scholarly journals Biocontrol of tomato bacterial wilt by a new strain Bacillus velezensis FJAT-46737 and its lipopeptides

2019 ◽  
Author(s):  
Meichun Chen ◽  
Jieping Wang ◽  
Bo Liu ◽  
Yujing Zhu ◽  
Rongfeng Xiao ◽  
...  
Keyword(s):  
Bacterial Wilt ◽  
Plant Pathogens ◽  
Antimicrobial Agents ◽  
Gene Clusters ◽  
Antimicrobial Activities ◽  
Whole Genome Sequence ◽  
Dependent Manner ◽  
Bacillus Velezensis ◽  
Tomato Bacterial Wilt

Abstract Background: There is an urgent need to discover alternative antimicrobial agents to control bacterial wilt. The objectives of this study were to report a new lipopeptide-producing biocontrol strain FJAT-46737 and its lipopeptides, and to investigate their antagonistic effects. Results: Based on the whole genome sequence analysis, the new strain FJAT-46737 was finally identified as Bacillus velezensis and seven gene clusters that responsible for synthesis of bioactive secondary metabolite in the FJAT-46737 were predicted. Antimicrobial results demonstrated that FJAT-46737 exhibited broad-spectrum antimicrobial activities in vitro against bacteria and fungi . The pot experiments showed that the tomato bacterial wilt control efficiencies of the whole cultures, the 2-fold diluted supernatants and the crude lipopeptide of FJAT-46737 were 66.2%, 82.0%, and 96.2%, respectively. The above results suggested that one of the antagonistic mechanisms of FJAT-46737 was secretion of lipopeptides that consisted of iturins, fengycins and surfactins. The crude lipopeptides had significant antagonistic activities on several pathogen in a dosage-dependent manner, and the fengycins confirmed to play a major role in antibacterial abilities in vitro of the lipopeptides against Ralstonia solanacearum . Furthermore, it was found that the rich organic nitrogen sources (especially yeast extracts) in the media were beneficial for FJAT-46737 to produce fengycin and surfactin. The secretion of these two lipopeptides were also adjusted by the culture temperature: the content of the fengycins decreased by 96.6% and that of the surfactins ascended by 59.9% from 20 o C to 40 o C. The temperature of 20 o C~25 o C is the suitable temperature for FJAT-46737 to produce lipopeptide. Conclusions: The B. velezensis strain FJAT-46737 and its lipopeptides would used as new sources of potential biocontrol agents against plant pathogens, especially the bacterial wilt pathogen R. solanacearum .

scholarly journals Biocontrol of tomato bacterial wilt by a new strain Bacillus velezensis FJAT-46737 and its lipopeptides

2019 ◽  
Author(s):  
Meichun Chen ◽  
Jieping Wang ◽  
Bo Liu ◽  
Yujing Zhu ◽  
Rongfeng Xiao ◽  
...  
Keyword(s):  
Bacterial Wilt ◽  
Plant Pathogens ◽  
Antimicrobial Agents ◽  
Gene Clusters ◽  
Antimicrobial Activities ◽  
Whole Genome Sequence ◽  
Dependent Manner ◽  
Bacillus Velezensis ◽  
Tomato Bacterial Wilt

Abstract Background There is an urgent need to discover alternative antimicrobial agents to control bacterial wilt. The objectives of this study were to report a new lipopeptide-producding biocontrol strain FJAT-46737 and its lipopeptides, and to investigate their antagonistic effects.Results Based on the whole genome sequence analysis, the new strain FJAT-46737 was finally identified as Bacillus velezensis and seven gene clusters that responsible for synthesis of bioactive secondary metabolite in the FJAT-46737 were predicted. Antimicrobial results demonstrated that FJAT-46737 exhibited broad-spectrum antimicrobial activities in vitro against bacteria and fungi. The pot experiments showed that the tomato bacterial wilt control efficiencies of the whole cultures, the 2-fold diluted supernatants and the crude lipopeptide of FJAT-46737 were 66.2%, 82.0%, and 96.2%, respectively. The above results suggested that one of the antagonistic mechanisms of FJAT-46737 was secretion of lipopeptides that consisted of iturins, fengycins and surfactins. The crude lipopeptides had significant antagonistic activities on several pathogen in a dosage-dependent manner, and the fengycins confirmed to play a major role in antibacterial abilities in vitro of the lipopeptides against Ralstonia solanacearum . Furthermore, it was found that the rich organic nitrogen sources (expecially yeast extracts) in the media were benificial for FJAT-46737 to produce fengycin and surfactin. The secretion of these two lipopeptides were also adjusted by the culture temperature: the content of the fengycins decreased by 96.6% and that of the surfactins ascended by 59.9% from 20 o C to 40 o C. And 20 o C~25 o C is the suitable temperature for FJAT-46737 to produce lipopeptide.Conclusions The B. velezensis strain FJAT-46737 and its lipopeptides would used as new sources of potential biocontrol agents against plant pathogens, especially the bacterial wilt pathogen R. solanacearum .

