Rhizosphere competent inoculants modulate the apple root-associated microbiome and plant phytoalexins.

Modulating the soil microbiome by applying microbial inoculants has gained increasing attention as eco-friendly option to improve soil disease suppressiveness. Currently, studies unraveling the interplay of inoculants, root-associated microbiome, and plant response are lacking for apple trees. Here, we provide insights into the ability of Bacillus velezensis FZB42 or Pseudomonas sp. RU47 to colonize apple root-associated microhabitats and to modulate their microbiome. We applied the two strains to apple plants grown in soils from the same site either affected by apple replant disease (ARD) or not (grass), screened their establishment by selective plating, and measured phytoalexins in roots 3, 16, and 28 days post inoculation (dpi). Sequencing of 16S rRNA gene and ITS fragments amplified from DNA extracted 28 dpi from different microhabitat samples revealed significant inoculation effects on fungal ß-diversity in root-affected soil and rhizoplane. Interestingly, only in ARD soil, most abundant bacterial amplicon sequence variants (ASVs) changed significantly in relative abundance. Relative abundances of ASVs affiliated with Enterobacteriaceae were higher in rhizoplane of apple grown in ARD soil and reduced by both inoculants. Bacterial communities in the root endosphere were not affected by the inoculants but their presence was indicated. Interestingly and previously unobserved, apple plants responded to the inoculants with increased phytoalexin content in roots, more pronounced in grass than ARD soil. Altogether, our results indicate that FZB42 and RU47 were rhizosphere competent, modulated the root-associated microbiome, and were perceived by the apple plants, which could make them interesting candidates for an eco-friendly mitigation strategy of ARD. KEY POINTS: • Rhizosphere competent inoculants modulated the microbiome (mainly fungi) • Inoculants reduced relative abundance of Enterobacteriaceae in the ARD rhizoplane • Inoculants increased phytoalexin content in roots, stronger in grass than ARD soil.

Anatomical Limitations in Adventitious Root Formation Revealed by Magnetic Resonance Imaging, Infrared Spectroscopy, and Histology of Rose Genotypes with Contrasting Rooting Phenotypes.

Adventitious root (AR) formation is one of the most important developmental processes in vegetative propagation. Although genotypic differences in rooting ability of rose are well known, the causing factors are not well understood. The rooting of two contrasting genotypes, 'Herzogin Friederike' and 'Mariatheresia', was compared following a multiscale approach. Using magnetic resonance imaging, we noninvasively monitored the inner structure of stem cuttings during initiation and progression of AR formation for the first time. Spatially resolved Fourier-transform infrared spectroscopy characterised the chemical composition of the tissues involved in AR formation. The results were validated through light microscopy and complemented by immunolabeling. The outcome demonstrated similarity of both genotypes in root primordia formation, which however, did not result in root protrusion through the shoot cortex in the difficult-to-root genotype 'Mariatheresia'. The biochemical composition of the contrasting genotypes highlighted main differences in cell wall-associated components. Further spectroscopic analysis of 15 contrasting rose genotypes confirmed the biochemical differences between easy- and difficult-to-root groups. Collectively, data indicates, that not the lack of root primordia limits AR formation in these rose genotypes, but the firmness of the outer stem tissue and/or cell wall modifications that pose a mechanical barrier and prevent root extension and protrusion.

GWAS of adventitious root formation in roses identifies a putative phosphoinositide phosphatase (SAC9) for marker-assisted selection.

Rose propagation by cuttings is limited by substantial genotypic differences in adventitious root formation. To identify possible genetic factors causing these differences and to develop a marker for marker-assisted selection for high rooting ability, we phenotyped 95 cut and 95 garden rose genotypes in a hydroponic rooting system over 6 weeks. Data on rooting percentage after 3 to 6 weeks, root number, and root fresh mass were highly variable among genotypes and used in association mappings performed on genotypic information from the WagRhSNP 68 K Axiom SNP array for roses. GWAS analyses revealed only one significantly associated SNP for rooting percentage after 3 weeks. Nevertheless, prominent genomic regions/peaks were observed and further analysed for rooting percentage after 6 weeks, root number and root fresh mass. Some of the SNPs in these peak regions were associated with large effects on adventitious root formation traits. Very prominent were ten SNPs, which were all located in a putative phosphoinositide phosphatase SAC9 on chromosome 2 and showed very high effects on rooting percentage after 6 weeks of more than 40% difference between nulliplex and quadruplex genotypes. SAC9 was reported to be involved in the regulation of endocytosis and in combination with other members of the SAC gene family to regulate the translocation of auxin-efflux PIN proteins via the dephosphorylation of phosphoinositides. For one SNP within SAC9, a KASP marker was successfully derived and used to select genotypes with a homozygous allele configuration. Phenotyping these homozygous genotypes for adventitious root formation verified the SNP allele dosage effect on rooting. Hence, the presented KASP derived from a SNP located in SAC9 can be used for marker-assisted selection in breeding programs for high rooting ability in the future.

