Identification and pathogenicity of Fusarium spp. associated with tea wilt in Zhejiang Province, China.

BACKGROUND: Tea is one of the most widely consumed beverages in the world, with significant economic and cultural value. However, tea production faces many challenges due to various biotic and abiotic stresses, among which fungal diseases are particularly devastating. RESULTS: To understand the identity and pathogenicity of isolates recovered from tea plants with symptoms of wilt, phylogenetic analyses and pathogenicity assays were conducted. Isolates were characterized to the species level by sequencing the ITS, tef-1α, tub2 and rpb2 sequences and morphology. Four Fusarium species were identified: Fusarium fujikuroi, Fusarium solani, Fusarium oxysporum, and Fusarium concentricum. The pathogenicity of the Fusarium isolates was evaluated on 1-year-old tea plants, whereby F. fujikuroi OS3 and OS4 strains were found to be the most virulent on tea. CONCLUSIONS: To the best of our knowledge, this is the first report of tea rot caused by F. fujikuroi in the world. This provides the foundation for the identification and control of wilt disease in tea plants.

First report of Colletotrichum fructicola causing anthracnose on Carya cathayensis Sarg. in China.

Carya cathayensis Sarg. (Chinese hickory) is one of the important economic forest plants, mainly distributed in Zhejiang and Anhui provinces in China. In September 2020, leaf spot disease occurred on 90% C. cathayensis in a 2.6 km2 plantation with 500 hickorys in Shangshu Village (30°26'N, 119°32'E), Huzhou, Zhejiang, China. Symptoms initially appeared as small brown spots. Later, the spots became dark brown, and joined into irregular shapes. Twenty diseased leaves with typical symptoms were collected and used to isolate the pathogen. The leaf tissues (5 × 5 mm) at junction of diseased and healthy portion were cut and surface-sterilized with 75% ethanol for 15 s, 0.1% NaClO for 2 min, and rinsed 3 times in sterile water, then placed on potato dextrose agar (PDA) plates and incubated at 25°C in the darkness for 3 days. Eight isolates with similar morphological characterizations were obtained after pure cultures by transferring hyphal tips. The colony growing on PDA for 7 days was circular, dense, white cotton-like hyphae, and light gray-black hyphae can be seen inside. The conidia were cylindrical, aseptate, hyaline, with rounded ends, and 12.5 to 20.0 × 5.0 to 7.5 µm (n = 50). The appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and were in the range of 6.4 to 10.2 × 5.0 to 6.7 µm (n = 50). The morphologies of the isolates were consistent with the genus description of Colletotrichum (Fuentes-Aragón et al. 2018; Liu et al. 2015). The internal transcribed spacer (ITS) regions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), chitin synthase (CHS-1), beta-tubulin 2 (TUB2) and actin (ACT) genes were amplified from genomic DNA for the isolates using the primers described by Weir et al. (2012). The sequences of eight isolates were consistent and the representative isolate CFZJ-64 were deposited in GenBank under the following accession numbers: ITS, OK145563; ACT, OK216738; CAL, OK216739; CHS-1, OK216740; GAPDH, OK216741; and TUB2, OK216742. A phylogenetic tree was generated by combining ITS, ACT, CAL, CHS-1, TUB2, and GAPDH sequences in MEGA11. Three representative isolates CFZJ-42, CFZJ-53 and CFZJ-64 clustered in the C. fructicola clade with 90% bootstrap support. Based on morphological characteristics and phylogenetic analysis, the isolates were identified as C. fructicola. To confirm pathogenicity, 9 detached healthy leaves and 9 healthy leaves on 3-year-old C. cathayensis seedlings were inoculated with conidial suspension of each isolate (20 µL, 1 × 106 conidia/mL). The control leaves were treated with distilled water (20 µL). Each tested leaf was covered with a clean ziplock bag and incubated for 48h at about 27°C, and 14h photoperiod. After five days, 7 of 8 isolates caused on all detached leaves or part of the leaves on the seedlings developed lesions similar to those observed in the field, whereas controls were asymptomatic. The same fungus was re-isolated from all the diseased leaves and identified by sequencing, confirming Koch's postulates. As far as we know, this is the first report of C. fructicola causing anthracnose on C. cathayensis. This study not only expands the knowledge on this important pathogen of C. cathayensis in China, but also provides the foundation to further investigate the biology, epidemiology, and control of the disease.

Synthesis, Structural Determination, and Antifungal Activity of Novel Fluorinated Quinoline Analogs.

