Volい8，No‖2（1957） 157
ANTIB‡OTIC PRODUCTIONINDUCED BY2，4，5−T AND
MC‡〕iN G且0属りぶ㌘0屈，g■び二財0ムg■v過度乙冴
CAUS‡NG OuVE ANTHRACNOSE
Nakato NAITO and Toshikazu TANr（LaboratoIy OfPhytopathol？gy） （Received Decembe‡20，1956）
1．Im七roduc七io王I
Thereis an extensiveliterature relating to the activity of various plant growth・・regulators on higher
plantsAs to their stimulative orinhibitoryeftect onmic【0・Organisms，manyinvestigationshavealsobeen
made especial1yin recent years．However，Our knowledge oithe mechanismir）their activityislargely
the result of studies on higher plants，and′1ittleis known o壬the pIOCeSSin funglInIeCentpape‡S（18719）， the authors have pointed out that Gloeo．5Po′・ium oltvarumAlm（CauSing the olive anthracnose）produces a
CeItain antibiotic substance whenit was grown on media suppliedwith sodium2，4−dichloIOphenoxyacetate
（2，4・D）h At the same time，theze have been pIOVidedfivelines of evidence whichled us to conclude that 2・4−Dmay glVe ane任ect on the pathogen by this antibioticlathe‡thanbyitsdirectactiononthe organism・ Therefore，it seemed desirable here to make clear whetheISuCh character ofinciting an antibiotic produc・ tionislimitedin2，4・D alone or not
From suchpoint of view，inthe presentinvestigation simi1ar studies weIe eXtended to seveIaldther
Chemicals，nOW Widely工eCOgmized as herbicides0IphytohoIrnOneSlike2，4・DlInconsequenQeO董theres一ユ1ts， We COuld con点rm that the phenomenonin questionis caused as wellby2，4，5uT and MCP both of whichhave st工uCturalIeSemblance to2，4」｝
2The Growth Curve of Gtoぐ0哩）OTiutTlOILvE7rtLm（m Riedia co！1taining 2．4．5LT or mCP Wi抽SpeeialRefeT・e！甘Ce O三l抽e．Compariso三IW孟蝕0竜心er且erb玉cides and NAK
A motive which
2，4・Dto produce a certainantibiotic substance r・eSpOnSible foritsinhibitoIyaCtivity，WaSinthefact（17）that the gIOWth curve of the pathogen on agar media contairung2，4qD had been the“staling”、typein spite of
being the㍑non・Staling”type on controlmedia withoutit… Consequently，for the purpose of obtainillg a preliminaryinioImationastothepossibilitythatanyotheIChemicalsthan2、4・Dmighthavethesamepropet・
ty，the growth curves of the causalfungus were studiedwith media containing di仔erent concentrations of
thefo1lowingchemicals≠1：2，4，5−tIichlorophencxyaceticacid（2，4，5・T），2・methyl・4−Chlorophenoxyaceticムcid
（MCP），isopIOpyl−n−（3・Chlorophenyl）carbamate（IPC），PentaChlorophenol（PCP）and potassiumα・naPhtha・
1eneacetate（NAK）。2，4，5・T and NAK used for tests were purematerial，butinMCPandIPC，POmme‡Cial
preparationswhichcontainthe eifective compound at4l“5and451B％respectively we工e prOVidedforuseex・ Cept When stated otherwise，SO that their concentrations being calculated on the basis of these 丘gures Since the percentage of the eぽective compound was not clearin PCP preparation，SuCh considerationlWaS
not paidinits dilution。Again，2，4，5・T，工PC and PCP were added to vessels as acetonesolutionbecause Of thei工insolubilityizIWater，and media were pouredinto them after being evaporated of thesoIvent
（ヱ）0几d紗”・刀電β戯α
Each of the丘ve chemicals chosen was added respectively to peptone−Salts agar mediaヰ！Ishowing5n4 in pH togive various concentrations，and the pathogen was cultured on themin Pet【idishes at250C for
lフ days exceptIPCin whichincubated for15 days。The diamete工 Of the respective colonies was
叫1We areindebted to MrKunikazu UEKIOf this university for obtaining the former four chemicals
Tech。BullFac．Agr．Xagawa Umiv．
158
measuIedeach day。Since，however，aCetOne WaSemployed as a soIvent for thIee Of the chemicals to betested，it wasthought possible that this acetone might affect the growth・Hence，media supplied with
acetonealone so as to be5ust the same as the highestlevelin thelots were also prepared as second control．
Tablel．．Diameterin mm of colonies o董Gloeo，SPorium olivwumgIOWnOn peptOne・Salts agar media containing different concentrations offive chemicals at250C
Concentra− tion （％） Culture duration（day） Cbemical 8 10 12 7▲︻b︵UOOU3 5﹁ノ556 6563︻b6 26540 5321止 7一64321 ︻b4 ︻ノ532 6︻ノ1649 つん534つん 033︻b23 2719 260U 6421﹂ 43つ‘1 6︻〇3つ山⊥1 33 0752⊥9 6︻b967▲ ︻〇⊥602︵J 527∩︶ 654フ6■L 24024 55つ︺627 6322 4321⊥ 3︵∠︵∠1﹂ 43211 22 42︻ノ969 50232 ⊥8010︻ノ 0804 059 3っ］l⊥ つ‘l l ユ 221 11 52404∩︶ 4606︵U 58︵∠216 059ワ▲ 3 ユ 1111 1 6︻ノ4387・440U︻′−3 5775フ2 09フ47 っ山︵○つん︵∠ 0い∝）2 0小（：の3 O 004 0．01 0…02 0‖04 T 〈 Mノ0696 45819