Low-cost and automated phenotyping system "Phenomenon" for multi-sensor in situ monitoring in plant in vitro culture.

BACKGROUND: The current development of sensor technologies towards ever more cost-effective and powerful systems is steadily increasing the application of low-cost sensors in different horticultural sectors. In plant in vitro culture, as a fundamental technique for plant breeding and plant propagation, the majority of evaluation methods to describe the performance of these cultures are based on destructive approaches, limiting data to unique endpoint measurements. Therefore, a non-destructive phenotyping system capable of automated, continuous and objective quantification of in vitro plant traits is desirable. RESULTS: An automated low-cost multi-sensor system acquiring phenotypic data of plant in vitro cultures was developed and evaluated. Unique hardware and software components were selected to construct a xyz-scanning system with an adequate accuracy for consistent data acquisition. Relevant plant growth predictors, such as projected area of explants and average canopy height were determined employing multi-sensory imaging and various developmental processes could be monitored and documented. The validation of the RGB image segmentation pipeline using a random forest classifier revealed very strong correlation with manual pixel annotation. Depth imaging by a laser distance sensor of plant in vitro cultures enabled the description of the dynamic behavior of the average canopy height, the maximum plant height, but also the culture media height and volume. Projected plant area in depth data by RANSAC (random sample consensus) segmentation approach well matched the projected plant area by RGB image processing pipeline. In addition, a successful proof of concept for in situ spectral fluorescence monitoring was achieved and challenges of thermal imaging were documented. Potential use cases for the digital quantification of key performance parameters in research and commercial application are discussed. CONCLUSION: The technical realization of "Phenomenon" allows phenotyping of plant in vitro cultures under highly challenging conditions and enables multi-sensory monitoring through closed vessels, ensuring the aseptic status of the cultures. Automated sensor application in plant tissue culture promises great potential for a non-destructive growth analysis enhancing commercial propagation as well as enabling research with novel digital parameters recorded over time.

Carnivory on demand: phosphorus deficiency induces glandular leaves in the African liana Triphyophyllum peltatum.

Triphyophyllum peltatum, a rare tropical African liana, is unique in its facultative carnivory. The trigger for carnivory is yet unknown, mainly because the plant is difficult to propagate and cultivate. This study aimed at identifying the conditions that result in the formation of carnivorous leaves. In vitro shoots were subjected to abiotic stressors in general and deficiencies of the major nutrients nitrogen, potassium and phosphorus in particular, to trigger carnivorous leaves' development. Adventitious root formation was improved to allow verification of the trigger in glasshouse-grown plants. Among all the stressors tested, only under phosphorus deficiency, the formation of carnivorous leaves was observed. These glandular leaves fully resembled those found under natural growing conditions including the secretion of sticky liquid by mature capture organs. To generate plants for glasshouse experiments, a pulse of 55.4 µM α-naphthaleneacetic acid was essential to achieve 90% in vitro rooting. This plant material facilitated the confirmation of phosphorus starvation to be essential and sufficient for carnivory induction, also under ex vitro conditions. Having established the cultivation of T. peltatum and the induction of carnivory, future gene expression profiles from phosphorus starvation-induced leaves will provide important insight to the molecular mechanism of carnivory on demand.

Combined nitrogen and drought stress leads to overlapping and unique proteomic responses in potato.