scholarly journals Biocontrol of tomato bacterial wilt by the new strain Bacillus velezensis FJAT-46737 and its lipopeptides

2020 ◽  
Author(s):  
Meichun Chen ◽  
Jieping Wang(New Corresponding Author) ◽  
Bo Liu(Former Corresponding Author) ◽  
Yujing Zhu ◽  
Rongfeng Xiao ◽  
...  
Keyword(s):  
Bacterial Wilt ◽  
Plant Pathogens ◽  
Antimicrobial Agents ◽  
Nitrogen Sources ◽  
Gene Clusters ◽  
Antimicrobial Activities ◽  
Whole Genome Sequence ◽  
Bacillus Velezensis ◽  
Tomato Bacterial Wilt

Abstract Background: There is an urgent need to discover alternative antimicrobial agents to control bacterial wilt. This study reports on a new lipopeptide-producing biocontrol strain FJAT-46737 and explores its lipopeptidic compounds, and this study investigates the antagonistic effects of these compounds. Results: Based on a whole genome sequence analysis, the new strain FJAT-46737 was identified as Bacillus velezensis, and seven gene clusters responsible for the synthesis of bioactive secondary metabolites in FJAT-46737 were predicted. The antimicrobial results demonstrated that FJAT-46737 exhibited broad-spectrum antimicrobial activities in vitro against three bacteria and three fungi. Pot experiments showed that the control efficiencies for tomato bacterial wilt of the whole cultures, the 2-fold diluted supernatants and the crude lipopeptide of FJAT-46737 were 66.2%, 82.0%, and 96.2%, respectively. The above results suggested that one of the antagonistic mechanisms of FJAT-46737 was the secretion of lipopeptides consisting of iturins, fengycins and surfactins. The crude lipopeptides had significant antagonistic activities against several pathogens (including Ralstonia solanacearum, Escherichia coli and Fusarium oxysporum) and fengycins were the major antibacterial components of the lipopeptides against R. solanacearum in vitro. Furthermore, the rich organic nitrogen sources (especially yeast extracts) in the media promoted the production of fengycin and surfactin by FJAT-46737. The secretion of these two lipopeptides was related to temperature fluctuations, with the fengycin content decreasing by 96.6% and the surfactins content increasing by 59.9% from 20 oC to 40 oC. The optimal temperature for lipopeptide production by FJAT-46737 varied between 20 oC and 25oC. Conclusions: The B. velezensis strain FJAT-46737 and its lipopeptides represent new sources of potential biocontrol agents against plant pathogens, especially the bacterial wilt pathogen R. solanacearum.

scholarly journals Biocontrol of tomato bacterial wilt by the new strain Bacillus velezensis FJAT-46737 and its lipopeptides

2020 ◽  
Author(s):  
Meichun Chen ◽  
Jieping Wang ◽  
Bo Liu ◽  
Yujing Zhu ◽  
Rongfeng Xiao ◽  
...  
Keyword(s):  
Bacterial Wilt ◽  
Plant Pathogens ◽  
Nitrogen Sources ◽  
Gene Clusters ◽  
Antimicrobial Activities ◽  
Biocontrol Agents ◽  
Whole Genome Sequence ◽  
The Rich ◽  
Tomato Bacterial Wilt

