Application of 15N natural abundance technique for evaluating biological nitrogen fixation in oil palm ecotypes at nursery stage in pot experiments and at mature plantation sites

A range of different species of diazotrophic bacteria has been found in tissues and the rhizosphere of oil palm plants, suggesting a potential to benefit from biological nitrogen fixation (BNF). A few studies have confirmed that plantlets at nursery stage can benefit significantly from BNF after inoculation with Azospirillum spp. but no data are available regarding the benefit from naturally-occurring diazotrophic bacteria in oil palm. The results described here were derived from two pot trials laid out under controlled conditions with plantlets from two important regions for palm oil production in Brazil, as well as from different field sites of mature oil palm plantations. The ¹⁵N natural abundance technique was employed to estimate plant dependence on BNF (%Ndfa) by the different ecotypes grown in soil and previously characterized as hosting diazotrophic bacteria. From both pot trials it was possible to identify some ecotypes of high potential for N₂-fixation that reached in some cases approximately 50%Ndfa. However, the accuracy of measurement still needs to be improved using more suitable reference plants for pot experiments. Values of δ ¹⁵N signals from oil palm and reference plants in the field were inconclusive concerning any benefit from BNF to oil palm, owing to apparently high temporal and spatial variability of δ ¹⁵N of the plant-available N in the heterogeneous soil matrix for the different palm and reference plant tested.     

Nitrogen fixation associated with grasses and cereals: Recent results and perspectives for future research

Over the last few years research in the area of biological nitrogen fixation (BNF) associated with cereals and grasses has become divided into two areas. On the one hand there have been a large number of reports of responses of field-grown plants to inoculation with N₂-fixing bacteria, principallyAzospirillum spp. On the other hand there have been several reports of significant contributions of associated BNF to the nutrition of several crops, including wetland rice, sugar cane and some forage grasses. However, where BNF contributions have definitely been established no certain information is available as to the diazotrophic organisms responsible. Furthermore, certain recent reports indicate that, at least in some cases, responses of plants to inoculation withAzospirillum spp. have been shown not to be due to BNF contributions. In this paper we review some recent progress in this field, particularly at our institute in Rio de Janeiro, concerning specificity of selected Azospirillum strains in the infection of cereal roots and the promotion of responses in the host plants. The possible mechanisms of plant response are discussed including the possibility that plant growth substances or bacterial nitrate reductase are involved. The application of¹⁵N and N balance techniques to the quantification of plant associated BNF are considered and the possible strategies that may be adopted to further the understanding of true N₂-fixing plant/diazotroph associations. The recent discovery of many more plant-associated N₂-fixing bacteria suggests that further research in this area may eventually lead to the development of such associations with applications for agricultural productivity.     

Infection and Colonization of Sugar Cane and Other Graminaceous Plants by Endophytic Diazotrophs

Agriculturally important grasses such as sugar cane (Saccharum sp.), rice (Oryza sativa), wheat (Triticum aestivum) sorghum (Sorghum bicolor), maize (Zea mays), Panicum maximum, Brachiaria spp., and Pennisetum purpureum contain numerous diazotrophic bacteria, such as, Acetobacter diazotrophicus, Herbaspirillum spp., Azospirillum spp. These bacteria do not usually cause disease symptoms in the plants with which they are associated and the more numerous of them, for example, Herbaspirillum spp. and A. diazotrophicus, are obligate or facultative endo-phytes that do not survive well (or at all) in native soil; these are thought to be spread from plant generation to plant generation via seeds, vegetative propagation, dead plant material, and possibly by insect sap feeders. By contrast, Azospirillum spp. are not wholly endophytic but are root-associated, soil-dwelling bacteria that are also often found within plants, probably entering host plants via seeds or via wounds/cracks at lateral root junctions. Endophytic diazotrophs have been isolated from a number of grasses in which significant biological N₂ fixation (BNF) has been demonstrated, particularly Brazilian sugar cane varieties, but also in rice, maize, and sorghum. However, although the endophytic diazotrophs are held to be the causative agents of the observed BNF, direct evidence for this is lacking. Therefore, in this review we examine probable sites of bacterial multiplication and/or BNF within endophyte-containing grasses and discuss these in terms of potential benefits (or not) to both host plants and bacteria. In particular, we examine how potentially large numbers of bacteria, especially Herbaspirillum spp., A. diazotrophicus, and Azospirillum spp., can exist extracellularly within non-specialized (for symbiotic purposes) regions such as xylem vessels and intercellular spaces. The processes of infection and colonization of various grasses (particularly sugar cane) by diazotrophic endophytes are also described, and these are compared with those of important (nondiazotrophic) endophytic sugar cane pathogens such as Clavibacter xyli subsp. xyli and Xanthomonas albilineans.     

