健 全 果
N． S．20，89−105（195る）．
（146）SIfiG‡‡，G P′ノ，ⅡusAIN，A。：Production of pectic and cellulolytic enzymes by arhar wilt fungus，
C〝γγ一郎け5最．（J〝d∠α），51，110一・112（19占2）；Cカβ∽A∂ざわ■，57，771るよ（19占2）．
（147）SMITH，W．K．：A suIVey Of the production of pectic enzymes by plant pathogenic and other bacteria，．／一G♂軌Aわ■c㌢〃∂≠〃J．，18，55−41（1958∴
（148）S6RENSEN，H．：On the specificityandproductsofactionof xylanasefrcm Chaetomiumglobosum
ⅨuNZE，タJα〝fα，5，185−198（1952）
（149）SRIVASIAVA，D．N…，EcHANDI，E．，WALKER，J．．C．：Pectinolyticandcellulclytic enzymes produced by朗査go♪〟55≠7の離／β′−，タカ．γわ♪α〃2♂わg．γ，49，145−・148（1959）．
（150）STROBEL，G小Aい：A xylanase system produced by Diblodia viticola，Ph．ytobクtholog．y，5る，592−
59る（19占5）
（151）VAN SuMERE，C．F．，VAN SLMERE−DE PRETER，C．，LEDING‡王AM，GA。：Celil・Wall−Spliting en−
ZymeS Of P〝CC去〝∠αgγ〃∽古刀jrs vaI‖ ≠γよわc∠，Cα乃．／り〟よcγ〃∂ioJ．．，る，7るト770（1957）
（152）龍谷 泉：試薬調製用法便覧，507−508，東京，太陽堂（1942）
（155）鈴木直治：甘藷紫傲羽病に関する研究 Ⅵ小 感染部位の組織化学的研究一感染組織の化学的変化，農技研 報，C8，d9−て5ロ（1957）．
（154）鈴木直治：植物病原菌の病原性，微生物生理学（植村定治郎ら編■），950，東京，朝倉苫店（19る0）．
（155）武田 晃，林 修・−L，宇佐美四郎，小沢瀾二郎：ペクチン轍酵に就て 第占報 ペクチン質の分解と maceI・ationの関係（1），虚学研究，58，45−45（1948）
（156）TALIEVA，M，N，PLOTNIXOVA，Y．M．：The rolein plant pathogenesis of pectolytic enzymes isolated from fungi，B．yul．Gl．BoianSada，1962，55p62（1962）；Chem。Abrstru，59，5091h（1965）
（157）田中正三：柿，化学，9，818−821（1954）
（158）谷 利一・：柿炭痕病菌の培地上の性質並びに．同菌有傷接種濫よる柿果上での病斑進展と分生胞子形成の品 種間差異について−，香川大農学報，8，115・−120（195占）
（159）谷 利一・：柿炭療病菌の菌糸体発育に及ぼすphenol姓物質の影響並びに同菌BAVEN工〉AMM反応について，
香川大農学報，8，1る8−171（1957）．
（1占0）谷 利一・：柿炭疫病薗によるCa工boIlydェaSeの分泌濫ついて，香川大彪学報，9，1r5ムー140（1958）．
（1占1）谷 利一・：柿炭疫病菌による柿果肉多糖類の酵系的分解について，香川大農学報，10，29−う5（1959）．
（162）TANI，T小：ごetection of the enzymes of GloeosboTium kakiin relation to tissue−maCeration，
A〝乃．タケγf∂♪αgゐ∂J．．50C．．ル♪α乃，28，て14−120（1965）
（165）TANI，TL‥Tissue・SOftening enzymesin the kaki血uit softened by theinvasion of GloeoS♪orium ゐ〃鳥孟，A乃〝．アみγ才0♪αfノわJ・・5〃C，・．／α♪α〝，28，187−194（19占5）．
（1る4）谷 利一・：未発表
（1占5）谷 利一・，内藤中人：柿炭疫病隈病果紅おける軟化起因物質の粗分離，香川大農学報，12，255−241
（19占1）．
（1るる）樽谷隆之‥カキ果実の貯蔵に関する研究，1−17る（19占1）
（1る7）栃内吾彦：植物病理学通論，252，東京，誠文堂（195る）u
（1虚）富樫浩吾：果樹病学，258，東京，朝倉古店（1950），
−78−
（169）TouzE，A．：Anthracnosein the melon．Comparison of pecticmetabolismin different organs of healthyandColleioirichumlagenクrium parasitized seedling，Combt．、Rend．，254，5025−5D26（19る2）；
C加働．A∂∫叶．，57，579 d（19占2）．
（17D）TRIBE，H．T．：Studiesin the physiology of parasitism XIX．On the k二11ing of plant cells by enzymes from BoLl−．yiis cinerea and Bacteriun aY oideae，Ann．Botan．y（Ondon），N．S．19，551−
5る8（1955）．
（171）TwEEZ）Y，B．G．，PowELL，D．：TheIOle of pectin・POlygalacturonasein expression of diseased SymptOmS Of coI・k rot，ContYib．Boyce Thombson hwi．，22，9・−16（19占5）
（172）Tw】‡ヱDY，B．G．，PowELL，D．：Production of pecti：いpOlygalacturonase by Alternaria mali，
タカγね♪〃タカ0わg．γ，52，50−51（19占2）．
（175）UzuTANI，Ⅰ．，STAIrMAトN，M‖A。：Pectclytic enzymes ofCcraioc．ysiis．fimbriaia，Ph．yiobatholog．γ，
51，277−・2e5（19引）．
（174）WAGGONER，P．E．，DIMOND，A…E．：Production and role of extracel二ular pectic enzymes of ダお射げ弟甜柁〃ガクざ♪∂γ〝∽ f．J．γC〃♪β′5さcざ，Pゐ．γね♪αfゐ0わg．γ，45，79・−87（1955）．
（175）wALLACE，Jい，Ku6，J．．，WILLIAMS，E∴B．，：Productionof extracellularenzymes by fourpathogens Of apple fluit，Ph．y10Paiholog．y，52，1004−1009（19る2）