43⊥94 43666
63つム 6421血 ︻ノ8つ］3 7一326 3つん1 96﹁ノ1 63⊥6 3︵∠1 42694 15205 フ3つ山⊥ 2，4，5− 85 01つん︵J O000﹁⊥ ∩〓UOOO ′−−−／＼−1・−1 hr C M 6904 48フ︻ノ 5︵∠⊥ 5583 2︵06フ ︻h︶21 90U53 0076︻ノ 4︵∠ュ 0．001 01．∞2 0り0025 0．004 0．0045 0．005 7474 070∩︶▲U OU3︵03 3449 9307つム 5︵∠9︻ノ フ︻b︵∠ 7542 フワ一エ 39フ3 29フ1﹂3 0U327 2 642 43 IPC ユ2．9 14．フ 9．0 ．10．1 2．8 3．4 61．2 66．5 3∠4．7 37い6 7．4 7い4 0435 444 93つムF︶ ︵J34 32⊥ 66︵ヒ 5497▲ 444 ﹁ノ683 巳︶57 ．4一44 21 457一5 054 69︰b2 7二7−OU ﹁⊥ 22へ∠ 649⊥ 69つん ︿b30︵U On︶・1 11﹂ヽ⊥ 1286 531 NAK Acetone contIOI Contr・Ol（Petri・dish） Control（Glass，tube）＋ 99．0 11．7．8 ．134．6 ユ．43．4＋Thefiguresrepresentingatwofoldlengthofcolonies ontheglass tubesweIe quOtedfrom a previous
I・epOIt（17）
As shownin Tablel，eaCh chemicalrestricted thegrowth at any givenlevel，PIOgreSSiveinhibition
being appearedwithincIeaSe Of concentration・Sincelittledi鮎renceingrowth wasdetected between the
acetonecontrolandthe prlnCipalcontrol，thereisno reasonto question the use of acetone for a soIvent・Averagedaily growth・rate On tWO COntrOIs，drawn fIOmthe負guresin Tablel，Showed thatitis nearly
constantthroughout thetestperiod．Onthe otherhand，thegrowthrateinthelotsof2，4，5−TandMCP
ingeneraldeclinedgraduaIIy asculture proceeded，WhiZethe reverse wastrue forotherchemicatswiththe
only exceptionofO“06％NAK”Consequently，thegrowthcurve forthe2，4，5・TandMCP seriesshowed
thetypeof”logaIithmic curve”（COnCaVeupWaIds），andthe one of other chemicalsgenerally resultedinthe type of“exponentialcurve”（COnVeX upWards）disregarding the excel）tionalinstance above stated
I）i鮎ringfromeitherofthetwotypes，neVertheless，COntIOIwas characterized by alinear growth・For
the sake of brevity，theirrepresentative data aregiveninFig・landFig12It should be noted here thatinal1tests not only pH value had beenalmostinvariable but also C
sources had probably remainedin a great dealeven at the end of cultureiudging from positive
Vol・8，No，2（195Lフ） 159
占岩二月山霊警莞−C
Fig‖2GrowthcurvesofGloeospori’umohvar
On peptOne・Salts agar media cont畠inlng
Variousc？nCentrations（％）ofNAKdurirg
16day，slnCubation at250C
〉 2 4 6 8 川 l：三 14 】G Days of incubation
Fig”1”Growthcurvesof Gloeosporiumolivarun？On peptone−Salts agaI media contajnlng VaIlOuS ？OnCentIations（％）of2，4，5−Tduzing16day’s incubation at 25oC
Fehling reaction of media．When the degrees of inhibition by these chemicals were expressed as index calculated by dividing diameteIOf their COlonies by that of control，multiplying the quo・ tient by100，theindexin the supply of 2，4．．5・・T and MCP was foundtodecrease progressivelyon
the whole with thelength ofincubation，but to increasein otheIChemicals，namelyindicating a gradualincIeaSe Ordec工eaSeOfinhibition degrees IeSpeCtively。 Fig3illustrates a paIt Of the data
（g）0花Jね〟ぎd朗e♂￡α
In order to make clear the growth cuIVe On
ち一芸OU︶岩馬欝H名書一言○占 0．005％クCJ＞ ヽI ヽl 。／■○
・・・・。／
0004％Jf℃・一←0／。一一
、 ＼へ ヽ ：で
ミ ぶモ 002％〟A．夕芸
●、、 ㌧ ・ ●、■・− 。・、・ −、 ■・●、．− ・、 60 002％〟A∬ 20 ′ ■1■●−−＿ 0Olク多2．4．5一丁 ■ ■ 】￠ バ 8 12 Days ofincubationFig・3Effect of5chemicals ongrowth index of Gloeo．spoYium oH2）arum．
peptone−Salts solution containingO・O15％2，4，5−T，Ol03％MCP andO．03％NAK respectively，theさathogen WaSincubated at 2フOCin EIlenmeyer 且asks of200 cc capacity，eaCh containing50cc of nutrients
SOlution Withdrawing4flasks at工andom per each chemicalat4・day−intervals，thegrowthIelativetothat in check was determined by dIy Weight（Table2）」InIegard to controlmedia，itis here seen thatthe CeaSe Of growth occured almost simultaneouslywith the riseinpHvaluefrom54to7A，andalsothat pre− Sumably carbon sources had been entirely exhaustedin view of Fehling reactionHowever，it seems un−
1ikely that this altered pHis responsible for resultinginhibition，becauseanearlierstudy（16）hasascertained that the gIOWth of the present fungusis almostidenticalat the pH range of5。4−3．0‖ Onthesegzounds，it is probable that比e mycelialgrowth of controIcontin11eS tOincrease with cultuledurationuptothepoint where autolysis owing to the exhaustionofnutrientsbeginstoinhibitgr’OWth‖ Onthecontrary，thegrowth
