Rivers, creeks, streams are integrators of biological, chemical and physical processes occurring in a catchment linking land cover from the headwaters to the outlet. The dynamics of human and animal pathogens in catchments have been widely studied in a large variety of contexts allowing the optimization of disease risk reduction. In parallel, there is an emerging awareness that crop pathogens might also be disseminated via surface waters especially when they are used for irrigation. However, there are no studies on the extent to which potential plant pathogens are present – nor about their dynamics - along the full course of a catchment. Here we have compared the seasonal dynamics of populations of the Pseudomonas syringae (Psy) and the Soft Rot Pectobacteriaceae (SRP) species complexes along a 270 km stretch of the Durance River from the upstream alpine reaches to the downstream agricultural production areas at the confluence with the Rhone River at Avignon. Among 168 samples collected at 21 sites in fall, winter, spring and summer of 2016 and 2017, Psy strains were detected at all sampling sites and in 156 of the samples at population densities up to 105 bacteria L-1. In contrast, SRP strains were detected in 98 of the samples, mostly from the southern part of the river, at population densities that did not exceed 3 ´ 104 bacteria L-1. Among the biological and chemical parameters that were characterized at each sampling site, temperature was the only factor that explained a significant amount of the variability in population size for both species complexes. Psy densities decreased with increasing temperature whereas SRP densities increased with increasing temperature. River-borne populations of SRP were composed mainly of Pectobacterium versatile and P. aquaticum that have little known epidemiological importance. Only a few strains of Pectobacterium and Dickeya species reputed for their epidemiological impact were observed. In contrast, Psy populations at all sites were dominated by a genetic lineage of phylogroup 2 known from other studies for its broad host range and its geographic and habitat ubiquity. Our observations suggest that surveillance of river water for SRP could be leveraged to signal diagnostic and management reactions to avoid disease outbreaks. In contrast, the constant presence of Psy throughout the catchment in absence of regular and widespread disease outbreaks due to this group of bacteria suggests that surveillance should focus on future changes in land use, river water conditions and agronomic practices that could destabilize the mechanisms currently holding Psy outbreaks in check.

ABSTRACTRivers, creeks, streams are integrators of biological, chemical and physical processes occurring in a catchment linking land cover from the headwaters to the outlet. The dynamics of human and animal pathogens in catchments have been widely studied in a large variety of contexts allowing the optimization of disease risk reduction. In parallel, there is an emerging awareness that crop pathogens might also be disseminated via surface waters especially when they are used for irrigation. However, there are no studies on the extent to which potential plant pathogens are present – nor about their dynamics - along the full course of a catchment. Here we have compared the seasonal dynamics of populations of thePseudomonas syringae(Psy) and the Soft RotPectobacteriaceae(SRP) species complexes along a 270 km stretch of the Durance River from the upstream alpine reaches to the downstream agricultural production areas at the confluence with the Rhone River at Avignon. Among 168 samples collected at 21 sites in fall, winter, spring and summer of 2016 and 2017, Psy strains were detected at all sampling sites and in 156 of the samples at population densities up to 105bacteria L−1. In contrast, SRP strains were detected in 98 of the samples, mostly from the southern part of the river, at population densities that did not exceed 3 × 104bacteria L−1. Among the biological and chemical parameters that were characterized at each sampling site, temperature was the only factor that explained a significant amount of the variability in population size for both species complexes. Psy densities decreased with increasing temperature whereas SRP densities increased with increasing temperature. River-borne populations of SRP were composed mainly ofPectobacterium versatileandP. aquaticumthat have little known epidemiological importance. Only a few strains ofPectobacteriumandDickeyaspecies reputed for their epidemiological impact were observed. In contrast, Psy populations at all sites were dominated by a genetic lineage of phylogroup 2 known from other studies for its broad host range and its geographic and habitat ubiquity. Our observations suggest that surveillance of river water for SRP could be leveraged to signal diagnostic and management reactions to avoid disease outbreaks. In contrast, the constant presence of Psy throughout the catchment in absence of regular and widespread disease outbreaks due to this group of bacteria suggests that surveillance should focus on future changes in land use, river water conditions and agronomic practices that could destabilize the mechanisms currently holding Psy outbreaks in check.

