Plant-bacteria interactions with special emphasis on the kallar grass association

Kallar grass is a highly salt-tolerant grass grown as a pioneer plant on alkaline, salt-affected soils in Pakistan. Nitrogen-fixing bacteria and kallar grass were found to be in close association, which was even root-zone specific: rhizoplane and endorhizosphere were colonized by two different populations. Among theAzospirillum isolates originating from the root surface, some were of a new species, now namedA. halopraeferens. To study plant-bacterium interactions, this natural kallar grass association was chosen. The possible role of bacterial chemotaxis and oxygen tolerance are discussed.     

Occurrence of effective nitrogen-scavenging bacteria in the rhizosphere of kallar grass

Bacteria occurring in high numbers on the rhizoplane of kallar grass grown at a natural site in Pakistan were effective scavengers of traces of combined nitrogen from the atmosphere. Bacteria grew under appropriate conditions in nitrogen-free semi-solid malate medium in the form of a typical subsurface pellicle which resulted in a significant nitrogen gain in the medium within 3 to 4 days of incubation; this could be also measured by¹⁵N-dilution. Bacteria grew and incorporated nitrogen under an atmosphere containing NH₃ and N₂O. A rapid and strong binding of strain W1 to roots of kallar grass grown in hydroponic culture was found by using a³²P-tracer technique. We obtained no evidence for diazotrophy of our strains because they failed to grow on nitrogen-free media when gases of high purity were used. No¹⁵N₂ was incorporated when bacteria were grown on¹⁵N₂ although a nitrogen gain was found, no acetylene reduction was observed and no homology with DNA containing sequences ofnifHDK structural genes for the nitrogenase components fromKlebsiella pneumoniae were detected. Owing to close contact of these bacteria with roots of kallar grass, utilization of scavenged nitrogen by the plant may have to be taken into account.     

Location of diazotrophs in the root interior with special attention to the kallar grass association

There is increasing evidence that nitrogen-fixing bacteria are able to colonize the interior of grass roots. Several techniques have been used to demonstrate the sites of colonization and are discussed. Among the techniques useful for specific labelling, gold-labelled reagents have been frequently successfully applied in other fields. We propose the use of the protein A-gold technique coupled with silver amplification and light microscopy to render diazotrophs visible in semi-thin sections of roots, and we have applied this technique to gnotobiotically grown kallar grass. LR white resin soft grade was a suitable resin for embedding. Diazotrophic rods predominating in the endorhizosphere of naturally growing kallar grass had the potential to colonize the aerenchyma of gnotobiotically-grown plants. Penetration by the bacteria probably occurred at epidermal cell junctions and at points of emergence of lateral roots, as also proposed forAzospirillum. Larger cell aggregates described by us for naturally occurring kallar grass plants were not detected in the gnotobiotic system. Physiological consequences of colonization of the aerenchyma are discussed.     

Survival and colonization of inoculated bacteria in kallar grass rhizosphere and quantification of N2-fixation

Two experiments have been conducted, one in semi-solid Hoagland nutrient medium and the other in shallow pots containing saline soil. N₂-fixing bacteria belonging toAzospirillum, Azotobacter, Klebsiella andEnterobacter were inoculated separately on kallar grass grown in semi-solid nutrient medium. It was shown that inoculation affects root proliferation and also results in¹⁵N isotopic dilution. The % Ndfa ranged from 47–70 whereas no significant effect on the total nitrogen uptake was observed. The bacterial colonization of the root surface and the presence of enteric bacteria inside the root hair cells is reported. In a soil pot experiment, non-N₂-fixingPolypogon monspeliensis was used as a reference plant (control). A treatment receiving a high rate of nitrogen was also used as a non-N₂-fixing control.¹⁵N-labelled ammonium sulphate at 20 kg N ha^–1 and 90 kg N ha^–1 was used. The % Ndfa in the aerial parts of kallar grass was 12–15 whenP. monspeliensis was used as reference plant whereas 37–39% Ndfa was estimated when the treatment receiving high nitrogen fertilizer was used as a non-N₂-fixing control. These investigations revealed some problems of methodology which are discussed.     

