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=Biotechnology and Bioprocess Engineering 2007,12:318-322 d=f=b=m=m=cI=Alternaria maliI=_=hJe=gG=Division of Food and Biotechnology,Chungju National University,Chungju 380-702,Korea=We investigated the growth inhibition effect of pyroligneous acid on the pathogenic fungus,=,which is=known to be the agent of Alternaria blotch of apple plants.Chemical control of K=could be achieved through the use of agro-chemical fungicides,while the substitute for agrochemical control is gradually increasing.It was observed that pyroligneous acid exhibited antifungal activity against some plant pathogenic fungi.More specifically,the growth of K=was com-pletely inhibited in pyroligneous acid at a dilution of 1:32.When its antifungal activity was compared to that of polyoxin B,which is used for the chemical control of Alternaria blotch of apple,it was observed that the antifungal activity of pyroligne-ous acid diluted at 1:32 corresponded to 2.0 mg/mL of polyoxin B.Consequently,it is concluded that the diluted pyroligne-ous acid can substitute for polyoxin B,thereby reducing the use of the agrochemical for the control of Alternaria blotch of apple.KSBB hW=,=,Alternaria mali,=,=Pyroligneous acid(also called wood vinegar)is a reddish-brown wood distillate that contains acetic acid,methanol,fualdehydes,and phenols 1-5.This distillate is particularly obtained from a by-product of charcoal production from woods such as oak(Quercus acutissima).The wood vinegar from sapwood consisted of methanol(15.34 mg/mL)and carboxylic acids(43.77 mg/mL)as a major component.Ad-ditionally,furaldehydes(2-furaldehyde,5-methyl-2-furaldehyde,5-hydroxymethyl-2-furaldehyde,etc.;2.34 mg/mL),phenols(phenol,pyrocatechol,p-cresol,3,5-xylenol,etc.;8.38 mg/mL),guaiacols(guaiacol,4-methyl guaiacol,vanillin,etc.;7.59 mg/mL)were observed as constituents of pyroligneous acid 6.Because of its antimicrobial activity 6,7 and the increasing necessity of the substitution of agro-chemical within the agricultural industry,pyroligneous acid is being studied for use in the improvement of productivity in the agricultural and livestock industries 8-12.The appli-cation of pyroligneous acid has been attempted in studies for food preservation 13 and particularly in the removal of odors 14.Recently,its medicinal use has been studied in-tensively in the field of oriental medical science 15-17,the way some natural resources has been done for investigating G=Tel:[phone] Fax:[phone] e-mail:khjungcjnu.ac.kr the biological activities 18-21.In this report,the growth inhibition effect of pyroligneous acid on pathogenic fungus,Alternaria mali(A.mali),was investigated.This fungus is known to be the agent of Alter-naria blotch of apple 22.While Alternaria is,in Europe,a very minor fruit-rotting fungus of apples,affecting only fruits that are already damaged,A.mali is important in the Far East because it causes disease of both the leaf and fruit.It can infect up to 85%of leaves on susceptible cultivars,compared with less than 1%on resistant cultivars 23.In the USA,the disease is only important in regions where Asian A.mali has been introduced(North Carolina)24.Lesions of Alternaria blotch in apple first appear on leaves or fruits in late spring or early summer;with plants showing small,round,purplish,or blackish spots,which gradually become enlarged to 1.55 mm in diameter,with a brownish-purple border 25.Chemical control of A.mali can be achieved through the use of fungicides such as iprodione,mancozeb,and captan 26,27.In addition,the use of fungicides such as mancozeb,ziram,thiram,oxine-copper,iminoctadine,di-fenoconazole,tebuconazole,chlorothalonil,and polyoxin B against Alternaria blotch has been reported 28-31.How-ever,alternative approaches for chemical control of A.mali have not been intensively studied,while the need for envi-ronmentally friendly methods of controlling A.mali continue to increase.In this report,we demonstrated that pyroligneous Biotechnol.Bioprocess Eng.PNV=q=N.Pathogenic fungi used in this study Pathogenic fungi Targets Diseases Abbreviations=KACC 40026 Apple Alternaria leaf spot AM s=KACC=40331 Apple Valsa canker VC d=KACC 40299 Apple,peach Bitter rot GC m=KACC 40839 Apple,peach Die-back PM _=KACC 40573 Apple,peach Gray mold BC cK=NK=Growth inhibitions of Gram(+)and Gram()bacteria in tryptic soy broth(TSB)with pyroligneous acid.1;TSB was prepared with distilled water(control),2;TSB was prepared only with pyroligneous acid,3;TSB was pre-pared with pyroligneous acid diluted 1:2,4;TSB was prepared with pyroligneous acid diluted 1:4,5;TSB was prepared with pyroligneous acid diluted 1:8.EC;=bJ=ATCC 11775.SA;p=subsp.ATCC 25923.OD at 600 nm was meas-ured after 24-h culture.acid inhibited the growth of the plant pathogenic fungus,A.mali.Pyroligneous acid was purchased from Geumga Char-coal Co.