Identification and population dynamics of bacteria in symptomatic oat leaves

Bacteria isolated from symptomatic oat leaves included pseudomonads,Erwinia herbicola, and others.Pseudomonas coronafaciens was isolated predominantly from leaves with halo blight symptoms or necrotic spots. Leaves with red leaf symptoms yielded many types of bacteria, including saprophytic pseudomonads,P. syringae, E. herbicola, Bacillus sp.,Micrococcus sp.,Corynebacterium sp., a yeast, and other unidentified species. Only isolates ofP. coronafaciens were pathogenic on the plant hosts tested. The bacteria associated with red leaf symptoms exist as saprophytes and/or epiphytes on leaves with those symptoms. It is concluded that bacteria do not contribute to red leaf symptom development in oats, but symptomatic leaves provide an environment for their growth.     

Paramecium calkinsi andP. putrinum (Ciliophora, Protista) harboring alpha-subgroup bacteria in the cytoplasm

Summary New intracellular bacteria were detected in the cytoplasm ofParamecium calkinsi andP. putrinum. Some of the bacteria were not evenly distributed in the cytoplasm of the host but were found in the center of the cell, eventually near the nuclei, but not in the cortex area, whereas another species was found in the cortex area. These peculiarities of intracellular bacteria localization in the host suggest that the conditions in various parts of the cytoplasm favor bacterial maintenance to different extent. Due to the results obtained by transmission electron microscopy and in situ hybridization using appropriate oligonucleotide probes, the bacteria, three or possibly four species, are Gram-negative and belong to the alpha-subgroup of proteobacteria. Bacteria from one stock ofP. calkinsi were found to be infectious for bacteria-free cells ofP. calkinsi andP. nephridiatum.     

Note: Colonization and invasion of leaves of the aquatic macrophyteCeratophyllum demersum L. by epiphytic bacteria

The colonization of leaves of the aquatic macrophyteCeratophyllum demersum L. by epiphytic bacteria, and the hypothesis that bacterial invasion causes leaf senescence, was studied using transmission and scanning electron microscopy and light microscopy. Population densities of epiphytic bacterial communities onCeratophyllum leaves were positively correlated with leaf age. Initial settlement of bacteria on young leaves appeared to favour the boundaries between epidermal cells. On older leaves, large populations of bacteria were present over the whole surface. One third of senescentCeratophyllum leaves examined by transmission electron microscopy showed signs of bacterial invasion. Of these, up to 54% of the leaf's epidermal cells contained bacteria. Areas of cell wall degradation were associated with invasive bacteria in senescent leaves. In healthy, nonsenescent leaves, no bacterial invasion was observed. These results suggest that epiphytic bacteria did not cause leaf senescence but probably colonized the internal tissues of leaves once senescence had occurred.     

Gut microflora of two saltmarsh detritivore thalassinid prawns,Upogebia africana andCallianassa kraussi

The presence and digestive capabilities of bacteria associated with the digestive systems and habitats of two saltmarsh-burrowing detritivore thalassinid prawns (Upogebia africana andCallianassa kraussi) was examined.U. africana is a filter-feeding prawn inhabiting muddy deposits, whereasC. kraussi, a deposit feeder, inhabits coarser more sandy deposits. Scanning electron microscopy was used to examine the gut lining and associated microflora and the nature of the ingested food of both prawns. The gut contents of both prawns included plant fragments, fragmented diatoms, partially degraded protozoa, and bacteria attached to organic matter. In bothU. africana andC. kraussi the midgut walls and gut contents were extensively coated by filamentous bacteria which were absent in the hindgut. The hindgut epithelium ofU. africana was coated by mats of rodshaped bacteria, not reported in marine invertebrates previously. The digestive glands of both species contained bacteria in the lumen. Isolation of gut and habitat bacteria suggests that bothU. africana andC. kraussi maintain a gut microflora distinct from the habitat microflora. Bacteria isolated from the guts of both species of prawn differed from those isolated from their respective habitats with regards to both the genera isolated and their digestive capabilities. The dominant genera isolated from the guts of bothU. africana andC. kraussi wereVibrio andPseudomonas, with an unidentified fermenter andPseudomonas, respectively dominating in the digestive glands. Bacteria of the genusAcinetobacter dominated the isolates from the habitats of both species of prawn. Resident gut bacteria isolated from the guts of both species of prawn exhibited lipase, protease, chitinase, and lysozyme, but not cellulase activity, and may contribute to nitrogen aquisition by the prawns. Isolates from the prawns' habitat exhibited alginase, gelatinase, and lipase activity, a few (3%) fromU. africana habitat having cellulases. In this study a distinction between resident gut bacteria and transient gut bacteria was made. Results suggest that some habitat bacteria remain viable in the guts ofU. africana, but not inC. kraussi.     

