The impact of various forms of silver nanoparticles on the rhizosphere of wheat (Triticum aestivum L.) - Shifts in microbiome structure and predicted microbial metabolic functions.

The study investigated the effects of different silver nanoparticles (AgNPs) on the soil microbiome and wheat growth. For comparison purposes, a commercial fungicide and silver nitrate (AgNO3) were used. The results revealed three distinct groups of nanoparticles based on their impacts. Small-size AgNPs (10 nm) with a negative charge, as well as fungicide had limited effects on the microbiome, similar to the no-treatment control. Bigger in size (30-60 nm) and a negative charge AgNPs showed the most beneficial effects on soil microbiota shifts. These AgNPs increased the abundance of bacteria with beneficial traits such as nitrogen-fixing, urease, protease, and lignin degradation bacteria. The third type of AgNPs had a positive charge of nanostructure and influenced specific microbial populations, increasing the abundance of anaerobic and autotrophic groups of microorganisms, which could be assessed as a harmful shift for plants growth promotions and was similar to the AgNO3 treatment. Overall, the study emphasized the potential of AgNPs in agriculture not only as biocidal. The conducted study proved that AgNPs with bigger size/negative charge, used in low concentration can have a surprisingly stimulating effect on the positive characteristics of the rhizosphere microbiome. Moreover, the surface charge of AgNPs is a significant factor affecting microbial activity of wheat rhizosphere soil, which in this treatment is significantly similar to the AgNO3 treatment.

Stigmas of holoparasitic Phelipanche arenaria (Orobanchaceae) - a suitable ephemeric flower habitat for development unique microbiome.

BACKGROUND: Microbial communities have occasionally been observed in part of the ephemeric reproductive structure of floral stigmas, but their prevalence, phylogenetic diversity and ecological roles are understudied. This report describes the first study of bacterial and fungal communities in immature and mature stigma tissue of the endangered holoparasitic plant Phelipanche arenaria. Culture-dependent methods coupled with next-generation sequencing indicated that a small surface of the flower stigma was an unexpectedly rich and diverse microhabitat for colonization of microbial. We also compared the enzymatic activity of the bacterial communities between immature and mature stigmas samples. RESULTS: Using high-throughput sequencing methods, we identified and classified 39 to over 51 OTUs per sample for bacterial OTUs represented by Pantoea agglomerans and P. ananatis, comprising 50.6%, followed by Pseudomonas, Luteibacter spp., Sphingomonas spp. with 17% of total frequency. The bacterial profile of immature stigmas of P. arenaria contained unique microorganisms (21 of the most numerous OTUs) that were not confirmed in mature stigmas. However, the enzymatic activity of bacteria in mature stigmas of P. arenaria showed more activity than observed in immature stigmas. In the fungal profile, we recorded even 80 OTUs in mature stigmas, consisting of Capnodiales 45.03% of the total abundance with 28.27% of frequency was created by Alternaria eichhorniae (10.55%), Mycosphaerella tassiana (9.69%), and Aureobasidium pullulans (8.03%). Additionally, numerous putative plant growth-promoting bacteria, fungal pathogens and pathogen-antagonistic yeasts were also detected. CONCLUSIONS: Our study uncovered that P. arenaria stigmas host diverse bacterial and fungal communities. These microorganisms are well known and have been described as beneficial for biotechnological and environmental applications (e.g., production of different enzymes and antimicrobial compounds). This research provided valuable insight into the parasitic plant-microbe interactions.

Diversity and potential plant growth promoting capacity of seed endophytic bacteria of the holoparasite Cistanche phelypaea (Orobanchaceae).

Salt marshes are highly dynamic, biologically diverse ecosystems with a broad range of ecological functions. We investigated the endophytic bacterial community of surface sterilized seeds of the holoparasitic Cistanche phelypaea growing in coastal salt marshes of the Iberian Peninsula in Portugal. C. phelypaea is the only representative of the genus Cistanche that was reported in such habitat. Using high-throughput sequencing methods, 23 bacterial phyla and 263 different OTUs on genus level were found. Bacterial strains belonging to phyla Proteobacteria and Actinobacteriota were dominating. Also some newly classified or undiscovered bacterial phyla, unclassified and unexplored taxonomic groups, symbiotic Archaea groups inhabited the C. phelypaea seeds. γ-Proteobacteria was the most diverse phylogenetic group. Sixty-three bacterial strains belonging to Bacilli, Actinomycetes, α-, γ- and ß-Proteobacteria and unclassified bacteria were isolated. We also investigated the in vitro PGP traits and salt tolerance of the isolates. Among the Actinobacteria, Micromonospora spp. showed the most promising endophytes in the seeds. Taken together, the results indicated that the seeds were inhabited by halotolerant bacterial strains that may play a role in mitigating the adverse effects of salt stress on the host plant. In future research, these bacteria should be assessed as potential sources of novel and unique bioactive compounds or as novel bacterial species.

