Carboxylesterase and Cytochrome P450 Confer Metabolic Resistance Simultaneously to Azoxystrobin and Some Other Fungicides in Botrytis cinerea.

Plant pathogens have frequently shown multidrug resistance (MDR) in the field, often linked to efflux and sometimes metabolism of fungicides. To investigate the potential role of metabolic resistance in B. cinerea strains showing MDR, the azoxystrobin-sensitive strain B05.10 and -resistant strain Bc242 were treated with azoxystrobin. The degradation half-life of azoxystrobin in Bc242 (9.63 days) was shorter than that in B05.10 (28.88 days). Azoxystrobin acid, identified as a metabolite, exhibited significantly lower inhibition rates on colony and conidia (9.34 and 11.98%, respectively) than azoxystrobin. Bc242 exhibited higher expression levels of 34 cytochrome P450s (P450s) and 11 carboxylesterase genes (CarEs) compared to B05.10 according to RNA-seq analysis. The expression of P450 genes Bcin_02g01260 and Bcin_12g06380, along with the CarEs Bcin_12g06360 in Saccharomyces cerevisiae, resulted in reduced sensitivity to various fungicides, including azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin, iprodione, and carbendazim. Thus, the mechanism of B. cinerea MDR is linked to metabolism mediated by the CarE and P450 genes.

Procymidone Application Contributes to Multidrug Resistance of Botrytis cinerea.

The necrotrophic pathogen Botrytis cinerea infects a broad range of plant hosts and causes substantial economic losses to many crops. Although resistance to procymidone has been observed in the field, it remains uncertain why procymidone is usually involved in multidrug resistance (MDR) together with other fungicides. Nine mutants derived from the B. cinerea strain B05.10 through procymidone domestication exhibited high resistance factors (RFs) against both procymidone and fludioxonil. However, the fitness of the mutants was reduced compared to their parental strain, showing non-sporulation and moderate virulence. Furthermore, the RFs of these mutants to other fungicides, such as azoxystrobin, fluazinam, difenoconazole, and pyrimethanil, ranged from 10 to 151, indicating the occurrence of MDR. Transcriptive expression analysis using the quantitative polymerase chain reaction (qPCR) revealed that the mutants overexpressed ABC transporter genes, ranging from 2 to 93.7-fold. These mutants carried single-point mutations W647X, R96X, and Q751X within BcBos1 by DNA sequencing. These alterations in BcBos1 conferred resistance to procymidone and other fungicides in the mutants. Molecular docking analysis suggested distinct interactions between procymidone and Bos1 in the B. cinerea standard strain B05.10 or the resistant mutants, suggesting a higher affinity of the former towards binding with the fungicide. This study provides a comprehensive understanding of the biological characteristics of the resistant mutants and conducts an initial investigation into its fungicide resistance traits, providing a reference for understanding the causes of multidrug resistance of B. cinerea in the field.

Multidrug resistance of Botrytis cinerea associated with its adaptation to plant secondary metabolites.

