Serendipita Fungi Modulate the Switchgrass Root Transcriptome to Circumvent Host Defenses and Establish a Symbiotic Relationship.

The fungal family Serendipitaceae encompasses root-associated lineages with endophytic, ericoid, orchid, and ectomycorrhizal lifestyles. Switchgrass is an important bioenergy crop for cellulosic ethanol production owing to high biomass production on marginal soils otherwise unfit for food crop cultivation. The aim of this study was to investigate the host plant responses to Serendipita spp. colonization by characterizing the switchgrass root transcriptome during different stages of symbiosis in vitro. For this, we included a native switchgrass strain, Serendipita bescii, and a related strain, S. vermifera, isolated from Australian orchids. Serendipita colonization progresses from thin hyphae that grow between root cells to, finally, the production of large, bulbous hyphae that fill root cells during the later stages of colonization. We report that switchgrass seems to perceive both fungi prior to physical contact, leading to the activation of chemical and structural defense responses and putative host disease resistance genes. Subsequently, the host defense system appears to be quenched and carbohydrate metabolism adjusted, potentially to accommodate the fungal symbiont. In addition, prior to contact, switchgrass exhibited significant increases in root hair density and root surface area. Furthermore, genes involved in phytohormone metabolism such as gibberellin, jasmonic acid, and salicylic acid were activated during different stages of colonization. Both fungal strains induced plant gene expression in a similar manner, indicating a conserved plant response to members of this fungal order. Understanding plant responsiveness to Serendipita spp. will inform our efforts to integrate them into forages and row crops for optimal plant-microbe functioning, thus facilitating low-input, sustainable agricultural practices.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Serendipita bescii promotes winter wheat growth and modulates the host root transcriptome under phosphorus and nitrogen starvation.

Serendipita vermifera ssp. bescii, hereafter referred to as S. bescii, is a root-associated fungus that promotes plant growth in both its native switchgrass host and a variety of monocots and dicots. Winter wheat (Triticum aestivum L.), a dual-purpose crop, used for both forage and grain production, significantly contributes to the agricultural economies of the Southern Great Plains, USA. In this study, we investigated the influence of S. bescii on growth and transcriptome regulation of nitrogen (N) and phosphorus (P) metabolism in winter wheat. Serendipita bescii significantly improved lateral root growth and forage biomass under a limited N or P regime. Further, S. bescii activated sets of host genes regulating N and P starvation responses. These genes include, root-specific auxin transport, strigolactone and gibberellin biosynthesis, degradation of phospholipids and biosynthesis of glycerolipid, downregulation of ammonium transport and nitrate assimilation, restriction of protein degradation by autophagy and subsequent N remobilization. All these genes are hypothesized to regulate acquisition, assimilation and remobilization of N and P. Based on transcriptional level gene regulation and physiological responses to N or P limitation, we suggest S. bescii plays a critical role in modulating stress imposed by limitation of these two critical nutrients in winter wheat.

Sebacina vermifera: a unique root symbiont with vast agronomic potential.

The Sebacinales belong to a taxonomically, ecologically, and physiologically diverse group of fungi in the Basidiomycota. While historically recognized as orchid mycorrhizae, recent DNA studies have brought to light both their pandemic distribution and the broad spectrum of mycorrhizal types they form. Indeed, ecological studies using molecular-based methods of detection have found Sebacinales fungi in field specimens of bryophytes (moss), pteridophytes (fern) and all families of herbaceous angiosperms (flowering plants) from temperate, subtropical and tropical regions. These natural host plants include, among others, liverworts, wheat, maize and Arabidopsis thaliana, the model plant traditionally viewed as non-mycorrhizal. The orchid mycorrhizal fungus Sebacina vermifera (MAFF 305830) was first isolated from the Australian orchid Cyrtostylis reniformis. Research performed with this strain clearly indicates its plant growth promoting abilities in a variety of plants, while demonstrating a lack of specificity that rivals or even surpasses that of arbuscular mycorrhizae. Indeed, these traits thus far appear to characterize a majority of strains belonging to the so-called "clade B" within the Sebacinales (recently re-classified as the Serendipitaceae), raising numerous basic research questions regarding plant-microbe signaling and the evolution of mycorrhizal symbioses. Further, given their proven beneficial impact on plant growth and their apparent but cryptic ubiquity, sebacinoid fungi should be considered as a previously hidden, but amenable and effective microbial tool for enhancing plant productivity and stress tolerance.