Methods to ensure the health of crops owe their efficacy to the extent to which we understand the ecology and biology of environmental microorganisms and the conditions under which their interactions with plants lead to losses in crop quality or yield. However, in the pursuit of this knowledge, notions of the ecology of plant-pathogenic microorganisms have been reduced to a plant-centric and agro-centric focus. With increasing global change, i.e. changes that encompass not only climate, but also biodiversity, the geographical distribution of biomes, human demographic and socio-economic adaptations and land use, new plant health problems will emerge via a range of processes influenced by these changes. Hence, knowledge of the ecology of plant pathogens will play an increasingly important role in the anticipation and response to disease emergence. Here, we present our opinion on the major challenges facing the study of the ecology of plant-pathogenic bacteria. We argue that the discovery of markedly novel insights into the ecology of plant-pathogenic bacteria is most likely to happen within a framework of more extensive scales of space, time and biotic interactions than those that currently guide much of the research on these bacteria. This will set a context that is more propitious for the discovery of unsuspected drivers of the survival and diversification of plant-pathogenic bacteria and of the factors most critical for disease emergence, and will set the foundation for new approaches to the sustainable management of plant health. We describe the contextual background of, justification for and specific research questions with regard to the following challenges: Development of terminology to describe plant-bacterial relationships in terms of bacterial fitness. Definition of the full scope of the environments in which plant-pathogenic bacteria reside or survive. Delineation of pertinent phylogenetic contours of plant-pathogenic bacteria and naming of strains independent of their presumed life style. Assessment of how traits of plant-pathogenic bacteria evolve within the overall framework of their life history. Exploration of possible beneficial ecosystem services contributed to by plant-pathogenic bacteria.

The assessment of the diversity of populations with low abundances in soil, as are most of the aerobic endospore-forming bacteria, is a real challenge in modern studies of microbial ecology. Besides the culture-independent approaches reported in De Vos, Studying the bacterial diversity of the soil by culture-independent approaches. In: Logan NA (ed) Endospore forming soil bacteria (Soil Biology 27). Springer, Heidelberg, 2011, doi:10.1007/978-3-642-19577-3, cultivation-based methods are still useful, as they allow the description of novel bacterial species, as well as extensive biological studies and biotechnological exploitation of the isolated strains. The common traditional approach to isolation, through spore selection followed by growth in selective conditions, is a very efficient strategy and can give much information on cultivable populations living in soils and other environments. To enlarge our view of the diversity of endospore-formers in soils, this chapter reviews the use and evaluation of methods with and without spore selection, including immunocapture or selective media in combination with molecular techniques.

Minimal standards for describing new taxa within the aerobic endospore-forming bacteria are proposed, following Recommendation 30b of the Bacteriological Code (1990 Revision). These minimal standards are recommended as guidelines to assist authors in the preparation of descriptions for novel taxa. They encourage broad polyphasic characterization and the construction of descriptions that are practically useful in routine diagnostic laboratories. The proposals have been endorsed by the Subcommittee on the Taxonomy of the Genus Bacillus and Related Organisms of the International Committee on Systematics of Prokaryotes.

The aim of this study was to investigate the role of microorganisms on the behaviour of selenium in natural soil maintained under strictly aerobic conditions. Six-day batch experiments were performed with soils constrained to different microbiological states, either by sterilisation or by adding organic substrates. Selenium was added to the soil as selenite. The distribution of selenium in the gaseous, liquid and solid phases of the batch was measured. Selenium partitioning between the various solid phases was investigated by chemical sequential extractions. Active microorganisms played major effects on the distribution of selenium within the soil. On the one hand, microorganisms could promote selenium volatilisation (in relatively small amounts), leading to the spreading of selenium compounds outside the soil. On the other hand, microbial activities increased both amount of selenium retained by the soil and the strength of its retention (less exchangeable selenium), making selenium less susceptible to remobilisation.

Effect of soil salinity on physico-chemical and biological properties renders the salt-affected soils unsuitable for soil microbial processes and growth of the crop plants. Soil aggregation around roots of the plants is a function of the bacterial exo-polysaccharides (EPS), however, such a role of the EPS-producing bacteria in the saline environments has rarely been investigated. Pot experiments were conducted to observe the effects of inoculating six strains of EPS-producing bacteria on growth of primary (seminal) roots and its relationship with saccharides, cations (Ca 2+ , Na + , K + ) contents and mass of rhizosheath soils of roots of the wheat plants grown in a salt-affected soil. A strong positive relationship of RS with different root growth parameters indicated that an integrated influence of various biotic and abiotic RS factors would have controlled and promoted growth of roots of the inoculated wheat plants. The increase in root growth in turn could help inoculated wheat plants to withstand the negative effects of soil salinity through an enhanced soil water uptake, a restricted Na + influx in the plants and the accelerated soil microbial process involved in cycling and availability of the soil nutrients to the plants. It was concluded that inoculation of the EPS-producing would be a valuable tool for amelioration and increasing crop productivity of the salt-affected soils.