Fungicides are an effective way to control gray mold of grapes, but the pathogen Botrytis cinerea can develop resistance, overcoming the effectiveness of a fungicide that is repeatedly applied. More importantly, the emergence of multidrug resistance (MDR) in the field, where multiple fungicides with different modes of action simultaneously lose their efficacies, is a significant concern. MDR is associated with ATP-binding cassette (ABC) transporters of the pathogen, and certain plant secondary metabolites (PSMs) stimulate the upregulation of ABC transporters, we hypothesized that the pathogen's preadaptation to PSMs might contribute to MDR development. To test this in B. cinerea, ten PSMs, namely, resveratrol, reserpine, chalcone, flavanone, eugenol, farnesol, anethene, camptothecin, salicylic acid, and psoralen, were selected based on their association with ABC transporters involved in fungicide resistance. B. cinerea strain B05.10 was continuously transferred for 15 generations on potato dextrose agar amended with a PSM (PDAP), and sensitivities to PSMs and fungicides were examined on the 5th, 10th, and 15th generations. RNA was extracted from B. cinerea from the selected generations. After 15 generations of culture transfers, an up-regulation was observed in the expression of ABC transporter-encoding genes BcatrB, BcatrD, and BcatrK using quantitative polymerase chain reaction (qPCR). This upregulation was found to contribute to MDR of B. cinerea against two or more fungicides, among azoxystrobin, boscalid, fludioxonil, difenoconazole, prochloraz, and pyrimethanil. This finding was confirmed through genetic transformation. The decreased sensitivity of B. cinerea to fungicides was confirmed as a subsequent MDR phenotype after exposure to camptothecin, flavanone, and resveratrol. Besides, transcriptome analysis also revealed the upregulation of transcription factors related to ABC expression following resveratrol exposure. This suggests that PSMs contributed to inducing preadaptation of B. cinerea, leading to subsequent MDR.IMPORTANCEThe emergence of MDR in plant pathogens is a threat to plant disease management and leads to the use of excessive fungicides. Botrytis cinerea is of particular concern because its MDR has widely emerged in the field. Understanding its genesis is the first step for controlling MDR. In this study, the contribution of PSMs to MDR has been examined. Effective management of this pathogen in agroecosystems relies on a better understanding of how it copes with phytochemicals or fungicides.

Fabrication of ROS-responsive nanoparticles by modifying the interior pore-wall of mesoporous silica for smart delivery of azoxystrobin.

The suboptimal efficiency in pesticide utilization may elevate residues, posing safety risks to human food and non-target organisms. To address this challenge, delivery systems, such as pathogen infection stimuli-responsive carriers, can be employed to augment the efficiency of fungicide utilization. The bursting of reactive oxygen species (ROS) is a common defense response of host plants to pathogenic infections. In this study, ROS-responsive mesoporous silica nanoparticles (MSN) modified with phenyl sulfide (PHS) as azoxystrobin (AZOX) carrier (MSN-PHS-AZOX) were fabricated. Results demonstrated that MSN-PHS-AZOX exhibited fungicide release kinetics dependent on ROS. In vitro inhibition experiments confirmed the fungicidal effect of MSN-PHS-AZOX on Botrytis cinerea, relying on external ROS. In vivo leaf experiments showcased the superior persistence of MSN-PHS-AZOX in compared to AZOX SC. Furthermore, MSN-PHS-AZOX exhibits favorable biosafety and lower toxicity to aquatic zebrafish compared to AZOX SC, with no adverse impact on cucumber leaf growth. These findings suggest the potential application of this ROS-responsive nano fungicide in managing plant disease in agricultural fields.

Multidrug resistance of Rhizoctonia solani determined by enhanced efflux for fungicides.

Plant pathogens can develop multidrug resistance (MDR) through metabolomic and efflux activities. Although MDR has been observed in the field, its mechanisms are yet to be further studied. MDR in Rhizoctonia solani induced by the uncoupler SYP-14288, which involved efflux transporters including ATP binding cassette (ABC) and major facilitator superfamily (MFS) have been reported in our previous study. To confirm this, corresponding genes of the wild-type R. solani X19 and its derived MDR mutant X19-7 were compared through transcriptomics, RNA-Seq data validation, and heterologous expression. Genes encoding six ABC transporters and seven MFS transporters were identified to be associated with MDR and mostly showed a constitutively higher expression in X19-7 than in X19 regardless of SYP-14288 treatment. Eight ABC transporter-encoding genes and eight MFS transporter-encoding genes were further characterized by transferring into Saccharomyces cerevisiae. The sensitivity of transformants containing either ABC transporter-encoding gene AG1IA_06082 and MFS transporter-encoding gene AG1IA_08645 was significantly decreased in responses to fungicides having various modes of action including SYP-14288, fluazinam, chlorothalonil, and difenoconazole, indicating that these two genes were related to MDR. The roles of two genes were further confirmed by successfully detecting their protein products and high accumulation of SYP-14288 in yeast transformants. Thus, ABC and MFS transporters contributed to the development of MDR in R. solani. The result helps to understand the cause and mechanisms that influence the efficient use of fungicide.

