Cellular Responses of the Late Blight Pathogen Phytophthora infestans to Cyclic Lipopeptide Surfactants and Their Dependence on G Proteins

Oomycete pathogens cause major yield losses for many crop plants, and their control depends heavily on agrochemicals. Cyclic lipopeptides (CLPs) were recently discovered as a new class of natural compounds with strong activities against oomycetes. The CLP massetolide A (Mass A), produced by Pseudomonas fluorescens, has zoosporicidal activity, induces systemic resistance, and reduces late blight in tomato. To gain further insight into the modes of action of CLPs, the effects of Mass A on pore formation, mycelial growth, sporangium formation, and zoospore behavior were investigated, as was the involvement of G proteins in the sensitivity of Phytophthora infestans to Mass A. The results showed that Mass A induced the formation of transmembrane pores with an estimated size of between 1.2 and 1.8 nm. Dose-response experiments revealed that zoospores were the most sensitive to Mass A, followed by mycelium and cysts. Mass A significantly reduced sporangium formation and caused increased branching and swelling of hyphae. At relatively low concentrations, Mass A induced encystment of zoospores. It had no effect on the chemotactic response of zoospores but did adversely affect zoospore autoaggregation. A loss-of-function transformant of P. infestans lacking the G-protein α subunit was more sensitive to Mass A, whereas a gain-of-function transformant required a higher Mass A concentration to interfere with zoospore aggregation. Results indicate that Mass A disturbs various developmental stages in the life cycle of P. infestans and suggest that the cellular responses of P. infestans to this CLP are, in part, dependent on G-protein signaling.Oomycetes cause devastating diseases of plants and animals. They are fungal look-alikes that grow as mycelium and propagate via spores but evolved independently from fungi (23). Among the plant pathogens are over 80 Phytophthora species, with the late blight pathogen Phytophthora infestans being the most renowned (12, 16). Late blight control relies heavily on fungicides that contain copper-, tin-, phenylamide-, or cyanocetamide-oximes as active ingredients. Public concerns about the adverse effects of these fungicides on food safety and the environment have led to an increased demand for novel control strategies, preferably based on natural products. In recent years, the destructive effects of cyclic lipopeptides (CLPs) on zoospores of oomycete plant pathogens have attracted considerable attention (8, 9, 33). CLPs are produced by a variety of bacterial genera including Bacillus and Pseudomonas (13, 29, 30, 33, 37). They are composed of a fatty acid tail linked to an oligopeptide, which is cyclized by a lactone ring between two amino acids in the peptide chain. Based on the length and composition of the fatty acid as well as the number, type, and configuration (L-D form) of the amino acids in the peptide moiety, their activity may change (29, 30, 33). CLPs can be chemically produced, and via structural or genetic modifications, their physicochemical properties and antimicrobial activities can be altered (1). Pseudomonas fluorescens strain SS101 produces nine cyclic lipopeptide surfactants, with massetolide A (Mass A) being the main cyclic lipopeptide (7, 9). The others are derivatives of Mass A differing in the amino acid compositions of the peptide ring (7).One of the main modes of action of natural and synthetic CLPs is interference with the membrane integrity of the target organism, leading to pore formation and cytolysis (4, 5, 18, 19, 27, 34). For example, the CLPs Mass A and viscosin, produced by P. fluorescens strains SS101 and SBW25, respectively, act on membranes of zoospores of plant-pathogenic oomycetes, including Pythium and Phytophthora species, and this leads to the complete elimination of these propagules within 1 min of exposure (6-9). The destructive effects of Mass A on zoospores may explain, at least in part, the activity of P. fluorescens SS101 against Pythium root rot of flower bulb crops (6, 9) and tomato late blight caused by P. infestans (44). However, CLPs not only act on zoospores but may also inhibit mycelial growth of oomycetes and fungi (29, 33). The observations that several CLPs induce systemic resistance in plants against fungal and oomycete pathogens including P. infestans (31, 44) further emphasize their versatile activities and potential for crop protection. To explore and exploit the use of CLPs as a novel supplementary strategy for disease control, fundamental knowledge on their modes of action and the cellular responses of target oomycete pathogens is required.In this study, we investigated the response of P. infestans to the CLP Mass A produced by P. fluorescens SS101. We examined various growth stages of P. infestans and performed dose-response experiments to determine the effects of Mass A on mycelial growth, sporangium formation, cyst germination, and zoospore behavior, including chemotaxis, autoaggregation, and encystment. We also examined the involvement of the α subunit of the heterotrimeric G protein in the cellular responses of P. infestans to Mass A. The results show differential sensitivities of the various growth stages to Mass A and suggest that G-protein signaling plays a role in mediating the response.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Vegetation and Soil Environment Influence the Spatial Distribution of Root-Associated Fungi in a Mature Beech-Maple Forest

