Azospirillum Strains Research Articles

The Structure of the O-Specific Polysaccharide and Lipid A of the Type Strain Azospirillum rugosum DSM-19657

The paper reports the structural study of the lipopolysaccharide from the type strain of the soil nitrogen-fixing bacterium Azospirillum rugosum isolated from oil-polluted soils. Based on the chemical analysis and 1D- and 2D 1H and 13С NMR spectroscopy data it has been demonstrated that the O-specific polysaccharide consists of the two types of repeating units identical to those previously described for the O-specific polysaccharide of the Azospirillum brasilense strain Jm125A2. Structural analysis of the lipid A using gas-liquid chromatography (GLC) and MALDI mass spectrometry revealed the presence of penta-, tetra-, and triacylated species. The primary fatty acids in the lipid A are the N-linked 16:0(3-OH) and О-linked 14:0(3-OH). Microheterogeneity within each species arises from the presence of different secondary fatty acids (16:0, 18:1, or 19:0) acylating the 16:0(3-OH) acid of the distal GlcN ring.

Genome Sequences of Novel Azospirillum sp. Strains B21 and Sh1, Isolated from Raised Sphagnum Bogs, and Type Strains Azospirillum lipoferum 59b and Azospirillum oryzae COC8.

Here, we report the genomic sequences of the novel Azospirillum sp. strains B21 and Sh1, isolated from raised bogs, along with the genome sequences of Azospirillum lipoferum 59bT, the type species of the genus, and Azospirillum oryzae COC8T, which were analyzed to get more knowledge about the genus Azospirillum.

Isolation and identification of Azospirillum and Azotobacter species from Acacia spp. at Riyadh, Saudi Arabia

Populations of Azospirillum and Azotobacter isolated and identified from the soil rhizosphere of Acacia ehernbergiana, A. gerrardii and A. tortilis from three sites varied widely irrespective of tree species, size and site. Azospirillum strains were purified and characterized through morphological, microscopic and biochemical tests for motility, cell shape, pigmentation, colony color, gram reaction, optimal temperatures, biotin requirement, pH growth, acidification of peptone glucose medium, ability of nitrogen fixation, growth on 3% NaCl, and D-glucose and D-ribose utilization. Twenty Azospirillum isolates were divided into four matching characters. They were identified as A. lipoferum, A. brasilense, A. dobereinerae and A. zeae. The same above tests with omission of temperatures, biotin requirement, and acidification of peptone and addition of starch hydrolysis, H2S, urease, oxidase and sucrose, maltose and mannitol as carbon source were applied to Azotobacter strains. Azotobacter isolates were divided into three matching characters. They were identified as A. chroococcum, A. beijerinckii and A. salinestris.

Extracellular Laccase Activity of Bacteria of the Genera Azospirillum and Niveispirillum

The ability to produce extracellular laccase by strains of Azospirillum (A. brasilense Sp7, Sp107, Sp245, SR80, A. lipoferum SR42, Sp59b, A. picis TAR-3, A. tiophilum Bv-S, and A. amazonense Am) and Niveispirillum (N. irakense KBC1 and N. irakense KA3) has been established. Induction of enzyme activity by a number of aromatic compounds and copper ions has been shown. A homogeneous preparation of the extracellular laccase of A. brasilense Sp245 strain has been isolated and partially characterized for the first time.

MORPHOLOGICAL, PRODUCTIVE, AND CHEMICAL TRAITS OF XARAÉS GRASS: NITROGEN TOPDRESSING VERSUS INOCULATION WITH DIAZOTROPHIC BACTERIA