On media supplied with2，4，5−T or MCPis nearly ceased as early as 8days afteIinoculation，nOtWith・ Standing the remaining of nutrients and the maintenance of constant pH value“Unlike these two chemi・
Tech。Bu11．Fac．Agr…Ragawa Univ Table2、Effect of three chemicalsllpOn grOWth oiG．oli21arZmgrOWnin peptone−Salts solution
at ca．2フOC 160
Reaction of filtrates CultuIeduration（day） after culture
4 8 12 16 20 24 Fehling Biuret 94．4 1010“7 1282“ユ 1336．8 1295‖1 ユ282．8 4．6 5．．8 74 ∴6 7．6 フ．6 161 64…6 60．9 52．3 46．．S 43い3 4日8 4．5 44 4，．4 4．4 4り3 21．フ 824 85．．2 86．2 993 80い0 46 45 4．ノ4 4…4 4．4 4．3 12．5 506 82。8 235．6 4eO．．2 654．5 4．8 4‖フ 4，7 4“6 4…6 50 ContI01 0‖015％2，4，5・T O‖03％MCP Ob3％NAK Dry weight of 壬nyCeユiu王n（mg） jpH of medium く ＋ ＋ ＋ ＋ 十 十 ∴￣
‥二．‥、
：・ ・ ‥‥二Cals，nO decrease of the growth rate was foundwithlegard to NAK‖〕h thisinstance，however，it seems
reasonableto assume that autolysISisnot asyet setin solely on account of thelower consumption of
nutIientscoming fromtheunfavourablegIOWthSo，the typeof this growth cu工Vemaybeacceptedtobeidenticalwith that of contIOl
Two series of experiments above statedindicate clearly that2，4，5・T andMCPa王euniquein showing the g【OWth−CurVeS O董the＝staling＝type onboth solid andliquid media，While those on mediawith or
without otherchemicalsprovedtobethe“non・Sta王ing，，type．Since，mOreOVer，thegrowthcurveoftheformer type dependsuponneither the change of pH value dming cultuIe nOr・the exhaustion of nutIients，theevi・
dence must be said to o圧er a strong suggestion foIthe mechamism bywhich2，4，5−T andMCPexeIttheir
inhibitory activity upon the causalfungus
3・TheProdlletion oぜClearZoneヽ守・駄en甘woC（〉ionies of G・・O言おαmm are即OWn tO酢蝕er
in蝕e Same Peね丘Di＄h o：lAgay班ed王aβupp】主edⅥr玉柏ei童心er2，4，5−T or光CP
In a previouspapel（19），it was reported that two colonies of G小Olivarum are separated byaninhibition ZOne Whenth9y are planted on the same agar plate containingO02％2，4・D，Whereas they comeinto con−
taet on controlmediawithoutit．Since，On the other hand，the type of the g工oWth curvesin2，4，5・T and MCP were essentia王Iy similar to that of2，4−D but rather an opposite trend occuredin NAK as we have
already seen，the comparative studies weIe Performed with these three chemicals as to aninhibition zone
pIOduction・Thus，tWO COlonies were grown together at25Oin the same Petridish on agar media with Or WithoutO006％2，4，5・T，0」m5％MCP and o03％NAK，bothinocula being set at a distance of14，28， 42arld7D mm apart respectively‖ 3di＄hes were used peIeaChinteIValfor each chemicalIn each of the two trials，tWO COlonies on media with orwithout NAKcameintodirectcontactnotlaterthanthe14th− 16th day，．In2，4，5・T and MCP，neVertheless，Vegetative growth stopped before they touch，thusleavinga clear zone of medium O／2−51mmwide even at the end of culture for30days
If theirinhibitory activityis entiIely due to the direct effect upon the present fungus，it seemsim−
probable that the clear zoneis maintained for solong period，eVenif the unfavourable growth shoud be
justiBable．In addition，the width of the clear zone tended toincrease as theinteIValofbothinoculawas
increasedThis may bein accordwith the 丘ndiIlgSlater seenin Table3which shows theincreasing
yield of an antibiotic with thelength ofinctlbation
4‡solation of a Crllde A；ltibiotic S11bstance from七he Culture Filtrates of GいOlivL＝r・am grOWn On艶edia conね方ning eitker2，4，5・T or拡C王＞
Vol8，No′2（ユ．95フ） 161
be usefulasincitants of antibiotic pIOductionlike2，4−D”Therefore，SOme−e圧0ItS Were madein ordez to isolate the pIObable substancein partially puIined form”Cultures wezemaintainedinquie七重0工12days．at 25O”300Cin丘asks containing50cc o董peptone・rSalts solution with aninitialsupply of O”O15％ 2，4，5・T or
O．C）3％MCPり Withdrawingatrandom4naskspez each chemical，the mycelialmats were removedby filtration，grOund with a mortaI，and then addedto fresh agar media of whichquantitywasquiteidenとical
