Integrated omics approach reveals the molecular pathways activated in tomato by Kocuria rhizophila, a soil plant growth-promoting bacterium.

Plant microbial biostimulants application has become a promising and eco-friendly agricultural strategy to improve crop yields, reducing chemical inputs for more sustainable cropping systems. The soil dwelling bacterium Kocuria rhizophila was previously characterized as Plant Growth Promoting Bacteria (PGPB) for its multiple PGP traits, such as indole-3-acetic acid production, phosphate solubilization capability and salt and drought stress tolerance. Here, we evaluated by a multi-omics approach, the PGP activity of K. rhizophila on tomato, revealing the molecular pathways by which it promotes plant growth. Transcriptomic analysis showed several up-regulated genes mainly related to amino acid metabolism, cell wall organization, lipid and secondary metabolism, together with a modulation in the DNA methylation profile, after PGPB inoculation. In agreement, proteins involved in photosynthesis, cell division, and plant growth were highly accumulated by K. rhizophila. Furthermore, "amino acid and peptides", "monosaccharides", and "TCA" classes of metabolites resulted the most affected by PGPB treatment, as well as dopamine, a catecholamine neurotransmitter mediating plant growth through S-adenosylmethionine decarboxylase (SAMDC), a gene enhancing the vegetative growth, up-regulated in tomato by K. rhizophila treatment. Interestingly, eight gene modules well correlated with differentially accumulated proteins (DAPs) and metabolites (DAMs), among which two modules showed the highest correlation with nine proteins, including a nucleoside diphosphate kinase, and cytosolic ascorbate peroxidase, as well as with several amino acids and metabolites involved in TCA cycle. Overall, our findings highlighted that sugars and amino acids, energy regulators, involved in tomato plant growth, were strongly modulated by the K. rhizophila-plant interaction.

Bioactive Metabolite Survey of Actinobacteria Showing Plant Growth Promoting Traits to Develop Novel Biofertilizers.

The use of chemical fertilizers and pesticides has caused harmful impacts on the environment with the increase in economic burden. Biofertilizers are biological products containing living microorganisms capable of improving plant growth through eco-friendly mechanisms. In this work, three actinobacterial strains Streptomyces violaceoruber, Streptomyces coelicolor, and Kocuria rhizophila were characterized for multiple plant growth promoting (PGP) traits such as indole acetic acid production, phosphate solubilization, N2-fixation, and drought and salt tolerance. Then, these strains were investigated for their secreted and cellular metabolome, revealing a rich arsenal of bioactive molecules, including antibiotics and siderophores, with S. violaceoruber being the most prolific strain. Furthermore, the in vivo assays, performed on tomato (Solanum lycopersicum L.), resulted in an improved germination index and the growth of seedlings from seeds treated with PGP actinobacteria, with a particular focus on S. violaceoruber cultures. In particular, this last strain, producing volatile organic compounds having antimicrobial activity, was able to modulate volatilome and exert control on the global DNA methylation of tomato seedlings. Thus, these results, confirming the efficacy of the selected actinobacteria strains in promoting plant growth and development by producing volatile and non-volatile bioactive molecules, can promote eco-friendly alternatives in sustainable agriculture.

Rootstocks with Different Tolerance Grade to Citrus Tristeza Virus Induce Dissimilar Volatile Profile in Citrus sinensis and Avoidance Response in the Vector Aphis gossypii Glover.

The citrus tristeza virus (CTV) is an agent of devastating epidemics of the citrus plant grafted on Citrus aurantium, one of the main rootstocks still used in the Mediterranean area. Consequently, CTV-tolerant alternative citrus rootstocks are considered necessary to manage this disease and/or its vector; that in Mediterranean countries is the aphid Aphis gossypii. In this study, we analyzed the VOCs emitted from Citrus sinensis plants grafted on the CTV-susceptible C. aurantium and on the CTV-tolerant Volkamer lemon, Forner-Alcaide no. 5, and Carrizo citrange. Furthermore, the aphid preference/avoidance response toward these combinations was evaluated in a semi-field experiment. The VOC profiles recorded on the leaves of C. sinensis grafted on the four rootstocks listed above showed significant differences in the abundances and ratios of the compounds emitted. The behavioral experiments indicated that A. gossypii prefers to orient and establish on the C. sinensis plants grafted on C. aurantium rather than on that grafted on the three CTV-tolerant varieties. The possibility that this avoidance mechanism is triggered by the different profile of the VOC emitted by the different combinations and the consequent susceptibility/tolerance shown toward CTV is discussed.

