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.

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.

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.

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.

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.

Apple Replant Disease: Causes and Mitigation Strategies.

After replanting apple (Malus domestica Borkh.) on the same site severe growth suppressions, and a decline in yield and fruit quality are observed in all apple producing areas worldwide. The causes of this complex phenomenon, called apple replant disease (ARD), are only poorly understood up to now which is in part due to inconsistencies in terms and methodologies. Therefore we suggest the following definition for ARD: ARD describes a harmfully disturbed physiological and morphological reaction of apple plants to soils that faced alterations in their (micro-) biome due to the previous apple cultures. The underlying interactions likely have multiple causes that extend beyond common analytical tools in microbial ecology. They are influenced by soil properties, faunal vectors, and trophic cascades, with genotype-specific effects on plant secondary metabolism, particularly phytoalexin biosynthesis. Yet, emerging tools allow to unravel the soil and rhizosphere (micro-) biome, to characterize alterations of habitat quality, and to decipher the plant reactions. Thereby, deep insights into the reactions taking place at the root rhizosphere interface will be gained. Counteractions are suggested, taking into account that culture management should emphasize on improving soil microbial and faunal diversity as well as habitat quality rather than focus on soil disinfection.

Transcriptomic analysis of molecular responses in Malus domestica 'M26' roots affected by apple replant disease.

KEY MESSAGE: Gene expression studies in roots of apple replant disease affected plants suggested defense reactions towards biotic stress to occur which did not lead to adequate responses to the biotic stressors. Apple replant disease (ARD) leads to growth inhibition and fruit yield reduction in replanted populations and results in economic losses for tree nurseries and fruit producers. The etiology is not well understood on a molecular level and causal agents show a great diversity indicating that no definitive cause, which applies to the majority of cases, has been found out yet. Hence, it is pivotal to gain a better understanding of the molecular and physiological reactions of the plant when affected by ARD and later to overcome the disease, for example by developing tolerant rootstocks. For the first time, gene expression was investigated in roots of ARD affected plants employing massive analysis of cDNA ends (MACE) and RT-qPCR. In reaction to ARD, genes in secondary metabolite production as well as plant defense, regulatory and signaling genes were upregulated whereas for several genes involved in primary metabolism lower expression was detected. For internal verification of MACE data, candidate genes were tested via RT-qPCR and a strong positive correlation between both datasets was observed. Comparison of apple 'M26' roots cultivated in ARD soil or γ-irradiated ARD soil suggests that typical defense reactions towards biotic stress take place in ARD affected plants but they did not allow responding to the biotic stressors attack adequately, leading to the observed growth depressions in ARD variants.

Genetic dissection of adventitious shoot regeneration in roses by employing genome-wide association studies.

KEY MESSAGE: We analysed the capacity to regenerate adventitious shoots in 96 rose genotypes and found 88 SNP markers associated with QTLs, some of which are derived from candidate genes for shoot regeneration. In an association panel of 96 rose genotypes previously analysed for petal colour, we conducted a genome-wide association study on the capacity of leaf petioles for direct shoot regeneration. Shoot regeneration rate and shoot ratio (number of shoots/total number of explants) were used as phenotypic descriptors for regeneration capacity. Two independent experiments were carried out with six replicates of ten explants each. We found significant variation between the genotypes ranging from 0.88 to 88.33% for the regeneration rate and from 0.008 to 1.2 for the shoot ratio, which exceeded the rates reported so far. Furthermore, we found 88 SNP markers associated with either the shoot regeneration rate or the shoot ratio. In this association analysis, we found 12 SNP markers from ESTs (expressed sequence tags) matching known candidate genes that are involved in shoot morphogenesis. The best markers explained more than 51% of the variance in the shoot regeneration rate and more than 0.65 of the variance in the shoot regeneration ratio between the homozygote marker classes. The genes underlying some of the best markers such as a GT-transcription factor or an LRR receptor-like protein kinase are novel candidate genes putatively involved in the observed phenotypic differences. The associated markers were mapped to the closely related genome of Fragaria vesca and revealed many distinct clusters, which also comprised the known candidate genes that functioned in the organogenesis of plant shoots. However, the validation of candidate genes and their functional relationship to shoot regeneration require further analysis in independent rose populations and functional analyses.