MAIN CONCLUSION: Nitrogen deficient and drought-tolerant or sensitive potatoes differ in proteomic responses under combined (NWD) and individual stresses. The sensitive genotype 'Kiebitz' exhibits a higher abundance of proteases under NWD. Abiotic stresses such as N deficiency and drought affect the yield of Solanum tuberosum L. tremendously. Therefore, it is of importance to improve potato genotypes in terms of stress tolerance. In this study, we identified differentially abundant proteins (DAPs) in four starch potato genotypes under N deficiency (ND), drought stress (WD), or combined stress (NWD) in two rain-out shelter experiments. The gel-free LC-MS analysis generated a set of 1177 identified and quantified proteins. The incidence of common DAPs in tolerant and sensitive genotypes under NWD indicates general responses to this stress combination. Most of these proteins were part of the amino acid metabolism (13.9%). Three isoforms of S-adenosyl methionine synthase (SAMS) were found to be lower abundant in all genotypes. As SAMS were found upon application of single stresses as well, these proteins appear to be part of the general stress response in potato. Interestingly, the sensitive genotype 'Kiebitz' showed a higher abundance of three proteases (subtilase, carboxypeptidase, subtilase family protein) and a lower abundance of a protease inhibitor (stigma expressed protein) under NWD stress compared to control plants. The comparably tolerant genotype 'Tomba', however, displayed lower abundances of proteases. This indicates a better coping strategy for the tolerant genotype and a quicker reaction to WD when previously stressed with ND.

Stimulation of adventitious root formation by laser wounding in rose cuttings: A matter of energy and pattern.

Adventitious root (AR) formation is the basis of vegetative propagation in rose, be it via stem cuttings or via stenting. During this process, wounding plays a pivotal role since cell reprogramming takes place at the tissue adjacent to the wound. We investigated the effects of wounding on AR formation on leafy single-node stem cuttings of the rose rootstock R. canina 'Pfänder' (codes R02-3 and R02-6) and the cut rose cultivar Rosa 'Tan09283' (Registration name 'Beluga'). Laser wounding treatments were based on the assisted removal of tissue layers located in the bark. The positioning of wounding was studied based on two marking directions: along the cutting base (strip pattern) and around the cutting base (ring pattern). Additionally, the effects of external supply of indole-butyric acid (IBA 1 mg L-1) on rooting were analyzed. Results showed that in order to remove specific tissue layers, the calculation of the laser energy density (J cm-2) in terms of cutting diameter was necessary. Interestingly, the application of energy densities from 2.5 J cm-2 up to approximately 8.5 J cm-2 were sufficient to expose the tissue layers of epidermis up to regions of phloem. Regarding AR formation for R. canina 'Pfänder', characterized by a low rooting response, an increase in the rooting percentage was registered when the laser treatment eliminated the tissue up to phloem proximities. Analysis of the nodal position showed that bud location was a preferential place for AR formation independently of wounding treatment. In case of Rosa 'Tan09283', laser treatments did not reduce its high rooting capacity, but an apparent reduction in rooting quality due to an investment in tissue healing was observed when wounding reached deeper layers such as parenchyma and sclerenchyma. Results also showed a strong AR formation directly from wounded regions in case of Rosa 'Tan09283' specifically when the wound was located below the axillary bud. In conclusion, wounding by assisted-elimination of layers by laser can induce positive effects on AR formation of single-node stem cuttings of the rose if energy applied is able to expose phloem proximities, a longitudinal orientation, and relative position to the axillary bud are considered.

Correction: Identification and validation of early genetic biomarkers for apple replant disease.

[This corrects the article DOI: 10.1371/journal.pone.0238876.].

Identification of Candidate Genes Associated With Tolerance to Apple Replant Disease by Genome-Wide Transcriptome Analysis.