Genome-wide detection of condition-sensitive alternative splicing in Arabidopsis roots.

Iron (Fe) deficiency is a world-wide nutritional disorder in both plants and humans, resulting from its restricted bioavailability for plants and, subsequently, low Fe concentration in edible plant parts. Plants have evolved sophisticated mechanisms to alleviate Fe deficiency, with the aim of recalibrating metabolic fluxes and maintaining cellular Fe homeostasis. To analyze condition-sensitive changes in precursor mRNA (pre-mRNA) splicing pattern, we mapped the transcriptome of Fe-deficient and Fe-sufficient Arabidopsis (Arabidopsis thaliana) roots using the RNA sequencing technology and a newly developed software toolbox, the Read Analysis & Comparison Kit in Java (RACKJ). In alternatively spliced genes, stress-related Gene Ontology categories were overrepresented, while housekeeping cellular functions were mainly transcriptionally controlled. Fe deficiency increased the complexity of the splicing pattern and triggered the differential alternative splicing of 313 genes, the majority of which had differentially retained introns. Several genes with important functions in Fe acquisition and homeostasis were both differentially expressed and differentially alternatively spliced upon Fe deficiency, indicating a complex regulation of gene activity in Fe-deficient conditions. A comparison with a data set for phosphate-deficient plants suggests that changes in splicing patterns are nutrient specific and not or not chiefly caused by stochastic fluctuations. In sum, our analysis identified extensive posttranscriptional control, biasing the abundance and activity of proteins in a condition-dependent manner. The production of a mixture of functional and nonfunctional transcripts may provide a means to fine-tune the abundance of transcripts with critical importance in cellular Fe homeostasis. It is assumed that differential gene expression and nutrient deficiency-induced changes in pre-mRNA splicing represent parallel, but potentially interacting, regulatory mechanisms.

Plant-associated fungi support bacterial resilience following water limitation.

Drought disrupts soil microbial activity and many biogeochemical processes. Although plant-associated fungi can support plant performance and nutrient cycling during drought, their effects on nearby drought-exposed soil microbial communities are not well resolved. We used H218O quantitative stable isotope probing (qSIP) and 16S rRNA gene profiling to investigate bacterial community dynamics following water limitation in the hyphospheres of two distinct fungal lineages (Rhizophagus irregularis and Serendipita bescii) grown with the bioenergy model grass Panicum hallii. In uninoculated soil, a history of water limitation resulted in significantly lower bacterial growth potential and growth efficiency, as well as lower diversity in the actively growing bacterial community. In contrast, both fungal lineages had a protective effect on hyphosphere bacterial communities exposed to water limitation: bacterial growth potential, growth efficiency, and the diversity of the actively growing bacterial community were not suppressed by a history of water limitation in soils inoculated with either fungus. Despite their similar effects at the community level, the two fungal lineages did elicit different taxon-specific responses, and bacterial growth potential was greater in R. irregularis compared to S. bescii-inoculated soils. Several of the bacterial taxa that responded positively to fungal inocula belong to lineages that are considered drought susceptible. Overall, H218O qSIP highlighted treatment effects on bacterial community structure that were less pronounced using traditional 16S rRNA gene profiling. Together, these results indicate that fungal-bacterial synergies may support bacterial resilience to moisture limitation.

Microbe to Microbiome: A Paradigm Shift in the Application of Microorganisms for Sustainable Agriculture.

