Characterization of Endophytic Fungi, Acremonium sp., from Lilium davidii and Analysis of Its Antifungal and Plant Growth-Promoting Effects.

The present study was aimed at isolating endophytic fungi from the Asian culinary and medicinal plant Lilium davidii and analyzing its antifungal and plant growth-promoting effects. In this study, the fungal endophyte Acremonium sp. Ld-03 was isolated from the bulbs of L. davidii and identified through morphological and molecular analysis. The molecular and morphological analysis confirmed the endophytic fungal strain as Acremonium sp. Ld-03. Antifungal effects of Ld-03 were observed against Fusarium oxysporum, Botrytis cinerea, Botryosphaeria dothidea, and Fusarium fujikuroi. The highest growth inhibition, i.e., 78.39 ± 4.21%, was observed for B. dothidea followed by 56.68 ± 4.38%, 43.62 ± 3.81%, and 20.12 ± 2.45% for B. cinerea, F. fujikuroi, and F. oxysporum, respectively. Analysis of the ethyl acetate fraction through UHPLC-LTQ-IT-MS/MS revealed putative secondary metabolites which included xanthurenic acid, valyl aspartic acid, gancidin W, peptides, and cyclic dipeptides such as valylarginine, cyclo-[L-(4-hydroxy-Pro)-L-leu], cyclo(Pro-Phe), and (3S,6S)-3-benzyl-6-(4-hydroxybenzyl)piperazine-2,5-dione. Other metabolites included (S)-3-(4-hydroxyphenyl)-2-((S)-pyrrolidine-2-carboxamido)propanoic acid, dibutyl phthalate (DBP), 9-octadecenamide, D-erythro-C18-Sphingosine, N-palmitoyl sphinganine, and hydroxypalmitoyl sphinganine. The strain Ld-03 showed indole acetic acid (IAA) production with or without the application of exogenous tryptophan. The IAA ranged from 53.12 ± 3.20 µg ml-1 to 167.71 ± 7.12 µg ml-1 under different tryptophan concentrations. The strain was able to produce siderophore, and its production was significantly decreased with increasing Fe(III) citrate concentrations in the medium. The endophytic fungal strain also showed production of organic acids and phosphate solubilization activity. Plant growth-promoting effects of the strain were evaluated on in vitro seedling growth of Allium tuberosum. Application of 40% culture dilution resulted in a significant increase in root and shoot length, i.e., 24.03 ± 2.71 mm and 37.27 ± 1.86 mm, respectively, compared to nontreated control plants. The fungal endophyte Ld-03 demonstrated the potential of conferring disease resistance and plant growth promotion. Therefore, we conclude that the isolated Acremonium sp. Ld-03 should be further investigated before utilization as a biocontrol agent and plant growth stimulator.

Strigolactones, from Plants to Human Health: Achievements and Challenges.

Strigolactones (SLs) are a class of sesquiterpenoid plant hormones that play a role in the response of plants to various biotic and abiotic stresses. When released into the rhizosphere, they are perceived by both beneficial symbiotic mycorrhizal fungi and parasitic plants. Due to their multiple roles, SLs are potentially interesting agricultural targets. Indeed, the use of SLs as agrochemicals can favor sustainable agriculture via multiple mechanisms, including shaping root architecture, promoting ideal branching, stimulating nutrient assimilation, controlling parasitic weeds, mitigating drought and enhancing mycorrhization. Moreover, over the last few years, a number of studies have shed light onto the effects exerted by SLs on human cells and on their possible applications in medicine. For example, SLs have been demonstrated to play a key role in the control of pathways related to apoptosis and inflammation. The elucidation of the molecular mechanisms behind their action has inspired further investigations into their effects on human cells and their possible uses as anti-cancer and antimicrobial agents.

Evaluation and Quantification of Natural Strigolactones from Root Exudates.

Strigolactones (SLs) in the root exudates can be detected by germination assays with root parasitic weed seeds, but precise and accurate evaluation and quantification are possible only by chemical analysis with the liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here we describe methods for root exudate collection, sample preparation, and LC-MS/MS analysis of SLs.

Isolation and Identification of Naturally Occurring Strigolactones.

