Nutrient availability and plant phenological stage influence the substrate microbiome in container-grown Impatiens walleriana 'Xtreme Red'.

BACKGROUND: The microbiome plays a fundamental role in plant health and performance. Soil serves as a reservoir of microbial diversity where plants attract microorganisms via root exudates. The soil has an important impact on the composition of the rhizosphere microbiome, but greenhouse ornamental plants are commonly grown in soilless substrates. While soil microbiomes have been extensively studied in traditional agriculture to improve plant performance, health, and sustainability, information about the microbiomes of soilless substrates is still limited. Thus, we conducted an experiment to explore the microbiome of a peat-based substrate used in container production of Impatiens walleriana, a popular greenhouse ornamental plant. We investigated the effects of plant phenological stage and fertilization level on the substrate microbiome. RESULTS: Impatiens plants grown under low fertilization rates were smaller and produced more flowers than plants grown under optimum and high fertilization. The top five bacterial phyla present in the substrate were Proteobacteria, Actinobacteria, Bacteriodota, Verrucomicrobiota, and Planctomycetota. We found a total of 2,535 amplicon sequence variants (ASV) grouped into 299 genera. The substrate core microbiome was represented by only 1.8% (48) of the identified ASV. The microbiome community composition was influenced by plant phenological stage and fertilizer levels. Phenological stage exhibited a stronger influence on microbiome composition than fertilizer levels. Differential abundance analysis using DESeq2 identified more ASVs significantly affected (enriched or depleted) in the high fertilizer levels at flowering. As observed for community composition, the effect of plant phenological stage on microbial community function was stronger than fertilizer level. Phenological stage and fertilizer treatments did not affect alpha-diversity in the substrate. CONCLUSIONS: In container-grown ornamental plants, the substrate serves as the main microbial reservoir for the plant, and the plant and agricultural inputs (fertilization) modulate the microbial community structure and function of the substrate. The differences observed in substrate microbiome composition across plant phenological stage were explained by pH, total organic carbon (TOC) and fluoride, and across fertilizer levels by pH and phosphate (PO4). Our project provides an initial diversity profile of the bacteria occurring in soilless substrates, an underexplored source of microbial diversity.

RNA-sequencing reveals early, dynamic transcriptome changes in the corollas of pollinated petunias.

BACKGROUND: Pollination reduces flower longevity in many angiosperms by accelerating corolla senescence. This response requires hormone signaling between the floral organs and results in the degradation of macromolecules and organelles within the petals to allow for nutrient remobilization to developing seeds. To investigate early pollination-induced changes in petal gene expression, we utilized high-throughput sequencing to identify transcripts that were differentially expressed between corollas of pollinated Petunia × hybrida flowers and their unpollinated controls at 12, 18, and 24 hours after opening. RESULTS: In total, close to 0.5 billion Illumina 101 bp reads were generated, de novo assembled, and annotated, resulting in an EST library of approximately 33 K genes. Over 4,700 unique, differentially expressed genes were identified using comparisons between the pollinated and unpollinated libraries followed by pairwise comparisons of pollinated libraries to unpollinated libraries from the same time point (i.e. 12-P/U, 18-P/U, and 24-P/U) in the Bioconductor R package DESeq2. Over 500 gene ontology terms were enriched. The response to auxin stimulus and response to 1-aminocyclopropane-1-carboxylic acid terms were enriched by 12 hours after pollination (hap). Using weighted gene correlation network analysis (WGCNA), three pollination-specific modules were identified. Module I had increased expression across pollinated corollas at 12, 18, and 24 h, and modules II and III had a peak of expression in pollinated corollas at 18 h. A total of 15 enriched KEGG pathways were identified. Many of the genes from these pathways were involved in metabolic processes or signaling. More than 300 differentially expressed transcription factors were identified. CONCLUSIONS: Gene expression changes in corollas were detected within 12 hap, well before fertilization and corolla wilting or ethylene evolution. Significant changes in gene expression occurred at 18 hap, including the up-regulation of autophagy and down-regulation of ribosomal genes and genes involved in carbon fixation. This transcriptomic database will greatly expand the genetic resources available in petunia. Additionally, it will guide future research aimed at identifying the best targets for increasing flower longevity by delaying corolla senescence.

Proteomic analysis of pollination-induced corolla senescence in petunia.

