Fresh Plant Tissue Research Articles

Revealing degradation mechanisms of archaeological flax textiles through the evolution of fibres’ parietal polymers by synchrotron deep-UV fluorescence

Autofluorescence generated by plant cells and tissues provides a label-free method for investigating them without altering their structure and biochemistry. While in the literature, research mainly focuses on investigating fresh plant tissues, this study examines flax yarns from five archaeological artefacts to understand how cell wall fibres degrade and how their biochemical composition is modified after natural ageing over millennia. Using Synchrotron-based deep UV fluorescence (S-DUV), we aim to correlate fibre autofluorescence with the environmental conditions of discovery (clothes of votive statuettes buried in a sanctuary or shrouds and bandages for mummification rituals found in tombs) or their intended purposes (fishing net, which underwent wet-dry cycles during its use). Out of the five archaeological objects studied, the two yarns sampled from the buried fabrics were found encrusted with minerals, but with significant differences in the fibres’ degradation. The yarn sampled from the fishing net displayed a great variability of the dataset, with some fibres biochemically similar to the buried samples while others were closer to the funerary linen shrouds. Lignin's predominant presence at 480 nm in the fishing net suggests extensive cellulose-enriched cell wall layer degradation while the lignin-enriched layer is still preserved. Additionally, second harmonic generation imaging microscopy (SHG) and 2-photon excitation fluorescence (2PEF) were used alongside S-DUV analyses to evaluate the flax fibres’ ultrastructure. SHG confirmed S-DUV findings and indicated poor crystalline cellulose preservation in one linen shroud, explaining the yarn's brittleness.

High-Performance Liquid Chromatographic Quantification of the Plant Hormone Abscisic Acid at ppb Levels in Plant Samples after a Single Immunoaffinity Column Cleanup.

High-performance liquid chromatography with ultraviolet detection (HPLC-UV) is a common analysis technique due to its high versatility and simple operation. In the present study, HPLC-UV detection was integrated with immunoaffinity cleanup (IAC) of the sample extracts. The matrix effect was greatly reduced, and the limit of detection was as low as 1 ng/g of free abscisic acid (ABA) in fresh plant tissues. A monoclonal antibody 3F1 (mAb 3F1) was developed to specifically recognize free ABA but not ABA analogues. The mAb 3F1-immobilized immunoaffinity column exhibited a capacity of 850 ng/mL and an elution efficiency of 88.8-105% for standards. The extraction recoveries of the column for ABA ranged from 80.4 to 108.9%. ABA content was detected in various plant samples with IAC-HPLC-UV. The results were verified with ultraperformance liquid chromatography-electrospray tandem mass spectrometry. IAC-HPLC-UV can be a sensitive and cost-efficient method for plant hormone analysis.

A novel colormetric and light-up fluorescent sensor from flavonol derivative grafted cellulose for rapid and sensitive detection of Hg2+ and its applications in biological and environmental system

Mercury ion (Hg2+) is one of harmful heavy metal ions that can accumulate inside the human organism and cause some health problems. In the article, a highly effective fluorescent probe named EC-T-PCBM was prepared by grafting flavonol derivatives onto ethyl cellulose for the specific recognition of Hg2+. EC-T-PCBM exhibited a remarkable fluorescence light-up response toward Hg2+ with excellent sensitivity. EC-T-PCBM possessed several prominent sensing properties for Hg2+, such as low detection limit (43.9 nM), short response time (5 min), and wide detection pH range (6–9). The response mechanism of EC-T-PCBM to Hg2+ has been verified through 1H NMR titration and DFT computation. Additionally, EC-T-PCBM not only can be used for accurately determining trace amount of Hg2+ in actual environmental water samples, but also can serve as a portable and rapid device by loading it on test strips for sensitive and selective visualization of Hg2+. More importantly, the confocal fluorescence imaging of onion cells suggested the favorable cell membrane permeability of EC-T-PCBM and its prominent ability to continuously monitor the enrichment from Hg2+ within fresh plant tissues.

