#METABOLOMICS WORKBENCH Andressa_Stuart_20210929_131232 DATATRACK_ID:2873 STUDY_ID:ST001927 ANALYSIS_ID:AN003134 VERSION 1 CREATED_ON 02-08-2024 #PROJECT PR:PROJECT_TITLE Fungal consortium of two Beauveria bassiana strains increases their virulence, PR:PROJECT_TITLE growth, and resistance to stress: a metabolomic approach. PR:PROJECT_TYPE Untargeted Metabolomics PR:PROJECT_SUMMARY Entomopathogenic fungi have been successfully used to control agricultural PR:PROJECT_SUMMARY pests. They infect insects by coming into direct contact with their cuticle or PR:PROJECT_SUMMARY when feeding on contaminated leaves or fruits. After contact with the insect, PR:PROJECT_SUMMARY the entomopathogenic fungus penetrates its body cavity, where it grows and PR:PROJECT_SUMMARY colonizes it from within, causing its death The use of two or more PR:PROJECT_SUMMARY microorganisms in a microbial consortium has been increasingly applied in the PR:PROJECT_SUMMARY biological control of diseases and pests. Beauveria bassiana is one of the most PR:PROJECT_SUMMARY widely studied fungal species in biological control, yet little is known about PR:PROJECT_SUMMARY its role in fungal consortiums. In a previous study, our group found that a PR:PROJECT_SUMMARY consortium formed by two strains of B. bassiana had significantly greater PR:PROJECT_SUMMARY biocontrol potential against the polyphagous caterpillars Duponchelia fovealis PR:PROJECT_SUMMARY (Lepidoptera: Crambidae) than either strain on its own. Despite recent PR:PROJECT_SUMMARY developments and growing efforts to better understand fungal metabolism and PR:PROJECT_SUMMARY metabolites, much remains unknown. Metabolomics therefore represents an PR:PROJECT_SUMMARY important field for evaluating the metabolites produced or modified by an PR:PROJECT_SUMMARY organism or its relationship with the environment. In the present study, we aim PR:PROJECT_SUMMARY to use untargeted metabolomics with gas and liquid chromatography coupled to PR:PROJECT_SUMMARY mass spectrometers (GC-MS and LC-MS/MS) to evaluate the metabolic alterations PR:PROJECT_SUMMARY caused by the co-cultivation of these strains and to correlate the metabolites PR:PROJECT_SUMMARY produced by this consortium with the increased mortality in D. fovealis observed PR:PROJECT_SUMMARY previously. PR:INSTITUTE Universidade Federal do Paraná PR:DEPARTMENT Patologia Básica PR:LABORATORY Laboratório de Microbiologia e Biologia Molecular PR:LAST_NAME Stuart PR:FIRST_NAME Andressa PR:ADDRESS Av. Cel. Francisco Heráclito dos Santos, 100, 81530000, Jardim das Américas, PR:ADDRESS Curitiba, Paraná, Brasil PR:EMAIL andressa.katiski@gmail.com PR:PHONE 5541991922779 PR:PROJECT_COMMENTS Two genetically distinct strains of B. bassiana (Bov 3 and Bov 2) were PR:PROJECT_COMMENTS cultivated in Agar Sabouraud culture medium, both separately and co-cultivated PR:PROJECT_COMMENTS to form a fungal consortium. The metabolomic analysis were performed at the PR:PROJECT_COMMENTS Laboratório de Genética de Plantas Max Feffer facility of the Escola Superior PR:PROJECT_COMMENTS de Agricultura Luiz de Queiroz of the Universidade de São Paulo (ESALQ/USP). PR:PROJECT_COMMENTS Three reads