Although the level of diversity of root-associated fungi can be quite high, the effect of plant distribution and soil environment on root-associated fungal communities at fine spatial scales has received little attention. Here, we examine how soil environment and plant distribution affect the occurrence, diversity, and community structure of root-associated fungi at local patch scales within a mature forest. We used terminal restriction fragment length polymorphism and sequence analysis to detect 63 fungal species representing 28 different genera colonizing tree root tips. At least 32 species matched previously identified mycorrhizal fungi, with the remaining fungi including both saprotrophic and parasitic species. Root fungal communities were significantly different between June and September, suggesting a rapid temporal change in root fungal communities. Plant distribution affected root fungal communities, with some root fungi positively correlated with tree diameter and herbaceous-plant coverage. Some aspects of the soil environment were correlated with root fungal community structure, with the abundance of some root fungi positively correlated with soil pH and moisture content in June and with soil phosphorous (P) in September. Fungal distribution and community structure may be governed by plant-soil interactions at fine spatial scales within a mature forest. Soil P may play a role in structuring root fungal communities at certain times of the year.In temperate forests, most trees form relationships with ectomycorrhizal (ECM) fungi, and the diversity of this fungal group alone can approach 100 species within a forest stand (17, 20, 60). The ECM mutualism may be necessary for the success of some native plant species, as approximately 90% of roots of some tree species are colonized by ECM fungi (65). Nevertheless, we still know surprisingly little about what controls the community structure and distribution of root-associated fungi in forest systems (44, 46). The occurrence of root-associated fungi may broadly reflect soil environmental conditions and the presence of preferred plant hosts (28, 61), but how these factors interact to influence the diversity, distribution, and community structure of these fungi within forest habitat patches at a local scale is uncertain.The distribution of root-associated fungi may be primarily a species response to local soil environmental conditions. For example, both the quality (i.e., nutrient content) and the quantity of soil organic matter are known to influence the diversity of ECM communities (18, 20, 32). ECM fungi also vary in drought tolerance (14, 36), resistance to fire (61, 65), and tolerance to soil acidity (19) and temperature (56). Changes in soil chemistry, especially as they relate to pH and the availability of nitrogen (N) and phosphorous (P), might favor selection of fungi most capable of tolerating environmental extremes (2, 28, 29).Plant distribution and identity may, however, play the strongest role in structuring the below-ground diversity of root-associated fungi. Many ECM fungi can colonize a wide range of plant species, and plant species can be host to a large number of ECM fungi (63), especially those in the families Russulaceae and Thelephoraceae (34, 35, 62). Moreover, some ECM fungi are also specific to certain tree species (e.g., Suillus and Rhizopogon species are specific to species in the family Pinaceae [38, 39]). At the local scale, fungal distribution and richness might be influenced by differences in root growth and architecture (30, 42), by the distance to the bole of the tree (11, 42, 49), or by the presence of neighboring trees (29, 64). Temporal changes in ECM communities could be associated with seasonal changes in plant physiology and phenology (3, 8, 17).An often overlooked factor influencing root-associated fungi of tree roots is the occurrence of herbaceous plant species within forest stands. Many species of parasitic, achlorophyllous angiosperms obtain carbon (C) from ECM fungi that colonize tree roots (43), and some autotrophic plants could also obtain C from ECM fungi during certain times of the year (58). Herbaceous plants also influence the cycling of nutrients, including N, P, and K (potassium) (31, 50), within forests, which could affect the distribution of root-associated fungi. Herbaceous plants can also produce secondary compounds that inhibit colonization of tree roots (68).In this study, we examine the effect of soil environment and plant distribution on root-associated fungi of tree roots in a mature beech-maple forest at two points in the growing season. We predict that plant distribution, both the distribution of host trees and that of herbaceous plants, influences fungi associated with tree roots in terms of both community structure and diversity. Molecular typing protocols, including a site-specific database of fungal sequences and fingerprints, were used to identify fungi on tree roots (i.e., beech or maple trees) to the species level.     

Influence of Pythium oligandrum Biocontrol on Fungal and Oomycete Population Dynamics in the Rhizosphere