Different Cell Types Affect the Transition from Juvenile to Mature Phase in Citrus Plants Regenerated through Somatic Embryogenesis.

Robust protocols for the regeneration of somatic embryos in vitro are essential for the efficient use of the most modern biotechnologies. Unfortunately, in perennial trees such as Citrus, plants regenerated from juvenile tissues usually exhibit strong, undesirable juvenile characters such as thorny habit and delayed flowering and fruit production. In this work, we tested whether the cell types (nucellar and stigma/style) used to regenerate Citrus plants through somatic embryogenesis affected the transition from the juvenile to mature phase. The results show that regenerants from nucellar cells presented persistent juvenile characters, whereas plants originating from stigma/style explants transited to the mature phase more rapidly. Our observations support the hypothesis that the totipotent cells originated from different cell types are not equivalent, possibly by maintaining memory of their previously differentiated state.

Autotetraploid Emergence via Somatic Embryogenesis in Vitis vinifera Induces Marked Morphological Changes in Shoots, Mature Leaves, and Stomata.

Polyploidy plays an important role in plant adaptation to biotic and abiotic stresses. Alterations of the ploidy in grapevine plants regenerated via somatic embryogenesis (SE) may provide a source of genetic variability useful for the improvement of agronomic characteristics of crops. In the grapevine, the SE induction process may cause ploidy changes without alterations in DNA profile. In the present research, tetraploid plants were observed for 9.3% of 'Frappato' grapevine somatic embryos regenerated in medium supplemented with the growth regulators ß-naphthoxyacetic acid (10 µM) and N6-benzylaminopurine (4.4 µM). Autotetraploid plants regenerated via SE without detectable changes in the DNA profiles were transferred in field conditions to analyze the effect of polyploidization. Different ploidy levels induced several anatomical and morphological changes of the shoots and mature leaves. Alterations have been also observed in stomata. The length and width of stomata of tetraploid leaves were 39.9 and 18.6% higher than diploids, respectively. The chloroplast number per guard cell pair was higher (5.2%) in tetraploid leaves. On the contrary, the stomatal index was markedly decreased (12%) in tetraploid leaves. The observed morphological alterations might be useful traits for breeding of grapevine varieties in a changing environment.

The endophytic microbiota of Citrus limon is transmitted from seed to shoot highlighting differences of bacterial and fungal community structures.

Citrus limon (L.) Burm. F. is an important evergreen fruit crop whose rhizosphere and phyllosphere microbiota  have been characterized, while seed microbiota is still unknown. Bacterial and fungal endophytes were isolated from C. limon surface-sterilized seeds. The isolated fungi-belonging to Aspergillus, Quambalaria and Bjerkandera genera-and bacteria-belonging to Staphylococcus genus-were characterized for indoleacetic acid production and phosphate solubilization. Next Generation Sequencing based approaches were then used to characterize the endophytic bacterial and fungal microbiota structures of surface-sterilized C. limon seeds and of shoots obtained under aseptic conditions from in vitro growing seedlings regenerated from surface-sterilized seeds. This analysis highlighted that Cutibacterium and Acinetobacter were the most abundant bacterial genera in both seeds and shoots, while Cladosporium and Debaryomyces were the most abundant fungal genera in seeds and shoots, respectively. The localization of bacterial endophytes in seed and shoot tissues was revealed by Fluorescence In Situ Hybridization coupled with Confocal Laser Scanning Microscopy revealing vascular bundle colonization. Thus, these results highlighted for the first time the structures of endophytic microbiota of C. limon seeds and the transmission to shoots, corroborating the idea of a vertical transmission of plant microbiota and suggesting its crucial role in seed germination and plant development.

Genetic Distinctiveness Highlights the Conservation Value of a Sicilian Manna Ash Germplasm Collection Assigned to Fraxinus angustifolia (Oleaceae).

