Boxwood phyllosphere fungal and bacterial communities and their differential responses to film-forming anti-desiccants.

BACKGROUND: Anti-desiccant is a class of agrochemicals widely used to protect plants from water stresses, rapid temperature variations, heat and sunburn, frost and freeze damages, transplant shock, and pathogen and pest attack. Although anti-desiccants are generally considered non-toxic to organisms, it is unclear whether they may impact the phyllosphere microbial communities. In this study, three film-forming anti-desiccant products, TransFilm, Vapor Gard, and Wilt-Pruf were applied to the canopy of two boxwood cultivars 'Vardar Valley' and 'Justin Brouwers' on April 13 and August 26, 2021. Shoot samples were collected from boxwood plants treated with each of the three products, as well as nontreated control on June 16, August 26 (before the second treatment), and October 18. Microbial and plant genomic DNA was isolated together and 16S rRNA gene and the extended internal transcribed spacer regions were amplified with PCR and sequenced on a Nanopore MinION platform for bacterial and fungal identification. RESULTS: Bacterial communities were more diverse than fungal communities. At the phylum level, the boxwood phyllosphere was dominated by Proteobacteria and Ascomycota; at the genus level, Methylobacterium and Shiraia were the most abundant bacteria and fungi, respectively. Among the three film-forming anti-desiccants, Vapor Gard and Wilt-Pruf had more impact than TransFilm on the microbial communities. Specifically, broader impacts were observed on fungal than bacterial community composition and structure, with most affected fungi being suppressed while bacteria promoted. CONCLUSION: This study addressed several major knowledge gaps regarding boxwood phyllosphere microbiota and the impact of anti-desiccants on plant microbiome. We identified diverse microbial communities of boxwood, a major evergreen woody crop and an iconic landscape plant. We also found differential effects of three film-forming anti-desiccants on the composition and structure of bacterial and fungal communities. These findings advanced our understanding of the associated microbiome of this landmark plant, enabling growers to fully utilize the potentials of microbiome and three anti-desiccants in improving boxwood health and productivity.

Characterization of Boxwood Shoot Bacterial Communities and Potential Impact from Fungicide Treatments.

Phyllosphere bacterial communities play important roles in plant fitness and growth. The objective of this study was to characterize the epiphytic and endophytic bacterial communities of boxwood shoots and determine how they may respond to commonly used fungicides. In early summer and early fall, shoot samples were collected immediately before and 1, 7, and 14 days after three fungicides containing chlorothalonil and/or propiconazole were applied to the canopy. Total genomic DNA from shoot surface washings and surface-sterilized shoot tissues was used as the template for 16S rRNA metabarcoding, and the amplicons were sequenced on a Nanopore MinION sequencer to characterize the epiphytic and endophytic communities. The bacterial communities were phylogenetically more diverse on the boxwood shoot surface than in the internal tissue, although the two communities shared 12.7% of the total 1,649 identified genera. The most abundant epiphytes were Methylobacterium and Pantoea, while Stenotrophomonas and Brevundimonas were the dominant endophytes. Fungicide treatments had strong impacts on epiphytic bacterial community structure and composition. Analysis of compositions of microbiomes with bias correction (ANCOM-BC) and analysis of variance (ANOVA)-like differential expression (ALDEx2) together identified 312 and 1,362 epiphytes changed in abundance due to fungicide treatments in early summer and early fall, respectively, and over 50% of these epiphytes were negatively impacted by fungicide. The two chlorothalonil-based contact fungicides demonstrated more marked effects than the propiconazole-based systemic fungicide. These results are foundational for exploring and utilizing the full potential of the microbiome and fungicide applications and developing a systems approach to boxwood health and production. IMPORTANCE Agrochemicals are important tools for safeguarding plants from invasive pathogens, insects, mites, and weeds. How they may affect the plant microbiome, a critical component of crop health and production, was poorly understood. Here, we used boxwood, an iconic low-maintenance landscape plant, to characterize shoot epiphytic and endophytic bacterial communities and their responses to contact and systemic fungicides. This study expanded our understanding of the above-ground microbiome in ornamental plants and is foundational for utilizing the full benefits of the microbiome in concert with different fungicide chemistries to improve boxwood health. This study also sets an example for a more thorough evaluation of these and other agrochemicals for their effects on boxwood microbiomes during production and offers an expanded systems approach that could be used with other crops for enhanced integrated pest management.