Trials to create artificial nitrogen-fixing symbioses and associations using in vitro methods: An outlook

Summary Biological nitrogen fixation is the most important process in which some prokaryotic organisms fix N₂ into ammonium. From an agricultural standpoint, biological nitrogen fixation (BNF) is critical because industrial production of nitrogen fertilizers seldom meets agricultural demands. To increase the BNF is one of the main challenges for the future. There are different possibilities for extending biological nitrogen fixation to the economically important plants. One of the possibilities is to create new artificial systems between diazotrophic bacteria and different higher plants. This is the main topic of the present review article which discusses the establishment of new associative and/or symbiotic systems, via introduction of diazotrophic bacteria into the roots by different methods; and incorporation of nitrogen-fixing bacteria in the entire plant by in vitro methods, through the establishment of intracellular endosymbioses via induced uptake of bacteria by plant protoplasts (endocytobiosis), and establishment of intercellular associations by forced introduction of bacteria into the plant tissues (exocytobiosis). The common characteristic of the methods to create artificial plant-microbe systems for atmospheric nitrogen fixation is the use of in vitro plant systems: cells, tissues and organ cultures. The review pays particular attention to new bacterial inoculation procedures for introduction of the diazotrophic bacteria inside the plant tissues.     

三株固氮类芽孢杆菌的特点及其对中国青菜的产量和土壤酶活性的影响

【目的】本研究分析三株固氮菌PGPR性状特征及其对中国青菜产量和土壤酶活的影响。【方法】氮(N)-修复(固氮)细菌被认为是一种能够促进植物生长和增产的施氮方式。在本研究中,我们用无氮培养基分离出了30株根际固氮细菌:11株来自小麦根际,16株来自中国青菜根际和3株来自莲花根际。基于16S r DNA序列分析,对小麦、中国青菜和莲花等植物根际中属于类芽孢杆菌属的主要固氮细菌进行研究。【结果】本研究从这30株固氮菌中筛选出三株属于类芽孢杆菌属(Paenibacillus)的细菌,分别命名为P-4、W-7和L-3,它们的固氮酶活性不但高于对照组(圆褐固氮菌),而且可以有效抑制两种或三种植物病原菌的生长,即核盘菌(Sclerotinia sclerotiorum)、玉蜀黍赤霉(Gibberella zeae)和棉花黄萎病菌(Verticillium dahliae)。菌株W-7还具有溶解难溶磷的能力,中国青菜在接种菌株W-7和L-3后,其鲜重显著增加,同时改变了田间土壤蔗糖酶、磷酸酶和过氧化氢酶的活性;而接种了菌株P-4对植物的生长和酶活性没有显著的影响。【结论】土壤蔗糖酶、磷酸酶和过氧化氢酶活性与中国青菜的生物量呈正相关。同时,菌株W-7和L-3具有促进植物产量和提高土壤质量的良好潜力。     

Endophytes of Grapevine Flowers,Berries, and Seeds: Identification of Cultivable Bacteria,Comparison with Other Plant Parts,and Visualization of Niches of Colonization

Endophytic bacteria can colonize various plants and organs. However, endophytes colonizing plant reproductive organs have been rarely analyzed. In this study, endophytes colonizing flowers as well as berries and seeds of grapevine plants grown under natural conditions were investigated by cultivation as well as by fluorescence in situ hybridization. For comparison, bacteria were additionally isolated from other plant parts and the rhizosphere and characterized. Flowers, fruits, and seeds hosted various endophytic bacteria. Some taxa were specifically isolated from plant reproductive organs, whereas others were also detected in the rhizosphere, endorhiza or grape inflo/infructescence stalk at the flowering or berry harvest stage. Microscopic analysis by fluorescence in situ hybridization of resin-embedded samples confirmed the presence of the isolated taxa in plant reproductive organs and enabled us to localize them within the plant. Gammaproteobacteria (including Pseudomonas spp.) and Firmicutes (including Bacillus spp.) were visualized inside the epidermis and xylem of ovary and/or inside flower ovules. Firmicutes, mainly Bacillus spp. were additionally visualized inside berries, in the intercellular spaces of pulp cells and/or xylem of pulp, but also along some cell walls inside parts of seeds. Analysis of cultivable bacteria as well as microscopic results indicated that certain endophytic bacteria can colonize flowers, berries, or seeds. Our results also indicated that some specific taxa may not only derive from the root environment but also from other sources such as the anthosphere.     

