Experimentally Induced Dieback Conditions Limit Phragmites australis Growth.

Phragmites australis is a cosmopolitan grass species common in wetland ecosystems across the world. In much of North America, the non-native subspecies of Phragmites threatens wetland biodiversity, hinders recreation, and is a persistent problem for natural resource managers. In other parts of the world, populations are in decline, as Reed Die-Back Syndrome (RDBS) plagues some Phragmites stands in its native range. RDBS is defined by a clumped growth form, stunted root and shoot growth, premature senescence, and shoot death. RDBS has been associated with a build-up of short-chain fatty acids (SCFAs) and altered bacterial and oomycete communities in soils, but the exact causes are unknown. To control invasive Phragmites populations, we sought to develop treatments that mimic the conditions of RDBS. We applied various SCFA treatments at various concentrations to mesocosm soils growing either Phragmites or native wetland plants. We found that the high-concentration SCFA treatments applied weekly induced strong significant declines in above- and belowground biomass of Phragmites. Declines were significant but slightly weaker in native species. In addition, soil bacterial abundance increased, diversity decreased, and bacterial community composition significantly differed following treatments, such that treated pots maintained a higher relative abundance of Pseudomonadaceae and fewer Acidobacteriaceae than untreated pots. Our results suggest that application of SCFAs to Phragmites can lead to stunted plants and altered soil bacterial communities similar to populations affected by RDBS. However, the lack of species-specificity and intensive application rate may not make this treatment ideal as a widespread management tool.

Optimization and application of non-native Phragmites australis transcriptome assemblies.

Phragmites australis (common reed) has a cosmopolitan distribution and has been suggested as a model organism for the study of invasive plant species. In North America, the non-native subspecies (ssp. australis) is widely distributed across the contiguous 48 states in the United States and large parts of Canada. Even though millions of dollars are spent annually on Phragmites management, insufficient knowledge of P. australis impeded the efficiency of management. To solve this problem, transcriptomic information generated from multiple types of tissue could be a valuable resource for future studies. Here, we constructed forty-nine P. australis transcriptomes assemblies via different assembly tools and multiple parameter settings. The optimal transcriptome assembly for functional annotation and downstream analyses was selected among these transcriptome assemblies by comprehensive assessments. For a total of 422,589 transcripts assembled in this transcriptome assembly, 319,046 transcripts (75.5%) have at least one functional annotation. Within the transcriptome assembly, we further identified 1,495 transcripts showing tissue-specific expression pattern, 10,828 putative transcription factors, and 72,165 candidates for simple sequence repeats markers. The identification and analyses of predicted transcripts related to herbicide- and salinity-resistant genes were shown as two applications of the transcriptomic information to facilitate further research on P. australis. Transcriptome assembly and selection would be important for the transcriptome annotation. With this optimal transcriptome assembly and all relative information from downstream analyses, we have helped to establish foundations for future studies on the mechanisms underlying the invasiveness of non-native P. australis subspecies.

Histochemical Evidence for Nitrogen-Transfer Endosymbiosis in Non-Photosynthetic Cells of Leaves and Inflorescence Bracts of Angiosperms.

We used light and confocal microscopy to visualize bacteria in leaf and bract cells of more than 30 species in 18 families of seed plants. Through histochemical analysis, we detected hormones (including ethylene and nitric oxide), superoxide, and nitrogenous chemicals (including nitric oxide and nitrate) around bacteria within plant cells. Bacteria were observed in epidermal cells, various filamentous and glandular trichomes, and other non-photosynthetic cells. Most notably, bacteria showing nitrate formation based on histochemical staining were present in glandular trichomes of some dicots (e.g., Humulus lupulus and Cannabis sativa). Glandular trichome chemistry is hypothesized to function to scavenge oxygen around bacteria and reduce oxidative damage to intracellular bacterial cells. Experiments to assess the differential absorption of isotopic nitrogen into plants suggest the assimilation of nitrogen into actively growing tissues of plants, where bacteria are most active and carbohydrates are more available. The leaf and bract cell endosymbiosis types outlined in this paper have not been previously reported and may be important in facilitating plant growth, development, oxidative stress resistance, and nutrient absorption into plants. It is unknown whether leaf and bract cell endosymbioses are significant in increasing the nitrogen content of plants. From the experiments that we conducted, it is impossible to know whether plant trichomes evolved specifically as organs for nitrogen fixation or if, instead, trichomes are structures in which bacteria easily colonize and where some casual nitrogen transfer may occur between bacteria and plant cells. It is likely that the endosymbioses seen in leaves and bracts are less efficient than those of root nodules of legumes in similar plants. However, the presence of endosymbioses that yield nitrate in plants could confer a reduced need for soil nitrogen and constitute increased nitrogen-use efficiency, even if the actual amount of nitrogen transferred to plant cells is small. More research is needed to evaluate the importance of nitrogen transfer within leaf and bract cells of plants.