of every biological replicate (five per treatment) were performed, PR:PROJECT_COMMENTS generating fifteen readings for each treatment. Pools of metabolites from each PR:PROJECT_COMMENTS group were created as a quality control. PR:DOI http://dx.doi.org/10.21228/M8D693 PR:CONTRIBUTORS Jason Lee Furuie, Thais Regiani Cataldi, Rodrigo Makowiecky Stuart, Maria PR:CONTRIBUTORS Aparecida Cassilha Zawadneak, Carlos Alberto Labate, Ida Chapaval Pimentel #STUDY ST:STUDY_TITLE Fungal consortium of two Beauveria bassiana strains increases their virulence, ST:STUDY_TITLE growth, and resistance to stress: a metabolomic approach. ST:STUDY_TYPE Untargeted Metabolomics ST:STUDY_SUMMARY Entomopathogenic fungi have been successfully used to control agricultural ST:STUDY_SUMMARY pests. They infect insects by coming into direct contact with their cuticle or ST:STUDY_SUMMARY when feeding on contaminated leaves or fruits. After contact with the insect, ST:STUDY_SUMMARY the entomopathogenic fungus penetrates its body cavity, where it grows and ST:STUDY_SUMMARY colonizes it from within, causing its death The use of two or more ST:STUDY_SUMMARY microorganisms in a microbial consortium has been increasingly applied in the ST:STUDY_SUMMARY biological control of diseases and pests. Beauveria bassiana is one of the most ST:STUDY_SUMMARY widely studied fungal species in biological control, yet little is known about ST:STUDY_SUMMARY its role in fungal consortiums. In a previous study, our group found that a ST:STUDY_SUMMARY consortium formed by two strains of B. bassiana had significantly greater ST:STUDY_SUMMARY biocontrol potential against the polyphagous caterpillars Duponchelia fovealis ST:STUDY_SUMMARY (Lepidoptera: Crambidae) than either strain on its own. Despite recent ST:STUDY_SUMMARY developments and growing efforts to better understand fungal metabolism and ST:STUDY_SUMMARY metabolites, much remains unknown. Metabolomics therefore represents an ST:STUDY_SUMMARY important field for evaluating the metabolites produced or modified by an ST:STUDY_SUMMARY organism or its relationship with the environment. In the present study, we aim ST:STUDY_SUMMARY to use untargeted metabolomics with gas and liquid chromatography coupled to ST:STUDY_SUMMARY mass spectrometers (GC-MS and LC-MS/MS) to evaluate the metabolic alterations ST:STUDY_SUMMARY caused by the co-cultivation of these strains and to correlate the metabolites ST:STUDY_SUMMARY produced by this consortium with the increased mortality in D. fovealis observed ST:STUDY_SUMMARY previosly. ST:INSTITUTE Universidade Federal do Paraná ST:DEPARTMENT Patologia Básica ST:LABORATORY Laboratório de Microbiologia e Biologia Molecular ST:LAST_NAME Stuart ST:FIRST_NAME Andressa ST:ADDRESS Av. Cel. Francisco Heráclito dos Santos, 100, 81530000, Jardim das Américas, ST:ADDRESS Curitiba, Paraná, Brasil ST:EMAIL andressa.katiski@gmail.com ST:PHONE 5541991922779 ST:SUBMIT_DATE 2021-09-29 #SUBJECT SU:SUBJECT_TYPE Fungi SU:SUBJECT_SPECIES Beauveria bassiana SU:GENOTYPE_STRAIN GenBank: KU751847; KU751848 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Bov2_1 Fungal species:Beauveria bassiana Strain Bov2 