（176）WALrON，G…S．，CAPPELLINI，R．A．，：Pectolytic and cellulolytic enzymes produced by Erwirnia
（αr〃ゎび♂γα，Pカ〆〃♪α才如才〃g．γ，52，927（19占2）．Absは
（177）WINSTEAD，N．N．，Mc〇oMBS，C．L．：Pectinolyticand cellulolytic enzyme productionbyP．ythium 卯血淵痛打肋細川，印γJ〃♪αf如Jog．γ，51，270−275（19る1）．
（178）WINSTEAD，N．N．，McCoMBS，C．L：Production of pectinolytic and cellulolytic enzymes by CllCuIbit anthracnose fungi，Ph．ytobaihoZog．y，5る，961M964（1965）．
（179）WINSTEAD，N．N．，WAlXER，J．Cい：Production of va㌣Cularb：・OWningbymetabolites from several pathogens，Ph．yiobaihology，44，1551−158（1954）．
（180）WooD，RいK．S．：Studiesin the physiol〇gy Of parasitism XV皿．Pectic enzymes secreted by 風那加カ鋸〝Z dけ・クgdβα，A〝乃．ββ≠α鱒γ（エ仇翫九潤），N．S．．19，1−27（1955）．
（181）WooD，R．K‖S ：Pathogen factorsin the physiology of disease−PeCtic enzymes，P）ant Patl10・
logy（ed．by HoLTON，C．S．et al ），100−109，Madison，Univ．Wisconsin Press（1959）．
（182）WooD，R∴K．S．：Pectic and cellulolytic enzymesin plant disease，Ann．Rev．PZant Physiol．，
11，299−522（19占0）．
（185）WooD，R．K．，S．：Chemicalability to bごeaCh the host barriers，Plant Pathoエogy（ed．by HoRSFALL，J．C．，DIMOND，A．E…），Ⅱ，255−272，New York，Academic Press（1960）．
（184）WooD，R．K．S．，GupLTA，S．C．：Studiesip the physiol〇gy Of parasitism XXV．Some properties Of the pectic enzymes secreted by Pythuim debarianum，Ann．Botan．y（London），N．S．22，509−519
（1958）．
（185）山崎保子：甘藷紫紋羽病に関する研究Ⅲ病原菌のペクチン分解力，段技研報，C8，57−51（1957）．
（18占）安盛 博：クリ頬たんそ病菌のキクリ葉侵入時に・串けろペクチン酸分解酵菜の分泌，島㌍農木研軌
（11）A，25−2る（19る5）！
ー79−
Studies on the PhytopatllOlogicalPhysiology Of Kaki
Anthracnose，With SpecialR⊂ference to the Role of Pecticin the Sympto二n Develop二31ent On KakiFruis
令Toshikazu TANI
EニeS
（Laboratory of Phyt）pathology）
SⅦmmary
As part of a studyinto pathologicaland physiologicalnatute Of kakianthracnose，the
author has made severalinvestigations from1955t〇19る2with specialre董erenc3tO the
roleofpecticenzymesinthesymptomdevelopmentonthefruitsofkaki（Dio3PyrosFakiLINN．f．）invaded by Gloeosporium kakiHoRI．
Theimportanceo董pectic enzyme systemin a widevariety of pathologicalprocessesis increasinglybecoming evident・UntilpresenttimemanylnVeStigationshavediscussedthe role ofpectic enzymesinrelationto the mechanismof paIaSitism，0土variet乙1resistance，
and of symptom developmentin soft rot as wellas wilt diseases．However，aS yet Very
little data are availableonthemechanismoftissuedisintegrationindryrotdiseases，andalsolittleisknown on the pectic enzymes produced by the affected hostitself・In the
earlystageofinvasionofthecausalfungusthekakifruits show the symptom of dry r・ot・
Atthis stage，therefore，investigatic，nS Should be c〇nducted with thefundamentalview−
point，thatis，how pectic enzymes of the pathogen promote the hyphaldevelopmentin
the host．In thelater stage ofinvasion the softeningoccurs on almost whole part of afruit with s11Ch an outward aspect as suggeStS the action of host enzymes．The c〇nfir−
mation of this presumption also would be a subjこCt Of muchimportance．
From thepointof view above mentioned，the present paperis devised to organize the
essentialdata obtained during thelast seven years，SOme O王whichhavebeenindividually p11blished．The prlnClpalresults are summa工ized as王01lows：
Ⅰ．Symptomsofthe disease andtypesofemlargememt￠f怯e diseas￠dl￠Si￠nS
