Performing Department
(N/A)
Non Technical Summary
Plant microbiomes, the microbes that live in and on plants, hold promise for sustainable disease protection in crops threatened by serious pandemic diseases. Although research has revealed many isolated beneficial microbes, each plant likely hosts thousands of different microbes that may work together to cooperatively protect plants. This project focuses on microbiomes in banana (genus Musa), which is one of the most globally valuable fruits. Commercially, bananas are among the highest production global crops, after wheat, rice, maize, and soybeans. They are the top fruit commodity globally, produced in 130 countries and valued at US$44 billion. About 85% of bananas and plantains serve as a staple food or support local economies, particularly in developing economies. However, pandemic plant diseases like Panama disease, which is caused by the fungus Fusarium oxysporum f. sp. cubense race 4 (Foc TR4), are a major threat to food security. Current predictions are that this fungus will decimate the leading Cavendish cultivar, in the near future, without alternative strategies to improve resistance. Therefore, the major goal of this project is to investigate disease-protective synergistic processes hidden in the banana plant's microbiome, to lay the foundations for sustainable disease control. Data show that different banana varieties and different cultivation histories affect disease-protective microbiomes. However, to date, molecular analyses have been limited, with few whole-community studies. The project objectives are to first characterize how microbiomes and plants form metabolic synergisms, and how these vary across banana varieties and different tissues from roots to leaves. Then, we seek to directly test the anti-fungal (i.e., anti-Foc) synergistic processes by transferring microbiomes between resistant and susceptible plants, both in petri plate cultures and in greenhouse plants. The central hypothesis is that less cultivated banana plants will have the greatest microbiome metabolic network complexity and synergistic disease-protective functions. Results will be communicated to the agricultural research communities, including student trainees, through high-impact publications, conference presentations, and in teaching graduate and undergraduate students. Our project will also train a postdoc, a PhD student, and undergraduates to be leaders in the study of agricultural microbiomes. We expect to develop new methods, such as microbiome enrichment, microbiome transfer by injection, and new analysis methods. The ultimate goal is to uncover diverse and novel synergies and bioactivities that will set the groundwork for development of sustainable microbiome-based strategies for disease management that may be relevant to a wide range of crop systems. Specific to banana, given the imminent disease threats to Cavendish, and its importance as a trade commodity and source of global food security, our project outcomes have potential for enormous impact that will inform modern sustainable biocontrol and biofortification technologies for this plant. This focus aligns with the goal of reducing use of chemicals (pesticides, antibiotics, and biocides) in banana production, and lead to the development of safer alternatives.
Animal Health Component
5%
Research Effort Categories
Basic
70%
Applied
5%
Developmental
25%
Goals / Objectives
The long-term goal of this project is to lay the foundations for engineering microbiomes that will provide low impact, sustainable pathogen control, specifically focusing on Musa spp. currently threatened by diseases. The overall objective is to evaluate how synergistic microbe-microbe and microbe-host metabolic activities differ between hosts and host tissues in response to host genotype and pathogen challenge. Additionally, we seek to develop approaches that will be broadly useful across all plant microbiomes. Our central hypotheses are that, (1) given the effects of cultivation, there will be greater metagenomic network complexity and synergistic disease-protective capacity in endemic diploid Musa genotypes than in commercial triploid cultivars, especially in key root tissues, and (2) there will be increased novel synergistic antifungal and antimicrobial compound synthesis making the 'holobiont' greater than the sum of its parts. Our rationale is that plant microbiomes hold promise for sustainable disease protection especially in crops threatened by serious pandemic disease, such as banana and plantains (Musa spp.), yet microbiome complexity has made this disease defense capacity difficult to study. Most often, plant microbiomes are assessed only superficially using single-gene approaches, or culture-based approaches that fail to characterize whole microbiome function. Conversely, the complexity of natural microbiomes is likely the key to their robust capacity to increase disease protection. This protection can be derived through ecological resource competition, enhanced defensive compound biosynthetic capacity, or fine-tuned regulatory control through microbe-host signaling, or microbe-microbe synergistic processes. Yet, long-cultivated crops can lose microbiome richness, and hence microbiome-derived protection. Given the global importance of banana and its high vulnerability to disease, it can serve as a model plan for studying synergistic defensive interactions. To date, surprisingly little is known about synergistic disease-protective microbiomes in Musa spp. Without this understanding, integrated