Interspecies and temporal dynamics of bacterial and fungal microbiomes of pistil stigmas in flowers in holoparasitic plants of the Orobanche series Alsaticae (Orobanchaceae).

Little is known about the microbiomes of flower parts, and even less information is available regarding these microorganisms' colonization of specific niches in parasitic plants. We investigate the temporal interspecies dynamics of the parasitic plants microbiome of flower stigmas in two stages of development: immature stigmas in flower buds and mature stigmas in opened flowers. We compared two related holoparasitic Orobanche species from localities approximately 90 km apart and characterize their bacterial and fungal communities using 16S rRNA gene and ITS sequences, respectively. We identified from 127 to over 228 OTUs per sample for fungi, sequences belonging to genera: Aureobasidium, Cladosporium, Malassezia, Mycosphaerella, and Pleosporales, constituting approximately 53% of the community in total. In the bacterial profile, we recorded 40 to over 68 OTUs per sample consisting of Enterobacteriaceae, and genera Cellulosimicrobium, Pantoea, and Pseudomonas spp., with an approximately 75% frequency. In microbial communities, higher numbers of OTUs colonizing mature stigmas were recorded than in immature. This implies that the dynamics and concurrence of microbial communities were different between O. alsatica and O. bartlingii and underwent significant changes during flower development. To the best of our knowledge, is the first study of the interspecies and temporal dynamics of the bacterial and fungal microbiomes of pistil stigmas in flowers.

Morphological diversity of pistil stigmas and its taxonomic significance of representatives of holoparasitic Orobanchaceae from Central Europe.

The stigma is the terminal part of the carpel which receives pollen during the pollination process. Although the role of the stigmas in pollination is the same for all angiosperms, stigmas structures are very diverse. This study aimed to evaluate intraspecific, interspecific and intergeneric stigmas variability and then find differences of the stigma morphology amongst 24 holoparasitic Orobanche and Phelipanche species and provide new insights into its potential taxonomic value. This paper was also focused on selecting the best diagnostic features that would be used for future stigma analysis in other species of Orobanchaceae. These analyses were conducted with fresh, dry and fixed material using stereomicroscopy from different locations from Central Europe. Twenty-one quantitative or qualitative morphological features were analysed. This study highlights the variation of stigma morphology and characters which are useful to improve the taxonomic understanding of problematic taxa. Thus, two main types of stigmas were established, based on tested features: 1-oval, rarely hemispherical in shape, most often one-coloured with lobes separated in Phelipanche stigmas; 2-spherical to hemispherical, rarely oval, multi-coloured with partially fused or separated lobes in Orobanche stigmas. The best diagnostic features of the stigmas for distinguishing the Orobanchaceae are the type and subtype of stigma, the length and area of the stigma, the width of single lobes, the width in the middle part of the stigma, the length of upper and lower separation in the middle part between lobes and the angle between lobes in the upper and lower part. The morphological features of the stigmas are important criteria for distinguishing genera, sections and subsections, as well as related species. In this study, we present the first stigma morphological studies for the most numerous genera from the tribe Orobancheae and this paper may determine features possible to use in solving certain taxonomic problems and evolutionary relationships of the species.

Tea plantations and their importance as host plants and hot spots for epiphytic cryptogams.

Bryophytes and lichens are outstanding bioindicators, not only of the plant community in which they develop, but also the substrates on which they grow. Some epiphytic cryptogams, particularly the rare ones, are stenotopic and require a long habitat continuity, for example substrates such as old trees. It could also be a tea plantation, this is because the shrubs are not felled, and most of them may have several dozen years. In addition, the shrubs are not subject to sudden changes in microclimatic conditions as only the young leaves are harvested. As the importance of tea plantations as host plants for mosses and lichens has not yet been studied, the present study examines the species diversity of cryptogams of two tea plantations in Georgia (Caucasus). The study also examines the phytogeography, spatial pattern, environmental conditions and ecological indicators of the cryptogams. Thirty-nine cryptogam taxa were identified; typical forest taxa dominated, even in the absence of typical forest communities. Some of these species are obligatory epiphytes, rare or even critically endangered in most European countries (e.g., Orthotrichum stellatum, O. stramineum, Lewinskya striata). The fairly abundant record of such species on tea plantations indicates the importance of these phytocoenoses for the preservation of rare species, and indicates that these habitats are hot spots for these cryptogams in otherwise changed envirnonment. Additionally, as indicated the analysis of the species composition of individual plantations and the mathematical analysis made on this basis, plantations differ from each other. Another interesting result is also the spatial distributions of cryptogams on tea bushes resemble those of forest communities and lichens seems to be more sensitive than bryophytes to antropogenic changes of environment.