Combating an Invasive Boxwood Pathogen - Calonectria pseudonaviculata - in the United States by Shifting Production to Less-Susceptible Cultivars.

Boxwood blight (BB) caused by Calonectria pseudonaviculata (Cps), an economically devastating disease affecting everyone in the supply chain from growers to gardeners, was first officially documented in the United States in 2011. This disease has taken a heavy toll on boxwood, an iconic landscape plant and the number one evergreen nursery crop. Instead of abandoning boxwood production and switching to other evergreens, growers in the United States, informed by the latest research, have elected to combat this disease. One of the strategies employed has been to shift boxwood production from highly susceptible to less-susceptible cultivars. The objective of this study was to investigate the ongoing shift by comparing boxwood sales of 17 selected nurseries from seven states across the country in 2011, 2016, and 2021. Results revealed an additional 21.24% (from 38.58 to 60.04%) in sales of less-susceptible boxwood cultivars in 2021 compared with 2016. The less-susceptible cultivars responsible for increased boxwood sales are 'Winter Gem', 'Wintergreen', 'SB 300' (Freedom), 'SB 108' (Independence), and 'Little Missy'. The potential for long-term positive impact on sustainable boxwood production and plantings in the United States through the use of less-susceptible cultivars is discussed. This shift in boxwood choices builds crop health into new plantings of this landmark plant and sustains growth in demand for boxwood. This sets a new example of sustainable protection for a crop that is under serious pressure from an invasive pathogen.

cDNA Transcriptome of Arabidopsis Reveals Various Defense Priming Induced by a Broad-Spectrum Biocontrol Agent Burkholderia sp. SSG.

Burkholderia sp. SSG is a potent biological control agent. Even though its survival on the leaf surface declined rapidly, SSG provided extended, moderate plant protection from a broad spectrum of pathogens. This study used Arabidopsis Col-0 and its mutants, eds16-1, npr1-1, and pad4-1 as model plants and compared treated plants with non-treated controls to elucidate whether SSG triggers plant defense priming. Only eds16-1 leaves with SSG became purplish, suggesting the involvement of salicylic acid (SA) in SSG-induced priming. cDNA sequencing of Col-0 plants and differential gene expression analysis identified 120 and 119 differentially expressed genes (DEGs) at 6- and 24-h post-treatment (hpt) with SSG, respectively. Most of these DEGs encoded responses to biotic and abiotic stimuli or stresses; four DEGs had more than two isoforms. A total of 23 DEGs were shared at 6 and 24 hpt, showing four regulation patterns. Functional categorization of these shared DEGs, and 44 very significantly upregulated DEGs revealed that SSG triggered various defense priming mechanisms, including responses to phosphate or iron deficiency, modulation of defense-linked SA, jasmonic acid, ethylene, and abscisic acid pathways, defense-related gene regulation, and chromatin modification. These data support that SSG is an induced systemic resistance (ISR) trigger conferring plant protection upon pathogen encounter.

Sexual dimorphism in the dioecious willow Salix purpurea.

PREMISE: The evolution of sex chromosomes is driven by sexual dimorphism, yet it can be challenging to document sexually dimorphic traits in dioecious plant species. At the genetic level, sexual dimorphism can be identified through sequence variation between females and males associated with sexually antagonistic traits and different fitness optima. This study aims to examine sexual dimorphism for 26 traits in three populations of Salix purpurea (a diversity panel and F1 and F2 populations) and determine the effect of the traits on biomass yield, a key trait in Salix bioenergy crops across multiple years, locations, and under manipulated growth conditions. METHODS: Sexual dimorphism was evaluated for morphological, phenological, physiological, and wood composition traits in a diversity panel of unrelated S. purpurea accessions and in full-sib F1 and F2 families produced through controlled cross pollinations and grown in replicated field trials. RESULTS: We observed sexual dimorphism in the timing of development for several traits that were highly predictive of biomass yield across three populations of S. purpurea. Across all populations and years surveyed, males had significantly shallower branching angle. Male plants highly predictive of biomass yield across three populations of S. purpurea also accumulated more nitrogen under fertilizer amendment as measured by SPAD in the diversity panel and had greater susceptibility to the rust fungus Melampsora americana in the F2 family. Allometric modelling of biomass yield showed an effect of sex and of location on the interaction between yield and stem height. CONCLUSIONS: These results provide evidence of sexual dimorphism for certain traits in S. purpurea that may be involved in sex chromosome evolution.