During a study in the Argentinian region of Chaco (Cordoba), some strains were isolated from the rhizosphere of grasses growing in semi-desertic arid soils. Two of these strains, one isolated from the rhizospheric soil of Chloris ciliata (strain CH01(T)) and the other from Pappophorum caespitosum (strain PA01), were Gram-negative, strictly aerobic rods, which formed yellow round colonies on nutrient agar. They produced a water-insoluble yellow pigment, and a fluorescent pigment was also detected. A polyphasic taxonomic approach was used to characterize the strains. Comparison of the 16S rRNA gene sequences showed a similarity of 99.3 % between them, and phylogenetic analysis revealed that the strains belong to the genus Pseudomonas, within the gamma-subclass of the Proteobacteria. The closest related species is Pseudomonas straminea IAM 1598(T) (similarity of 99.0 % to strain CH01(T) and 98.8 % to strain PA01), clustering in a separate branch with the various methods of tree building used. Strains CH01(T) and PA01 both had a single polar flagellum, like other yellow pigment-producing pseudomonads related to them. Both strains produced catalase and oxidase. Similar to P. straminea, they did not hydrolyse gelatin or casein. The G+C DNA contents determined were 57.5 mol% for CH01(T) and 58.0 mol% for PA01. DNA-DNA hybridization results showed 81 % relatedness between them, and only 40-44 % relatedness with respect to the type strain of P. straminea. These results, together with other phenotypic characteristics, support the conclusion that both isolates belong to the same species, and should be described as representing a novel species within the genus Pseudomonas, for which the name Pseudomonas argentinensis sp. nov. is proposed. The type strain is CH01(T) (=LMG 22563(T) = CECT 7010(T)).

SUMMARY Research interest in microbial biodiversity over the past 25 years has increased markedly as microbiologists have become interested in the significance of biodiversity for ecological processes and as the industrial, medical, and agricultural applications of this diversity have evolved. One major challenge for studies of microbial habitats is how to account for the diversity of extremely large and heterogeneous populations with samples that represent only a very small fraction of these populations. This review presents an analysis of the way in which the field of microbial biodiversity has exploited sampling, experimental design, and the process of hypothesis testing to meet this challenge. This review is based on a systematic analysis of 753 publications randomly sampled from the primary scientific literature from 1975 to 1999 concerning the microbial biodiversity of eight habitats related to water, soil, plants, and food. These publications illustrate a dominant and growing interest in questions concerning the effect of specific environmental factors on microbial biodiversity, the spatial and temporal heterogeneity of this biodiversity, and quantitative measures of population structure for most of the habitats covered here. Nevertheless, our analysis reveals that descriptions of sampling strategies or other information concerning the representativeness of the sample are often missing from publications, that there is very limited use of statistical tests of hypotheses, and that only a very few publications report the results of multiple independent tests of hypotheses. Examples are cited of different approaches and constraints to experimental design and hypothesis testing in studies of microbial biodiversity. To prompt a more rigorous approach to unambiguous evaluation of the impact of microbial biodiversity on ecological processes, we present guidelines for reporting information about experimental design, sampling strategies, and analyses of results in publications concerning microbial biodiversity.

Sixteen Gram-positive endospore-forming bacteria previously isolated from soil, plant rhizospheres, plant roots and pasteurized pureed vegetables were studied to determine their taxonomic positions. The isolates were formerly identified as Bacillus circulans based on their biochemical characters using API galleries. Two of these strains, RSA19(T) and TOD45(T), were recently assigned to the genus Paenibacillus based on phylogenetic analysis of their 16S rRNA (rrs) gene sequence. In the present work, the sixteen isolates were assigned to two genomospecies using DNA-DNA hybridization, in agreement with rrs gene sequence analysis. These genomospecies can also be differentiated on the basis of their cultural and biochemical characters into two novel species, for which the names Paenibacillus graminis sp. nov. (type strain RSA19(T) = ATCC BAA-95(T) = LMG 19080(T)) and Paenibacillus odorifer sp. nov. (type strain TOD45T = ATCC BAA-93(T) = LMG 19079(T)) are proposed.