disease management strategies may be significantly less effective: current control strategies are only partly effective and present serious challenges such as synthetic chemical toxicity, resistant pathogen evolution, and slow development of resistant cultivars. Our project will achieve the following specific objectives:Objective 1. Characterize how endophytic microbiome synergies vary across banana (Musa spp.) genotypes and tissues before and after pathogen challenge. We hypothesize that Musa genotypes with the least history of cultivation will support the richest microbiomes, metagenome repertoires, and predicted antimicrobial functions and facilitate synergistic microbe-microbe associations in resistant cultivars. We predict that plant tissues at the key interfaces with pathogens, such as rhizome surface-cortex tissue, will be richest in synergistic microbes, and are most impacted by environmental source/soils of origin. We predict metagenomic metabolic models will uncover defense synergies with co-synthesized or co-regulated antimicrobial compounds.Obj. 1.1 Collect ~30 Musa genotypes along with soil/rhizosphere for each plant and non-destructively characterize root, leaf, and rhizosphere microbiomes. This will (1) determine the baseline diversity, complexity, and function of native microbiomes (and surrounding soil/rhizosphere) (2) enable tests of the effects of environment and source for these plants that will later be grown and challenged in Obj. 1.2 and 1.3.Obj. 1.2 Transplant Musa plants to greenhouse and allow to acclimatize, then sample non-destructively again, to characterize root, leaf, and rhizosphere microbiomes. This will (1) stabilize plants for pathogen challenge and (2) determine possible effects of acclimatization and transplantation to uniform greenhouse soil.Obj. 1.3 Challenge Musa plants with two Foc pathogen strains, assessing disease phenotypes and analyzing microbiome changes. This will (1) evaluate changes in endophytic microbiomes driven by fungal pathogen challenge, and (2) assess disease phenotypes for holobionts.Obj. 1.4 Sequence and analyze microbiomes from Obj. 1.1, 1.2, 1.3 to compare diversity, function, metabolic synergism, and responses to pathogens. This will characterize and analyze data from the preceding sub-objectives.Objective 2. Evaluate the effects of microbiome transfers between resistant and susceptible Musa genotypes. By transferring microbiomes between plants, we will disentangle the effects of host and microbiota on disease resistance observed in Obj 1. We hypothesize that transferred microbiomes from diploid or less cultivated plants, or from roots will confer increased fungal pathogen resistance in recipient plants, showing microbiomes play a major role. We predict some combinations will show synergistic host-microbe associations in resistant cultivars. Metatranscriptomic functional analyses and changes driven host and pathogen will inform the selection of microbes for Obj 3.Obj. 2.1 Establish donor and recipient Musa plant genotypes with different susceptibilities to Foc TR1 and TR4 and evaluate their microbiomes. This will characterize initial microbiome profiles for comparison with Obj 2.3.Obj. 2.2 Inoculate recipient Musa plants with donor endophytic microbiomes and assess the donor microbiome's growth and persistence. This will assess the effectiveness of microbial enriched layer taxa at establishing in new plants.Obj. 2.3 Challenge recipient Musa plants with pathogens (Foc TR1 and TR4) and assess disease phenotypes and microbiome changes. This will test the effects of microbiome on the host (holobiont) response to Fusarium wilt disease.Objective 3. Directly test synergistic interactions among subsets of microbes and Musa plants in vitro and in planta. By analyzing how select subsets of the microbiome act in vivo and in planta, we will disentangle defensive synergism at several levels. We hypothesize that challenge with fungal pathogens will cause changes in microbial gene expression and metabolite synthesis that is more than additive, i.e., showing metabolic complementarity or co-regulation. Novel in planta holobiont antimicrobial synergistic defense processes and confirm in silico and in planta predictions from Obj 1 and Obj 2, providing candidate synergistic associations for developing new bio-amendments.Obj. 3.1 Assess synergistic interactions by measuring novel gene expression and metabolomic profiles in vitro with and without plant homogenate and Foc. This will follow up on our preliminary and ongoing experiments with whole microbiome and plant communities, with the critical addition that we will test individual and combined microbes under different host-like backgrounds.Obj. 3.2 Analyze effects of in planta conditions on synergistic interactions for selected Musa endophyte sub-communities introduced to microbe-depleted plants. Given the interest in microbial inocula for plant pathogen protection, we seek to develop in planta tests to assess our findings from Obj 3.1 in the context of disease. Expected outcomes include new critical knowledge of microbiome diversity and functional capacity in the context of in planta communities and synergistic antimicrobial compound synthesis. While outcomes will be important for addressing a serious global pandemic disease of Musa spp., this system serves as a model. Results will also have a positive impact through setting the groundwork for development of sustainable microbiome-based strategies to enhance integrated disease management for a wide range of crop systems.
