Colonization of peanut roots by biofilm-forming Paenibacillus polymyxa initiates biocontrol against crown rot disease

Aim: To investigate the role of biofilm‐forming Paenibacillus polymyxa strains in controlling crown root rot disease. Methods and Results: Two plant growth‐promoting P. polymyxa strains were isolated from the peanut rhizosphere, from Aspergillus niger‐suppressive soils. The strains were tested, under greenhouse and field conditions for inhibition of the crown root rot pathogen of the peanut, as well as for biofilm formation in the peanut rhizosphere. The strains’ colonization and biofilm formation were further studied on roots of the model plant Arabidopsis thaliana and with solid surface assays. Their crown root rot inhibition performance was studied in field and pot experiments. The strains’ ability to form biofilms in gnotobiotic and soil systems was studied employing scanning electron microscope. Conclusion: Both strains were able to suppress the pathogen but the superior biofilm former offers significantly better protection against crown rot. Significance and Impact of the Study: The study highlights the importance of efficient rhizosphere colonization and biofilm formation in biocontrol.     

A MORPHOLOGICAL AND TAXONOMIC CONSIDERATION OF TABELLARIA (BACILLARIOPHYCEAE) FROM THE NORTHCENTRAL UNITED STATES1,2

The taxonomy, distribution, and ecology of the diatom genus Tabellaria Ehr. from a portion of the northcentral United States were studied from collections made from August 1968 to October 1971. Using the transmission electron microscope with carbon replicas of the frustule surfaces, taxonomically significant characters were identified and related to those visible with the light microscope, four morphologically distinguishable taxa were recognized. The frustular morphology of T. fenestrata (Lyngbye) kütz. em. Knudson var. fenestrata was confirmed by both light and electron microscopy. T. flocculosa (Roth) Kütz. var. flocculosa is composed of 3 morphologically distinguishable strains. One of which is sufficiently different from the nominate variety to be described as T. flocculosa var. linearis var. nov. Also, a planktonic ecotype of one of the strains was identified.     

BACTERIAL ENDOSYMBIONTS IN VOLVOX CARTERI (CHLOROP HYCEAE)1

The morphology of the bacterial endosymbiont of Volvox carteri Stein (Clone KA-1) was studied with the electron microscope. Endosymbionts were found in the cytoplasm of somatic cells, gonidia and sperm, but never in nuclei, chloroplasts or mitochondria. DNA preparations contained, an extra DNA species assumed to be endosymbiont DNA. Attempts to isolate the endosymbionts or to “cure” the alga with antibiotics were unsuccessful. All progeny from crosses of infected and noninfected strains contained the endosymbiont.     

Cell morphology and growth characteristics ofPorphyridium aerugineum (Rhodophyta)

Four unialgal strains of the freshwater coccoid red algaPorphyridium aerugineum Geitler were cultivated under laboratory conditions. Cell morphology was studied with the light microscope. The cell surface was examined by means of electron microscopy in order to contribute to the knowledge of polysaccharide sheaths and cytoplasmic membranes. Optimum growth conditions were determined. The range of cell sizes and the average dry masses of single cells were compared in all four strains cultivated at exactly defined temperatures and irradiances. Photosynthetic pigment maxima were measured in intact cells. The red-coloured phycobiliprotein phycoerythrin was not found in any of the examined strains.Dedicated to Prof. DrLothar Geitler on the occasion of the 90th anniversary of his birthday.     

Fine Structure of the Schizont Stage of the Testate Marine Ameba,Trichosphaerium sp.*

SYNOPSIS. The schizont stage of 3 strains of the testate marine ameba, Trichosphaerium sp., was examined in the transmission electron microscope. The cytoplasm of this multinucleate organism contained the usual organelles; the test was covered by spicules. Pseudopodial types included broad ectoplasmic lobopods that assisted in locomotion, and thin dactylopods that probably had a sensory function. Nuclear division, observed in one of the strains, was characterized by an intact nuclear envelope (at least through anaphase), and the absence of centrioles. Nuclei in an ameba divided synchronously. An unusual intranuclear body of unknown function was found in another of the strains examined.     