Light, water and healthy soil are three essential natural resources required for agricultural productivity. Industrialization of agriculture has resulted in intensification of cropping practices using enormous amounts of chemical pesticides and fertilizers that damage these natural resources. Therefore, there is a need to embrace agriculture practices that do not depend on greater use of fertilizers and water to meet the growing demand of global food requirements. Plants and soil harbor millions of microorganisms, which collectively form a microbial community known as the microbiome. An effective microbiome can offer benefits to its host, including plant growth promotion, nutrient use efficiency, and control of pests and phytopathogens. Therefore, there is an immediate need to bring functional potential of plant-associated microbiome and its innovation into crop production. In addition to that, new scientific methodologies that can track the nutrient flux through the plant, its resident microbiome and surrounding soil, will offer new opportunities for the design of more efficient microbial consortia design. It is now increasingly acknowledged that the diversity of a microbial inoculum is as important as its plant growth promoting ability. Not surprisingly, outcomes from such plant and soil microbiome studies have resulted in a paradigm shift away from single, specific soil microbes to a more holistic microbiome approach for enhancing crop productivity and the restoration of soil health. Herein, we have reviewed this paradigm shift and discussed various aspects of benign microbiome-based approaches for sustainable agriculture.

Correlation between organic acid exudation and metal uptake by ectomycorrhizal fungi grown on pond ash in vitro.

Experiments were conducted to investigate the effect of coal ash on organic acid exudation and subsequent metal uptake by ectomycorrhizal fungi. Four isolates of ectomycorrhizal fungi namely, Pisolithus tinctorius (EM-1293 and EM-1299), Scleroderma verucosum (EM-1283) and Scleroderma cepa (EM-1233) were grown on pond ash moistened with Modified Melin-Norkans medium in vitro. Exudation of formic acid, malic acid and succinic acid by these fungi were detected by HPLC. Mycelial accumulation of Al, As, Cd, Cr, Ni and Pb by these fungi was assayed by atomic absorption spectrophotometer. Relationship between organic acid exudation and metal uptake was determined using classical multivariate linear regression model. Correlation between organic acid exudation and metal uptake could be substantiated when several metals are considered collectively. The finding supports the widespread role of low molecular weight organic acid as a function of tolerance, when exposed to metals in vitro.

Scavenging organic nitrogen and remodelling lipid metabolism are key survival strategies adopted by the endophytic fungi, Serendipita vermifera and Serendipita bescii to alleviate nitrogen and phosphorous starvation in vitro.

Serendipitaceae represents a diverse fungal group in the Basidiomycota that includes endophytes and lineages that repeatedly evolved ericoid, orchid and ectomycorrhizal lifestyle. Plants rely upon both nitrogen and phosphorous, for essential growth processes, and are often provided by mycorrhizal fungi. In this study, we investigated the cellular proteome of Serendipita vermifera MAFF305830 and closely related Serendipita vermifera subsp. bescii NFPB0129 grown in vitro under (N) ammonium and (P) phosphate starvation conditions. Mycelial growth pattern was documented under these conditions to correlate growth-specific responses to nutrient starvation. We found that N-starvation accelerated hyphal radial growth, whereas P-starvation accelerated hyphal branching. Additionally, P-starvation triggers an integrated starvation response leading to remodelling of lipid metabolism. Higher abundance of an ammonium transporter known to serve as both an ammonium sensor and stimulator of hyphal growth was detected under N-starvation. Additionally, N-starvation led to strong up-regulation of nitrate, amino acid, peptide, and urea transporters, along with several proteins predicted to have peptidase activity. Taken together, our finding suggests S. bescii and S. vermifera have the metabolic capacity for nitrogen assimilation from organic forms of N compounds. We hypothesize that the nitrogen metabolite repression is a key regulator of such organic N assimilation.

Toward a Resilient, Functional Microbiome: Drought Tolerance-Alleviating Microbes for Sustainable Agriculture.

In recent years, the utilization of novel sequencing techniques opened a new field of research into plant microbiota and was used to explore a wide diversity of microorganisms both inside and outside of plant host tissues, i.e., the endosphere and rhizosphere, respectively. An early realization from such research was that species richness and diversity of the plant microbiome are both greater than believed even a few years ago, and soil is likely home to the most abundant and diverse microbial habitats known. In most ecosystems sampled thus far, overall microbial complexity is determined by the combined influences of plant genotype, soil structure and chemistry, and prevailing environmental conditions, as well as the native "bulk soil" microbial populations from which membership is drawn. Beneficial microorganisms, traditionally referring primarily to nitrogen-fixing bacteria, plant growth-promoting rhizobacteria, and mycorrhizal fungi, play a key role in major functions such as plant nutrition acquisition and plant resistance to biotic and abiotic stresses . Utilization of plant-associated microbes in food production is likely to be critical for twenty-first century agriculture, where arable cropland is limited and food, fiber, and feed productivity must be sustained or even improved with fewer chemical inputs and less irrigation.

