Biological and Chemical Control of Sclerotinia sclerotiorum using

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Bioactive Constituents, Metabolites, and Functions

Bio- and Chemicalcontrol of Sclerotinia sclerotiorum using Stachybotrys levispora and its secondary metabolite griseofulvin Alany Ingrid Ribeiro, Eveline Soares Costa, Sergio Scherrer Thomasi, Dayson Fernando Ribeiro Brandão, Paulo Cezar Vieira, Joao Batista Fernandes, Moacir Rossi Forim, Antonio Gilberto Ferreira, Sérgio Florentino Pascholati, Luis Fernando Pascholati Gusmão, and Maria Fátima das Graças Fernandes Da Silva J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04197 • Publication Date (Web): 26 Jun 2018 Downloaded from http://pubs.acs.org on June 27, 2018

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Journal of Agricultural and Food Chemistry

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Bio- and Chemicalcontrol of Sclerotinia sclerotiorum using Stachybotrys levispora

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and its secondary metabolite griseofulvin

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Alany Ingrid Ribeiro, Eveline Soares Costa, Sergio Scherrer Thomasi,§ Dayson

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Fernando Ribeiro Brandão,# Paulo Cesar Vieira, João Batista Fernandes, Moacir Rossi

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Forim, Antonio Gilberto Ferreira, Sérgio Florentino Pascholati,# Luis Fernando

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Pascholati Gusmão,¶ Maria Fátima das Graças Fernandes da Silval*,

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Departamento de Química, Universidade Federal de São Carlos, CP 676, São Carlos-

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SP 13565-905, Brazil

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§

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000, Brazil

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#

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900, Piracicaba, SP, Brasil

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¶

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116, Km 03, 44031-460, Feira de Santana, BA, Brasil

Departamento de Química, Universidade Federal de Lavras, CP 3037, Lavras 37200-

Universidade de São Paulo, Escola Superior de Agricultura Luiz de Queiroz, 13418-

Universidade Estadual de Feira de Santana, Departamento de Ciências Biológicas, BR

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ABSTRACT: Sclerotinia sclerotiorum is responsible for white mold of soybean, and

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the difficulty to control the disease in Brazil is causing million-dollar damages.

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Stachybotrys levispora showed activity against S. sclerotiorum. In our present

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investigation, we analyzed the chemical basis of this inhibition. Eight compounds were

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isolated and using spectroscopic methods their structures were identified as the known

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substances: 7-Dechlorogriseofulvin, 7-dechlorodehydrogriseofulvin, griseofulvin,

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dehydrogriseofulvin, 3,13-dihydroxy-5,9,11-trimethoxy-1-methylbenzophenone,

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griseophenone A, 13-hydroxy-3,5,9,11-tetramethoxy-1-methylbenzophenone and 12-

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chloro-13-hydroxy-3,5,9,11-tetramethoxy-1-methylbenzophenone. Griseofulvin

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inhibited the mycelial growth of S. sclerotiorum at 2 µg mL-1. Thus, the antagonistic

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effect of S. levispora to S. sclerotiorum may well be due to the presence of

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griseofulvins. Our results stimulate new work on biosynthesis of griseofulvins, to locate

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genes that encode key enzymes in these routes, and use them to increase the production

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of these compounds and thus potentiate the fungicide effect of this fungus. S. levispora

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represents an agent for biocontrol and griseofulvin a fungicidal to S. sclerotiorum.

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KEYWORDS: Stachybotrys levispora; Sclerotinia sclerotiorum; Memnoniella;

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griseofulvins; 1-methylbenzophenones.

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INTRODUCTION Sclerotinia sclerotiorum is responsible for white mold of soybean (or Sclerotinia

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stem rot), and the high severity and the difficulty to control the disease in Southern

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Brazil are causing million-dollar damages. In Brazil soybean producers have been

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developing strategies to control this fungus, including the removal of diseased plants

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from the field, avoiding the transfer of the microorganism to the others in the plantation,

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the production of resistant cultivars, and chemical control. The economic importance of

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the soybean in Brazil has resulted in a resistant varieties selection research program and

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in the use of biological control, mainly using Saprobic fungi, which have shown

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potential for disease control.1, 2 Stachybotrys levispora (= Memnoniella levispora)3 has

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been shown to possess activity against S. sclerotiorum, which was found to be

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particularly affected and its growth was inhibited by 20% in in vitro assay. In a growth

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chamber, soybean plants treated with S. levispora were later inoculated with S.