Genetic diversity and plant growth promoting traits of diazotrophic bacteria isolated from two Pennisetum purpureum Schum. genotypes grown in the field

Background and aims

Some elephant grass (Pennisetum purpureum) genotypes are able to produce large amounts of biomass and accumulate N derived from BNF when growing in soil with low N levels. However, information about the diazotrophic bacteria colonizing this C4 plant is still very scarce. This study aimed to characterize the plant growth promoting traits of a fraction of culturable diazotrophs colonizing the genotypes CNPGL F06-3 and Cameroon.

Methods

A total of 204 isolates were obtained from surface sterilized leaves, stems and roots after culturing on five different N-free semisolid media. These were then analyzed by BOX-PCR, and the 16S rRNA and nifH sequences of representative isolates were obtained. The functional ability of the isolates to reduce acetylene, produce indole and to solubilize phosphate was also determined.

Results

The diazotrophic bacterial population varied from 10² up to 10⁶ bacteria g^?1 fresh tissues of both genotypes. The BOX-PCR analysis suggested a trend in the genetic diversity among the 204 diazotrophic strains colonizing the different genotypes and plant tissues. Sequencing of 16S rRNA fragments confirmed the presence of Azospirillum brasilense and Gluconacetobacter diazotrophicus and revealed for the first time the occurrence of G. liquefaciens, G. sacchari, Burkholderia silvatlantica, Klebsiella sp., Enterobacter cloacae and E. oryzae in elephant grass. Interestingly, several nifH sequences from isolates identified as G. liquefaciens and G. sacchari showed homologies with nifH sequences of Enterobacter species. The majority of the isolates (97%) produced indole compounds, 22% solubilized phosphate and 6.4% possessed both characteristics.

Conclusions

The results showed the occurrence of novel diazotrophic bacterial species colonizing different tissues of both genotypes of elephant grass. In addition, the study revealed the presence of several bacteria with growth promoting traits, and highlighted their potential to be exploited as biofertilizers.     

The contribution of nitrogen fixation to sugarcane (Saccharum officinarum L.), and the identification and characterization of part of the associated diazotrophic bacterial community

Background and aims

Rhizospheric, epiphytic and endophytic bacteria are associated with several non-legumes, colonizing their surface and inner tissues. Many of these bacteria are beneficial to their hosts, and are collectively termed plant growth-promoting rhizobacteria (PGPR). Recent interest has focused particularly upon PGPR that are endophytic (i.e. PGPE), and which have been reported to be associated with important crops such as rice, wheat and sugarcane. Different mechanisms are involved in bacteria-induced plant growth promotion (PGP), including biological nitrogen fixation (BNF), mineral solubilization, production of phytohormones and pathogen biocontrol. In Uruguay, sugarcane (Saccharum officinarum L.) is considered a strategic multipurpose crop, used for bioenergy, feed, sugar and bioethanol production. The aim of this work was to estimate the BNF contribution to Uruguayan sugarcane cultivars, as well as to identify and characterize the (culturable) putatively endophytic diazotrophic bacteria associated with these varieties.

Methods and results

Results using the ¹⁵N-dilution technique have shown that these sugarcane varieties obtain significant inputs of N from BNF (34.8–58.8% Ndfa). In parallel, a collection of 598 isolates of potentially endophytic diazotrophs was obtained from surface-sterilized stems using standard isolation techniques, and nifH ⁺ isolates from these were the subject of further studies. The bacteria were shown to belong to several genera, including Pseudomonas, Stenotrophomonas, Xanthomonas, Acinetobacter, Rhanella, Enterobacter, Pantoea, Shinella, Agrobacterium and Achromobacter. Additionally, some PGP features were studied in 35 selected isolates. The data obtained in this study represent the initial steps in a program aimed at determining the mechanisms of PGP of non-legume crops in Uruguay (such as sugarcane) with potentially beneficial plant-associated bacteria.     