RAW_FILE_NAME=Bov2_1; RAW_FILE_NAME=Bov2_1_R001 Bov2_1_R002 Bov2_1_R003; RAW_FILE_NAME=Bov2_1_R001_NEG Bov2_1_R002_NEG Bov2_1_R003_NEG; Group=Bov2 SUBJECT_SAMPLE_FACTORS - Bov2_2 Fungal species:Beauveria bassiana Strain Bov2 RAW_FILE_NAME=Bov2_2; RAW_FILE_NAME=Bov2_2_R001 Bov2_2_R002 Bov2_2_R003; RAW_FILE_NAME=Bov2_2_R001_NEG Bov2_2_R002_NEG Bov2_2_R003_NEG; Group=Bov2 SUBJECT_SAMPLE_FACTORS - Bov2_3 Fungal species:Beauveria bassiana Strain Bov2 RAW_FILE_NAME=Bov2_3; RAW_FILE_NAME=Bov2_3_R001 Bov2_3_R002 Bov2_3_R003; RAW_FILE_NAME=Bov2_3_R001_NEG Bov2_3_R003_NEG Bov2_3_R003_NEG; Group=Bov2 SUBJECT_SAMPLE_FACTORS - Bov2_4 Fungal species:Beauveria bassiana Strain Bov2 RAW_FILE_NAME=Bov2_4; RAW_FILE_NAME=Bov2_4_R001 Bov2_4_R002 Bov2_4_R003; RAW_FILE_NAME=Bov2_4_R001_NEG Bov2_4_R002_NEG Bov2_4_R003_NEG; Group=Bov2 SUBJECT_SAMPLE_FACTORS - Bov2_5 Fungal species:Beauveria bassiana Strain Bov2 RAW_FILE_NAME=Bov2_5; RAW_FILE_NAME=Bov2_5_R001 Bov2_5_R002 Bov2_5_R003; RAW_FILE_NAME=Bov2_5_R001_NEG Bov2_5_R002_NEG Bov2_5_R003_NEG; Group=Bov2 SUBJECT_SAMPLE_FACTORS - Bov3_1 Fungal species:Beauveria bassiana Strain Bov3 RAW_FILE_NAME=Bov3_1; RAW_FILE_NAME=Bov3_1_R001 Bov3_1_R002 Bov3_1_R003; RAW_FILE_NAME=Bov3_1_R001_NEG Bov3_1_R002_NEG Bov3_1_R003_NEG; Group=Bov3 SUBJECT_SAMPLE_FACTORS - Bov3_2 Fungal species:Beauveria bassiana Strain Bov3 RAW_FILE_NAME=Bov3_2; RAW_FILE_NAME=Bov3_2_R001 Bov3_2_R002 Bov3_2_R003; RAW_FILE_NAME=Bov3_2_R001_NEG Bov3_2_R002_NEG Bov3_2_R003_NEG; Group=Bov3 SUBJECT_SAMPLE_FACTORS - Bov3_3 Fungal species:Beauveria bassiana Strain Bov3 RAW_FILE_NAME=Bov3_3; RAW_FILE_NAME=Bov3_3_R001 Bov3_3_R002 Bov3_3_R003; RAW_FILE_NAME=Bov3_3_R001_NEG Bov3_3_R002_NEG Bov3_3_R003_NEG; Group=Bov3 SUBJECT_SAMPLE_FACTORS - Bov3_4 Fungal species:Beauveria bassiana Strain Bov3 RAW_FILE_NAME=Bov3_4; RAW_FILE_NAME=Bov3_4_R001 Bov3_4_R002 Bov3_4_R003; RAW_FILE_NAME=Bov3_4_R001_NEG Bov3_4_R002_NEG Bov3_4_R003_NEG; Group=Bov3 SUBJECT_SAMPLE_FACTORS - Bov3_5 Fungal species:Beauveria bassiana Strain Bov3 RAW_FILE_NAME=Bov3_5; RAW_FILE_NAME=Bov3_5_R001 Bov3_5_R002 Bov3_5_R003; RAW_FILE_NAME=Bov3_5_R001_NEG Bov3_5_R002_NEG Bov3_5_R003_NEG; Group=Bov3 SUBJECT_SAMPLE_FACTORS - Consortium_1 Fungal species:Consortium formed by Bov2 and Bov3 strains RAW_FILE_NAME=Cons_1; RAW_FILE_NAME=Cons_1_R001 Cons_1_R002 Cons_1_R003; RAW_FILE_NAME=Cons_1_R001_NEG Cons_1_R002_NEG Cons_1_R003_NEG; Group=Cons SUBJECT_SAMPLE_FACTORS - Consortium_2 Fungal species:Consortium formed by Bov2 and Bov3 strains RAW_FILE_NAME=Cons_2; RAW_FILE_NAME=Cons_2_R001 Cons_2_R002 Cons_2_R003; RAW_FILE_NAME=Cons_2_R001_NEG Cons_2_R002_NEG Cons_2_R003_NEG; Group=Cons SUBJECT_SAMPLE_FACTORS - Consortium_3 Fungal species:Consortium formed by Bov2 and Bov3 strains RAW_FILE_NAME=Cons_3; RAW_FILE_NAME=Cons_3_R001 Cons_3_R002 Cons_3_R003; RAW_FILE_NAME=Cons_3_R001_NEG Cons_3_R002_NEG Cons_3_R003_NEG; Group=Cons SUBJECT_SAMPLE_FACTORS - Consortium_4 Fungal species:Consortium formed by Bov2 and Bov3 strains RAW_FILE_NAME=Cons_4; RAW_FILE_NAME=Cons_4_R001 Cons_4_R002 Cons_4_R003; RAW_FILE_NAME=Cons_4_R001_NEG Cons_4_R002_NEG Cons_4_R003_NEG; Group=Cons SUBJECT_SAMPLE_FACTORS - Consortium_5 Fungal