Distribution of Vicine, Convicine and Levodopa in Faba Bean Plant Tissues Determined by UltraHigh Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry

Faba bean is an emerging pulse crop for the Canada Prairies and is becoming an increasingly important source of plant sourced protein. It also has a high content of levodopa (up to 0.1 % of the dry seed weight), a nutraceutical compound for treating Parkinson's Disease (PD). The presence of pyrimidine glycosides, vicine and convicine, however, restricts the use of faba due to their potential to cause favism in susceptible individuals. For the rapid separation and quantification of levodopa, vicine and convicine, a 5-min ultrahigh performance liquid chromatography-electrospray ionization mass spectrometry method (UHPLC-ESI MS) was developed. This method provided a wide linear range of quantification (4.8 nM to 60 µM for vicine, and 6.0 nM to 24 µM for convicine and levodopa), and low limit of detection (1.2 to 3.0 nM). The method was applied in the analysis of diverse faba bean samples, from dehulled mature faba bean and seed coat, to fresh plant tissues such as flowers, leaves, stems, roots and pods. The results indicated that fresh faba bean plant tissues, such as leaves and flowers, had the highest concentration of levodopa and low level of VC, which makes them an alternative source of plant-based l-DOPA with potential as a food-based treatment for Parkinson's Disease.

Synthetic and analytical routes to the L-amino acid conjugates of cis-OPDA and their identification and quantification in plants

Cis-(+)-12-oxophytodienoic acid (cis-(+)-OPDA) is a bioactive jasmonate, a precursor of jasmonic acid, which also displays signaling activity on its own. Modulation of cis-(+)-OPDA actions may be carried out via biotransformation leading to metabolites of various functions. This work introduces a methodology for the synthesis of racemic cis-OPDA conjugates with amino acids (OPDA-aa) and their deuterium-labeled analogs, which enables the unambiguous identification and accurate quantification of these compounds in plants. We have developed a highly sensitive liquid chromatography-tandem mass spectrometry-based method for the reliable determination of seven OPDA-aa (OPDA-Alanine, OPDA-Aspartate, OPDA-Glutamate, OPDA-Glycine, OPDA-Isoleucine, OPDA-Phenylalanine, and OPDA-Valine) from minute amount of plant material. The extraction from 10 mg of fresh plant tissue by 10% aqueous methanol followed by single-step sample clean-up on hydrophilic–lipophilic balanced columns prior to final analysis was optimized. The method was validated in terms of accuracy and precision, and the method parameters such as process efficiency, recovery and matrix effects were evaluated. In mechanically wounded 30-day-old Arabidopsis thaliana leaves, five endogenous (+)-OPDA-aa were identified and their endogenous levels were estimated. The time-course accumulation revealed a peak 60 min after the wounding, roughly corresponding to the accumulation of cis-(+)-OPDA. Our synthetic and analytical methodologies will support studies on cis-(+)-OPDA conjugation with amino acids and research into the biological significance of these metabolites in plants.

A Fast and Cost-Effective Genotyping Method for CRISPR-Cas9-Generated Mutant Rice Lines.

With increasing throughput in both the generation and phenotyping of mutant lines in plants, it is important to have an efficient and reliable genotyping method. Traditional workflows, still commonly used in many labs, have time-consuming and expensive steps, such as DNA purification, cloning and growing E. coli cultures. We propose an alternative workflow where these steps are bypassed, using Phire polymerase on fresh plant tissue, and ExoProStar treatment as preparation for sequencing. We generated CRISPR-Cas9 mutants for ZAS (ZAXINONE SYNTHASE) in rice with two guide RNAs. Using both a traditional workflow and our proposed workflow, we genotyped nine T1 plants. To interpret the sequencing output, which is often complex in CRISPR-generated mutants, we used free online automatic analysis systems and compared the results. Our proposed workflow produces results of the same quality as the old workflow, but in 1 day instead of 3 days and about 35 times cheaper. This workflow also consists of fewer steps and reduces the risk of cross contamination and mistakes. Furthermore, the automated sequence analysis packages are mostly accurate and could easily be used for bulk analysis. Based on these advantages, we encourage academic and commercial labs conducting genotyping to consider switching over to our proposed workflow.

Optimization of Targeted Plant Proteomics Using Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS).