Plant effects on and response to soil microbes in native and non-native Phragmites australis.

Plant-soil feedbacks (PSFs) mediate plant community dynamics and may plausibly facilitate plant invasions. Microbially mediated PSFs are defined by plant effects on soil microbes and subsequent changes in plant performance (responses), both positive and negative. For microbial interactions to benefit invasive plants disproportionately, native and invasive plants must either (1) have different effects on and responses to soil microbial communities or (2) only respond differently to similar microbial communities. In other words, invasive plants do not need to cultivate different microbial communities than natives if they respond differently to them. However, effects and responses are not often explored separately, making it difficult to determine the underlying causes of performance differences. We performed a reciprocal-transplant PSF experiment with multiple microbial inhibition treatments to determine how native and non-native lineages of Phragmites australis affect and respond to soil bacteria, fungi, and oomycetes. Non-native Phragmites is a large, fast-growing, cosmopolitan invasive plant, whereas the North American native variety is comparatively smaller, slower growing, and typically considered a desirable wetland plant. We identified the effects of each plant lineage on soil microbes using DNA meta-barcoding and linked plant responses to microbial communities. Both Phragmites lineages displayed equally weak, insignificant PSFs. We found evidence of slight differential effects on microbial community composition, but no significant differential plant responses. Soils conditioned by each lineage differed only slightly in bacterial community composition, but not in fungal composition. Additionally, native and non-native Phragmites lineages did not significantly differ in their response to similar soil microbial communities. Neither lineage appreciably differed when plant biomass was compared between those grown in sterile and live soils. Targeted microbial inhibitor treatments revealed both lineages were negatively impacted by soil bacteria, but the negative response was stronger in non-native Phragmites. These observations were opposite of expectations from invasion theory and imply that the success of non-native Phragmites, relative to the native lineage, does not result from its interaction with soil microorganisms. More broadly, quantifying plant effects on, and responses to soil microbes separately provides detailed and nuanced insight into plant-microbial interactions and their role in invasions, which could inform management outcomes for invasive plants.

Novel genome characteristics contribute to the invasiveness of Phragmites australis (common reed).

The rapid invasion of the non-native Phragmites australis (Poaceae, subfamily Arundinoideae) is a major threat to native wetland ecosystems in North America and elsewhere. We describe the first reference genome for P. australis and compare invasive (ssp. australis) and native (ssp. americanus) genotypes collected from replicated populations across the Laurentian Great Lakes to deduce genomic bases driving its invasive success. Here, we report novel genomic features including a Phragmites lineage-specific whole genome duplication, followed by gene loss and preferential retention of genes associated with transcription factors and regulatory functions in the remaining duplicates. Comparative transcriptomic analyses revealed that genes associated with biotic stress and defence responses were expressed at a higher basal level in invasive genotypes, but native genotypes showed a stronger induction of defence responses when challenged by a fungal endophyte. The reference genome and transcriptomes, combined with previous ecological and environmental data, add to our understanding of mechanisms leading to invasiveness and support the development of novel, genomics-assisted management approaches for invasive Phragmites.

Differences in rhizosphere microbial communities between native and non-native Phragmites australis may depend on stand density.