Fungal and oomycete populations and their dynamics were investigated following the introduction of the biocontrol agent Pythium oligandrum into the rhizosphere of tomato plants grown in soilless culture. Three strains of P. oligandrum were selected on the basis of their ability to form oospores (resting structures) and to produce tryptamine (an auxin-like compound) and oligandrin (a glycoprotein elicitor). Real-time PCR and plate counting demonstrated the persistence of large amounts of the antagonistic oomycete in the rhizosphere throughout the cropping season (April to September). Inter-simple-sequence-repeat analysis of the P. oligandrum strains collected from root samples at the end of the cropping season showed that among the three strains used for inoculation, the one producing the smallest amount of oospores was detected at 90%. Single-strand conformational polymorphism analysis revealed increases in the number of members and the complexity of the fungal community over time. There were no significant differences between the microbial ecosystems inoculated with P. oligandrum and those that were not treated, except for a reduction of Pythium dissotocum (ubiquitous tomato root minor pathogen) populations in inoculated systems during the last 3 months of culture. These findings raise interesting issues concerning the use of P. oligandrum strains producing elicitor and auxin molecules for plant protection and the development of biocontrol.In soilless cultures, the recycling of drainage water within a system is the consequence of new laws concerning water saving and limitation of pollution. Such closed systems minimize costs by conserving water and reducing fertilizer input; however, they may favor the dissemination of pathogens (13). When pathogens manage to enter recirculation systems, they are rapidly disseminated and may cause disease epidemics, particularly during periods of stress, e.g., stress due to high temperatures and/or to low levels of dissolved oxygen in the nutrient solution. Thus, numerous facultative pathogens commonly found in conventional cultures may become economically significant (53). Several of them, e.g., Pythium spp. and Phytophthora spp., are well adapted to the aquatic environment of hydroponic systems: they produce flagellate zoospores which enable them to swim in the nutrient solution, facilitating the spread of infection (18, 21, 36, 54, 61).Various methods are used to reduce the risks to plant health. Over recent years, the disinfection of nutrient solutions by physical or chemical treatments, e.g., ozonization, UV irradiation, chlorination, and thermo-disinfection, has been developed (13, 38). These methods effectively destroy pathogenic microorganisms but are harmful to species liable to benefit the plant, to be used as biocontrol agents, or both. Indeed, recirculation of nutrient solutions in closed hydroponic systems favors the establishment of a potentially suppressive microflora besides the pathogenic microflora (16, 28, 39, 41). The development of a beneficial microflora may thus be impeded by treatments used to destroy pathogenic microorganisms. Consequently, interest has been focused on the management of microorganisms in soilless cultures (12). Postma and coworkers (40) found that the extent of root disease is increased by the use of autoclaved rock wool. Tu and coworkers (59) observed that root rot disease was less severe in closed hydroponic systems than in open cultures and suggested that the difference was due to a higher density of bacteria in the closed systems. According to Paulitz (34), the diversity of microorganisms in soilless cultures is more limited than that in conventional soil cultures, such that conditions are more suitable for beneficial microorganisms, and consequently for effective biological control, in soilless than in conventional soil cultures.Biocontrol strategies are promising (7, 35). However, both biotic and abiotic factors may affect the performance of biocontrol methods. Relevant biotic factors include interactions with nontarget microorganisms (6), poor implantation of the biocontrol agent due to nonadaptation to the hydroponic system or resistance from the native microflora, shelf life and formulation, and host plant species and cultivar effects. Abiotic factors include climatic, chemical, and physical conditions of the soil or rhizosphere.Despite the limitations, various studies report evidence of the suppression of disease following the inoculation of hydroponic systems with antagonistic microorganisms. In particular, Pythium oligandrum is an effective biocontrol agent (2, 14, 49, 64). This oomycete colonizes roots without damaging the host plant cells (24, 45) and survives in the rhizosphere, where it exerts its biocontrol (57). P. oligandrum acts through both direct effects (mycoparasitism, antibiosis, and competition for nutrients and space) and indirect effects (stimulation of plant defense reactions and plant growth promotion) (49). The operating effects seem to depend on the type of pathogenic fungi being controlled (3, 48, 49). Le Floch and coworkers suggested that mycoparasitism is not the main mode of action (23). Root colonization by P. oligandrum may induce systemic resistance associated with the synthesis of elicitors protecting the plant from its aggressors (4, 17, 31, 37, 56). Several studies have investigated formulations of P. oligandrum oospores applied to soil or seeds, and their production and use, to optimize the efficacy of biocontrol (9, 30).Effective biocontrol by P. oligandrum may be limited by its heterogeneous implantation in the rhizosphere (46). Therefore, enhanced implantation and persistence of P. oligandrum in the rhizosphere should improve plant protection. We report an investigation of the persistence of P. oligandrum and its impact on the native fungal microflora of the roots. Three strains with characteristic traits were selected to constitute an inoculum applied to tomato plant roots. The characteristics of the strains were the production of oospores to allow root colonization and favor persistence, the synthesis of tryptamine, a plant growth enhancer (22), and the production of oligandrin, a plant-protective elicitor (37). The inoculated rhizospheres were monitored to evaluate the persistence of the strains and their effects on the microflora. The populations of the common tomato root pathogen P. dissotocum (endemic in the studied systems) and of P. oligandrum were both assessed by plate counting and real-time PCR. The strain(s) of P. oligandrum responsible for the colonization of the rhizosphere was identified by inter-simple-sequence-repeat (ISSR) methodology. Single-strand conformational polymorphism (SSCP) investigations were used to study the effects of P. oligandrum on the fungal populations colonizing the rhizosphere and the fungal dynamics throughout the cropping season.     

Calcium Oxalate Biomineralization by Piloderma fallax in Response to Various Levels of Calcium and Phosphorus