with theinitialsolution（200cc）u When the fungus was againinoculated on them，nOinhibition followed theseinoculationsin each case，SuggeSting that the probable slユbstanCein questionislocatedi王1theculture iiltratesFor this reason，the丘Itrates were Lractionatedin o‡der with a variety of soIvents accoIding to the procedur・epreSentedin Fig．4，eaCh fraction being assayed for aninhibitory activitywith、the aid ofa dilution agar media technique oIpaper disk method
l車重二f 辰抽ate ーーー【￣ J Mycelial mat 万iltIate 隋諾急ぎ莞孟￡ヤb ￣▲￣￣￣￣声 EtheトpaIt
】Add4％NaOH
H2SO4二paIt ■ Ether“pa比 桓vaporateetherl・し．1ミ州・∴il
NaOH・paIt隈紙慧票㍊With
EtbeI■＿pa王t H2SO4・・paIt 「師前ntibiotic） vaporateether CIude cIyStals BFig”4。The fractionation of culture filtrates
The results showed that the fraction A，a yellowish oil，alone develops a markedinhibitionin either case of both chemicals．J11dging from the negligible effectiveness of fraction B to which both 2，4，5・T and MCP move actua11y，itis probable that they had been either conveItedinto mateIialdevoid of fungi・ toxicity orthey constitute precursor of the active mateIialin questionlHowever that may be，it seems reasonable to assume that the c工ude substance of a yellowish oilplays a main role for theinhibitoIy activity of the culture丘1trates，Doubtless，the appea工anCe Of the substanceis not a result of the simple chemicalreactionbetweenbothchemicalsandnutIientsinitiailyadded，Since none of thatwas produced even when the same mediahadbeenkeptinthepresenceofeachchemicalat2SOCfor24dayswi’tholユtinoculation Moreover，it has been already ascertained by the previous studies（19）that fungitoxicity against the causal fungus couldbedetectedinneitheraltratesnormyceliahaIVeStedfrom the controIcultuIe。Laboratorytests indicate that the respective crude substanceis very activeagainst a number of plant pathogenic fungitoo， including Cochliobol鉱SmiyabeamLS and GibbeY■ella sau）in2iii，SO thatit may be considered a certain antibiotic substance untiladditionalinformationis availablelDetai1ed data on the e＃ect against fungiand bacteria
Willbe discussed on thelater pages”NAXこ failed toincite the p工Oduction of detectable amounts of antibiotic by the similaICulture
5h The Relation of（】ul蝕re Duration and the Concentratio：10f2．4，5−T or 如こC
to the Antibiotic ProdtlCt主0王I
ユ62 Tecn．Bull．Fac．Agr．Kagawa Univ． pliedwithOlO15％2，4，5−T andO．03％MCPrespectively，initialpH5．4 Withdrawing4且askspereachchem・ icalevery4days，the crude antibiotic，designated preparatio王IAin Fig4，WaS eXtraCtedfrom their own 鋸t王ateSreSpeCtively，and thenincorporatedinthe same newagarmediaofwhichquantitywasquitesimilar to the originalsolutions”The2nd culture of the causalfungus was conducted at25OC on these media
The relative potency of the antibiotic pIOducedin thelst culture was determined by the diameter of colo−
nies on the2nd cultuIe afterincubation of7days…In this manner，the relation of the cuituIe durationtotheantibioticproductioncouldbeestablishedineachchemical（Table3）．The results show anincreasing
titeIOf the respective antibiotic according to the advance of culture
In similarexpeIimentstoo，Which werepeIformedwith a supply ofO，Ol，0．02％2，4，5−T or O．Ol，0．02， 0小04％MCPin thelst culture of12day’sincubation，the crude substance developed virulencein anycase
（Table4）・Atthe same time，the yield of theantibioticis assumedto rise withincreasing concentration Ofeachchemicalinitially added．atleastwithinthecor）．Centration・・工ange teSted
Table3．Diameterin mm of colonies of GOlirvwum on agar media containing the crude antibiotic
isolated f工Om Culture filtrates of the causalfungus which had beenincubated at can 270C for・different periods on peptone・Salts solutionin the presence of2，4，5・・T or M〇P
Culture duration oflst culture（day）
Chemical ContIOl 0＋ 4 8 12 16 20 24 0015％2，4，5−T O03％MCP 48．．6 4フ．．8 フ“2 0．0 0．0 0nO 49 50．9 52．6 5之5 ユ5．8 14．4 6…フ ユ3．0 10。0 50．9
had been kept董or24 ＋The sameisolation process was carried outwith nutrients・SOlution which
tIle PreSenCe Of2，4，5・T or MCP withoutinoculation days in
Tabh4〃I）iameterin mm of colonies of G．olivarum on agar media containing the crude antibiotic isolated from culturefiltrateswhich had beenincubated at250C for12daysonpeptone・SattS so工utionin the presence of vaIious concentrations of2，4，5−T or MCP
