The screening method for 39 phytotoxins and mycotoxins in blood and urine with liquid chromatography-high resolution mass spectrometry.

BACKGROUND: Poisonings caused by plant toxins and mycotoxins occur frequently, which do great harm to human health and social public health safety. When a poisoning incident occurs, biological samples are commonly be used to conduct the detection of toxic substances and their metabolites for targeted clinical treatment and incident analysis. OBJECTIVE: To establish an efficient and accurate analysis method of 39 phytotoxins and mycotoxins in blood and urine by high performance liquid chromatography quadrupole tandem orbitrap mass spectrometry (HPLC-Orbitrap MS). METHOD: After 3 mL of methanol being added to 1 mL blood and urine respectively for extraction and protein precipitation, the supernatant was injected into HPLC-Orbitrap MS for analysis. The phytotoxins and mycotoxins were separated by Hypersil GOLD PFP column with gradient elution using methanol-5 mmol/L ammonium acetate as mobile phase. The data were collected in ESI positive ion mode using Full MS/dd-MS2 for mass spectrometry detection. RESULT: The mass database of 39 phytotoxins and mycotoxins was developed, and accurate qualitative analysis can be obtained by matching with the database using the proposed identification criteria. Limit of detections (LODs) were 1.34 × 10-4 âˆ¼ 1.92 ng/mL and 1.92 × 10-4 âˆ¼ 9.80 ng/mL for blood and urine samples, respectively. Limits of quantification (LOQ) of toxins in blood and urine ranged from 4.47 × 10-4 âˆ¼ 6.32 ng/mL and 6.39 × 10-4 âˆ¼ 32.67 ng/mL, respectively. Intra-day relative standard deviations (RSDs) were 0.79 % âˆ¼ 10.90 %, and inter-day RSDs were 1.08 % âˆ¼ 18.93 %. The recoveries can reach 90 % âˆ¼ 110 % with matrix matching calibration curves. CONCLUSION: The established method is simple and rapid to operate, which can complete the sample analysis within 30 min, providing technical support for clinical poisoning treatment and public health poisoning analysis.

Genetic dissection of the tissue-specific roles of type III effectors and phytotoxins in the pathogenicity of Pseudomonas syringae pv. syringae to cherry.

When compared with other phylogroups (PGs) of the Pseudomonas syringae species complex, P. syringae pv. syringae (Pss) strains within PG2 have a reduced repertoire of type III effectors (T3Es) but produce several phytotoxins. Effectors within the cherry pathogen Pss 9644 were grouped based on their frequency in strains from Prunus as the conserved effector locus (CEL) common to most P. syringae pathogens; a core of effectors common to PG2; a set of PRUNUS effectors common to cherry pathogens; and a FLEXIBLE set of T3Es. Pss 9644 also contains gene clusters for biosynthesis of toxins syringomycin, syringopeptin and syringolin A. After confirmation of virulence gene expression, mutants with a sequential series of T3E and toxin deletions were pathogenicity tested on wood, leaves and fruits of sweet cherry (Prunus avium) and leaves of ornamental cherry (Prunus incisa). The toxins had a key role in disease development in fruits but were less important in leaves and wood. An effectorless mutant retained some pathogenicity to fruit but not wood or leaves. Striking redundancy was observed amongst effector groups. The CEL effectors have important roles during the early stages of leaf infection and possibly acted synergistically with toxins in all tissues. Deletion of separate groups of T3Es had more effect in P. incisa than in P. avium. Mixed inocula were used to complement the toxin mutations in trans and indicated that strain mixtures may be important in the field. Our results highlight the niche-specific role of toxins in P. avium tissues and the complexity of effector redundancy in the pathogen Pss 9644.

Regulation of virulence mechanisms in plant-pathogenic Streptomyces.