Integrative genomics reveals paths to sex dimorphism in Salix purpurea L.

Sex dimorphism and gene expression were studied in developing catkins in 159 F2 individuals from the bioenergy crop Salix purpurea, and potential mechanisms and pathways for regulating sex development were explored. Differential expression, eQTL, bisulfite sequencing, and network analysis were used to characterize sex dimorphism, detect candidate master regulator genes, and identify pathways through which the sex determination region (SDR) may mediate sex dimorphism. Eleven genes are presented as candidates for master regulators of sex, supported by gene expression and network analyses. These include genes putatively involved in hormone signaling, epigenetic modification, and regulation of transcription. eQTL analysis revealed a suite of transcription factors and genes involved in secondary metabolism and floral development that were predicted to be under direct control of the sex determination region. Furthermore, data from bisulfite sequencing and small RNA sequencing revealed strong differences in expression between males and females that would implicate both of these processes in sex dimorphism pathways. These data indicate that the mechanism of sex determination in Salix purpurea is likely different from that observed in the related genus Populus. This further demonstrates the dynamic nature of SDRs in plants, which involves a multitude of mechanisms of sex determination and a high rate of turnover.

Multiple independent recombinations led to hermaphroditism in grapevine.

Hermaphroditic (perfect) flowers were a key trait in grapevine domestication, enabling a drastic increase in yields due to the efficiency of self-pollination in the domesticated grapevine (Vitis vinifera L. ssp. vinifera). In contrast, all extant wild Vitis species are dioecious, each plant having only male or female flowers. In this study, we identified the male (M) and female (f) haplotypes of the sex-determining region (SDR) in the wild grapevine species V. cinerea and confirmed the boundaries of the SDR. We also demonstrated that the SDR and its boundaries are precisely conserved across the Vitis genus using shotgun resequencing data of 556 wild and domesticated accessions from North America, East Asia, and Europe. A high linkage disequilibrium was found at the SDR in all wild grape species, while different recombination signatures were observed along the hermaphrodite (H) haplotype of 363 cultivated accessions, revealing two distinct H haplotypes, named H1 and H2. To further examine the H2 haplotype, we sequenced the genome of two grapevine cultivars, 'Riesling' and 'Chardonnay'. By reconstructing the first two H2 haplotypes, we estimated the divergence time between H1 and H2 haplotypes at ∼6 million years ago, which predates the domestication of grapevine (∼8,000 y ago). Our findings emphasize the important role of recombination suppression in maintaining dioecy in wild grape species and lend additional support to the hypothesis that at least two independent recombination events led to the reversion to hermaphroditism in grapevine.

Using acetone for rapid PCR-amplifiable DNA extraction from recalcitrant woody plant taxa.

PREMISE: Quick and effective DNA extraction from plants for subsequent PCR amplification is sometimes challenging when working across diverse plant taxa that may contain a variety of inhibitory compounds. Time-consuming methods may be needed to overcome these inhibitory effects as well as the effects of various preservation and collection methods to extract DNA from leaf samples. Our objective was to develop a rapid DNA extraction protocol that could be used with diverse plant taxa to produce high-quality DNA suitable for downstream PCR applications. METHODS AND RESULTS: We tested the efficacy of acetone in extracting DNA from fresh, frozen, oven-dried, acetone-fixed, and herbarium leaf material of 22 species from 16 woody and herbaceous plant families. An improved simplified DNA extraction protocol was developed using acetone-fixed leaf material. The addition of 1% sodium dodecyl sulfate solution resulted in the optimal extraction from all tissue samples. The DNA resulting from the extraction protocol was readily amplified using real-time PCR assays. CONCLUSIONS: The protocol described here resulted in the extraction of DNA from recalcitrant plant species that was of sufficient quality and quantity for PCR amplification, as indicated by the low threshold cycle values from real-time assays. This method is simple, fast, and cost-effective, and is a reliable tool for extracting high-quality DNA from plant material containing PCR inhibitors.