Microorganisms surrounding plant roots may benefit invasive species through enhanced mutualism or decreased antagonism, when compared to surrounding native species. We surveyed the rhizosphere soil microbiome of a prominent invasive plant, Phragmites australis, and its co-occurring native subspecies for evidence of microbial drivers of invasiveness. If the rhizosphere microbial community is important in driving plant invasions, we hypothesized that non-native Phragmites would cultivate a different microbiome from native Phragmites, containing fewer pathogens, more mutualists, or both. We surveyed populations of native and non-native Phragmites across Michigan and Ohio USA, and we described rhizosphere microbial communities using culture-independent next-generation sequencing. We found little evidence that native and non-native Phragmites cultivate distinct bacterial, fungal, or oomycete rhizosphere communities. Microbial community differences in our Michigan survey were not associated with plant lineage but were mainly driven by environmental factors, such as soil saturation and nutrient concentrations. Intensive sampling along transects consisting of dense monocultures of each lineage and mixed zones revealed bacterial community differences between lineages in dense monoculture, but not in mixture. We found no evidence of functional differences in the microbial communities surrounding each lineage. We extrapolate that the invasiveness of non-native Phragmites, when compared to its native congener, does not result from the differential cultivation of beneficial or antagonistic rhizosphere microorganisms.

Growth and Behavior of North American Microbes on Phragmites australis Leaves.

Phragmites australis subsp. australis is a cosmopolitan wetland grass that is invasive in many regions of the world, including North America, where it co-occurs with the closely related Phragmites australis subsp. americanus. Because the difference in invasive behavior is unlikely to be related to physiological differences, we hypothesize that interactions with unique members of their microbiomes may significantly affect the behavior of each subspecies. Therefore, we systematically inoculated both plant lineages with a diverse array of 162 fungal and bacterial isolates to determine which could (1) differentiate between Phragmites hosts, (2) infect leaves at various stages of development, or (3) obtain plant-based carbon saprophytically. We found that many of the microbes isolated from Phragmites leaves behave as saprophytes. Only 1% (two taxa) were determined to be strong pathogens, 12% (20 taxa) were weakly pathogenic, and the remaining 87% were nonpathogenic. None of the isolates clearly discriminated between host plant lineages, and the Phragmites cuticle was shown to be a strong nonspecific barrier to infection. These results largely agree with the broad body of literature on leaf-associated phyllosphere microbes in Phragmites.

Review: Endophytic microbes and their potential applications in crop management.

Endophytes are microbes (mostly bacteria and fungi) present asymptomatically in plants. Endophytic microbes are often functional in that they may carry nutrients from the soil into plants, modulate plant development, increase stress tolerance of plants, suppress virulence in pathogens, increase disease resistance in plants, and suppress development of competitor plant species. Endophytic microbes have been shown to: (i) obtain nutrients in soils and transfer nutrients to plants in the rhizophagy cycle and other nutrient-transfer symbioses; (ii) increase plant growth and development; (iii) reduce oxidative stress of hosts; (iv) protect plants from disease; (v) deter feeding by herbivores; and (vi) suppress growth of competitor plant species. Because of the effective functions of endophytic microbes, we suggest that endophytic microbes may significantly reduce use of agrochemicals (fertilizers, fungicides, insecticides, and herbicides) in the cultivation of crop plants. The loss of endophytic microbes from crop plants during domestication and long-term cultivation could be remedied by transfer of endophytes from wild relatives of crops to crop species. Increasing atmospheric carbon dioxide levels could reduce the efficiency of the rhizophagy cycle due to repression of reactive oxygen used to extract nutrients from microbes in roots. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Rhizophagy Cycle: An Oxidative Process in Plants for Nutrient Extraction from Symbiotic Microbes.

