Interactions between bacteriophages and bacteria in soil

The population biology of certain phages for Bacillus species in soil is described. In soil at 37°C the growth profile of B. circulans phage ST1 shows a lag phase, a phase of exponential growth and a plateau phase, much as occurs in monoculture except that the time scale is transposed into hours rather than minutes. Individual strains of replicating bacteria have been identified by a phage typing technique. At 37°C the number of different phage-sensitive strains falls sharply during the first 6 h but has risen again by 10h. After 10 h at 37°C, 76% of the bacterial types scored represent new strains, as judged by their susceptibility to the eleven phages used for typing. The data presented indicate that a sensitive equilibrium exists between phage and host bacterial cells in soil and that this may be altered very quickly under selective pressures.     

The ecology of viruses attacking Bacillus stearothermophilus in soil

The ecology of bacteriophages attacking the thermophilic soil organism Bacillus stearothermophilus has been investigated. Phage was found in every soil tested including a sample from the geothermal area of Taupo. Since the numbers of plaque-forming units (pfu) in most soils were often low a technique for enriching soil with substrate for the host cells and factors promoting phage proliferation was developed; this enabled statistically significant numbers of pfu to be scored under a variety of test conditions. Phage development in soil was greatest at 45°C while proliferation at 55°C was usually much less pronounced. These results indicate that in soil these bacteria and their associated phages grow optimally at mesophilic rather than thermophilic temperatures. It is proposed that in the field the bulk of the metabolizing population of B. stearothermophilus is mesophilic in its biochemistry and that the ‘thermophilic’ strains of this organism held by many laboratories represent selection by the isolation procedure of a minority of atypical cells.     

Morphology and general characteristics of phages specific to Lens culinaris rhizobia

Four lytic phages, namely LRP-1, LRP-4, LRP-13, and LRP-15, active against indigenous rhizobial strains of Lens culinaris were isolated and characterized for their individual morphology, host range, plaque characteristics, lytic behavior, and restriction endonuclease profiling of phage DNA. All phages had a typical polyhedral head and long non-contractile tail, representing the bacteriophage family close to Siphoviridae. Phages produced distinct types of plaques on their indicator bacterial strains. The host range of the phage isolates was restricted to Rhizobium leguminosarum biovars and no cross infectivity among susceptible strains was observed. A study on the lytic cycle of the phages under identical conditions exhibited distinct latent period and burst size. Inactivation pattern of phages with temperature and UV light was quite distinct. Phage LRP-1 showed higher thermal resistance, though greater sensitivity to UV light, as compared to other phages. Genome sizes of the phages were estimated to vary between 50–72 kbp. The 16S rRNA sequence analysis of the phage indicator rhizobial strains revealed 81% to 100% similarity with R. leguminosarum bv. viciae. The phages could thus prove to be considerably useful in typing and investigating into the genetic variability which might exist amongst the soil rhizobia nodulating Lens culinaris.     

From genetic diversity and structure to conservation: Genetic signature of recent population declines in three mouse lemur species (Microcebus spp.)

The exceptional biodiversity of Madagascar is threatened by anthropogenic landscape changes that took place during the 2000 years of human colonization. This study focuses on the influence of geographic distance and forest fragmentation on genetic diversity and population differentiation of three rare, nocturnal, arboreal lemur species in northwestern Madagascar. Historic declines in population sizes as a consequence of forest fragmentation are quantified and dated. Eighteen sites were visited, and a total of 205 Microcebus ravelobensis, 45 M. bongolavensis and 78 M. danfossi were genotyped with eight microsatellite loci. Genetic differentiation among the sites, as measured by F_ST, ranged from 0.01 to 0.19. These values were significant in almost all cases and indicated genetic structure in the samples. Isolation-by-distance was detected in one species and a STRUCTURE analysis indicated that fragmentation further promoted genetic differentiation. Bayesian methods revealed that populations from all three species underwent a major demographic collapse of around two orders of magnitude. This decrease probably began after the arrival of humans, most likely within the last 500 years. This result suggests that anthropogenic changes may have been limited during the first 1500 years of human colonization in all three ranges. Two of the study species (M. danfossi, M. bongolavensis) lack effectively protected areas in their ranges. Consequently, quick conservation actions are now needed in order to secure the remaining genetic diversity of these species.     