Abstract: Nitrogen topdressing is essential for forage production, but the high cost of fertilizers stimulates the search for new technologies, such as the use of diazotrophic bacteria, which can fix the atmospheric nitrogen. To evaluate the potential of this technology in providing the amount of nitrogen requested by xaraés grass as topdressing, plants were fertilized at planting with an N rate of 100 kg ha-1, inoculated with strains of diazotrophic bacteria and Azospirillum brasilense, and compared with plants that received 100 kg ha-1 of N at planting and as topdressing during two periods of shoot growth (58 days in the first period and 26 days in the second period) in 20-dm3 pots. The data were subjected to analysis of variance and means compared by the Scott-Knott test at 5% significance. The leaf area of xaraés grass inoculated with strain UN42 was similar to that of plants that received nitrogen topdressing. However, grass biomass production was higher when nitrogen topdressing was applied. Plant inoculation with Azospirillum brasilense and strains UN16, UN17, UN21, UN33, UN49, and UN78 increased crude protein contents of xaraés grass when compared to nitrogen fertilization at planting without inoculation. However, grass inoculation with diazotrophic bacteria does not replace nitrogen topdressing completely.

Structural studies of O-specific polysaccharide(s) and biological activity toward plants of the lipopolysaccharide from Azospirillum brasilense SR8

Lipopolysaccharide (LPS) was extracted from dry bacterial cells of plant-growth-promoting bacterium Azospirillum brasilense SR8 (IBPPM 5). The O-specific polysaccharide (OPS) was obtained by mild acid hydrolysis of the lipopolysaccharide and studied by sugar analysis, 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC and HMBC experiments, computational NMR-based structure analysis, and Smith degradation. The OPS was shown to contain two types of repeating units of the following structure: [Display omitted] Both OPS structures are present in A. brasilense 54, from which structure 1 has been reported earlier (Fedonenko et al., 2011), whereas to our knowledge structure 2 has not been hitherto found in bacterial saccharides. Treatment of wheat seedling roots with LPS of A. brasilense SR8 increased the number of root hair deformations as compared to seedlings grown without LPS, but had no effect on adsorption of the bacteria to the root surface. A. brasilense SR8 was able to utilize LPS of several structurally related Azospirillum strains.

New insights into indole-3-acetic acid metabolism in Azospirillum brasilense.

The aim of this research was to analyse the global indole-3-acetic acid (IAA) metabolism in three commercially used strains of Azospirillum brasilense. Azospirillum brasilense Sp245, Az39 and Cd, containing a plasmid with the ipdC-gusA fusion (pFAJ64), were cultured in minimal medium MMAB with or without 10mgl-1 of l-trp till exponential or stationary growth phase. The cultures were then split into 10ml tubes and individually treated with 10mgml-1 IAA, IBA or NAA (auxin catabolism and homeostasis); IAPhe, IALeu, IAA-ala, IAA-glucose (IAA conjugate hydrolysis); or l-lys, l-leu, l-ileu, l-phe, l-ala, l-val, l-arg, l-glu, l-his, l-met, l-asp, l-cys, l-ser, l-pro, l-thr and l-trp (regulation of IAA biosynthesis and IAA conjugation). Bacterial growth, IAA production and ipdC expression were evaluated. None of the A. brasilense strains were able to hydrolyse IAA conjugates, catabolize auxins, or conjugate IAA with amino acids or glucose. l-amino acids l-met, l-val, l-cys and l-ser inhibited bacterial growth and decreased IAA biosynthesis. The expression of ipdC and IAA biosynthesis but not bacterial growth was affected by l-leu, l-phe, l-ala, l-ile, l-pro. l-arg, l-glu, l-his, l-lys, l-asp and l-thr did not affect any of the measured parameters. In this paper, we confirmed that A. brasilense produces IAA only in presence of l-trp is not able to degrade auxins, conjugate IAA with sugars and/or l-amino acids, or hydrolyse such conjugates to release free IAA. Finally, we found that bacterial growth and/or IAA biosynthesis were inhibited by the presence of several l-amino acids probably by diversion of the cellular metabolism. We propose a renewed model to explain IAA metabolism in A. brasilense, one of the most studied phytostimulatory bacteria.

Isolation and Characterization of Azospirillum Isolates from Soil and Their Effect on Growth and Yield of Wheat (Triticum aestivum L.) under Different Levels of Nitrogen Fertilizer

This study involved isolation of Azospirillum from soils that are different in texturefrom Aswan governorate. Also, study the response of wheat (Triticum aestivum L.) to inoculation with the isolated Azospirillum strain (M-1, M-2 and M-3) in presence of different levels of nitrogen fertilization (40, 60 and 80 kg N/fed.) was tested in field experiments in the two seasons 2014/2015 and 2015/2016.

