Antifungal activity of selected indigenous pseudomonas and bacillus from the soybean rhizosphere.

The purpose of this study was to isolate and select indigenous soil Pseudomonas and Bacillus bacteria capable of developing multiple mechanisms of action related to the biocontrol of phytopathogenic fungi affecting soybean crops. The screening procedure consisted of antagonism tests against a panel of phytopathogenic fungi, taxonomic identification, detection by PCR of several genes related to antifungal activity, in vitro detection of the antifungal products, and root colonization assays. Two isolates, identified and designated as Pseudomonas fluorescens BNM296 and Bacillus amyloliquefaciens BNM340, were selected for further studies. These isolates protected plants against the damping-off caused by Pythium ultimum and were able to increase the seedling emergence rate after inoculation of soybean seeds with each bacterium. Also, the shoot nitrogen content was higher in plants when seeds were inoculated with BNM296. The polyphasic approach of this work allowed us to select two indigenous bacterial strains that promoted the early development of soybean plants.

Assessment of the role of chemotaxis and biofilm formation as requirements for colonization of roots and seeds of soybean plants by Bacillus amyloliquefaciens BNM339.

This article correlates colonization with parameters, such as chemotaxis, biofilm formation, and bacterial growth, that are believed to be connected. We show here, by using two varieties of soybean plants that seeds axenically produced exudates, induced a chemotactic response in Bacillus amyloliquefaciens, whereas root exudates did not, even when the exudates, also collected under axenic conditions, were concentrated up to 200-fold. Root exudates did not support bacterial cell division, whereas seed exudates contain compounds that support active cell division and high cell biomass at stationary phase. Seed exudates of the two soybean varieties also induced biofilm formation. B. amyloliquefaciens colonized both seeds and roots, and plant variety significantly affected bacterial root colonization, whereas it did not affect seed colonization. Colonization of roots in B. amyloliquefaciens occurred despite the lack of chemotaxis and growth stimulation by root exudates. The data presented in this article suggest that soybean seed colonization, but not root colonization, by B. amyloliquefaciens is influenced by chemotaxis, growth, and biofilm formation and that this may be caused by qualitative changes of the composition of root exudates.

Oxygen and light effects on the expression of the photosynthetic apparatus in Bradyrhizobium sp. C7T1 strain.

Photosynthetic bradyrhizobia are nitrogen-fixing symbionts colonizing the stem and roots of some leguminous plants like Aeschynomene. The effect of oxygen and light on the formation of the photosynthetic apparatus of Bradyrhizobium sp. C7T1 strain is described here. Oxygen is required for growth, but at high concentration inhibits the synthesis of bacteriochlorophyll (BChl) and of the photosynthetic apparatus. However, we show that in vitro, aerobic photosynthetic electron transport occurred leading to ADP photophosphorylation. The expression of the photosynthetic apparatus was regulated by oxygen in a manner which did not agree with earlier results in other photosynthetic bradyrhizobia since BChl accumulation was the highest under microaerobic conditions. This strain produces photosynthetic pigments when grown under cyclic illumination or darkness. However, under continuous white light illumination, a Northern blot analysis of the puf operon showed that, the expression of the photosynthetic genes of the antenna was considerable. Under latter conditions BChl accumulation in the cells was dependent on the oxygen concentration. It was not detectable at high oxygen tensions but became accumulated under low oxygen (microaerobiosis). It is known that in photosynthetic bradyrhizobia bacteriophytochrome photoreceptor (BphP) partially controls the synthesis of the photosystem in response to light. In C7T1 strain far-red light illumination did not stimulate the synthesis of the photosynthetic apparatus suggesting the presence of a non-functional BphP-mediated light regulatory mechanism.

Genetic and functional characterization of a Bacillus sp. strain excreting surfactin and antifungal metabolites partially identified as iturin-like compounds.

AIMS: A bacterial strain producing antifungal compounds active against the plant pathogenic fungi Fusarium, Rhizoctonia and Sclerotinia has been characterized and shown to control Rhizoctonia root rot of soya bean. METHODS AND RESULTS: The metabolites excreted by Bacillus BNM 122 remained active after autoclaving, were resistant over a wide pH range and to hydrolytic enzymes. By (1)H-NMR and thin-layer chromatography analyses surfactin and iturin-like compounds were partially identified. Moreover, soya bean seeds bacterization with BNM 122 in a compost-based formulation was as effective controlling Rhizoctonia solani as pentachloronitrobenzene. According to its 16S rDNA sequence BNM 122 was closely related to Bacillus amyloliquefaciens and Bacillus subtilis. PCR analysis of the 16S-23S rRNA intergenic spacer region and repetitive sequence-based PCR (rep-PCR) genomic fingerprinting revealed a close genetic relationship to B. amyloliquefaciens. However, by physiological characterization using API tests, this strain resembled more B. subtilis. CONCLUSIONS: This is the first report describing the co-production of surfactin and iturin-like compounds by a putative strain of B. amyloliquefaciens. The synergistic effect of both lipopetides is a remarkable trait for a candidate biocontrol agent. SIGNIFICANCE AND IMPACT OF THE STUDY: This kind of research has relevance in order to minimize the use of synthetic fungicides and surfactants, contributing to the preservation of the environment.