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sclerotiorum. Control plants were treated with the culture medium without the fungus

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and the acibenzolar-S-methyl resistance inducer. The symptoms of white mold disease

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were evaluated after 8, 14, and 21 days. Plants treated with S. levispora showed a

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reduction in symptomatic area of 21%, higher than that found in plants treated with the

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resistance inducer (acibenzolar-S-methyl 15%).2

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Stachybotrys spp. are saprotrophic fungi widely distributed and generally have

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been found in soil. Eight species have been reported from Brazilian soil, and S.

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levispora is a new record from South America.1, 4 Some review articles are available

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concerning the secondary metabolites isolated from the genera Stachybotrys and

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Memnoniella, and the last one is very recent.3 This review and the search in Chemical

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Abstracts (SciFinder, 2016) and Web of Science showed that the natural products of S.

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levispora have not been previously studied. Thus, we studied this fungus in order to 3 ACS Paragon Plus Environment


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isolate the compounds in higher concentrations in the extracts and to verify if they

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would be responsible for the antagonistic activity of it against S. sclerotiorum. Most

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chemicals act very fast and when selected properly they are highly effective in

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eliminating the phytopathogen. Additionally, chemicals are easy to use in controlling

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phytopathogen, which implies that a farmer can always mix the right amount of solvent

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with the chemical and then spray the crop. Thus, in our present investigation, we

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analyzed the chemical basis of S. levispora antagonism to S. sclerotiorum.

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MATERIALS AND METHODS Biological material. The experiments were performed with Memnoniella

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levispora Subram. previously collected in Brazil, Bahia, São Felix do Coribe, on dead

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petiole of unidentified dicotyledonous plant, 12 Dec. 2008, leg. S.M. Leão-Ferreira

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(HUEFS 136884). In previous work, the microorganism was initially characterized as

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M. levispora, however, in an investigation using molecular phylogenetic analysis the

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genus was included in Stachybotrys.2 Thus, the worked strain was S. levispora. The

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fungal strains were preserved according to Castellanis’s modified method,5 the pieces of

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fungal cultures are introduced in rubber cap tubes with sterile distilled water and left at

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room temperature (25-28 0C) in darkness.

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Instrumentation. High performance liquid chromatography (HPLC) analyses

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were performed using an Agilent chromatograph (1200 series; GmbH), which contained

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a quaternary pump (G1311A), a degasser (G1322A) and an auto-sampler (G1329A).

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The detector used was a variable wavelength diode array (G1315D). Hystar 2.3

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Software (Bruker) was used in the analyses and an automatic cartridge exchanger

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(Bruker Biospin GmbH) was directly coupled to the chromatograph. The flow was 4 ACS Paragon Plus Environment

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automatically directed to the cartridges, which contained different stationary phases.

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NMR Spectra: Nuclear magnetic resonance analysis was performed using a Bruker

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Avance III instrument (14.1 Tesla / 600 MHz). A TCI 5-mm triple resonance z-field

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gradient cryoprobe was used, which allows obtaining 1H, 13C and 15N spectra, whose

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signals (δ) were expressed in ppm relative to the internal standard Me4Si, and the

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respective J in Hz. In the High Resolution Electron Impact Mass Spectrometry

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(HREIMS) analyzes the system used was Waters Xevo G2-XS QTOF (Waters

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Corporation, Milford, USA) and the spectra were recorded in positive ionization mode.

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Fungal cultivation. The fungus preserved in modified Castellani medium was

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reactivated on potato dextrose agar (PDA). The media were autoclaved at 121 oC for 20

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minutes. After reaching room temperature, 20 mL of this medium was placed into

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sterile Petri dishes 80 mm in diameter. These were inoculated in the center with a single

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plug of diameter 5 mm containing mycelium, and subsequently incubated at 25 °C for

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21 days, with 3 replicates. Based on initial conidial morphology and mycelial growth

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characteristics, and comparison with those in the literature, S. levispora was confirmed.