This study was conducted to optimize a targeted plant proteomics approach from signature peptide selection and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analytical method development and optimization to sample preparation method optimization. Three typical protein extraction and precipitation methods, including trichloroacetic acid (TCA)/acetone method, phenol method, and TCA/acetone/phenol method, and two digestion methods, including trypsin digestion and LysC/trypsin digestion, were evaluated for selected proteins related to the impact of engineered nanomaterials (ENMs) on wheat (Triticum aestivum) plant growth. In addition, we compared two plant tissue homogenization methods: grinding freeze-dried tissue and fresh tissue into a fine powder using a mortar and pestle aided with liquid nitrogen. Wheat plants were grown under a 16 h photoperiod (light intensity 150 μmol·m-2·s-1) for 4 weeks at 22 °C with a relative humidity of 60% and were watered daily to maintain a 70-90% water content in the soil. Processed samples were analyzed with an optimized LC-MS/MS method. The concentration of selected signature peptides for the wheat proteins of interest indicated that the phenol extraction method using fresh plant tissue, coupled with trypsin digestion, was the best sample preparation method for the targeted proteomics study. Overall, the optimized approach yielded the highest total peptide concentration (68,831 ng/g, 2.4 times the lowest concentration) as well as higher signature peptide concentrations for most peptides (19 out of 28). In addition, three of the signature peptides could only be detected using the optimized approach. This study provides a workflow for optimizing targeted proteomics studies.

Complementation between microbial necromass and plant debris governs the long-term build-up of the soil organic carbon pool in conservation agriculture

Plant residue is ultimately the source of soil organic matter but occurs as a continuum from relatively fresh plant tissue to highly evolved components. Nevertheless, the extent to which crop residue accumulates as plant tissue versus microbial necromass in conservation agricultural systems is still uncertain. A 12-year conservation tillage experiment with maize additions of zero, half and full harvest was conducted in Northeast China. Soil samples in the 0–10 cm depth were collected at 1–4 year intervals. The microbial residue biomarker amino sugar and plant component biomarker lignin phenol were quantified to trace the dynamics of microbial necromass and plant debris. Compared with the initial value, the content of amino sugar increased exponentially to 21–45% and approached steady-state equilibrium after 12 years of maize straw mulching, indicating the existence of “microbial carrying capacity” under repeated plant residue addition. With prolonged duration of conservation tillage and increasing rate of maize straw return, the ratio of amino sugar to lignin phenol decreased from 6.2 to 1.7, accompanied by a decreased degree of lignin side-chain oxidation, indicating that organic input enhanced the selective retention of fresh plant lignin to a larger extent than microbial necromass. The build-up of the SOC pool under conservation tillage was critically controlled by the functional complementation of microbial- and plant-derived components. Maize straw input was able to enhance the contribution of microbial necromass to SOC stabilization by increasing fungal necromass accumulation, while the retention of lignin-like plant debris enlarged the SOC pool capacity and potentially maintained the decomposability of SOC in the long term.

Effects of Fungi on Soil Organic Carbon and Soil Enzyme Activity under Agricultural and Pasture Land of Eastern Türkiye

Soil organic matter (SOM) is a heterogeneous mixture of materials ranging from fresh plant tissues to highly decomposed humus at different stages of decomposition. Soil organic carbon (SOC) status is directly related to the amount of organic matter in soil and therefore is generally used to measure it. Soil carbon sequestration refers to the removal of carbon (C) containing substances from the atmosphere and its storage in soil C pools. The soil microbial community (SMC) plays an important role in the C cycle, and its activity is considered to be the main driver of differences in C storage potential in soil. The composition of SMC is crucial for maintaining soil ecosystem services, as the structure and activity of SMC also regulate the turnover and distribution of nutrients, as well as the rate of soil organic matter (SOM) decomposition. Here, we applied fungi on the soils taken from two fields, one used as a pasture and one for agriculture (wheat cultivation), in a histosol in the eastern part of Türkiye and investigated the changes in the organic carbon and enzyme activity contents of the soils at the end of the 41-day incubation period. In the study, four different fungal species (Verticillium dahliae (SOR-8), Rhizoctonia solani (S-TR-6), Fusarium oxysporum (HMK2-6), and Trichoderma sp.) were used and catalase, urease, and alkaline phosphatase activities were examined. Results showed that the values of SOC were V. dahliae (7.46%), Trichoderma sp. (7.27%), R. solani (7.03%), Control (6.97%), and F. oxysporum (6.7%) in pastureland and were V. dahlia (4.72%), control (4.69%), F. oxysporum (4.65%), R. solani (4.37%), and Trichoderma sp. (4.14%) in agricultural land, respectively. SOC and soil enzyme activities were significantly affected by land use types (p < 0.05). The higher SOC and enzyme activities were observed in pastureland. Finally, it was determined that soil organic carbon and soil enzyme activities were affected by fungi. This study is important in terms of revealing that the effects of fungi on soil organic carbon and enzyme activities are different in various land types.