Endophytic Establishment of Diazotrophic Bacteria in Auxin-Induced Tumors of Cereal Crops

Gramineous crops such as wheat (triticum oestivum), maize (zea mays), and rice (oryza sativa) develop tumorous structures (para-nodules) along primary and secondary roots when treated with low concentrations of various auxins. Rice forms additional tumors along its hypocotyle. Histologically, auxin-induced tumors appear as cancerous grown out root meristems and thus are comparable in origin and structure to stem nodules of the legume sesbania rostrata. Auxin-affected root meristems do not recover and develop further to large nodule-like organs. Introduced diazotrophs (Azospirillum spp., Azorhizobium caulinodans, Rhizobium spp.) potentially inhabit tissues of both stem and root tumors with the central meristem as a major colonization niche. Evidence is given that infecting bacteria follow a ‘crack entry’ invasion at sites where developing tumors have emerged through the root cortex and epidermis. Bacteria are shown to establish with high cell numbers inside intercellular spaces of cortical and meristematic tissues. Plant-cell infection of tumor cells takes place with bacteria found inside the cell-cytoplasm surrounded by membrane-like structures. Once inhabiting induced tumor tissues introduced diazotrophs colonize endophytically with high cell numbers. Mutant, ammonium-excreting and thus ecologically disadvantaged A. brasilense is shown to survive inside para-nodulating maize and rice plants with a dense population. Micro-aerobic nitrogenase activities of tumor inhabiting diazotrophic bacteria (A. brasilense, Azotobacter vinelandii, A. caulonidans) are in general highly increased when compared with untreated control plants. Additionally, bacterial nitrogenase activity is less sensitive to an increased oxygen tension in the root environment. The host plants benefit from the enhanced nitrogen fixation in their para-nodulating roots. Highest rates of incorporation of fixed nitrogen into host plant material is reported for para-nodule inhabiting ammonium excreting A. brasilense strain C3. The host plant potentially stimulates the nitrogenase activity of endophytically colonizing diazotrophs by providing energy in the form of a suitable carbon source. In conclusion, it is demonstrated that gramineous plants are potentially capable of developing an endophytical diazotrophic symbiosis through para-nodule formation.     

Arbuscular Mycorrhizal Assemblages in Native Plant Roots Change in the Presence of Invasive Exotic Grasses

Plant invasions have the potential to significantly alter soil microbial communities, given their often considerable aboveground effects. We examined how plant invasions altered the arbuscular mycorrhizal fungi of native plant roots in a grassland site in California and one in Utah. In the California site, we used experimentally created plant communities composed of exotic (Avena barbata, Bromus hordeaceus) and native (Nassella pulchra, Lupinus bicolor) monocultures and mixtures. In the Utah semi-arid grassland, we took advantage of invasion by Bromus tectorum into long-term plots dominated by either of two native grasses, Hilaria jamesii or Stipa hymenoides. Arbuscular mycorrhizal fungi colonizing roots were characterized with PCR amplification of the ITS region, cloning, and sequencing. We saw a significant effect of the presence of exotic grasses on the diversity of mycorrhizal fungi colonizing native plant roots. In the three native grasses, richness of mycorrhizal fungi decreased; in the native forb at the California site, the number of fungal RFLP patterns increased in the presence of exotics. The exotic grasses also caused the composition of the mycorrhizal community in native roots to shift dramatically both in California, with turnover of Glomus spp., and Utah, with replacement of Glomus spp. by apparently non-mycorrhizal fungi. Invading plants may be able to influence the network of mycorrhizal fungi in soil that is available to natives through either earlier root activity or differential carbon provision compared to natives. Alteration of the soil microbial community by plant invasion can provide a mechanism for both successful invasion and the resulting effects of invaders on the ecosystem.     

Plant Growth-Promoting Effects of Diazotrophs in the Rhizosphere

Because of their ability to transform atmospheric N₂ into ammonia that can be used by the plant, researchers were originally very optimistic about the potential of associative diazotrophic bacteria to promote the growth of many cereals and grasses. However, multiple inoculation experiments during recent decades failed to show a substantial contribution of Biological Nitrogen Fixation (BNF) to plant growth in most cases. It is now clear that associative diazotrophs exert their positive effects on plant growth directly or indirectly through (a combination of) different mechanisms. Apart from fixing N₂, diazotrophs can affect plant growth directly by the synthesis of phytohormones and vitamins, inhibition of plant ethylene synthesis, improved nutrient uptake, enhanced stress resistance, solubilization of inorganic phosphate and mineralization of organic phosphate. Indirectly, diazotrophs are able to decrease or prevent the deleterious effects of pathogenic microorganisms, mostly through the synthesis of antibiotics and/or fungicidal compounds, through competition for nutrients (for instance, by siderophore production) or by the induction of systemic resistance to pathogens. In addition, they can affect the plant indirectly by interacting with other beneficial microorganisms, for example, Azospirillum increasing nodulation of legumes by rhizobia. The further elucidation of the different mechanisms involved will help to make associative diazotrophs a valuable partner in future agriculture.     