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The growth characters of S. levispora were studied on five solid media namely PDA,

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the latter containing 1% (m/m) of yeast extract (PDAY), PDA containing 1% (m/m) of

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malt extract (PDAM), Czapeck (CZA) and Carrot-Corn Agar (CCA). All media were

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prepared following the producer's preparation notes. For the preparation of CCA the

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fresh vegetables after being purchased from the market they were washed thoroughly

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with tap water and then reduced to small particles by hand on metal grater. Macerated

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carrot (15 g) and corn (15 g) were then mixed with 500 mL water in which the agar has

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been dissolved. All media were autoclaved at 121 °C for 20 minutes. Upon cooling, 20

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mL of medium was poured into 60 mm diameter sterile disposable Petri dishes. The 5 ACS Paragon Plus Environment


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plates were centrally inoculated with a single plug of diameter 5 mm containing

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mycelium from PDA culture and incubated at 25 °C for 21 days. There were three

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replicate plates of each medium. One plate of each medium without inoculating the

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fungus was used for control purposes. Six plugs (5 mm diameter) were removed

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following a straight line from one border to the other of the plate passing through the

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center of the fungal colony, and these were transferred to an eppendorf and extracted

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with 500 µL of MeOH/CH2Cl2/EtOAc (1:2:3, v/v). The mixture of these solvents was

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prepared at the time of extraction, which were performed at an ultrasonic water bath for

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60 minutes and three times. The extract obtained was filtered into a second clean flask

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using a Pasteur pipette containing pre-sterilized cotton inside. The solvent was

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evaporated under nitrogen, re-suspended in 1:9 H2O/ACN (500 µL) and filtrated using a

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fluoropore PTFE filter membrane (polytetrafluoroethylene, Millipore filter, 15 mm,

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0.20 µm) for HPLC and NMR analysis. The extraction was conducted according to the

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micro-extraction method developed by Smedsgaard.6

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Scaled up and metabolites extraction. 1H NMR spectra were obtained for the

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above extracts and these compared with those of the control (extracts of the medium

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without the fungus), and Carrot-Corn Agar showed more features concerning metabolite

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production compared to the control. Thus, CC medium was chosen to scale up fungal

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growth according to the best response in the NMR spectra analysis for production of the

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secondary metabolites. Thus, macerated carrots (15 g), macerated corn (14 g) and 250

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ml of distilled water were placed in 500 ml Erlenmeyer flasks, in a total of 25 vials and

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all were autoclaved twice at 120 °C for 20 min. Six small (0.5 cm) disks of S. levispora

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mycelium were removed from a Petri dish containing this fungus in PDA medium

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(obtained as described above) and transferred to each of the above 25 vials, all under 6 ACS Paragon Plus Environment

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sterile conditions. In three Erlenmeyer flasks were not placed the fungus and these were

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used as control. The fungus was allowed to grow statically for 21 days at 25 °C, then the

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mycelium was filtered off under vacuum and the liquid phase was analyzed. This was

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partitioned with AcOEt (3 x 500 mL) and the organic phase at concentration in vacuum

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led to 2.3 g of extract.

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Separation and trapping of S. levispora metabolites. Part of the dry extract of

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AcOEt obtained above (20 mg) was dissolved in 10.0 mL of a 8:2 mixture of water and

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acetonitrile (ultrapure H2O was produced in house by Milli-Q System), and thereafter

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filtered using a syringe filter PVDF membrane (Polyvinyl Difluoride; 25 mm, 0.45 lm;

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Tedia Brasil), for further HPLC analysis. The chromatographic conditions for the

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analysis of these samples were: Zorbax C-18 column (250 × 4.6 mm, 5 µm, Agilent),

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mobile phase solvent A- Milli-Q H2O with 0.01% trifluoroacetic acid (TFA) and B-

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acetonitrile with 0.01% TFA in the flow 1 mL min-1 and UV detection at 200 nm. The

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gradient profile was: 0-5 min, 20% B; 40 min, 68% B; 41 min, 84% B; 51-52 min,

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100% B; 57-61 min, 20% B; 60 mg mL-1, injection volume of 15 µL. The HPLC was

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coupled to the automated cartridge exchange system (ACETM Spark Holland,

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Germany), which has a pump (Knauer) that diluted the mobile phase with water,

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decreasing the force of the mobile phase, before the fractions were retained in the

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cartridge, in the SPE Prospekt II® unit. The HPLC-SPE was controlled by computer

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and software HyStar® 2.3 (Bruker). Using the above conditions twenty-eight

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consecutive chromatographic runs with 1.0 mL min-1 flow, injections of 15 µL and at a

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concentration of 60 mg mL-1 were performed. Fraction collection was performed using

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UV absorption at 200 nm, which was used to monitor the separation and to set an

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absorbance threshold to initiate SPE trapping. The compounds were trapped in 7 ACS Paragon Plus Environment


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HySphere Resin GP cartridge (10 mm x 2 mm 10 µm) spherical phase consisting of

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polyvinylbenzene. After the adsorption process, the cartridges were dried with nitrogen

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gas (N2) for 30 min to remove the residual solvent from the chromatographic run and

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SPE trapping. Deuterated methanol-d4 (250 µL, 99.8%) was used to elute the SPE

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cartridge compounds (1-10) directly into NMR tubes (Deutero, 3 mm o.d) and 1H-NMR

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and 2D spectra (HSQC, HMBC) were obtained. After these experiments the solvents

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were evaporated under N2 flow, and then, submitted for HREIMS analysis.