A series of new fluorinated quinoline analogs were synthesized using Tebufloquin as the lead compound, 2-fluoroaniline, ethyl 2-methylacetoacetate, and substituted benzoic acid as raw materials. Their structures were confirmed by 1H NMR, 13C NMR, and HRMS. The compound 8-fluoro-2,3-dimethylquinolin-4-yl 4-(tert-butyl)benzoate (2b) was further determined by X-ray single-crystal diffraction. The antifungal activity was tested at 50 µg/mL, and the bioassay results showed that these quinoline derivatives had good antifungal activity. Among them, compounds 2b, 2e, 2f, 2k, and 2n exhibited good activity (>80%) against S. sclerotiorum, and compound 2g displayed good activity (80.8%) against R. solani.

Synthesis, Herbicidal Activity, Mode of Action, and In Silico Analysis of Novel Pyrido[2,3-d]pyrimidine Compounds.

Natural products are a main source of new chemical entities for use in drug and pesticide discovery. In order to discover lead compounds with high herbicidal activity, a series of new pyrido[2,3-d] pyrimidine derivatives were designed and synthesized using 2-chloronicotinic acid as the starting material. Their structures were characterized with 1H NMR, 13C NMR and HRMS, and the herbicidal activities against dicotyledonous lettuce (Lactuca sativa), field mustard (Brassica campestris), monocotyledonous bentgrass (Agrostis stolonifera) and wheat (Triticum aestivum) were determined. The results indicated that most of the pyrido[2,3-d] pyrimidine derivatives had no marked inhibitory effect on lettuce at 1 mM. However, most of the pyrido[2,3-d] pyrimidine derivatives possessed good activity against bentgrass at 1 mM. Among them, the most active compound, 3-methyl-1-(2,3,4-trifluorophenyl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione (2o), was as active as the positive controls, the commercial herbicides clomazone and flumioxazin. Molecular simulation was performed with molecular docking and DFT calculations. The docking studies provided strong evidence that 2o acts as an herbicide by inhibition of protoporphyrinogen oxidase. However, the physiological results indicate that it does not act on this target in vivo, implying that it could be metabolically converted to a compound with a different molecular target.

Synthesis, crystal structure, herbicidal activity and mode of action of new cyclopropane-1,1-dicarboxylic acid analogues.

A new series of cyclopropane-1,1-dicarboxylic (CPD) acid analogues were designed and synthesized. CPD is an inhibitor of ketol-acid reductoisomerase (KARI), an enzyme of the branched chain amino acid pathway in plants. The structures of CPD analogues were characterized by 1H NMR and HRMS. The structure of N,N'-bis(4-(tert-butyl)phenyl)cyclopropane-1,1-dicarboxamide was further elucidated by X-ray diffraction. The herbicidal activities of these compounds were tested against lettuce (Lactuca sativa) and bentgrass (Agrostis stolonifera). Most of these compounds exhibited low herbicidal activity against both plant species. Among them, N,N'-bis(2-ethylphenyl)cyclopropane-1,1-dicarboxamide displayed moderate activity against bentgrass. Inhibition of KARI activity by the CPD analogues was also assessed experimentally and by molecular docking simulation with results supporting inhibition of KARI as their mode of action. These results provide the basis for design of more effective KARI inhibitors.

Synthesis and Pesticidal Activity of New Niacinamide Derivatives Containing a Flexible, Chiral Chain.

Natural products are a source for pesticide or drug discovery. In order to discover lead compounds with high fungicidal or herbicidal activity, new niacinamide derivatives derived from the natural product niacinamide, containing chiral flexible chains, were designed and synthesized. Their structures were confirmed by 1H NMR, 13C NMR and HRMS analysis. The fungicidal and herbicidal activities of these compounds were tested. The fungicidal activity results demonstrated that the compound (S)-2-(2-chloronicotinamido)propyl-2-methylbenzoate (3i) exhibited good fungicidal activity (92.3% inhibition) against the plant pathogen Botryosphaeria berengriana at 50 µg/mL and with an EC50 of 6.68 ± 0.72 µg/mL, which is the same as the positive control (fluxapyroxad). Compound 3i was not phytotoxic and could therefore be used as a fungicide on crops. Structure-activity relationships (SAR) were studied by molecular docking simulations with the succinate dehydrogenase of the fungal mitochondrial respiratory chain.

Novel Pyrazole Acyl(thio)urea Derivatives Containing a Biphenyl Scaffold as Potential Succinate Dehydrogenase Inhibitors: Design, Synthesis, Fungicidal Activity, and SAR.