Biological nitrogen fixation associated with sugar cane and rice: Contributions and prospects for improvement

¹⁵N isotope and N balance studies performed over the last few years have shown that several Brazilian varieties of sugarcane are capable of obtaining over 60% of their nitrogen (<150 kg N ha^-1 year^-1) from biological nitrogen fixation (BNF). This may be due to the fact that this crop in Brazil has been systematically bred for high yields with low fertilizer N inputs. In the case of wetland rice, N balance experiments performed both in the field and in pots suggest that 30 to 60 N ha^-1 crop^-1 may be obtained from plant-associated BNF and that different varieties have different capacities to obtain N from this source. ¹⁵N₂ incorporation studies have proved that wetland rice can obtain at least some N from BNF and acetylene reduction (AR) assays also indicate differences in N₂-fixing ability between different rice varieties. However in situ AR field estimates suggest plant-associated BNF inputs to be less than 8 kg N ha^-1 crop^-1. The problems associated with the use of the ¹⁵N dilution technique for BNF quantification are discussed and illustrated with data from a recent study performed at EMBRAPA-CNPAB. Although many species of diazotrophs have been isolated from the rhizosphere of both sugarcane and wetland rice, the recent discovery of endophytic N₂-fixing bacteria within roots, shoots and leaves of both crops suggests, at least in the case of sugarcane, that these bacteria may be the most important contributors to the observed BNF contributions. In sugarcane both Acetobacter diazotrophicus and Herbaspirillum spp. have been found within roots and aerial tissues and these microorganisms, unlike Azospirillum spp. and other rhizospheric diazotrophs, have been shown to survive poorly in soil. Herbaspirillum spp. are found in many graminaceous crops, including rice (in roots and aerial tissue), and are able to survive and pass from crop to crop in the seeds. The physiology, ecology and infection of plants by these endophytes are fully discussed in this paper. The sugarcane/endophytic diazotroph association is the first efficient N₂-fixing system to be discovered associated with any member of the gramineae. As yet the individual roles of the different diazotrophs in this system have not been elucidated and far more work on the physiology and anatomy of this system is required. However, the understanding gained in these studies should serve as a foundation for the improvement/development of similar N₂-fixing systems in wetland rice and other cereal crops.     

Yield of micropropagated sugarcane varieties in different soil types following inoculation with diazotrophic bacteria

It is well described that the beneficial interactions between plants and bacteria are genotype and site specific. Brazilian sugarcane varieties can obtain up to 70% of their nitrogen requirement from biological nitrogen fixation (BNF), and this contribution is related to the Brazilian breeding and selection processes, by example of the variety SP70-1143. In this study the effect of two inoculation mixtures containing diazotrophic bacteria in our earlier pot experiment was evaluated with two sugarcane varieties, a known responder, SP70-1143, and a newly selected variety, SP81-3250, to investigate the sugarcane genotype effect and the role of the mixtures. The sugarcane varieties SP70-1143 and SP81-3250 were grown under commercial field conditions at three sites with contrasting soil types: an Alfisol, an Oxisol and an Ultisol that means a low, medium and high natural fertility respectively. The stem yield and BNF contribution in response to bacterial inoculation were influenced by the strain combinations in the inoculum, the plant genotype, and the soil type and nitrogen fertilization, confirming the genetic and environmental influence in PGP-bacteria interactions. Inoculation effects on the BNF contribution and stem yield increased in the variety SP70-1143 grown in the Alfisol without nitrogen fertilization for three consecutive crops, and it was equivalent to the annual nitrogen fertilization. The plants grown in the Oxisol showed small increases in the productivity of the variety SP70-1143, and in the Ultisol the sugarcane plants presented even decreases in the stem productivity due to inoculation with diazotrophic bacteria mixtures. The results demonstrate the feasibility of the inoculation technology using diazotrophic bacteria in micropropagated sugarcane varieties grown in soils with low to medium levels of fertility. In addition, the results also indicated that specific plant – bacteria – environment combinations are needed to harness the full benefits of BNF. Section Editor: C. P. Vance     