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Spectroscopic data. 13-Hydroxy-3,5,9,11-tetramethoxy-1-methylbenzophenone

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(7). Yellow amorphous powder; Table 1 shows the data of 1H and 13C NMR; Mass of

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the protonated ion of the compound HREIMS: m/z: calcd for C18H21O6 [M+H]+:

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333.1333; found 333.1338.

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12-Chloro-13-hydroxy-3,5,9,11-tetramethoxy-1-methylbenzophenone (8).

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Yellow amorphous powder; Table 1 shows the data of 1H and 13C NMR; Mass of the

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protonated ion of the compound HREIMS: m/z: calcd for C18H20ClO6 [M+H]+:

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367.0943; found 367.0942.

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Screening of antifungal activity. Sclerotinia sclerotiorum was cultured on

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potato dextrose agar (PDA), and maintained in B.O.D (incubated on biochemical

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oxygen demand; Fanem 347 CD, Guarulhos, SP, Brazil) under constant light at 21 °C

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for 7 days. The inhibition of fungal growth was observed by agar well diffusion method.

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A well borer of 1.3 cm diameter was properly sterilized by flame and used to make 5

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uniform wells, 3 cm away from the center of each 9-cm polystyrene plates containing

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20 mL of PDA. Griseofulvin solutions were prepared in methanol at serial dilutions 0.2,

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2.0, 200.0 and 2000.0 µg mL-1, and 100 µL was introduced into wells, respectively, to 8 ACS Paragon Plus Environment

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obtain a final volume of 500 µL per plate. The plates were kept in a sterile laminar flow

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chamber to diffuse for 1 h and the methanol was then evaporated. Mycelia disc of 0.5

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cm diameter of the fungal pathogen was transferred from PDA onto the center of PDA

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plates, which were maintained for 5 days at 21 °C. The 100 µL of methanol were placed

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into wells, also 3 cm away from the fungal discs as a negative control. Five Petri dishes

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were prepared containing methanol, 0.2, 2.0, 200.0, and 2000.0 µg mL-1 of griseofulvin

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respectively. The rays of the zones of inhibition were measured in each experiment and

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averaged 4 replicates. Thus, the plates were marked with two straight lines crossing in

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the center of the disc of 0.5 cm containing the fungus, aiming to measure the radial

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growth daily. The four rays were measured daily starting at the edge of the inoculum

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until the end of the mycelial development of the fungus. Analysis of variance was

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performed using the Scott-Knott and Duncan tests at 5% error probability, the Tukey

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test, which showed a confidence level of 0.05, and Student's T test. The growth of S.

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sclerotiorum was not significantly affected by MeOH alone.

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RESULTS AND DISCUSSIONS

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Structural identification of compounds. Initially the metabolic profile of S.

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levispora was evaluated in different culture mediums and in the Carrot-Corn (CC)

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medium the production of secondary metabolites was higher, so it was chosen to scale

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up fungal growth. A method has been developed using hyphenated HPLC-UV-SPE-

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NMR to separate and identify all interested compounds contained in the fraction of

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ethyl acetate obtained in extractions from the Carrot-Corn culture medium.

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The HPLC-MS hyphenation is a much more sensitive method of analysis than

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the HPLC-NMR, but the latter is non-destructive, allowing collecting the sample and 9 ACS Paragon Plus Environment


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obtaining the mass spectrum later. In addition, HPLC-SPE hyphenation is controlled by

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a computer and software (HyStar® 2.3, Bruker) optimizing the analysis. Fraction

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collection was performed using UV absorption at 200 nm to set an absorbance threshold

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to initiate SPE trapping. The major technical improvement in HPLC-SPE-NMR

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hyphenation is the 5 mm triple resonance cryoprobe inverse (TCI, 1N/13C/15N) equipped

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with a z-field gradient and automatic tuning and matching (ATMA®) , which allows the

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recording of NMR spectra of low-concentration analytes (below 1 mg). Ten peaks were

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detected with adequate intensity but at low concentration, however, using TCI the