Streptomyces have a uniquely complex developmental life cycle that involves the coordination of morphological differentiation with the production of numerous bioactive specialized metabolites. The majority of Streptomyces spp. are soil-dwelling saprophytes, while plant pathogenicity is a rare attribute among members of this genus. Phytopathogenic Streptomyces are responsible for economically important diseases such as common scab, which affects potato and other root crops. Following the acquisition of genes encoding virulence factors, Streptomyces pathogens are expected to have specifically adapted their regulatory pathways to enable transition from a primarily saprophytic to a pathogenic lifestyle. Investigations of the regulation of pathogenesis have primarily focused on Streptomyces scabiei and the principal pathogenicity determinant thaxtomin A. The coordination of growth and thaxtomin A production in this species is controlled in a hierarchical manner by cluster-situated regulators, pleiotropic regulators, signalling and plant-derived molecules, and nutrients. Although the majority of phytopathogenic Streptomyces produce thaxtomins, many also produce additional virulence factors, and there are scab-causing pathogens that do not produce thaxtomins. The development of effective control strategies for common scab and other Streptomyces plant diseases requires a more in-depth understanding of the genetic and environmental factors that modulate the plant pathogenic lifestyle of these organisms.

Comparative Analysis of Secondary Metabolites Produced by Ascochyta fabae under In Vitro Conditions and Their Phytotoxicity on the Primary Host, Vicia faba, and Related Legume Crops.

Ascochyta blight, caused by Ascochyta fabae, poses a significant threat to faba bean and other legumes worldwide. Necrotic lesions on stems, leaves, and pods characterize the disease. Given the economic impact of this pathogen and the potential involvement of secondary metabolites in symptom development, a study was conducted to investigate the fungus's ability to produce bioactive metabolites that might contribute to its pathogenicity. For this investigation, the fungus was cultured in three substrates (Czapek-Dox, PDB, and rice). The produced metabolites were analyzed by NMR and LC-HRMS methods, resulting in the dereplication of seven metabolites, which varied with the cultural substrates. Ascochlorin, ascofuranol, and (R)-mevalonolactone were isolated from the Czapek-Dox extract; ascosalipyrone, benzoic acid, and tyrosol from the PDB extract; and ascosalitoxin and ascosalipyrone from the rice extract. The phytotoxicity of the pure metabolites was assessed at different concentrations on their primary hosts and related legumes. The fungal exudates displayed varying degrees of phytotoxicity, with the Czapek-Dox medium's exudate exhibiting the highest activity across almost all legumes tested. The species belonging to the genus Vicia spp. were the most susceptible, with faba bean being susceptible to all metabolites, at least at the highest concentration tested, as expected. In particular, ascosalitoxin and benzoic acid were the most phytotoxic in the tested condition and, as a consequence, expected to play an important role on necrosis's appearance.

Determination of ptaquiloside in ten species of Pteris in northwestern Argentina.

The toxic effect of ferns of the genus of Pteris in bovines is caused by ptaquiloside, the main carcinogenic toxin. In this study, ten species of Pteris fern in different phenologic stages and plant conditions were collected in northwest Argentina. The phytochemical analysis showed the presence of Pt in the recent collected samples (adults and young plants) but not in the herbarium specimens. The results show a great variation of Pt concentration that depends on the phenologic stage, plant condition, and collection site. Pt was measured in 6-4326 µg/g concentration, with a mean concentration of 644 µg/g. No Pt was detected in eight species of Pteris collected from herbarium samples; such results may be a false negative. It is important to notice that analysis of herbarium samples for Pt may not be a reliable method to determine its presence. It is important to further understand the potential toxicity caused by these ferns because of their effect on animals, public health, and the environment.

Genetic rearrangements in Pseudomonas amygdali pathovar aesculi shape coronatine plasmids.

Plant pathogenic Pseudomonas species use multiple classes of toxins and virulence factors during host infection. The genes encoding these pathogenicity factors are often located on plasmids and other mobile genetic elements, suggesting that they are acquired through horizontal gene transfer to confer an evolutionary advantage for successful adaptation to host infection. However, the genetic rearrangements that have led to mobilization of the pathogenicity genes are not fully understood. In this study, we have sequenced and analyzed the complete genome sequences of four Pseudomonas amygdali pv. aesculi (Pae), which infect European horse chestnut trees (Aesculus hippocastanum) and belong to phylogroup 3 of the P. syringae species complex. The four investigated genomes contain six groups of plasmids that all encode pathogenicity factors. Effector genes were found to be mostly associated with insertion sequence elements, suggesting that virulence genes are generally mobilized and potentially undergo horizontal gene transfer after transfer to a conjugative plasmid. We show that the biosynthetic gene cluster encoding the phytotoxin coronatine was recently transferred from a chromosomal location to a mobilizable plasmid that subsequently formed a co-integrate with a conjugative plasmid.