Phosphorylation of LHI beta during membrane synthesis in the photosynthetic bacterium Rhodovulum sulfidophilum.

Cells of Rhv. sulfidophilum were grown under different conditions in the presence of 32P-phosphate and the corresponding H and L membrane fractions obtained and fractionated by SDS-PAGE. Both membranes showed almost identical polypeptide composition. The bacteriochlorophyll (Bchl) specific content in H was always lower that in L. As described before, oxygen did not regulate gene expression. Under high light, an almost two- to threefold decrease of the cellular specific Bchl content was observed. Pulse and chase experiments showed that transitions from aerobiosis to light-anaerobiosis did not quantitatively affect the Bchl content of the membranes, although a turnover of the 32P-phosphate and 35S-methionine was observed. LHI beta was the only polypeptidic subunit of the Bchl-binding polypeptides that was phosphorylated in vivo, and phosphotyrosine was the only phosphorylated amino acid detectable. The phosphorylated LHI beta was determined to be insoluble in the organic solvent mixture of (vol/vol) 1:1 chloroform-methanol containing ammonium acetate (0.1 m final concentration). Treatment with a chaotropic agent such as Na2CO3 solubilized the phosphorylated LHI beta, indicating that part of this posttranslationally modified polypeptide was not inserted in a transmembrane position. These results were used to speculate about the regulatory properties of this posttranslational modification of LHI beta on membrane differentiation.

The LHIalpha and LHIIalpha complexes in association with phospholipids are able to be inserted in heavy membranes of Rhodobacter capsulatus B10.

We show in this paper that a complex constituted by phospholipids and LHI and LHII alpha polypeptides was inserted in a heavy membrane fraction in a nonextractable form, indicating a transmembrane localization. The best accepting membranes originated from aerobically grown cells. Addition of ATP during the insertion inhibited this reaction 25 to 30% in heavy membranes isolated from aerobically grown cells (HMaer) and a higher inhibition (60 to 65%) was detected when using heavy membranes isolated from photosynthetically grown cells (HMpho). Purification by gel filtration of a crude Na2CO3 extract yielded three phosphate-labeled fractions. Two of them contained protein and phospholipids in a stable association. However, only fractions containing phosphatidylethanolamine were shown to be reconstituted. The third radioactive fraction contained labeled ATP and protein, but no phospholipids and could not be reassociated to the heavy membranes of any origin. A model for the insertion of the LH polypeptides is presented in which the recently synthesized polypeptides are phosphorylated and become associated to anionic phospholipids. The interaction of this complex to the membrane spontaneously leads to stable insertion.

Association of LHIalpha(B870) polypeptide with phospholipids during insertion in the photosynthetic membrane of an LHII- mutant of Rhodobacter capsulatus.

Membranes from in vivo labeled cells of Rhodobacter capsulatus U43[pTX35] grown photosynthetically carried 60% of the [32P]-Pi in the "heavy" fraction (HM) after sucrose gradient sedimentation. Metal-chelating chromatography of either"heavy" or "light" (LM) membrane fractions rendered similar Bchl-protein complex profiles after octyl-glucoside treatment,including most of the radioactivity in the same corresponding elution fraction (F II). Similar labeling distribution of pigment-protein complexes was obtained for membranes of dark-grown cells induced by lowering oxygen tension. Fractions derived from HM showed highly labeled LHIalpha, whereas the same complex from LM was essentially [32P]-Pi-free, as revealed by SDS-PAGE followed by autoradiography. Phospholipid analysis showed a similar pattern for membranes isolated from cells photosynthetically or semiaerobically grown, being the most abundant: phosphatidylglycerol,phosphatidylethanolamine, cardiolipin, and phosphatidylcholine. Part of the phospholipids from HM comigrated with LHIalpha during SDS-PAGE and dissociated from the complexes only after solvent extraction and hydrophobic chromatography. However, a small amount remained always attached to LHIalpha,indicating an unusual strong interaction. These results suggest the existence of two operationally defined membrane regions carrying LHIalpha complexes differing in phosphorylation status and protein-phospholipid interaction.

Short communication: Influence of inoculum preparation on citric acid production by Aspergillus niger.

The type of sporulation medium and time of incubation had an effect on spore viability and citric acid production by mycelia grown from Aspergillus niger spores. Shu & Johnson agar (SJA) and potato dextrose agar gave higher citric acid titres than malt-extract agar. SJA also gave better germinability than the other media. Viability increased with time of incubation, but higher production of citric acid was achieved with spores incubated for less than 7 days.

Nitrate reductase from Rhodopseudomonas sphaeroides.