Evaluation of reproductive barriers contributes to the development of novel interspecific hybrids in the Kalanchoë genus.

BACKGROUND: Interspecific hybridization is a useful tool in ornamental breeding to increase genetic variability and introduce new valuable traits into existing cultivars. The successful formation of interspecific hybrids is frequently limited by the presence of pre- and post-fertilization barriers. In the present study, we investigated the nature of hybridization barriers occurring in crosses between Kalanchoë species and evaluated possibilities of obtaining interspecific hybrids. RESULTS: The qualitative and quantitative analyses of pollen tube growth in situ were performed following intra- and interspecific pollinations. They revealed occurrence of pre-fertilization barriers associated with inhibition of pollen germination on the stigma and abnormal growth of pollen tubes. Unilateral incongruity related to differences in pistil length was also observed. The pollen quality was identified as a strong factor influencing the number of pollen tubes germinating in the stigma. In relation to post-fertilization barriers, endosperm degeneration was a probable barrier hampering production of interspecific hybrids. Moreover, our results demonstrate the relation of genetic distance estimated by AFLP marker analysis of hybridization partners with cross-compatibility of Kalanchoë species. At the same time, differences in ploidy did not influence the success of interspecific crosses. CONCLUSIONS: Our study presents the first comprehensive analysis of hybridization barriers occurring within Kalanchoë genus. Reproductive barriers were detected on both, pre- and post-fertilization levels. This new knowledge will contribute to further understanding of reproductive isolation of Kalanchoë species and facilitate breeding of new cultivars. For the first time, interspecific hybrids between K. nyikae as maternal plant and K. blossfeldiana as well as K. blossfeldiana and K. marnieriana were generated.

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.

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.

From callus to embryo: a proteomic view on the development and maturation of somatic embryos in Cyclamen persicum.

In this study, the proteome structures following the pathway in somatic embryogenesis of Cyclamen persicum were analysed via high-resolution 2D-SDS-PAGE with two objectives: (1) to identify the significant physiological processes during somatic embryogenesis in Cyclamen and (2) to improve the maturation of somatic embryos. Therefore, the effects of maturation-promoting plant growth regulator abscisic acid (ABA) and high sucrose levels on torpedo-shaped embryos were investigated. In total, 108 proteins of differential abundance were identified using a combination of tandem mass spectrometry and a digital proteome reference map. In callus, enzymes related to energy supply were especially distinct, most likely due to energy demand caused by fast growth and cell division. The switch from callus to globular embryo as well as from globular to torpedo-shaped embryo was associated with controlled proteolysis via the ubiquitin-26S proteasome pathway. Storage compound accumulation was first detected 21 days after transfer to plant growth regulator (PGR)-free medium in early torpedo-shaped embryos. Increase in abundance of auxin-amidohydrolase during embryogenesis suggests a possible increase in auxin release in the late embryo stages of Cyclamen. A development-specific isoelectric point switch of catalases has been reported for the first time for somatic embryogenesis. Several proteins were identified to represent markers for the different developmental stages analysed. High sucrose levels and ABA treatment promoted the accumulation of storage compounds in torpedo-shaped embryos. Additionally, proteins of the primary metabolic pathways were decreased in the proteomes of ABA-treated embryos. Thus, ABA and high sucrose concentration in the culture medium improved maturation and consequently the quality of somatic embryos in C. persicum.

Thermotolerant cyclamen with reduced acrolein and methyl vinyl ketone.