Build up of patches caused by Rhizoctonia solani

Rhizoctonia solani is a complex species that is composed of different anastomosis groups (AG). Although these different AGs show differences in their host ranges, generally R. solani is a phytopathogenic species with a wide spectrum of hosts. It has the ability to grow as a saprotroph, which further complicates its behaviour as a parasite. The losses caused by R. solani are very important and need a sustainable management strategy. The patchy appearance of the disease caused by this pathogen is well-known. The patches show within and between season dynamics. The factors which affect the spread of the disease can be grouped into three main categories: host plant, pathogen and environment. However, each of the categories in its detail may depend on or react with the other categories. There are a number of factors that may be involved in dynamics of patches. These potential mechanisms are discussed. It is essential to know about the mechanisms involved to develop an effective control strategy. Although more work is needed to investigate different mechanisms of parasitism deployed by different AGs in different hosts, it seems that many mechanisms external to the host are operating at the same time which necessitates an integrative research approach to study and control the diseases caused by R. solani.     

Replant diseases: Bacterial community structure and diversity in peach rhizosphere as determined by metabolic and genetic fingerprinting

Peach tree replant disease, though reported on in the literature for more than two centuries, has yet to have its causes clearly defined. Decline in peach productivity has been attributed to toxic agents, insects, nutritional disturbances, spray residues, fungi and nematodes. Bacteria has also been indicated as a contributing factor.Peach replant disease was reproduced by using two successive cultures on the same soil. Bacterial communities were isolated and characterized from healthy and diseased peach trees. The potential role of cyanide production by rhizobacteria in the replant problem of peaches was studied. Culture-dependent (evaluation of the number of culturable bacteria, metabolic activities, Biolog^® GN2) and independent (ribosomal intergenic spacer analysis, RISA) methods were used, in order to compare bacterial community structure and diversity in healthy and sick soils and to evaluate the possible role of cyanide.Bacterial densities were significantly increased in sick soils. Metabolic activities (Biolog^® GN2) and genetic structure, observed through RISA, were also significantly modified in sick soils. Changes in the composition of individual microbial groups in the rhizosphere of peach trees excavated from healthy or sick soil indicated the involvement of rhizobacteria in the etiology of the replant sickness of peach soil. More than 60% of the strains isolated from healthy soils corresponded to Pseudomonas sp. and 58% of the isolates from sick soils were Bacillus sp. This study determined that Bacillus were able to produce in vitro HCN. It also appeared that in sick soil, there was a shift in the structure of bacterial communities with an increase noted in phytotoxic microorganisms capable of producing HCN compounds.     

Identification of 12 EST-derived SSR markers in Lumbricus rubellus

While many species of earthworms are globally distributed, very little is known about the genetic population dynamics of this diverse group. We present the characterization of novel simple sequence repeat (SSR) markers, including primer information, number of alleles, repeat motif, and approximate size ranges, to be used in population genetic analyses of the earthworm Lumbricus rubellus Hoffmeister 1843. Specifically, we designed and characterized 12 novel, polymorphic markers derived from published expressed sequence tags (EST) for amplification in L. rubellus. The mean number of alleles per locus was 6.25 ± 1.91, indicating these markers will be sufficiently polymorphic for population genetic studies of this species.     

Isolation and characterisation of phages against Pseudomonas syringae pv. actinidiae

Purpose: Pseudomonas syringae pv. actinidiae causes bacterial canker of kiwifruit and is responsible for severe economic losses and emergence of drug-resistant bacteria. Bacteriophages are viruses that infect target bacterial hosts and may be the best strategy to prevent and control kiwifruit canker disease. The objective of this experiment was to monitor the prevalence of Pseudomonas syringae pv. actinidiae and provide insight for the use of phages in biological control.

Materials and methods: In this study, 52 strains of Pseudomonas syringae pv. actinidiae were isolated from 68 stem samples of kiwi plant (cv. Hongyang & Jinkui). Following polymerase chain reaction (PCR) analysis, 15 isolates belonging to biovar 3 were identified, one of which was named XWY0007 and used as the target strain to isolate the phages. Thirty-six phages were isolated and purified from a total of 51 surface water samples collected in Shanghai. All phages were identified by transmission electron microscopy (TEM) and their host ranges were evaluated. Three phages, designated φXWY0013, φXWY0014 and φXWY0026 were selected and further characterised using one-step growth curve and stability at different temperatures and pH.