Correlational nutritional relationships and interactions between expansive holoparasite Orobanche laxissima and woody hosts on metal-rich soils.

Plant parasitism by other plants, combined with abiotic environmental stress, offers a unique opportunity to study correlational nutritional relationships in terms of parasite-host interactions and their functional roles in nutrient cycling in ecosystems. Our study analysed the transfer of selected mineral elements, including heavy metals, from soil to different organs in hosts (Punica granatum and Fraxinus angustifolia) and from hosts to the expansive holoparasite (Orobanche laxissima) in cinnamonic soil habitats in Georgia (Caucasus). We also identified other correlated trophic and bioactive effects in the parasite-host relationship. O. laxissima was characterized by a high accumulation tendency for micro- and macroelements, such as K and Ca, and heavy metals, such as Zn, Ni, and Cd. Parasites can reduce the concentration of heavy metals in host tissues owing to this high accumulation tendency. In total, 85 compounds were identified in the examined parasite and its hosts. Despite the distinct phytochemical content of species of the infected host, the parasite produced specific metabolites with dominant phenylethanoid glycosides (PhGs), with acteoside and crenatoside being the primary dominant compounds - ca. 98% of all polyphenols. Polyphenols in parasite specimens that are correlated with Cu and Zn are antagonistic to polyphenols correlated with Fe, Pb, Cr, and Ni. The profile of polyphenols in the host species was both qualitatively and quantitatively distinct from the profile of the compounds in the parasite and between hosts (only acteoside in group PhGs was common between the parasite and Fraxinus host), which indicates the existence of a unique compound biosynthesis pathway in the parasite. Our results demonstrated that the parasite, particularly in its flowers, exhibited higher polyphenol content, antioxidative effects (ABTS-+, DPPH, and FRAP), and inhibitory effects.

Phylogeny and historical biogeography analysis support Caucasian and Mediterranean centres of origin of key holoparasitic Orobancheae (Orobanchaceae) lineages.

The extensive diversity of the tribe Orobancheae, the most species-rich lineage of holoparasitic Orobanchaceae, is concentrated in the Caucasus and Mediterranean regions of the Old World. This extant diversity has inspired hypotheses that these regions are also centres of origin of its key lineages, however the ability to test hypotheses has been limited by a lack of sampling and phylogenetic information about the species, especially in the Caucasus region. First, we assessed the phylogenetic relationships of several poorly known, problematic, or newly described species and host-races of four genera of Orobancheae occurring in the Caucasus region-Cistanche, Phelypaea, Phelipanche and Orobanche-using nuclear ribosomal (ITS) and plastid (trnL-trnF) sequence data. Then we applied a probablistic dispersal-extinction-cladogenesis model of historical biogeography across a more inclusive clade of holoparasites, to explicitly test hypotheses of Orobancheae diversification and historical biogeography shifts. In sum, we sampled 548 sequences (including 196 newly generated) from 13 genera, 140 species, and 175 taxa across 44 countries. We find that the Western Asia (particularly the Caucasus) and the Mediterranean are the centre of origin for large clades of holoparasitic Orobancheae within the last 6 million years. In the Caucasus, the centres of diversity are composed both of long-branch taxa and shallow, recently diversified clades, while Orobancheae diversity in the Mediterranean appears to represent mainly recent diversification.

The First Report of Orobanche laxissima Parasitizing Pomegranate (Punica granatum) in Georgia.