2，4，5−T（％） MCP（％）
叫 Control 0．01 0け02 001 0．02 0．03
30．0 0…0 44り5 19．2 4．フ
8．PhysicalandCtlemicalProperties oft血eAntibio抽pⅦrelyisoiatedfrom抽eCtl号ねre
Filtra七es of G．Ogねαr〟m grOWnin七he t）resence o君雄C呈｝
l，364．6mgofthe crude antibiotic co111d be extracted from4・5L of the12−day quiet・Culture altr年teSOf
G．ohvarumwhich had been growninpeptone・SaltsliquidmediumcontainingO‥03％MCPfor12daysat25OC
by the use of100aasks‖ When the c工ude antibiotic wasleftin the acetone・Water mixtures（3：7v／V），CryS− ta11ization has oIiginatedin severaIdays・After being further一 工eCIyStallized from ether，the above treat−
mentwasrepeated severaltimestoisc・1ate丘nal1y apure antibiotic substance of white needle・Shaped cryS・ tals melting at55OC′ The yield was816mg”There occured he工e a reaSOnable question whether the
antibiotic thus obtainedmight had beeninduced byimpurities because the experiments hitheItOmentioned
had been conductedwith the commercialmaterialof MCP‖ Since，however，p11re material埠alsop工OVed to be similarly e鮎ctiveinincitingtlleinhibition of growthas wellas the antibiotic production，itis beyonddoubt that the antibiotic productionconcernedisnotattributabletotheimpur’ities Althoughattempts have also been made to purify the crude antibiofic obtained by12，4・5−T suppiy，nOne have so far been suc・
cessful．Thephysicaland chemicalpropertiesof the pure antibiotic are as fo1lows
Vol．8，No．2（195フ） 163
（1）Thernwstabili砂 Because nolossof activity was detected even after heating to150QCfor，2hIS．，
the antibiotic seems to be thermostable
（2）Quantitative tests The antibiotic red11CeS Nessle工’s reagent，anditis positive for alkalixanthate
reaction．The Fehling，To11ens，Ⅹanthoprotein，diazotized benzidine，Millon、and Liebermann（for nitroso COmpOunds）test aIeal1negative‖ Bromine wateris slightly decolorized by the test sol11tion。Beilstein
test（7）gives a green color to aflame，Characteristic to organic compounds containing halogen・Lime test
and Lassaigne，s test for nitzogen（T），Lassaigne’s test etc．for sulfur（1，7），a teSt for P（7〉LWere allnegative．
（3）Solubility The antibioticis nearlyinsolublein cold water，butsoiubleinpetroieumether，ligroin and hot wateIand e？Silysolubleinmethanol，ethanol，butanol，aCetOne，ether．，Chloroform，aCeticacid，benzol，
xylol and toluol
（4）勒erchl・抄matOgTqPhg One・dimensionalpaperchIOmatOgIaphy studies were made・Ascending chromatograms wereIun foI7hrsat200Con“T6y6roshj”Nol5Alterpaperstrips40cmlongandユCmWide
Strips weIe SpOtted with the testing materialwhich were caIriedin dilute methan01，and they were dried
and suspendedin a sealed chamber，the atmosphere of which had been saturated with the developing
miⅩture．The mixture used contained30per cent acetone，5per cent methanoland65per cent water，by volume．Rf value was determined from the position o董inhibjtion zone，by placlng the developed chro−matograms foI48 hrs．at260C on alarge assay plate seeded with conidia of G．olivarum．The results
showed that theinhibition zone presentedin stIips of the crude materialwas only one andalsothatRfof
the spot was quiteidenticalwith that of the pure antibiotic，both being O73．Hencethepu工e，antibioticisconsidered to play a principal role in the virulence of the crudc material
（5）Ta＄te The pure substance has a bitter taste on touchingthe tongue・T王1e Similar taste of the
crude substance might be due to this puIe antibiotic
7り Fungis七atic and Bacteriostatic Activity of the Antibiotic pllrelyisolated from the Clllture Filtrate＄Of GOlluarLLm grOWnin the Presence of M．CP
The crystalline antibioticpIOduced by MCP cultuIe Of GOlivaY・um WaS teSted against a number of
microorganismsリ
（J）肌cf叫pOルfゐeダrO紗fゐ
Theantibioticwasincorporatedintopeptone・Saltsagarmediatogivevariousdilutionsranglnghoml：3，000 tol：50，000and10cclots of assay agaIWereaddedto5つcc負asks，thegrowthof3speciesofphytopathogenic fungion them being compared by the diameter of colonies afteIincubation at250C for3 days小 On ac−
count ofinsolubilityin cold water，the antibiotic was added to魚asks asacetonesolution，aftetitsevapora−