Abstract Background: There is an urgent need to discover biological biocontrol agents to control bacterial wilt. This study reports on a new lipopeptide-producing biocontrol strain FJAT-46737 and explores its lipopeptidic compounds, and this study investigates the antagonistic effects of these compounds.Results: Based on a whole genome sequence analysis, the new strain FJAT-46737 was identified as Bacillus velezensis, and seven gene clusters responsible for the synthesis of bioactive secondary metabolites in FJAT-46737 were predicted. The antimicrobial results demonstrated that FJAT-46737 exhibited broad-spectrum antimicrobial activities in vitro against three bacteria and three fungi. Pot experiments showed that the control efficiencies for tomato bacterial wilt of the whole cultures, the 2-fold diluted supernatants and the crude lipopeptide of FJAT-46737 were 66.2%, 82.0%, and 96.2%, respectively. The above results suggested that one of the antagonistic mechanisms of FJAT-46737 was the secretion of lipopeptides consisting of iturins, fengycins and surfactins. The crude lipopeptides had significant antagonistic activities against several pathogens (including Ralstonia solanacearum, Escherichia coli and Fusarium oxysporum) and fengycins were the major antibacterial components of the lipopeptides against R. solanacearum in vitro. Furthermore, the rich organic nitrogen sources (especially yeast extracts) in the media promoted the production of fengycin and surfactin by FJAT-46737. The secretion of these two lipopeptides was related to temperature fluctuations, with the fengycin content decreasing by 96.6% and the surfactins content increasing by 59.9% from 20 oC to 40 oC. The optimal temperature for lipopeptide production by FJAT-46737 varied between 20 oC and 25oC. Conclusions: The B. velezensis strain FJAT-46737 and its secreted lipopeptides could be used as new sources of potential biocontrol agents against several plant pathogens, and especially the bacterial wilt pathogen R. solanacearum.

scholarly journals Biocontrol of tomato bacterial wilt by the new strain Bacillus velezensis FJAT-46737 and its lipopeptides

2020 ◽  
Author(s):  
Meichun Chen ◽  
Jieping Wang ◽  
Bo Liu ◽  
Yujing Zhu ◽  
Rongfeng Xiao ◽  
...  
Keyword(s):  
Bacterial Wilt ◽  
Plant Pathogens ◽  
Nitrogen Sources ◽  
Gene Clusters ◽  
Antimicrobial Activities ◽  
Biocontrol Agents ◽  
Whole Genome Sequence ◽  
The Rich ◽  
Tomato Bacterial Wilt

Abstract Background: There is an urgent need to discover alternative Bacillus biocontrol agents to control bacterial wilt. This study reports on a new lipopeptide-producing biocontrol strain FJAT-46737 and explores its lipopeptidic compounds, and this study investigates the antagonistic effects of these compounds.Results: Based on a whole genome sequence analysis, the new strain FJAT-46737 was identified as Bacillus velezensis, and seven gene clusters responsible for the synthesis of bioactive secondary metabolites in FJAT-46737 were predicted. The antimicrobial results demonstrated that FJAT-46737 exhibited broad-spectrum antimicrobial activities in vitro against three bacteria and three fungi. Pot experiments showed that the control efficiencies for tomato bacterial wilt of the whole cultures, the 2-fold diluted supernatants and the crude lipopeptide of FJAT-46737 were 66.2%, 82.0%, and 96.2%, respectively. The above results suggested that one of the antagonistic mechanisms of FJAT-46737 was the secretion of lipopeptides consisting of iturins, fengycins and surfactins. The crude lipopeptides had significant antagonistic activities against several pathogens (including Ralstonia solanacearum, Escherichia coli and Fusarium oxysporum) and fengycins were the major antibacterial components of the lipopeptides against R. solanacearum in vitro. Furthermore, the rich organic nitrogen sources (especially yeast extracts) in the media promoted the production of fengycin and surfactin by FJAT-46737. The secretion of these two lipopeptides was related to temperature fluctuations, with the fengycin content decreasing by 96.6% and the surfactins content increasing by 59.9% from 20 oC to 40 oC. The optimal temperature for lipopeptide production by FJAT-46737 varied between 20 oC and 25oC. Conclusions: The B. velezensis strain FJAT-46737 and its secreted lipopeptides could be used as new sources of potential biocontrol agents against several plant pathogens, and especially the bacterial wilt pathogen R. solanacearum.