Isolation, molecular characterization and growth-promoting activities of endophytic sugarcane diazotroph Klebsiella sp. GR9

A group of endophytic diazotrophs were isolated from surface-sterilized roots and stems of different sugarcane varieties in the Tamilnadu region of India. From these, four isolates were selected, based on the highest acetylene reduction activity. Gene-specific PCR amplification confirmed the presence of nif-D genes in those isolates. The 16S rRNA sequence of isolates GR4 and GR7 had a 99.5% sequence similarity to the Pseudomonas sp. pDL01 (AF125317) and 16S rDNA sequence of isolate GR3 had a 100% similarity to that of Burkholderia vietnamiensis (AY973820). The 16S rDNA sequence of isolate GR9 was 99.79% similar to that of the Klebsiella pneumoniae type strain (KPY17657). Colonization by the isolates was confirmed using micropropagated sugarcane and sterile rice seedlings. Isolate GR9, identified as Klebsiella pneumoniae, was consistently more active in reducing acetylene as compared with the other isolates. The effects of GR9 and the sugarcane diazotroph Gluconacetobacter diazotrophicus were compared in inoculated micropropagated sugarcane plantlets. The effects of K. pneumoniae GR9, and four other diazotrophs, G. diazotrophicus, Herbaspirillum seropedicae, Azospirillum lipoferum 4B, and Burkholderia vietnamiensis in inoculated rice seedlings were compared. GR9 alone or in combination with the other diazotrophs performed best under pot conditions. The combined effects of nitrogen fixation and endophytic colonization of this diazotroph may be useful for the development of bio-inoculants.     

Salinicoccus salitudinis sp. nov., a new moderately halophilic bacterium isolated from a saline soil sample

A novel pale-yellow-pigmented, moderately halophilic, facultatively alkaliphilic, non-motile, non-spore-forming, catalase- and oxidase-positive, obligately aerobic Gram-positive coccus, strain YIM-C678^T was isolated from a saline soil sample collected from a hypersaline habitat in the Qaidam basin, northwest China. The organism grew at 4–37°C and pH 6.0–11.0, with optimum growth at 25°C and pH 8.0. Strain YIM-C678^T grew optimally in the presence of 10–12% (w/v) NaCl and growth was observed in 1–25% (w/v) NaCl. The cell wall murein type was l-Lys-Gly₅. Major cellular fatty acids were anteiso-C_15:0, iso-C_15:0, iso-C_16:0 and C_16:0. Menaquinone 6 (MK-6) was the major respiratory quinone. The DNA G + C content was 46.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain YIM-C678^T belonged to the family Staphylococcaceae and was most closely related to the eight described species of the genus Salinicoccus with sequence similarities from 92.2 (S. luteus YIM 70202^T) to 97.5% (S. kunmingensis YIM Y15^T). The DNA–DNA relatedness between strain YIM-C678^T and S. kunmingensis YIM Y15^T was 35.4%. Chemotaxonomic data and 16S rRNA gene sequence analysis supported the affiliation of strain YIM-C678^T with the genus Salinicoccus. The combination of phylogenetic analysis, phenotypic characteristics, chemotaxonomic differences and DNA–DNA hybridization data supported the view that the bacterium represents a novel species of the genus Salinicoccus, for which the name Salinicoccus salitudinis sp. nov. is proposed, with YIM-C678^T (=DSM 17846 = CGMCC 1.6299) as the type strain.     

Methylovorus mays sp. nov.: A new species of aerobic, obligately methylotrophic bacteria associated with plants

A bacterial strain utilizing methanol as the sole source of carbon and energy was isolated from the maize phyllosphere. Cells are nonpigmented gram-negative motile rods that do not form spores or prosthecae and reproduce by binary fission. The strain does not require vitamins or supplementary growth factors. It is obligately aerobic and urease-, oxidase-, and catalase-positive. The optimum growth temperature is 35–40°C; the optimum pH is 7.0–7.5. The doubling time is 2 h. The bacterium implements the ribulose monophosphate pathway and possesses NAD⁺-dependent 6-phosphogluconate dehydrogenase and enzymes of the glutamate cycle. α-Ketoglutarate dehydrogenase and enzymes of the glyoxylate cycle (isocitrate lyase and malate synthase) are absent. Fatty acids are dominated by palmitic (C_16:0) and palmitoleic (C_16:1) acids. The major phospholipids are phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine. Cardiolipin is present in minor amounts. The dominant ubiquinone is Q₈ The bacterial genome contains genes controlling the synthesis and secretion of cytokinins. The G+C content of DNA is 57.2 mol %, as determined from the DNA thermal denaturation temperature T_m. The bacterium shows low DNA homology (<10%) with restricted facultative methylotrophic bacteria of the genusMethylophilus (M. methylotrophus NCIMB 10515^T andM. leisingerii VKM B-2013¹) and with the obligate methylotrophic bacterium (Methylobacillus glycogenes ATCC 29475^T). DNA homology with the type representative of the genusMethylovorus, M. glucosetrophus VKM B-1745^T, is high (58%). The new isolate was classified as a new species,Methylovorus mays sp. nov.     