ABSTRACT One hundred nineteen isolates from a commercial zucchini purée stored at 4, 10, and 20 to 25°C were fingerprinted using repetitive sequence-based PCR (REP-PCR) and classified into 35 REP types. One representative isolate of each REP type was subsequently identified by API50CHB/20E profile and partial rrs gene sequence analysis. Nine REP types were misidentified by the API system. Strains were misidentified as being in the Bacillus circulans (group 2) API taxon or in taxa with a low number of positive API characters such as Brevibacillus brevis . A phylogenetic analysis pointed to one new species of Bacillus and three new species of Paenibacillus among the misidentified REP types. Bacterial components in zucchini purée were compared phenotypically with those obtained in previous work on broccoli, carrot, leek, potato, and split pea purées, based on simple matching coefficient and unweighted pair group method with averages cluster analysis. Out of 254 strains, 69 strains previously identified as B. circulans (group 2) or B. circulans/B. macerans/B. polymyxa were assigned to a new Paenibacillus taxon phylogenetically related to P. azotofixans . Storage conditions at 4°C favored the development of “ B. macroides/B. maroccanus ” and Paenibacillus spp. in zucchini purées and Paenibacillus spp. in other purées. Storage conditions at 20 to 25°C favored the development of B. subtilis group ( B. licheniformis and B. subtilis ) and B. cereus group strains. At 10°C, Paenibacillus spp. were always present at high frequencies, whereas the occurrence of B. macroides/B. maroccanus (in zucchini purées), B. cereus , and B. pumilus varied with the experiment.

TVV75, a strain of Burkholderia vietnamiensis, was isolated from an acid sulphate soil of south Vietnam, and selected for its high in vitro nitrogen fixation potential. This plant growth-promoting rhizobacterium (PGPR) had been used in a previously reported pot experiment. It was used in two new pot experiments and four field experiments to inoculate lowland rice at sowing and at transplanting, in three different South Vietnam acid sulphate soils. We first studied the effect of inoculation during early plant growth in nurseries. Seedlings were then transplanted both to field and pots. Treatments included two levels of inoculation (inoculated vs uninoculated) and three levels of N fertilizer (0, recommended rate and half this rate), in a randomized block design with six replicates. In all four experiments nitrogen appeared to be the limiting factor for yield. Inoculation had already had a strong beneficial effect at the transplanting stage (day 24), as measured by shoot weight (+33%) root weight (+57%), and leaf surface (+30% at day 14). Final results indicated that inoculation of rice with B. vietnamiensis TVV75 significantly increased several yield components, resulting in a final 13 to 22% increase in grain yield. A late yield component, 1,000 grain weight, was significantly increased by inoculation, but not by nitrogen fertilizers, in all pot and field experiments, indicating a long-lasting effect of the inoculated bacteria. It was possible to evaluate the nitrogen fertilizer equivalent of inoculation (NFEI): at the medium rate of N fertilizer, inoculation ensured a yield equivalent to that obtained in the uninoculated control with 25 to 30 kg more nitrogen fertilizer. Comparison of the local cost of NFEI kg N-fertilizer and the cost of inoculation would help in making the decision to inoculate.

Inoculation of wheat roots with Paenibacillus (formerly Bacillus) polymyxa CF43 increases the mass of root-adhering soil. We tested the role of levan, a fructosyl polymer produced by strain CF43, in the aggregation of soil adhering to wheat roots. The P. polymyxa gene homologous to the Bacillus subtilis sacB gene encoding levansucrase was cloned and sequenced. The corresponding gene product synthesises high molecular weight levan. A P. polymyxa mutant strain, SB03, whose sacB gene is disrupted, was constructed using heterogramic conjugation. Effects of wheat inoculation with the wild type and the mutant strain were compared using two different cultivated silt loam soils in four independent pot experiments. Roots of wheat plantlets inoculated with CF43 or SB03 were colonized after 7-14 days at the same level, and root and shoot masses were not significantly different from those of the non-inoculated control plants. The ratio of root-adhering soil dry mass to root tissue dry mass was significantly higher for plants inoculated with strain CF43 than for those inoculated with mutant strain SB03: +30% in Orgeval soil and +100% in Dieulouard soil. Thus the levan produced by P. polymyxa is implicated in the aggregation of root-adhering soil on wheat.