(Chungju,Korea).Polyoxin B(Dongbu Hannong Chemicals,Korea)was compared to pyroligneous acid in terms of its antifungal activity.The antibacterial effect was tested by using Staphylococcus aureus subsp.aureus ATCC 25923 and Escherichia coli ATCC 11775 as Gram(+)and Gram()bacteria,respectively.Liquid cul-tures for seed preparation and antibacterial testing were prepared in 3%(w/v)tryptic soy broth(TSB)(Becton Dickinson,USA)at 37oC and pH 7.0.The pH of pyrolig-neous acid was adjusted to 7.0 by the addition of 5 M NaOH.Liquid culture media were prepared by mixing both pyroligneous acid and 3%(w/v)TSB;media were then sterilized using a 0.22 m filter.Five milliliters of the ster-ilized media were dispensed into a sterilized 15 mL test tube.For seed preparation of S.aureus subsp.aureus ATCC 25923 and E.coli ATCC 11775,two shake flask cultures with volumes of 100 mL were carried out at 37oC and 150 rpm overnight,in which one colony of each of the bacteria that had been grown on TSB agar plates was used as seed for seed flask culture.Five L of the seed cultures were then inoculated into 5 mL of TSB media containing pyroligneous acid.After 24 h culturing of these test tubes in a shaking incubator at 37oC and 150 rpm,the OD at 600 nm of each test tube was measured.The antifungal effect of pyroligneous acid was investi-gated against five plant pathogenic fungi(Table 1),which were kindly donated by KACC(Korean Agricultural Culture Collection).For seed cultures of these fungi,a loop of fungi mycelium cultured on a malt extract(Becton Dickinson)agar(MEA,1.7%,w/v malt extract and 2%,w/v agar)plate(60 15 mm)was inoculated into 10 mL of malt extract liquid medium in a sterile 50 mL conical tube(Greiner Bio-One,USA).These five seed cultures were cultured in a shaking incubator at 25oC and 150 rpm for 23 days.After the mycelium growth was confirmed visually,a sterilized filter paper disc(8 mm,Advantec,Japan)was soaked in 10 mL of malt extract liquid medium,followed by gentle shak-ing for one minute in order to allow effective absorption of fungi mycelium.Filter paper was then placed on the center of the pyroligneous acid-containing MEA plate(60 15 mm).The MEA plates were incubated at 25oC for 6 days,and the degree of mycelia growth on MEA plate was ob-served.As shown in Fig.1,the antibacterial effect of pyroligne-ous acid was insignificant,as indicated by the inhibition of growth of Gram(+)and Gram()bacteria when TSB was prepared with only pyroligneous acid.When TSB was pre-pared with the diluted pyroligneous acid,the antibacterial effect decreased in a dose-dependent manner.However,it was observed that pyroligneous acid exhibited antifungal activity against some plant pathogenic fungi(Table 2).The growth of A.mali was completely inhibited at a 1:32 dilution of pyroligneous acid,and the growth of Botrytis cinerea(agent of gray mold)was completely inhibited at a dilution of 1:4.Complete inhibition of the growths of Glomerella cingulata,Valsa ceratosperma,and Phomopsis mali oc-curred at pyroligneous acid dilutions of 1:16,1:8,and 1:16,respectively.These results demonstrated that pyroligneous acid inhibited the growth of A.mali more effectively at a lower concentration,compared to other fungi.Fig.2 shows the growth inhibition of pathogenic fungus,A.mali,on malt extract agar(MEA)with pyroligneous acid(diluted 1:16 to 1:64).To compare the antifungal activity of pyroligneous acid to that of an agrochemical,polyoxin B,POM=q=O.Degree of growth inhibition of pathogenic fungi on malt extract agar(MEA)with pyroligneous acid Degree of growth inhibitiona Dilution of PLAb AMc BC GC VC PM 1:2 1:4 1:8 +1:16 +1:32 +1:64+Controld+a,No growth;+,radial growth from disc filter paper is less than half of the radius of MEA plate,+;radial growth from disc filter paper is more than half of the radius of MEA plate and did not cover the entire MEA plate,+;growth from disc filter paper covered the entire MEA plate.bPLA is an abbreviation for pyroligneous acid,and PLA at dilutions from 1:2 to 1:64 were investigated in this experiment.cPathogenic fungi are described as in Table 1.dMEA was prepared only with distilled water.