In this paper, we describe a mechanism for the transfer of nutrients from symbiotic microbes (bacteria and fungi) to host plant roots that we term the 'rhizophagy cycle.' In the rhizophagy cycle, microbes alternate between a root intracellular endophytic phase and a free-living soil phase. Microbes acquire soil nutrients in the free-living soil phase; nutrients are extracted through exposure to host-produced reactive oxygen in the intracellular endophytic phase. We conducted experiments on several seed-vectored microbes in several host species. We found that initially the symbiotic microbes grow on the rhizoplane in the exudate zone adjacent the root meristem. Microbes enter root tip meristem cells-locating within the periplasmic spaces between cell wall and plasma membrane. In the periplasmic spaces of root cells, microbes convert to wall-less protoplast forms. As root cells mature, microbes continue to be subjected to reactive oxygen (superoxide) produced by NADPH oxidases (NOX) on the root cell plasma membranes. Reactive oxygen degrades some of the intracellular microbes, also likely inducing electrolyte leakage from microbes-effectively extracting nutrients from microbes. Surviving bacteria in root epidermal cells trigger root hair elongation and as hairs elongate bacteria exit at the hair tips, reforming cell walls and cell shapes as microbes emerge into the rhizosphere where they may obtain additional nutrients. Precisely what nutrients are transferred through rhizophagy or how important this process is for nutrient acquisition is still unknown.

Fungal Disease Prevention in Seedlings of Rice (Oryza sativa) and Other Grasses by Growth-Promoting Seed-Associated Endophytic Bacteria from Invasive Phragmites australis.

Non-cultivated plants carry microbial endophytes that may be used to enhance development and disease resistance of crop species where growth-promoting and protective microbes may have been lost. During seedling establishment, seedlings may be infected by several fungal pathogens that are seed or soil borne. Several species of Fusarium, Pythium and other water moulds cause seed rots during germination. Fusarium blights of seedlings are also very common and significantly affect seedling development. In the present study we screened nine endophytic bacteria isolated from the seeds of invasive Phragmites australis by inoculating onto rice, Bermuda grass (Cynodon dactylon), or annual bluegrass (Poa annua) seeds to evaluate plant growth promotion and protection from disease caused by Fusarium oxysporum. We found that three bacteria belonging to genus Pseudomonas spp. (SLB4-P. fluorescens, SLB6-Pseudomonas sp. and SY1-Pseudomonas sp.) promoted seedling development, including enhancement of root and shoot growth, and stimulation of root hair formation. These bacteria were also found to increase phosphate solubilization in in vitro experiments. Pseudomonas sp. (SY1) significantly protected grass seedlings from Fusarium infection. In co-culture experiments, strain SY1 strongly inhibited fungal pathogens with 85.71% growth inhibition of F. oxysporum, 86.33% growth inhibition of Curvularia sp. and 82.14% growth inhibition of Alternaria sp. Seedlings previously treated with bacteria were found much less infected by F. oxysporum in comparison to non-treated controls. On microscopic observation we found that bacteria appeared to degrade fungal mycelia actively. Metabolite products of strain SY1 in agar were also found to inhibit fungal growth on nutrient media. Pseudomonas sp. (SY1) was found to produce antifungal volatiles. Polymerase chain reaction (PCR) amplification using specific primers for pyrrolnitirin synthesis and HCN (hydrogen cyanide) production suggested presence of genes for both compounds in the genome of SY1. HCN was detected in cultures of SY1. We conclude that microbes from non-cultivated plants may provide disease protection and promote growth of crop plants.

Advancing the science of microbial symbiosis to support invasive species management: a case study on Phragmites in the Great Lakes.

A growing body of literature supports microbial symbiosis as a foundational principle for the competitive success of invasive plant species. Further exploration of the relationships between invasive species and their associated microbiomes, as well as the interactions with the microbiomes of native species, can lead to key new insights into invasive success and potentially new and effective control approaches. In this manuscript, we review microbial relationships with plants, outline steps necessary to develop invasive species control strategies that are based on those relationships, and use the invasive plant species Phragmites australis (common reed) as an example of how development of microbial-based control strategies can be enhanced using a collective impact approach. The proposed science agenda, developed by the Collaborative for Microbial Symbiosis and Phragmites Management, contains a foundation of sequential steps and mutually-reinforcing tasks to guide the development of microbial-based control strategies for Phragmites and other invasive species. Just as the science of plant-microbial symbiosis can be transferred for use in other invasive species, so too can the model of collective impact be applied to other avenues of research and management.