Maize Inoculation with Azospirillum brasilense Ab-V5 Cells Enriched with Exopolysaccharides and Polyhydroxybutyrate Results in High Productivity under Low N Fertilizer Input.

Although Azospirillum strains used in commercial inoculant formulations presents diazotrophic activity, it has been reported that their ability to produce phytohormones plays a pivotal role in plant growth-promotion, leading to a general recommendation of its use in association with regular N-fertilizer doses. In addition, a high variability in the effectiveness of Azospirillum inoculants is still reported under field conditions, contributing to the adoption of the inoculation technology as an additional management practice rather than its use as an alternative practice to the use of chemical inputs in agriculture. To investigate whether the content of stress-resistance biopolymers would improve the viability and performance of Azospirillum inoculants when used as substitute of N-fertilizers, biomass of A. brasilense strain Ab-V5 enriched in exopolysaccharides (EPS) and polyhydroxybutirate (PHB) was produced using a new culture medium developed by factorial mixture design, and the effectiveness of resulting inoculants was evaluated under field conditions. The culture medium formulation extended the log phase of A. brasilense cultures, which presented higher cell counts and increased EPS and PHB contents than observed in the cultures grown in the OAB medium used as control. An inoculation trial with maize conducted under greenhouse conditions and using the biopolymers-enriched Ab-V5 cells demonstrated the importance of EPS and PHB to the long term bacterial viability in soil and to the effectiveness of inoculation. The effectiveness of liquid and peat inoculants prepared with Ab-V5 cells enriched with EPS and PHB was also evaluated under field conditions, using maize as target crop along different seasons, with the inoculants applied directly over seeds or at topdressing under limiting levels of N-fertilization. No additive effect on yield resulted from inoculation under high N fertilizer input, while inoculated plants grown under 80% reduction in N fertilizer showed yields at levels compared to fully fertilized plants, regardless the inoculation method. The presented data highlights the feasibility to partially substitute the N-fertilizer demand in non-legume crops using high-quality inoculant formulations, prepared with diazotrophic bacteria enriched with stress-resistance biopolymers that confer increased viability an effectiveness to the bacterial cells.

AZOSPIRILLUM STRAINS AS BIOFERTILIZERS AND BIOCONTROL AGENTS -A PRACTICAL REVIEW

Azospirillum biofertilizers have a history going back to the beginning of the XXth However, researches still have to find new and competitive strains and improve biofertilizers production and application techniques. They are one of te most used biofertilizers in organic farming. The present paper review and analyses the development of the new agriculture using such microorganism, and showing not only the biofertilization effect but some biocontrol properties of the presented strains. The products containing Azospirillum strains and their use begin to play an important role in modern agriculture and in special in some countries in course of development as an alternative to chemical expensive and some time not environmental friendly products. Countries from Asia, South America, use it in huge quantities. In developed area of the lobe, there are only some tiny initiative, mainly because the multinational companies, and because of the legislation in use.

Long-term aromatic rice cultivation effect on frequency and diversity of diazotrophs in its rhizosphere