=_=a cK=OK Growth inhibition of the pathogenic fungus,=,=on malt extract agar(MEA)with pyroligneous acid.(A)MEA was prepared with pyroligneous acid diluted 1:16,(B)MEA was prepared with pyroligneous acid diluted 1:32,(C)MEA was prepared with pyroligneous acid diluted 1:64,(D)MEA was prepared with distilled water(control).which is used for the control of Alternaria blotch of apple,the antifungal activity of polyoxin B against A.mali was investigated using the same method.As shown in Table 3,the minimum inhibitory concentration(MIC)of polyoxin B was at least 2.0 mg/mL.However,at a polyoxin B concen-tration of 1.0 mg/mL,the recommended concentration de-scribed in an insert of this agrochemical product,the growth q=PK=Degree of growth inhibition of=on malt extract agar(MEA)with polyoxin B Polyoxin B(mg/mL)Degree of growth inhibition of AMa2.0 1.0b+0.5+0.25+0.125+0.0625+0.03125+Controlc+aAM is an abbreviation for=.Degree of growth inhibition was determined as the principle described in the footnote for Table 2.bPolyoxin B at 1.0 mg/mL is a recommended concentration described in an insert of this agrochemical product.cMEA was prepared only with distilled water.of A.mali was not fully inhibited.Therefore,it was noted that the antifungal activity of a 1:32 dilution of pyroligneous acid against A.mali is the same as that of polyoxin B of 2.0 mg/mL.Consequently,it is concluded that polyoxin B,an agrochemical used for the control of Alternaria blotch of ap-ple,can be substituted by a 1:32 dilution of pyroligneous acid solution.A 1:32 dilution of pyroligneous acid corresponds to 30.3 mg/mL as MIC.Hwang et al.6 and Lee et al.11 had reported MICs of wood vinegar against some plant patho-genic fungi as 5 to 20(Rolstonia solanacearum,Phy-tophthora capsici,Fusarium oxysporum,Pythium splendens)and 10 to 100 mg/mL(Trichoderma harzianum,Rhizina un-dulata,Rhizoctonia solani),respectively.These MIC values have very similar to that of a 1:32 dilution of pyroligneous acid.However,those could be not compared quantitatively with polyoxin B as MIC,because pyroligneous acid is aque-ous solution containing several chemicals and polyoxin B is single agrochemical.When compared the constituents of pyroligneous acid with several antifungal phytochemicals in databases,it could be deduced that furaldehydes and phenols in pyroligneous acid were major constituents for antifungal activity in view of their MICs 6,7,32,33.The inland area of Korea(Chungju,Moonkyung,etc.)is a major region of apple production,and apple is the represen-tative fruit of this region.Because the production of apples in this region sits at the center of the regional economy,the control of A.mali is of significance to increase apple produc-tion yields and produce high-quality apples.It is concluded that the diluted pyroligneous acid will be helpful for the con-trol of plant pathogenic fungus,particularly because it can be substituted for the agrochemical and thereby reduce the use of the agrochemical for the control of Alternaria blotch of apple.However,some constituents in pyroligneous acid might have harmful effect on the environment.Then it is necessary that the use and dosage of the diluted pyroligneous acid will be evaluated throughout the field test.=The author thanks Ms.Jeong-Sun Biotechnol.Bioprocess Eng.PON=Lee(Chungju Agricultural Technology Center,Chungju,Korea)for her technical support on the plant pathogenic fungi of apple.Received December 12,2006;accepted March 31,2007 obcbobkbp=1.Demirba,A.(2000)A Recent advances in biomass con-version technologies.Energy Educ.Sci.Technol.6:19-40.2.Demirbas,A.and D.Gullu(1998)Acetic acid,metha-nol and acetone from lignocellulosics by pyrolysis.En-ergy Educ.Sci.Technol.1:111-115.3.Gullu,D.and A.Demirbas(2001)Biomass to methanol via pyrolysis process.Energy Convers.Manage.42:1349-1356.4.Kim,Y.H.,S.K.Kim,K.S.Kim,and Y.H.Lee(2001)Composition of constituents of commercial wood vine-gar liquor in 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