Results and conclusions: The isolated phages are promising for use as antimicrobials against bacterial canker in kiwi. This report is regarding Pseudomonas syringae pv. actinidiae and its phages from major areas of kiwifruit cultivation.     

Endophytic bacteria from selenium-supplemented wheat plants could be useful for plant-growth promotion,biofortification and Gaeumannomyces graminis biocontrol in wheat production

In this study, we isolated putative plant-growth-promoting endophytic bacteria from selenium-supplemented wheat grown under field conditions. These bacterial strains belonged to Bacillus, Paenibacillus, Klebsiella, and Acinetobacter genera and showed genetic similarly with rhizospheric bacteria isolated in the same Andisol soil and with other endophytic strains previously reported. Strains isolated from selenium-supplemented wheat were highly tolerant to elevated selenium concentration (ranged from 60 to 180 mM), and showed potential plant-growth-promoting capabilities (auxin and siderophore production, phytate mineralization, and tricalcium phosphate solubilization). In addition, some strains like Acinetobacter sp. (strain E6.2), Bacillus sp. (strain E8.1), Bacillus sp., and Klebsiella sp. (strains E5 and E1) inhibited the growth of Gaeumannomyces graminis mycelia in vitro at 100, 50, and 30 %, respectively. These endophytic microorganisms would be useful for dual purposes: selenium biofortification of wheat plants and control of G. graminis, the principal soil-borne pathogen in volcanic soils from southern Chile.     

Response of the soil bacterial community to the addition of toluene and toluene-degrading bacteria

Changes in soil bacterial communities after toluene and/or toluene degrading bacteria were added were monitored by growth on various media, and by the culture-independent method of Reverse Sample Genome Probing (RSGP). A total of 397 isolates that were cultured from toluene-amended and non-amended soil were identified using fatty acid methyl ester (FAME) analysis. In 75% of the soil samples, gram-positive bacteria dominated, primarily Bacillus sp. and Cellulomonas sp. In contrast, RSGP revealed Proteobacteria (α, β, and γ subgroups) to be the dominant species, with Bacillus sp. dominant in only one soil sample.In toluene-treated soil, FAME and RSGP analyses indicated that by day 5 the major bacterial populations were gram-negative bacteria, in particular, Pseudomonas sp., Acinetobacter sp., and Alcaligenes sp. When toluene and Pseudomonas putida D8 were coincidentally introduced, the proportion of Pseudomonas sp. in the bacterial population recovered using non-selective medium increased from 16 to 62% and then rapidly decreased to about 9%. When we used selective medium to monitor the population of P. putida D8, a rapid initial increase followed by a gradual decline was also observed. In samples analyzed by RSGP, D8 was one of the major species of the bacterial community at day 2, but its signal intensity dropped to 9.5% by day 5.The influence of D8 addition on the bacterial profile was monitored in growth-based examinations. Bacillus sp. and Pseudomonas sp. were initially dominant. By day 5, Bacillus sp. decreased while the Proteobacteria, (including Acinetobacter sp., Agrobacterium sp., Alcaligenes sp., Burkholderia sp., Erwinia sp., and Pseudomonas sp.) increased. At the same time, D8 decreased to a level indistinguishable from background. Conversely, RSGP analysis revealed the population dominance of P. putida (including D8) and Rhizobium fredii at day 2. Populations shifted toward Agrobacterium tumefaciens, Bacillus subtilis, R. fredii, and D8 by day 5.P. putida D8 levels could be monitored using RSGP when cultivation failed. However, cultivation of Bacillus sp. was always successful, while the organism was only occasionally detected by RSGP. While cultivation and RSGP methods comparably detected the same major bacterial populations, the overall bacterial diversity was greater with RSGP than with growth-based testing.     