Project Methods
Efforts will advance knowledge through determining how synergistic interactions contribute to disease protective metabolic activities through (Obj.1) challenging plants with fugal pathogens for Musa cultivars and tissues, (Obj. 2) testing microbiome transplants to investigate host-microbiome synergism, and (Obj. 3) directly testing defensive synergism in subsets of microbes. Impacts will contribute critical knowledge of processes essential to microbiome engineering for sustainable pathogen control.Methods Obj. 1: We will compare Musa genotypes and tissues, assessing shifts in microbiomes after plants are challenged by F. oxysporum f. sp. cubense (Foc). For ~30 Musa genotypes we will non-destructively characterize root, leaf, and rhizosphere microbiomes. Then, we will challenge plants with Foc, assessing disease phenotypes and analyzing microbiome metagenome changes discover function and metabolic synergism in response to pathogens.Plant collection and processing: We will collect plantlets from the Tropical Research and Education Center, U. Florida, for ~30 cultivars of Musa including A- B- diploids, triploids, and tetraploids, and their hybrids, with 12 biological replicates each. DNA will be isolated from ~1 g of adherent soil with the DNeasy PowerSoil Pro Kit, then plants will be surface sterilized and DNA isolated from the (1) rhizome outer cortex, (2) rhizome central vascular tissue, and (3) leaf/pseudostem.Transplanting and inoculation: Plants will be potted in sterilized soil and acclimatized before challenging with Fusarium oxysporum f. sp. cubense isolate 23532 (VCG 0124) Race 1 (ATCC 96285) and isolate 23486 (VCG 0120) Race 4 (ATCC 96289) (Foc TR1 and Foc TR4). PD Brown has an APHIS permit for Foc TR1 and will apply for the same for TR4. 1/3 of replicates will be inoculated with TR1, 1/3 with TR4, and 1/3 with sterile water as a control. Progress of Fusarium wilt disease will be assessed each 2 days.Characterizing endophytic microbiomes: Plant tissues will be sampled as before, then microbiomes isolated via a protocol that removes plant cellular matter using Nycodenz gradients. DNA will be isolated from Nycodenz using the DNeasy kit. A sub-sample will be submitted for long-read sequencing. Libraries will be sequenced on Illumina NovaSeq 6000. Hybrid assembly will be performed with HASLR. Quality will be checked with QUAST/CheckM and DIAMOND before annotation with PROKKA or JGI/MycoCosm, FunGap.Diversity analysis and network modeling: Diversity will be assessed using MetaPhlan4, Phyloseq and Chao, ACE, Shannon, and Simpson indices and Bray-Curtis dissimilarity will be plotted with PCoA and PERMANOVA tests in'Adonis'. Network analyses in Cytoscape with NetworkAnalyzer will integrate metadata in FlashWeave. Orthologs will be clustered with Roary and gene ontology (GO) enrichment tests will be done with topGO.Predict antimicrobial biosynthetic gene clusters (BGCs): We will search for antimicrobial function using DeepBGC, antiSMASH and BiG-SCAPE for product classes (NRPS, PKS I, PKS other, PKS-NRPS hybrids, RiPPs, saccharides and terpenes, antifungals, antibacterials, and antivirals).Genome-scale metabolic models to investigate metabolic synergism: We will analyze genome-scale metabolic models (GEMs) in modelSEED and metagenome-scale metabolic models (MEMs) with MicrobioLink, Pathway Tools, and MetaFlux.Methods Obj. 2: We will test if transferred microbiomes from diploid or less cultivated plants confer increased fungal pathogen resistance (i.e., that resistance is at least partially conferred by the microbiome). We will establish donor and recipient Musa plant genotypes with different susceptibilities to Foc TR1 and TR4 and inoculate recipient Musa plants with donor endophytic microbiomes and assess growth and persistence. We will then challenge recipient Musa plants with pathogens (Foc TR1 and TR4) and assess disease and microbiome changes.Select treatments and challenge with Foc