Diversity and capacity to promote maize growth of bacteria isolated from the Amazon region

ABSTRACT Maize plants can establish beneficial associations with plant growth-promoting bacteria. However, few studies have been conducted on the characterization and inoculation of these bacteria in the Amazon region. This study aimed to characterize endophytic bacteria isolated from maize in the Amazon region and to assess their capacity to promote plant growth. Fifty-five bacterial isolates were obtained from maize grown in two types of ecosystems, i.e., a cerrado (savanna) and a forest area. The isolates were characterized by the presence of the nifH gene, their ability to synthesize indole-3-acetic acid (IAA) and solubilize calcium phosphate (CaHPO4), and 16S rRNA partial gene sequencing. Twenty-four bacteria contained the nifH gene, of which seven were isolated from maize plants cultivated in a cerrado area and seventeen from a forest area. Fourteen samples showed the capacity to synthesize IAA and only four solubilized calcium phosphate. The following genera were found among these isolates: Pseudomonas; Acinetobacter; Enterobacter; Pantoea; Burkholderia and Bacillus. In addition, eight isolates with plant growth-promoting capacity were selected for a glasshouse experiment involving the inoculation of two maize genotypes (a hybrid and a variety) grown in pots containing soil. Inoculation promoted the development of the maize plants but no significant interaction between maize cultivar and bacterial inoculation was found. A high diversity of endophytic bacteria is present in the Amazon region and these bacteria have potential to promote the development of maize plants.

Use of the pyrophosphatase activity as a reliable tonoplast marker in maize roots.

Membranes of maize (Zea mays L., cv LG 11) roots were fractionated by sucrose (in presence or absence of Mg2+) or dextran density gradient centrifugations and the locations of organelles were determined using marker enzymes. Latent UDPase was used as a Golgi marker, catalase for the peroxysomes, cytochrome c oxidase for the mitochondria, UDP-Gal-galactosyltransferase for the amyloplast membranes and NADH-cytochrome c reductase for the ER. Two markers were selected for the plasmalemma, the vanadate-sensitive ATPase and UDP-Glc-sterolglucosyltransferase. The distributions of the PPase and vacuolar ATPase were found to be similar after density gradient centrifugation. The PPase and vacuolar ATPase activities were clearly separated from almost all the other markers tested, however, a partial association of both activities with the ER cannot be completely ruled out. The PPase of maize roots is more active and easier to measure than the vacuolar ATPase and is therefore an excellent candidate for use as a tonoplast marker.

Characterization of the tonoplast Ca2+/H+ antiport system from maize roots.

The tonoplast calcium Ca2+/H+ antiport system of maize (Zea mays L. cv LG 11) roots was characterized using the ''pH jump'' technique in order to avoid interference from the tonoplast proton and Ca2+ pumps. Ca2+ uptake was recorded in the presence of different inhibitors and divalent ions. Chemical modification of amino acid residues of the antiport was used to elucidate the amino acid residues participating in the Ca2+ transport activity. The Ca2+/H+ antiport activity was found to be strongly inhibited by ruthenium red and verapamil, whereas diethylstilbestrol was less effective. Vanadate, erythrosin B, cyclopiazonic acid, bafilomycin, thapsigargin, N,N'-dicyclohexylcarbodiimide (DCCD) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) were without effect. Lanthanum and divalent ions were strongly inhibitory (Cd2+ > Mn2+ > Sr2+ > Ba2+). While reagents modifying sulfhydryl groups (N-ethylmaleimide and 5,5'-dithio-bis(2-nitrobenzoate)) did not affect the antiport activity, modification of trytophan residues (N-bromosuccinimide) was strongly inhibitory. We conclude that ruthenium red, verapamil, lanthanum and divalent cations directly inhibit Ca2+ uptake independent of the function of the proton and Ca2+ pumps. Moreover, the results of chemically modified amino acid residues suggest that sulfhydryl groups are not involved in Ca2+ transport, while tryptophan residues seem important for this translocation.