Molecular and cytogenetic characterization of a collection of bahiagrass (Paspalum notatum Flügge) native to Uruguay

Paspalum notatum is a subtropical grass present throughout America, and one of the main constituents of the natural grasslands in Uruguay. An apomictic autotetraploid (2n?=?4x?=?40) is the most frequent cytotype. The occurrence of sexual diploids (2n?=?2x?=?20) has also been reported as well as the occasional presence of apomictic triploids and pentaploids in Argentina. In this study, ISSR (inter simple sequence repeats) molecular markers were used to analyze the genetic variability of 210 P. notatum individuals from a collection from Uruguay. Cytometric analyses and chromosome counts were used to assess the ploidy level of the individuals. All plants from Uruguay analyzed were tetraploid. Intra- and inter-population variability was found both in genomic DNA content and at the genotypic level. Several multilocus genotypes were shared among individuals within populations and among populations over moderate geographical ranges, at the same time, very dissimilar genotypes were found within the same population. Part of the genetic variance among populations can be explained by a broader scale geographic structure which is partly coincident with the traditionally recognized grassland management regions. In spite of the apparently high degree of genetic admixture within populations, groups of related genotypes seem to follow a broader geographical structure in the area under study. These results suggest that an efficient collection strategy for this apomictically reproducing species should include carefully planned intra- and inter-population sampling. A broader scale regional sampling strategy should also be considered although further studies will be required to define genetic structure at this level.     

Amphibian-killing fungus loses genetic diversity as it spreads across the New World

Emergent infectious diseases are a severe threat to global biodiversity, thus conservation biologists need to understand the emergence, spread, and evolution of pathogens to identify factors driving disease outbreaks. Amphibian chytridiomycosis is a recently emerged amphibian disease caused by the fungus Batrachochytrium dendrobatidis (Bd) that has led to species extinctions and declines worldwide. The spatio-temporal dynamics of pathogen occurrence and disease outbreaks, and comparative genomic analyses of global Bd strains, support the spreading pathogen hypothesis (SPH) with pandemics occurring after introduction of Bd into naïve host populations. Here we used population genetics of the amphibian-killing fungus to test for genetic consequences of pathogen spread. Our population genetic analyses are consistent with the spread of Bd from North to Central America, based on low genetic diversity, reduction in heterozygosity, and increased allele fixation in Bd from recently infected populations. Our findings confirm the spread of Bd in the New World, and indicate that future conservation efforts should focus on: (i) functional consequences (such as changes in pathogenicity) of these genetic changes, and (ii) public education and restrictions on wildlife trade to help slow spread of the pathogen at the invasion front.     

Changes in Thermophilic Bacteria Population and Diversity during Composting of Coal Fly Ash and Sewage Sludge

The thermophilic bacteria in compost made from coal flyash-amended sewage sludge were isolated and identified using theBiolog system to investigate the effect of coal fly ash on thethermophilic decomposition of sewage sludge during composting. Atotal of 8 species of Bacillus were isolated from thecompost and Bacillus brevis was the dominant speciesduring the entire composting process. The present resultsdemonstrate that the Biolog system is a fast and simple methodfor identifying bacterial species in compost, provided thatoptimum conditions could be achieved for the Bacillusculture. Adding coal fly ash as an amendment did not change thedominant bacteria species during composting, but decreased thepopulation and diversity of thermophilic bacteria species due tothe high alkalinity and salinity. Fewer thermophilic bacteriawere detected in ash-amended sewage sludge compost than insludge compost. There was also reduced metabolic activityobserved in the ash-amended sludge compost from the data ofCO₂ evolution and weight loss. Although ash amendmentdemonstrated a negative effect on the population and diversityduring thermophilic phase, it did not cause any significanteffect on compost maturity.     

Effects of certain full and partial sterilization treatments on leaf litter

Experiments are described which tested the effects of X-rays, gamma-irradiation, autoclaving, and propylene oxide on the organisms and chemical composition of hazel and oak leaf litter.X-ray treatment at 0.02 Mrad produced no major changes in the composition of the microbial populations, nor significant changes in the chemical composition of the litters. However, this treatment has been used to kill animals in litter, and it is a suggested treatment of litter if micro-organisms are to be studied in the absence of animals, or if a different population of animals is to be introduced.Autoclaving, gamma-irradiation, and propylene oxide, were effective as total sterilizing treatments: a Bacillus species was the only survivor detected. The results confirm that autoclaving is the most drastic of these treatments, and it appears to reduce the content of soluble tannins in the litter. Propylene oxide has certain disadvantages, which are discussed. Gamma-irradiation is at least as effective as autoclaving in killing organisms, and may he more so, as the Bacillus species appeared to be damaged more by irradiation than by the other treatments. Gamma-irradiation appears to leave at least some enzyme systems functioning, but storage of the treated material before use allows free enzyme activity to decline. The effect of storage time on free enzyme systems needs further study.     