Dynamic Changes in Plant Secondary Metabolites Induced by Botrytis cinerea Infection.

In response to pathogen infection, some plants increase production of secondary metabolites, which not only enhance plant defense but also induce fungicide resistance, especially multidrug resistance (MDR) in the pathogen through preadaptation. To investigate the cause of MDR in Botrytis cinerea, grapes 'Victoria' (susceptible to B. cinerea) and 'Shine Muscat' (resistant to B. cinerea) were inoculated into seedling leaves with B. cinerea, followed by extraction of metabolites from the leaves on days 3, 6, and 9 after inoculation. The extract was analyzed using gas chromatography/quadrupole time-of-flight mass (GC/QTOF) combined with solid-phase microextraction (SPME) for volatile and nonvolatile metabolomic components. Nonvolatile metabolites γ-aminobutyric acid (GABA), resveratrol, piceid, and some carbohydrates or amino acids, coupled with volatile metabolites ß-ocimene, α-farnesene, caryophyllene, germacrene D, ß-copaene, and alkanes, accumulated at a higher level in grape leaves infected with B. cinerea compared to in noninoculated leaves. Among the established metabolic pathways, seven had greater impacts, including aminoacyl-tRNA biosynthesis, galactose metabolism, valine, leucine, and isoleucine biosynthesis. Furthermore, isoquinoline alkaloid biosynthesis; phenylpropanoid biosynthesis; monobactam biosynthesis; tropane, piperidine, and pyridine alkaloid biosynthesis; phenylalanine metabolism; and glucosinolate biosynthesis were related to antifungal activities. Based on liquid chromatography/quadrupole time-of-flight mass (LC/QTOF) detection and bioassay, B. cinerea infection induced production of plant secondary metabolites (PSMs) including eugenol, flavanone, reserpine, resveratrol, and salicylic acid, which all have inhibitory activity against B. cinerea. These compounds also promoted overexpression of ATP-binding cassette (ABC) transporter genes, which are involved in induction of MDR in B. cinerea.

Functional validation of ZbFAD2 and ZbFAD3 in the alkylamide biosynthesis pathway from Zanthoxylum bungeanum Maxim.

The spicy taste and medicinal properties of Zanthoxylum bungeanum are imparted by several alkylamides. Although most studies have focused on their isolation and identification, few have reported their biosynthesis pathways. Among the differentially expressed genes (DEGs) reported in the numerous varieties of Z. bungeanum, some might contribute to alkylamide biosynthesis. However, they are not yet functionally validated. The present study explored the function of two genes, ZbFAD2 and ZbFAD3, in the alkylamide biosynthesis pathway, and their stable and transient expression in Arabidopsis thaliana and Nicotiana benthamiana were also analyzed. As compared with the wild-type (WT), the fatty acid content analysis indicated that ZbFAD2-A. thaliana transgenic seeds had lower oleic acid and higher linoleic acid contents, while the ZbFAD3-A. thaliana transgenic seeds showed lower linoleic acid and higher α-linolenic acid levels. Moreover, hydroxy-α-sanshool, a major alkylamide, was considerably higher in the ZbFAD2-N. benthamiana transgenic plants (0.2167 ± 0.0026 mg/g) than in the WT (0.0875 ± 0.0049 mg/g), while it was lower in the ZbFAD3-N. benthamiana transgenic plants (0.0535 ± 0.0037 mg/g). These results suggest that both ZbFAD2 and ZbFAD3 are vital alkylamide biosynthesis enzymes in Z. bungeanum. Our study not only helps to scale up the alkylamide production, but also establishes the role of the uncharacterized genes.

Effects of Low Temperature Stress on Source-Sink Organs in Wheat and Phosphorus Mitigation Strategies.