Peach palm growth and heart-of-palm yield responses to liming

The effects of liming rates on growth and heart-of-palm yield of peach palm plants (Bactris gasipaes Kunth) were studied in a two-year field experiment conducted in Pariquera-Açu, State of Sao Paulo, Brazil. Soils in this region are allic (sub group Ultic Haplorthox), with base saturation ranging from 15 to 26 % of the cation exchange capacity (CEC). A randomized complete block design, with five rates of dolomitic limestone (0, 0.7, 4.7, 8.7, and 14.6 Mg ha-1) and five replications was utilized. Individual plots were composed of 80 plants but only the inner rows (24 plants) were used for data recording. Planting spacing was 2 x 1 m. There was a cubic effect of liming rates on growth and yield. Maximum heart-of-palm yield was estimated to be achieved at 4.3 Mg ha-1 of limestone application, corresponding to 51.4 % soil base saturation. A significant decrease in growth and yield was observed when large amounts of limestone were applied (8.7 and 14.6 Mg ha-1), probably due to a decreased micronutrient availability.

Relationships between grain yield and accumulation of biomass, nitrogen and phosphorus in common bean cultivars

Shoot biomass is considered a relevant component for crop yield, but relationships between biological productivity and grain yield in legume crops are usually difficult to establish. Two field experiments were carried out to investigate the relationships between grain yield, biomass production and N and P accumulation at reproductive stages of common bean (Phaseolus vulgaris) cultivars. Nine and 18 cultivars were grown on 16 m² plots in 1998 and 1999, respectively, with four replications. Crop biomass was sampled at four growth stages (flowering R6, pod setting R7, beginning of pod filling R8, and mid-pod filling R8.5), grain yield was measured at maturity, and N and P concentrations were determined in plant tissues. In both years, bean cultivars differed in grain yield, in root mass at R6 and R7 stages, and in shoot mass at R6 and R8.5, whereas at R7 and R8 differences in shoot mass were significant in 1998 only. In both years, grain yield did not correlate with shoot mass at R6 and R7 and with root mass at R6. Grain yield correlated with shoot mass at R8 in 1999 but not in 1998, with shoot mass at R8.5 and with root mass at R7 in both years. Path coefficient analysis indicated that shoot mass at R8.5 had a direct effect on grain yield in both years, that root mass at R7 had a direct effect on grain yield in 1998, and that in 1999 the amounts of N and P in shoots at R8.5 had indirect effects on grain yield via shoot mass at R8.5. A combined analysis of both experiments revealed that biomass accumulation, N and P in shoots at R6 and R7 as well as root mass at R6 were similar in both years. In 1998 however bean accumulated more root mass at R7 and more biomass and N and P in shoots at R8 and R8.5, resulting in a 57 % higher grain yield in 1998. This indicates that grain yield of different common bean cultivars is not intrinsically associated with vegetative vigor at flowering and that mechanisms during pod filling can strongly influence the final crop yield. The establishment of a profuse root system during pod setting, associated with the continuous N and P acquisition during early pod filling, seems to be relevant for higher grain yields of common bean.

Effect of abscisic acid on maize root growth. A critical examination

The effect of abscisic acid (ABA) on the growth of maize roots maintained in the dark is investigated in relation to the root varieties and the root age, the mode of application, the concentration used and the duration of both the treatment and the culture. In all the assays, when ABA produces a significant change in root elongation, it shows an inhibitory effect which is enhanced with increasing ABA concentration. The data strongly support the hypothesis that ABA could be one of the growth inhibitors which are formed in or released from the root cap.

Effect of potassium fertilization on yield and nutrition of yerba mate (Ilex paraguariensis)

Yerba mate (Ilex paraguariensis) is a tree species native to the subtropical regions of South America, and is found in Brazil predominantly in the southern region. Despite the historical importance in this region, so far, studies on crop nutrition to improve yields are scarce. Thus, this study evaluated the effect of potassium rates on K soil availability, and the yield and nutritional status of yerba mate. The experiment was conducted in São Mateus do Sul, State of Paraná, on a Humox soil, where K2O rates of 0, 20, 40, 80, 160, and 320 kg ha-1 were tested on 7-year-old plantations. The experiment was harvested 24 months after installation by removing approximately 95 % of the canopy that had sprouted from the previous harvest. The soil was evaluated for K availability in the layers 0-10, 0-20, 10-20, and 20-40 cm. The plant parts leaf fresh matter (LM), twigs (TW), thick branches (BR) and commercial yerba mate (COYM), i.e., LM+TW, were analyzed. In addition, the relationship between fresh matter/dry matter (FM/DM) and K concentration in LM, AG and BR were evaluated. The fertilization increased K availability in all evaluated soil layers, indicating good mobility of the nutrient even at low rates. Yerba mate responded positively to increasing K2O rates with higher yields of all harvested components. The crop proved K-demanding, with a maximum COYM yield of 28.5 t ha-1, when 72 mg dm-3 K was available in the 0-20 cm layer. Yerba mate in the plant production stage requires soil K availability at medium to high level; in clayey soil with low K availability, a rate of 300 kg ha-1 K2O should be applied at 24 month intervals to obtain high yields. A leaf K concentration of 16.0 g ha-1 is suitable for yerba mate in the growth stage.