Systematic Position of Ptilophorus dufourii Inferred from Its Primary Larva, with Notes on Ptilophorinae (Coleoptera: Ripiphoridae)

Since the description of Ptilophorus dufourii (Latreille, 1818), the unknown immature stages and biology of Ptilophorinae is one of the longest persisting gap in the research of the wedge-shaped beetles (Ripiphoridae). Here we describe the primary larva of P. dufourii based on SEM and CLSM images. Its modified mouthparts unsuitable to grasp prey rule out predaceous habits and also processing any solid substrates (e.g., wood or other fresh plant tissues). In contrast, the weakly sclerotized and very small body, the enlargement of the sensorial appendage of the antennae, and simplified mouthparts strongly suggest parasitism and consumption of liquid food. We provide a comparison with the only known larva of Pelecotominae, another possible basal lineage of Ripiphoridae, and discuss potential synapomorphies and differences between larvae of both subfamilies. A sistergroup relationship between Ptilophorinae and Pelecotominae is suggested by the presence of only two stemmata, spatulate femoral setae, a scale-like lateral cranial surface, and an enlarged antennal sensorial appendage, and a clade Ripidiini + Ripiphorinae by a characteristic boat-shape of the primary larvae. The host of P. dufourii remains unknown. We reviewed published host records, but none of them is based on traceable evidence and none of them appears credible. Observations of adult behaviour of P. dufourii are documented. Taxonomy and systematics of Ptilophorus and Ptilophorinae are discussed. The Cretaceous Spinotoma ruicheni Hsiao et Huang, 2017 is transferred from Pelecotominae to Ptilophorinae in accordance with diagnostic characters given in the original description and current definitions of both subfamilies.

Recent studies on Polyphenols: Extraction technologies and prospects

Polyphenols constitute a group of the most important bioactive compounds widely represented naturally in many plant species. Fresh plant tissues, plant residues, and agro-wastes contain consequent amounts of polyphenolic compounds. In recent years, there is a developing interest in utilizing these bioactive compounds for nutraceutical and pharmaceutical applications due to their beneficial effects on human health and their potential in improving the quality of food. However, their effectiveness is depending on many factors such as the extraction technique, the plant tissue, and species origin, environmental factors, the solvent used for the extraction, etc. The purpose of this critical review is to address and give an insight into the latest review and research articles regarding extraction methods and highlights the prospects of bioactive compounds found in several plant tissues.

Tree Fresh Leaf- and Twig-Leached Dissolved Organic Matter Quantity and Biodegradability in Subtropical Plantations in China

Extreme weather events often cause the input of fresh plant tissues into soils in forests. However, the interspecific patterns of tree fresh plant tissue-leached dissolved organic matter (DOM) characteristics are poorly understood. In this study, we collected fresh leaves and twigs of two broadleaf trees (Liquidambar formosana and Schima superba) and two coniferous trees (Pinus massoniana and Pinus elliottii) in subtropical plantations in China, and measured tree fresh tissue-leached DOM quantity and biodegradability. The interspecific patterns of fresh plant tissue-leached DOM production varied with organ types. Broadleaf tree leaves leached greater amounts of dissolved organic carbon (DOC), dissolved total nitrogen (DTN), and dissolved total phosphorus (DTP) than coniferous tree leaves, but an opposite pattern of DOC and DTN productions was observed between broadleaf and coniferous tree twigs. Regardless of tree species, leaves often leached greater quantities of DOC, DTN, and DTP than twigs. For both leaves and twigs, broadleaf tree tissue-leached DOM had greater aromaticity and lower biodegradability than coniferous tree tissue-leached DOM. Moreover, leaf-leached DOM had greater aromaticity and lower biodegradability than twig-leached DOM. In addition, DOM biodegradability negatively correlated with the initial aromaticity and DOC:DTN ratio, despite no relationship between DOM biodegradability and DOC:DTP ratio. These findings highlight the pivotal roles of leaf habit and organ type in regulating fresh tree tissue-leached DOM production and biodegradability and reveal that the substantial variations of fresh tissue-leached DOM biodegradability are co-driven by DOM aromaticity and N availability in subtropical plantations in China.