species:Consortium formed by Bov2 and Bov3 strains RAW_FILE_NAME=Cons_5; RAW_FILE_NAME=Cons_5_R001 Cons_5_R002 Cons_5_R003; RAW_FILE_NAME=Cons_5_R001_NEG Cons_5_R002_NEG Cons_5_R003_NEG; Group=Cons #COLLECTION CO:COLLECTION_SUMMARY Two genetically distinct strains of Beauveria bassiana (Bov 3 and Bov 2) were CO:COLLECTION_SUMMARY cultivated both separately and co-cultivated to form a fungal consortium. After CO:COLLECTION_SUMMARY the colonies had grown, the mycelium of each treatment (Bov 2, Bov 3, and the CO:COLLECTION_SUMMARY fungal consortium) was scraped from the culture medium with a spatula and then CO:COLLECTION_SUMMARY macerated separately in liquid nitrogen (N2) CO:COLLECTION_PROTOCOL_FILENAME MetaboliteExtraction CO:SAMPLE_TYPE Fungal mycelium CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Two genetically distinct strains of B. bassiana (Bov 3 and Bov 2) were TR:TREATMENT_SUMMARY cultivated in Petri dishes containing Agar Sabouraud culture medium, both TR:TREATMENT_SUMMARY separately and co-cultivated to form a fungal consortium. The cultures were TR:TREATMENT_SUMMARY incubated in the dark in a biological oxygen demand (BOD) oven for 14 days at TR:TREATMENT_SUMMARY 28°C. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Extraction was performed in microtubes, from 200 mg of fungal macerate to which SP:SAMPLEPREP_SUMMARY 1 mL of 6:2:2 methanol:chloroform:water ice-cold extraction solution was added. SP:SAMPLEPREP_SUMMARY These extraction microtubes were vigorously vortexed and placed in an ultrasonic SP:SAMPLEPREP_SUMMARY low-temperature bath at 20 Hz s-1 for 15 min. The samples were then centrifuged SP:SAMPLEPREP_SUMMARY (Eppendorf, Germany) at 4°C for 10 min at 14,000 rpm. Then, the supernatant was SP:SAMPLEPREP_SUMMARY filtered using a 0.22 μm Whatman® filter (Merck, Germany) and transferred to a SP:SAMPLEPREP_SUMMARY chromatographic vial where the extracts were lyophilized (Thermo Fischer SP:SAMPLEPREP_SUMMARY Scientific, MA, USA) until completely dry. Finally, the lyophilized samples were SP:SAMPLEPREP_SUMMARY resuspended in 200 μL of extraction solution and aliquoted for use in the GC-MS SP:SAMPLEPREP_SUMMARY and LC-MS/MS. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Positive Mode (.raw) CH:METHODS_FILENAME LCMSMS CH:INSTRUMENT_NAME Waters Acquity UPLC CH:COLUMN_NAME Waters Acquity UPLC HSS (100 x 2.1mm, 1.7um) CH:COLUMN_TEMPERATURE 35 ºC CH:FLOW_GRADIENT 95% solvent A and 5% B. The gradient increased linearly to 75% A and 25% B over CH:FLOW_GRADIENT the next 6 min. The polarity was reversed to 25% A and 75% B for 6 min, and CH:FLOW_GRADIENT finally 5% A and 95% B for 1 min CH:FLOW_RATE 0.5 mL·min-1 CH:SOLVENT_A Water; formic acid CH:SOLVENT_B 100% acetonitrile; formic acid. CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Ultima QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:MS_COMMENTS Generated data were pre-processed using MassLynx 4.1 software MS:ION_MODE POSITIVE MS:CAPILLARY_VOLTAGE 3 kV MS:SOURCE_TEMPERATURE 150 ºC MS:DESOLVATION_GAS_FLOW 550 L/hr. MS:MS_RESULTS_FILE ST001927_AN003134_Results.txt UNITS:Relative intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END