Phenotypic differences among single-oospore cultures of Phytophthora palmivora and P. botryosa from Heavea brasiliensis

Oospores ofPhytophthora palmivora andP. botryosa fromHevea brasiliensis were produced when complementary strains of the same species were incubated on V-8 juice agar in continuous darkness, with or without a subsequent period of continuous light. The oospores germinated at a rate of 15–30 % in demineralised water at 26 °C in normal daylight conditions. Other substrates did not improve the germination rate. Single-zoospore colonies derived from sporangia formed by a single oospore were similar to each other in morphology and in pathogenicity toHevea leaves. Over 400 single-oospore isolates from four intraspecific matings ofP. palmivora, and 102 from one pairing ofP. botryosa, were examined. The progeny differed in morphological appearance, mating behaviour, temperature-growth relations, pathogenicity toHevea leaf petioles and cacao pods, rate of production, shape and size of sporangia and in the abundance of chlamydospores. The progeny from an intraspecific cross ofP. botryosa was more variable, with a few isolates being similar in appearance toP. palmivora, having permanently lost their parental characteristic of producing small oval sporangia in clumps. One isolate in particular was indistinguishable fromP. palmivora in morphology and in its ability to produce functional oospores when mated withP. palmivora. Oospores formed by interspecific crosses could not be germinated. With both species, many progeny was less pathogenic than the parents, and many completely non-infective isolates with respect toHevea, cacao and other host plants were produced. Sexual reproduction resulted in a diversity of phenotypes, and both parental types and recombinants were recovered.     

Enterobacter cloacae is an endophytic symbiont of corn

The bacteriumEnterobacter cloacae is presently used for biocontrol of postharvest diseases of fruits and vegetables and as a preplant seed treatment for suppression of damping-off. This bacterium has apparent affinities for several grass species, but it is not considered to be an endophyte. While screening corn for fungi and bacteria with potential for biocontrol of various corn diseases, the surface-sterilized kernels of one unknown Italian corn cultivar produced fungus-free corn seedlings with roots endophytically infected byE. cloacae. This paper describes the microscopic nature ofE. cloacae RRC 101 with corn, and the in vitro control ofFusarium moniliforme and other fungi with this bacterium. Light and electron microscopy determined that this isolate ofE. cloacae was biologically associated with corn seedling roots, where it was distributed intercellularly within the cortex and stele. This is a first report of a strain of this bacterium as an endophytic symbiont of roots. Following a topical application ofE. cloacae to kernels, and upon germination this bacterium readily infected roots of two other corn cultivars. The bacterium was observed within the endosperm of germinating corn seedling, but germination was not affected. Further, the bacterium was isolated from leaves and stems of 3- to 6-week-old seedlings indicating that the above ground portions of corn were also colonized. There was no evidence of damage to cells of the root during a three to four week observation period. This bacterium was antagonistic to several isolates of the corn pathogenFusarium moniliforme, and to two other species of fungi, all of which produce mycotoxins on corn.     

DESMID-BACTERIAL ASSOCIATIONS IN SPHAGNUM-DOMINATED WISCONSIN PEATLANDS1

In a survey employing epifluorescence microscopy with the DNA fluorochrome DAPI, associations between bacteria and filamentous desmids were found to be commonplace in acidic, Sphagnum-dominated Wisconsin peat-lands. Bacteria were associated with all genera of filamentous desmids encountered including Desmidium, Hyalotheca, Onychonema, Spondylosium, and Teilingia. Although only associations involving filamentous desmids having mucilaginous sheaths are illustrated here, bacteria were also noted on taxa lacking sheaths as well as some unicellular forms. Bacteria on Desmidium majus Lagerheim, D. grevillii (Kütz.) De Bary, and Hyalotheca dissiliens (Smith) Bréb. ex Ralfs tended to be concentrated in small pockets in the sheath material located near the isthmus and in the region between adjacent cells in the filament, whereas those associated with Spondylosium pulchrum (Bail.) Archer were more evenly distributed throughout the sheath. Most bacteria were rodshaped. Those associated with S. pulchrum, D. grevillii, and D. majus ranged from 1.1 to 11.2 μm in length. Bacteria within the sheaths of H. dissiliens and D. grevillii were Gram-negative. A second morphologically distinct population of bacteria was found at the sheath margin in D. majus and D. grevillii. Field collections containing filamentous desmids were examined with scanning electron microscopy and bacteria associated with Desmidium majus were investigated with transmission electron microscopy.     