Piloderma fallax is an ectomycorrhizal fungus commonly associated with several conifer and hardwood species. We examined the formation of calcium oxalate crystals by P. fallax in response to calcium (0.0, 0.1, 0.5, 1, and 5 mM) and phosphorus (0.1 and 6 mM) additions in modified Melin-Norkrans agar medium. Both calcium and phosphorus supplementation significantly affected the amount of calcium oxalate formed. More calcium oxalate was formed at high P levels. Concentrations of soluble oxalate in the fungus and medium were higher at low P levels. There was a strong positive linear relationship between Ca level and calcium oxalate but only under conditions of phosphorus limitation. Calcium oxalate crystals were identified as the monohydrate form (calcium oxalate monohydrate [COM] whewellite) by X-ray diffraction analysis. Prismatic, styloid, and raphide forms of the crystals, characteristic COM, were observed on the surface of fungal hyphae by scanning electron microscopy. P. fallax may be capable of dissolving hyphal calcium oxalate under conditions of limited Ca. The biomineralization of calcium oxalate by fungi may be an important step in the translocation and cycling of Ca and P in soil.Many fungi from forest litter, including ectomycorrhizal fungi, exhibit calcium oxalate (CaOx) crystals on their hyphae. The ubiquity of CaOx crystals on fungal hyphae suggests that their formation may provide a selective advantage to the organism (4). CaOx formation is hypothesized to regulate intracellular pH and levels of oxalate and Ca and, hence, serves as a major sink for toxic amounts of Ca in soil and other environments (52, 53, 61). In plants, CaOx crystals have also been proposed to serve as a calcium source under conditions of calcium limitation (14, 18, 41), but such a process has yet to be established among fungi.CaOx on fungal hyphae is formed from soil-derived calcium and biologically synthesized oxalate. Oxalate released by ectomycorrhizae has been correlated with increased phosphorus bioavailability in the rhizosphere (V. Casarin, cited by Hinsinger in reference 25). The ability of oxalate to chelate metal ions makes it important in the solubilization and transport of metals in soil, the weathering and diagenesis of rocks and soil minerals (9, 23, 31, 57), and, consequently, the transport of nutrients. It is generally presumed that CaOx crystals form on the surface of fungal hyphae as a result of precipitation when released oxalic acid interacts with calcium cations (23, 43). However, the regularity of the CaOx crystals suggests that their formation is regulated and that they may be formed within the fungal hyphae at specific sites of origin (3, 5, 7).CaOx crystals vary in morphology, ranging from plates to raphides, druses, tetragonal bipyramids, and prisms. This variation in morphology can be seen among fungal genera and species (4). The crystals also usually occur either as CaOx monohydrate (COM; whewellite) (29) or CaOx dihydrate (weddellite) (3, 5, 28, 35, 60). Either crystal form or both may be present on fungal hyphae at the same time.In earlier studies (8, 9), we reported that Piloderma fallax is one of the major species of ectomycorrhizal fungi in subboreal forests. In addition, Piloderma sp. is found in temperate forest soils in association with conifer and hardwood species (34). Piloderma influences nutrient uptake and modifies mineral transformation in rock and soil systems (3, 33). In this study, we chose P. fallax because of (i) its ability to produce oxalate and form CaOx crystals (8, 56), (ii) its presence in many types of forest ecosystems, and (iii) its significant role in the breakdown and formation of soil minerals (9).The objective of this study was to quantify and characterize the formation of CaOx by P. fallax in response to various P and Ca levels in agar medium. We tested the hypothesis that P limitation will induce the production of oxalate and that increased concentrations of Ca will result in greater CaOx formation. This study also examined the dissolution of CaOx on P. fallax when it is grown on Ca-deficient medium and determined whether CaOx can serve as temporary Ca storage. Our study was conducted to add to knowledge of the ecological significance of CaOx, especially of its influence in biogeochemical cycling of P and Ca in soils.     

Diversity of Dissimilatory Sulfite Reductase Genes (dsrAB) in a Salt Marsh Impacted by Long-Term Acid Mine Drainage

Sulfate-reducing bacteria (SRB) play a major role in the coupled biogeochemical cycling of sulfur and chalcophilic metal(loid)s. By implication, they can exert a strong influence on the speciation and mobility of multiple metal(loid) contaminants. In this study, we combined DsrAB gene sequencing and sulfur isotopic profiling to identify the phylogeny and distribution of SRB and to assess their metabolic activity in salt marsh sediments exposed to acid mine drainage (AMD) for over 100 years. Recovered dsrAB sequences from three sites sampled along an AMD flow path indicated the dominance of a single Desulfovibrio species. Other major sequence clades were related most closely to Desulfosarcina, Desulfococcus, Desulfobulbus, and Desulfosporosinus species. The presence of metal sulfides with low δ³⁴S values relative to δ³⁴S values of pore water sulfate showed that sediment SRB populations were actively reducing sulfate under ambient conditions (pH of ∼2), although possibly within less acidic microenvironments. Interestingly, δ³⁴S values for pore water sulfate were lower than those for sulfate delivered during tidal inundation of marsh sediments. 16S rRNA gene sequence data from sediments and sulfur isotope data confirmed that sulfur-oxidizing bacteria drove the reoxidation of biogenic sulfide coupled to oxygen or nitrate reduction over a timescale of hours. Collectively, these findings imply a highly dynamic microbially mediated cycling of sulfate and sulfide, and thus the speciation and mobility of chalcophilic contaminant metal(loid)s, in AMD-impacted marsh sediments.Salt marshes exhibit high primary production rates (1, 101) and form biogeochemical “transition zones” for nutrient production, transport, and cycling between terrestrial and coastal marine environments (41, 66, 100). These zones also serve to reduce the flux of potentially toxic metals in contaminated groundwater to estuaries (12, 99, 106). Both functions depend strongly on microbial activity, especially that of sulfate-reducing bacteria (SRB) (42, 62, 67). SRB recycle much of the sedimentary organic carbon pool in marsh sediments (42-44) and indirectly inhibit production of the greenhouse gas methane (37, 71). They can restrict the mobility of dissolved contaminant metals by inducing precipitation of poorly soluble metal sulfides, and studies have examined their use in constructed wetlands to bioremediate acid mine drainage (AMD) and other metalliferous waste streams (11, 35, 40, 46, 50, 76, 90, 94, 104). However, the high acidity and metal concentrations inherent to AMD can inhibit SRB growth (15, 88, 98), and preferential growth of iron- and sulfur-oxidizing bacteria over SRB has been observed in some treatment wetlands (39).For natural salt marshes, 16S ribosomal nucleic acid- and phospholipid fatty acid (PLFA)-based analyses have shown that SRB commonly comprise a significant fraction of the microbial community (13, 24, 31, 34, 51, 58). Studies of salt marsh dissimilatory sulfite reductase genes (dsrAB), a highly conserved functional phylogenetic marker of prokaryotic sulfate reducers (49, 57, 102, 103, 107), have revealed both novel and deeply branching clades (3). Studies of mining-impacted sites at pH 2.0 to 7.8 (5, 7, 39, 70, 72, 77, 84), of soils and geothermal settings at a pH of ∼4 (55, 68), of metal-contaminated estuaries at pH 6.8 to 7.2 (65), and of hypersaline lakes at pH 7.5 (56) further outline the distribution and tolerance of specific groups and species of SRB under geochemically stringent conditions. Other findings point toward the existence of deltaproteobacteria in environments at a pH of ∼1 (10), although it is unknown if these include SRB. SRB diversity in salt marshes under long-term contamination by AMD has not been well investigated. Such studies may provide useful information for bioremediation projects in estuarine environments, as well as general insights into relationships between SRB physiology and the geochemistry of AMD.We studied the diversity of SRB, based on phylogenetic analysis of recovered DsrAB gene sequences (∼1.9 kb), in natural salt marsh sediments of the San Francisco Bay impacted by AMD for over 100 years. Sulfur isotope ratio and concentration measurements of pore water sulfate and metal sulfide minerals provided information about the spatial and temporal extent of active bacterial sulfate reduction (BSR) in sediment cores taken from specific sites along an AMD flow path. Collectively, the results revealed a tidal marsh system characterized by rapidly cycling bacterial sulfate reduction and sulfide reoxidation associated with oscillating tidal inundation and groundwater infiltration.     