The roots of the halophyte Salicornia brachiata are a source of new halotolerant diazotrophic bacteria with plant growth-promoting potential

Soil salinity is the major cause limiting plant productivity worldwide. Nitrogen-fixing bacteria were enriched and characterised from roots of Salicornia brachiata, an extreme halophyte which has substantial economic value as a bioresource of diverse and valuable products. Nitrogen-free semisolid NFb medium with malate as carbon source and up to 4% NaCl were used for enrichment and isolation of diazotrophic bacteria. The isolates were tested for plant growth-promoting traits and 16S rRNA, nifH and acdS genes were analysed. For selected strains, plant growth-promoting activities were tested in axenically grown Salicornia seedlings at different NaCl concentrations (0–0.5M). New halotolerant diazotrophic bacteria were isolated from roots of S. brachiata. The isolates were identified as Brachybacterium saurashtrense sp. nov., Zhihengliuella sp., Brevibacterium casei, Haererehalobacter sp., Halomonas sp., Vibrio sp., Cronobacter sakazakii, Pseudomonas spp., Rhizobium radiobacter, and Mesorhizobium sp. Nitrogen fixation as well as plant growth-promoting traits such as indole acetic acid (IAA) production, phosphate solubilisation, and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity were demonstrated. For Brachybacterium saurashtrense and Pseudomonas sp., significant plant growth-promoting activities were observed in Salicornia in salt stress conditions. Salicornia brachiata is a useful source of new halotolerant diazotrophic bacteria with plant growth-promoting potential.     

Nematodes associated with date palm and their control measures

Date palm, Phoenix dactylifera L., is dioecious and can be artificially pollinated by man, and one-third of all the dates of the world are grown in Iraq. In Egypt, there are about 12?million date palm trees grown in 99,867?feddans (fed.?=?4200?m²). Productivity is 1352,954?million?tons with yield 111.7?kg/tree. Plant parasitic nematodes associated with date palm are Criconemoides spp., Helicotylenchus spp., Hemicriconemoides spp., Hemicycliophora spp., Hoplolaimus spp., Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne javanica, Pratylenchus brachyurus, Pratylenchus jordanensis, Pratylenchus coffeae, Pratylenchus neglectus, Pratylenchus thornei, Trichodorus spp., Tylenchorhynchus goffarti, Tylenchorhynchus latus and Xiphinema spp.; Meloidogyne incognita-infected roots of susceptible cultivar favoured giant cell and galls formation. Date palm roots infected with Pratylenchus penetrans showed puncture of epidermal cells and disarrangement of cortical cells with large empty abnormal cavities. As control measures, it is advised to; 1 – plant immune or resistant cultivars against pathogenic nematodes, 2 – use oil cakes or poultry manure as organic amendments and a nematicide, carbofuran.These were tested and found effective in the control of Helicotylenchus multicinctus and P. penetrans, 3 – treat nematode-infested date palm seedlings with hot water at a suitable temperature for a given period before transplanting to open field, 4 – plant nematode -free date palm seedlings, 5 – soil solarisation and tillage before planting, 6 – weed control, 7 – intercrop with nematode-resistant horticultural crops and 8 – induce resistance in susceptible date palm cultivars against root knot nematode.     

A combination of humic substances and Herbaspirillum seropedicae inoculation enhances the growth of maize (Zea mays L.)

Background

Endophytic diazotrophic bacteria colonize several non-leguminous plants and promote plant growth. Different mechanisms are involved in bacteria-induced plant growth promotion, including biological nitrogen fixation (BNF), mineral solubilization, production of phytohormones, and pathogen biocontrol. Herbaspirillum seropedicae is a broad-host-range endophyte that colonizes sugarcane, rice, wheat, sorghum, and maize, and has been used as a biofertilizer. Contrasting results between greenhouse and field experiments have prompted efforts to improve the consistency of the plant response to microbial stimulation.

Aims

The aim of this study was to evaluate the effect of the presence of humic substances on inoculation of maize (Zea mays L.) with H. seropedicae.

Methods

Two experiments were conducted: one in the greenhouse using sand and nutrient solution and the other a field trial in soil with low natural fertility and to which was applied N in the form of urea (50 kg ha^?1). In the greenhouse, pre-emerging seeds were inoculated with a solution of H. seropedicae (10⁹ cells mL^?1) in the presence of humic substances isolated from vermicompost (10, 20, or 30 mg C?L^?1); in the field trial, bacteria combined with humate were added as a foliar spray (450 L?ha^?1).