tion media being pouredinto themThe mean value of5flasks per each dilutionisIeCOrded foIeaCh
fungusin Table5lIThese dataindicate thatGtOlivarum，Co：hliobolusmi．yabeanuSandCoYiiciumcenirif8qum are completely or nearly completely preventedin the growth at thelevelofl：5，000，and also that the highest dilution of the antibioticin whichanyinhibitory eだectiveness could be demonstIated seems tolie
about atl：40，COO，l：10，OOO andl：30，000respectiveiy against the respective fungi Table5”Diameterinmm of colomies of three pathogenic fungion peptone・Salts agar media
COntainlng the pure antibiotic o董different dilutions
Antibiotic dilution FdnguS Control 1：3，00〇．1：5，∝D l：10，∝氾 1．：2〇，0〇0 1：30，0〇0 1：40，0（刀 1：50，000 C o／f（−dJ・〟〃J C戒．γd∂βα〝鉱ゞ C川畑折角g弘明 ± 11い4 13．2 16．0 18．4 20り1 20い9 ± 10．8 13．2 13…2 16．5 15．2 13リ1● 0 ＋ trace 2．1 11．7 8，8 e．2 0 0 0
Tech BullFacAg工…Kagawa Univ
164
Another examination was also done by paper disk method．In the case of fungl，Square 魚IteIpape【
Oflcm2containing O．3，0”l，0，J3and O．Olmg of this substance respectively was placed onagarplateata distance of3mm from、the ridge of each colony which had been previously gIOWn untilculture platesin
PetridisheswereJhalf coveredAfter beingincubated ther6after at250Cfor24hrs，thewidthof・aC】ear zone was estimatedり Because o董itsinsolubiliIyin coldwater，thesubstanceconcefnedwasaddedtopLapeI asacetone solution，afterits evaporation the paper being providedforuse…Inbacteria，Ontheotherhand， the roundfilter paperユImmin diameter was treatedinthesamemanner，andthenlaidonagaIplatesoon after their：suspeIisions had beenpoured onthe Bouillon，s agar plate，the supe工natantfluid being removed
at〉once，The diameter of theinhibition zone surrounding the disksweIemeaSuredafteIincubatingfor24 hr・S at300C．The results are shownin Table6．Conside王ing allthe date，it appears that the substance is active againstal1the organismstested at thelevelof O・03mg or higher，With only oneeXCeption of
洗面切払財勿励頑戯J〝研
Table 6 The width or diameter in mm of the clear zone formed belween the paper disk containing
thd p11re antibiotic and the colonies ofmicroorganisms＋
Micrograms of the antibiotic per disk Micr00Igamism 0．1 0．03 0．01 0 0 ︻b O 5 3 ︻b O つJ 3 つム ︵∠ ⊥ つム つん 3 SぐJぐ′￠J‖〝〃恒・d川♪鋸／J〟〃 Gわβ0．映励綱川妨如汐祉閻 Cocゐ加わoJ〝ゞ∽よ，γα∂♂α邦〟√S CoJ如‖〃〃r川JJ小（gJ… 且忽cタ05β07∠〟椚如γγg P抄よc〟Jαγ査α0′．γZαβ PI・油症椚￠〆刷血正一JJく〟〝肌 Gわ，彬γ♂〟b cg喝励頻 一〇．55．〇．2．5．〇 1 0 1 1⊥ ⊥2 0 ⊥ 一J ± ± ±5 ±．5 一一一一一〇 l 14り1（22．8） 12い5（23い2） 211．6 16け0 20．0 15．5 20．．0 1フ“5 13．3 ± β′・gd戒α．S〝鋸よJよぶ＋ヰ 且α㌢・0査dβαβ 且cαタ・0ねび0γ・α P細微九㈲制帽S（ね．sか′〝Cね乃5 β5Cゐ♂㌢・舘財αい溝 ±O J 詫 ± 4 3 つ、︶ l l l 一〇 ±〇一 3 3 ⊥ ﹁⊥
＋ ThefiguresinfunglpreSCntthe widthofthe clear zone andthoseinbacteria p工eSentthe diameterof that，．±Clear zone，absent；inhibitive action，Slightl−Inhibitive action，nOne
＋＋lnhibition zone were free fron growth of the assay oIganismin the central area of spa工Se grOWth
Meas！lrementSin parenthesesincluded thisincompletelyinhibited area
（2）脈￠f叫pO柁fゐe co花王dヱαZ♂er朗如α如花
Inprder toconarmtheeHectoftheantibjoticuponthegerminationofconidia，withG olivarumasthe
testorganism，the conidialsuspensionwasdroppedonathinlayeIOfagarmediatowhichthepuIeanti・ biotic had been addedin various dilutionslAfte工 being auowed to dry with the removalof the
supernatants，Slideswerekeptinamoistconditionat250Cfo工12hISlbeforgerminationcountsweremade Itis obvious hOmthe datain Tableプthatnogerminationtakesplaceatl：3，OC）〕，andalsothatthegermina・
tionpercentage aswellasthe elongationofgerm−tubesareIelativelypoorevenatthehighestdilutionof
l：20，0∝）．
8り Disenssion and Co‡lelusion
Studiesinthepresent pape＝eVealthat both2，4・5・TandMCPalsofavorantibiotic productionasin
the case of2，4−D，When G．olivarumisgIOWninthe presenceof each ofthemAlthoughallattemptsto ieolateapureantibioticfromthe c：udepreparationobtaineまbythe addition of2，4，5・T have yet been
Vol・8，No・2（1957）
Table7．Germination of conidia of G。Ohvarum on peptone・SaltsagaImedia containing the p11re斧ntibiotic ofdifferent dilutions
165 NumbeI COunted Mean length of germ・tubes（p） Dilution of theantibiotic l：3，000 Number
germinated geI■mination Per cent