我国沿海拟菱形藻属的2新记录种及其产毒特征分析

为澄清我国沿海拟菱形藻属(Pseudo-nitzschia)的物种多样性,并确认中国海域拟菱形藻属是否具有产生多莫酸(Domoicacid)的能力,采用毛细管显微操作技术从我国沿海水体中分离、纯化拟菱形藻细胞,建立了单克隆培养株系,并基于核糖体转录间隔区ITS1-5.8S-ITS2 序列构建了分子系统树。结果表明,结合在光学显微镜和透射电镜下观察的形态学特征和分子系统发育分析数据,鉴定到我国拟菱形藻属的2新记录种:银河拟菱形藻(P. galaxiae Lundholm & Moestrup)和微孔拟菱形藻(P. micropora Priisholm, Moestrup & Lundholm),对其形态学特征进行了详细描述,并与相似种类进行了比较研究。利用高效液相色谱(HPLC)技术对多莫酸特征进行了检测,结果表明培养株系并不产生多莫酸。这些为我国拟菱形藻属物种多样性和产毒特征研究提供了基础数据。     

Effect of the composition of the growth medium on morphology and reproduction ofAzotobacter chroococcum

Summary The growth of threeAzotobacter chroococcum strains was investigated in a number of media. The cultures were incubated at 29°C. and studied by means of a Wild phase contrast microscope. The experiments have clearly demonstrated that the morphology and reproduction ofA. chroococcum are influenced greatly on the one hand by conditions of nutrition and on the other hand also by the properties of the strains. Although all strains used in this work were able to produce gonidia and regenerative rods, these were not found in all media. The formation of filamentous cells, and vegetative cells inside such cells, depended also on the medium and, in some cases, on the strain. Cysts could sometimes resist a temperature of 50°C. for 5.5 minutes, but not a single strain with cysts survived temperatures of 60°C. or higher. Spores could not be detected by the usual staining methods.     

Confocal scanning light microscopy of the Escherichia coli nucleoid: comparison with phase-contrast and electron microscope images.

The nucleoid of living and OsO4- or glutaraldehyde-fixed cells of Escherichia coli strains was studied with a phase-contrast microscope, a confocal scanning light microscope, and an electron microscope. The trustworthiness of the images obtained with the confocal scanning light microscope was investigated by comparison with phase-contrast micrographs and reconstructions based on serially sectioned material of DNA-containing and DNA-less cells. This comparison showed higher resolution of the confocal scanning light microscope as compared with the phase-contrast microscope, and agreement with results obtained with the electron microscope. The effects of fixation on the structure of the nucleoid were studied in E. coli B/r H266. Confocal scanning light micrographs and electron microscopic reconstructions showed that the shape of the nucleoid remained similar after OsO4 or glutaraldehyde fixation; however, the OsO4 nucleoid appeared to be somewhat smaller and more centralized within the cell.     

A cixiid survey for natural potential vectors of ‘Candidatus Phytoplasma phoenicium’ in Lebanon and preliminary transmission trials

Almond witches'‐broom (AlmWB) disease, associated with ‘Candidatus Phytoplasma phoenicium’, is an emerging threat with real risk of introduction in Euro‐Mediterranean Countries. Its rapid spread over large geographical areas suggests the presence of efficient insect vector(s). In the present work, a survey on cixiids was carried out in Lebanon in the years 2010–2013 in AlmWB‐infested almond and nectarine orchards. Insects were collected by means of different methods, identified with a stereo microscope, and analysed for phytoplasma identification through 16S rDNA PCR‐based amplification and nucleotide sequence analyses. Preliminary transmission trials were performed with the most abundant species. A list of the cixiid genera and species present in the studied area is given as well as some information about their biology. ‘Ca. Phytoplasma phoenicium’ strains were detected in the genera Cixius, Tachycixius, Eumecurus and Hyalesthes. Preliminary trials revealed that Tachycixius specimens were able to transmit the detected strains to healthy peach potted seedlings. Further studies are required to better clarify the taxonomic status and the bio‐ethology of collected planthoppers and deeply study their role as phytoplasma vectors.     

Molecular typing and distribution of filamentous heterocystous cyanobacteria isolated from two distinctly located regions in North-Eastern India

The diversity of cyanobacteria in the North-Eastern region of India has not been studied except for a few sporadic and inconclusive reports. Loktak Lake is a huge reservoir for various kinds of organisms, including cyanobacteria. The present study describes the isolation and molecular diversity of 72 filamentous, heterocystous cyanobacterial strains isolated from samples collected from Loktak Lake, its adjoining rice fields and rice fields in Indian Council of Agricultural Research (ICAR) complex, Shillong, Meghalaya, India. The isolated strains belonged to the genera Anabaena, Nostoc, Calothrix, Cylindrospermum and Mastigocladus. The molecular analysis of isolates revealed the occurrence of certain strains being present in the sample collected from the rice fields falling in the catchment area of Loktak Lake, Manipur and rice fields in ICAR complex, Shillong, Meghalaya both. A polyphasic approach based on morphological features and PCR based molecular polymorphism revealed enormous level of molecular diversity. Out of three primers targeted regions used for determining genetic polymorphism, STRR1A produced best fingerprint profile of cyanobacterial strains. The morphological diversity of isolates was assured by light microscope whereas PCR based multiple fingerprint profile was used for molecular characterization. Molecular typing using short tandemly repeated repetitive STRR1A sequences as primer provided strain specific fingerprint profiles of the isolates.     

