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环境分析

Volatile profilesoffungi –Chemotyping ofspecies and ecological functions

1.Introduction
Volatile organic compound (VOC)emissions play important eco- logical and physiolog  ical roles for many organisms.Also fungi release alarge spectrum ofVOCs (Splivallo etal.,2011;Kramer and Abraham,2012  ).Fungal VOC emission  sbelong toseveral chemical groups with different biochemi  cal origins such as monoterpenes, sesquiterpenes, alcohols, aldehydes , aromatic compounds,esters,furans,hydrocarbons,ketones,aswell asnitrogen- and sulfur-con- taining compounds (Splivallo etal.,2007a;Campos etal.,2010;Kramer and Abraham  ,2012  ).

2.Materials and methods
2.1.Fungal species and growth

The measure  ments inthis study were performed onthe mycelia ofnine fungal strains,four ofwhich belonged tothe EMgroup (Cenococcum geophilum  Fries, Laccaria bicolor  (Maire)P.D.Orton and the Paxillus involutus (Batsch)Fr.strains MAJ and NAU),three topathogens (Armillaria mellea (Vahl)P.Kumm., Pholiota squarrosa (Fries)Kummer and  Verticillium longispor  um (C.Stark)Karapapa,strain VL43)and two tosaprophy  tes (Stropharia rugosoannulata Farlow exMurrill and Trichoderma viride Pers).

2.2.Sampling offungal VOCs
Volatile compound  swere collected inthe headspace from all fungi grown inglass Petri dishes (diameter10cm)containing 30mlofthe modifiedMelin-Nor  krans synthetic medium described above.Each Petri dish was inoculated inthe center with asingle fungal plug (diameter1cm)and incubated at24 Cinpermanent darkness (exceptfor the short moment the cultures were taken tolight for measuring the diameter ofthe colonies).Control plates without fungal inocula were incubated under the same conditions.The fungal diameter was measured every six days.

2.3.Analyses ofVOCs
The samples were analyzed with athermo-des  orption unit (GerstelGmbH &Co.KG,Mülheimander Ruhr,Germany)coupled toagas chromatograph-  mass spectromete  r(GC–MS;GCmodel:7890A;MSmodel:5975C;Agilent Technologie  s,Santa Clara,CA,USA).The fungal volatiles inthe samples were desorbed from 30 Cto250 Catarate of400 Cmin 1
,followed byaholding time of2min.The compounds were refocused onTenax (cryo-cooling technique)at 50 Cand desorbed to250 Catarate of12 Cs1,followed byaholding time of3min.Compounds were separated using acapillary GCcolumn ((14%-Cyanopropyl-phe- nyl)-methylpolysiloxan  e;70m  250 lm,filmthickness 0.25 lm;Agilent J&W 122-5562G,DB-5MS +10mDG).The carrier gas was helium with aconstant flowrate of1.2 mlmin 1.The GCoven temperat  ure was held at40 Cfor 2min,then increased to80 Catarate of6 Cmin 1,then to170 Catarate of3.4 Cmin 1
and finallyto300 Catarate of12 Cmin 1.

2.4.Statistical analyses
The differences between the VOC concentrations ofthe fungal species and functional fungal groups were tested using the linear mixed model procedure ofthe IBM SPSS Statistics version 19.0 for Windows (Armonk,NY,USA).The procedures were performed inrepeated measureme  nts onthe VOC groups and included thefungalspeciesorfunctional fungal group and collection asfixed factors,with the factor collection taking the repeated measurements into account.The interactions between the fixedfactors were kept with a P-value <0.15 (Faubert etal.,2012  ).The grouped VOC emissions were compare  dbetween fungal species orfunctional fungal groups using a post hocBonferroni t test,following the mixed model analysis.The fungal plate represented the unit ofreplicatio  n(n =5).Differences between fungal species orfunc- tional fungal groups were considered statistical  lysignificantwith
P-values <0.05.The visualization ofthe relationship  sbetween the concentratio  nsofindividua  lcompound  sand the different functional fungal groups was conducted with aVenn diagram using the website Venny(Oliveros,2007  ).APearson correlation test was performed toevaluate the correlation between the sum ofVOCs emit-
ted byfungal species and their mycelium surface area atthe end of the growing period.


3.Results

3.1.Fungal growth and ITS sequence.

3.2.The volatile pattern from eight fungal species

3.3.Chemotypin  goffungal species and functional fungal groups

4.Discussion

4.1.Diversity offungal VOC emissions

4.2.VOC emissions from EMfungi

4.3.VOC emissions from pathogen

4.4.VOC emissions from saprophytes

4.5.Chemotypin  gbased onVOC emissions

References

Agger,S.,Lopez-Gallego,F.,Schmidt-Dannert,C.,2009.Diversity ofsesquiterpene synthases inthe basidiomycete  Coprinus cinereus  .Mol.Microbiol.72,1181–1195.

Bäck,J.,Aaltonen,H.,Hellén,H.,Kajos,M.,Patokoski,J.,Taipale,R.,Pumpanen,J.,Heinonsalo,J.,2010.Variable emissions ofmicrobial volatile organic compounds (MVOCs)from root-associated fungi isolated from Scotspine.Atmos.Environ.44,3651–3659.