More Than the Sum of Its Parts: Unlocking the Power of Network Structure for Understanding Organization and Function in Microbiomes.

Plant and soil microbiomes are integral to the health and productivity of plants and ecosystems, yet researchers struggle to identify microbiome characteristics important for providing beneficial outcomes. Network analysis offers a shift in analytical framework beyond "who is present" to the organization or patterns of coexistence between microbes within the microbiome. Because microbial phenotypes are often significantly impacted by coexisting populations, patterns of coexistence within microbiomes are likely to be especially important in predicting functional outcomes. Here, we provide an overview of the how and why of network analysis in microbiome research, highlighting the ways in which network analyses have provided novel insights into microbiome organization and functional capacities, the diverse network roles of different microbial populations, and the eco-evolutionary dynamics of plant and soil microbiomes.

Development of Microbiome Biobanks - Challenges and Opportunities.

The microbiome research field is rapidly evolving, but the required biobanking infrastructure is currently fragmented and not prepared for the biobanking of microbiomes. The rapid advancement of technologies requires an urgent assessment of how biobanks can underpin research by preserving microbiome samples and their functional potential.

Densities and inhibitory phenotypes among indigenous Streptomyces spp. vary across native and agricultural habitats.

Streptomyces spp. perform vital roles in natural and agricultural soil ecosystems including in decomposition and nutrient cycling, promotion of plant growth and fitness, and plant disease suppression. Streptomyces densities can vary across the landscape, and inhibitory phenotypes are often a result of selection mediated by microbial competitive interactions in soil communities. Diverse environmental factors, including those specific to habitat, are likely to determine microbial densities in the soil and the outcomes of microbial species interactions. Here, we characterized indigenous Streptomyces densities and inhibitory phenotypes from soil samples (n = 82) collected in 6 distinct habitats across the Cedar Creek Ecosystem Science Reserve (CCESR; agricultural, prairie, savanna, wetland, wet-woodland, and forest). Significant variation in Streptomyces density and the frequency of antagonistic Streptomyces were observed among habitats. There was also significant variation in soil chemical properties among habitats, including percent carbon, percent nitrogen, available phosphorus, extractable potassium, and pH. Density and frequency of antagonists were significantly correlated with one or more environmental parameters across all habitats, though relationships with some parameters differed among habitats. In addition, we found that habitat rather than spatial proximity was a better predictor of variation in Streptomyces density and inhibitory phenotypes. Moreover, habitats least conducive for Streptomyces growth and proliferation, as determined by population density, had increased frequencies of inhibitory phenotypes. Identifying environmental parameters that structure variation in density and frequency of antagonistic Streptomyces can provide insight for determining factors that mediate selection for inhibitory phenotypes across the landscape.

Microbiome Networks: A Systems Framework for Identifying Candidate Microbial Assemblages for Disease Management.

Network models of soil and plant microbiomes provide new opportunities for enhancing disease management, but also challenges for interpretation. We present a framework for interpreting microbiome networks, illustrating how observed network structures can be used to generate testable hypotheses about candidate microbes affecting plant health. The framework includes four types of network analyses. "General network analysis" identifies candidate taxa for maintaining an existing microbial community. "Host-focused analysis" includes a node representing a plant response such as yield, identifying taxa with direct or indirect associations with that node. "Pathogen-focused analysis" identifies taxa with direct or indirect associations with taxa known a priori as pathogens. "Disease-focused analysis" identifies taxa associated with disease. Positive direct or indirect associations with desirable outcomes, or negative associations with undesirable outcomes, indicate candidate taxa. Network analysis provides characterization not only of taxa with direct associations with important outcomes such as disease suppression, biofertilization, or expression of plant host resistance, but also taxa with indirect associations via their association with other key taxa. We illustrate the interpretation of network structure with analyses of microbiomes in the oak phyllosphere, and in wheat rhizosphere and bulk soil associated with the presence or absence of infection by Rhizoctonia solani.

Rhizosphere bacterial communities associated with long-lived perennial prairie plants vary in diversity, composition, and structure.