The cosmopolitan genus Fraxinus comprises about 40 species occupying several habitats in the Northern Hemisphere. With some species hybridizing and sharing genetic variants, questions remain on the species assignment of germplasm within the genus Fraxinus despite numerous species-specific assessments. A multidisciplinary approach was employed to provide a definitive insight into the genetics of an endangered Fraxinus "manna ash" collection, located in a rich plant biodiversity hotspot of the Madonie Mountains (Sicily). Although the collection size was small, genetic diversity, assessed by chloroplast (cpSSR) and nuclear (nSSR) microsatellites (SSR-Simple Sequence Repeats), allowed identifying three different chloroplast haplotypes, with one (H5) dominant, and several polymorphic loci, able to discriminate most of the local accessions studied. Molecular data were linked to cytofluorimetric and phenotypic evaluations and, contrary to popular belief that manna ash is Fraxinus ornus L., the germplasm currently used for manna production belongs to Fraxinus angustifolia Vahl. Interestingly, joint analysis of our genetic panel with a large European dataset of Fraxinus spp. suggested the presence of a possible glacial refuge in Sicily, confirming its importance as biodiversity source. Our results will be helpful for the design of long-term conservation programs for genetic resources, such as in situ and ex situ conservation, seed collection and tree reintroduction.

Correction to: SNP genotyping elucidates the genetic diversity of Magna Graecia grapevine germplasm and its historical origin and dissemination.

An amendment to this paper has been published and can be accessed via the original article.

Water relations of two Sicilian grapevine cultivars in response to potassium availability and drought stress.

We investigated the response of two Sicilian grapevine cultivars, Catarratto and Nero d'Avola, to potassium deficiency and drought stress. Two-year-old plants grafted on 1103 Paulsen were grown in agriperlite, with or without potassium in the fertigation solution for six weeks, and subjected to moderate drought stress by suspending irrigation for one week. Potassium content of leaves, roots and xylem sap were measured with an ion-selective electrode. Changes in stomatal conductance, stem and leaf water potential and hydraulic conductance were compared between genotypes and treatments. Potassium deficiency led to significant decreases in leaf potassium content in both cultivars and under both well-watered and drought stress conditions. Potassium content in xylem sap showed no significant differences between cultivars and was correlated with stem hydraulic conductance, particularly in the drought stress treatments. Under drought stress conditions, potassium availability led to an increase in stomatal conductance, particularly in Nero d'Avola. Both cultivars showed a rather isohydric behavior under these experimental conditions, and the level of isohydry varied with potassium availability. These results can be useful for the development of optimal fertigation practices and the selection of drought tolerant varieties.

Characterization of Sicilian rosemary (Rosmarinus officinalis L.) germplasm through a multidisciplinary approach.

In Sicily, small differences exist between wild and cultivated rosemary biotypes; VOCs and genetic profiles may be a useful tool to distinguish them. A germplasm collection of Rosmarinus officinalis L. was harvested from 15 locations in Sicily. Eleven wild and four cultivated populations were collected and, due to the surveyed area covered, they can be considered as a representative panel of Sicilian genetic background of the species. Ex situ plant collection was transferred to the field cultivation in homogeneous conditions for characterizing through a multidisciplinary approach. The study included morphological traits observations (growth habitus, flower color, number and size of leaves, length and number of internodes), VOC profiles using HS-SPME, genome size by flow cytometry analysis, and genetic characterization by means of DNA and nuclear microsatellite (nSSR) investigation. To detect any pattern within- and among-populations variability, all morphological and chemical data were submitted to ANOVA, while clustering and structure population analysis were carried out using genetic profiles. The present work allowed us to distinguish rather well between wild and cultivated genotypes and to underline the biodiversity richness among rosemary Sicilian germplasm, never highlighted, useful for future breeding programs addressed to exploit this important resource.

Phylogenetic Relationship Among Wild and Cultivated Grapevine in Sicily: A Hotspot in the Middle of the Mediterranean Basin.

Grapevine (Vitis vinifera ssp. sativa) is a perennial crop especially important for wine and fruit production. The species is highly polymorphic with thousands of different varieties selected by farmers and clonally propagated. However, it is still debated whether grapevine domestication from its wild ancestor (V. vinifera ssp. sylvestris) has been a single event or rather it occurred on multiple occasions during the diffusion of its cultivation across the Mediterranean. Located in the center of the Basin, Sicily is its largest island and has served as a hotspot for all civilizations that have crossed the Mediterranean throughout history. Hundreds of unique grapevine cultivars are still cultivated in Sicily and its surrounding minor islands, though most of them are menaced by extinction. Wild grapevine is also present with isolated populations thriving along riverbanks. With the aim to evaluate the phylogenetic relationships among Sicilian varieties, and to assess the possible contribution of indigenous wild populations to the genetic makeup of cultivated grapevine, we analyzed 170 domestic cultivars and 125 wild plants, collected from 10 different populations, with 23 SSR markers. We also compared our data with published dataset from Eurasia. Results show that Sicilian wild populations are related to the cultivated Sicilian and Italian germplasm, suggesting events of introgression and/or domestication of local varieties.