A large collection of bacterial strains, immunotrapped from soil and from the wheat rhizoplane, was subjected to polyphasic taxonomy by examining various pheno- and genotypic parameters. Strains were grouped on (inter) repetitive extragenic palindromic DNA (REP) PCR profiles at the intraspecies level. Pheno- and genotypic characters were assessed for representatives from 13 different REP groups. Strains of nine REP groups constituting two physiological BIOLOG clusters fell in the coherent DNA-DNA reassociation group of Ochrobactrum anthropi. Strains of two REP groups constituting a separate BIOLOG cluster fell in the coherent DNA-DNA reassociation group of Ochrobactrum intermedium. Additional phenotypic characters differentiating O. anthropi and O. intermedium were found. REP group K strains constituted a different BIOLOG cluster, a separate DNA-DNA reassociation group and a distinct phylogenetic lineage in 165 rDNA homology analysis, indicating that REP group K strains represent a new species. Diagnostic phenotypic characters were found. Closest relatives were Ochrobactrum species. The name Ochrobactrum grignonense sp. nov. is proposed (type strain OgA9aT = LMG 18954T = DSM 13338T). REP group J strains again constituted a different BIOLOG cluster, a separate DNA-DNA reassociation group and showed, as a biological particularity, a strict preference for the rhizoplane as habitat. Diagnostic phenotypic characters were found. This indicated that REP group J strains represent a further new species, although phylogenetic analyses using 16S rDNA homology were not able to separate the cluster of REP group J sequences significantly from 16S rDNA sequences of Ochrobactrum anthropi. The name Ochrobactrum tritici sp. nov. is proposed (type strain SCII24T = LMG 18957T = DSM 13340T).

The diversity of Paenibacillus polymyxa populations associated with the rhizosphere of durum wheat was investigated in Algerian soils sampled in regions where wheat had been cultivated for 5 and 26 years (Hamiz, H5 and Z26), 70 years (Algiers, D70), and more than 2 000 years (Tiaret, T2000 and K2000). A total of 111 strains were isolated by immunotrapping and identified as P. polymyxa using an AP150CHB kit and restriction analysis of the amplified 16S rDNA gene. The phenotypic characteristics of the P. polymyxa populations were compared and the strains found not to cluster according to their origin. The longer the history of wheat cultivation, the lower the phenotypic diversity and the higher the frequency of nitrogen-fixing strains. Population genetic diversity, evaluated by ERIC-PCR, showed the same trends as phenotypic diversity. The distribution of ERIC genotypes among the different populations studied were compared using Pearson's Chi-squared test. The strains isolated in D70, 226, and H5 derived from soil populations sharing the same genetic structure, but those isolated in T2000 and K2000 each stemmed from a population with a specific genetic structure. These data suggest that the genetic structure of P. polymyxa populations has been affected by long-term wheat cultivation. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

This study was conducted to isolate and identify the EPS-producing bacteria associated with the roots of three wheat lines grown in saline and non-saline soils. Results indicated the presence of various EPS-producing bacterial genera in unplanted saline and non-saline soil, rhizosphere and rhizoplane of the three wheat fines. Bacillus sp. were more variable in unplanted saline than non-saline soil. In contrast to uniform distribution of Bacillus sp. in unplanted soils, other EPS-producing bacterial genera isolated from rhizosphere and rhizoplane were more numerous and diversified. Frequent occurrence of Microbacterium sp. in the soil and rhizosphere of the plants invoke the desire for more research work to explore the role of this bacterium in the saline environment.

A collection of 300 isolates of fluorescent pseudomonads was established from Douglas fir-Laccaria bicolor mycorrhizas and mycorrhizosphere and from adjacent bulk soil. These isolates were first phenotypically characterized with the Biolog method. Taxonomic identification assigned 90% of the isolates to the different biovars of Pseudomonas fluorescens, with inverted frequencies of biovars V and I from the bulk soil to the mycorrhizas, suggesting that the mycorrhizas exert a selective stimulation of the P. fluorescens bv. I and a counterselection of the P. fluorescens bv. V present in the soil. Multivariate analyses of the carbon source utilization data led to the definition of homogenous metabolic groups and to the identification of the most discriminating substrates for each group. The isolates from the mycorrhizosphere and from the mycorrhizas seem to preferentially utilize carbohydrates, in particular trehalose, which is the most abundant carbohydrate accumulated in the mycelium of L. bicolor. The results suggest that L. bicolor exerts a trehalose-mediated selection on the fluorescent pseudomonads present in the vicinity of the mycorrhizas. Isolates of P. fluorescens from the mycorrhizosphere and mycorrhizas were then genotypically characterized by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes and enterobacterial repetitive intergenic consensus-PCR DNA fingerprinting. Both methods revealed a high genetic polymorphism within the population studied, which was well correlated with the phenotypic characterization.