Selection of a Bacillus pumilus Strain Highly Active against Ceratitis capitata (Wiedemann) Larvae

Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), the Mediterranean fruit fly (medfly), is one of the most important fruit pests worldwide. The medfly is a polyphagous species that causes losses in many crops, which leads to huge economic losses. Entomopathogenic bacteria belonging to the genus Bacillus have been proven to be safe, environmentally friendly, and cost-effective tools to control pest populations. As no control method for C. capitata based on these bacteria has been developed, isolation of novel strains is needed. Here, we report the isolation of 115 bacterial strains and the results of toxicity screening with adults and larvae of C. capitata. As a result of this analysis, we obtained a novel Bacillus pumilus strain, strain 15.1, that is highly toxic to C. capitata larvae. The toxicity of this strain for C. capitata was related to the sporulation process and was observed only when cultures were incubated at low temperatures before they were used in a bioassay. The mortality rate for C. capitata larvae ranged from 68 to 94% depending on the conditions under which the culture was kept before the bioassay. Toxicity was proven to be a special characteristic of the newly isolated strain, since other B. pumilus strains did not have a toxic effect on C. capitata larvae. The results of the present study suggest that B. pumilus 15.1 could be considered a strong candidate for developing strategies for biological control of C. capitata.The Mediterranean fruit fly (medfly), Ceratitis capitata, is considered a highly invasive agricultural and economically important pest throughout the world. In less than 200 years the range of this species has expanded from its native habitat in sub-Saharan Africa, and it has become a cosmopolitan species (26) that is present on five continents (14, 46). The wide distribution of the medfly is attributed, among other things, to its remarkably polyphagous behavior (more than 300 host plants have been reported) (43), to its resistance to cold climates (65), and to successful establishment after multiple introductions (30, 49) as a result of the increasing frequency of global trade (46).Medfly infestations cause serious economic losses and sometimes result in complete loss of crops (76). Numerous methods have been tried to control medfly populations, including chemical products, such as malathion and other organophosphate insecticides (4, 8), classic biological control programs based on the release of some of parasitoids and predators (38, 41, 44), toxic baits (2, 13, 31, 32, 35, 56), mass trapping systems (24, 51), the sterile insect technique (7, 34, 61, 63, 72, 73), and development of integrated strategies of management (71). In spite of all of these attempts, control of Mediterranean fruit fly populations has been ineffective, and losses associated with this pest worldwide are constantly increasing (21, 46).Insecticides based on microbial agents (bacteria, fungi, and viruses) are a promising alternative that has received a great deal of attention for control of C. capitata (5, 13, 18, 40, 55), but so far no such insecticide has reached a commercial stage. Among the microbial insecticides, bacteria are very successful agents in biological control programs (17, 29). The entomopathogenic bacteria belonging to the genus Bacillus are natural agents used for biological control of invertebrate pests and are the basis of many commercial insecticides. Three species of the genus Bacillus have been mass produced and commercialized: Bacillus sphaericus, Bacillus thuringiensis, and Paenibacillus popilliae (formerly Bacillus popilliae) (29, 54). These organisms have different spectra and levels of activity that are correlated with the nature of the toxins, which are very frequently produced during sporulation (16, 17). B. thuringiensis was the first Bacillus species used in biological control programs for pests and human vector disease insects (17, 62). During its stationary phase, this Gram-positive, aerobic, ubiquitous, endospore-forming bacterium produces parasporal crystalline inclusions composed mainly of two types of insecticidal proteins (Cry and Cyt toxins) (62) that are toxic to a variety of insects, in some cases at the species level.There have been some reports of B. thuringiensis strains active against other fruit flies (3, 37, 58, 59, 67), but there has been no report of any Bacillus strain with activity against C. capitata.The aim of this study was to search for novel bacteria belonging to the genus Bacillus, specifically B. thuringiensis, with activity against adults and larvae of C. capitata that could be used as biological control agents. Isolation of 115 bacterial strains, evaluation of the insecticidal activities of these strains, and identification of a novel strain of Bacillus pumilus that is highly toxic to C. capitata larvae are reported here. In addition, we found that toxicity was observed only when cultures of B. pumilus strain 15.1 were exposed to low temperatures. The isolation of this novel pathogenic strain could be important for future development of biotechnological strategies aimed at reducing the economic losses caused by C. capitata.     