Diazotrophs are one of the most important microbial communities which play a key role in rhizosphere to enhance plant growth-promotion by supplying fixed nitrogen to the plants. The aim of present study was to analyze the frequency and diversity of diazotrophs in the rhizospheres of five genotypes of aromatic rice (Oryza sativa cultivars Nua Kalajeera, Ketekijoha, Nua Desheri, Nua Chandan and Kala Namak) grown in ten years old long-term aromatic rice-rice (LARR) cropping system under sub-humid tropical condition. In this study, it was observed that very less frequency of active diazotrophs (0–14.28%) in the rhizosphere of aromatic rice genotypes based on polymerase chain reaction (PCR)-based nifH gene amplification, dot blot hybridization and acetylene reduction assay (ARA). The following five isolates (B10, B12, B29, BK16, NKR16) from rhizosphere of aromatic rice and the reference strain Azospirillum brasilence showed nifH amplification and positive signal for dot blot. None of these isolates showed positive in ARA test except A. brasilence. Basic local alighnment search tool (BLAST) homology of nifH sequences of B10, B12, B29, BK16 and NKR16 matched with Gluconoacetobacter diazotrophicus, Pseudomonas stutzeri, Klebsiella sp., Klebsiella pneumoniae and Sinorhizobium meliloti, respectively. Whereas, isolates B10, B12 and BK16 were identified as Bacillus cereus, Enterobacter sp., and Lysinibacillus sp., respectively based on 16S-rDNA sequencing. As per nifH sequences, these diazotrophs (B10, B12, B29, BK16, NKR16) fall under α and γ-proteobacter, whereas 16S-rDNA sequences revealed that they belonged to firmicutes and γ-proteobacter. Multiple sequence alignment of nifH gene sequences showed wider variation among diazotrophs, however 3D structure of NifH protein revealed the less diversity among isolates. Moreover, restriction digestion of 16S-rDNA using tetracutters (MspI, MboI and HaeIII) deciphered a unique pattern among small population of diazotrophs.The results of this finding provided an interesting data and novel information of diazotroph diversity in LARR cropping system. Finally, the present study indicated that the continuous application of high dose of inorganic nitrogenous fertilizers (nitrogen at the rate of 60 & 80kgNha−1year−1 in wet and dry seasons, respectively) limit the frequency and diversity of rhizospheric diazotrophs in the LARR cropping system.

Uso de Azospirrillum spp. como biofertilizante en la producción de estrella africana (Cynodon nlemfuensis)

El pasto estrella africana es importante en algunas regiones tropicales y puede ser beneficioso el uso de Azospirillum como biofertilizante. Probamos tres cepas de Azospirrilum en Turrialba, Costa Rica, entre el 2011 y 2013. Utilizamos una dosis única de 5 litros por hectárea para las tres cepas (108 UFC.ml-1) y la mezcla de las tres cepas en partes iguales y las comparamos con una dosis de 10 t/ha de vermicompost y la fertilización química (78 kg/N/ha). En la segunda prueba evaluamos Azospirillum por su potencial para sustituir el fertilizante químico en proporciones de 0, 25, 50 y 75% una dosis de 5 L / ha (108 UFC.ml-1) utilizamos para todos los tratamientos y en comparación con la dosis de 100% de los fertilizantes químicos. Dos de las cepas y tres de las mezclas produjo biomasa forrajera similar (3,65 a 3,93 t / ha) como el fertilizante químico (4,12 t / ha). La fertilización con humus de lombriz fue menor (3,56 t / ha). Una disminución de las lluvias 2012-2013 redujo la biomasa del forraje 25% cuando se aplicó fertilización química (4,07 vs 3,08 t / ha). En el segundo experimento, no se encontraron diferencias significativas en los rendimientos de biomasa para todos los tratamientos con Azospirillum o fertilizantes químicos (2,97 vs 3,08 t / ha). Azospirillum tiene potencial para mantener la productividad del pasto estrella africana bajo un sistema de pastoreo, pero se requiere más investigación para definir el número de aplicaciones por año para mantener un efecto constante.

Characterization of Root-Nodule Bacteria Isolated from Hedysarum spinosissimum L, Growing in Mining Sites of Northeastern Region of Morocco

