi on growth and nutrition of mung beans [Vigna radiata (L.) Wilczek] in an arid soil

Summary The efficiency of different phosphatase-producing fungi in arid soils was studied in pot experiments in order to evaluate the effect of these organisms on the enhancement of rhizosphere enzyme activities, the uptake of different nutrients, dry matter production, and grain yields of the mung bean. A significant increase in phosphatase (acid and alkaline), nitrogenase, and dehydrogenase activities was observed upon inoculation. Inoculation with phosphatase-producing fungi significantly increased dry matter production and grain yields compared with uninoculated controls. In general, there was a significant improvement in the uptake of N, P, K, Ca, Mg, Fe, and Zn while no effect on Na, Mn, and Cu was recorded. A significant positive correlation was observed between the activities of various enzymes in the rhizosphere on the one hand and N and P uptake, dry matter production, and grain yields of the mung bean on the other. Aspergillus rugulosus was the best of the phosphatase-producing fungi that were tested, followed by A. terreus. The experiment confirmed that phosphatase-producing fungi have a significant effect on growth and nutrient uptake in the mung bean and indicated that these fungi are particularly important in arid soils.     

Effect of fungicides on three vesicular-arbuscular mycorrhizal fungi associated with winter wheat (Triticum aestivum L.)

Summary We studied the effects of field application rates of three fungicides on spore germination and root infection in three species of Glomus. Bavistin (carbendazim) prevented germination of spores of G. monosporum and G. mosseae whereas Calixin (tridemorph) had no effect. Tilt Turbo (propiconazole) inhibited spore germination less than Bavistin. Spores of G. geosporum germinated in the presence of all three fungicides. Foliar applications of Bavistin alone and in a triple-spray programme significantly reduced infection of all three Glomus species in wheat roots grown in conditions of low P availability (2 mg P 1^–1). Two fungicides with triazole derivatives, Sportak and Tilt Turbo, also decreased infection and the former significantly reduced spore production of all three Glomus species. Calixin-treated plants, however, showed increased infection levels, resulting in greater yields and greater foliar-P concentrations than control plants. Milgo (ethirimol) was not detrimental to the vesicular-arbuscular mycorrhizal (VAM) symbiosis when applied as a foliar spray.     

Reduction of bacterial growth by a vesicular-arbuscular mycorrhizal fungus in the rhizosphere of cucumber (Cucumis sativus L.)

Summary Cucumber was grown in a partially sterilized sand-soil mixture with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum or left uninoculated. Fresh soil extract was places in polyvinyl chloride tubes without propagules of mycorrhizal fungi. Root tips and root segments with adhering soil, bulk soil, and soil from unplanted tubes were sampled after 4 weeks. Samples were labelled with [³H]-thymidine and bacteria in different size classes were measured after staining by acridine orange. The presence of VAM decreased the rate of bacterial DNA synthesis, decreased the bacterial biomass, and changed the spatial pattern of bacterial growth compared to non-mycorrhizal cucumbers. The [³H]-thymidine incorporation was significantly higher on root tips in the top of tubes, and on root segments and bulk soil in the center of tubes on non-mycorrhizal plants compared to mycorrhizal plants. At the bottom of the tubes, the [³H]-thymidine incorporation was significantly higher on root tips of mycorrhizal plants. Correspondingly, the bacterial biovolumes of rods with dimension 0.28–0.40×1.1–1.6 m, from the bulk soil in the center of tubes and from root segments in the center and top of tubes, and of cocci with a diameter of 0.55–0.78 m in the bulk soil in the center of tubes, were significantly reduced by VAM fungi. The extremely high bacterial biomass (1–7 mg C g^-1 dry weight soil) was significant reduced by mycorrhizal colonization on root segments and in bulk soil. The incorporation of [³H]-thymidine was around one order of magnitude lower compared to other rhizosphere measurements, probably because pseudomonads that did not incorporate [³H]-thymidine dominated the bacterial population. The VAM probably decreased the amount of plant root-derived organic matter available for bacterial growth, and increased bacterial spatial variability by competition. Thus VAM plants seem to be better adapted to compete with the saprophytic soil microflora for common nutrients, e.g., N and P, compared to non-mycorrhizal plants.     

Competition and persistence of Rhizobium tropici and Rhizobium etli in tropical soil during successive bean (Phaseolus vulgaris L.) cultures

Strains of Rhizobium tropici IIB, CIAT899 and F98.5, both showing good N₂ fixation, and a R. etli strain W16.3SB were introduced into a field which had no history of bean culture. Plant dilution estimates showed that in the presence of its host (Phaseolus vulgaris cv. Carioca) during the cropping seasons and the subsequent fallow summer periods, the bean rhizobial populations increased from less than 30 to 10³ g^–1 dry soil after 1 year and to 10⁴ g^–1 dry soil after 2 years. In the 1st year crop, the inoculated strains occupied most of the nodules, which resulted in a higher nodulation and C₂H₂ reduction activity. Without reinoculation for the second and third crops, however, little R. tropici IIB was recovered from the nodules and the bean population consisted mainly of R. etli, R. leguminosarum bv. phaseoli, and R. tropici IIA. Reinoculation with our superior R. tropici IIB strains before the second crop resulted in R. tropici IIB occupying the main part of the nodules and a positive effect on nodulation and C₂H₂ reduction activity, but reintroduction of the inoculant strain in the third season did not have any effect.     

Influence of soluble phosphorus fertilizer on the interaction between a vesicular-arbuscular mycorrhizal fungus and Azospirillum brasilense in barley (Herdeum vulgare L.)