INOCULATION AND ISOLATION OF PLANT GROWTH-PROMOTING BACTERIA IN MAIZE GROWN IN VITÓRIA DA CONQUISTA, BAHIA, BRAZIL

Maize is among the most important crops in the world. This plant species can be colonized by diazotrophic bacteria able to convert atmospheric N into ammonium under natural conditions. This study aimed to investigate the effect of inoculation of the diazotrophic bacterium Herbaspirillum seropedicae (ZAE94) and isolate new strains of plant growth-promoting bacteria in maize grown in Vitória da Conquista, Bahia, Brazil. The study was conducted in a greenhouse at the Experimental Area of the Universidade Estadual do Sudoeste da Bahia. Inoculation was performed with peat substrate, with and without inoculation containing strain ZAE94 of H. seropedicae and four rates of N, in the form of ammonium sulfate (0, 60, 100, and 140 kg ha-1 N). After 45 days, plant height, dry matter accumulation in shoots, percentage of N, and total N (NTotal) were evaluated. The bacteria were isolated from root and shoot fragments of the absolute control; the technique of the most probable number and identification of bacteria were used. The new isolates were physiologically characterized for production of indole acetic acid (IAA) and nitrogenase activity. We obtained 30 isolates from maize plants. Inoculation with strain ZAE94 promoted an increase of 14.3 % in shoot dry mass and of 44.3 % in NTotal when associated with the rate 60 kg ha-1 N. The strains N11 and N13 performed best with regard to IAA production and J06, J08, J10, and N15 stood out in acetylene reduction activity, demonstrating potential for inoculation of maize.

Soil tillage affects the community structure of mycorrhizal fungi in maize roots

In this study we tested whether communities of arbuscular mycorrhizal fungi (AMF) colonizing the roots of maize (Zea mays L.) were affected by soil tillage practices (plowing, chiseling, and no-till) in a long-term field experiment carried out in Tanikon (Switzerland). AMF were identified in the roots using specific polymerase chain reaction (PCR) markers that had been developed for the AMF previously isolated from the soils of the studied site. A nested PCR procedure with primers of increased specificity (eukaryotic, then, fungal, then AMF species or. species-grouop specific) was used. Sequencing of amplified DNA confirmed that the DNA obtained from the maize roots was of AMF origin. Presence of particular AMF species or species-group was scored as a presence of a DNA product after PCR with specific primers. We also used single-strand conformation polymorphism analysis (SSCP), of amplified DNA samples to-check if the amplification of the DNA from maize roots matched the expected profile for a particular AMF isolate with a given specific primer pair. Presence of the genus Scutellospora, in maize roots was strongly reduced in plowed and chiseled soils. Fungi from the suborder Glomineae were more prevalent colonizers of maize roots growing in plowed soils, but were also present in the roots from other tillage treatments. These changes in community of AMF colonizing maize roots might be due to (1), the differences in tolerance to the tillage-induced disruption of the hyphae among the different AMF species, (2) changes in nutrient content of the soil, (3) changes in microbial activity, or (4) changes in weed populations in response to soil tillage. This is the first report on community composition of AMF in the roots of a field-grown crop plant (maize) as affected by soil tillage.

Phenotyping maize for adaptation to drought

The need of a better adaptation of crops to drought is an issue of increasing urgency. However, enhancing the tolerance of maize has, therefore, proved to be somewhat elusive in terms of plant breeding. In that context, proper phenotyping remains as one of the main factors limiting breeding advance. Topics covered by this review include the conceptual framework for identifying secondary traits associated with yield response to drought and how to measure these secondary traits in practice.

