Phenolic and Antioxidant Compound Accumulation of Quercus robur Bark Diverges Based on Tree Genotype, Phenology and Extraction Method.

Oak bark is a rich niche for beneficial bioactive compounds. It is known that the amount of the compounds found in plant tissues can depend on species, genotype, growth site, etc., but it is unclear whether oak phenology, i.e., late or early bud burst, can also influence the amount of phenols and antioxidants that can be extracted. We tested two Quercus robur populations expressing different phenology and five half-sib families in each population to see how phenology, genotype, as well as extrahent differences (75% methanol or water) can determine the total phenol, total flavonoid content, as well as antioxidant activity. Significant statistical differences were found between half-sib families of the same population, between populations representing different oak phenology and different extrahents used. We determined that the extraction of flavonoids was more favorable when using water. So was antioxidant activity using one of the indicators, when significant differences between extrahents were observed. Furthermore, in families where there was a significant difference, phenols showed better results when using methanol. Overall, late bud burst families exhibited higher levels in all parameters tested. Thus, we recommend that for further bioactive compound extraction, all these factors be noted.

Genotype-Dependent Jasmonic Acid Effect on Pinus sylvestris L. Growth and Induced Systemic Resistance Indicators.

Due to temperature changes, forests are expected to encounter more stress than before, both in terms of biotic factors, such as increased insect attacks, and abiotic factors, such as more frequent droughts. Priming trees to respond to these changes faster and more effectively would be beneficial. Induced systemic resistance (ISR) is a mechanism that is turned on when plants encounter unfavorable conditions. Certain elicitors, such as jasmonic acid (JA) are known to induce plants' metabolic response. However, even though studies on ISR in herbaceous species are common and varied ISR elicitors can be used in agriculture, the same cannot be said about trees and forestry enterprises. We aimed to investigate whether JA used in different concentrations could induce metabolic changes (total phenol content, total flavonoid content, photosynthesis pigment content, antioxidant enzyme activity) in Pinus sylvestris seedlings and how this varies between different pine half-sib families (genotypes). After six weeks with a single application of JA, pine seedlings in several pine genetic families exhibited increased antioxidant enzyme activity, total phenol content and carotenoid content that correlated positively with JA concentrations used. Results from other genetic families were varied, but in many cases, there was a significant response to JA, with a noticeable increase as compared to the unaffected group. The impact on chlorophyll content and flavonoids was less noticeable overall. A positive effect on seedling growth parameters was not observed in any of the test cases. We conclude that JA can induce systemic resistance after a single application exogenously in P. sylvestris seedlings and recommend that the use of JA needs to be optimized by selecting appropriate concentrations.

Traceability of bilberries (Vaccinium myrtillus L.) of the Baltic-Nordic region using surface-enhanced Raman spectroscopy (SERS): DFT simulation-based DNA analysis.

Food traceability is a major issue in the industry. We investigated whether bilberries (Vaccinium myrtillus L.) from 4 different locations within the Baltic-Nordic region could be effectively differentiated using surface-enhanced Raman scattering (SERS) based spectral data and chemometric analyses. Furthermore, we aimed to determine if nucleobase (adenine and cytosine) methylation could be responsible for any observed variation. Our experiment was successful in that both principal component (PCA) and discriminant function analyses (DFA) showed differentiation between bilberry DNA from all 4 geographical regions. Density functional theory (DFT) based simulations allowed us to analyze whether DNA's spectral data dissimilarities may be due to nucleobase methylation. Although results were inconclusive on this, our investigation provides valuable data on simulated versus experimental DNA and DNA component spectra. Further research will be directed towards understanding what other epigenetic changes could be responsible for the observed DNA variation as well as determining the optimal parameters for using DFT simulations in upcoming projects.

Monilinia fructigena Suppressing and Plant Growth Promoting Endophytic Pseudomonas spp. Bacteria Isolated from Plum.