Adherence of clinical isolates ofPseudomonas aeruginosa to hamster trachael epithelium in vitro

The adherence to hamster tracheal epithelium, of mucoid and nonmucoid clinical isolates ofPseudomonas aeruginosa from cystic fibrosis patients, was studied using tracheal organ cultures. Tracheal cultures were infected with 10⁷ colony-forming units per ml of either mucoid or nonmucoid clinical isolates ofP aeruginosa. The tracheal explants were rinsed at various time intervals to remove nonadherent bacteria, fixed, and prepared for transmission-and scanning-electron microscopy. Mucoid isolates were seen adhering to the ciliated epithelium as early as 4 h after initiation of infection, whereas nonmucoid isolates were only observed adhering at 6 to 8 h after infection. Mucoid organisms were found as clusters of bacteria embedded in an extensive extracellular matrix. The nonmucoid isolates were generally found as single organisms with no evidence of an extracellular matrix. These results suggest that the prevalence of mucoid isolates ofP. aeruginosa in cystic fibrosis may be due to adherent properties of the mucoid organism.     

Morphological and physiological comparisons ofHelicobasidium mompa andH. purpureum

Morphological and physiological comparisons were made of sevenHelicobasidium mompa isolates and fourH. purpureum isolates. Colonies of theH. mompa isolates were thin, dense, or hard and dense, and most were pale brown to brown or dark brown, while that of isolate 344c was pinkish. Colonies ofH. purpureum isolates were hard and dense, and their colonies were dark brown. Diameters of hyphae were similar forH. mompa andH. purpureum. Dimensions of conidia and morphology of conidiophores ofH. mompa isolate 344c were close to those ofH. purpureum reported previously.H. mompa isolates grew well at 23°C, 25°C or 27°C, while all isolates ofH. purpureum grew well at 23°C. Growth rates ofH. purpureum isolates was almost the same as those ofH. mompa isolates with slow growth. Polygaracturonase activity at pH 3 was variable among the isolates for bothH. mompa andH. purpureum. Itaconic acid was produced abundantly by three isolates ofH. mompa but not produced by isolate AH130, whereas all isoaltes ofH. purpureum produced a small amount of itaconic acid.     

Populations of bacteria and actinomycetes associated with sclerotia ofPhymatotrichum omnivorum buried in Houston black clay

Sclerotia ofPhymatotrichum omnivorum (Shear) Duggar (the causal agent of root rot of cotton) were produced in the laboratory and then buried at a depth of 45 cm at three sites in Texas situated on Houston black clay soils with various cropping histories. The sites included a native grassland prairie, a field in continuous cotton production, and a field in which cotton, corn, and sorghum were grown in rotation. Samples of sclerotia were retrieved monthly over a 12 month period. Populations of bacteria and actinomycetes were enumerated using dilution-plate techniques and isolates were screened (in vitro) for their ability to produce substances inhibitory toP. omnivorum. The sclerotia supported large numbers of bacteria (including fluorescent pseudomonads) and actinomycetes. Numbers associated with sclerotia ranged from 10⁶–10⁹ cells per gram of sclerotia plus adherent soil and were 2–3 orders of magnitude greater than numbers from soil at the same depth but free of sclerotia. Bacteria and actinomycetes antagonistic toP. omnivorum were isolated from sclerotia buried at each of the three sites. Up to 26% of the isolates inhibited growth ofP. omnivorum.     

Production of cytochalasins C,D & E from dematiaceous hyphomycetes

One hundred isolates of 27 species belonging to 13 genera of dematiaceous hyphomycetes were screened for production of cytochalasins C, D and E. Only two isolates ofBipolaris neergaardii and one isolate ofPhoma herbarum produced cytochalasins C and D. Also, cytochalasin E was produced by two isolates ofAlternaria chlamydospora, and one isolate ofCochliobolus tuberculatus. This is the first report about the production of cytochalasins C, D and E by these species of dematiaceous hyphomycetes.     

Dinitrogen fixation and infection of grass leaves byPseudomonas rubrisubalbicans andHerbaspirillum seropedicae

Bacteria causing mottled stripe disease in sugar cane, known asPseudomonas rubrisubalbicans, were shown to be able to fix molecular N₂ and to grow on it. The root associated diazotroph known asHerbaspirillum seropedicae, after artificial inoculation caused mottled stripe disease symptoms on sorghum and Napier grass but not on sugar cane. Both bacteria could be reisolated from leaves even 60 days after. Sugar cane leaves contained large numbers of these bacteria even in the uninoculated controls. Additional physiological characteristics of six strains ofP. rubrisubalbicans were compared with those of twoH. seropedicae strains and were shown to be very similar.     