Klebsiella variicola, a novel species with clinical and plant-associated isolates

A new Klebsiella species, K. variicola, is proposed on the basis of total DNA-DNA hybridization, on the monophyly observed in the phylogenetic analysis derived from the sequences of rpoB, gyrA, mdh, infB, phoE and nifH genes and on distinct phenotypic traits. The bacteria from this new species seem to be genetically isolated from K. pneumoniae strains, do not ferment adonitol and were obtained from plants (such as banana, rice, sugar cane and maize) and hospitals. The type strain is F2R9T (= ATCC BAA-830T = CFNE 2004T).     

Cochliobolus heveicola sp. nov. (Bipolaris heveae) causes brown stripe of bermudagrass and Zoysia grass

The pathogen of brown stripe in leaves of Cynodon dactylon (bermudagrass) and Zoysia japonica (Zoysia grass) in Japan is identical with Bipolaris heveae, a rubber tree pathogen, based on morphological and phylogenetic characteristics, following pathogenicity studies. Crossing isolates used in the study with each other, the obtained teleomorph is described as Cochliobolus heveicola sp. nov.     

A novel low-temperature active alkaline pectate lyase from Klebsiella sp. Y1 with potential in textile industry

Alkaline pectate lyases are favorable for the textile industry. Here we report the cloning of a pectate lyase gene (pl A), from Klebsiella sp. Y1, and its heterologous expression in Escherichia coli. The full-length pl A consists of 1710 bp and encodes for a 569-amino acid polypeptide including a putative 22-residue signal peptide and a catalytic domain belonging to pectate lyase family 2. The recombinant enzyme (r-PL A) was purified to electrophoretic homogeneity by single-step Ni²⁺-NTA affinity chromatography and showed an apparent molecular weight of ∼60 kDa. The pH and temperature optima of r-PL A were found to be 9.0 and 30–50 °C, respectively. r-PL A was highly active at low temperatures, exhibiting >60% of the maximal activity at 20 °C and >20% activity even at 0 °C. The enzyme was stable in a broad alkaline pH range of 7.0–12.0 for 1 h at 37 °C. The values of K_m(app) and V_max(app) of r-PL A for polygalacturonic acid were 2.47 mg/ml and 11.94 μmol/min/mg, respectively. Compared with the commercial compound pectinase from Novozymes, purified r-PL A showed similar efficacy in reducing the intrinsic viscosity of polygalacturonic acid (68.8% vs. 67.1%) and in bioscouring of jute (7.38% vs. 7.58%). Thus r-PL A is a valuable material for the textile industry.     

Nitrogen fixation associated with roots of Kallar grass (Leptochloa fusca L. Kunth)

Summary An ecological survey was made to measure the N₂-fixing activity in the rhizosphere of a salt-tolerant grass,Leptochloa fusca (L.) Kunth. Samples were obtained every month at two sites over a period of one year. Soil cores, unwashed, washed and surface-sterilized roots were subjected to acetylene reduction assay (ARA). ARA values up to 50 nmoles h^–1 for soil cores, 1095 nmoles g dry root^–1 h^–1 for unwashed roots, 4929 nmoles g dry root^–1 h^–1 for washed roots and 2494 nmoles g dry root^–1 h^–1 for surface-sterilized roots were observed but for most samples the range was 1–200 nmoles g dry root^–1 h^–1. A lag period of 5–7 h was observed before the onset of N₂-fixing activity by excised roots and O₂ levels had no effect on this lag. Values for roots incubated without preincubation were similar to those for unwashed preincubated roots. Activity was highest in September, October and November when the temperature is not very high and photosynthetic activity is reasonably good. N₂-fixing-bacteria were counted on the same samples by plate count and MPN methods, the latter being estimated on the basis of ARA and pellicle formation. Fairly high numbers of bacteria (10⁴–10⁷) were recorded in the histoplane fraction which indicates the presence of diazotrophs in the inner cells of grass roots.     