The goal of this research was to investigate the variation in rhizosphere microbial community composition, diversity, and structure among individual Andropogon gerardii Vitman (big bluestem) and Lespedeza capitata Michx. (bush clover). Bacterial communities from the rhizosphere of 10 plants of each species (n = 20 plants total) were explored using a culture-independent pipeline. Microbial communities associated with both host plants had high bacterial diversity within individual plant rhizosphere and taxa unique to individual rhizospheres. Bacterial communities associated with the rhizosphere of A. gerardii were consistently more diverse than those associated with L. capitata, and there were significant differences between plant species in rhizosphere bacterial community composition. Differences included microbial taxa with no known functional relationship with their preferred host species, including sulfide-methylating obligate anaerobes (Holophaga), complete denitrifiers (Rhodoplanes), sludge inhabitants (Ktedonobacter), and nitrate oxidizers (Nitrospira). These results suggest the potential for plant species to have significant impacts on a broad array of ecosystem functions (e.g., cycling of carbon, nitrogen sulfurs, metals, and trace elements) via their selective impacts on soil microbes. However, sequence-based community analysis and the corresponding lack of intact microbial cultures limits understanding of the potential influences of enriched microbial taxa on plant hosts and their roles in ecosystem functioning.

Pathogen variation and urea influence selection and success of Streptomyces mixtures in biological control.

Success in biological control of plant diseases remains inconsistent in the field. A collection of well-characterized Streptomyces antagonists (n = 19 isolates) was tested for their capacities to inhibit pathogenic Streptomyces scabies (n = 15 isolates). There was significant variation among antagonists in ability to inhibit pathogen isolates and among pathogens in their susceptibility to inhibition. Only one antagonist could inhibit all pathogens, and antagonist-pathogen interactions were highly specific, highlighting the limitations of single-strain inoculum in biological control. However, the collection of pathogens could be inhibited by several combinations of antagonists, suggesting the potential for successful antagonist mixtures. Urea generally increased effectiveness of antagonists at inhibiting pathogens in vitro (increased mean inhibition zones) but its specific effects varied among antagonist-pathogen combinations. In greenhouse trials, urea enhanced the effectiveness of antagonist mixtures relative to individual antagonists in controlling potato scab. Although antagonist mixtures were frequently antagonistic in the absence of urea, all n= 2 and n = 3 antagonist-isolate combinations were synergistic in the presence of urea. This work provides insights into the efficacy of single- versus multiple-strain inocula in biological control and on the potential for nutrients to influence mixture success.

Green manures and crop sequences influence potato diseases and pathogen inhibitory activity of indigenous streptomycetes.

ABSTRACT A 2-year field trial was conducted to determine the effects of green manures and crop sequences on potato scab and Verticillium wilt. In addition, indigenous streptomycete densities and in vitro pathogen inhibitory activity were measured and their relationships to plant disease were determined. Green manures (buckwheat, canola, or fallow controls) were tested in conjunction with three crop sequences (alfalfa-potato, cornpotato, and potato-potato). Compared with fallow controls, tubers grown in buckwheat-treated soil had significantly lower Verticillium wilt ratings, and tubers grown in buckwheat- or canola-treated soil had greater yields. Potatoes grown in soil planted to corn or alfalfa the previous year had significantly lower Verticillium wilt and potato scab ratings as well as higher yields than potatoes grown in soil previously planted to potato. Streptomycetes from soils collected from green manure-treated plots tended to have greater in vitro pathogen inhibitory activity than streptomycetes from fallow-treated plots. Furthermore, streptomycete pathogen inhibitory activity was frequently negatively correlated with plant disease and positively correlated with potato yield. These results indicate that green manure treatments may contribute to active management of the pathogen inhibitory activity of the streptomycete community to achieve plant disease control.

Spatial variation in Streptomyces genetic composition and diversity in a prairie soil.