Distinct glycoconjugate cell surface structures make the pelagic diatom Thalassiosira rotula an attractive habitat for bacteria

Interactions between marine diatoms and bacteria have been studied for decades. However, the visualization of physical interactions between these diatoms and their colonizers is still limited. To enhance our understanding of these specific interactions, a new Thalassiosira rotula isolate from the North Sea (strain 8673) was characterized by scanning electron microscopy and confocal laser scanning microscopy (CLSM) after staining with fluorescently labeled lectins targeting specific glycoconjugates. To investigate defined interactions of this strain with bacteria the new strain was made axenic and co-cultivated with a natural bacterial community and in two- or three-partner consortia with different bacteria of the Roseobacter group, Gammaproteobacteria and Bacteroidetes. The CLSM analysis of the consortia identified six out of 78 different lectins as very suitable to characterize glycoconjugates of T. rotula. The resulting images show that fucose-containing threads were the dominant glycoconjugates secreted by the T. rotula cells but chitin and to a lesser extent other glycoconjugates were also identified. Bacteria attached predominantly to the fucose glycoconjugates. The colonizing bacteria showed various attachment patterns such as adhering to the diatom threads in aggregates only or attaching to both the surfaces and the threads of the diatom. Interestingly the colonization patterns of single bacteria differed strikingly from those of bacterial co-cultures, indicating that interactions between two bacterial species impacted the colonization of the diatom. Our observations help to better understand physical interactions and specific colonization patterns of distinct bacterial mono- and co-cultures with an abundant diatom of costal seas.     

Characterization of endophytic bacteria associated with rose plant (Rosa damascena trigintipeta) during flowering stage and their plant growth promoting traits

Little is known about the bacterial communities associated with the rose plants inhabiting dry desert ecosystems. The aim of this study was to isolate and characterize endophytic bacteria from different organs of rose plant. Endophytic bacteria were observed in healthy roots, stems, leaves, and flowers of rose plant, with a significantly higher density in roots, followed by stems, leaves, and petals. A total of 38 bacterial endophytes were isolated and are closely related phylogenetically to Acetobacter, Acinetobacter, Methylococcus, Bacillus, Micrococcus, Planococcus by 16S rRNA sequence analysis. Six endophytic bacteria were found to produce IAA, solubilize Ca₃(PO₄)₂ and produce siderophore. The six endophytic bacteria all had the capacity to produce hydrolytic enzyme such as cellulase, xylanase, pectinase, amylase, protease, lipase, and chitinase, but difference existed among these isolates.     

Molecular genetic characterization of bacterial isolates causing brown blotch on cultivated mushrooms in Japan

The polymerase chain-reaction (PCR) was used to amplify 16S ribosomal DNA (16S rDNA) from bacteria, identified asPseudomonas tolaasii orP. fluorescens, causing brown blotch on cultivated mushrooms in Japan. PCR-amplified 16S rDNA was analyzed on the basis of nucleotide sequence and restriction fragment length polymorphisms (RFLP) to determine the specific identity of isolates. Banding patterns obtained through PCR using primers corresponding to repetitive extragenic palindromic sequences of enteric bacteria (REP-PCR) were used to determine the relatedness of conspecific isolates. AllP. tolaasii isolates and a mushroom pathogen identified asP. fluorescens had identical RFLP patterns and partial 16S sequences, and are considered conspecific. An isolate ofP. fluorescens from creamery wastes (IFO 3507) differed slightly from isolates ofP. tolaasii in both 16S sequence (0.8%) and RFLP patterns (d=0.08), and had almost entirely different REP-PCR bands (d=0.88–1.0). Phylogenetic analyses based on 16S sequences indicated thatP. tolaasii andP. fluorescens are close members ofPseudomonas sensu stricto. REP-PCR shows promise in characterizing isolates pathogenic on different mushroom crops. Two isolates ofP. tolaasii pathogenic onPleurotus ostreatus had identical banding patterns, but three isolates fromLentinula edodes showed the greatest diversity. Contribution No. 312 of the Tottori Mycological Institute, Totori, Japan.     