The facultative phototroph Rhodopseudomonas sphaeroides DSM158 was incapable of either assimilating or dissimilating nitrate, although the organism could reduce it enzymatically to nitrite either anaerobically in the light or aerobically in the dark. Reduction of nitrate was mediated by a nitrate reductase bound to chromatophores that could be easily solubilized and functioned with chemically reduced viologens or photochemically reduced flavins as electron donors. The enzyme was solubilized, and some of its kinetic and molecular parameters were determined. It seemed to be nonadaptive, ammonia did not repress its synthesis, and its activity underwent a rapid decline when the cells entered the stationary growth phase. Studies with inhibitors and with metal antagonists indicated that molybdenum and possibly iron participate in the enzymatic reduction of nitrate. The conjectural significance of this nitrate reductase in phototrophic bacteria is discussed.

Isolation and purification of reaction center from Rhodopseudomonas viridis NHTC 133 by means of LDAO.

Two different procedures are described to isolate and purify the reaction center complex from Rhodopseudomonas viridis NHTC 133 by means of the non-ionic detergent dodecyldimethylamine oxide. Both reaction center particles thus obtained were active, as shown by a photobleaching centered at 975 nm. The reaction center also contained, in addition to bacteriochlorophyll, bacteriopheophytin. Other components were also found in this particle: cytochromes C553 and C558 and a menaquinone-like substance. The SDS gel electrophoresis of reaction centers is shown. The molecular weights of the subunits forming the reaction center in 0.5% sodium dodecyl sulfate and 1% mercaptoethanol were calculated as being: 45 +/- 1.5 and 37 +/- 1.5 kdalton, 29 +/- 1.5 and 23 +/- 1.5 kdalton. The molecular weight of the complex determined by means of gel filtration (Sepharose 6-B and Bio-Gel P-300) gives a value of approximately 240 kdalton. The minimum molecular weight of the complex calculated by disc gel electrophoresis was 231 kdalton.

Possible mechanism of photophosphorylation in Rhodopseudomonas viridis.

Photophosphorylation in the presence of (32P) inorganic phosphate (Pi) and ADP or Pi and (beta 32P) -ADP was carried out in membranes of Rhodopseudomonas viridis. When Pi and (32P)-ADP were used, 32Pi and (32P)-ATP were detected by ion exchange chromatography and the specific activity of the (beta 32P)-ADP recovered was substantially diminished. Moreover, when the (32P)-ATP was analyzed, the radioactivity was equally distributed between its beta and gamma position. When 32Pi and ADP were used instead, most of the radioactivity was found as (32P)-ATP although consistently (beta-32P)-ADP was detected. When the (32P)-ATP was analyzed, 90% of the label was found in its gamma phosphate and the remaining in its beta position. These results cannot be explained as a direct addition of phosphate to ADP as being the sole mechanism of photophosphorylation, and we propose that this pattern of labeling may be explained by a transphosphorylation reaction between one bound and one free ADP.

Possible mechanism of photophosphorylation in Rhodopseudomonas viridis

Photophosphorylation in the presence of (32P) inorganic phosphate (Pi) and ADP or Pi and (beta 32P) -ADP was carried out in membranes of Rhodopseudomonas viridis. When Pi and (32P)-ADP were used, 32Pi and (32P)-ATP were detected by ion exchange chromatography and the specific activity of the (beta 32P)-ADP recovered was substantially diminished. Moreover, when the (32P)-ATP was analyzed, the radioactivity was equally distributed between its beta and gamma position. When 32Pi and ADP were used instead, most of the radioactivity was found as (32P)-ATP although consistently (beta-32P)-ADP was detected. When the (32P)-ATP was analyzed, 90

of the label was found in its gamma phosphate and the remaining in its beta position. These results cannot be explained as a direct addition of phosphate to ADP as being the sole mechanism of photophosphorylation, and we propose that this pattern of labeling may be explained by a transphosphorylation reaction between one bound and one free ADP.

Possible mechanism of photophosphorylation in Rhodopseudomonas viridis.

Photophosphorylation in the presence of (32P) inorganic phosphate (Pi) and ADP or Pi and (beta 32P) -ADP was carried out in membranes of Rhodopseudomonas viridis. When Pi and (32P)-ADP were used, 32Pi and (32P)-ATP were detected by ion exchange chromatography and the specific activity of the (beta 32P)-ADP recovered was substantially diminished. Moreover, when the (32P)-ATP was analyzed, the radioactivity was equally distributed between its beta and gamma position. When 32Pi and ADP were used instead, most of the radioactivity was found as (32P)-ATP although consistently (beta-32P)-ADP was detected. When the (32P)-ATP was analyzed, 90

of the label was found in its gamma phosphate and the remaining in its beta position. These results cannot be explained as a direct addition of phosphate to ADP as being the sole mechanism of photophosphorylation, and we propose that this pattern of labeling may be explained by a transphosphorylation reaction between one bound and one free ADP.