In Situ Phylogenetic Structure and Diversity of Wild Bradyrhizobium Communities

Bacteria often infect their hosts from environmental sources, but little is known about how environmental and host-infecting populations are related. Here, phylogenetic clustering and diversity were investigated in a natural community of rhizobial bacteria from the genus Bradyrhizobium. These bacteria live in the soil and also form beneficial root nodule symbioses with legumes, including those in the genus Lotus. Two hundred eighty pure cultures of Bradyrhizobium bacteria were isolated and genotyped from wild hosts, including Lotus angustissimus, Lotus heermannii, Lotus micranthus, and Lotus strigosus. Bacteria were cultured directly from symbiotic nodules and from two microenvironments on the soil-root interface: root tips and mature (old) root surfaces. Bayesian phylogenies of Bradyrhizobium isolates were reconstructed using the internal transcribed spacer (ITS), and the structure of phylogenetic relatedness among bacteria was examined by host species and microenvironment. Inoculation assays were performed to confirm the nodulation status of a subset of isolates. Most recovered rhizobial genotypes were unique and found only in root surface communities, where little bacterial population genetic structure was detected among hosts. Conversely, most nodule isolates could be classified into several related, hyper-abundant genotypes that were phylogenetically clustered within host species. This pattern suggests that host infection provides ample rewards to symbiotic bacteria but that host specificity can strongly structure only a small subset of the rhizobial community.Symbiotic bacteria often encounter hosts from environmental sources (32, 48, 60), which leads to multipartite life histories including host-inhabiting and environmental stages. Research on host-associated bacteria, including pathogens and beneficial symbionts, has focused primarily on infection and proliferation in hosts, and key questions about the ecology and evolution of the free-living stages have remained unanswered. For instance, is host association ubiquitous within a bacterial lineage, or if not, do host-infecting genotypes represent a phylogenetically nonrandom subset? Assuming that host infection and free-living existence exert different selective pressures, do bacterial lineages diverge into specialists for these different lifestyles? Another set of questions addresses the degree to which bacteria associate with specific host partners. Do bacterial genotypes invariably associate with specific host lineages, and is such specificity controlled by one or both partners? Alternatively, is specificity simply a by-product of ecological cooccurrence among bacteria and hosts?Rhizobial bacteria comprise several distantly related proteobacterial lineages, most notably the genera Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium (52), that have acquired the ability to form nodules on legumes and symbiotically fix nitrogen. Acquisition of nodulation and nitrogen fixation loci has likely occurred through repeated lateral transfer of symbiotic loci (13, 74). Thus, the term “rhizobia” identifies a suite of symbiotic traits in multiple genomic backgrounds rather than a taxonomic classification. When rhizobia infect legume hosts, they differentiate into specialized endosymbiotic cells called bacteroids, which reduce atmospheric nitrogen in exchange for photosynthates from the plant (35, 60). Rhizobial transmission among legume hosts is infectious. Rhizobia can spread among hosts through the soil (60), and maternal inheritance (through seeds) is unknown (11, 43, 55). Nodule formation on hosts is guided by reciprocal molecular signaling between bacteria and plant (5, 46, 58), and successful infection requires a compatible pairing of legume and rhizobial genotypes. While both host and symbiont genotypes can alter the outcome of rhizobial competition for adsorption (34) and nodulation (33, 39, 65) of legume roots, little is known about how this competition plays out in nature.Rhizobia can achieve reproductive success via multiple lifestyles (12), including living free in the soil (14, 44, 53, 62), on or near root surfaces (12, 18, 19, 51), or in legume nodules (60). Least is known about rhizobia in bulk soil (not penetrated by plant roots). While rhizobia can persist for years in soil without host legumes (12, 30, 61), it appears that growth is often negligible in bulk soil (4, 10, 14, 22, 25). Rhizobia can also proliferate in the rhizosphere (soil near the root zone) of legumes (4, 10, 18, 19, 22, 25, 51). Some rhizobia might specialize in rhizosphere growth and infect hosts only rarely (12, 14, 51), whereas other genotypes are clearly nonsymbiotic because they lack key genes (62) and must therefore persist in the soil. The best-understood rhizobial lifestyle is the root nodule symbiosis with legumes, which is thought to offer fitness rewards that are superior to life in the soil (12). After the initial infection, nodules grow and harbor increasing populations of bacteria until the nodules senesce and the rhizobia are released into the soil (11, 12, 38, 40, 55). However, rhizobial fitness in nodules is not guaranteed. Host species differ in the type of nodules they form, and this can determine the degree to which differentiated bacteroids can repopulate the soil (11, 12, 38, 59). Furthermore, some legumes can hinder the growth of nodules with ineffective rhizobia, thus punishing uncooperative symbionts (11, 27, 28, 56, 71).Here, we investigated the relationships between environmental and host-infecting populations of rhizobia. A main objective was to test the hypothesis that rhizobia exhibit specificity among host species as well as among host microenvironments, specifically symbiotic nodules, root surfaces, and root tips. We predicted that host infection and environmental existence exert different selective pressures on rhizobia, leading to divergent patterns of clustering, diversity, and abundance of rhizobial genotypes.     