Apple replant disease (ARD) is a worldwide economic risk in apple cultivation for fruit tree nurseries and fruit growers. Several studies on the reaction of apple plants to ARD are documented but less is known about the genetic mechanisms behind this symptomatology. RNA-seq analysis is a powerful tool for revealing candidate genes that are involved in the molecular responses to biotic stresses in plants. The aim of our work was to find differentially expressed genes in response to ARD in Malus. For this, we compared transcriptome data of the rootstock 'M9' (susceptible) and the wild apple genotype M. ×robusta 5 (Mr5, tolerant) after cultivation in ARD soil and disinfected ARD soil, respectively. When comparing apple plantlets grown in ARD soil to those grown in disinfected ARD soil, 1,206 differentially expressed genes (DEGs) were identified based on a log2 fold change, (LFC) ≥ 1 for up- and ≤ -1 for downregulation (p < 0.05). Subsequent validation revealed a highly significant positive correlation (r = 0.91; p < 0.0001) between RNA-seq and RT-qPCR results indicating a high reliability of the RNA-seq data. PageMan analysis showed that transcripts of genes involved in gibberellic acid (GA) biosynthesis were significantly enriched in the DEG dataset. Most of these GA biosynthesis genes were associated with functions in cell wall stabilization. Further genes were related to detoxification processes. Genes of both groups were expressed significantly higher in Mr5, suggesting that the lower susceptibility to ARD in Mr5 is not due to a single mechanism. These findings contribute to a better insight into ARD response in susceptible and tolerant apple genotypes. However, future research is needed to identify the defense mechanisms, which are most effective for the plant to overcome ARD.

Dynamics of Bacterial Root Endophytes of Malus domestica Plants Grown in Field Soils Affected by Apple Replant Disease.

Apple replant disease (ARD) is a worldwide problem for tree nurseries and orchards leading to reduced plant growth and fruit quality. The etiology of this complex phenomenon is poorly understood, but shifts of the bulk soil and rhizosphere microbiome seem to play an important role. Since roots are colonized by microbes from the rhizosphere, studies of the endophytic microbiome in relation to ARD are meaningful. In this study, culture-independent and culture-dependent approaches were used in order to unravel the endophytic root microbiome of apple plants 3, 7, and 12 months after planting in ARD-affected soil and ARD-unaffected control soil at two different field sites. Next to a high diversity of Pseudomonas in roots from all soils, molecular barcoding approaches revealed an increase in relative abundance of endophytic Actinobacteria over time in plants grown in ARD and control plots. Furthermore, several amplicon sequence variants (ASVs) linked to Streptomyces, which had been shown in a previous greenhouse ARD biotest to be negatively correlated to shoot length and fresh mass, were also detected in roots from both field sites. Especially in roots of apple plants from control soil, these Streptomyces ASVs increased in their relative abundance over time. The isolation of 150 bacterial strains in the culture-dependent approach revealed a high diversity of members of the genus Pseudomonas, confirming the data of the molecular barcoding approach. However, only partial overlaps were found between the two approaches, underlining the importance of combining these methods in order to better understand this complex disease and develop possible countermeasures. Overall, this study suggests a key role of Streptomyces in the etiology of ARD in the field.

Removing the major allergen Bra j I from brown mustard (Brassica juncea) by CRISPR/Cas9.

Food allergies are a major health issue worldwide. Modern breeding techniques such as genome editing via CRISPR/Cas9 have the potential to mitigate this by targeting allergens in plants. This study addressed the major allergen Bra j I, a seed storage protein of the 2S albumin class, in the allotetraploid brown mustard (Brassica juncea). Cotyledon explants of an Indian gene bank accession (CR2664) and the German variety Terratop were transformed using Agrobacterium tumefaciens harboring binary vectors with multiple single guide RNAs to induce either large deletions or frameshift mutations in both Bra j I homoeologs. A total of 49 T0 lines were obtained with up to 3.8% transformation efficiency. Four lines had large deletions of 566 up to 790 bp in the Bra j IB allele. Among 18 Terratop T0 lines, nine carried indels in the targeted regions. From 16 analyzed CR2664 T0 lines, 14 held indels and three had all four Bra j I alleles mutated. The majority of the CRISPR/Cas9-induced mutations were heritable to T1 progenies. In some edited lines, seed formation and viability were reduced and seeds showed a precocious development of the embryo leading to a rupture of the testa already in the siliques. Immunoblotting using newly developed Bra j I-specific antibodies revealed the amount of Bra j I protein to be reduced or absent in seed extracts of selected lines. Removing an allergenic determinant from mustard is an important first step towards the development of safer food crops.

Formation and exudation of biphenyl and dibenzofuran phytoalexins by roots of the apple rootstock M26 grown in apple replant disease soil.