Reduced levels of trienoic fatty acids (TAs) in chloroplast membranes induce thermotolerance in several plant species, but the underlying mechanisms remain unclear. TA peroxidation in plant cell membranes generates cytotoxic, TA-derived compounds containing α,ß-unsaturated carbonyl groups. The relationship between low TA levels and the amounts of cytotoxic TA-derived compounds was examined using thermotolerant transgenic cyclamen (Cyclamen persicum Mill.) with low TA contents. Changes in the levels of the cytotoxic TA-derived acrolein (ACR), methyl vinyl ketone (MVK), (E)-2-hexenal, 4-hydroxy-2-nonenal, and malondialdehyde were analysed in the leaf tissues of wild-type (WT) and thermotolerant transgenic cyclamen under heat stress. Levels of ACR and MVK in the WT increased in parallel with the occurrence of heat-induced tissue damage, whereas no such changes were observed in the thermotolerant transgenic lines. Furthermore, exogenous ACR and MVK infiltrated into leaves to concentrations similar to those observed in heat-stressed WT leaves caused similar disease symptoms. These results suggest that thermotolerance in transgenic cyclamen depends on reduced production rates of ACR and MVK under heat stress, due to the low level of TAs in these plants.

Interspecific somatic hybrids between Cyclamen persicum and C. coum, two sexually incompatible species.

By applying polyethylene glycol (PEG)-mediated protoplast fusion, the first somatic hybrids were obtained between Cyclamen persicum (2n = 2x = 48) and C. coum (2n = 2x = 30)-two species that cannot be combined by cross breeding. Heterofusion was detected by double fluorescent staining with fluorescein diacetate and scopoletin. The highest heterofusion frequencies (of about 5%) resulted from a protocol using a protoplast density of 1 × 10(6)/mL and 40% PEG. The DNA content of C. coum was estimated for the first time by propidium iodide staining to be 14.7 pg/2C and was 4.6 times higher than that of C. persicum. Among 200 in vitro plantlets regenerated from fusion experiments, most resembled the C. coum parent, whereas only 5 plants showed typical C. persicum phenotypes and 46 had a deviating morphology. By flow cytometry, six putative somatic hybrids were identified. A species-specific DNA marker was developed based on the sequence of the 5.8S gene in the ribosomal nuclear DNA and its flanking internal transcribed spacers ITS1 and ITS2. The hybrid status of only one plant could be verified by the species-specific DNA marker as well as sequencing of the amplification product. RAPD markers turned out to be less informative and applicable for hybrid identification, as no clear additivity of the parental marker bands was observed. Chromosome counting in root tips of four hybrids revealed the presence of the 30 C. coum chromosomes and 2-41 additional ones indicating elimination of C. persicum chromosomes.

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.

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.

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.

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

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

Enolases: storage compounds in seeds? Evidence from a proteomic comparison of zygotic and somatic embryos of Cyclamen persicum Mill.

Somatic embryogenesis is well established for the economic relevant ornamental crop Cyclamen and thus could supplement the elaborate propagation via seeds. However, the use of somatic embryogenesis for commercial large scale propagation is still limited due to physiological disorders and asynchronous development within emerged embryos. To overcome these problems, profound knowledge of the physiological processes in Cyclamen embryogenesis is essential. Thus, the proteomes of somatic and zygotic embryos were characterised in a comparative approach. Protein separation via two dimensional IEF-SDS PAGE led to a resolution of more than 1,000 protein spots/gel. Overall, 246 proteins were of differential abundance in the two tissues compared. Mass spectrometry analysis of the 300 most abundant protein spots resulted in the identification of 247 proteins, which represent 90 distinct protein species. Fifty-five percent of the 247 proteins belong to only three physiological categories: glycolysis, protein folding and stress response. The latter physiological process was especially predominant in the somatic embryos. Remarkably, the glycolytic enzyme enolase was the protein most frequently detected and thus is supposed to play an important role in Cyclamen embryogenesis. Data are presented that indicate involvement of "small enolases" as storage proteins in Cyclamen. A digital reference map was established via a novel software tool for the web-based presentation of proteome data linked to KEGG and ExPasy protein-databases and both were made publicly available online.

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.