The accurate structure determination of strigolactones (SLs) that are produced by plants leads to the precise understanding of the biosynthesis and functions of their molecules. SLs need to be isolated and purified from the plant roots or root exudates in a hydroponic solution using appropriate methods in order to determine the structures. In this chapter, we describe a small-scale extraction method for chromatographic analysis of known SLs and a large-scale purification method for isolation of unknown SLs, together with methods for the hydroponic culture of plants and collection of root exudates. Finally, we present spectroscopic data that are helpful in identifying SLs.

Magnetic boron nitride nanosheets as a novel magnetic solid-phase extraction adsorbent for the determination of plant growth regulators in tomatoes.

A novel magnetic boron nitride nanosheets (Fe3O4@BNNSs) composite-based magnetic solid-phase extraction (MSPE) method was employed to analyse six plant growth regulators (PGRs) in tomatoes combined with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The novel Fe3O4@BNNSs composite was prepared via an in situ chemical coprecipitation process and characterized by scanning electron microscopy (SEM), Fourier transform infrared spectrometry (FT-IR), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and vibrating sample magnetometry (VSM). Several factors that may affect the extraction efficiencies were optimized. Under the optimal factors, low limits of detection (0.002-0.010 ng g-1), good linear ranges (0.05-10 ng g-1) and satisfactory precisions (intra-day: 1.2%-3.9%; inter-day: 2.1%-6.9%) were achieved. The established approach was successfully employed to extract and determine PGRs in tomatoes, and the spiked recoveries were between 85.2 and 109.0%.

Discovery of novel potential plant growth regulators from Corydalis mucronifera.

Three pairs of enantiomers mucroniferals A-C (1-3), with a novel skeleton of 1,4-epoxynaphthalene-2,3-dicarboxylic acid first reported from nature source, were isolated from Corydalis mucronifera. Their structures were elucidated based on extensive spectroscopic data analysis of MS, 1D and 2D NMR, and their absolute configurations were confirmed by single-crystal X-ray diffraction analysis and comparison of the experimental and calculated ECD data. Mucroniferals A-C showed broad-spectrum inhibitory activities on seedling growth of all plants tested (Lepidium apetalum, Raphanus sativus, Lactuca sativa, and Arabidopsis thaliana) with a dose-dependent relationship. Additionally, mucroniferals A and B exhibited significant inhibitory effects on germination of most seeds at concentration of 80 µg/mL, and the inhibition was reversible.

Plant growth regulators from mushrooms.

Plants interact with fungi in their natural growing environments, and relationships between plants and diverse fungal species impact plants in complex symbiotic, parasitic, and pathogenic ways. Over the past 10 years, we have intensively investigated plant growth regulators produced by mushrooms, and we succeeded in finding various regulators from mushroom-forming fungi: (1) fairy chemicals as a candidate family of new plant hormones from Lepista sordida, (2) agrocybynes A to E from fungus Agrocybe praecox that stimulate strawberry growth, (3) armillariols A to C and sesquiterpene aryl esters from genus Armillaria that are allelopathic and cause Arimillaria root disease, and (4) other plant growth regulators from other mushrooms, such as Stropharia rugosoannulata, Tricholoma flavovirens, Hericium erinaceus, Leccinum extremiorientale, Russula vinosa, Pholiota lubrica and Cortinarius caperatus.

Improved Solid-Phase Extraction for Simple, Sensitive, and Efficient Determination of Trace Plant Growth Regulators in Cherry Tomatoes by High-Performance Liquid Chromatography.

The overuse of plant growth regulators (PGRs) in agricultural products has gradually increased in recent years, resulting in hazardous effects on food safety and human health. For the first time, a sensitive, accurate, and low-cost analytical method involving improved solid-phase extraction coupled with high-performance liquid chromatography was developed to determine trace PGRs in cherry tomatoes. Thereafter, the extraction mechanism and conditions were elucidated. Under optimized conditions, good linearity (0.04-400 ng g-1; r ≥ 0.9996) and lower limits of detection (0.005-0.006 ng g-1) were observed. The recoveries were 81.4-90.1%, with relative standard deviations of ≤6.7% (three levels). Finally, the developed method was successfully used to detect trace PGRs in cherry tomatoes. The results illustrated that this sensitive method shows great potential for application to monitor trace PGRs in agricultural products and, thus, provide technical support for food safety and public health.