Comparison of g23 gene sequence diversity between Novosphingobium and Sphingomonas phages and phage communities in the floodwater of a Japanese paddy field

Our previous study indicated that the diversity of the major capsid gene (g23) of T4-type bacteriophages (phages) of Novosphingobium and Sphingomonas strains isolated from the floodwater of a Japanese paddy field is comparable to those of the clones obtained from other Japanese paddy fields. For more strict comparison of the diversity, this study examined g23 sequences between Novosphingobium and Sphingomonas phages and phage communities in the identical floodwater of a Japanese paddy field. The clones were obtained by applying g23-specific primers to DNA extracted from the floodwaters. Many 23 clones in the floodwater were grouped into the same clusters of Paddy Groups I-VI with g23 genes of Novosphingobium/Sphingomonas phages with some clones belonging to an additional cluster. In addition, the remaining clones belonged to the clusters of marine clones and T4-type enterophages. These findings indicate that the g23 genes in the floodwater are more diversified than those of Novosphingobium/Sphingomonas phages including g23 genes closely related to the genes of enterophages and marine origins.     

Genetic variation in captive population of chinese alligator, Alligator sinensis, revealed by random amplified polymorphic DNA (RAPD)

Chinese alligator (Alligator sinensis) is an endemic species in China. The likely extinction of it in the wild has been recognised. To prevent this species becoming extinct, the Anhui Research Centre of Chinese Alligator Reproduction (ARCCAR) was established in Xuanzhou, Anhui Province in 1979, where has been established the largest captive population of Chinese alligator (XZSP) in the world. Another farm (CXSP) was established by villagers in Changxin, Zhejiang Province. The results of an investigation of the two captive subpopulation structures by genetic analysis are presented in this paper. We examined the genetic variation in the two captive subpopulations using RAPDs. Thirty-one random primers were selected among 199 random primers screened. A total of 193 reproducible RAPD fragments were scored among 43 individuals, of which 21 (10.88%) were polymorphic. The genetic distances between 43 individuals ranged from 0 to 0.0376 with average of 0.0104±0.0055 S.E. The genetic similarity in CXSP (0.9948±0.0029 S.E.) was higher than that in XZSP (0.9894±0.0055 S.E.). The founder effect is a possible explanation for very low genetic variation in CXSP. Analysis of the RAPD data showed that the mean phenotypic band frequencies of each polymorphic loci was 0.6656±0.3730 S.E. The lowest phenotypic band frequency (0.0233) was found in four of those polymorphic loci. There was no genetic difference between the two subpopulations (D_ij=0.0009). According to the dendrogram and the distribution of polymorphic fragments in two subpopulations, CXSP originated genetically from XZSP. This paper summarises a preliminary research on genetic structure in populations of Chinese alligator. Although there is higher genetic similary (0.9896±0.0055 S.E.) in captive population of A. sinensis, we did not determine whether or not loss of genetic variation had occurred in relation to a wild control population. The data of malformed offspring will be collected carefully, and wild samples be added to set up a control population in future study.     

Occurrence of both Casuarina-infective and Elaeagnus-infective Frankia strains within actinorhizae of Casuarina collina,endemic to New Caledonia

New Caledonia is characterized by an exceptional concentration of endemic Casuarinaceae species including eight Gymnostoma species and Casuarina collina. Frankia strains isolated from nodules from C. collina have been studied through a range of techniques including molecular typing, infectivity/effectivity and host spectrum. All these isolates had characteristics similar to those of atypical Frankia from Casuarina previously described. This work evidenced several original features within these 26 isolates: (i) they are localized inside the nodule; (ii) they fit into the Elaeagnus-infective Frankia group; (iii) they belonged to four ITS groups already described for Frankia from Gymnostoma. In fact, it is highly probable that atypical Frankia from C. collina and Frankia from Gymnostoma are the same. We can formulate the hypothesis that the Frankia from Gymnostoma could use C. collina as an alternative host in absence of Gymnostoma spp. or when the ecological conditions are favourable to C. collina.     