Uncovering Phytotoxic Compounds Produced by Colletotrichum spp. Involved in Legume Diseases Using an OSMAC-Metabolomics Approach.

Different fungal species belonging to the Colletotrichum genus cause anthracnose disease in a range of major crops, resulting in huge economic losses worldwide. Typical symptoms include dark, sunken lesions on leaves, stems, or fruits. Colletotrichum spp. have synthesized, in vitro, a number of biologically active and structurally unusual metabolites that are involved in their host's infection process. In this study, we applied a one strain many compounds (OSMAC) approach, integrated with targeted and non-targeted metabolomics profiling, to shed light on the secondary phytotoxic metabolite panels produced by pathogenic isolates of Colletotrichum truncatum and Colletotrichum trifolii. The phytotoxicity of the fungal crude extracts was also assessed on their primary hosts and related legumes, and the results correlated with the metabolite profile that arose from the different cultural conditions. To the best of our knowledge, this is the first time that the OSMAC strategy integrated with metabolomics approaches has been applied to Colletotrichum species involved in legume diseases.

(±)-3-Deoxyradicinin Induces Stomata Opening and Chloroplast Oxidative Stress in Tomato (Solanum lycopersicum L.).

Radicinin is a phytotoxic dihydropyranopyran-4,5-dione isolated from the culture filtrates of Cochliobolus australiensis, a phytopathogenic fungus of the invasive weed buffelgrass (Cenchrus ciliaris). Radicinin proved to have interesting potential as a natural herbicide. Being interested in elucidating the mechanism of action and considering radicinin is produced in small quantities by C. australiensis, we opted to use (±)-3-deoxyradicinin, a synthetic analogue of radicinin that is available in larger quantities and shows radicinin-like phytotoxic activities. To obtain information about subcellular targets and mechanism(s) of action of the toxin, the study was carried out by using tomato (Solanum lycopersicum L.), which, apart from its economic relevance, has become a model plant species for physiological and molecular studies. Results of biochemical assays showed that (±)-3-deoxyradicinin administration to leaves induced chlorosis, ion leakage, hydrogen peroxide production, and membrane lipid peroxidation. Remarkably, the compound determined the uncontrolled opening of stomata, which, in turn, resulted in plant wilting. Confocal microscopy analysis of protoplasts treated with (±)-3-deoxyradicinin ascertained that the toxin targeted chloroplasts, eliciting an overproduction of reactive singlet oxygen species. This oxidative stress status was related by qRT-PCR experiments to the activation of transcription of genes of a chloroplast-specific pathway of programmed cell death.

Specialized Metabolites Produced by Phytotopatogen Fungi to Control Weeds and Parasite Plants.

Weeds such as parasite plants are one of the most serious pests that farmers are forced to combat since the development of agriculture using different methods including mechanic and agronomy strategies. These pests have generated significant losses of agrarian and herding production, constituting a serious impediment for agricultural activities in reforestation practices and in important infrastructures. All these serious problems have induced the expansive and massive use of synthetic herbicides, which represents one of the main cause of environmental pollution, as well as serious risks for human and animal health. An alternative environmental friendly control method could be the use of bioherbicides based on suitably bioformulated natural products, of which the main ones are fungal phytotoxins. This review covers the literature from 1980 to the present (2022) and concerns fungal phytotoxins with potential herbicidal activity in order to obtain their efficacy as bioherbicides for practical application in agriculture. Furthermore, some bioherbicides based on microbial toxic metabolites are commercially available, and their application in field, mode of action and future perspectives are also discussed.

The barrier to radial oxygen loss protects roots against hydrogen sulphide intrusion and its toxic effect.