Simultaneous detection of plant growth regulators jasmonic acid and methyl jasmonate in plant samples by a monoclonal antibody-based ELISA.

Methyl jasmonate (MeJA) and its free-acid form, jasmonic acid (JA), collectively referred to as jasmonates (JAs), are natural plant growth regulators that are widely present in higher plants. Simultaneous detection of JA and MeJA in plant samples is of significance and is a great challenging issue. In this study, coupling with two extraction methods, a sensitive monoclonal antibody (mAb) based enzyme-linked immunosorbent assay (ELISA) for simultaneous detection of JA and MeJA in plant samples was developed. The JA-bovine serum albumin (BSA) conjugate was used as an immunogen for the production of mAb. As the produced mAb exhibited higher recognition ability towards MeJA than towards JA, ELISA was established using MeJA as the standard. Under optimal experimental conditions, the IC50 and LOD values of ELISA for MeJA were 2.02 ng mL-1 and 0.20 ng mL-1, respectively. In the first extraction method, MeJA in plant samples was evaporated and only JA was extracted. In the second extraction method, both JA and MeJA were extracted. After methylation, JA in the extracts was converted into MeJA, and the whole MeJA in the extracts was measured by ELISA. Plant samples including the leaves of Salvia splendens, the flowers of Salvia splendens and the fruit of grapes were collected. JA and MeJA in these samples were detected by the proposed ELISA. It was found that the concentrations of JA in these three plant samples were about 3-5 times higher than those of MeJA in those samples. ELISA was also confirmed by HPLC. There was a good correlation between ELISA and HPLC.

A New Endophytic Fusarium Oxysporum Gibberellic Acid: Optimization of Production Using Combined Strategies of Experimental Designs and Potency on Tomato Growth under Stress Condition.

This study reports the potential of the endophytic fungi identified as a Fusarium oxysporum to produce gibberellic acid (GA3). The GA3 production was confirmed by high performance liquid chromatography. To improve the production of this phytohormone under solid state fermentation (SSF), successive optimization strategies were used. Firstly, Plackett-Burman design was applied for screening medium components and culture condition. Under the optimized condition, GA3 yield (7.14 g/kg) was 2.62-fold higher than by the use of the initial condition (2.72 g/kg). The concentration of the most influential parameters and their interaction were optimized with a Box-Behnken experimental design. The optimized condition led to a 1.14-fold enhancement in GA3 production, reaching 8.16 g/kg. The GA3 crude extract obtained by SSF was then used to study its ameliorative role on adverse salinity effect on tomato plants (Solanum lycopersicum L.). The interactive effects of different GA3 concentrations were examined on morphological and physiological parameters of tomato plants. The application of GA3 (10-6 M) under salt stress condition (100 mM) was found to improve growth and physiological parameters including plant height, total chlorophyll, starch, and proline contents. The exogenous application of GA3 is a potent strategy to reverse abiotic stress that affect the agricultural productivity and limit plant growth and yield.

Concise synthesis of heliolactone, a non-canonical strigolactone isolated from sunflower.

Heliolactone is one of the earliest identified non-canonical strigolactones. Its concise synthesis was achieved by employing Knoevenagel-type condensation and semi-reduction of a malonate intermediate as the key steps. This synthesis was performed in a non-stereoselective manner, and thus a racemic and diastereomeric mixture of heliolactone was obtained. The developed synthetic route is fairly concise and straightforward.

Production, extraction and characterization of Chlorella vulgaris soluble polysaccharides and their applications in AgNPs biosynthesis and biostimulation of plant growth.

