#METABOLOMICS WORKBENCH Andressa_Stuart_20210929_131232 DATATRACK_ID:2873 STUDY_ID:ST001927 ANALYSIS_ID:AN003134 PROJECT_ID:PR001217
VERSION             	1
CREATED_ON             	October 18, 2021, 10:26 am
#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: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:NUM_GROUPS                    	3
ST:TOTAL_SUBJECTS                	15
ST:STUDY_COMMENTS                	Two genetically distinct strains of B. bassiana (Bov 3 and Bov 2) were
ST:STUDY_COMMENTS                	cultivated in Agar Sabouraud culture medium, both separately and co-cultivated
ST:STUDY_COMMENTS                	to form a fungal consortium. The metabolomic analysis were performed at the
ST:STUDY_COMMENTS                	Laboratório de Genética de Plantas Max Feffer facility of the Escola Superior
ST:STUDY_COMMENTS                	de Agricultura Luiz de Queiroz of the Universidade de São Paulo (ESALQ/USP).
ST:STUDY_COMMENTS                	Three reads of every biological replicate (five per treatment) were performed,
ST:STUDY_COMMENTS                	generating fifteen readings for each treatment. Pools of metabolites from each
ST:STUDY_COMMENTS                	group were created as a quality control.
#SUBJECT
SU:SUBJECT_TYPE                  	Fungi
SU:SUBJECT_SPECIES               	Beauveria bassiana
SU:GENOTYPE_STRAIN               	GenBank: KU751847; KU751848
#FACTORS
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and 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:CHROMATOGRAPHY_TYPE           	LC
CH:INSTRUMENT_NAME               	Waters Acquity UPLC
CH:COLUMN_NAME                   	Waters Acquity UPLC HSS (2.1 x 100mm, 1.7um)
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:COLUMN_TEMPERATURE            	35 ºC
CH:METHODS_FILENAME              	LCMSMS
CH:SOLVENT_A                     	Water with formic acid
CH:SOLVENT_B                     	Acetonitrile with formic acid.
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Waters Ultima QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Generated data were pre-processed using MassLynx 4.1 software
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