Morphology,phylogeny, growth rate and nodularin production of Nodularia spumigena from Brazil

Blooms of the toxic cyanobacterium Nodularia spumigena occur in various locations worldwide, but have not been observed in Brazil until recently. Three Nodularia strains were isolated from summer blooms in experimental shrimp production ponds of Penaeus vannamei in Rio Grande, in southern Brazil; these strains were characterized by morphology, phylogeny, growth rate and toxicity. The strains were identified as N. spumigena based on the size of vegetative cells, heterocytes and akinetes under a light microscope and based on the number of gas vesicles per μm² under a transmission electron microscope. The 16S rRNA gene sequences of the three strains showed high identity (> 99%) with N. spumigena sequences available on the NCBI database but were grouped closer in the phylogenetic tree with N. spumigena strains from Australia and USA than those from the Baltic Sea. The growth rate in batch culture varied between 0.2 and 0.6 μ?d^?1 based on cell density, optical density and chlorophyll-a content. The three strains produced the hepatotoxin nodularin (ELISA plate kit) with similar toxicity values (4.8–4.9?µg?l^?1). We conclude that the three isolated strains are N. spumigena with similar rates of growth and nodularin production. The presence of N. spumigena now represents a potential problem in aquaculture and estuarine environments in Brazil.     

Flow cytometric discrimination between Acinetobacter calcoaceticus 69-V and Alcaligenes eutrophus JMP134 by fluorescently labelled rRNA-targeted oligonucleotide probes and DNA staining

In situ hybridization with fluorescently monolabelled rRNA-targeted oligonucleotide probes (17 to 18 nucleotides) was used to discriminate between Alcaligenes eutrophus JMP 134 and Acinetobacter calcoaceticus 69-V by flow cytometry. The strains were grown in batch experiments in a mixed population. The forward light scatter and fluorescence of each bacterial cell were measured with a single laser cytometer. The intensity of fluorescence after rRNA staining depended on the content of ribosomes, which correlated with the growth rate of bacteria. Therefore exponentially growing cells could be clearly detected. For other growth phases, signal amplification was necessary using multiple probes. The two bacterial strains were identified with differently labelled probes under an epifluorescent microscope. Using a single laser cytometer, rRNA based identification was possible nut not ideal. Better discrimination between the two strains of the mixed population was achieved by DNA staining, combined with the different forward light scatter signals. Due to the significantly different cellular DNA and GC content of both strains, the fluorescent dye DAPI (4′, 6-diamidino-2-phenylindole), preferring AT-rich regions of DNA, was found to be a supplementary tool for population analysis. The abundance ratios of the two strains in mixed culture determined by DNA or rRNA staining were similar.     

Differences in life history traits between alcohol dehydrogenase genotypes of Drosophila mercatorum: background and maternal genotype effects

The effects of genotype at the alcohol dehydrogenase (ADH) locus on developmental time and fecundity were studied using three strains of Drosophila mercatorum. All three strains had similar genetic backgrounds and two were homozygous for the same electrophoretic allele (F for fast). The third strain was homozygous for the slow allele (S). The life history traits of all possible progeny of these strains were studied. The results showed that for both mean developmental time and pattern of eclosion, strong maternal genotype and background effects were present. Fecundity among genotypes was not different, although there was an effect on the total number of offspring suggesting differences in egg-to-adult survivorship.     

Effect of Bacterial Chemotaxis on Biodegradation in a Porous Medium

This research investigates the effect of bacterial chemotaxis on biodegradation rate in an experimental model for in situ bioremediation. The novel experimental methodology of this work has provided for the systematic evaluation of the effect of the chemotaxis phenomenon in a saturated porous medium. The methodology has been developed to measure enhancement of degradation rate of serine, a simulated contaminant and chemoattractant. Escherichia coli RP437 was used as a representative, chemotactic, in situ bacterium, whereas E. coli RP5700, a tsr? mutant strain of RP437 that lacks the serine chemoreceptor, was used as the specifically nonchemotactic control strain. These two strains were highly characterized for this work. Swimming speeds, run lengths, and turn angles were compared using a tracking microscope and were statistically similar, as were serine uptake rates, making this pair of strains an excellent choice for chemotaxis studies. For these experiments, a model aquifer introduces bacteria to serine in saturated sand via a sharp gradient. The aquifer successfully achieved biodegradation at an 18% level; however, the degradation rate of serine was similar for both strains over 21 h, indicating that chemotaxis enhancement did not occur. This result is in agreement with certain prior works which did not detect enhanced chemotactic migration.     