Construction of a transposon containing a gene for polygalacturonate trans-eliminase from Klebsiella oxytoca

A DNA fragment containing a Klebsiella oxytoca gene for polygalacturonate trans-eliminase was cloned into the kanamycin resistance transposon Tn5. This new transposon, designated Tn5-Pga ⁺, had a transposition frequency of 1×10^-6. The broad host range plasmid pR751::Tn5-Pga ⁺ was conjugally transferred to a variety of genetic backgrounds. The ability to degrade polygalacturonate was expressed in Aeromonas hydrophila, Alcaligenes eutrophus, Azotomonas insolita, Escherichia coli, Pseudomonas putida and Rhodopseudomonas sphaeroides, but not in Zymomonas mobilis.Abbreviations PGA polygalacturonate - UGA unsaturated galacturonic acid - PATE polygalacturonate trans-eliminase - PG polygalacturonase - CVP crystal-violet pectate     

A new purple sulfur bacterium from saline littoral sediments,Thiorhodovibrio winogradskyi gen. nov. and sp. nov.

Two strains of a new purple sulfur bacterium were isolated in pure culture from the littoral sediment of a saline lake (Mahoney Lake, Canada) and a marine microbial mat from the North Sea island of Mellum, respectively. Single cells were vibrioid-to spirilloid-shaped and motile by means of single polar flagella. Intracellular photosynthetic membranes were of the vesicular type. As photosynthetic pigments, bacteriochlorophyll a and the carotenoids lycopene, rhodopin, anhydrorhodovibrin, rhodovibrin and spirilloxanthin were present.Hydrogen sulfide and elemental sulfur were used under anoxic conditions for phototrophic growth. In addition one strain (06511) used thiosulfate. Carbon dioxide, acetate and pyruvate were utilized by both strains as carbon sources. Depending on the strain propionate, succinate, fumarate, malate, tartrate, malonate, glycerol or peptone may additionally serve as carbon sources in the light. Optimum growth rates were obtained at pH 7.2, 33 °C, 50 mol m^-2 s^-1 intensity of daylight fluorescent tubes and a salinity of 2.2–3.2% NaCl. During growth on sulfide, up to ten small sulfur globules were formed inside the cells. The strains grew microaerophilic in the dark and exhibited high specific respiration rates. No vitamins were required for growth. The DNA base composition was 61.0–62.4 mol% G+C.The newly isolated bacterium belongs to the family chromatiaceae and is described as a member of a new genus and species, Thiorhodovibrio winogradskyi gen. nov. and sp. nov. with the type strain SSP1, DSM No. 6702.     

Cylindrocladium angustatum sp. nov., a new leaf spot pathogen ofTillandsia capitata from Florida, U.S.A.

Cylindrocladium angustatum is described as a new species fromTillandsia introduced with plant material into the U.S.A. from Central America. Koch's postulates are established to prove its pathogenicity to this host. The species is compared with and distinguished fromC. heptaseptatum andC. rumohrae based on morphology, cultural characteristics and phylogenetic analysis of sequence data of the beta-tubulin gene.     

A new genus of alonine chydorid cladocerans from athalassic saline waters of New South Wales,Australia

A new genus (Celsinotum) and three new species are described from intermittent athalassic saline waters in the Paroo region of northwest New South Wales, which have TDS varying from 2.8 to 14.0 g l^-1. The species are large, thin, high-bodied, and have a pronounced keel on the shell but not on the head. The marginal armature of the postabdomen consists entirely of clusters of fine spinules. Most strikingly, the males have 12 terminal aesthetascs on the antennule, and in addition have 5 or 6 equally long accessory aesthetascs arising laterally. Alona taraporevalae from India presumably also has this character, but a comparison of the two species indicates they are not closely related.The taxa bear a general similarity to Alona diaphana from Australia, but a close comparison shows that they, too, are unrelated. A. diaphana has a prominent spine arising from the basal segment of the antennal exopod, the two IDL (= inner distal lobe) setae on trunklimb I have a much coarser setulation distally than in Celsinotum, and on the postabdomen the postanal portion is always considerably longer than the anal, and the marginal armament consists of single setae distally and clusters of only a few setae proximally. Besides, the species is smaller, the body is wider and less high, and the shell although weakly ridged is not keeled. The male has 9 terminal aesthetascs and no accessory lateral ones.In athalassic saline waters around the world the number of anomopod species declines negative exponentially in relation to salinity. Many of the freshwater species persist into the 3 to 10 range without any difficulty, some occur in abundance up to 30, and a few even extend into the hypersaline range. Chydorids, though, except for a very few species, do not tolerate salinities higher than about 15, and none of them seem adapted to these salinities and confined to them. Celsinotum, though, possibly is so adapted, as it did not occur in other basins of the region with lower salinities, and it is not presently known from other regions.Having different but closely related congeneric taxa in nearby lakes of the same region seems surprising. The three species of Celsinotum are very similar in most details of morphology, but they vary significantly in body proportions, the number of denticle clusters on the postanal portion of the postabdomen, and in such seemingly minor details as the amount of expansion of the labral plate. Such taxonomic differentiation, which has been observed in other groups of organisms as well, is believed to have arisen from the extreme temporal isolation of these waterbodies, those in the Paroo region normally containing water only once every 5 to 20 years.     