scholarly journals Streptomyces sp. JCK-6131 Protects Plants Against Bacterial and Fungal Diseases via Two Mechanisms

2021 ◽  
Vol 12 ◽  
Author(s):  
Khanh Duy Le ◽  
Jeun Kim ◽  
Hoa Thi Nguyen ◽  
Nan Hee Yu ◽  
Ae Ran Park ◽  
...  
Keyword(s):  
Bacterial Wilt ◽  
Foliar Application ◽  
Antagonistic Activity ◽  
Fungal Diseases ◽  
Dependent Manner ◽  
Minimum Concentration ◽  
Bacteria And Fungi ◽  
Tomato Bacterial Wilt

Plant bacterial and fungal diseases cause significant agricultural losses and need to be controlled. Beneficial bacteria are promising candidates for controlling these diseases. In this study, Streptomyces sp. JCK-6131 exhibited broad-spectrum antagonistic activity against various phytopathogenic bacteria and fungi. In vitro assays showed that the fermentation filtrate of JCK-6131 inhibited the growth of bacteria and fungi with minimum concentration inhibitory (MIC) values of 0.31–10% and 0.31–1.25%, respectively. In the in vivo experiments, treatment with JCK-6131 effectively suppressed the development of apple fire blight, tomato bacterial wilt, and cucumber Fusarium wilt in a dose-dependent manner. RP-HPLC and ESI-MS/MS analyses indicated that JCK-6131 can produce several antimicrobial compounds, three of which were identified as streptothricin E acid, streptothricin D, and 12-carbamoyl streptothricin D. In addition, the disease control efficacy of the foliar application of JCK-6131 against tomato bacterial wilt was similar to that of the soil drench application, indicating that JCK-6131 could enhance defense resistance in plants. Molecular studies on tomato plants showed that JCK-6131 treatment induced the expression of the pathogenesis-related (PR) genes PR1, PR3, PR5, and PR12, suggesting the simultaneous activation of the salicylate (SA) and jasmonate (JA) signaling pathways. The transcription levels of PR genes increased earlier and were higher in treated plants than in untreated plants following Ralstonia solanacearum infection. These results indicate that Streptomyces sp. JCK-6131 can effectively control various plant bacterial and fungal diseases via two distinct mechanisms of antibiosis and induced resistance.

scholarly journals Isolation, Identification, and Complete Genome Assembly of an Endophytic Bacillus velezensis YB-130, Potential Biocontrol Agent Against Fusarium graminearum

2020 ◽  
Vol 11 ◽  
Author(s):  
Wen Xu ◽  
Liyong Zhang ◽  
Paul H. Goodwin ◽  
Mingcong Xia ◽  
Jie Zhang ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
Biological Control Agent ◽  
Gene Clusters ◽  
Whole Genome Sequence ◽  
Yield Reduction ◽  
Rrna Gene ◽  
Whole Genome ◽  
Bacillus Velezensis ◽  
Fungal Plant Pathogens ◽  
Dual Cultures

Wheat scab caused by F. graminearum is a highly destructive disease that leads to yield reduction and mycotoxin contamination of grains. In this study, an endophytic bacterium of strain YB-130 was isolated from surface sterilized wheat spikes with scab symptoms and identified as Bacillus velezensis by whole genome annotation, 16S rRNA gene and average nucleotide identities analysis. The whole-genome sequence of strain YB-130 was obtained by PacBio sequencing. 88 putative Carbohydrate-Active Enzymes and 12 gene clusters encoding for secondary metabolites were identified in the YB-130 genome, including one gene cluster for the synthesis of lanthipeptide only found in strain YB-130 genome. In dual cultures, strain YB-130 significantly inhibited the growth of F. graminearum PH-1 and other eight fungal plant pathogens, indicating a broad antifungal activity. Furthermore, strain YB-130 was able to significantly inhibit spore morphology and hyphal development of F. graminearum PH-1. Strain YB-130 also reduced deoxynivalenol production by F. graminearum PH-1 in dual cultures, possibly due to its ability to suppress the expression of tri5, tri3, and tri8 that are required for deoxynivalenol production in F. graminearum. Overall, B. velezensis YB-130 is a promising biological control agent of both F. graminearum infection and mycotoxin production.

scholarly journals Stachytarpheta indica Leaf Extract: Oral Acute Toxicity, In vitro Phytochemical and Antimicrobial Potentials