10．0 0．．8 16‖9 51，2 532 491 4フ寧 534 1：5，000 1：10，00〇 1：20，000 ContIOl 65 13 コ 346 フ2…フ 530 99L．3 unsuccessful，inthecaSeOfO・03％MCPsu■pplytheyieldofthepure？ntibioticdqs9ri鞄Pdinsectiop6repて
resentsthatits concentzationin the L2・day culturefi1trateIeaChes caい0”02％小 Since，On the other h？nd，
this concentration of the antibioticisnearly su魚cient toinhibit the growth of the pathogen 甲 Seenin Table5，this factisinterpreted as support for the view that the工eSults of Table2showing the cease of vegetative growthin originalcultureisdue to this antibioticlOn these grounds，We may dzaw a conclu・
sionthat theinhibitory activity of MCPinvit；IOdn the growth of the pathogenis mainlyinvited by this
antibioticIatherthanbyitsdirect e＃ectupon the fungus．Comparison of the physicalandchemicalpro・pertie琴Of the antibioticin question with th弓m Of the one repo工ted for2，4−I）（18〉shows that the both anti・
biotics differ distinctlyin mp，Rf and xanthoprotein reaction，althoughit may be that they are closely related each othez onacco叩tOf the striking resemblancein other respects− Furthermっre，judging from
thefactsthat2，4・q，2，4，5・T and MCP allinduc、ed an antibiotic production b申NAK fai1edto doso，SuCh
nature of antibiotic productionis considered to be per’hapsspeci負c董or2，4−Dan（＝elatedcompounds，instead of ageneTalphenomenon of the gT’OWth−regulators．
Ourpresentknowiedg弓Ofthe fneChanism bywhichthe antibiotics are producedisfarfromcomplete InIegard to2，4・D especial1y，however，there have been known a number of publications which appear to
give a valuable suggestion for the futuIe Studies concerning this subiectThe oneis an opinion thatits
herbicidalactivityonhigherplants depends on the accumulationoftoxicsubstancesinthem，the substances
being believed to be abnormalmetabolic products of plantsFo工eXample，FuLTS et al．（8〉demonstrated thiswith scppoletin andtheide？WaSSuppOrtedby Ov甲BEEX et al（23）Such assumption agrees withthe report of AuDUS et al”（2）who stafed that the toxic actiqn of2，4・I）resembles that of coumarin and
alsoagreeswiththeinvestigation obtainedby HAMNER et al小｛10），Whonoted thatβ−methyl・Tlmbe11iferone，a COumarine de【ivative，is more toxic to bro？肘eaved plantS than to Gr牟mineae”Sinceitisalso known，On theotherhand，thatinplants寧甲eral1ya S甲allquantity of coumaIin derivativesis detected（？・21）andthat
they areIoxic to plants as wellasphytopathogenic f11ngi（LO，13・2O・22），this assumption would cal1forspecial
notice，althoughitis uncertain now whether the antibiotic早in question are coumarin derivatives or not Besidesthese studiesabove stated，reCeTltinvestigationscc＞nCernlng decomposition of2，4−D by organ− isms mustやe takenin considerationh芦relFor example，BaニIeriumglobiforme3，4），FlabobaGterium aq
anda？qrtain CoYこyn寧baeieYium・1ike species（14）iso！？ted f：Om SOilrespectively are known to be capable of
decomposing2，4−D，the more favourat〉1e for the growth of microc＞rganisms the soilcondition，the sooner beingitsdeqomposition bytheml・Especially，the formest sp？Ciesis said to be able toutilize2，4・Dalone asCsources・As2・4・D・decompositionproduct芦bymicroorganisms，2，4・dichlorophenol（5）andcertainphenolic COmpOunds（15）have been reported untilnow，althoughMuNEKATA etal．（15）are doubtfulas to the former
Chemical．In theinitialstage of a study，2，4−D absorbedinto higher plants was coユSidered to move inthemasanoriginalform（24）l・However，relatively recent studiesperformelby the use of C14labeled
TecIl・駄瓜Fac．Aか．Xagawa Univ． 166
2，4・D have ascertained thatit undergoes a grad11at breakdown by higher plants too（ll），and that radio，
active carbon dioxideis pr・Oducted by soybeanplazltSWhichhavebeentreatedwiththesamechemicalcon− taining C14in either the carboxylor themethyleneposition？，Whereasnocarbon dioxideisevoIvedfromthe
ring carbon at positionl（6・25）い As 2，4−D・decomposit三on prcdl】CtS by soy bean（26）and kidney bean
plantsく12），tWO kinds of ether soluble substancesand a water soluble substance havebeenisolatedrespect− ively・However，itis quite unknown yet as to whether such decomposilion products by microoIganjsms
and higher・planfs are related to the physiologicalaction of the growth・regulator or not．