Role of Geobacter sulfurreducens Outer Surface c-Type Cytochromes in Reduction of Soil Humic Acid and Anthraquinone-2,6-Disulfonate

Deleting individual genes for outer surface c-type cytochromes in Geobacter sulfurreducens partially inhibited the reduction of humic substances and anthraquinone-2,6,-disulfonate. Complete inhibition was obtained only when five of these genes were simultaneously deleted, suggesting that diverse outer surface cytochromes can contribute to the reduction of humic substances and other extracellular quinones.Humic substances can play an important role in the reduction of Fe(III), and possibly other metals, in sedimentary environments (6, 34). Diverse dissimilatory Fe(III)-reducing microorganisms (3, 5, 7, 9, 11, 19-22, 25) can transfer electrons onto the quinone moieties of humic substances (38) or the model compound anthraquinone-2,6-disulfonate (AQDS). Reduced humic substances or AQDS abiotically reduces Fe(III) to Fe(II), regenerating the quinone. Electron shuttling in this manner can greatly increase the rate of electron transfer to insoluble Fe(III) oxides, presumably because soluble quinone-containing molecules are more accessible for microbial reduction than insoluble Fe(III) oxides (19, 22). Thus, catalytic amounts of humic substances have the potential to dramatically influence rates of Fe(III) reduction in soils and sediments and can promote more rapid degradation of organic contaminants coupled to Fe(III) reduction (1, 2, 4, 10, 24).To our knowledge, the mechanisms by which Fe(III)-reducing microorganisms transfer electrons to humic substances have not been investigated previously for any microorganism. However, reduction of AQDS has been studied using Shewanella oneidensis (17, 40). Disruption of the gene for MtrB, an outer membrane protein required for proper localization of outer membrane cytochromes (31), inhibited reduction of AQDS, as did disruption of the gene for the outer membrane c-type cytochrome, MtrC (17). However, in each case inhibition was incomplete, and it was suggested that there was a possibility of some periplasmic reduction (17), which would be consistent with the ability of AQDS to enter the cell (40).The mechanisms for electron transfer to humic substances in Geobacter species are of interest because molecular studies have frequently demonstrated that Geobacter species are the predominant Fe(III)-reducing microorganisms in sedimentary environments in which Fe(III) reduction is an important process (references 20, 32, and 42 and references therein). Geobacter sulfurreducens has routinely been used for investigations of the physiology of Geobacter species because of the availability of its genome sequence (29), a genetic system (8), and a genome-scale metabolic model (26) has made it possible to take a systems biology approach to understanding the growth of this organism in sedimentary environments (23).     

Bat Guano Virome: Predominance of Dietary Viruses from Insects and Plants plus Novel Mammalian Viruses