Summary Pot-culture studies were carried out to examine the response of barley (Hordeum vulgare L.) to inoculation with Azospirillum brasilense and Glomus versiforme, singly and/or in combination, under varying levels of nitrogenous [(¹⁵NH₄)₂SO₄] and soluble phosphatic (single superphosphate) fertilizers. The interaction between both the endophytes led to increased growth and nutrition of the barley plants. Roots from plants inoculated with Azospirillum brasilense and Glomus versiforme exhibited very low acetylene reduction activity. N₂ fixation in the plants increased with the increase in plant growth but the mycorrhiza alone gave a low level of N₂ fixation in the plants compared to combined inoculation with both the endophytes.     

Variable effectivity of three vesicular-arbuscular mycorrhizal endophytes in Hedysarum coronarium and Medicago sativa

Summary Plants of Hedysarum coronarium L. and Medicago sativa L., inoculated with Glomus caledonium, were grown for 6 weeks in soils with increasing amounts of available phosphorus (P). H. coronarium showed no or very low levels of infection even at very low soil P content (4 ppm), while in M. sativa the highest soil P level (20 ppm) was associated with the lowest percentage of infection. Medicago sativa and H. coronarium grown for 16 weeks in a sandy soil showed clear effects of Glomus inoculation on shoot growth and P uptake. In M. sativa inoculation increased shoot growth and P uptake in all treatments considerably, while the P concentration in the shoot was depressed by G. caledonium. In H. coronarium inoculation with G. mosseae and G. occultum had only a modest impact on shoot growth; G. caledonium even depressed shoot growth significantly. Glomus inoculation had a positive impact on the P concentrations in the shoots of H. coronarium. The growth-depressing effect of G. caledonium on H. coronarium is discussed.     

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 humic acids on laurel growth,associated rhizospheric microorganisms,and mycorrhizal fungi

Summary Increasing concentrations of humic acids were tested in order to determine their effects on the microbial rhizosphere and the growth of laurel (Laurus nobilis L.). Plants that were treated with 300 mg kg^-1 of humic acids had the heaviest weights of both fresh and dry shoots; however, doses of 3000 mg kg^-1 were highly phytotoxic and inhibited the growth of laurel. Total aerobic bacteria and actinomycetes were stimulated by doses of 1500 and 3000 mg kg^-1 of humic acids at the first harvest. Nevertheless, at the end of the experiment no significant differences were found among the different doses. The number of fungi living in the laurel rhizosphere was not affected by any concentration of humic acids. Vesicular-arbuscular mycorrhizal (VAM) colonization was only slightly affected by the addition of increasing concentrations of humic substances to the soil, while the hyphal growth of Glomus mosseae was reduced.     

Effects of herbicides on three vesicular-arbuscular fungi associated with winter wheat (Riticum aestivum L.)

Summary We studied the effects of field application rates of four herbicides (Avenge, Ceridor, Dicurane, and Harrier) on spore germination and infection of wheat roots by three species of Glomus grown under conditions of low P availability. Low concentrations of Ceridor (bifenox, mecoprop) and Harrier (mecoprop, ioxynil, clopyralid) inhibited spore germination while higher concentrations were stimulatory. Avenge (difenzoquat methyl sulphate) prevented spore germination completely, while Dicurane (chlortoluron) had no effect. The herbicide applications had no significant effect on the infection rates of any of the three fungi except Harrier on G. geosporum. The herbicide treatments did, however, affect plant growth and ear yields. Ceridor and Harrier increased ear yields, while Dicurane showed marked phytotoxic effects.     

Arbuscular mycorrhizal fungi mediated uptake of nutrient elements by Chinese milk vetch (<Emphasis Type="Italic">Astragalus sinicus</Emphasis> L.) grown in lanthanum spiked soil

A greenhouse experiment was conducted to study the effect of mycorrhizal colonization by Gigaspora margarita, Glomus intraradices, and Acaulospora laevis on nutrient uptake of K, Ca, Mg, Cu, Zn, Fe, and Mn by Astragalus sinicus L. in soils spiked with lanthanum at five rates (0, 1, 5, 10, and 20 mg kg⁻¹). Lanthanum application significantly decreased the concentrations of K, Ca, Mg, Cu, Zn, and Fe in shoots and the concentrations of Cu and Zn in roots. Mycorrhizal treatments markedly improved uptake of nutrients, and these results are important since nutrient deficiency often occurs in contaminated sites.     

The influence of inorganic nutrient fertilization on the growth,nutrient composition and vesicular-arbuscular mycorrhizal colonization of pretransplant rice (Oryza sativa L.) plants

Summary The effects of P, N and Ca+Mg fertilization on biomass production, leaf area, root length, vesiculararbuscular mycorrhizal (VAM) colonization, and shoot and root nutrient concentrations of pretransplant rice (Oryza sativa L.) plants were investigated. Mycorrhizal plants generally had a higher biomass and P, N, K, Ca, Mn, Fe, Cu, Na, B, Zn, Al, Mg, and S shoot-tissue nutrient concentrations than non-mycorrhizal plants. Although mycorrhizal plants always had higher root-tissue nutrient concentrations than non-mycorrhizal plants, they were not significantly different, except for Mn. N fertilization stimulated colonization of the root system (colonized root length), and increased biomass production and nutrient concentrations of mycorrhizal plants. Biomass increases due to N were larger when the plants were not fertilized with additional P. P fertilization reduced the colonized root length and biomass production of mycorrhizal plants. The base treatment (Ca+Mg) did not significantly affect biomass production but increased the colonized root length. These results stress the importance of evaluating the VAM rice symbiosis under various fertilization regimes. The results of this study suggest that pretransplant mycorrhizal rice plants may have a potential for better field establishment than non-mycorrhizal plants.