Hence，the evidencein this pape【Showing that theinhibitory activity of2，4，5・TandMCP mainlyde− pends on the an†ibidticsis of particular jnterest．Moreover，unpublisheddatathatthevirulenceoffraclion BinFig．4towhich2，4，5−TandMCPmovepractical1yisfound todeclinegradual1ywiththblength of incubation whenit was harvested，in contrast with theincreasingyieldofthean＝bjoIjcs，SuggeStSthat bolh chemicals are morelikely to consIjtu†e precursors of the respective antibjotic．Further studies are，how・
ever，neCeSSary aS tO Whether the antibioticsare by・prOducts of an〔abnormalf11ngalmetabolisminduced
by the addition of these chemicals，Or prOducts resulting from thetransformationofthosechemicalsbythe
causal fungus Referenees eited l．．ARIMA，J．：Yukikagaku−bunshekiho，46−47，To− kyo，1wanamishyoten（1950）（inJapanese）・ 2‖ AuDUS∴LJ，QuASTEL，J．H”：Naiur’e15g，320 324（．1947） 3… u＿．．∴∴月毎扉（川ガふ姐，2，31−36（1．949） 4“㌦：肋は，3，1フ0・192（195け 5．＿：．J．5cf．釣odAgγ小，5，268・274（1952）・ 6．FANG，SC、，JAWORSKI，E．G，LoGAN，A・V…， FREED，ⅤいH．，如TrS，．丁．S∴ノ如・cゐ戯0ごゐβ朋・，32， 249・255（1951） フ．FEIGL，F，：Spot testsII．Organicapplications， FourthEnglishedition，64−65，フ0・7l，72−73，73− 75，フフ・7∂，New York，EIsevier・PublCo・（1954）・ 8．FuLTS，J。，Jo王INSON，M：West．WeedConfo Denver，January（1950） 9．GooDWIN，RH小，TRAVES，C∴Amer” 37，224・231（1950） 10．、HAMNER，C、L，SELL，H”M・，KLO丸正PARENS， WM、，VAtJGHN，．J．R．‥βクね〝，ノGβg，112（2），135 13フ（1950）． ⊥1。HoLLBY，R−W■‖，B〇YLE，F．P．，HAND，D、B・： Arcゐ．βまocゐβ沼，27，1イ3・151（195つ）小 12い ＿＿＿：乃∼♂“，35，171（1952） 13．．IsHII，Yl：．／4㌢・ノC鹿朋5〃C−♪♪α〝，27∴777・ 780（1953）（inJapanese） 14．．JENSEN，HL，PETERSON，H‖Ⅰ∴Ⅳ〃ねγg，170， 39・40（1952）． 1鼠r MuNA琴ATA，Ku，KAWATA，St：Npgyo qγObi
鞄〆（Agficu加ヱeandHo工・tieu王血・e），28，693−
69フ（1953）（inJapanese）． 16・NAITO，N・，TANI，T．：釣cゐ・助仏∬αgα紺ムAgタ． Coll・，5（2），164・174（1953）（jnJapanese） 17‘ ：RuNIXATA，H∴ 釣′・∫Cゐ．ヱチノい 節．， Ⅹyoto，Japan，5（3），一113〝ユユ8（ユ9S5）（inJapa・nse）
墳・柵：TANI，T・：乃紘，5（4）ト127・138（1956）巾 1畠．＿ 】 ＿．，＿＿．：榊〃〝．√助川y，15（2），152− 163（1956）・ 20．SAN AヱヾTONエ0，JけPい：βofリGαg・，114（1），フ9・95 （195∼） 2l“S王oLL，A，PERIERA，A。，RENZ，J。：Ltelv．Chim βCね，33（6），1637・1647（1950） 22n T‡1IMANN，E、Ⅴ，，BoNNER，W‖D．：勒．Ⅳα払 A川感お古，35，2フ2・2フ6（1949） 23．．VAN OvERB苫EK，．J・：点扉いR紺・，5，655−681（ユ．939） 24．WEINTRAUB，RL．，BROWN，J．W．，YEATMAN， J．N．：烈甜＝勒畑〃J．，25，140・149（1．950）・ 25小 ㌣，∵，FIELDS∴M．，Rof‡AN，J．： 鬼才♂．，2ワ，293・301（1952）● 26．．＿，YEATMAN，JりN，，LocxHART，J，A」， REINHARで，一丁，H，FIELDS，M∴Aγ（ゐ．β査ocカβ桝， 40，2フフ（1952）．〕67
Vo18，No・2（195フ〉
R色sllm畠
When GloeosPoriumolibarumAlm”CauSing the olive anIhracnose wasgIOWn Onmediawithvariouscon・
centrationsoffivechemicals，2，4，5−†richlorophenoxyace†主c acid（2，4，5・T），2−merhyl−Chlorophenoxyaceticacid （MCP），isopIOpyl・n・（3・ChloIOphenyl）carbamate（IPC），pentaChlorophenol（PCP）andpotassiumα・naphtha・1eneacetate（NAK），thegrowthcuIVefoI2，4，5−TandMCPwasfound†obethe“staling”type，Whilethat
on mediawitho工without other chemicals proved to be the＝non−Staljng，，type‖ TwocoloniesofthecausalfunguscameintodiIeCt COntaCtWhentheywere culturedinthe same Petrj dish on agaIplatewith or
withoutO．03％NAK．InOhO06％2，4，5・TaムdO．O15％MCP，neVertheless，theyseparatedfromeachother
leaving aninhibition zone．
SoIventiractionaIionof cul†ure蝕IateSOf thepathogenwhichhadbeengIOWninthepresenceof2，4，5・
TorMCPyieldedacrudeantibioticofayellowisl10ilrespectively，theIelativetiteIbeinggraduallyin・
creasedwi†h the culture duraIion and alsowith theincrease of concentIation ofeach chemicalatleastwithinthe concentrationrangetested，Inthe caseofMCPsupply，apuIeantibioticofwhiteneedle・Shaped
crys†alsmeltingat550CcouldbeisolatedfromthecrudesubstancelItisthermostable，nearlyinsoluble
jn cold wateI，but solublein hotwaterandorganic soIvents”It containsC，H，0，Cland the hydIOXyl
groupcharacteris−ictoaprima工y OrSeCOndaryalcohol・butdoes notcontainS，P，andN”Thegrowth
ofG．olivarum，Cochliobolusmi．yabeanusandCorliciumcenirifygumweIealmostcompletelyinhjbi†ed bythis
antibioticaIthelevelofl：5，00〕，the dilutionend pointagainsttherespeclivefungibeingaboutatl：40・000・l：10，∝）○andl：30，000respecrively．
Judgingfromtheyjeldandthefungitoxicityoftheantibiotic，theinhjbitoryactivityofMCPin vilYO
on G．olivarumisconsidered tobe attributable to this antibioticIatheIthan toits direct e鮎ct on the
pathogen．