Brown rot caused by Monilinia spp. fungi causes substantial losses in stone and pome fruit production. Reports suggest that up to 90% of the harvest could be lost. This constitutes an important worldwide issue in the food chain that cannot be solved by the use of chemical fungicides alone. Biocontrol agents (BCAs) based on microorganisms are considered a potential alternative to chemical fungicides. We hypothesized that endophytic bacteria from Prunus domestica could exhibit antagonistic properties towards Monilinia fructigena, one of the main causative agents of brown rot. Among the bacteria isolated from vegetative buds, eight isolates showed antagonistic activity against M. fructigena, including three Pseudomonas spp. isolates that demonstrated 34% to 90% inhibition of the pathogen's growth when cultivated on two different media in vitro. As the stimulation of plant growth could contribute to the disease-suppressing activity of the potential BCAs, plant growth promoting traits (PGPTs) were assessed for bacterial isolates with M. fructigena-suppressing activity. While all isolates were capable of producing siderophores and indole-3-acetic acid (IAA), fixating nitrogen, mineralizing organic phosphate, and solubilizing inorganic phosphate and potassium, only the Pseudomonas spp. isolates showed 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity. Overall, our study paves the way for the development of an eco-friendly strategy for managing M. fructigena pathogens by using BCAs including Pseudomonas spp. bacteria, which could also serve as growth stimulators.

Endophytes from blueberry (Vaccinium sp.) fruit: Characterization of yeast and bacteria via label-free surface-enhanced Raman spectroscopy (SERS).

Blueberries (Vaccinium sp.) are consumed all around the globe, however, their endophytic community has not been thoroughly researched, specifically their fruit endophytes. We aimed to isolate and analyze easily cultivable blueberry fruit endophytes to help in future research, concerning probiotic microorganisms. Twelve strains were isolated in this pilot study, genetically homologous with Staphylococcus hominis, Staphylococcus cohnii, Salmonella enterica, Leuconostoc mesenteroides, and [Candida] santamariae. To determine the molecular composition of these isolates we used label-free surface-enhanced Raman spectroscopy (SERS). To our knowledge, this is the first time that SERS spectra for L. mesenteroides and C. santamariae are presented, as well as the first report of Candida yeast, isolated specifically from blueberry fruits. Our findings suggest that the differences in tested yeast and bacteria SERS spectra and subsequent differentiation are facilitated by minor shifts in spectral peak positions as well as their intensities. Moreover, we used principal component and discriminant function analyses to differentiate chemotypes within our isolate group, proving the sensitivity of the technique and its usefulness to recognize different strains in plant-associated microbe samples, which will aid to streamline future studies in biofertilizers and biocontrol agents.

Priming of Resistance-Related Phenolics: A Study of Plant-Associated Bacteria and Hymenoscyphus fraxineus.

European ash (Fraxinus excelsior) is highly affected by the pathogenic fungus Hymenoscyphus fraxineus in all of Europe. Increases in plant's secondary metabolite (SM) production is often linked tol enhanced resistance to stress, both biotic and abiotic. Moreover, plant-associated bacteria have been shown to enhance SM production in inoculated plants. Thus, our hypothesis is that bacteria may boost ash SM production, hence priming the tree's metabolism and facilitating higher levels of resilience to H. fraxineus. We tested three different ash genotypes and used Paenibacillus sp. and Pseudomonas sp. for inoculation in vitro. Total phenol (TPC), total flavonoid (TFC) and carotenoid contents were measured, as well as the chlorophyll a/b ratio and morphometric growth parameters, in a two-stage trial, whereby seedlings were inoculated with the bacteria during the first stage and with H. fraxineus during the second stage. While the tested bacteria did not positively affect the morphometric growth parameters of ash seedlings, they had a statistically significant effect on TPC, TFC, the chlorophyll a/b ratio and carotenoid content in both stages, thus confirming our hypothesis. Specifically, in ash genotype 64, both bacteria elicited an increase in carotenoid content, TPC and TFC during both stages. Additionally, Pseudomonas sp. inoculated seedlings demonstrated an increase in phenolics after infection with the fungus in both genotypes 64 and 87. Our results indicate that next to genetic selection of the most resilient planting material for ash reforestation, plant-associated bacteria could also be used to boost ash SM production.

Impact of Plant-Associated Bacteria on the In Vitro Growth and Pathogenic Resistance against Phellinus tremulae of Different Aspen (Populus) Genotypes.