Senescence represents the last phase of petal development during which macromolecules and organelles are degraded and nutrients are recycled to developing tissues. To understand better the post-transcriptional changes regulating petal senescence, a proteomic approach was used to profile protein changes during the senescence of Petuniaxhybrida 'Mitchell Diploid' corollas. Total soluble proteins were extracted from unpollinated petunia corollas at 0, 24, 48, and 72 h after flower opening and at 24, 48, and 72 h after pollination. Two-dimensional gel electrophoresis (2-DE) was used to identify proteins that were differentially expressed in non-senescing (unpollinated) and senescing (pollinated) corollas, and image analysis was used to determine which proteins were up- or down-regulated by the experimentally determined cut-off of 2.1-fold for P <0.05. One hundred and thirty-three differentially expressed protein spots were selected for sequencing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the identity of these proteins. Searching translated EST databases and the NCBI non-redundant protein database, it was possible to assign a putative identification to greater than 90% of these proteins. Many of the senescence up-regulated proteins were putatively involved in defence and stress responses or macromolecule catabolism. Some proteins, not previously characterized during flower senescence, were identified, including an orthologue of the tomato abscisic acid stress ripening protein 4 (ASR4). Gene expression patterns did not always correlate with protein expression, confirming that both proteomic and genomic approaches will be required to obtain a detailed understanding of the regulation of petal senescence.

Virus-Induced Gene Silencing for Functional Analysis of Flower Traits in Petunia.

Virus-induced gene silencing (VIGS) uses recombinant viruses to knock down the expression of endogenous plant genes, allowing for rapid functional analysis without generating stable transgenic plants. The Tobacco rattle virus (TRV) is a popular vector for VIGS because it has a wide host range that includes Petunia × hybrida (petunia), and it induces minimal viral symptoms. Using reporter genes like chalcone synthase (CHS) in tandem with a gene of interest (GOI; pTRV2-PhCHS-GOI), it is possible to visually identify silenced flowers so that phenotyping is more accurate. Inoculation methods and environmental conditions need to be optimized for each host plant-virus interaction to maximize silencing efficiency. This chapter will provide detailed protocols for VIGS in petunia, with an emphasis on the investigation of flower phenotypes.

Exogenous abscisic acid enhances physiological, metabolic, and transcriptional cold acclimation responses in greenhouse-grown grapevines.

Previous studies have demonstrated that the freezing tolerance (FT) of grapevine was enhanced by foliar application of exogenous abscisic acid (exo-ABA), a treatment which might be incorporated into cultural practices to mitigate cold damage in vineyards. To investigate the underlying mechanisms of this response, a two-year (2017 and 2018) study was conducted to characterize the effects of exo-ABA on greenhouse-grown 'Cabernet franc' grapevine. In control grapevines, both physiological (deeper dormancy) and biochemical (sugar accumulation in buds) changes occurred, indicating that grapevines initiated cold acclimation in the greenhouse. Compared to control, exo-ABA decreased stomatal conductance 2 h after application. Two weeks post application, exo-ABA treated grapevines showed accelerated transition of grapevine physiology during cold acclimation (increased depth of dormancy, decreased bud water content and enhanced bud FT), relative to control. Exo-ABA induced the accumulation of several sugars in buds including the raffinose family oligosaccharides (RFOs), and the RFO precursor, galactinol. The expression of raffinose and galactinol synthase genes was higher in exo-ABA treated grapevine buds, compared to control. The new findings from this study have advanced our understanding of the role of ABA in grapevine FT, which will be useful to develop future strategies to protect grapevines from cold damage.

Ethylene regulates phosphorus remobilization and expression of a phosphate transporter (PhPT1) during petunia corolla senescence.

The programmed degradation of macromolecules during petal senescence allows the plant to remobilize nutrients from dying to developing tissues. Ethylene is involved in regulating the timing of nucleic acid degradation in petunia, but it is not clear if ethylene has a role in the remobilization of phosphorus during petal senescence. To investigate ethylene's role in nutrient remobilization, the P content of petals (collectively called the corolla) during early development and senescence was compared in ethylene-sensitive wild type Petunia x hybrida 'Mitchell Diploid' (MD) and transgenic petunias with reduced sensitivity to ethylene (35S::etr1-1). When compared to the total P content of corollas on the day of flower opening (the early non-senescing stage), P in MD corollas had decreased 74% by the late stage of senescence (advanced wilting). By contrast, P levels were only reduced by an average of 32% during etr1-1 corolla (lines 44568 and Z00-35-10) senescence. A high-affinity phosphate transporter, PhPT1 (PhPht1;1), was cloned from senescing petunia corollas by RT-PCR. PhPT1 expression was up-regulated during MD corolla senescence and a much smaller increase was detected during the senescence of etr1-1 petunia corollas. PhPT1 mRNA levels showed a rapid increase in detached corollas (treated at 1 d after flower opening) following treatment with low levels of ethylene (0.1 microl l(-1)). Transcripts accumulated in the presence of the protein synthesis inhibitor, cycloheximide, indicating that PhPT1 is a primary ethylene response gene. PhPT1 is a putative phosphate transporter that may function in Pi translocation during senescence.