Zygozyma suomiensis,sp. nov. (Lipomycetaceae), a new species from Finland

A new species of the genus Zygozyma, Z. suomiensis, is described, based on the study of a single strain, derived from a bovine skin lesion.     

Influence of host plant-derived and abiotic environmental parameters on the composition of the diazotroph assemblage associated with roots of Juncus roemerianus

Environmental factors governing the distributions of plant root-associated bacteria are poorly understood. Most plant species occurring in salt marsh estuaries are restricted to very specific habitats within the marsh and plant-derived and abiotic environmental features covary. We examined diazotrophic bacteria inhabiting the rhizoplanes of different populations of the black needlerush, Juncus roemerianus , growing in two different habitats, in order to examine the relative influence of plant-derived and abiotic environmental parameters on diazotroph assemblage composition. Juncus roots were collected from a monotypic Juncus patch in the low intertidal marsh, and from the main monotypic Juncus stand in the high marsh. A total of 235 bacterial pure cultures were isolated from the roots using combined nitrogen-free media. Physiologically similar strains were grouped, producing 58 different groups. Strains representing 49 of these groups tested positive for nifH , and substrate utilization profiles of these strains were compared quantitatively. Three major substrate utilization clusters were identified and all contained both Juncus patch and main stand isolates. Denaturing gradient gel electrophoresis analysis of nifH amplicons recovered from roots and from vegetated sediments taken from the main stand and from two patches was also performed. Juncus root nifH amplicon profiles from all three sampling sites were very similar. Profiles of amplicons from vegetated sediments were also similar across sites, but less similar than the root profiles. Results from two independent methodological approaches indicated a strong impact of the plant host relative to that of the abiotic environment on the composition of the root-associated diazotroph assemblage.     

Bullera anomala sp. nov. and Bullera pseudovariabilis sp. nov., two new ballistoconidium-forming yeast species from Yunnan,China

Two ballistoconidium-forming yeast strains, CH 2.504 and CH 2.509, were isolated from the wilting leaves of Parthenocissus sp. collected in Yunnan, China in 1996. These two strains were assigned to the genus Bullera Derx by the characteristics of forming rotationally symmetric ballistoconidia, containing xylose in the cell hydrolysates and having Q-10 as the major ubiquinone. Molecular phylogenetic analysis based on small subunit ribosomal RNA gene (18S rDNA) and internal transcribed spacer (ITS) region sequences revealed that CH 2.504 is closely related to B. variabilis Nakase & Suzuki, while CH 2.509 is located in a clearly separate clade among the described Bullera species and related taxa. The results indicated that CH 2.509 represents a new species, for which Bullera anomala sp. nov. is the proposed name (type strain AS 2.2094^T). Further DNA-DNA reassociation data showed that CH 2.504 also represents a distinct species, for which the name Bullera pseudovariabilis sp. nov. is given (type strain AS 2.2092^T). This revised version was published online in June 2006 with corrections to the Cover Date.     

Thermococcus litoralis sp. nov.: A new species of extremely thermophilic marine archaebacteria

We describe a new species, Thermococcus litoralis, which is different from the type species Thermococcus celer in molecular, morphological and physiological characteristics.Abbreviations 3 x SSC (standard saline citrate) - 0.45 M NaCl 0.045 M Na₃-citrate     

Nitrogen fixation by Spirillum sp. from rice roots

Nitrogen fixation by Spirillum sp. obtained from the roots of rice plants grown at different levels of combined nitrogen was studied. The roots of rice plants exposed to low levels of combined nitrogen (20–40 kg N/ha) harboured Spirillum sp. possessing higher nitrogen-fixing efficiency as compared to the cultures from plants receiving 60–100 kg N/ha. More-over, the nitrogen-fixing efficiency of these Spirillum spp. varied with age of the plant, irrespective of the dosage of combined nitrogen.