Understanding how microbial genotypes are arrayed in space is crucial for identifying local factors that may influence the spatial distribution of genetic diversity. In this study we investigated variation in 16S rDNA sequences and rep-PCR fingerprints of Streptomyces stains isolated from prairie soil among three locations and four soil depths. Substantial variation in Streptomyces OTU (operational taxonomic unit) and BOX-PCR fingerprint diversity was found among locations within a limited spatial area (1 m2). Further, phylogenetic lineages at each location were distinct. However, there was little variation in genetic diversity among isolates from different soil depths and similar phylogenetic lineages were found at each depth. Some clones were found at a localized scale while other clones had a relatively widespread distribution. There was poor correspondence between 16S rDNA groupings and rep-PCR fingerprint groupings. The finding of distinct phylogenetic lineages and the variation in spatial distribution of clones suggests that selection pressures may vary over the soil landscape.

Competition and antibiosis in the biological control of potato scab.

Nonpathogenic, antibiotic-producing streptomycetes have been shown to reduce potato scab when added to disease-conducive soil. Spontaneous mutants of the pathogenic Streptomyces scabies RB4 that are resistant to at least one antibiotic activity produced by the nonpathogenic suppressive isolates Streptomyces diastatochromogenes strain PonSSII and S. scabies PonR have been isolated. To determine the importance of antibiosis in this biocontrol system, these mutants were investigated for their ability to cause disease in the presence of the two pathogen antagonists in a greenhouse assay. Disease caused by one of the mutant strains was reduced in the presence of both suppressive isolates, whereas disease caused by the other five mutants was not significantly reduced by either suppressive strain. In addition, a nonpathogenic mutant of S. scabies RB4 was isolated, which produced no detectable in vitro antibiotic activity and reduced disease caused by its pathogenic parent strain when the pathogen and mutant were coinoculated into soil. Population densities of the pathogen were consistently lower than those of the suppressive strains when individual strains were inoculated into soil. When a pathogen was coinoculated with a suppressive strain, the total streptomycete population density in the pot was always less than that observed when the suppressive isolate was inoculated alone. When the pathogens were inoculated individually into soil, a positive correlation was seen between population density and disease severity. In coinoculation experiments with pathogen and suppressive strains, higher total streptomycete population densities were correlated with lower amounts of disease.

Relationship Between Phyllosphere Population Sizes of Xanthomonas translucens pv. translucens and Bacterial Leaf Streak Severity on Wheat Seedlings.

ABSTRACT The relationship between leaf-associated population sizes of Xanthomonas translucens pv. translucens on asymptomatic leaves and subsequent bacterial leaf streak (BLS) severity was investigated. In three experiments, X. translucens pv. translucens was spray-inoculated onto 10-day-old wheat seedlings over a range of inoculum densities (10(4), 10(5), 10(6), 10(7), and 10(8) CFU/ml). Lesions developed most rapidly on plants inoculated with higher densities of X. translucens pv. translucens. Leaf-associated pathogen population sizes recovered 48 h after inoculation were highly predictive of BLS severity 7 days after inoculation (R(2) = 0.970, P < 0.0001). The relationship between pathogen population size on leaves and subsequent BLS severity was best described by the logistic model. Leaf-associated X. translucens pv. translucens population size and BLS severity from a particular pathogen inoculum density often varied among experiments; however, the disease severity level caused by a particular leaf-associated X. translucens pv. translucens population size was not significantly different among experiments. Biological and disease control implications of the X. translucens pv. translucens population size-BLS severity relationship are discussed.

Quantitative relationships among thaxtomin A production, potato scab severity, and fatty acid composition in Streptomyces.

Thaxtomin A production in culture, potato common scab severity (percentage of tuber surface infected or number of lesions per tuber), and fatty acid profiles were determined for 78 Streptomyces isolates. Only pathogenic Streptomyces spp. (n = 17) produced thaxtomin A in culture. Thaxtomin A production in culture (microgram/mL) was significantly positively correlated with the percentage of tuber surface infected (R = 0.60; p = 0.017) but not with the number of lesions per tuber (R = 0.37; p = 0.17). An increase of 1 microgram/mL in thaxtomin A production corresponded to an 11% increase in disease severity (percentage of tuber surface infected). The data indicate that quantitative information on the ability of a particular pathogen isolate or population to produce thaxtomin A may be critical to understanding and predicting the disease potential of that population. Using cluster analysis of fatty acid data, 94% of 67 unknown field isolates grouped with other field isolates having the same pathogenicity (plus or minus).