Rhizosphere Sampling Protocols for Microbiome (16S/18S/ITS rRNA) Library Preparation and Enrichment for the Isolation of Drought Tolerance-Promoting Microbes.

Natural plant microbiomes are abundant and have a remarkably robust composition, both as epiphytes on the plant surface and as endophytes within plant tissues. Microbes in the former "habitat" face limited nutrients and harsh environmental conditions, while those in the latter likely lead a more sheltered existence. The most populous and diverse of these microbiomes are associated with the zone around the plant roots, commonly referred to as the rhizosphere. A majority of recent studies characterize these plant-associated microbiomes by community profiling of bacteria and fungi, using amplicon-based marker genes and next-generation sequencing (NGS). Here, we collate a group of protocols that incorporate current best practices and optimized methodologies for sampling, handling of samples, and rRNA library preparation for variable regions of V5-V6 and V9 of the bacterial 16S ribosomal RNA (rRNA) gene, and the ITS2 region joining the 5.8S and 28S regions of the fungal rRNA gene. Samples collected for such culture-independent analyses can also be used for the actual isolation of microbes of interest, perhaps even those identified by the libraries described above. One group of microbes that holds promise for mediating plant stress incurred by drought are bacteria that are capable of reducing or eliminating the plant's perception of the stress through degradation of the gaseous plant hormone ethylene, which is abundantly produced in response to drought stimuli. This is accomplished by some types of soil bacteria that can produce the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which is the immediate precursor to ethylene. Here we provide a high-throughput protocol for screening of ACC deaminase-producing bacteria for the applied purpose of mitigating the impact of plant drought stress.

Non-targeted Colonization by the Endomycorrhizal Fungus, Serendipita vermifera, in Three Weeds Typically Co-occurring with Switchgrass.

Serendipita vermifera (=Sebacina vermifera; isolate MAFF305830) is a mycorrhizal fungus originally isolated from the roots of an Australian orchid that we have previously shown to be beneficial in enhancing biomass yield and drought tolerance in switchgrass, an important bioenergy crop for cellulosic ethanol production in the United States. However, almost nothing is known about how this root-associated fungus proliferates and grows through the soil matrix. Such information is critical to evaluate the possibility of non-target effects, such as unintended spread to weedy plants growing near a colonized switchgrass plant in a field environment. A microcosm experiment was conducted to study movement of vegetative mycelia of S. vermifera between intentionally inoculated switchgrass (Panicum virgatum L.) and nearby weeds. We constructed size-exclusion microcosms to test three different common weeds, large crabgrass (Digitaria sanguinalis L.), Texas panicum (Panicum texanum L.), and Broadleaf signalgrass (Brachiaria platyphylla L.), all species that typically co-occur in Southern Oklahoma and potentially compete with switchgrass. We report that such colonization of non-target plants by S. vermifera can indeed occur, seemingly via co-mingled root systems. As a consequence of colonization, significant enhancement of growth was noted in signalgrass, while a mild increase (albeit not significant) was evident in crabgrass. Migration of the fungus seems unlikely in root-free bulk soil, as we failed to see transmission when the roots were kept separate. This research is the first documentation of non-targeted colonization of this unique root symbiotic fungus and highlights the need for such assessments prior to deployment of biological organisms in the field.

Pseudomonas putida KT2440 response to nickel or cobalt induced stress by quantitative proteomics.

Nickel and cobalt are obligate nutrients for the gammaproteobacteria but when present at high concentrations they display toxic effects. These two metals are present in the environment, their origin being either from natural sources or from industrial use. In this study, the effect of inhibitory concentrations of Ni or Co was assessed on the soil bacterium Pseudomonas putida KT2440 using a proteomic approach. The identification of more than 400 spots resulted in the quantification of 160 proteins that underwent significant variations in cells exposed to Co and Ni. This analysis allowed us to depict the cellular response of P. putida cells toward metallic stress. More precisely, the parallel comparison of the two proteomes showed distinct responses of P. putida to Ni or Co toxicity. The most striking effect of Co was revealed by the accumulation of several proteins involved in the defense against oxidative damage, which include proteins involved in the detoxification of the reactive oxygen species, superoxides and peroxides. The up-regulation of the genes encoding these enzymes was confirmed using qRT-PCR. Interestingly, in the Ni-treated samples, sodB, encoding superoxide dismutase, was up-regulated, indicating the apparition of superoxide radicals due to the presence of Ni. However, the most striking effect of Ni was the accumulation of several proteins involved in the synthesis of amino acids. The measurement of the amount of amino acids in Ni-treated cells revealed a strong accumulation of glutamate.