on kaki fruits
l．Whenmyceliaofthecausal土ungusareinoculatedonkaki土ruits（var．Fuyu）wouz＝ded with a sterilized needle，the necroticlesi〇nS，Which appearwithiユtWO tO four days，
gradua11y extend tillseven to tenincubation days without softening・During this peri〕d
theinfected tissue of hostislittle disintegraded and the cellwallin parenchymatoustissueis we11stained with pectic reagents．Whenincubati〇n days are more prolonged，
however，the softeningoccurs onalmost whole part of the fruit・At this periodthehost tissueis vlgOrOuSly degradedby the probable dissoluti〇n Of pectic substancesin middle lamellae．Therefore，the author has made a di二tiユCtion between the disease symptoms of both periods，designatingthe former dry rot stage‖and thelatter soft rot stage＝，
respectively．
2．Theinoculation expe工iments as mentioned above werefurther made with kakifruits
of twenty five kinds of horticulturalvarieties．The extension of necroticlesions during一80−
dry r・ot Stage COuld be classi王iedinto three groups on the basis of the size oflesions，
and alsoint〇tWO grOupS from the relationship between the enlargement ratio oflesions andthele工】gtho董inc11bation period．InmostvarietiesincludingFuyu（susceptiblevar．），
the enlargement ratio oflesions was constant．On the other hand，the necroticlesion on Goban（resistant var．）which appears by twoto three days incubation hardly enlarged therea董terthroughoutthe experimentalp3riodof ten days．Onthe basis of these董indings，
symptom of Gobanfruits was described as ceased type ，distinguishingitf工Om enlarg−
1ng tyPe of other vaIieties．As representatives of both types，fruits of Fuyu and Goban have been employed for the followinginvestigati〇nS C〇nCerningdry rot stage・
I［．Secretion of polys8CCharascs by the causalf11ngミ1S
5／The fungus grew we1lon both fruit slices of Fuyu and Goban，Which had been p工ePared by steam sterilization．The juices expressed out ofinoculated slices revealed
saccharifying activities on pectic acid，SOdium carboxymethylcellulose（Na−CMC），and xylan．Liquefying activity on Na−CMC was also found．Allof the activities detected were moreintensivein the c；ユ1turalextrac七from the slice of Fu．yu thani皿that of Goban．
4．Pectin−1iquefyingaCtivityin Fuyu slice culture was distinct onlyin the extract Obtained from three daysinc：1bation．
5．Both of pectin・1iquefying and pectic acid・SaCChariiying activities we工e COnSiderably greaterin cコユ1ture filtrates from soy，p3PtJ）ne Salts，Richユr・d3，and Czapek solutions than ir）the extract from Fuy11Slic3Culture．In order to make clear why enzymic ac乞ivityis low on Fuyu cユIture，SO：ne eXperim3ユt3 Were mユd3 With various c】ユ1turalc〇nditions，
and following results were obtained・
（a）The secretionoflique王ying activityishigher onanaerobiccondition thanon aerobic
One．
（b）Bcth ofliquefying and saccharifying activities come up to the maximum at the last stage oflogarithr）ic phase of mycelialgrowth．
（c）The secretion of both activities are maximalwhen aninitialpHvalue of medium is adjusted at4．2．
（d）Direct c〇rrelation was hardly found between sucrose c〇nCentrationin medium and
the secretion of both enzymic activities．