Joint linkage and association mapping of complex traits in shrub willow (Salix purpurea L.).

BACKGROUND AND AIMS: Increasing energy demands and the necessity to reduce greenhouse gas emissions are key motivating factors driving the development of lignocellulosic crops as an alternative to non-renewable energy sources. The effects of global climate change will require a better understanding of the genetic basis of complex adaptive traits to breed more resilient bioenergy feedstocks, like willow (Salix spp.). Shrub willow is a sustainable and dedicated bioenergy crop, bred to be fast-growing and high-yielding on marginal land without competing with food crops. In a rapidly changing climate, genomic advances will be vital for the sustained improvement of willow and other non-model bioenergy crops. Here, joint genetic mapping was used to exploit genetic variation garnered from both recent and historical recombination events in S. purpurea. METHODS: A panel of North American naturalized S. purpurea accessions and full-sib F2S. purpurea population were genotyped and phenotyped for a suite of morphological, physiological, pest and disease resistance, and wood chemical composition traits, collected from multi-environment and multi-year replicated field trials. Controlling for population stratification and kinship in the association panel and spatial variation in the F2, a comprehensive mixed model analysis was used to dissect the complex genetic architecture and plasticity of these important traits. KEY RESULTS: Individually, genome-wide association (GWAS) models differed in terms of power, but the combined approach, which corrects for yearly and environmental co-factors across datasets, improved the overall detection and resolution of associated loci. Although there were few significant GWAS hits located within support intervals of QTL for corresponding traits in the F2, many large-effect QTL were identified, as well as QTL hotspots. CONCLUSIONS: This study provides the first comparison of linkage analysis and linkage disequilibrium mapping approaches in Salix, and highlights the complementarity and limits of these two methods for elucidating the genetic architecture of complex bioenergy-related traits of a woody perennial breeding programme.

Characterization of a large sex determination region in Salix purpurea L. (Salicaceae).

Dioecy has evolved numerous times in plants, but heteromorphic sex chromosomes are apparently rare. Sex determination has been studied in multiple Salix and Populus (Salicaceae) species, and P. trichocarpa has an XY sex determination system on chromosome 19, while S. suchowensis and S. viminalis have a ZW system on chromosome 15. Here we use whole genome sequencing coupled with quantitative trait locus mapping and a genome-wide association study to characterize the genomic composition of the non-recombining portion of the sex determination region. We demonstrate that Salix purpurea also has a ZW system on chromosome 15. The sex determination region has reduced recombination, high structural polymorphism, an abundance of transposable elements, and contains genes that are involved in sex expression in other plants. We also show that chromosome 19 contains sex-associated markers in this S. purpurea assembly, along with other autosomes. This raises the intriguing possibility of a translocation of the sex determination region within the Salicaceae lineage, suggesting a common evolutionary origin of the Populus and Salix sex determination loci.

Early selection of novel triploid hybrids of shrub willow with improved biomass yield relative to diploids.

BACKGROUND: Genetic improvement of shrub willow (Salix), a perennial energy crop common to temperate climates, has led to the development of new cultivars with improved biomass yield, pest and disease resistance, and biomass composition suitable for bioenergy applications. These improvements have largely been associated with species hybridization, yet little is known about the genetic mechanisms responsible for improved yield and performance of certain willow species hybrids. RESULTS: The top performing genotypes in this study, representing advanced pedigrees compared with those in previous studies, were mostly triploid in nature and outperformed current commercial cultivars. Of the genotypes studied, the diploids had the lowest mean yield of 8.29 oven dry Mg ha-1 yr-1, while triploids yielded 12.65 Mg ha-1 yr-1, with the top five producing over 16 Mg ha-1 yr-1. Triploids had high stem area and height across all three years of growth in addition to greatest specific gravity. The lowest specific gravity was observed among the tetraploid genotypes. Height was the early trait most correlated with and the best predictor of third-year yield. CONCLUSIONS: These results establish a paradigm for future breeding and improvement of Salix bioenergy crops based on the development of triploid species hybrids. Stem height and total stem area are effective traits for early prediction of relative yield performance.