Electron microscopy of methanol-utilizing bacteria

Two different groups of methanol-utilizing bacteria were studied by electron microscopy. Bacteria using the serine pathway for the assimilation of methanol were found to have a thin cell envelope (outer membrane, periplasmic area and cytoplasmic membrane). Those using the assimilatory ribulose monophosphate pathway of formaldehyde fixation had a much thicker cell envelope and in the case ofPseudomonas C protrusions of the outer membrane were found.     

Isolation and characterization of <Emphasis Type="Italic">Pseudoalteromonas ruthenica</Emphasis> (SBT033), an EPS-producing biofilm bacterium from the seawater intake point of a tropical power station

Biofilm formation in bacteria is closely linked with production of exopolysaccharides (EPS). This study examined the quantitative variations in EPS production and biofilm-forming ability among bacteria isolated from the seawater intake point of a power station located on the east coast of India. Of the 233 isolates obtained from the intake site, 71 bacterial isolates displayed different colony morphological characteristics. Thirteen isolates that produced wide and thick mucoid colonies were further tested for their ability to attach and form biofilms by microtitre plate assay and confocal microscopy. EPS production among the selected bacterial isolates ranged from 826 to 1838 μg ml⁻¹. Strain SBT033, which produced the maximum amount of EPS also displayed the maximum biofilm-forming ability among the 13 isolates. This strain was selected for further characterization using biochemical and molecular methods. The pale orange-pigmented isolate was a Gram negative, aerobic, short rod-shaped and grew well only in the presence of 2% NaCl. On the basis of phenotypic characteristics the isolate SBT033 is shown to belong to the genus Pseudoalteromonas. Analysis of 16S rRNA of the isolate revealed 99% homology with Pseudoalteromonas ruthenica.     

Occurrence and ultrastructural characterization of bacteria in association with and isolated from Azolla caroliniana.

The occurrence and ultrastructure of bacteria in leaf cavities of symbiotic Azolla caroliniana were examined by transmission electron microscopy. Bacteria were observed in all leaf cavities of Azolla cultures. Five ultrastructurally distinct types of bacteria were observed in each individual leaf cavity. Features used to characterize the bacteria included morphology, cell wall structure, and cytoplasmic organization. At least one gram-positive and as many as four gram-negative types of bacteria reside in leaf cavities of A. caroliniana. The morphological and ultrastructural characteristics of the gram-positive bacterium suggest that it is an Arthrobacter sp. The gram-negative bacteria could not be cultured; therefore, they have not been classified further. Bacterial cell shape and cell wall structure were similar in leaf cavities of different ages, but cell size and cytoplasmic composition varied. The relative contributions of each bacterial type to the total community within individual leaves was determined. Ultrastructural characteristics of bacterial isolates cultured from A. caroliniana in a free-living state were also examined.     

Extraction and properties of nonfimbrial mannose-resistant hemagglutinin from a urinary isolate ofEscherichia coli

A mannose-resistant hemagglutinin (MRH) was extracted from an agar-grown urinary isolate ofEscherichia coli (827) by heating the organisms at 65°C for 1 h. Both MRH and the organisms agglutinated erythrocytes from human (group A) but not from seven other animal species tested, but were devoid of any detectable fimbriae as examined by electron microscopy. MRH was sensitive to pronase or 100°C heating, but not to trypsin or periodate, could not be sedimented by ultracentrifugation 149,000g, and passed the void volume of Sepharose-4B column. MRH inhibited the adherence of bacteria to tissue culture cells. The results suggest thatE. coli human isolate produces mannose-resistant hemagglutinin, which is located on the cell surface in nonfimbrial form and probably mediates adherence of the bacteria to eukaryotic cells.     

Vegetative compatibility of an isolate ofVerticillium dahliae pathogenic to both tomato and pepper

An isolate ofVerticillum dahliae Vdp-4, pathogenic to both tomato and pepper (tomato-pepper pathotype), was examined for its vegetative compatibility with testers of the Japanese vegetative compatibility group (subgroups J1, J2, and J3). Seven isolates ofV. dahliae from the same field as Vdp-4 in Misato, Nagano Pref. and two isolates from Hokkaido were separately determined as either tomato pathotype (B) or pepper pathotype (C). Isolate 5922 previously reported as tomato-pepper pathotype was also examined. Compatiblenit1 and NitM mutants were obtained from all isolates except for isolates Vdp-3 and Vdt-10. The isolate of tomato-pepper pathotype Vdp-4 showed a strong reaction with VCGJ1 and J3 and was thus assigned to J3. Seven of these isolates showed compatibility and were assigned into three provisional subgroups. The isolate 5922 was self-incompatible.