（e）The differezICe Ofmycelialgrowthin severalkindsof media hadno c）nneCtion with
the secretion of enzymic activities．
In these experimentalcc）nditions，the prlnCipalfactor of an enzymic secretion was pre−
Sumed to be a finalpH of culturalmedia because the filtrates of high activity were
alwayslowin pH value．However，theiact that the secretioI】islow on Fuyu culture COuld not be we11explaiユed only by the results above st3ted Therefore，further experir
ments were made with juices of Fuyu fruits．According to the resultsitis probable
that the fruit tissue may c）ntain a certain substance which gives aninhibitory effect on the enzyme prDduction．
6．Using the crude enzyme preparation from soyliquid culture，the respective presence Of etldo−pOlymethylgalact：ユrOnaSe（end〇−PMG），endo−pOlygalacturonase（end〇−PG），eXO・
ー81−
polygalacturonase（exo−PG），and cellulase Cx was demonstrated by means of zone electrophoretic examination． Pectinesterase activity was not detected．Activities of e工Ido−PMG，endo−PG，and exo・PG were greatest at pH4．5，55，and5．0，reSpeCtive王y・
Probable presence of these three pectic enzymes wasalso elucidated by the pH−aCtivity curves of the crude enzyme preparation from Fuyu slice culture．
7．The funguS Seemed to prod11Ce Cellulase Cl，Ce11ulase Cx，and cellobiase on artifi−
Cialmedia，but ce1lobiase was considered to be useless for tissue disintegration．
Ⅱ．Polysaccharides and polysaccharaseinhibitors foumdin the paretLCllymatOuS tissue of kakifruits
8．Constructive polysaccharidesin parenchymatous tiss11eS Of Fuyu and Goban fruits
are composed of pectin，Cell11lose，and hemicellulose hydrolyzableinto glucose，Ⅹylose，
and galactose．
9．Crude pectin of Fuyu was much degraded by董ungalenzymes to the same degree as citrus pectic acid was，Whereas that of Goban c〇uld hardly be hydrolyzed．The fact that the enzymic activity for crude Goban pectinislow was not attェibuted to the pectin itself，but to an enzymeinhibitor Which was closely connected with pectin．
1ロ．Xylanase of the fung・us WaS thought to belユSeless for the breakdown of tissue cell
Wal1s，because xylosein kakiholocellulose did not seem to form xylan．Hemicellulose in kaki王ruits，however，WaS SaCChari董ied by the fungalenzyme whichisinterpreted as
hemicellulosein a wide sense．Ce11ulolytic enzymes produced by the fungus appeared to
be concernedin the degradation of kakiholoce11ulose．
11．Thepectin，hemice11ulose，and ce1111lose of Goban were almost the same with those Of Fuyu respectivelyin view−pOint of enzymic substrates．
12．Although kakipectin and holoce1111lose wereutilizedbythefungus土orthe mycelial growth，the ef王iciencies of them werelower than those of glucose and xylose．
15．The concentration ofinhibitorsin the cold wateIinsoluble fraction of Gobanfruits
WaS found to be sufficient toinhibit completely the activity of fungalpectic al】d ce11ulo−1ytic enzymes．
14．The cold waterinsoluble matter could be fractionatedinto four kinds ofinhibitors
Which closely resembled one anotherin ultraviolet absorption curves．They were pre−
Sumed to be analogOuS Substances o董relatively high molecule weight originated from the polymerization of same monome工．
15．Theinhibitors from Goban fruits we工e COmpared with various chemicalsininhibi−