The aim of this study was to identify bacteria present in the nodules of the legume Hedysarum spinosissimum growing in metal-contaminated soils; and to test whether these root-nodule bacteria are able to promote host plant growth and enhance their phytostabilization potential. Seventy-four bacteria were isolated from nodules of H. spinosissimum growing in 3 different mining sites in Morocco and were identified by 16S rDNA gene sequencing. They belonged to 8 genera affiliated to Pseudomonas (49 strains), Pantoea (11), Rhizobium (6), Herbaspirillum (3), Bacillus (2), and one strain of Serratia, Agrobacterium and Azospirillum. Seven strains, presenting a high tolerance to Zn and Pb, exhibited capacity of inorganic phosphate solubilization and ammonia production from peptone degradation. The inoculation of H. spinosissimum, growing in 2000 μM of Zn by Pseudomonas putida and Herbaspirillum huttiense suppresses Zn toxicity symptoms in plants and also enhances plant growth by significantly increasing plant shoot and root fresh weights. The maximal Zn accumulation was observed in roots of plants inoculated by Pseudomonas putida with a translocation factor of 0.05(\textpm 0.006). Our results, evidence that the selection of metal-resistant bacteria is a key step in polluted soils for the use of plants like Hedysarum spinosissimum for in situ phytoremediation.

Interspecific cooperation: enhanced growth, attachment and strain-specific distribution in biofilms through Azospirillum brasilense-Pseudomonas protegens co-cultivation.

Plant-growth-promoting bacteria belonging to Azospirillum and Pseudomonas genera are major inhabitants of the rhizosphere. Both are increasingly commercialized as crops inoculants. Interspecific interaction in the rhizosphere is critical for inoculants aptness. The objective of this work was to evaluate Azospirillum and Pseudomonas interaction in mixed biofilms by co-cultivation of the model strains Azospirillum brasilense Sp245 and Pseudomonas protegens CHA0. The results revealed enhanced growth of both strains when co-cultured in static conditions. Moreover, Sp245 biofilm formed in plastic surfaces was increased 2-fold in the presence of CHA0. Confocal microscopy revealed highly structured mixed biofilms showing Sp245 mainly on the bottom and CHA0 towards the biofilm surface. In addition, A. brasilense biofilm was thicker and denser when co-cultured with P. protegens. In a colony-colony interaction assay, Sp245 changed nearby CHA0 producing small colony phenotype, which accounts for a diffusible metabolite mediator; though CHA0 spent medium did not affect Sp245 colony phenotype. Altogether, these results point to a cooperative interaction between A. brasilense Sp245 and P. protegens CHA0 in which both strains increase their static growth and produce structured mixed biofilms with a strain-specific distribution.

Pengaruh Hormon Asam Indol Asetat yang Dihasilkan Azospirillum sp. terhadap Perkembangan Akar Padi

<p>Free-living bacteria of the<br />genus Azospirillum live in close association with rice roots.<br />This bacteria produced indole acetic acid (IAA), a plant<br />growth hormon, to the environment. IAA was isolated from<br />cultures of Azospirillum strains and investigated for its effect<br />on root development and plant height of rice variety IR64 in<br />vitro. Rice cultures of variety IR64 were grown in vitro and<br />inoculated with cultures of Azospirilllum. Production of IAA<br />by the bacterium during its growth period in rice culture medium<br />containing different levels of nitrogen was observed.<br />Results of the experiment showed that strains Azospirillum<br />Az15 and Az44 had a high ability to produce IAA, i.e., 57.93<br />μg/ml at 12 days after incubation (DAI) and 40.42 μg/ml at 7<br />DAI, respectively. The IAA production pattern of Azospirillum<br />Az15 and Az44 in the liquid medium were fluctuative<br />until the end of the incubation period, while that of the strain<br />Az7 was linier. Strain Az7 gave a better effect on the root<br />development and plant height than strains Az15 and Az44.<br />Treatment combination of strain Az7 and 100% nitrogen gave<br />highest root development. High level of nitrogen increased<br />IAA content in the uninoculated culture, while low IAA<br />content on the inoculated one. Inoculation the culture with<br />strain Az7 together with 50% nitrogen application resulted in<br />the IAA content, root dry weight, root length, fiber root number,<br />and plant height as high as those on cultures containing<br />100% nitrogen (1 mM NH4NO3) without inoculation. Inoculation<br />of rice culture with Azospirillum is expected to reduce<br />nitrogen application on rice IR64 by the IAA production as<br />indicated by significant changes in the root growth and<br />development. A higher concentrations of IAA tend to give<br />better effects on the root growth and development of rice<br />IR64.</p>