2019 ◽  
pp. 163-173
Author(s):  
Achimugu Dickson Musa ◽  
Cyril Ogbiko ◽  
Musa Usman Dabai ◽  
Ibeabuchi Jude Ali ◽  
Abubakar Sani Yelwa ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Acute Toxicity ◽  
Leaf Extract ◽  
Antimicrobial Agents ◽  
Antimicrobial Activities ◽  
Phytochemical Analysis ◽  
Dependent Manner ◽  
Antimicrobial Drugs ◽  
Oral Acute Toxicity

This study was designed to ascertain the safety profile as well as explore new antimicrobial agents from the methanol leaf extract of Stachytarpheta indica. The methanol extract was prepared and screened for its phytochemical composition, oral acute toxicity profile as well as challenged with common pathogenic microorganisms for its antimicrobial activities using standard procedures. The phytochemical analysis revealed the presence of various pharmaceutically active secondary metabolites like alkaloids, saponins, carbohydrate, cardiac glycosides, terpenoid, tannin, anthraquinones, phenolics and flavonoid. P. aeruginosa and S. aureus showed the best and least antibacterial activity respectively. The extract exhibited antifungal activity in a dose dependent manner with the inhibition more pronounced with A. niger than with C. albicans. The standard drugs ciprofloxacin and fluconazole exhibited a near 100% activity. There was no mortality at 8 g/kg p.o. after 24 hours and no sign of delayed toxicity or mortality after 14 days of observation. There were no statistically significant differences (p > 0.05) observed in the examined organs as well as body weights in both 24 h and 14 d study. This research holds promise for the exploration of various potentially active secondary metabolites which would help in developing pharmaceuticals especially antimicrobial drugs as well as recommend the short-term oral administration of the extract up to a dose of 8 g/kg body weight.

Synthesis of Novel 3(N,N-dialkylamino)alkyl/phenyl Substituted Thieno [2,3-d]pyrimidinones as H1-Anti-Histaminic and Antimicrobial Agents

2019 ◽  
Vol 15 (1) ◽  
pp. 63-70
Author(s):  
Shiv Dev Singh ◽  
Arvind Kumar ◽  
Firoz Babar ◽  
Neetu Sachan ◽  
Arun Kumar Sharma
Keyword(s):  
Antimicrobial Activity ◽  
Potassium Hydroxide ◽  
Antimicrobial Agents ◽  
Pyrimidine Ring ◽  
Antimicrobial Activities ◽  
Screening Methods ◽  
Chlorpheniramine Maleate ◽  
In Vivo Screening

Background: Thienopyrimidines are the bioisoster of quinazoline and unlike quinazoline exist in three isomeric forms corresponding to the three possible types annulation of thiophene to the pyrimidine ring viz thieno[2,3-d] pyrimidine, thieno[3,2-d] pyrimidine and thieno[3,4-d]pyrimidine. Heterocyclic containing the thienopyrimidinone moiety exhibits various pronounced activities such as anti-hypertensive, analgesic and anti-inflammatory, antiviral, platelet aggregation inhibitory, antiprotozoal bronchodilatory, phosphodiesterase inhibitory, antihistaminic, antipsychotic and antimicrobial activity. Objective: Synthesis of novel 3(N,N-dialkylamino)alkyl/phenyl substituted thieno[2,3-d]pyrimidinones as H1-anti-histaminic and antimicrobial agents. Methods: A series of 3-[(N,N-dialkylamino)alkyl/phenyl]-2-(1H)thioxo-5,6,7,8-tetrahydrobenzo(b) thieno(2,3-d)pyrimidine-4(3H)-ones[4a-d], their oxo analogous [5a-d] and 3-[(N,N-dialkylamino)alkyl]- 2-chlorophenyl-5,6,7,8-tetrahydrobenzo(b)thieno(2,3-d)pyrimidine- 4 (3H)-ones[6a-d]derivative were synthesized from 2-amino-4,5,6,7-tetrahydrobenzo(b)thiophene-3-carboxylic acid by nucleophilic substitution of different N,N-dialkyl alkylene/phenylene diamines on activated 3-acylchloride moiety followed by cyclocondensation with carbon disulfide and ethanolic potassium hydroxide to get [4a-d] and in second reaction by condensation with 4-chlorobenzoyl chloride to get [6a-d] by single pot novel innovative route. The oxo analogous [5a-d] were prepared by treating derivatives [4a-d] with potassium permagnate in ethanolic KOH. The synthesized compound were evaluated for H1-antihistaminic and antimicrobial activities. Results: All synthesized compounds exhibited significant H1-antihistaminic activity by in vitro and in vivo screening methods and data were verified analytically and statistically. The compound 4a, 4b, 5a and 5b showed significant H1-antihistaminiic activity than the reference standard chlorpheniramine maleate. The compound 6d, 6c, 5c and 4c exhibited significant antimicrobial activity.