Chlorella vulgaris, like a wide range of other microalgae, are able to grow mixotrophically. This maximizes its growth and production of polysaccharides (PS). The extracted polysaccharides have a complex monosaccharide composition (fructose, maltose, lactose and glucose), sulphate (210.65 ± 10.5 mg g-1 PS), uronic acids (171.97 ± 5.7 mg g-1 PS), total protein content (32.99 ± 2.1 mg g-1 PS), and total carbohydrate (495.44 ± 8.4 mg g-1 PS). Fourier Transform infrared spectroscopy (FT-IR) analysis of the extracted polysaccharides showed the presence of N-H, O-H, C-H, -CH3, >CH2, COO-1, S=O and the C=O functional groups. UV-Visible spectral analysis shows the presence of proteins, nucleic acids and chemical groups (ester, carbonyl, carboxyl and amine). Purified polysaccharides were light green in color and in a form of odorless powder. It was soluble in water but insoluble in other organic solvents. Thermogravimetric analysis demonstrates that Chlorella vulgaris soluble polysaccharide is thermostable until 240°C and degradation occurs in three distinct phases. Differential scanning calorimetry (DSC) analysis showed the characteristic exothermic transition of Chlorella vulgaris soluble polysaccharides with crystallization temperature peaks at 144.1°C, 162.3°C and 227.7°C. The X-ray diffractogram illustrated the semicrystalline nature of these polysaccharides. Silver nanoparticles (AgNPs) had been biosynthesized using a solution of Chlorella vulgaris soluble polysaccharides. The pale green color solution of soluble polysaccharides was turned brown when it was incubated for 24 hours with 100 mM silver nitrate in the dark, it showed peak maximum located at 430 nm. FT-IR analysis for the biosynthesized AgNPs reported the presence of carbonyl, -CH3, >CH2, C-H,-OH and -NH functional groups. Scanning and transmission electron microscopy show that AgNPs have spherical shape with an average particle size of 5.76. Energy-dispersive X-ray (EDX) analysis showed the dominance of silver. The biosynthesized silver nanoparticles were tested for its antimicrobial activity and have positive effects against Bacillus sp., Erwinia sp., Candida sp. Priming seeds of Triticum vulgare and Phaseolus vulgaris with polysaccharides solutions (3 and 5 mg mL-1) resulted in significant enhancement of seedling growth. Increased root length, leaf area, shoot length, photosynthetic pigments, protein content, carbohydrate content, fresh and dry biomass were observed, in addition these growth increments may be attributed to the increase of antioxidant activities.

Ionic liquid-organic-functionalized ordered mesoporous silica-integrated dispersive solid-phase extraction for determination of plant growth regulators in fresh Panax ginseng.

The massive accumulation of plant growth regulators (PGRs) in Panax ginseng causes serious harm to human health. A new analytical method for the simultaneous determination of multiple PGRs in 19 types of fresh Panax ginseng is developed by a new designed wool cluster-inspired ionic liquid-functionalized ordered mesoporous silica-integrated dispersive solid-phase extraction coupled to high performance liquid chromatography (IL-WFOMS-I-DSPE-HPLC). The proposed method combines the advantages of the multiple adsorption mechanisms, high mass transfer rate and large adsorption capacity of the synthesized IL-WFOMS adsorbent with the safe, convenient operation of the new designed I-DSPE method. Under optimized conditions, the recoveries at three spike levels were in a range of 77.6-98.3% for 3-indole acetic acid (IAA), 3-indole propionic acid (IPA), 3-indole butyric acid (IBA), and 1-naphthaleneacetic acid (NAA) with the relative standard deviations (RSD) ≤8.6%, n = 3. This method exhibits the advantages of safety, convenience, reliability, and has great potential for simultaneous determination of multiple trace PGRs in complex sample matrices.

Newly designed molecularly imprinted 3-aminophenol-glyoxal-urea resin as hydrophilic solid-phase extraction sorbent for specific simultaneous determination of three plant growth regulators in green bell peppers.

A method based on solid-phase extraction (SPE) coupled with high-performance liquid chromatography (HPLC) was developed to determine the content of 4-chlorophenoxy acetic acid (PCPA), naphthylacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) in green bell peppers. A molecularly imprinted 3-aminophenol-glyoxal-urea resin (MIAGUR) was first synthesized as a specific SPE adsorbent, which exhibited special selectivity and multiple adsorption interactions including hydrogen bonding and π-π interactions. The quantities of PCPA, NAA and 2,4-D in green bell peppers from 20 samples scattered throughout Baoding city (Hebei province, China) were determined using the proposed method. The results suggested that the use of PCPA, NAA and 2,4-D in green bell peppers from Baoding's markets is almost in fair. Moreover, MIAGUR-SPE-HPLC is suitable for the specific simultaneous monitoring of PCPA, NAA, and 2,4-D in green bell peppers and has potential applications in determination of PCPA, NAA, and 2,4-D of other plant products.