A comparative study on the formation of citric acid and polyols and on morphological changes of three strains of free and immobilized Aspergillus niger

Summary The formation of citric acid, oxalic acid, erythritol and glycerol by three strains of Aspergillus niger immobilized in calcium alginate was investigated and compared with that of free cells when cultivated in shake flasks under phosphate limitation. Morphological changes were followed using an electron microscope. The production of acids and polyols, the consumption of glucose and fructose, and also the morphological changes were strain-dependent. The results also reflected the influence of long storage of a strain on productivity, morphological behaviour and phosphate consumption. Offprint requests to: H.-J. Rehm     

Molecular relationships among plasmids of Bacillus thuringiensis: conserved sequences through 11 crystalliferous strains

Summary Screening for the plasmid content of 11 strains belonging to nine different serotypes ofB. thuringiensis was carried out by electron microscope examination and electrophoresis in agarose gels. All the strains contained at least two covalently closed, circular (CCC) DNA species. In one strain (berliner 1715), 17 extrachromosomal elements could be distinguished with regard to their size, ranging from 3.9 to 180 Mdal. Southern hybridisation experiments showed that most of these plasmids fell into two categories (inferior to 15 Mdal and superior to 15 Mdal) which have no homology between them. Within these two size groups there is partial conservation of DNA sequences through various serotypes. Further relationships among the plasmids were investigated by a two dimensional version of the Southern's blotting technique.Possible homology between plasmids and the chromosomal DNA was studied. It was shown that the smaller plasmids from the berliner 1715 and kurstaki HD₁ strains contained no sequence related to chromosomal DNA, whereas among the larger plasmids a few showed homologous sequences.Abbreviations cry^- tacrystalliferous mutant - GCC covalently closed circular DNA - OC open circular DNA - Mdal megadalton - kb 1,000 base pairs     

Colonization behavior of bacterium Burkholderia cepacia inside the Oryza sativa roots visualized using green fluorescent protein reporter

Colonization behavior of endophytic bacteria Burkholderia cepacia strains RRE-3 and RRE-5 was studied in the seedlings of rice variety NDR97 using confocal laser scanning microscopy under controlled laboratory and greenhouse conditions. For studying colonization pattern, bacterial strains were tagged with pHRGFPGUS plasmid. The role of bacterial strains (both gfp/gus-tagged and untagged) in growth promotion was also studied. After coming into contact with the host root system the bacteria showed an irregular spreading. Dense colonization was observed on the primary and secondary roots and also on the junction of emergence of the lateral roots. Results showed that the colonization pattern of Burkholderia cepacia strains was similar to that of other endophytic bacteria isolated from non-legumes. Burkholderia cepacia got entry inside the root at the sites of emergence of lateral roots, without formation of infection threads as in the case of symbiotic rhizobacteria. Observations suggested that the endophytic bacterial strains RRE-3 and RRE-5 entered inside the rice roots in a progressive manner. Bacteria were found to line up along the intercellular spaces of adjoining epidermal cells adjacent to the lateral root junction, indicating endophytic colonization pattern of Burkholderia cepacia strains. Experiments with the rice seedlings inoculated with RRE-3 and RRE-5 strains revealed that both strains enhanced plant growth considerably when observed under laboratory and greenhouse conditions and produced significantly higher plant biomass. No considerable difference was observed between the gfp/gus-tagged and non-gfp/gus-tagged strains in the plant growth experiments both in the laboratory and greenhouse conditions.     

Changes in lignocellulolytic enzyme activites in six <Emphasis Type="Italic">Pleurotus</Emphasis> spp. strains cultivated on coffee pulp in confrontation with <Emphasis Type="Italic">Trichoderma</Emphasis> spp.

Summary The antagonistic effect of six Pleurotus spp. strains was studied in confrontation with three strains of Trichoderma spp. Pleurotus strains were cultivated on sterile coffee pulp, with and without a Trichoderma inoculant. Laccase, Mn peroxidase and endoglucanase activities were determined during incubation. Laccase production was also studied by PAGE analysis to detect enzymatic isoforms. Results show that the presence of Trichoderma induced a significant increase in oxidase production by the Pleurotus strains. Nevertheless, Trichoderma was not observed to induce laccase isoforms.