scholarly journals A plant endophyte Staphylococcus hominis strain MBL_AB63 produces a novel lantibiotic, homicorcin and a position one variant

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
M. Aftab Uddin ◽  
Shammi Akter ◽  
Mahbuba Ferdous ◽  
Badrul Haidar ◽  
Al Amin ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Antimicrobial Activities ◽  
Whole Genome Sequence ◽  
Antimicrobial Substances ◽  
The Core ◽  
Rp Hplc ◽  
Ring Structures ◽  
Core Peptide ◽  
Staphylococcus Hominis ◽  
Epilancin 15X

AbstractHere we report a jute endophyte Staphylococcus hominis strain MBL_AB63 isolated from jute seeds which showed promising antimicrobial activity against Staphylococcus aureus SG511 when screening for antimicrobial substances. The whole genome sequence of this strain, annotated using BAGEL4 and antiSMASH 5.0 to predict the gene clusters for antimicrobial substances identified a novel antimicrobial peptide cluster that belongs to the class I lantibiotic group. The predicted lantibiotic (homicorcin) was found to be 82% similar to a reported peptide epicidin 280 having a difference of seven amino acids at several positions of the core peptide. Two distinct peaks obtained at close retention times from a RP-HPLC purified fraction have comparable antimicrobial activities and LC–MS revealed the molecular mass of these peaks to be 3046.5 and 3043.2 Da. The presence of an oxidoreductase (homO) similar to that of epicidin 280- associated eciO or epilancin 15X- associated elxO in the homicorcin gene cluster is predicted to be responsible for the reduction of the first dehydrated residue dehydroalanine (Dha) to 2-hydroxypropionate that causes an increase of 3 Da mass of homicorcin 1. Trypsin digestion of the core peptide and its variant followed by ESI–MS analysis suggests the presence of three ring structures, one in the N-terminal and other two interlocking rings at the C-terminal region that remain undigested. Homicorcin exerts bactericidal activity against susceptible cells by disrupting the integrity of the cytoplasmic membrane through pore formation as observed under FE-SEM.

scholarly journals Microbial Transformations of Isophorone by Alternaria alternata and Neurospora crassa

2013 ◽  
Vol 8 (1) ◽  
pp. 1934578X1300800 ◽  
Author(s):  
Ismail Kiran ◽  
Özge Özşen ◽  
Turgay Çelik ◽  
Semra İlhan ◽  
Bükay Yenice Gürsu ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Alternaria Alternata ◽  
Antimicrobial Agents ◽  
Antimicrobial Activities ◽  
Anti Oxidant ◽  
Agar Dilution ◽  
Important Field ◽  
Pharmacology And Toxicology ◽  
Derivatives Of

Isophorone (3,5,5-trimethyl-2-cyclohexen-1-one), a monoterpene, and the structurally related 1,8-cineole and camphor, have demonstrated a protective effect against cancer, biological activity against a variety of microorganisms, and anti-oxidant properties. The derivatization of isophorone is, therefore, an important field of xenobiochemistry, pharmacology and toxicology. The aim of this study was to obtain derivatives of isophorone through microbial biotransformation and evaluate the biotransformation metabolites as potential antimicrobial agents. Incubation of isophorone with the fungi Alternaria alternata and Neurospora crassa afforded 4α-hydroxy- and 7-hydroxy-isophorone as transformation metabolites. The antimicrobial activities of isophorone and the metabolites were evaluated in vitro both by using agar dilution and microdilution methods. However, no significant antibacterial activity was observed when compared with those of standard substances.

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