As part of a program to discover novel succinate dehydrogenase inhibitor fungicides, a series of new pyrazole acyl(thio)urea compounds containing a diphenyl motif were designed and synthesized. Their structures were confirmed by 1H NMR, HRMS, and single X-ray crystal diffraction analysis. Most of these compounds possessed excellent activity against 10 fungal plant pathogens at 50 µg mL-1, especially against Rhizoctonia solani, Alternaria solani, Sclerotinia sclerotiorum, Botrytis cinerea, and Cercospora arachidicola. Interestingly, compounds 3-(difluoromethyl)-1-methyl-N-((3',4',5'-trifluoro-[1,1'-biphenyl]-2-yl)carbamoyl)-1H-pyrazole-4-carboxamide (9b, EC50 = 0.97 ± 0.18 µg mL-1), 1,3-dimethyl-N-((3',4',5'-trifluoro-[1,1'-biphenyl]-2-yl)carbamoyl)-1H-pyrazole-4-carboxamide (9a, EC50 = 2.63 ± 0.41 µg mL-1), and N-((4'-chloro-[1,1'-biphenyl]-2-yl)carbamoyl)-1,3-dimethyl-1H-pyrazole-4-carboxamide (9g, EC50 = 1.31 ± 0.15 µg mL-1) exhibited activities against S. sclerotiorum that were better than the commercial fungicide bixafen (EC50 = 9.15 ± 0.05 µg mL-1) and similar to the positive control fluxapyroxad (EC50 = 0.71 ± 0.11 µg mL-1). These compounds were not significantly phytotoxic to monocotyledonous and dicotyledonous plants. Structure-activity relationships (SAR) are discussed by substituent effects/molecular docking, and density functional theory analysis indicated that these compounds are succinate dehydrogenase inhibitors.

Discovery of Highly Efficient Novel Antifungal Lead Compounds Targeting Succinate Dehydrogenase: Pyrazole-4-carboxamide Derivatives with an N-Phenyl Substituted Amide Fragment.

Developing environmentally friendly fungicides is crucial to tackle the issue of rising pesticide resistance. In this study, a series of novel pyrazole-4-carboxamide derivatives containing N-phenyl substituted amide fragments were designed and synthesized. The structures of target compounds were confirmed by 1H NMR, 13C NMR, and HRMS, and the crystal structure of the most active compound N-(1-(4-(4-(tert-butyl)benzamido)phenyl)propan-2-yl)-3-(difluoromethyl)-N-methoxy-1-methyl-1H-pyrazole-4-carboxamide (U22) was further determined by X-ray single-crystal diffraction. The bioassay results indicated that the 26 target compounds possessed good in vitro antifungal activity against Sclerotinia sclerotiorum with EC50 values for compounds U12, U13, U15, U16, U18, U22, and U23 being 4.17 ± 0.46, 8.04 ± 0.71, 7.01 ± 0.71, 12.77 ± 1.00, 8.11 ± 0.70, 0.94 ± 0.11, and 9.48 ± 0.83 µg·mL-1, respectively, which were the similar to controls bixafen (6.70 ± 0.47 µg·mL-1), fluxapyroxad (0.71 ± 0.14 µg·mL-1), and pydiflumetofen (0.06 ± 0.01 µg·mL-1). Furthermore, in vivo antifungal activity results against S. sclerotiorum indicated that compounds U12 (80.6%) and U22 (89.9%) possessed excellent preventative efficacy at 200 µg·mL-1, which was the same as the control pydiflumetofen (82.4%). Scanning electron microscopy and transmission electron microscopy studies found that the compound U22 could destroy the hyphal morphology and damage mitochondria, cell membranes, and vacuoles. The results of molecular docking of compound U22 and pydiflumetofen with succinate dehydrogenase (SDH) indicated they interact well with the active site of SDH. This study validated our approach and design strategy to produce compounds with an enhanced biological activity as compared to the parent structure.

Novel Dioxolane Ring Compounds for the Management of Phytopathogen Diseases as Ergosterol Biosynthesis Inhibitors: Synthesis, Biological Activities, and Molecular Docking.