First report of banana freckle disease caused by Phyllosticta cavendishii in Mauritius

Banana (Musa sp.) is among the most important fruit crops grown in Mauritius with significant socioeconomic and cultural considerations. The annual production of banana approximates to around 8000 tonnes (Statistics Mauritius, 2018). Dwarf Cavendish is the main variety grown commercially in Mauritius. During the hot and humid month of February 2014, symptoms consisting of dark black spots were observed on unripe banana of Dwarf Cavendish from the region of Nouvelle Découverte (super humid zone). A disease incidence of more than 50% with a moderate to severe level of severity across the field was reported by the extension services of the Food and Agricultural Research and Extension Institute. On the fruits, the initial symptom was tiny reddish-brown spots, bordered by dark green water-soaked margins (Figure 1). As the spots enlarged and coalesced, the surface became rough and turned black. These spots had a rough texture like sand-paper, similar to the description described by Wong et al. (2012). Comparable symptoms were observed on the leaves (Figure 2). Results from a rapid island-wide survey showed that only the Cavendish-type banana varieties displayed symptoms of infection. Out of 98 banana fields surveyed (50.9 ha), 65% were affected. Morphological identification of the suspected fungal pathogen was done using a compound light microscope. A sterile needle was used to tease pycnidia on leaves in a drop of sterile water to release conidia, before being mounted in clear lactic acid for microscopic examination. Conidia (Figure 3) formed singly at the apex of the conidiogenous cell were hyaline, aseptate, oblong to ellipsoidal, coarsely guttulate and measured 7.1-15.6 × 4.4-8.3 μm. The apical mucilaginous appendage arising from the conidium was straight to curved and was 5.1-12.9 μm long. Spermagonia were 70–125 μm in diameter and 50–85 μm high. Dumbbell-shaped spermatia (Figure 4) were hyaline, aseptate, biguttulate and measured 7.5-9.5 × 1.3-1.8 μm. These morphological characters are consistent with the species description of Phyllosticta cavendishii (Wong et al., 2012). Lesions from 11 samples of leaf and fruit with fruiting bodies were ground in liquid nitrogen followed by genomic DNA extraction. Extraction of DNA was done on c. 50 mg fresh plant tissue using the Biosprint 15 DNA Plant Kit (Qiagen, Germany). Tissues were first homogenized using a Tissuelyser (Qiagen, Germany) in a 2 ml microcentrifuge tube containing a 3 mm metal bead for two 30 second bursts at 30Hz. Two microliters of the eluted DNA was used as template in an ITS PCR amplification using AmpliTaq Gold 360 Mastermix (Applied Biosystems, USA) and 0.5 μM each of primers GMF1 and GMR2 (Wong et al., 2012). Thermal cycling conditions were 95°C for 10 min followed by 40 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 1 min. Final extension of PCR products was done at 72°C for 10 min. Amplicons (550bp) were directly sequenced using the same primer pair. Resulting sequences were analysed using BlastN as well as by manual comparison to known sequences of Phyllosticta isolates which cause freckle disease of banana. The causal agent from all 11 samples tested was found to be Phyllosticta cavendishii. The ITS sequences were deposited in GenBank (Accession Nos. MW520341-MW520351) and at the Queensland Plant Pathology Herbarium (BRIP 72048 to 72058). Pathogenicity was tested using twenty mature green healthy fruits of banana variety Dwarf Cavendish. Fruits were surface sterilised using 0.2% sodium hypochlorite for 2 mins, rinsed with sterilised distilled water and allowed to dry before inoculation. Ten fruits were artificially inoculated on the outer surface, clearly marked, with a conidial suspension of 106 conidia/ml, prepared from pycnidia extracted from field infected tissue. Control fruits were treated with sterile distilled water. All the fruits were covered with plastic bags for 48 hours and incubated at room temperature, 80% relative humidity and with a 12-hour photoperiod. After 7 days, typical minute black spots identical to those observed in the field, appeared on inoculated fruits, whereas controls remained healthy. The morphological characteristics of the fungus were consistent with those previously observed, hence fulfilling Koch's postulates. To our knowledge, this is the first report of Phyllosticta cavendishii causing banana freckle disease in Mauritius. The authors acknowledge the Centre for Plant Science, Queensland Alliance for Agriculture and Food Innovation for the molecular identification of Phyllosticta cavendishii and also Dr Yu Pei Tan from the Brisbane Plant Pathology Herbarium for accessioning isolates and submission into GenBank.

