Background: Botrytis cinerea causes serious gray mold disease in many plants. This pathogen has developed resistance to many fungicides. Thus, it has become necessary to look for new safe yet effective compounds against B. cinerea. Methods: Essential oils (EOs) from 17 plant species were assayed against B. cinerea, of which Origanum vulgare essential oil (OVEO) showed strong antifungal activity, and accordingly its main components were detected by GC/MS. Further study was conducted on the effects of OVEO, carvacrol and thymol in vitro on mycelium growth and spore germination, mycelium morphology, leakages of cytoplasmic contents, mitochondrial injury and accumulation of reactive oxygen species (ROS) of B. cinerea. The control efficacies of OVEO, carvacrol and thymol on tomato gray mold were evaluated in vivo. Results: Of all the 17 plant EOs tested, Cinnamomum cassia, Litsea cubeba var. formosana and O. vulgare EOs had the best inhibitory effect on B. cinerea, with 0.5 mg/mL completely inhibiting the mycelium growth of B. cinerea. Twenty-one different compounds of OVEO were identified by gas chromatography–mass spectrometry, and the main chemical components were carvacrol (89.98%), β-caryophyllene (3.34%), thymol (2.39%), α-humulene (1.38%) and 1-methyl-2-propan-2-ylbenzene isopropyl benzene (1.36%). In vitro experiment showed EC₅₀ values of OVEO, carvacrol and thymol were 140.04, 9.09 and 21.32 μg/mL, respectively. Carvacrol and thymol completely inhibited the spore germination of B. cinerea at the concentration of 300 μg/mL while the inhibition rate of OVEO was 80.03%. EC₅₀ of carvacrol and thymol have significantly (P < 0.05) reduced the fresh and dry weight of mycelia. The collapse and damage on B. cinerea mycelia treated with 40 μg/mL of carvacrol and thymol was examined by scanning electron microscope (SEM). Through extracellular conductivity test and fluorescence microscope observation, it was found that carvacrol and thymol led to increase the permeability of target cells, the destruction of mitochondrial membrane and ROS accumulation. In vivo conditions, 1000 μg/mL carvacrol had the best protective and therapeutic effects on tomato gray mold (77.98% and 28.04%, respectively), and the protective effect was significantly higher than that of 400 μg/mL pyrimethanil (43.15%). While the therapeutic and protective effects of 1,000 μg/mL OVEO and thymol were comparable to chemical control. Conclusions: OVEO showed moderate antifungal activity, whereas its main components carvacrol and thymol have great application potential as natural fungicides or lead compounds for commercial fungicides in preventing and controlling plant diseases caused by B. cinerea.

Background: Phytophthora capsici Leonian (P. capsici) can cause wilting and roots rotting on pepper and other cash crops. The new fungicide cinnamaldehyde (CA) has high activity against this pathogen. However, its potential mechanism is still unknown. Methods: In order to gain insights into the mechanism, isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics was used to analyze P. capsici treated with CA. The iTRAQ results were evaluated by parallel reaction monitoring (PRM) analysis and quantitative real-time PCR (qRT-PCR) analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to speculate the biochemical pathways that the agent may act on. Results: The results showed that 1502 differentially expressed proteins were identified, annotated and classified into 209 different terms (like metabolic process, cellular process, single-organism process) based on Gene Ontology (GO) functional enrichment analysis and nine different pathways (glyoxylate and dicarboxylate metabolism, fatty acid metabolism and so on) based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. This study suggested that CA disordered fatty acid metabolism, polysaccharide metabolism and leucine metabolism. Based on PRM analysis, five proteins including CAMK/CAMK1 protein kinase, glucan 1,3-beta-glucosidase, 1,3-beta-glucanosyltransferase, methylcrotonoyl-CoA carboxylase subunit alpha and isovaleryl-CoA dehydrogenase were down-regulated in P. capsici treated with CA. Furthermore, the qRT-PCR analysis showed that the gene expression level of the interested proteins was consistent with the protein expression level, except for CAMK/CAMK1 protein kinase, acetyl-CoA carboxylase and fatty acid synthase subunit alpha. Conclusions: CA destroyed the metabolic homoeostasisof P. capsici, which led to cell death. This is the first proteomic analysis of P. capsici treated with CA, which may provide an important information for exploring the mechanism of the fungicide CA against P. capsici.

The aim of this study was to determine the extraction technique of supercritical fluid carbon dioxide (SF-CO₂) for the essential oil from Inula britannica flowers and its antifungal activities against plant pathogenic fungi for its potential application as botanical fungicide. The effects of factors, including extraction temperature, extraction pressure, SF-CO₂ flow rate, flower powder size, and time on the essential oil yield were studied using the single factor experiment. An orthogonal experiment was conducted to determine the best operating conditions for the maximum extraction oil yield. Adopting the optimum conditions, the maximum yield reached 10.01% at 40°C temperature, 30 MPa pressure, 60 mesh flower powder size, 20 L h⁻¹ SF-CO₂ flow rate, and 90 min extraction time. The antifungal activities of I. britannica essential oil using the SF-CO₂ against the most important plant pathogenic fungi were also examined through in vitro and in vivo tests. Sixteen plant pathogenic fungi were inhibited to varying degrees at 1 mg mL⁻¹ concentration of the essential oil. The mycelial growth of Gaeumannomyces graminis var. tritici was completely inhibited. The radial growths of Phytophthora capsici and Fusarium monilifome were also inhibited by 83.76 and 64.69%, respectively. In addition, the essential oil can inhibit the spore germination of Fusarium oxysporum f. sp. Vasinfectum, Phytophthora capsici, Colletotrichum orbiculare, and Pyricularia grisea, and the corresponding inhibition rates were 98.26, 96.54, 87.89, and 87.35% respectively. The present study has demonstrated that the essential oil of I. britannica flowers extracted through the SF-CO₂ technique is one potential and promising antifungal agent that can be used as botanical fungicide to protect crops.