Results

At early stages (7 and 45 days old) in the greenhouse, the treatment activated plant metabolism including enhancement of plasma membrane H⁺-ATPase activity, alteration of sugar and N metabolism, and greater net photosynthesis. The number of viable bacterial cells was higher in root tissues when inoculation was in the presence of soluble humic substances. Foliar application of endophytic diazotrophic bacteria and humic substances increased maize grain production 65 % under field conditions. These results show a promising use of humic substances to improve the benefit of endophytic diazotrophic inoculation.     

Gas exchange, growth, and antioxidant activity in sugarcane under biological nitrogen fixation

The aim of this study was to characterize the key physiological aspects of three sugarcane cultivars (RB92579, RB867515 and RB872552) under biological nitrogen fixation (BNF). Plants were generated in tubes containing aseptic substrates and these plants were transferred to pots containing washed sand, but watered with a mineral fertilizer, and inoculated with a mixture of five diazotrophic bacteria three times at seven-day intervals. Under BNF, all of the cultivars contained half of their total leaf nitrogen content and 50% less shoot dry mass. The leaves of plants under BNF showed approximately 65% less of the total protein content (TP). The gas-exchange control plants had twice the CO₂ assimilation rates than the BNF plants. The activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX) was increased in all cultivars under BNF when compared with the control; thus, the content of hydrogen peroxide (H₂O₂) was also increased in these plants. The results of this study indicate that after acclimatization, the inoculation of young plants from tissue culture with diazotrophic bacteria could supply approximately 50% of their nitrogen requirement.     

Occurrence and survival ofAzospirillum spp. in temperate regions

In order to evaluate the suitability ofAzospirillum spp. as a crop inoculant in temperate regions, the natural occurrence, distribution and survival ofAzospirillum after seed inoculation in Belgian agricultural soils was studied.Azospirillum was present in most of the fields examined, but concentrations never exceeded 1000 cfu per g soil or per g roots. Under field conditions none of the known species was found to be localized inside the roots of barley, wheat, rye, maize or grasses. Also, the distribution ofA. brasilense SpBr 14 within the root system of hydroponic-grown wheat was studied by immunofluorescence. From the rhizosphere samples of the field crops investigated, a number of microaërophilic, diazotrophic bacteria were isolated and identified asA. lipoferum, found only on maize and grass roots, andA. brasilense, present under all crops. In contrast toA. brasilense, A. lipoferum was able to use different amino-acids and some derivatives as sole carbon and nitrogen sources. Use of a peat-based seed inoculant resulted in the establishment of theAzospirillum spp. in the rhizosphere of field-grown winter barley and winter wheat. The established population survived during winter without appreciable change in numbers, but there was no indication of active growth during spring or summer.     

Interspecific competition among grasshoppers and their effect on plant abundance in experimental field environments

Summary We tested whether grasshoppers in experimental field environments, i.e. cages (40×40 cm) placed on existing old field vegetation, (1) were limited in density by plant abundance and/or nitrogen content, (2) exhibited interspecific competition, and (3) altered the relative abundance of different plant species. We examined interactions among a pair of early season grasshopper species (May–June; Arphia conspersa and Pardalophora apiculata) and a late season pair (July–August; Melanoplus femur-rubrum and Melanoplus bivittatus). Each grasshopper species was placed in cages by itself and with another grasshopper species. Grasshoppers generally survived at higher density in fertilized cages and they reduced plant abundance relative to empty cages, suggesting that grasshoppers may be food limited at these densities. In unfertilized plots, early season grasshoppers preferred grasses (Schizachyrium scoparium and Poa pratensis) and favored the growth of forbs (especially Solidago spp.). However, late in summer, Melanoplus spp. preferred Solidago spp. and favored the growth of grasses.The pattern of grasshopper survivorship and plant reduction within these experimental environments provide preliminary support for some of the predictions of resource competition theory. Grasshoppers exhibited interspecific competition only if they significantly reduced plant biomass. If two species competed, a grasshopper species was eliminated only if the superior competitor, when living by itself, could reduce plant biomass to a significantly lower level than the inferior competitor. Competitors persisted only if they did not differ in their ability to reduce plant biomass or reduced the abundance of different plant species.