The root barrier to radial O2 loss (ROL) is a key root trait preventing O2 loss from roots to anoxic soils, thereby enabling root growth into anoxic, flooded soils. We hypothesized that the ROL barrier can also prevent intrusion of hydrogen sulphide (H2 S), a potent phytotoxin in flooded soils. Using H2 S- and O2 -sensitive microsensors, we measured the apparent permeance to H2 S of rice roots, tested whether restricted H2 S intrusion reduced its adverse effects on root respiration, and whether H2 S could induce the formation of a ROL barrier. The ROL barrier reduced apparent permeance to H2 S by almost 99%, greatly restricting H2 S intrusion. The ROL barrier acted as a shield towards H2 S; O2 consumption in roots with a ROL barrier remained unaffected at high H2 S concentration (500 µM), compared to a 67% decline in roots without a barrier. Importantly, low H2 S concentrations induced the formation of a ROL barrier. In conclusion, the ROL barrier plays a key role in protecting against H2 S intrusion, and H2 S can act as an environmental signalling molecule for the induction of the barrier. This study demonstrates the multiple functions of the suberized/lignified outer part of the rice root beyond that of restricting ROL.

Metabolite Formation by Fungal Pathogens of Potatoes (Solanum tuberosum L.) in the Presence of Bioprotective Agents.

The potato is a crop of global importance for the food industry. This is why effective protection against pathogens is so important. Fungi as potato pathogens are responsible for plant diseases and a significant reduction in yields, as well as for the formation of mycotoxins. This study focuses on the effect of three natural biocides, yeast Metschnikowia pulcherrima, lactic acid bacteria Lactiplantibacillus plantarum, and aqueous garlic extract, on the improvement of the physiology of planted potato tubers and the reduction in mycotoxin formation. The secondary metabolites produced by the fungal pathogens of genera Fusarium, Alternaria, Colletotrichum, Rhizoctonia, and Phoma in the presence of these biocontrol agents were compared to profiles obtained from contaminated potatoes. Analysis of liquid chromatography coupled with tandem mass spectrometry data showed the presence of 68 secondary metabolites, including the mycotoxins: alternariol, alternariol methyl ether, altertoxin-I, aurofusarin, beauvericin, diacetoxyscirpenol, enniatin B, and sterigmatocystin. The studies showed that the applied biocontrol agents had a positive effect on the physiological parameters of potatoes (including root growth, stem growth, gas exchange, and chlorophyll content index) and on the reduction in the production of mycotoxins and other secondary metabolites by Fusarium, Alternaria, and Phoma.

Status of Phytotoxins Isolated from Necrotrophic Fungi Causing Diseases on Grain Legumes.

Fungal phytotoxins can be defined as secondary metabolites toxic to host plants and are believed to be involved in the symptoms developed of a number of plant diseases by targeting host cellular machineries or interfering with host immune responses. As any crop, legumes can be affected by a number of fungal diseases, causing severe yield losses worldwide. In this review, we report and discuss the isolation, chemical, and biological characterization of fungal phytotoxins produced by the most important necrotrophic fungi involved in legume diseases. Their possible role in plant-pathogen interaction and structure-toxicity relationship studies have also been reported and discussed. Moreover, multidisciplinary studies on other prominent biological activity conducted on reviewed phytotoxins are described. Finally, we explore the challenges in the identification of new fungal metabolites and their possible applications in future experiments.

Biotechnological detoxification: an unchanging source-sink balance strategy for crop improvement.

The wide occurrence of natural phytotoxins renders many crops unfit for human consumption. To overcome this problem and produce detoxified crop varieties, we propose the use of biotechnological strategies that can enhance the harvest index without the need to increase crop biomass or alter whole plant architecture.

Mutagenic potential and structural alerts of phytotoxins.