Construction of probe of the plant growth-promoting bacteria Bacillus subtilis useful for fluorescence in situ hybridization

Strains of Bacillus subtilis are plant growth-promoting bacteria (PGPB) of many crops and are used as inoculants. PGPB colonization is an important trait for success of a PGPB on plants. A specific probe, based on the 16s rRNA of Bacillus subtilis, was designed and evaluated to distinguishing, by fluorescence in situ hybridization (FISH), between this species and the closely related Bacillus amyloliquefaciens. The selected target for the probe was between nucleotides 465 and 483 of the gene, where three different nucleotides can be identified. The designed probe successfully hybridized with several strains of Bacillus subtilis, but failed to hybridize not only with B. amyloliquefaciens, but also with other strains such as Bacillus altitudinis, Bacillus cereus, Bacillus gibsonii, Bacillus megaterium, Bacillus pumilus; and with the external phylogenetic strains Azospirillum brasilense Cd, Micrococcus sp. and Paenibacillus sp. The results showed the specificity of this molecular probe for B. subtilis.

Isolation and identification by 16S rRNA sequence analysis of plant growth-promoting azospirilla from the rhizosphere of wheat.

The main objective of the present study was to isolate phytohormone-producing, phosphate-solubilizing strains of Azospirillum from wheat to be used as inoculants for plant growth promotion. Five Azospirillum strains were isolated from the rhizosphere of field-grown wheat (Triticum aestivum L.), and it was confirmed by BOX-polymerase chain reaction (PCR) that the isolates were different and not re-isolates of the same strain. Sequence analysis of the PCR-amplified 16S rRNA gene indicated that four isolates showed maximum similarity to Azospirillum brasilense and one isolate showed maximum similarity to Azospirillum zeae. This is the first report indicating the presence of an A. zeae like isolate in the wheat rhizosphere in Pakistan. The bacterial isolates were characterized for their plant growth-promoting traits, phosphate solubilization, and indole-3-acetic acid (IAA) production. None of the isolates showed phosphate solubilization activity in the commonly used Pikovskaya medium. However, all strains (except AzoK4) exhibited ability to solubilize tricalcium phosphate (TCP) in modified Pikovskaya medium in which sucrose was replaced by Na-malate, as well as in TCP-supplemented Luria-Bertani (LB) medium. Organic acids, such as acetic, citric, lactic, malic, and succinic acids, were detected in culture supernatants of the tested Azospirillum strains. All strains exhibited ability to produce IAA in the growth medium, except Azospirillum sp. AzoK1. Among the strains tested, the maximum IAA production (30.49 ± 1.04 mg L−1) and phosphate solubilization (105.50 ± 4.93 mg L−1) were shown by a pure culture of Azospirillum sp. AzoK2. In pot experiments, single-strain inocula of Azospirillum sp. AzoK1 and AzoK2 improved wheat plant growth.

English

Optimization of land use can be attained by incorporating technologies to crop production, such as the use of diazotrophic bacteria, fertilizers, and pesticides. Seed inoculation with Azospirillum is an alternative that favors the incorporation of green agriculture in regions of conventional farming, such as the Brazilian savannah (Cerrado). However, limited information is available about this bacterium’s contribution to agriculture when other technologies are also incorporated. This study evaluated the performance of maize hybrids inoculated, or not, with Azospirillum brasilense, with or without fungicide applications, and subjected to different nitrogen rates under Cerrado field conditions. Each factor analyzed contributes to the increased maize grain yield. The use of inoculants containing plant growth promoting bacteria is a good option to ensure high yield of maize. Still, nitrogen should not be replaced, neither totally nor partially, by seed inoculation with Azospirillum. Fungicide applications should be done, as required, during maize cycle. Moreover, specific maize breeding programs should consider the affinity between Azospirillum strains and maize hybrids, mainly for regions with nitrogen deficient soils, like Cerrado. Thus, by incorporating additional technologies, maize crop farmers can optimize land use and, consequently, reduce the expansion into new agricultural areas. Key words: Foliar protection, nitrogen use, plant growth promoting bacteria, sustainability, Zea mays L.