Apple replant disease (ARD) is a severe soil-borne disease frequently observed in apple tree nurseries and orchards worldwide. One of the responses of apple trees to ARD is the formation of biphenyl and dibenzofuran phytoalexins in their roots. However, there is no information on whether or not these phytoalexins are exuded into the soil. To answer this open question, a model system was established using the ARD-sensitive apple rootstock M26 (Malus × domestica Borkh. Rosaceae) and GC-MS analysis in combination with an in-house GC-MS database including retention indices. We have detected a total of 35 phytoalexins, i.e. 10 biphenyls and 25 dibenzofurans in root samples, thereby adding eight compounds to the previously reported 27 phytoalexins of Malinae species. When in vitro cultured M26 plantlets were treated with yeast extract, all the 35 phytoalexins were formed in the roots and 85.2% of the total phytoalexin amount was exuded into the culture medium. In roots of M26 plants grown in ARD soil in pot, 26 phytoalexins were detected and their exudation was demonstrated using two independent approaches of collecting root exudates. In a modified dipping experiment and a soil-hydroponic hybrid setup, the exudation rate was 39.5% and 20.6%, respectively. The exudation rates for individual phytoalexins differed, indicating controlled exudation processes. The exuded phytoalexins may play an important role in shaping the soil microbiome, which appears to greatly influence the development and severity of ARD.

The Orphan Crop Crassocephalum crepidioides Accumulates the Pyrrolizidine Alkaloid Jacobine in Response to Nitrogen Starvation.

Crassocephalum crepidioides is an African orphan crop that is used as a leafy vegetable and medicinal plant. Although it is of high regional importance in Sub-Saharan Africa, the plant is still mainly collected from the wild and therefore efforts are made to promote its domestication. However, in addition to beneficial properties, there was first evidence that C. crepidioides can accumulate the highly toxic pyrrolizidine alkaloid (PA) jacobine and here it was investigated, how jacobine production is controlled. Using ecotypes from Africa and Asia that were characterized in terms of their PA profiles, it is shown that the tetraploid C. crepidioides forms jacobine, an ability that its diploid close relative Crassocephalum rubens appears to lack. Evidence is provided that nitrogen (N) deficiency strongly increases jacobine in the leaves of C. crepidioides, that this capacity depends more strongly on the shoot than the root system, and that homospermidine synthase (HSS) activity is not rate-limiting for this reaction. A characterization of HSS gene representation and transcription showed that C. crepidioides and C. rubens possess two functional versions, one of which is conserved, that the HSS transcript is mainly present in roots and that its abundance is not controlled by N deficiency. In summary, this work improves our understanding of how environmental cues impact PA biosynthesis in plants and provides a basis for the development of PA-free C. crepidioides cultivars, which will aid its domestication and safe use.

Corrigendum: Genes Involved in Stress Response and Especially in Phytoalexin Biosynthesis Are Upregulated in Four Malus Genotypes in Response to Apple Replant Disease.

[This corrects the article DOI: 10.3389/fpls.2019.01724.].

Root exposure to apple replant disease soil triggers local defense response and rhizoplane microbiome dysbiosis.

A soil column split-root experiment was designed to investigate the ability of apple replant disease (ARD)-causing agents to spread in soil. 'M26' apple rootstocks grew into a top layer of Control soil, followed by a barrier-free split-soil layer (Control soil/ARD soil). We observed a severely reduced root growth, concomitant with enhanced gene expression of phytoalexin biosynthetic genes and phytoalexin content in roots from ARD soil, indicating a pronounced local plant defense response. Amplicon sequencing (bacteria, archaea, fungi) revealed local shifts in diversity and composition of microorganisms in the rhizoplane of roots from ARD soil. An enrichment of operational taxonomic units affiliated to potential ARD fungal pathogens (Ilyonectria and Nectria sp.) and bacteria frequently associated with ARD (Streptomyces, Variovorax) was noted. In conclusion, our integrated study supports the idea of ARD being local and not spreading into surrounding soil, as only the roots in ARD soil were affected in terms of growth, phytoalexin biosynthetic gene expression, phytoalexin production and altered microbiome structure. This study further reinforces the microbiological nature of ARD, being likely triggered by a disturbed soil microbiome enriched with low mobility of the ARD-causing agents that induce a strong plant defense and rhizoplane microbiome dysbiosis, concurring with root damage.