Toxic plant-produced chemicals, so-called phytotoxins, constitute a category of natural compounds belonging to a diversity of chemical classes. Some of them (e.g., alkaloids, terpenes, saponins) are associated with high toxic potency, while for many of others no toxicological data is available. In this study, the mutagenic potential of 1586 phytotoxins, as obtained from a publicly available database, was investigated applying different in silico approaches. (Q)SAR models (including statistical-based and rule-based systems) were used for the prediction of bacterial in vitro mutagenicity (Ames test) and the results from multiple tools were combined to assign consensus predicted values (i.e., positive, negative, inconclusive). The overall consensus outcome was then employed to investigate relationships between structural features of classes of phytotoxins and potential mutagenicity, allowing the identification of structural alerts raising a specific concern. The results highlighted that about 10% of the screened compounds were predicted to have mutagenic potential and the critical classes of concern, such as alkaloids, were further investigated in terms of subclasses (e.g., indole alkaloids, isoquinoline alkaloids), getting a deeper insight into the mutagenic potential of possible naturally occurring chemicals in plant materials and their structural alerts.

Activation of Ustilaginoidin Biosynthesis Gene uvpks1 in Villosiclava virens Albino Strain LN02 Influences Development, Stress Responses, and Inhibition of Rice Seed Germination.

Villosiclava virens (anamorph: Ustilaginoidea virens) is the pathogen of rice false smut (RFS), which is a destructive rice fungal disease. The albino strain LN02 is a natural white-phenotype mutant of V. virens due to its incapability to produce toxic ustilaginoidins. In this study, three strains including the normal strain P1, albino strain LN02, and complemented strain uvpks1C-1 of the LN02 strain were employed to investigate the activation of the ustilaginoidin biosynthesis gene uvpks1 in the albino strain LN02 to influence sporulation, conidia germination, pigment production, stress responses, and the inhibition of rice seed germination. The activation of the ustilaginoidin biosynthesis gene uvpks1 increased fungal tolerances to NaCl-induced osmotic stress, Congo-red-induced cell wall stress, SDS-induced cell membrane stress, and H2O2-induced oxidative stress. The activation of uvpks1 also increased sporulation, conidia germination, pigment production, and the inhibition of rice seed germination. In addition, the activation of uvpks1 was able to increase the mycelial growth of the V. virens albino strain LN02 at 23 °C and a pH from 5.5 to 7.5. The findings help in understanding the effects of the activation of uvpks1 in albino strain LN02 on development, pigment production, stress responses, and the inhibition of rice seed germination by controlling ustilaginoidin biosynthesis.

Phytotoxic Metabolites Produced by Fungi Involved in Grapevine Trunk Diseases: Progress, Challenges, and Opportunities.

Grapevine trunk diseases (GTDs), caused by fungal pathogens, are a serious threat to vineyards worldwide, causing significant yield and economic loss. To date, curative methods are not available for GTDs, and the relationship between the pathogen and symptom expression is poorly understood. Several plant pathologists, molecular biologists, and chemists have been investigating different aspects of the pathogenicity, biochemistry, and chemical ecology of the fungal species involved in GTDs. Many studies have been conducted to investigate virulence factors, including the chemical characterization of phytotoxic metabolites (PMs) that assist fungi in invading and colonizing crops such as grapevines. Moreover, multidisciplinary studies on their role in pathogenicity, symptom development, and plant-pathogen interactions have also been carried out. The aim of the present review is to provide an illustrative overview of the biological and chemical characterization of PMs produced by fungi involved in Eutypa dieback, Esca complex, and Botryosphaeria dieback. Moreover, multidisciplinary investigations on host-pathogen interactions, including those using cutting-edge Omics techniques, will also be reviewed and discussed. Finally, challenges and opportunities in the role of PMs for reliable field diagnosis and control of GTDs in vineyards will also be explored.

Synthesis and Activity of 2-Acyl-cyclohexane-1,3-dione Congeners Derived from Peperomia Natural Products against the Plant p-Hydroxyphenylpyruvate Dioxygenase Herbicidal Molecular Target Site.