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following compounds were separated and identified: 7-Dechlorogriseofulvin (1), 7-

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dechlorodehydrogriseofulvin (2), griseofulvin (3), dehydrogriseofulvin (4), 3,13-

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dihydroxy-5,9,11-trimethoxy-1-methylbenzophenone (5), griseophenone A (6),11-13 13-

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hydroxy-3,5,9,11-tetramethoxy-1-methylbenzophenone (7) and 12-chloro-13-hydroxy-

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3,5,9,11-tetramethoxy-1-methylbenzophenone (8) (Figure 2). HSQC, and HMBC

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correlation maps, and High-Resolution Mass Spectrometry (HRMS) of recovered

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fractions confirmed the proposed structures. The known fatty acid 9(Z),12(Z)-

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octadecadienoic (RT 50.31 min) and 9(Z),11(E)-octadecadienoic (RT 50.67 min) were

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also obtained.14 HREIMS spectra of recovered fractions confirmed the presence of 7-

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dechlorogriseofulvin (1) C17H18O6 at m/z 319.1173 [M+H]+, 7-

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dechlorodehydrogriseofulvin (2) C17H16O6 at m/z 317.1033 [M+H]+, griseofulvin (3)

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C17H17ClO6 at m/z 353.0789 [M+H]+, dehydrogriseofulvin (4) C17H15ClO6 at m/z

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351.0657 [M+H]+, griseophenone A (6) C17H17ClO6 at m/z 353.0811 [M+H]+, and two

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new 1-methylbenzophenones (7) and (8). Figures S1-37 (Supplementary material)

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show NMR and HREIMS spectra of compounds 1-10.

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Compounds 7 and 8 appear to be new natural products. The HREIMS spectrum

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indicated a molecular formula C18H20O6 (m/z 333.1333 [M+H]+), and showed a 10 ACS Paragon Plus Environment

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fragment at m/z 179.0959 (C10H11O3) resulted from cleavage at aryl bond adjacent to the

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oxygen atom of ketone group, suggesting that methylbenzoyl ring carried two

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methoxyls (Figure 3). These data allow placing the hydroxyl in the second ring. The

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identification of the compound 7 as 13-hydroxy-3,5,9,11-tetramethoxy-1-

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methylbenzophenone was also supported by comparison of the 1H and 13C NMR spectra

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(Table 1, Figure S25 and S26) with those of compound 5.13

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Compound 8 exhibited similar NMR spectra to 7 (Table 1) except for the

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presence of the signals for one aryl ring pentasubstituted (δ 6.16 s, 1H, δC 90.1). The

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HREIMS spectrum indicated molecular formula C18H19ClO6 (m/z 367.0943 [M+H]+)

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for compound 8, and showed fragments at m/z 179.0986 (C10H11O3) and 215.0322

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(C9H8ClO4) due to cleavage at aryl bond adjacent to the oxygen atom of ketone group

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(Figure 3), which strongly indicated the presence of chlorine in aryl ring

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pentasubstituted. HSQC experiments showed signals only for methoxyl groups attached

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to a carbon bearing one ortho substituent (chemical shift below δC 60),15 thus placing

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chlorine group at C-12. Thus, the structure of compound 8 was concluded to be 12-

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chloro-13-hydroxy-3,5,9,11-tetramethoxy-1-methylbenzophenone. Complete 13C NMR

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assignments for 8 were made using HSQC and HMBC experiments (Table 1; Figures

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S29 and S30).

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Other species in the Stachybotrys genus have been shown to produce griseofulvin derivatives.13

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The antagonistic effect of S. levispora on S. sclerotiorum. Biological control

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has been studied by numerous researchers and they comment that within a given

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integrated pest management strategy, the biological control are strongly disfavored

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when compared with chemical control, and the first results in an reduction in a pest 11 ACS Paragon Plus Environment


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population, while the latter is potent to eliminate all of them, and this fact may be

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considered a disadvantage by some farmers. In addition, an organism that has been

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introduced from another area to control a pest may become a pest itself, especially if it

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has no natural predators in its new habitat. Thus, previous experiences are needed to

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better assess if the introduced predator, or pathogen can cause a problem.16 These

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comments stimulate new studies that could show if compounds of secondary

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metabolism play an important role in the biological control of S. levispora on S.

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sclerotiorum. If a class of compounds had fungicidal effect against S. sclerotiorum,

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genetic studies to activate the biosynthesis of this class would be the strategy to follow.