Genetic diversity and potential for promotion of plant growth detected in nodule endophytic bacteria of soybean grown in Heilongjiang province of China

A total of 98 non-symbiotic endophytic bacterial strains isolated from soybean root nodules were classified into eight rDNA types in ARDRA analysis and 21 BOX types in BOX-PCR. The phylogenetic analysis of 16S rDNA identified these strains as Pantoea, Serratia, Acinetobacter, Bacillus, Agrobacterium, and Burkholderia. Limited genetic diversity was revealed among these bacteria since most of the strains (85.7%) were found in three very similar rDNA types corresponding to Pantoea agglomerans, and many strains shared the same BOX-PCR patterns. The inoculation of nodule endophytes had no significant effects on the growth and nodulation of soybean, but most of the strains produced indoleacetic acid (IAA), could solubilize mineral phosphate, and could fix nitrogen, implying that they are a valuable pool for discovering plant growth promoting bacteria. Our results demonstrated that the nodule endophytes were common in soybean and their diversity was affected by the plant's character and the soil conditions. The 99% similarities found in the nifH genes of Bradyrhizobium japonicum and of the endophytic Bacillus strains strongly indicated that horizontal transfer of symbiotic genes happened between the symbiotic bacteria and the endophytes.     

Assessing genetic diversity of wild southeastern North American <Emphasis Type="Italic">Vaccinium</Emphasis> species using microsatellite markers

Wild species representatives from Northwestern, Central and Southern Florida, and neighboring U.S. states were collected in multiple United States Department of Agriculture (USDA) exploration expeditions and are being preserved at the USDA, Agricultural Research Service, National Clonal Germplasm Repository in Corvallis, Oregon. Germplasm from these southeastern regions of North America is particularly vulnerable to loss in the wild due to encroachment of human development in key habitats and biotic and abiotic stresses from climate change. Fourteen simple sequence repeats (SSRs), previously developed from the highbush blueberry (Vaccinium corymbosum) cultivar ‘Bluecrop’, were used to estimate genetic diversity and genetic differentiation of 67 diploid individuals from three species, including 19 V. elliottii, 12 V. fuscatum, and 35 V. darrowii accessions collected throughout the species’ ranges. Results from our analyses indicated that the samples from each species could be reliably resolved using genetic distance measures with ordination and neighbor joining approaches. In addition, we estimated admixture among these species by using Bayesian assignment tests, and were able to identify a mis-labeled accession of V. darrowii ‘Johnblue’, two mis-classified accessions (CVAC 735.001 and CVAC 1223.001), and four accessions of previously undescribed hybrid origin (CVAC 734.001, CVAC 1721.001, CVAC 1741.001, and Florida 4B CVAC 1790). Allele composition at the 14 SSRs confirmed that Florida 4B CVAC 1790, the donor of low chilling for the southern highbush blueberry, was the critical parent of US 74. Genetic diversity assessment and identification of these wild accessions are crucial for optimal germplasm management and expand opportunities to utilize natural variation in breeding programs.     

Some effects of montmorillonite on the growth of mixed microbial cultures

A defined starch urea-mineral-salts medium was used to study the effect of montmorillonite on the growth of pairs of microorganisms. The pairs of microorganisms used were Bacillus 3B-Klebsiella U34 and Bacillus 3B-Candida N2. When Bacillus 3B and Klebsiella U34 were grown together between montmorillonite concentrations of 0.4 mg·ml^?1 and 10 mg·ml^?1, linear relationships existed between logio (montmorillonite concentration) and the maximum yield of both Bacillus 3B and Klebsiella U34 and the final $\text{3B}\text{U34}$ ratio. At montmorillonite concentrations of greater than 10 mg·ml^?1 growth of both Bacillus 3B and Klebsiella U34 was reduced, possibly due to complete binding of starch to montmorillonite. Montmorillonite reduced the growth of Bacillus 3B and Candida N2 when they were grown together, possibly due to reduced urea hydrolysis.In these well defined systems, the analysis of the effects of clay minerals on microbial growth was complex. This complexity was due to the large number of points in the system at which the mineral could affect the system. The use of mathematical tools for analysis helped to quantify the exact relationships between Bacillus 3B, Klebsiella U34 and montmorillonite.