tory activity．The results obtained are as土01lows：
（a）Metals：Polyvalent metals which coagulates the substrates，and heavy metals are
bothinhibitory．
（b）Enzymeinhibitors：Only KCN and some chemicals are slightlyinhibitory．
（c）PhenoIs：Tho11ghlow molecule phenoIs arelittleinhibitory，plant phenoIs appar−
entlyinhibit exo−PG activity．By an oxidation treatment，thelatter considerablyin−
CreaSe theinhibitory potency against endo−PMG azld endo・PG activities．
The inhibitors from Goban fIuits corIespond in their activities to HgClz and
ー82−
b−benzoquinone treated with alkalireagent，Which revealthe highest activity among the chemicals used．
16．Theinhibitoriraction，Which had been obtained fIOm Goban fruits prevel】ting auto・0Ⅹidation during theisolation process，did notinhibit exo】PG activity so much．
When auto−0Ⅹidized to dark blackin color，however，this preparationinhibited the activities of endoqPMG and exo−PG by about80per cent at a finalc〇nCentration of50
ppm．
17．From theェesults of the sections14，15−C，and16，aS Wellas theisolation process andquantitative test，itwaspresumedthat acertainprecurso軍Characteristicofpolyphenol is pT・eSentin parenchymatous cellwalls o董 Goban fruits，and that qulnOne POlyme工S Of precursor actsinhibitory upon the董ungalpectic and cellulolytic enzymes・
Theinhibitors produced seemed to have no effect onthe mycelialgrowthof G・kaki ・
Ⅳ．Relationship betweenttle aCtionof fungalpecticenzymes andthe
eAlargement ofnecroticlesionsin dry rot stage
18．In the examination made with elevenisolates of G．kakino correlation was found
betweenthesecretion ofpectic enzymesonaItificialmediaandthe enlargement of necro−
ticlesions on kakifruits．
19．In the a土fectedlesions of Fuyu fruits，there existed the activities of endo−PMG
and exo−PG but no orlittle ones could be detectedin the healthy parts of affected Fuyu
fruits and the diseasedlesions of Goban fruits．
20．When eleven vegetables wereinfected with G．kakiby needleinoculation，there
was a definte correlation between the quantity of enzyme secretion on host tissues and
thelesion enlargement On them．
21．Microscopicalobservations were made with various portioDS Of Fuyu f工uit where
ahyphacontactedwitha cellwallofparenchymatoustiss11e・TheresultsseemtosuggeSt
thatthe penetration of fungusizltO Cellwallmay d11e tO a meChanicalpush o董 growing hypha co−Operated with fungalenzymic action．
22．On Fuyu fruits the discolored area of parenchymatous tissue was samein size to the areainvaded by hyphae，Whereas on Goban fruits the tissue cellwal1s were black in color even throughout severallayers contiguous toinvaded cells．
25．．The histochemicalinvestigations have shown that at preinfectionalstage alarge
amount of phenolic compounds characteristic of tanninis presentin both parenchyma−
tous cellwalls of Fuyu and Goban，and that only at Goban they convertinto quinone polymers prior to the approach of hypha．This phenomenonis considered to be a nature specific to Goban variety．
24．Probably the discoloration characteristic to Goban fruits would not beinduced by the funga11accase b11t by a specificinstigation of the pathogen against host．