SNP genotyping elucidates the genetic diversity of Magna Graecia grapevine germplasm and its historical origin and dissemination.

BACKGROUND: Magna Graecia is the ancient name for the modern geopolitical region of South Italy extensively populated by Greek colonizers, shown by archeological and historical evidence to be the oldest wine growing region of Italy, crucial for the spread of specialized viticulture around Mediterranean shores. Here, the genetic diversity of Magna Graecia grape germplasm was assessed and its role in grapevine propagation around the Mediterranean basin was underlined. RESULTS: A large collection of grapevines from Magna Graecia was compared with germplasm from Georgia to the Iberian Peninsula using the 18 K SNP array. A high level of genetic diversity of the analyzed germplasm was determined; clustering, structure analysis and DAPC (Discriminant Analysis of Principal Components) highlighted the genetic relationships among genotypes from South Italy and the Eastern Mediterranean (Greece). Gene flow from east (Georgia) to west (Iberian Peninsula) was identified throughout the large number of detected admixed samples. Pedigree analysis showed a complex and well-structured network of first degree relationships, where the cultivars from Magna Graecia were mainly involved. CONCLUSIONS: This study provided evidence that Magna Graecia germplasm was shaped by historical events that occurred in the area due to the robust link between South Italian and Greek genotypes, as well as, by the availability of different thermal resources for cultivars growing in such different winegrowing areas. The uniqueness of this ampelographic platform was mainly an outcome of complex natural or human-driven crosses involving elite cultivars.

An integrated proteomic and metabolomic study to evaluate the effect of nucleus-cytoplasm interaction in a diploid citrus cybrid between sweet orange and lemon.

KEY MESSAGE: Our results provide a comprehensive overview how the alloplasmic condition might lead to a significant improvement in citrus plant breeding, developing varieties more adaptable to a wide range of conditions. Citrus cybrids resulting from somatic hybridization hold great potential in plant improvement. They represent effective products resulting from the transfer of organelle-encoded traits into cultivated varieties. In these cases, the plant coordinated array of physiological, biochemical, and molecular functions remains the result of integration among different signals, which derive from the compartmentalized genomes of nucleus, plastids and mitochondria. To dissect the effects of genome rearrangement into cybrids, a multidisciplinary study was conducted on a diploid cybrid (C2N), resulting from a breeding program aimed to improve interesting agronomical traits for lemon, the parental cultivars 'Valencia' sweet orange (V) and 'femminello' lemon (F), and the corresponding somatic allotetraploid hybrid (V + F). In particular, a differential proteomic analysis, based on 2D-DIGE and MS procedures, was carried out on leaf proteomes of C2N, V, F and V + F, using the C2N proteome as pivotal condition. This investigation revealed differentially represented protein patterns that can be associated with genome rearrangement and cell compartment interplay. Interestingly, most of the up-regulated proteins in the cybrid are involved in crucial biological processes such as photosynthesis, energy production and stress tolerance response. The cybrid differential proteome pattern was concomitant with a general increase of leaf gas exchange and content of volatile organic compounds, highlighting a stimulation of specific pathways that can be related to observed plant performances. Our results contribute to a better understanding how the alloplasmic condition might lead to a substantial improvement in plant breeding, opening new opportunities to develop varieties more adaptable to a wide range of conditions.

Somatic embryogenesis of muskmelon (Cucumis melo L.) and genetic stability assessment of regenerants using flow cytometry and ISSR markers.