Development of molecular markers of ectomycorrhizal fungi based on ITS region.

Four heavy-metal-tolerant isolates of ectomycorrhizal fungi namely Laccaria fraterna (Cooke & Massee) Pegler (EM-1083) and Pisolithus tinctorius (Mich. Ex Pers) Coker & Couch (EM-1290, EM-1293 and EM-1299) were selected on the basis of the previously performed experiments. DNA extraction and purification was done by following standard protocols. Subsequently, polymerase chain reaction (PCR) amplification of the ITS region was done using universal primer ITS 1 and ITS 4. The amplicons were digested with HinfI. The prominent band of the restricted fragments was excised and purified using geneEXIT. Cloning and sequencing of the excised fragment was done following the standard method. Primers were designed with sequence information using the Gene Fisher Interactive PCR Primer design software. Amplification was successfully obtained using the designed primers in Pisolithus tinctorius (EM-1293). The size of the amplicon was 200 bp.

Effect of coal ash on growth and metal uptake by some selected ectomycorrhizal fungi in vitro.

Six isolates of ectomycorrhizal fungi namely, Laccaria fraterna (EM-1083), Pisolithus tinctorius (EM-1081), Pisolithus tinctorius (EM-1290), Pisolithus tinctorius (EM-1293), Scleroderma verucosum (EM-1283), and Scleroderma cepa (EM-1233), were grown on three variants of coal ash, namely electrostatically precipitated (ESP) ash, pond ash, and bottom ash moistened with Modified Melin-Norkans (MMN) medium in vitro The colony diameter reflected the growth of the isolates on the coal ash. Metal accumulation in the mycelia was assayed by atomic absorption spectrophotometry. Six metals, namely aluminum, cadmium, chromium, iron, lead, and nickel, were selected on the basis of their abundance in coal ash and toxicity potential for the present work. Growth of vegetative mycelium on fly ash variants and metal accumulation data indicated that Pisolithus tinctorius (EM-1290) was the most tolerant among the isolates tested for most of the metals. Since this isolate is known to be mycorrhizal with Eucalyptus, it could be used for the reclamation of coal ash over burdened sites.

Prediction of delayed renal allograft function using an artificial neural network.

BACKGROUND: Delayed graft function (DGF) is one of the most important complications in the post-transplant period, having an adverse effect on both the immediate and long-term graft survival. In this study, an artificial neural network was used to predict the occurrence of DGF and compared with traditional logistical regression models for prediction of DGF. METHODS: A total of 304 cadaveric renal transplants performed at the Jewish Hospital, Louisville were included in the study. Covariate analysis by artificial neural networks and traditional logistical regression were done to predict the occurrence of DGF. RESULTS: The incidence of DGF in this study was 38%. Logistic regression analysis was more sensitive to prediction of no DGF (91 vs 70%), while the neural network was more sensitive to prediction of yes for DGF (56 vs 37%). Overall prediction accuracy for both logistic regression and the neural network was 64 and 63%, respectively. Logistic regression was 36.5% sensitive and 90.7% specific. The neural network was 63.5% sensitive and 64.8% specific. The only covariate with a P < 0.001 was the transplant of a white donor kidney to a black recipient. Cox proportional hazard regression was used to test for the negative effect of DGF on long-term graft survival. One year graft survival in patients without DGF was 92 +/- 2% vs 81 +/- 3% in patients with DGF. The 5-year graft survival was not affected by DGF in this study. CONCLUSION: Artificial neural networks may be used for prediction of DGF in cadaveric renal transplants. This method is more sensitive but less specific than logistic regression methods.