25．Puttingvarious resultsof ChapterIpIV together，the author has made a folloⅥ ing
presumption on the relationship between the enlargement of diseased lesions and the
fungalpolysaccharases．
Inkakifruits，G．kakisecretes endo−PMG，endo−PG，eXO−PG，Cellulase Cl，Ce11111ase
−8．5−
Cx，and hemicellulase．These enzymes begin to attackthe parenchymatous cellwall at the portion contacted with the hypha・The pathogen then penetrates the portion
with the pushingPOWer Of hypha andinvades the host cell・In case of enlarglng type＝，thisprocessis continuously performed so thatthe enlargement ratioofthedis−
easedlesionis constant．At Goban fruit，On the other hand，OWlngtO SPeCificinstiga−
tion of the pathogen a phenolic precursor presentin cellwalls o董host changesinto oxidative polymers which areinhibitory agalnSt fungalenzymes・Thisisthecause that theenla羊gementOf thediseasedlesionceasesatGobanfruit・
Ⅴ．朗e（血anism of softemimg・imdiseased kakifrnits
26．When the funguSisinoculated on kaki土工uit wounded with a steIili2＝ed needle，
so王tening occurs on almost whole part of a fruit within seven to ten days，Whilein
controIwithoutinoculationitis very feeble evenin abo11t thirty days．Macerating activity of the juice expressed outofinoculated fruits graduallyincreased accordingtOthe degree o王so董tening．
27．As thecausalagent of fruit−SOfteming，a tissue−maCerating substance was crudely isolated from expressed juice through salting out，dialysis，and acetone treatment・
MaceratingaCtivityof the resultedpreparation was pェ・eSumedto be causedby aprotein，
because the activity was completelyinhibited by a protease treatment・
28．The crudepreparationalso revealedtheactivitiesof endo−PMG，endo・PG，eXO・PG，
pectineste工aSe，and cellulase Cx・Theliquefying activities onpectic solutions，howeve工，
were considerablylower as compared with tissue−maCerating activity・
29．Inorder to determinethe property of fungaltissue−maCerating enzyme，the crude preparation obtained f工Om SOyliquid c111ture of the fungus was examined by means
of zone electrophoresis．Endo−PMG and endo−PG were工eCOgnized to be concernedin maceration，and no other macerating factors could be demonstrated．However，it was hardly presumable that these fungaldepolyme工ic enzymes caused the softeningof kaki
Lruitin vivo，because they were slightly active on kakifruit tissue・
50．Two kinds of macerating enzymes having a characteristic ofproteinorprotein−1ike substancewereseparatedfromso王t rottedfruits byzone electrophoresis・Theiractivities
were maximalatpHrange of4．0−5．O and45−5，0，reSpeCtively．Both enzymes revealed
little or no activities on pectic solutions and gave no effect on the Oz uptake of kakidisks at an early stage Ofincubation．Theseproperties resemble those of macerating enzyme of Closiridiumfdsineumvar．sikokianum reported by KAJI（1959）・
51．The occurrence of twokinds of specific macerating enzymesinthe extract of kaki Lruits was also demonstrated using young fIuits artificia11y softened by crumpling them
Lirmly with董ingers．
52．Similarity was confirmed between the properties of expressed juices from both
血ealthy and diseased fruits aDd those of the macerating enzymes purified with zone
electrophoresis．