Plastoquinone is a key electron carrier in photosynthesis and an essential cofactor for the biosynthesis of carotenoids. p-Hydroxyphenylpyruvate dioxygenase (HPPD) is a vital enzymatic step in plastoquinone biosynthesis that is the target of triketone herbicides, such as those derived from the pharmacophore backbone of the natural product leptospermone. In this work, the inhibitory activity of a series of 2-acyl-cyclohexane-1,3-diones congeners derived from Peperomia natural products was tested on plant HPPD. The most active compound was a 2-acyl-cyclohexane-1,3-dione with a C11 alkyl side chain (5d; I50app: 0.18 ± 0.02 µM) that was slightly more potent than the commercial triketone herbicide sulcotrione (I50app: 0.25 ± 0.02 µM). QSAR analysis and docking studies were performed to further characterize the key structural features imparting activity. A 1,3-dione feature was required for inhibition of HPPD. Molecules with a side chain of 11 carbons were found to be optimal for inhibition, while the presence of a double bond, hydroxy, or methyl beyond the required structural features on the cyclohexane ring generally decreased HPPD inhibiting activity.

In Vitro Effects of Fungal Phytotoxins on Cancer Cell Viability: First Insight into Structure Activity Relationship of a Potent Metabolite of Cochliobolus australiensis Radicinin.

Natural compounds have always represented an important source for new drugs. Although fungi represent one such viable source, to date, no fungal metabolite has been marketed as an anticancer drug. Based on our work with phytotoxins as potential chemical scaffolds and our recent findings involving three phytopathogenic fungi, i.e., Cochliobolus australiensis, Kalmusia variispora and Hymenoscyphus fraxineus, herein, we evaluate the in vitro anti-cancer activity of the metabolites of these fungi by MTT assays on three cancer cell models harboring various resistance levels to chemotherapeutic drugs. Radicinin, a phytotoxic dihydropyranopyran-4,5-dione produced by Cochliobolus australiensis, with great potential for the biocontrol of the invasive weed buffelgrass (Cenchrus ciliaris), showed significant anticancer activity in the micromolar range. Furthermore, a SAR study was carried out using radicinin, some natural analogues and hemisynthetic derivatives prepared by synthetic methods developed as part of work aimed at the potential application of these molecules as bioherbicides. This investigation opens new avenues for the design and synthesis of novel radicinin analogues as potential anticancer agents.

Documentation of Phytotoxic Compounds Existing in Parthenium hysterophorus L. Leaf and Their Phytotoxicity on Eleusine indica (L.) Gaertn. and Digitaria sanguinalis (L.) Scop.

The utilization of the invasive weed, Parthenium hysterophorus L. for producing value-added products is novel research for sustaining our environment. Therefore, the current study aims to document the phytotoxic compounds contained in the leaf of parthenium and to examine the phytotoxic effects of all those phytochemicals on the seed sprouting and growth of Crabgrass Digitaria sanguinalis (L.) Scop. and Goosegrass Eleusine indica (L.) Gaertn. The phytotoxic substances of the methanol extract of the P. hysterophorus leaf were analyzed by LC-ESI-QTOF-MS=MS. From the LC-MS study, many compounds, such as terpenoids, flavonoids, amino acids, pseudo guaianolides, and carbohydrate and phenolic acids, were identified. Among them, seven potential phytotoxic compounds (i.e., caffeic acid, vanillic acid, ferulic acid, chlorogenic acid, quinic acid, anisic acid, and parthenin) were documented, those are responsible for plant growth inhibition. The concentration needed to reach 50% growth inhibition in respect to germination (ECg50), root length (ECr50), and shoot length (ECs50) was estimated and the severity of phytotoxicity of the biochemicals was determined by the pooled values (rank value) of three inhibition parameters. The highest growth inhibition was demarcated by caffeic acid, which was confirmed and indicated by cluster analysis and principal component analysis (PCA). In the case of D. sanguinalis, the germination was reduced by 60.02%, root length was reduced by 76.49%, and shoot length was reduced by 71.14% when the chemical was applied at 800 µM concentration, but in the case of E. indica, 100% reduction of seed germination, root length, and shoot length reduction occurred at the same concentration. The lowest rank value was observed from caffeic acids in both E. indica (rank value 684.7) and D. sanguinalis (909.5) caused by parthenin. It means that caffeic acid showed the highest phytotoxicity. As a result, there is a significant chance that the parthenium weed will be used to create bioherbicides in the future.