The 21st century presents many challenges to mankind, including climate change, fast growing human population, and serious concerns over food security. Wheat is a leading cereal crop that largely fulfills the global food needs. Low temperature stress accompanied by nutrient-starved soils is badly disrupting the source-sink relationship of wheat, thus causing an acute decline in final yield and deteriorating the grain quality. This review paper aimed to understand how low temperature stress affects wheat source-sink organs (i.e., leaves, roots, and spikes) and how phosphorus application reliefs in alleviating its harmful consequences. Also, we discussed mitigation strategies to enhance wheat capacity to adapt to varying temperature extremes and made rational recommendations based on modern agronomic and breeding approaches. Therefore, this study is likely to establish a solid foundation for improving the tolerance to low temperature stress and to improve its phosphorus utilization efficiency in wheat.

Metabolic Mechanism of Plant Defense against Rice Blast Induced by Probenazole.

The probenazole fungicide is used for controlling rice blast (Magnaporthe grisea) primarily by inducing disease resistance of the plant. To investigate the mechanism of induced plant defense, rice seedlings were treated with probenazole at 15 days post emergence, and non-treated plants were used for the control. The plants were infected with M. grisea 5 days after chemical treatment and incubated in a greenhouse. After 7 days, rice seedlings were sampled. The metabolome of rice seedlings was chemically extracted and analyzed using gas chromatography and mass spectrum (GC-MS). The GC-MS data were processed using analysis of variance (ANOVA), principal component analysis (PCA) and metabolic pathway elucidation. Results showed that probenazole application significantly affected the metabolic profile of rice seedlings, and the effect was proportionally leveraged with the increase of probenazole concentration. Probenazole resulted in a change of 54 metabolites. Salicylic acid, γ-aminobutyrate, shikimate and several other primary metabolites related to plant resistance were significantly up-regulated and some metabolites such as phenylalanine, valine and proline were down-regulated in probenazole-treated seedlings. These results revealed a metabolic pathway of rice seedlings induced by probenazole treatment regarding the resistance to M. grisea infection.

Tracking pesticide exposure to operating workers for risk assessment in seed coating with tebuconazole and carbofuran.

BACKGROUND: Coating seed with pesticides is an effective way to control plant pests, however, factory-based coating processes may carry a potential risk to operational workers of chemical exposure. To study the risk, carbofuran and tebuconazole were used to coat corn seed and their subsequent distribution on the bodies of workers was measured at manufacturers XFS and LS (Shanxi, China). Clothing was collected from workers during operations and analyzed using high-performance liquid chromatography. RESULTS: At XFS, dermal exposure to carbofuran was 4.83, 3.31 and 1.48 mg kg-1 , and exposure to tebuconazole was 6.88, 5.16 and 1.72 mg kg-1 for coating, packing and transport workers, respectively. At LS, dermal exposure to carbofuran was 2.32, 0.46 and 0.55 mg kg-1 , and exposure to tebuconazole was 1.69, 0.46 and 0.70 mg kg-1 , for coating, packing and transport workers, respectively. The level of pesticide exposure was significantly higher for seed-coating workers than for packing and transport workers. The main area of exposure was the hands for all workers and the lower limbs for packers; exposure was relatively uniform for pesticide handlers. Occupational risk was assessed based on margin of exposure (MOE). In seed-coating, the MOE was greater than 100 for tebuconazole, indicating no potential risk, but ranged from 0.25 to 2.88 for carbofuran, indicating the risk of a health impact. CONCLUSION: The level of exposure varied depending on type of operation undertaken and body parts of workers' body, but the risk of a health impact was highly associated with pesticide toxicity. This provides a guideline for workers in pesticide manufacturing to ensure safe operation of the seed-coating process. © 2021 Society of Chemical Industry.

Comparative Transcriptome Analysis and Expression of Genes Reveal the Biosynthesis and Accumulation Patterns of Key Flavonoids in Different Varieties of Zanthoxylum bungeanum Leaves.