Aspens (Populus tremula and its hybrids), economically and ecologically important fast-growing trees, are often damaged by Phellinus tremulae, a rot-causing fungus. Plant-associated bacteria can be used to increase plant growth and resistance; however, no systematic studies relating the activity of symbiotic bacteria to aspen resistance against Phellinus tremulae have been conducted so far. The present pioneer study investigated the responses of two Populus tremula and two P. tremula × P. tremuloides genotypes to in vitro inoculations with, first, either Pseudomonas sp. or Paenibacillus sp. bacteria (isolated originally from hybrid aspen tissue cultures and being most closely related to Pseudomonas oryzihabitans and Paenibacillus tundrae, respectively) and, in the subsequent stage, with Phellinus tremulae. Both morphological parameters of in vitro-grown plants and biochemical content of their leaves, including photosynthesis pigments and secondary metabolites, were analyzed. It was found that both Populus tremula × P. tremuloides genotypes, whose development in vitro was significantly damaged by Phellinus tremulae, were characterized by certain responses to the studied bacteria: decreased shoot development by both Paenibacillus sp. and Pseudomonas sp. and increased phenol content by Pseudomonas sp. In turn, these responses were lacking in both Populus tremula genotypes that showed in vitro resistance to the fungus. Moreover, these genotypes showed positive long-term growth responses to bacterial inoculation, even synergistic with the subsequent fungal inoculation. Hence, the studied bacteria were demonstrated as a potential tool for the improved in vitro propagation of fungus-resistant aspen genotypes.

Differentiation of Closely Related Oak-Associated Gram-Negative Bacteria by Label-Free Surface Enhanced Raman Spectroscopy (SERS).

Due to the harmful effects of chemical fertilizers and pesticides, the need for an eco-friendly solution to improve soil fertility has become a necessity, thus microbial biofertilizer research is on the rise. Plant endophytic bacteria inhabiting internal tissues represent a novel niche for research into new biofertilizer strains. However, the number of species and strains that need to be differentiated and identified to facilitate faster screening in future plant-bacteria interaction studies, is enormous. Surface enhanced Raman spectroscopy (SERS) may provide a platform for bacterial discrimination and identification, which, compared with the traditional methods, is relatively rapid, uncomplicated and ensures high specificity. In this study, we attempted to differentiate 18 bacterial isolates from two oaks via morphological, physiological, biochemical tests and SERS spectra analysis. Previous 16S rRNA gene fragment sequencing showed that three isolates belong to Paenibacillus, 3-to Pantoea and 12-to Pseudomonas genera. Additional tests were not able to further sort these bacteria into strain-specific groups. However, the obtained label-free SERS bacterial spectra along with the high-accuracy principal component (PCA) and discriminant function analyses (DFA) demonstrated the possibility to differentiate these bacteria into variant strains. Furthermore, we collected information about the biochemical characteristics of selected isolates. The results of this study suggest a promising application of SERS in combination with PCA/DFA as a rapid, non-expensive and sensitive method for the detection and identification of plant-associated bacteria.

Oak (Quercus robur) Associated Endophytic Paenibacillus sp. Promotes Poplar (Populus spp.) Root Growth In Vitro.

Soil fertilization is necessary for high-demand crop production in agriculture and forestry. Our current dependence on chemical fertilizers has significant harmful side effects. Biofertilization using microorganisms is a sustainable way to limit the need for chemical fertilizers in various enterprises. Most plant endophytic bacteria have thus far been unstudied for their plant growth promoting potential and hence present a novel niche for new biofertilizer strains. We isolated English oak (Quercus robur) endophytic bacteria and tested them for plant growth promoting traits (PGPTs) such as nitrogen fixation, phosphate mineralization/solubilization, siderophore and indole-3-acetic acid (IAA) production. We also investigated the effect the selected isolate had on poplar (Populus spp.) microshoot vegetative growth parameters in vitro. In total 48 bacterial strains were isolated, attributed to Bacillus, Delftia, Paenibacillus, Pantoea and Pseudomonas genera. All the isolates displayed at least three PGPTs, with 39.6% of the isolates displaying all five (all were Pseudomonas spp.) and 18.75% displaying four. Based on relative abundance, Paenibacillus sp. isolate was selected for the poplar microshoot inoculation study. The isolate had a significant positive effect on poplar microshoot root growth and development. Two tested poplar genotypes both had increased lateral root number and density, fresh and dry root biomass. Furthermore, one genotype had increased length and number of adventitious roots as well as a decrease in fresh aboveground biomass. The root enhancement was attributed to IAA production. We propose this isolate for further studies as a potential biofertilizer.