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The antagonistic effect of S. levispora in vitro to S. sclerotiorum1,2 may well be owing

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to the presence of griseofulvins 1-4 and their benzophenone precursors 5-8. However, it

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is still necessary to find more experimental evidence on the role of griseofulvin

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derivatives 1, 2 and 4 and the benzophenones 5-8 on S. sclerotiorum, such as isolating

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these compounds in sufficient quantity to test them against this fungus.

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Unfortunately, these compounds were obtained in very small amount. Griseofulvin was

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the most abundant in S. levispora, thus it was tested for in vitro activity against S.

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sclerotiorum. Griseofulvin at 2.0 µg mL-1 or higher effectively inhibited S. sclerotiorum

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mycelial growth (Figure 4). The results indicated that griseofulvin tested in vitro were

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active at concentrations in the micromolar range (5.7 µM). The literature lacks data

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comparing MIC values of antifungal in vitro and subsequent in vivo. However, there are

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data showing that when griseofulvin assays show a MIC of 3 µg mL-1 against a fungus,

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this is indicative of a good candidate for in vivo evaluation.17 Good activity seen here

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with griseofulvin (2.0 µg mL-1) agreed with the other studies, and validated the results

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from this paper.


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Griseofulvin (3) has long been used in the treatment of mycosis in animals and

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humans, but its mode of action has been contested since its discovery.12 There is not

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enough experimental evidence to permit identify the structural requirements for

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antifungal activity, which would be important as a model for the synthesis of new

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griseofulvins.12,18

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Y. Tang and his group characterized genes and corresponding enzymes involved in the

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biosynthesis of griseofulvins, and they isolated derivatives of this compound in gene

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deletion experiments, suggesting its potential for biosynthetic engineering. Our

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researches appoint new possibilities for genetic studies to activate the biosynthesis of

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griseofulvin (3) in S. levispora, to improve its activity and selectivity. Finally, S.

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levispora represent a novel agent for biocontrol and griseofulvin a fungicide to S.

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sclerotiorum.

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In addition, micro-encapsulated particles containing conidia S. levispora using

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biodegradable polymers 20 are in progress. Conidia S. levispora encapsulated after the

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drying process is useful for direct applications in plants, soil or target microorganisms,

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without the need to prepare them in aqueous dispersions, with lower environmental

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impact and more economical than isolated griseofulvin.

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ASSOCIATED CONTENT

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Supporting Information

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Figures S1, S5, S9, S13, S17, S20, S24, S28, S32, S35: 1H NMR spectra of compounds

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1-10 and expansion of a selected region (methano-d4, 600 MHz); Figures S2, S6, S10,

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S14, S18, S21, S25, S29, S33, S36: g-HSQC of compounds 1-10 (methano-d4, 600

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MHz); Figures S3, S7, S11, S15, S19, S22, S26, S30, S34, S37: g-HMBC of

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compounds 1-10 (methano-d4, 600 MHz); Figures S4, S8, S12, S16, S23, S27, S31: 13 ACS Paragon Plus Environment


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HREIMS spectra of compounds 1-4, 6-8 (ESI+, 6eV). The Supporting Information is

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available free of charge on the ACS Publications website at http://pubs.acs.org.

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AUTHOR INFORMATION

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Corresponding Author

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*

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[email protected]

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Funding

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The authors thank the Brazilian agencies: National Council for Scientific and

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Technological Development (CNPq - INCT,465357/2014-8), FAPESP – São Paulo

335

Research Foundation (FAPESP-INCT, 14/50918-7; Temático 2012/25299-6, APR

336

2016/16117-2), and CAPES – Higher Education Improvement Coordination.

337

Notes

338

The authors declare no competing financial interest.

(MFGFS) Telephone: + 55 (16) 3351.8093; Fax: + 55 (16) 3351.8350; E-mail:

339 340

REFERENCES

341

(1) Leão-Ferreira, S. M.; Gusmão, L. F. P.; Ruiz, R. F. C. Conidial fungi from the semi-

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arid Caatinga biome of Brazil. Three new species and new records. Beih. Nova

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Hedwigia 2013, 96, 479-494.

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(2) Barros, D. C. M.; Fonseca, I. C. B.; Balbi-Peña, M. I.; Pascholati, S. F.; Peitl, D. C.;.

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Biocontrol of Sclerotinia sclerotiorum and white mold of soybean using saprobic fungi

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from semi-arid areas of Northeastern Brazil. Summa Phytopathol. 2015, 41, 251-255.