Simultaneous determination of multiclass plant growth regulators in fruits using the quick, easy, cheap, effective, rugged, and safe method and ultra-high performance liquid chromatography-tandem mass spectrometry.

In this study, a modified quick, easy, cheap, effective, rugged, and safe method combined with ultra-high performance liquid chromatography and tandem mass spectrometry was developed for the multiclass determination of 28 plant growth regulators in various fruits. Different extraction solvents and adsorbents, including primary secondary amine, octadecylsilyl, graphitized carbon black, and zirconia-based sorbent, were investigated. Internal calibration and isotope internal standards, chlormequat chloride-d4 , mepiquat chloride-d6 , indole-3-acetic acid-d2 , and forchlorfenuron-d5 were used to improve accuracy. For method validation, good linearity, low limits of detection and quantification were obtained. At three spiked concentrations (10, 50, and 100 µg/kg), satisfactory recoveries with relative standard deviations of 2.4-17.5% were obtained for strawberries (75.2-119.8%), grapes (70.5-114.0%), tangerines (71.7-115.4%), apples (72.7-115.4%), and kiwi fruits (71.7-119.2%). Samples analysis revealed that 15.6% of the samples (n = 96) were contaminated with one or two kinds of plant growth regulators, including chlormequat chloride, forchlorfenuron, paclobutrazol, 2,4-dichlorophenoxyacetic acid, 2-diethylaminoethyl hexanoate, and mepiquat chloride. Similar results were obtained by ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry, indicating the robustness, effectiveness, and suitability of the developed method for routine monitoring of plant growth regulator residues in fruits.

Determination of Major Phytohormones in Fourteen Different Seaweeds Utilizing SPE-LC-MS/MS.

Analysis of plant growth regulators (PGRs) should be approached by considering their extremely low concentrations and serious interfering effects that result from the matrix of various plant tissues. In the current research, the separation and simultaneous determination of different classes of phytohormones in 14 seaweeds collected from Turkey seashores were achieved by using solid-phase extraction (SPE) followed by a rapid and sensitive liquid chromatography tandem mass detection method. OASIS HLB (Hydrophilic-Lipophilic Balance) cartridges were successfully used for SPE process to eliminate the matrix effect and enhance the PGRs including zeatin, benzyl amino purine, indole-3-acetic acid, abscisic acid and gibberellic acid within partially different polarities. Based on the optimized experimental conditions, the method presented excellent performance related to linearity (r, 0.9996-0.9999) within the ranges of 0.5-500 ng/mL, relative standard deviation values ((1.43-2.01) for intraday and (2.36-3.50) for interday)), the limit of detection (0.01-0.84 µg/L) and the limit of quantification (0.02-2.76 µg/L). The obtained results confirm that the SPE-liquid chromatography/tandem mass spectrometry method performed is highly effective and convenient for routine analyses of trace amounts of the tested phytohormones in seaweeds and any other plant samples as well.

Simultaneous extraction of four plant growth regulators residues in vegetable samples using solid phase extraction based on thiol-functionalized nanofibers mat.

A simple and environmentally friendly sample pretreatment method was established for extraction of four plant growth regulators (PGRs) residues in vegetable samples. In order to effectively extract targets from complex sample matrices, three types of polar groups (COOH, NH2, or SH) functionalized polyacrylonitrile nanofibers mats (PAN NFsM) were prepared and evaluated, respectively. The superior -SH functionalized PAN NFsM was further selected as a novel adsorbent of solid phase extraction (SPE). Under optimal conditions, the proposed method could achieve high sensitivity (0.2-2 ng g-1) and acceptable recovery (88.94%-106.42%) by consuming lower amount of adsorbent (only 5 mg) and organic reagent (only 1.4 mL), and performing much fewer steps than current methods. In addition, the prepared SH-PAN NFsM possessed excellent reusability which could be reused for 6 times without degradation in adsorption capability. These results presented the great potential of the established method for environmental-friendly and efficiently detecting PGRs residues.