Orobanche laxissima Uhlich & Rätzel (Orobanchaceae) is a polyphagous root parasitic plant distributed in the Caucasus Mountains and Transcaucasia; especially Russia, Georgia, Armenia, Azerbaijan, N.E. Turkey (Piwowarczyk et al. 2019). It infects many wild or sometimes cultivated trees and shrubs, such as Betulaceae, Oleaceae, Fagaceae, Aceraceae, Cornaceae, usually Fraxinus L., Fagus L., Carpinus L. (Piwowarczyk et al. 2019, 2020). Punica granatum L. (Lythraceae), commonly known as pomegranate, is native to the Caucasus, the Himalayas in North Pakistan and Northern India, and is widely cultivated, e.g. in USA and throughout the Mediterranean. Pomegranate is one of the first domesticated fruits and have been used in folk medicine or as a food for centuries. Fruit, seed, leaves, flower, root, or barks extracts have extensive medicinal properties (Shaygannia et al. 2015). Field surveys conducted in south-eastern Georgia in May 2019 revealed extensive infestations of O. laxissima on the roots of P. granatum in one locality in Kakheti Province, near Sighnaghi (41°37,4 N, 45°56,3 E, 480 m elevation), in roadside or hills scrub and cultivated areas of pomegranate. The infection was confirmed by verifying the attachment of the Orobanche to the Punica root. The population of the parasite consisted of at least a ca. thousand shoots, sometimes in one clump was ca. 100 individuals. A single plant of pomegranate was parasitized by few to c.a. a hundred of broomrape plants, and 10 to 20% of the ca. 1 ha location was infested. The main botanical features of the O. laxissima are: i) stem simple, (10-)25-40(-100) cm high, with haustoria; ii) inflorescence usually long to short cylindrical or lax, usually many-flowered; iii) calyx-segments entire or bidentate, rarely with 4 teeth; iv) corolla (16-)22-24(-31) mm long, tubular-bell-shaped; purple, pink, rarely dirty yellow, light brown; v) stigma purple, orange, or yellow (Piwowarczyk et al. 2019, 2020). For molecular analysis, total genomic DNA was extracted from the sample and the plastid gene rbcL (rubisco large subunit) was sequenced and amplified as described in Piwowarczyk et al. (2015). The sequence (1231 bp) was deposited in GenBank (MN384886). BLAST search found that it was most similar to (Query Cover 100%, Per Ident. 100%) O. laxissima (KR260928). To the best of our knowledge, this is the first report of a O. laxissima parasitizing P. granatum. O. laxissima appearing in large numbers on singles pomegranate shrubs can weaken the plants, and reduce flowering and fruiting. In the Caucasus region, O. laxissima was observed in mesophilic forests and shrubs, but our report suggests the possibility of a potential spread to neighboring cultivated areas, especially fruit trees and shrubs. Until now, only one report of pathogenic plants was documented for P. granatum, included Phelipanche aegyptiaca (Pers.) Pomel and O. crenata Forssk. in Israel (Dor et al. 2014).

Fatty acid composition in seeds of holoparasitic Orobanchaceae from the Caucasus region: Relation to species, climatic conditions and nutritional value.

The specialization of parasitic plants from the Orobanchaceae family to the heterotrophic lifestyle caused several morphological, physiological and molecular changes. One of the adaptations to the parasitic lifestyle is the production of a large number of the smallest seeds in world flora, also called "dust-seeds". Seeds of 34 holoparasitic species from the Cistanche, Orobanche, Phelipanche, and Phelypaea genera were collected in the Caucasus region (54 samples) and their fatty acid content and compositions analysed. Of these seeds, 28 were investigated for the first time, and 12 are endemic to the Caucasus (one of the most important biodiversity hotspots in the world). The influence of different hosts, populations, habitats, and climatic conditions on the fatty acid content and composition, as well as some connections of taxonomic classification are discussed. The fatty acid content in the species varied between 0.9 and 42.5%, and showed quantitative differences at generic and infrageneric levels, while displaying uniform fatty acid composition. Thirteen fatty acids were identified, of which nine were undescribed for Orobanchaceae. The fatty acid composition of the Orobanchaceae seeds represented a mixture of saturated fatty acids (SFAs) (average 7.8%) and unsaturated fatty acids (UFAs) (average 92.2%). The fatty acid content in the Orobanchaceae seeds was directly unrelated to taxonomy, while the n-6/n-3 fatty acid ratio supported the clear separation of the Orobanche and Phelipanche genera. Orobanchaceae seeds contained mainly linoleic and oleic acids, thus they could be a potential nutritional source of the unsaturated fatty acids. Additionally, the studies confirmed the hypothesis that the degree of seed oil fatty acid unsaturation increased in colder climatic conditions, especially for the Orobanche genus.