Identification of Pseudomonas Spp. That Increase Ornamental Crop Quality During Abiotic Stress.

The sustainability of ornamental crop production is of increasing concern to both producers and consumers. As resources become more limited, it is important for greenhouse growers to reduce production inputs such as water and chemical fertilizers, without sacrificing crop quality. Plant growth promoting rhizobacteria (PGPR) can stimulate plant growth under resource-limiting conditions by enhancing tolerance to abiotic stress and increasing nutrient availability, uptake, and assimilation. PGPR are beneficial bacteria that colonize the rhizosphere, the narrow zone of soil in the vicinity of the roots that is influenced by root exudates. In this study, in vitro experiments were utilized to screen a collection of 44 Pseudomonas strains for their ability to withstand osmotic stress. A high-throughput greenhouse experiment was then utilized to evaluate selected strains for their ability to stimulate plant growth under resource-limiting conditions when applied to ornamental crop production systems. The development of a high-throughput greenhouse trial identified two pseudomonads, P. poae 29G9 and P. fluorescens 90F12-2, that increased petunia flower number and plant biomass under drought and low-nutrient conditions. These two strains were validated in a production-scale experiment to evaluate the effects on growth promotion of three economically important crops: Petunia × hybrida, Impatiens walleriana, and Viola × wittrockiana. Plants treated with the two bacteria strains had greater shoot biomass than untreated control plants when grown under low-nutrient conditions and after recovery from drought stress. Bacteria treatment resulted in increased flower numbers in drought-stressed P. hybrida and I. walleriana. In addition, bacteria-treated plants grown under low-nutrient conditions had higher leaf nutrient content compared to the untreated plants. Collectively, these results show that the combination of in vitro and greenhouse experiments can efficiently identify beneficial Pseudomonas strains that increase the quality of ornamental crops grown under resource-limiting conditions.

Distribution and Fungicide Sensitivity of Fungal Pathogens Causing Anthracnose-like Lesions on Tomatoes Grown in Ohio.

The primary causal agents of anthracnose-like fruit rots in Ohio and their potential resistance to fungicides commonly used to control these fungal pathogens were determined. Nineteen tomato production fields throughout the state were sampled in 2002 and 2003 for fruit with anthracnose-like lesions. Fungi were isolated from these samples, classified using restriction fragment length polymorphism analysis, and identified by internal transcribed spacer sequence analysis. Some of the fungi isolated may represent secondary invaders of preexisting wounds or lesions. Colletotrichum spp. were most abundant in our collection, representing 136 of the 187 isolates. In addition, there were 23 Alternaria, 12 Fusarium, 12 Phomopsis, and 4 Mucor isolates. Colletotrichum, Alternaria, and Fusarium spp. were found throughout the major tomato production areas in the state. In a laboratory investigation, a subset of the Colletotrichum, Alternaria, and Fusarium isolates caused symptoms similar to early development of anthracnose on wounded tomato fruit. In vitro inhibition assays indicated that most Colletotrichum isolates were sensitive to labeled rates of azoxystrobin, chlorothalonil, and mancozeb. However, some Alternaria isolates were less sensitive to azoxystrobin and chlorothalonil than the Colletotrichum isolates. In addition, most Fusarium isolates were also more insensitive to azoxystrobin and mancozeb, and most Phomopsis isolates were not inhibited by azoxystrobin at the levels tested. The patterns of insensitivity to azoxystrobin and chlorothalonil were also observed in situ with excised fruit. Because the fungicides tested are not currently labeled for control of tomato diseases caused by Fusarium or Phomopsis, these results indicate that some pathogen species that can cause anthracnose-like symptoms may not be entirely sensitive to fungicides commonly used in tomato production.