Determinants of density- and frequency-dependent fitness in competing plant pathogens.

ABSTRACT Using mathematical models, we investigated how infection and sporulation characteristics of competing plant pathogens determine the density and frequency dependence of relative fitness. Two models, one for the infection stage and one for the sporulation stage of a pathogen's life cycle, describe reproductive output of pathogen strains in mixture as a function of the strains' population densities. Model parameters include infection and sporulation efficiencies, carrying capacities on leaves for sporulating lesions and spore production, and coefficients of interstrain competitive effects in both life cycle stages. Although the models were originally developed for rust fungi, they are generally applicable to any organism with distinct colonization (e.g., infection) and propagative (e.g., sporulation) life cycle stages. In this work, paired hypothetical strains were assigned equal baseline parameter values. Parameters were then altered one at a time for one or both strains, and relative fitness was calculated over a range of densities and strain frequencies. Except for infection efficiency, the fitness benefit conferred by an advantage in a single parameter was always density dependent. Relative fitness was frequency dependent whenever inter- and intrastrain competitive effects were not equal. These results suggest that the fitness of pathogens in nature is rarely fixed, but, rather, may typically be highly dependent on the densities and frequencies of all coexisting strains in a habitat.

Microbial population dynamics on leaves.

Microbial population dynamics on leaves in time and space are a function of immigration, emigration, growth, and death. Insight into the relative significance of each population process to the generation of specific dynamics for individual microorganisms is necessary to understanding the ecology and life history strategy of the microorganism and to developing effective control strategies. Additionally, information on the significance of within-leaf versus extra-leaf processes to the generation of phyllosphere dynamics is important to determining the range of spatial scales over which a population should be studied. Unfortunately, such information is difficult to obtain due to the lack of effective methodologies for distinguishing these processes within phyllosphere populations. Future research efforts should focus on the quantification of immigration, emigration, growth, and death relative to the population dynamics of phyllosphere microorganisms.

Utility of microcosm studies for predicting phylloplane bacterium population sizes in the field.

Population sizes of two ice nucleation-active strains of Pseudomonas syringae were compared on leaves in controlled environments and in the field to determine the ability of microcosm studies to predict plant habitat preferences in the field. The P. syringae strains investigated were the parental strains of recombinant deletion mutant strains deficient in ice nucleation activity that had been field tested for their ability to control plant frost injury. The population size of the P. syringae strains was measured after inoculation at three field locations on up to 40 of the same plant species that were studied in the growth chamber. There was seldom a significant relationship between the mean population size of a given P. syringae strain incubated under either wet or dry conditions in microcosms and the mean population size which could be recovered from the same species when inoculated in the field. Specifically, on some plant species, the population size recovered from leaves in the field was substantially greater than from that species in a controlled environment, while for other plant species field populations were significantly smaller than those observed under controlled conditions. Population sizes of inoculated P. syringae strains, however, were frequently highly positively correlated with the indigenous bacterial population size on the same plant species in the field, suggesting that the ability of a particular plant species to support introduced bacterial strains is correlated with its ability to support large bacterial populations or that indigenous bacteria enhance the survival of introduced strains. Microcosm studies therefore seem most effective at assessing possible differences between parental and recombinant strains under a given environmental regime but are limited in their ability to predict the specific population sizes or plant habitat preferences of bacteria on leaves under field conditions.

Use of repetitive intergenic DNA sequences to classify pathogenic and disease-suppressive Streptomyces strains.

PCR DNA fingerprinting using repetitive intergenic DNA sequences (rep-PCR) was investigated as a means of differentiating between closely related strains of Streptomyces which were, in some cases, indistinguishable by other classification methods. Our results demonstrated that the majority of strains had unique rep-PCR DNA fingerprints and established that the technique could be a very useful tool in rapidly determining strain identity.

Influence of disease-suppressive strains of Streptomyces on the native Streptomyces community in soil as determined by the analysis of cellular fatty acids.