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(3) Wang, Y.; Hyde, K. D.; McKenzie, E. H. C.; Jiang, Y. -L.; Li, D. -W.; Zhao, D. -G.

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Overview of Stachybotrys (Memnoniella) and current species status. Fungal Divers.

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O.; Gusmão, L. F. P. The genus Stachybotrys (anamorphic fungi) in the semi-arid

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region of Brazil. Revista Brasil. Bot. 2010, 33, 479-487.

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(5) Figueiredo M. B. Estudo sobre a aplicação do método de Castellani para

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conservação de fungos patógenos em plantas. O Biológico 1967, 33, 9-13.

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fungal metabolite production in cultures. J. Chromatogr. A. 1997, 760, 264-270.

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(7) Jaroszewski, J. W. Hyphenated NMR methods in natural products research, Part 2:

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HPLC-SPENMR and other new trends in NMR hyphenation. Planta Med. 2005, 71,

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795-802.

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(8) Pan, Z.; Raftery, D. Comparing and combining NMR spectroscopy and mass

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spectrometry in metabolomics. Anal. Bioanal. Chem. 2007, 387, 525-527.

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(9) Koskela, H.; Ervasti, M.; Björk, H.; Vanninen, P. On-Flow Pulsed field gradient

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heteronuclear correlation spectrometry in off-Line LC-SPE-NMR analysis of chemicals

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related to the chemical weapons convention. Anal. Chem. 2009, 81, 1262-1269.

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(10) Daolio, C.; Schneider, B. Coupling Liquid Chromatography and Other Separation

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Techniques to Nuclear Magnetic Resonance Spectroscopy. In Hyphenated and

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Alternative Methods of Detection in Chromatography; Shalliker, R.A., Ed.; Taylor &

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Francis–CRC Press: Oxford, UK, Chromatographic Science Series, 2012. Vol. 104, pp.

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61-98.

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(11) Rhodes, A.; Somerfield, G. A.; McGonagle, M. P. Biosynthesis of griseofulvin.

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Observations on the incorporation of [14C] Griseophenone C and [36Cl] Griseophenones

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B and A. Biochem. J. 1963, 88, 349-357.

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(12) Petersen, A. B.; Ronnest, M. H.; Larsen, T. O.; Clausen, M. H. The Chemistry of

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Griseofulvin. Chem. Rev. 2014, 114, 12088-12107. 15 ACS Paragon Plus Environment


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Sesquiterpenoids and xanthones derivatives produced by sponge-derived fungus

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Stachybotry sp. HH1 ZSDS1F1-2. J. Antibiot. (Tokyo) 2014, 68, 121-125.

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(14) Jie, M. S. F. L. K.; Pasha, M. K.; Alam, M. S. Synthesis and nuclear magnetic

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resonance properties of all geometric isomers of conjugated linoleic acids. Lipids 1997,

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32, 1041-1044.

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(15) Agrawal, P. K. Carbon-13 NMR of flavonoids, Elsevier Science Publishers B. V.,

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Amsterdam, The Netherlands, 1989.

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(16) Monte, E. Understanding Trichoderma: between biotechnology and microbial

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ecology. Int. Microbiol. 2001, 4, 1-4.

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(17) Goh, C-L; Tay, Y. K.; Ali, K. B.; Koh, M. T.; Seow, C. S. In vitro evaluation of

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griseofulvin, ketoconazole, and itraconazole against various dermatophytes in

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(18) Araujo, O. E.; Flowers, F. P.; King, M. M. Griseofulvin: A new look at an old

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drug. DlCP Ann. Pharmacother. 1990, 24, 851-4.

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(19) Cacho, R. A.; Chooi, Y. -H.; Zhou, H.; Tang, Y. Complexity generation in fungal

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polyketide biosynthesis: A spirocycle-forming P450 in the concise pathway to the

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antifungal drug griseofulvin. Chem. Biol. 2013, 8, 2322-2330.

393

(20) Rodrigues, I. M. W.; Forim, M. R.; Silva, M. F. G. F.; Fernandes, J. B.; Batista-

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Filho, A. Effect of ultraviolet radiation on fungi Beauveria bassiana and Metarhizium

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anisopliae, pure and encapsulated, and bio-insecticide action on Diatraea saccharalis.

396

Adv. Entomol. 2016, 4, 151-162.

397 398 399


Page 17 of 23


400

FIGURE CAPTIONS

401

Fig. 1. Chromatogram of ethyl acetate extract obtained after S. levispora growth, by

402

LC-SPE-NMR; (Retention times 21.90, 22.04, 24.71, 24.89, 26.40, 30.61, 37.22, 38.81

403

min, respectively).