First record of Pachybrachis Chevrolat, 1836 (Coleoptera: Chrysomelidae: Cryptocephalinae) on Spermacoce verticillata L. (Rubiaceae) and synthesis of its plant associations in Brazil

This study reports Pachybrachis sp. association with a new host, Spermacoce verticillata L. (Rubiaceae). It synthesizes Brazil locality records and host plant records from literature. Specimens of adults and larvae were collected on the host in an urban area of Maceió, Alagoas, Brazil. Larvae and adults were reared on the host plant in the laboratory, using fresh plant tissue daily.

The Influence of Locality on Phenolic Profile and Antioxidant Capacity of Bud Extracts.

Gemmotherapy represents the most recent therapeutic technique that uses the properties of extracts from fresh meristematic plant tissues, mainly buds and sprouts, by macerating them in ethanol and glycerol. The harvesting time and the location can significantly affect the chemical composition of the buds. Therefore, this work aimed to point out the possible variability in the phenolic content and the antioxidant potential of extracts prepared from commonly grown trees in the Czech Republic. Extracts from buds collected during autumn and spring in three different localities were analysed using UHPLC-MS (ultra-high-pressure liquid chromatography) for the phenols profile. Five tests assays were used for the evaluation of the extract antioxidant potential. The sampling time positively affected the content of total phenols, flavonoids, and phenolic acids. The increased levels of total phenols and flavonoids in localities with high and medium pollution may be the result of the higher levels of NO and SO2, the main air pollutants. However, surprisingly, the content of phenolic acid showed the highest values in the area with the lowest pollution. The results of antioxidant tests did not completely correlate with the levels of phenolic metabolites, which may be due to the involvement of other active molecules (e.g., ascorbate, tocopherol, or proline) in the antioxidant machinery.

Waltheria indica is a New Host of Phytoplasma Belongs to 16SrI-B Subgroup Associated with Virescence Symptoms in China.