Comparative Physiological Analysis of Methyl Jasmonate in the Delay of Postharvest Physiological Deterioration and Cell Oxidative Damage in Cassava.

The short postharvest life of cassava is mainly due to its rapid postharvest physiological deterioration (PPD) and cell oxidative damage, however, how to effectively control this remains elusive. In this study, South China 5 cassava slices were sprayed with water and methyl jasmonate (MeJA) to study the effects of MeJA on reactive oxygen species, antioxidant enzymes, quality, endogenous hormone levels, and melatonin biosynthesis genes. We found that exogenous MeJA could delay the deterioration rate for at least 36 h and alleviate cell oxidative damage through activation of superoxide dismutase, catalase, and peroxidase. Moreover, MeJA increased the concentrations of melatonin and gibberellin during PPD, which had a significant effect on regulating PPD. Notably, exogenous MeJA had a significant effect on maintaining cassava quality, as evidenced by increased ascorbic acid content and carotenoid content. Taken together, MeJA treatment is an effective and promising way to maintain a long postharvest life, alleviate cell oxidative damage, and regulate storage quality in cassava.

Labdane-Type Diterpenoids from Leonurus japonicus and Their Plant-Growth Regulatory Activity.

Thirteen new labdane-type diterpenoids 1-6, 9-11, 13, 14, 18, and 19 and seven known ones were isolated from the aerial parts of Leonurus japonicus. Compounds 1-5 represent rare examples of labdane-type diterpenoids, of which compounds 1-4 carry an N-chain linked at C-7 in their B-ring and compound 5 featured an α,ß-unsaturated-γ-lactam moiety. The structures and absolute configurations of these new diterpenoids were characterized by a combination of spectroscopic techniques, X-ray crystallography, electronic circular dichroism, and calculated specific rotations. The plant-growth regulatory activity of these compounds on the growth of the roots and shoots of Lactuca sativa and Lolium perenne seedlings were evaluated. Compound 3 showed a broad-spectrum inhibitory activity with the inhibition rates ranging from 60 to 83.5% at a concentration of 200 µg/mL, which were as active as those of glyphosate. Compound 8 had a selective inhibitory activity against the growth of the roots of L. perenne seedlings with an inhibition rate of 81.7%. However, compounds 11 and 16 exhibited significant stimulation effects on the roots of L. sativa with stimulation rates of 59.8 and 65.3%, respectively. In addition, compounds 3 and 8 exhibited inhibitory effects on the germination of L. perenne seeds.

Isolation, characterization and the effect of indigenous heavy metal-resistant plant growth-promoting bacteria on sorghum grown in acid mine drainage polluted soils.

The research purpose was the characterization of indigenous heavy metal-resistant plant growth-promoting bacteria (PGPB) from the farmlands located on the Le'an River basin contaminated by acid mine drainage and their effects on plant growth, nutrient uptake, antioxidant enzyme activities and metal accumulation. The plant growth-promoting (PGP) traits, including 1-aminocyclopropane-1-carboxylic acid deaminase, indoleacetic acid, siderophore, ammonia production and phosphate solubilization, as well as antibiotics, acid/alkali and salt resistance were determined. Ten isolates with relatively high PGP activities were identified to belong to the genera Burkholderia, Paraburkholderia, Cupriavidus, Pseudomonas and Ralstonia. The numerical classification based on bacterial resistant characteristics was mostly consistent with their phylogenetic positions. Burkholderia sp. strain S6-1 and Pseudomonas sp. strain S2-3 possessed both greater PGP activities and resistant characteristics in overall comparison. Compared with non-inoculated plants, strains S6-1 and S2-3 significantly increased the height, dry weight and N uptake of sorghum (Sorghum bicolor L.). The presence of S6-1 significantly increased Pb accumulation and enhanced the translocation of Zn from root to shoot in sorghum. Strain S2-3 helped sorghum to uptake Cu and Zn and improved the remediation effect of sorghum on Cu and Zn. Overall, indigenous PGPB did not show better advantages in improving plant growth than non-indigenous P. putida UW4. Nevertheless, indigenous PGPB can be used as better candidates in heavy metal phytoremediation to minimize the potential risks of introducing invasive microbial species into an agricultural ecosystem.