Analysis of cellular fatty acid profiles was used to distinguish among introduced pathogen- suppressive strains and indigenous strains of Streptomyces spp. isolated from soil of field plots established to test the efficacy of Streptomyces strains PonSSII and PonR in the biological control of potato scab. Reference libraries of fatty acid profiles were developed for a collection of known pathogenic strains and the introduced suppressive strains. Population densities of pathogen-related, suppressive, and saprophytic Streptomyces strains were determined from the relationship of field isolates to mean library profiles using cluster analysis and the unweighted pair-group method using arithmetic averages. Community diversity was similarly determined. Streptomyces strains PonSSII and PonR were distinguished from each other and from the pathogen group (which clustered together) based on fatty acid profiles. The introduced, suppressive strains successfully colonized the soil and represented 2-19% of the isolates sampled over 2 years. The introduction of the suppressive strains inhibited the population of strains related to the pathogen library at each sample date; the pathogen population was substantially lower in soil from treatments where the suppressive strains were introduced compared with the nonamended control. At harvest, the pathogen-related population was suppressed 85-93 and 36-44% in 1991 and 1992, respectively, in treatments with the suppressive strains compared with the nonamended control. Diversity of the community was not affected by the introduced strains, and diversity and equitability indices were similar among treatments at any sample time. The inhibition of the pathogen-related population was correlated with a reduction of scab symptoms observed in the field plots into which the suppressive strains were introduced. Implications of a fundamental shift in the pathogen-related population in response to the introduction of the suppressive strains for long-term biological control of potato scab are encouraging.

Population Sizes, Immigration, and Growth of Epiphytic Bacteria on Leaves of Different Ages and Positions of Field-Grown Endive (Cichorium endivia var. latifolia).

Total, fluorescent, and pectolytic epiphytic bacterial population sizes were quantified on leaves of different age groups of broad-leaved endive during field cultivation from leaf emergence until harvest. Greater bacterial population densities (log(inf10) CFU per square centimeter) were observed on outer leaves than on inner leaves of the plants throughout the growing season. These differences were statistically significant for total bacterial populations at all sampling times and were often significant for fluorescent and pectolytic bacterial populations. At harvest, a linear gradient of decreasing densities of epiphytic bacteria from outer (older) to inner (younger) leaves of the head was significant. Leaf age influenced the frequency distribution and variability of bacterial population sizes associated with leaves of broad-leaved endive. Total bacterial population sizes were greater at leaf emergence for leaves emerging during the second half of the cultivation period than for leaves emerging earlier. The size of fluorescent and pectolytic bacterial populations on newly emerged leaves increased throughout the season as plants aged. To assess the importance of plant age on bacterial immigration at leaf emergence, bacterial densities were quantified on leaves emerging simultaneously on plants of different ages. In two of the three experiments, greater bacterial population sizes were observed on leaves emerging on younger plants. This indicates that factors other than an increase in concentration of airborne bacteria can lead to increases in population sizes at leaf emergence as plants age in the field. Results of leaf pruning experiments suggested that adjacent leaves may act as a barrier for immigration of fluorescent bacteria on newly emerged leaves. Survival of an inoculated strain of Pseudomonas fluorescens on newly emerged leaves generally did not vary with the age of plants. However, these effects were not consistent among experiments, suggesting that interactions among micro- and macroenvironmental conditions, physiological condition of leaves, and accessibility of leaves to airborne bacteria are important in controlling epiphytic bacterial population sizes.

Effect of sampling scale on the assessment of epiphytic bacterial populations.

Bacterial populations on above-ground plant surfaces were estimated at three different biological scales, including leaflet disks, entire leaflets, and whole plants. The influence of sample scale on the estimation of mean bacterial population size per unit and per gram and on the variability among sampling units was quantified at each scale. Populations were highly variable among sampling units at every scale examined, suggesting that there is no optimal scale at which sample variance is reduced. The distribution of population sizes among sample units was sometimes, but not consistently, described by the lognormal. Regardless of the sampling scale, expression of population sizes on a per gram basis may not reduce variance, because population size was not generally a function of sample unit weight within any single sampling scale. In addition, the data show that scaling populations on a per gram basis does not provide a useful means of comparing population estimates from samples taken at different scales. The implications of these results for designing sampling strategies to address specific issues in microbial ecology are discussed.