Zanthoxylum bungeanum (Rutaceae), a popular food flavoring and traditional Chinese medicine ingredient, is an important cash crop. Its leaves are rich in flavonoids with multiple bioactivities. However, the transcriptional sequencing has not been investigated, and the molecular basis for the flavonoid biosynthesis remains unclear in this plant. This paper, the key flavonoids (epicatechin, rutin, hyperoside, trifolin, quercitrin, and afzelin) contents were determined in the leaves of 10 Z. bungeanum varieties from a common garden. Results show the leaves of Z. bungeanum mainly contained hyperoside (11.410-21.721 mg/g) and quercitrin (9.401-18.016 mg/g). The total content of these key components was the highest in Fengxian Dahongpao (66.012 mg/g) and the lowest in Fugu (32.223 mg/g). Three varieties (Hancheng stingless, Fugu, and Fengxian Dahongpao) with significant differences in the total content of key flavonoids were selected for transcriptome analysis to obtain flavonoid biosynthesis-related genes. In total, 83 522 unigenes were obtained, 40 668 (48.69%) unigenes were annotated, and 6656 differentially expressed genes (DEGs) were identified. Comparison of the other two varieties, Fugu had many differentially expressed genes indicating the particularity of its variety. Flavonoid-related DEGs of 22 structural genes, including three PALs, one CYP73A, three 4CLs, six CHSs, one CHI, one F3H, one DFR, two ANSs, one ANR, one FLS, and two CYP75B1s, as well as nine MYBs were obtained. These structural genes had different expression patterns in different Z. bungeanum varieties. It is worth noting that the genes expressing the flavonoid 3'5' hydroxylase are absent in Z. bungeanum. Furthermore, quantitative real-time PCR experiment showed consistent results in transcriptome analysis. The RNA-Seq data set of this study sheds lights on the molecular mechanism of flavonoid biosynthesis in Z. bungeanum, provides valuable information for the metabolic regulation of flavonoids, and may serve as a guide for future breeding programs.

Simultaneous Enrichment and Separation of Four Flavonoids from Zanthoxylum bungeanum Leaves by Ultrasound-Assisted Extraction and Macroporous Resins with Evaluation of Antioxidant Activities.

Quercitrin, hyperoside, rutin, and afzelin are the dominant flavonoids compounds from Zanthoxylum bungeanum leaves, and they play major roles in the antioxidant activity. Macroporous adsorption resin (MAR) treatment, a simple, low-cost and efficient method, was combined with ultrasound-assisted extraction (UAE) to enrich and purify these four flavonoids from Z. bungeanum leaves efficiently. The optimal conditions for UAE based on Response Surface Methodology (RSM) were determined to be an ethanol concentration of 60%, leaves size of 40 mesh, temperature of 50 °C and ultrasonic power of 400 W with four flavonoids contents of 120.84 mg/g. After the extraction process, five kinds of MARs (D4020, D-101, NKA-9, AB-8, and X-5) were tested through static adsorption/desorption to enrich and purify the ultrasonic-assisted extracts, and D-101 was selected as the most suitable resin. The optimal adsorption conditions were 5 bed volumes (BV) of sample solution with an initial concentration of 7.5 mg/mL and pH 5.0. Meanwhile, the optimal desorption parameters were 5 BV each of deionized water and 30% ethanol, then 10 BV 70% ethanol, and a flow rate of 2 BV/hr. Under the optimized conditions, the contents of quercitrin, hyperoside, rutin, and afzelin increased by 276.39%, 187.46%, 221.81%, and 288.45%, respectively, and the recovery yields were 85.47%, 73.53%, 81.35%, and 65.06%. In addition, laboratory preparative-scale separation indicated that the preparative separation of four flavonoids was feasible and easy. Moreover, the antioxidant activities of the purified products were significantly increased after enrichment. In conclusion, all of the results indicated that these methods are highly efficient, low cost, environmentally friendly and easy to scale up. PRACTICAL APPLICATION: This study provided an environmentally friendly, rapid, and highly productive method for the extraction and purification of four active compounds from Zanthoxylum bungeanum leaves. The results can be used for the utilization of Z. bungeanum leaves as a kind of food supplement in an industrial setting.