Thirty novel dioxolane ring compounds were designed and synthesized. Their chemical structures were confirmed by 1H NMR, HRMS, and single crystal X-ray diffraction analysis. Bioassays indicated that these dioxolane ring derivatives exhibited excellent fungicidal activity against Rhizoctonia solani, Pyricularia oryae, Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum, Physalospora piricola, Cercospora arachidicola and herbicidal activity against lettuce (Lactuca sativa), bentgrass (Agrostis stolonifera), and duckweed (Lemna pausicostata). Among these compounds, 1-((2-(4-chlorophenyl)-5-methyl-1,3-dioxan-2-yl)methyl)-1H-1,2,4-triazole (D17), 1-(((4R)-2-(4-chlorophenyl)-4-methyl-1,3-dioxolan-2-yl)methyl)-1H-1,2,4-triazole (D20), 1-((5-methyl-2-(4-(trifluoromethyl)phenyl)-1,3-dioxan-2-yl)methyl)-1H-1,2,4-triazole (D22), and 1-((2-(4-fluorophenyl)-1,3-dioxolan-2-yl)methyl)-1H-1,2,4-triazole (D26) had broad spectrum fungicidal and herbicidal activity. The IC50 values against duckweed were 20.5 ± 9.0, 14.2 ± 6.7, 24.0 ± 11.0, 8.7 ± 3.5, and 8.0 ± 3.1 µM for D17, D20, D22, and D26 and the positive control difenoconazole, respectively. The EC50 values were 7.31 ± 0.67, 9.74 ± 0.83, 17.32 ± 1.23, 11.96 ± 0.98, and 8.93 ± 0.91 mg/L for D17, D20, D22, and D26 and the positive control difenoconazole against the plant pathogen R. solani, respectively. Germination experiments with Arabidopsis seeds indicated that the target of these dioxolane ring compounds in plants is brassinosteroid biosynthesis. Molecular simulation docking results of compound D26 and difenoconazole with fungal CYP51 P450 confirmed that they both inhibit this enzyme involved in ergosterol biosynthesis. The structure-activity relationships (SAR) are discussed by substituent effect, molecular docking, and density functional theory analysis, which provided useful information for designing more active compounds.

Response of the microbial community to phosphate-solubilizing bacterial inoculants on Ulmus chenmoui Cheng in Eastern China.

Phosphate-solubilizing bacteria (PSB) have beneficial effects on plant health and soil composition. To date, studies of PSB in soil have largely been performed under field or greenhouse conditions. However, less is known about the impact of introducing indigenous PSB in the field, including their effects on the local microbial community. In this study, we conducted greenhouse and field experiments to explore the effects of the addition of indigenous PSB on the growth of Chenmou elm (Ulmus chenmoui) and on the diversity and composition of the bacterial community in the soil. We obtained four bacterial isolates with the highest phosphate-solubilizing activity: UC_1 (Pseudomonas sp.), UC_M (Klebsiella sp.), UC_J (Burkholderia sp.), and UC_3 (Chryseobacterium sp.). Sequencing on the Illumina MiSeq platform showed that the inoculated PSB did not become the dominant strains in the U. chenmoui rhizosphere. However, the soil bacterial community structure was altered by the addition of these PSB. The relative abundance of Chloroflexi decreased significantly in response to PSB application in all treatment groups, whereas the populations of several bacteria, including Proteobacteria and Bacteroidetes, increased. Network analysis indicated that Chloroflexi was the most strongly negatively correlated with Proteobacteria, whereas Proteobacteria was strongly positively correlated with Bacteroidetes. Our findings indicate that inoculation with PSB (UC_1, UC_M, UC_J, and UC_3) can improve the growth of U. chenmoui and regulate its rhizosphere microbial community. Therefore, inoculation with these bacterial strains could promote the efficient cultivation and production of high-quality plant materials.

Synthesis and Pesticidal Activities of New Quinoxalines.

Natural products are a source of many novel compounds with biological activity for the discovery of new pesticides and pharmaceuticals. Quinoxaline is a fused N-heterocycle in many natural products and synthetic compounds, and seven novel quinoxaline derivatives were designed and synthesized via three steps. Pesticidal activities of title quinoxaline derivatives were bioassayed. Most of these compounds had herbicidal, fungicidal, and insecticidal activities. The compounds 2-(6-methoxy-2-oxo-3-phenylquinoxalin-1(2H)-yl)acetonitrile (3f) and 1-allyl-6-methoxy-3-phenylquinoxalin-2(1H)-one (3g) were the most active herbicides and fungicides. Mode-of-action studies indicated that 3f is a protoprophyrinogen oxidase-inhibiting herbicide. Compound 3f also possessed broad-spectrum fungicidal activity against the plant pathogen Colletotrichum species. Some of these compounds also had insecticidal activity. Molecular docking and DFT analysis can potentially be used to design more active compounds.