A need for disclosure of the identity of microorganisms, constituents, and application methods when reporting tests with microbe-based or pesticide-based products

Plant growth-promoting microorganisms, biological control agents, pesticides, and a variety of stimulants to enhance plant performance are commonly tested and commercially applied worldwide on increasing scales each year (Bashan et al. 2014; Calvo et al. 2014; Lugtenberg and Kamilova 2009). In recent years, inoculation of plants with microorganisms to enhance productivity has become widespread, with more sophisticated and complex formulations and application methods. These formulations use a variety of application techniques, such as using natural and synthetic polymers or add more than one microorganism to the inoculants. These microbial consortia in inoculants may have over ten different microorganisms. Inoculants may have synthetic vitamins, plant hormones, and humic and fulvic acids. Other inoculants add different plant or algae or seaweed extracts, protein hydrolytes and amino acids as stimulants, as well as bulking materials, such as clays, minerals (vermiculite and perlite), talcum, or undisclosed soil fractions. Numerous adhesives, surfactants, stabilizing and dispersing materials, and preservatives to enhance survival and dispersal of the microorganisms are commonly added. The identity of these components in microbial formulations is often not disclosed in the product label. Specific details of application are usually explained by the manufacturer on the container label, but rarely in a formal publication. As product formulations change, performance of bacterial strains in the product may change. Hence, it might not be possible to objectively compare the effects of an inoculated microorganism in laboratory experiments with the effects of the same microbe in a commercial formulation. This results from some components in the formulations having synergistic bioactivities that are greater than a single strain of microorganism. As an example, the plant growth-promoting bacterium Azospirillum brasilense strain AZ39 is one of the most common Azospirillum strains used in Latin America. Yet, its performance significantly varies, depending on the specific formulation and the method of inoculation. Similar phenomenon occurs with rhizobia. Pesticides are commonly used in agriculture and are classified, according to the pest organism, in insecticides, fungicides, herbicides (weeds killers), nematocides, and rodenticides. The first three pesticides are those most used. Pesticides differ greatly for their chemical structures, and this result in a great variety of toxicological properties, most of them contain aromatic rings and some are chlorinated, just to mention a few chemical characteristics. Field rates depend on several factors including soil type (Bremner and Mulvaney 1976; Cervelli et al. 1976). Usually, pesticides are applied in formulations containing several other constituents often not disclosed in the product label, as mentioned above for the microbial inocula. These additives may include solvents, by-products of the target compounds, salts, chemical vehicles of the target compound, etc. Obviously toxicological studies of the target pesticide should also consider the effect of all other constituents; these effects may be synergic or antagonist with those by the target pesticide (Cervelli et al. 1976). * Yoav Bashan ybb0001@auburn.edu

Development of sequence-characterized amplified region (SCAR) markers as a quality standard of inoculants based on Azospirillum.

An attempt was made in this work to develop a strain-level molecular marker for unambiguous authentication of two Azospirillum inoculants, viz. A. lipoferum (strain Az204) and A. brasilense (strain Sp7). The sequence-characterized amplified region (SCAR) markers obtained from DNA fingerprints were designed for discrete detection of the strains. The SCAR primers could successfully amplify the target strain without cross-reaction with other Azospirillum strains, native isolates and other inoculants. The detection limit of SCAR primer for Az204 was 8.00 pg of DNA (approximately 10(5) cells per mL), and for Sp7, it was 0.49 pg of DNA (equal to 10(4) cells per mL). A simplified Sephadex G100-based crude DNA extraction protocol developed in this study was found suitable for SCAR marker-based strain authentication. Further, SCAR primers were assessed for simultaneous authentication as well as quantification of commercially prepared Azospirillum inoculants by quantitative real-time PCR (RT-PCR) and most-probable-number PCR (MPN-PCR). The RT-PCR assay can be able to quantify the commercial formulations as equal to culturable MPN method, while MPN-PCR failed for Az204. The SCAR marker-based strain authentication and presumptive quantification developed in the present work can contribute to improving the quality standard of commercial inoculants.