Exploring microbial determinants of apple replant disease (ARD): a microhabitat approach under split-root design.

Apple replant disease (ARD) occurs worldwide in apple orchards and nurseries and leads to a severe growth and productivity decline. Despite research on the topic, its causality remains unclear. In a split-root experiment, we grew ARD-susceptible 'M26' apple rootstocks in different substrate combinations (+ARD: ARD soil; -ARD: gamma-irradiated ARD soil; and Control: soil with no apple history). We investigated the microbial community composition by 16S rRNA gene amplicon sequencing (bacteria and archaea) along the soil-root continuum (bulk soil, rhizosphere and rhizoplane). Significant differences in microbial community composition and structure were found between +ARD and -ARD or +ARD and Control along the soil-root continuum, even for plants exposed simultaneously to two different substrates (-ARD/+ARD and Control/+ARD). The substrates in the respective split-root compartment defined the assembly of root-associated microbial communities, being hardly influenced by the type of substrate in the respective neighbor compartment. Root-associated representatives from Actinobacteria were the most dynamic taxa in response to the treatments, suggesting a pivotal role in ARD. Altogether, we evidenced an altered state of the microbial community in the +ARD soil, displaying altered alpha- and beta-diversity, which in turn will also impact the normal development of apple rhizosphere and rhizoplane microbiota (dysbiosis), concurring with symptom appearance.

Identification and validation of early genetic biomarkers for apple replant disease.

Apple replant disease (ARD) is a serious threat to producers of apple trees and fruits worldwide. The ARD etiology is not unraveled and managing options are either economically not applicable or environmentally harmful. Thus, interest is given in biomarkers that allow to indicate ARD situations at early time points in order to classify soils according to ARD severity but also to analyze the effectiveness to potential countermeasures. This study aimed at (i) identifying ARD biomarkers on the transcriptional level in root tissue by analyzing the expression of previously identified candidate genes in ARD soils of different origin and texture and (ii) testing the specificity of these marker genes to ARD. In vitro propagated M26 plantlets were submitted to a bio-test with three ARD soils, either untreated or disinfected by γ-irradiation. Expression of seven candidate genes identified in a previous transcriptomic study was investigated by RT-qPCR in a time course experiment. Already three days after planting, a prominent upregulation of the phytoalexin biosynthesis genes biphenyl synthase 3 (BIS3) and biphenyl 4-hydroxylase (B4Hb) was observed in the untreated ARD variants of all three soils. The phytoalexin composition in roots was comparable for all three soils and the total phytoalexin content correlated with the expression of BIS3 and B4Hb. The third promising candidate gene that was upregulated under ARD conditions was the ethylene-responsive transcription factor 1B-like (ERF1B). In a second experiment M26 plantlets were exposed to different abiotic stressors, namely heat, salt and nutrient starvation, and candidate gene expression was determined in the roots. The expression levels of BIS3 and B4Hb were highly and specifically upregulated in ARD soil, but not upon the abiotic stress conditions, whereas ERF1B also showed higher expression under heat stress. In conclusion, BIS3 and B4Hb are recommended as early ARD biomarkers due to their high expression levels and their high specificity.

Genomes of the Venus Flytrap and Close Relatives Unveil the Roots of Plant Carnivory.

Most plants grow and develop by taking up nutrients from the soil while continuously under threat from foraging animals. Carnivorous plants have turned the tables by capturing and consuming nutrient-rich animal prey, enabling them to thrive in nutrient-poor soil. To better understand the evolution of botanical carnivory, we compared the draft genome of the Venus flytrap (Dionaea muscipula) with that of its aquatic sister, the waterwheel plant Aldrovanda vesiculosa, and the sundew Drosera spatulata. We identified an early whole-genome duplication in the family as source for carnivory-associated genes. Recruitment of genes to the trap from the root especially was a major mechanism in the evolution of carnivory, supported by family-specific duplications. Still, these genomes belong to the gene poorest land plants sequenced thus far, suggesting reduction of selective pressure on different processes, including non-carnivorous nutrient acquisition. Our results show how non-carnivorous plants evolved into the most skillful green hunters on the planet.