A new protocol for in vitro regeneration through direct somatic embryogenesis for two muskmelon cultivars (Cucumis melo L., "Mashhadi" and "Eivanaki") is reported. Somatic embryos were obtained culturing 4- and 8-day-old cotyledons, seeds, and hypocotyls on Murashige and Skoog medium supplemented with three different hormonal combinations never tested so far for melon (naphthoxyacetic acid (NOA) + thidiazuron (TDZ), NOA + 6-banzylaminopurine (BAP), and 2,4-dichlorophenoxyacetic acid (2,4-D) + N-(2-chloro-4-pyridyl)-N'-phenylurea (4-CPPU)). Results were compared with those obtained when explants were cultivated in the presence of 2,4-D + BAP, previously used on melon. Embryogenesis occurred more successfully in 4-day-old cotyledons and seeds than hypocotyls and 8-day-old cotyledons. The best result was achieved with NOA + BAP. Genotypes significantly affected embryogenesis. The number of embryos in "Eivanaki" was significantly higher than that in "Mashhadi." Embryo proliferation when explants were maintained in jars (9.3%) was found to be higher compared to that in petri dishes. For the first time, genetic stability of regenerated melon plants was evaluated using inter-simple sequence repeat markers. Polymerase chain reaction (PCR) products demonstrated a total of 102 well-resolved bands, and regenerants were 93% similar compared to the mother plant. Somaclonal changes during embryogenesis were evaluated by flow cytometry, showing 91% of the same patterns in regenerated plants. The results suggest that the new hormone components are effective when applied for in vitro embryogenesis of muskmelon as they show a high frequency in regeneration and genetic homogeneity.

Retrotransposon Proliferation Coincident with the Evolution of Dioecy in Asparagus.

Current phylogenetic sampling reveals that dioecy and an XY sex chromosome pair evolved once, or possibly twice, in the genus Asparagus Although there appear to be some lineage-specific polyploidization events, the base chromosome number of 2n = 2× = 20 is relatively conserved across the Asparagus genus. Regardless, dioecious species tend to have larger genomes than hermaphroditic species. Here, we test whether this genome size expansion in dioecious species is related to a polyploidization and subsequent chromosome fusion, or to retrotransposon proliferation in dioecious species. We first estimate genome sizes, or use published values, for four hermaphrodites and four dioecious species distributed across the phylogeny, and show that dioecious species typically have larger genomes than hermaphroditic species. Utilizing a phylogenomic approach, we find no evidence for ancient polyploidization contributing to increased genome sizes of sampled dioecious species. We do find support for an ancient whole genome duplication (WGD) event predating the diversification of the Asparagus genus. Repetitive DNA content of the four hermaphroditic and four dioecious species was characterized based on randomly sampled whole genome shotgun sequencing, and common elements were annotated. Across our broad phylogenetic sampling, Ty-1 Copia retroelements, in particular, have undergone a marked proliferation in dioecious species. In the absence of a detectable WGD event, retrotransposon proliferation is the most likely explanation for the precipitous increase in genome size in dioecious Asparagus species.

Polyphenols distribution in juices from Citrus allotetraploid somatic hybrids and their sexual hybrids.

The polyphenol profile of an interspecific allotetraploid somatic hybrid, achieved by protoplast fusion, the 'Valencia' sweet orange ( Citrus sinensis L. Osbeck) + 'Femminello' lemon ( Citrus limon L. Burm), and three sexual hybrids obtained by backcrosses between Femminello lemon and the allotetraploid somatic hybrid ('Valencia' + 'Femminello') was studied by liquid chromatography-ultraviolet-diode array detector-mass spectrometry (LC-UV-DAD-MS). The aim of the work was to evaluate whether superior traits and improved performance can be observed in these new genotypes. Ten flavonoids (TF), comprising seven flavanones and three flavones, and four hydroxycinnamic acids (HCA) have been characterized and quantified in all hybrids and compared with those of the respective parents. The 'Valencia' + 'Femminello' somatic hybrid shows an intermediate polyphenol composition with respect to those of the parents, with a slight prevalence of lemon influence. The three sexual hybrids show, instead, different and more complex chromatographic profiles.

Identification of zygotic and nucellar seedlings in citrus interploid crosses by means of isozymes, flow cytometry and ISSR-PCR.

'Milam' (a purported hybrid of Citrus jambhiri Lush) + 'Femminello' lemon (Citrus limon L. Burm. f.) allotetraploid somatic hybrids were used as pollen parents in interploid crosses with diploid 'Femminello' lemon to achieve mal secco tolerance in different populations of seedless triploid lemon types with good fruit quality. A total of 137 plantlets were obtained and subjected to screening experiments, in order to distinguish zygotic embryos from nucellars. Here we report on and discuss the results obtained with three techniques: flow cytometry, isozyme analysis and ISSR-PCR (the inter-simple sequence repeats-polymerase chain reaction). ISSR-PCR resulted to be a very efficient and reliable technique for the identification of zygotic plantlets.