TR1/TR4: We will select ~8 Musa genotypes displaying a range of Foc TR1 and TR4 susceptibilities with 16 plants per genotype per set (set 1 = 4 per treatment with each of 4 donor genotypes, no pathogen; set 2 = 4 per treatment with each donor, with TR1; set 3 = same for TR4; set 4 = same with control/self-donor). We will destructively sample resistant donors and use their microbiomes to inoculate susceptible recipients using a sterile puncture tool, closing the wound with a tissue plug and grafting tape.Metatranscriptomics: Plant mRNA will be captured with polyDT in the NEBNext Ultra Directional RNA Kit, and non-captured RNA will be rRNA depleted with RiboZero Plus Kit before standard library preparation. Comparative metatranscriptomic analysis will be performed with Kallisto and SAMSA2. Differential expression analysis will be performed with DEseq2 and functional enrichment, pathway analysis, and co-expression analysis will be performed using BlastKoala, KEGG, and String, assessing network connectivity and co-abundance networks with Cytoscape and WGCNA.Methods Obj. 3: We will directly test synergistic interactions among microbes and Musa plants in vitro and in planta by measuring gene expression and metabolomic profiles in vitro with and without plant homogenate and Foc. Then we will analyze in planta conditions on synergistic interactions for selected endophyte sub-communities introduced to microbe-depleted plants.Musa microbiome-depletion and culturing: We will deplete plant microbiomes using standard propagation protocols, slicing rhizome/pseudostem meristematic tissue radially. For 6 focal Musa genotypes, 6 types of agar plate will be prepared: nutrient agar (NA); potato dextrose agar (PDA); microbiome-depleted plant-homogenate NA; microbiome-depleted plant-homogenate PDA; full holobiont-homogenate NA; and full holobiont-homogenate PDA. We will inoculate test plates with cultured Foc TR1 and TR4 on agar and streak the enriched microbiome layers from selected plants derived from Obj 1 and 2 onto full holobiont-homogenate NA and PDA plates. We will pick ~50-100 colonies of interest and transfer each to a fresh plate. One side will be inoculated with Foc TR1 or TR4 to test for inhibition.Inoculation of experimental plates and metatranscriptomics and metabolomics: Candidates that induce a zone of inhibition will proceed to metatranscriptomics and metabolomics. Cells will be pelleted and processed immediately for metatranscriptomics and metabolomics. Replicate samples will be processed for untargeted metabolomics in orthogonal design using MS2 data to create molecular networks and Venn diagrams through the GNPS database. We will identify compounds via mass GC-QTOF, LC-MS, and LC-QTOF. MS/MS fragmentation will be compared with Metlin, MassBank, HMDB, and GNPS. Pathway analysis of metabolites will be conducted using MetaboAnalyst.Plant inoculation and analysis: Enriched communities of interest based on results above will be isolated from Musa plants and introduced to plants with at least 4 replicates each. Disease assessment and monitoring will follow methods above. Metatranscriptomic and metabolomic analyses will be performed on samples showing signs of synergistic disease-protective effects as above.Evaluation and milestones: Project success will be evaluated based on results showing (1) significant genome data on hundreds of protective microbiota from ~30 Musa cultivars; (2) discovery of microbial community structure and function driving endophyte responses to Fusarium pathogens; (3) multi-omic sequence profiles for thousands of endophytic microbes and hundreds of thousands of genes; (4) discovery of hundreds of disease-protective host-microbe and microbe-microbe synergistic biosynthetic gene clusters; (5) obtaining direct metabolomic data revealing in vitro and in vivo processes of microbiome-based pathogen resistance; (6) developing new laboratory and computation methods for plant microbiome analysis.