The effect of cover crop and crop rotation on soil water storage and on sorghum yield

Crop rotation and cover crop can be important means for enhancing crop yield in rainfed areas such as the lower Coastal Bend Region of Texas, USA. A trial was conducted in 1995 as part of a long-term cropping experiment (7 years) to investigate the effect of oat (Avena sativa L.) cover and rotation on soil water storage and yield of sorghum (Sorghum bicolor L.). The trial design was a RCB in a split-plot arrangement with four replicates. Rotation sequences were the main plots and oat cover crop the subplots. Cover crop reduced sorghum grain yield. This effect was attributed to a reduced concentration of available soil N and less soil water storage under this treatment. By delaying cover termination, the residue with a high C/N acted as an N sink through competition and/or immobilization instead of an N source to sorghum plants. Crop rotation had a significantly positive effect on sorghum yield and this effect was attributed to a significantly larger amount of N concentration under these rotation sequences.

Influence of micronutrients on dry matter yield and interaction with other nutrients in annual crops

The objective of this work was to determine the influence of Zn, Mn and Cu on shoot dry matter yield and uptake of macro and micronutrients in upland rice, common bean and corn. Six greenhouse experiments were conducted using a Dark Red Latosol (Typic Haplusthox). Treatments consisted of application of Zn at 0, 5, 10, 20, 40, 80 and 120 mg kg-1, of Mn at 0, 10, 20, 40, 80, 160, 320 and 640 mg kg-1 and of Cu application at 0, 2, 4, 8, 32, 64 and 96 mg kg-1. Zinc increased yield of rice, Mn increased yields of corn and bean and Cu improved yields of rice and bean. Uptake of N, Ca, and Cu in rice was decreased by zinc treatment. In common bean, uptake of N, Mg, and Cu was increased by zinc application, whereas, uptake of P was decreased. Manganese increased uptake of Mg, Zn and Fe and decreased uptake of Ca, in corn. Uptake of K, Zn and Mn was increased and uptake of P and Cu was decreased by Mn application, in bean. Copper had positive and negative interactions in the uptake of macro and micronutrients, depending on crop species and nutrients involved.

Grain yield and seed quality of soybean selected for high protein content

The objective of this work was to evaluate the effects of selection for high protein on seed physiological quality and grain yield of soybean. Four populations of BC1F4 and four of F4, each from a cross between a commercial variety and a line bearing high protein seeds, were used. The high protein content selection has a tendency to affect negatively the seed physiological quality. Estimates of correlation coefficients between protein content and grain yield were mostly negative but varied among populations. It is possible to obtain lines with high protein content, keeping the grain yield and the seed physiological quality of their respective recurrent progenitors.

Optimization of particle bombardment parameters for the genetic transformation of Brazilian maize inbred lines

The objective of this work was to develop a genetic transformation system for tropical maize genotypes via particle bombardment of immature zygotic embryos. Particle bombardment was carried out using a genetic construct with bar and uidA genes under control of CaMV35S promoter. The best conditions to transform maize tropical inbred lines L3 and L1345 were obtained when immature embryos were cultivated, prior to the bombardment, in higher osmolarity during 4 hours and bombarded at an acceleration helium gas pressure of 1,100 psi, two shots per plate, and a microcarrier flying distance of 6.6 cm. Transformation frequencies obtained using these conditions ranged from 0.9 to 2.31%. Integration of foreign genes into the genome of maize plants was confirmed by Southern blot analysis as well as bar and uidA gene expressions. The maize genetic transformation protocol developed in this work will possibly improve the efficiency to produce new transgenic tropical maize lines expressing desirable agronomic characteristics.

Simulating maize phenology as a function of air temperature with a linear and a nonlinear model

The objective of this study was to adapt a nonlinear model (Wang and Engel - WE) for simulating the phenology of maize (Zea mays L.), and to evaluate this model and a linear one (thermal time), in order to predict developmental stages of a field-grown maize variety. A field experiment, during 2005/2006 and 2006/2007 was conducted in Santa Maria, RS, Brazil, in two growing seasons, with seven sowing dates each. Dates of emergence, silking, and physiological maturity of the maize variety BRS Missões were recorded in six replications in each sowing date. Data collected in 2005/2006 growing season were used to estimate the coefficients of the two models, and data collected in the 2006/2007 growing season were used as independent data set for model evaluations. The nonlinear WE model accurately predicted the date of silking and physiological maturity, and had a lower root mean square error (RMSE) than the linear (thermal time) model. The overall RMSE for silking and physiological maturity was 2.7 and 4.8 days with WE model, and 5.6 and 8.3 days with thermal time model, respectively.