404

Fig. 2. Compounds isolated from ethyl acetate extract obtained after S. levispora

405

growth.

406 407

Fig. 3. Fragmentation pathways of compounds 7 and 8 by HREIMS.

408

Fig. 4. In vitro antifungal effect of serial dilutions of griseofulvin (µg mL-1) on S.

409

sclerotiorum expressed as zone of inhibition (diameter in mm). Data are shown as mean

410

± SD for quadruplicate. Methanol (a) and griseofulvin (b): letters a and b indicate that

411

the values are significantly different (Scott-Knott test at 5% error probability) from each

412

other.

413 414 415 416 417 418 419 420 421 422



423

Table 1. 1H and 13C NMR spectroscopic data for 7 and 8 H

424 425 426 427 428 429

Page 18 of 23

7

8

2

6.37 (d, J= 2.0, 1H)

6.39 (brs, 2H)

4

6.39 (d, J=2.0, 1H)

10

C

7

8

1

138.1

138.3

6.39 (brs, 2H)

2

108.8

108.9

5.92 (d, J=1.9, 1H)

6.16 (s, 1H)

3

163.5

163.7

12

6.10 (d, J= 1.9, 1H)

-

4

98.0

97.5

1-Me

2.09 (s, 3H)

2.10 (s, 3H)

5

160.0

160.1

3-OMe

3.80 (s, 3H)

3.81 (s, 3H)

6

129.0

128.8

5-OMe

3.66 (s, 3H)

3.66 (s, 3H)

7

-

203.2

9-OMe

3.83 (s, 3H)

3.96 (s, 3H)

8

110.6

110.4

11-OMe

3.39 (s, 3H)

3.48 (s, 3H)

9

170.4

165.0

10

93.0

90.1

11

166.5

165.4

12

96.4

103.5

13

169.6

-

1-Me

20.8

20.8

3-OMe

57.4

57.0

5-OMe

57.5

58.3

9-OMe

57.5

58.0

11-OMe

57.5

58.6

1

H NMR spectrum was acquired in CD3OD at 600 MHz. Chemical shifts are shown in the δ scale with J values (Hz) in parentheses. Assignments are based on COSY, HSQC and HMBC experiments. 13C NMR data were obtained from HSQC and HMBC experiments because the compounds were isolated in low amounts. Thus, the signals of some totally substituted sp2 carbons were not detected.

430 431 432 433 434


Page 19 of 23


435

Figures.

436 437

Figure 1.

438

439 440 441 442 443 444 445 446 447 448 449



Page 20 of 23

450 451 452

Figure 2.

453 O 4 7

O 6

O

O 2'

3a 3 1

O

6'

O

7a

O

O

O

O O

O

5'

O

O

O O

1

Cl

2

O

O

O

O

O

O 9

O 11

O

O

O

O

Cl

O

454

O

OH 7

OH

OH

O

O

O

O

7

O 6

5 1

13

OH

12

3

OH

14

6 O

OH Cl

O

O 3 O

8

Cl

5

4 O

O

3'

O

O

8

455 456 457 458 459 460 461 462 463 464 465 466


Page 21 of 23


467 468 469

Figure 3. O

O

O

+ O

O

OH

O

O

O m/z 179.0959

7 m/z 333.1338 [M+H]+ O

O

O

+

O

OH Cl

470

O

O

O

O

+

O

O

OH Cl

8

+ O

m/z 179.0986

m/z 215.0322

m/z 367.0942 [M+H]

471 472 473 474 475 476 477 478 479 480 481 482 483 484 485



Page 22 of 23

486 487 488

Figure 4.

489 60 Methanol

Griseofulvin

Mycelial growth (mm)

50 40 30 20 10 0 0.2

490

2 200 2000 Serial dilutions (µg mL-1)

491 492 493 494 495 496

Table of Contents Graphic

497 60

499 500 501 502

Mycelial growth (mm)

498

Griseofulvin inhibited the mycelial growth of S. sclerotiorum Methanol Griseoful.

40

20

0 0.2

2

200

2000 µg mL-1



60 Mycelial growth (mm)

Page 23 of 23

Griseofulvin inhibited the mycelial growth of S. sclerotiorum Methanol Griseoful.

40

20

0 0.2

2

200

ACS Paragon Plus Environment

2000 µg mL-1

Biological and Chemical Control of Sclerotinia sclerotiorum using

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