Bats are hosts to a variety of viruses capable of zoonotic transmissions. Because of increased contact between bats, humans, and other animal species, the possibility exists for further cross-species transmissions and ensuing disease outbreaks. We describe here full and partial viral genomes identified using metagenomics in the guano of bats from California and Texas. A total of 34% and 58% of 390,000 sequence reads from bat guano in California and Texas, respectively, were related to eukaryotic viruses, and the largest proportion of those infect insects, reflecting the diet of these insectivorous bats, including members of the viral families Dicistroviridae, Iflaviridae, Tetraviridae, and Nodaviridae and the subfamily Densovirinae. The second largest proportion of virus-related sequences infects plants and fungi, likely reflecting the diet of ingested insects, including members of the viral families Luteoviridae, Secoviridae, Tymoviridae, and Partitiviridae and the genus Sobemovirus. Bat guano viruses related to those infecting mammals comprised the third largest group, including members of the viral families Parvoviridae, Circoviridae, Picornaviridae, Adenoviridae, Poxviridae, Astroviridae, and Coronaviridae. No close relative of known human viral pathogens was identified in these bat populations. Phylogenetic analysis was used to clarify the relationship to known viral taxa of novel sequences detected in bat guano samples, showing that some guano viral sequences fall outside existing taxonomic groups. This initial characterization of the bat guano virome, the first metagenomic analysis of viruses in wild mammals using second-generation sequencing, therefore showed the presence of previously unidentified viral species, genera, and possibly families. Viral metagenomics is a useful tool for genetically characterizing viruses present in animals with the known capability of direct or indirect viral zoonosis to humans.Bats belong to one of the most diverse, abundant, and widely distributed group of mammals. More than 1,100 bat species belong to the order of Chiroptera, representing approximately 20% of all mammalian species (54). Most bat species feed on insects and other arthropods, while others feed on fruit nectar, bird or mammal blood, and small vertebrates such as fish, frogs, mice, and birds (30). Of the 47 species of bats reported in the United States, most of them are insectivorous (http://www.batcon.org/).Bats are considered the natural reservoir of a large variety of zoonotic viruses causing serious human diseases such as lyssaviruses, henipaviruses, severe acute respiratory syndrome coronavirus, and Ebola virus (6, 38, 46, 59, 63, 65). Characteristics of bats, including their genetic diversity, broad geological distribution, gregarious habits, high population density, migratory habits, and long life span (30, 58), likely endow them with the ability to host diverse viruses, some of which are also able to infect humans and other mammals (41, 63).More than 80 virus species have been isolated or detected in bats using nucleic acid-based methods (6, 38, 59, 65). Viruses that have been recently discovered in bats include astroviruses, adeno-associated viruses (AAVs), adenoviruses, herpesviruses, and polyomavirus (8, 9, 13, 31, 32, 35, 37, 39, 40, 42, 61, 62, 68). For example, it was recently reported that a newly identified adenovirus isolated from bat guano was capable of infecting various vertebrate cell lines, including those of humans, monkeys, dogs, and pigs (35). With increasing human populations in previously wild areas, contact of bats with humans and with wild and domestic animals has increased, providing greater opportunities for cross-species transmissions of potentially pathogenic bat viruses. To better understand the range of viruses carried by bats, we undertook an initial characterization of the guano viromes of several common bat species in the United States.The development of massively parallel sequencing technology makes is possible to reveal uncultured viral assemblages within biological or environmental samples (11, 28). To date, this approach has been used to characterize viruses in equine feces (7), human blood (5), tissue (14), human feces (3, 4, 15, 45, 60, 67), and human respiratory secretions (64), which in turn has facilitated the discovery of many novel viruses (18, 20, 25, 33, 47, 50). In the present study, we analyzed the viruses present in guano from several bat species in California and Texas, using sequence-independent PCR amplification, pyrosequencing, and sequence similarity searches.     

Use of Ichip for High-Throughput In Situ Cultivation of “Uncultivable” Microbial Species

One of the oldest unresolved microbiological phenomena is why only a small fraction of the diverse microbiological population grows on artificial media. The “uncultivable” microbial majority arguably represents our planet''s largest unexplored pool of biological and chemical novelty. Previously we showed that species from this pool could be grown inside diffusion chambers incubated in situ, likely because diffusion provides microorganisms with their naturally occurring growth factors. Here we utilize this approach and develop a novel high-throughput platform for parallel cultivation and isolation of previously uncultivated microbial species from a variety of environments. We have designed and tested an isolation chip (ichip) composed of several hundred miniature diffusion chambers, each inoculated with a single environmental cell. We show that microbial recovery in the ichip exceeds manyfold that afforded by standard cultivation, and the grown species are of significant phylogenetic novelty. The new method allows access to a large and diverse array of previously inaccessible microorganisms and is well suited for both fundamental and applied research.It has been known for over a century that the overwhelming majority of microbial species do not grow on synthetic media in vitro and remain unexplored (13, 32, 37, 39, 40, 43). The rRNA and metagenomics approaches demonstrated a spectacular diversity of these uncultivated species (11, 21, 25-27, 30, 36). Accessing this “missing” microbial diversity is of significant interest for both basic and applied sciences and has been recognized as one of the principal challenges for microbiology today (12, 29, 41). In recent years, technical advances in cultivation methodologies have recovered a diverse set of ecologically relevant species (1, 3, 5, 7, 15, 20, 24, 28, 33, 42). However, by and large the gap between microbial diversity in nature and that in culture collections remains unchanged, and most microbial phyla still have no cultivable representatives (25, 29). Earlier, we developed a novel method of in situ cultivation of environmental microorganisms inside diffusion chambers (15). The rationale for such an approach was that diffusion would provide cells inside the chamber with naturally occurring growth components and enable those species that grew in nature at the time of the experiment to also grow inside the diffusion chambers. Expectedly, this method yields a rate of microbial recovery many times larger than those of standard techniques. Even so, this method is laborious and does not allow an efficient, high-throughput isolation of microbial species en masse. This limits the method''s applicability, for example, in the drug discovery effort. Here we transform this methodology into a high-throughput technology platform for massively parallel cultivation of “uncultivable” species. Capitalizing on earlier microfluidics methods developed for microbial storage and screening (4, 16), we have designed and tested an isolation chip, or ichip for short, which consists of hundreds of miniature diffusion chambers. If each diffusion minichamber is loaded with a single cell, the resulting culture is monospecific. The ichip thus allows microbial growth and isolation into pure culture in one step. Here we demonstrate that cultivation of environmental microorganisms inside the ichip incubated in situ leads to a significantly increased colony count over that observed on synthetic media. Perhaps even more significantly, species grown in ichips are different from those registered in standard petri dishes and are highly novel.