Waltheria indica L. is a kind of medicinal plants belonging to the family of Sterculiaceae distributed in China, which extracts with many active compounds used for treatment of rheumatism and sore pains (Hua et al., 2019). During September to November 2020, the plants showing abnormal symptoms including floral virescence, leaf chlorosis and leaflet, as shown in Fig.1, were found in Dingan county of Hainan province, China, with about 70% incidence. The disease symptoms which were suspected to be infected by the phytoplasma, a phloem-limited cell-wall-less prokaryotic pathogen could not be cultured in vitro, severely impacted Waltheria indica growth resulting in financial loss and ecological damage in the location. For identification of the causal pathogen, the total DNA of symptom or symptomless Waltheria indica samples were extracted using 0.10 g fresh plant tissues using CTAB method. PCR reactions were performed using primers R16mF2/R16mR1 (Lee et al., 1993) and AYgroelF/AYgroelR (Mitrović et al., 2011) specific for phytoplasma 16S rRNA and groEL gene fragments. The target productions of the two gene fragments of phytoplasma were detected in the DNA from four symptomatic plant samples whereas not in the DNA from the symptomless plant samples. The PCR productions were sequenced and the data were deposited in GenBank. The two gene fragments of the DNA extracted from the symptom plant samples were all identical, with the length of 1340 bp 16S rRNA (GenBank accession: MW353909) and 1312 bp groEL (MW353709) gene sequence fragments, putatively encoding 437 (groEL) amino acids sequence. The phytoplasma strain was named as Waltheria indica virescence (WiV) phytoplasma, WiV-hnda strain. A Blast search based on the 16S rRNA gene fragment of WiV-hnda phytoplasma strain revealed the highest level of sequence identities (99.85%) with that of 16SrI aster yellows group members (16SrI-B subgroup), such as Onion yellows phytoplasma strain OY-M (AP006628) from Japan (Oshima et al., 2004); Periwinkle virescence phytoplasma strain PeV-hnhk (KP662136), Chinaberry witches'-broom phytoplasma strain CWB-hnsy1 (KP662119) and CWB-hnsy2 (KP662120), all the strains from Hainan island of China (Yu et al., 2017). A Blast search based on the groEL gene sequence fragment of WiV-hnda indicated 99.92% sequence identity with that of 16SrI aster yellows group members (16SrI-B subgroup) such as Onion yellows phytoplasma strain OY-M (AP006628). Homology and phylogenetic analysis by DNAMAN 5.0 and MEGA 7.0 software indicated that the phytoplasma strains of WiV-hnda, OY-M, PeV-hnhk, CWB-hnsy1 and CWB-hnsy2 were clustered into one clade based on the 16S rRNA gene fragments. WiV-hnda, OY-M and Aster yellow witches'-broom (AYWB) (CP000061) phytoplasma strains were clustered into one clade based on the groEL gene fragments. To our knowledge, this was the first time that Waltheria indica virescence disease induced by 16SrI-B subgroup phytoplasma strain was reported in China. Genetic analysis showed that WiV-hnda was closely related to the phytoplasma strains causing Onion yellows in Japan, Periwinkle virescence and Chinaberry witches'-broom disease in China.

Sisymbrium Officinale (the Singers’ Plant) as an Ingredient: Analysis of Somatosensory Active Volatile Isothiocyanates in Model Food and Drinks

Sisymbrium officinale (L.) Scop. (hedge mustard) is a wild common plant of the Brassicaceae family. It is known as “the singers’ plant” for its traditional use in treating aphonia and vocal disability. The plant is rich in glucosinolates and isothiocyanates; the latter has been demonstrated to be a strong agonist in vitro of the Transient Receptor Potential Ankirine 1 (TRPA1) channel, which is involved in the somatosensory perception of pungency as well as in the nociception pathway of inflammatory pain. Volatile ITCs are released by the enzymatic or chemical hydrolysis of GLSs (glucosinolates) during sample crushing and/or by the mastication of fresh plant tissues when the plant is used as an ingredient. Some functional food and drink model preparations have been realised: honey enriched with seeds and flowers, infusions, cold drink (voice drink), artisanal beer, and a fermented tea (kombucha). Using SPME-GCMS chromatography, we analysed samples of the plant and of the food preparations adopting conditions that simulate the release of isothiocyanates (ITCs) during oral assumption. Two active compounds, iso-propylisothiocyanate and 2-butylisothiocyanate, have been assayed. The concentration of ITCs varies according to temperature, pH, grinding conditions, and different plant organs used. Kombucha-type fermentation seems to eliminate the ITCs, whereas they are retained in beer. The ITCs’ concentration is higher when entire seeds and flowers are used.

HPLC coupled with quadrupole time of flight tandem mass spectrometry for analysis of glycosylated components from the fresh flowers of two congeneric species: Robinia hispida L. and Robinia pseudoacacia L.

Developing methods for the systematic and rapid identification of the chemical compositions of fresh plant tissues has long attracted the attention of phytochemists and pharmacologists. In the present study, based on highly efficient sample pretreatment and high-throughput analysis of high-performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry data using molecular networks, a method was developed for systematically analyzing the chemical constituents of the fresh flowers of Robinia hispida L. and Robina pseudoacacia L., two congeneric ornamental species that lack prior consideration. A total of 44 glycosylated structures were characterized. And on the basis of establishing of the fragmentation pathways of 11 known flavonoid glycosides, together with the molecular networking analysis, 18 other ions of flavonoid glycosides in five classes were clustered. Moreover, 15 soyasaponins/triterpenoid glycosides were tentatively identified by comparison of their tandem mass spectrometry characteristic ions with those reported in the literature or the online Global Natural Product Social Molecular Networking database. The water extracts were separated by flash chromatography, which resulted in the discovery of one new compound, named rohispidascopolin, along with five known entities. The pharmacological targets were predicted by SwissTargetPrediction.

Extractability, stability, and accumulation of nepetoidins in Ocimum basilicum L. leaves and cell cultures

Antioxidant polyphenols nepetoidin A and B are widely spread in Lamiaceae family yet often remain undetected due to their purported instability and poor extractability with polar solvents typically used for phenolics extraction. Coextraction of nepetoidins may confer additional, or enhance existing, bioactivities of polyphenol extracts. This study includes a detailed analysis of nepetoidin extractability from sweet basil (Ocimum basilicum L.) leaves, and a survey of their occurrence in basil tissue cultures and peltate trichomes. Coextraction of nepetoidins with the major polyphenol rosmarinic acid was possible with solvent mixtures, yet substantially less efficient than that with the respective preferred solvents. Nepetoidin, but not rosmarinic acid levels in extracts from dried leaves were dramatically reduced. However, this was not due to degradation suggested by earlier reports, but to reduced extractability, which could be restored by wetting the tissue with water prior to addition of nonpolar solvent. Nepetoidins were found to accumulate at high levels in basil callus and suspension cultures. Correlation analysis revealed three novel compounds that are coextracted with nepetoidins and are proposed to represent nepetoidin dimers. Candidate signals for nepetoidin and rosmarinic acid glycosides were also detected in dedifferentiated basil cells. By contrast, nepetoidins were not enriched in basil peltate trichomes. This study establishes optimized recovery procedure for nepetoidins, reports their production by dedifferentiated basil cells, and illustrates how sample preparation methodology affects the phytochemical findings. This study reports an improved method for the recovery of polyphenolic nepetoidins from fresh and dry plant tissue and demonstrates their high abundance in dedifferentiated Ocimum basilicum cells.

Phytochemicals from Castanea spp. buds and green extraction technologies: the Finnover project

Green economy is a sustainable development tool based on the valorization of economic, natural and social resources. It is recognized as a tool to be applied to all the production sectors (goods and services), as well as for the conservation and sustainable use of natural resources. The FINNOVER project (“Innovative strategies for the development of cross-border green supply chains”) proposes a technical-economic path for the creation and development of new supply chains for the eco-sustainable extraction and use of natural bioactive compounds. In this study, an innovative extraction and re-use strategy to obtain value-added products from botanical by-products was developed as an alternative to waste incineration or composting. It was applied to Castanea sativa bud extract production as a case study, but it could be analogously applied for other herbal preparations. Castanea spp. and their preparations have been widely used for hundreds of years as medicinal plants in composite formulae. Bioactive compounds (botanicals) are quite variable in the plant material, according to genotype (intraspecific chemodiversity), different collection stages, pedoclimatic conditions of sampling sites (wild or cultivation zones), agrotechniques, and post-harvest handling. This research aimed to compare the bioactive compound pattern of Castanea spp. bud preparations (herbal preparations derived from embryonic fresh plant tissues as buds and sprouts) with the composition of extracts derived from the bud-waste management process. Molecules were extracted by the encoded traditional method (maceration in hydroglyceroalcoholic solution) and by green extraction technologies (Pulsed Ultrasound-Assisted Extraction). HPLC methods were used to identify and quantify the main bioactive compounds, and to obtain a specific profile to assess the contribution of every single bioactive class to the total phytocomplex. The established protocol was simple, sensitive and reliable and it could be used for the evaluation and quality control of natural products and relative eco-sustainable extracts. The valorization of bud marcs, which remain after the bud-preparation production, could have a significant economic impact for the commercial producers, representing an important innovation in this sector.