Received December 26, 1956 (Communicated by Dr. Shri Ranjan, r.^.sc.) INTRODUCTION

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ASSIMILATION OF DISACCHARIDES BY SOME FUNGI CAUSING "LEAF SPOT "" DISEASES BY R N TANDON AND K S BILGRAMI (Department f Btany, University f Allahabad) Received December 26, 1956 (Cmmunicated by Dr

1 ASSIMILATION OF DISACCHARIDES BY SOME FUNGI CAUSING "LEAF SPOT "" DISEASES BY R N TANDON AND K S BILGRAMI (Department f Btany, University f Allahabad) Received December 26, 1956 (Cmmunicated by Dr Shri Ranjan, r^sc) INTRODUCTION ASSIMILATION f disaccharides by the micr-rganisms generally depends upn the prductin f necessary hydrlytic enzymes Nn-utilizatin f these cmplex sugars is usually assciated with lack f such enzymes Instances are knwn where the cmplex sugars may nt be utilized by an rganism even thugh its hydrlytic prducts may supprt gd grwth Thus Marglin (1942) fund that Syncephalastrum racemsum culd utilize the hydrlytic prducts f lactse (viz, glucse and galactse), but it was incapable f using lactse Accrding t him the nn-utilizatin f lactse was due t the inability f that rganism t synthesize lactase Mandels (I954), hwever, suspected that in sme cases the disaccharides culd be assimilated even withut being hydrlysed He suggested that the spres f the fungus Myrthecium errucaria did nt use sucrse thrugh a hydrlytic pathway Smith (1949) als btained almst similar results with Marasmius chrdalis and accrding t him, this rganism attacked cellbise by a rute that invlved neither preliminary hydrlysis nr phsphrylatin The bservatins f Smith (1949) and Mandels (1954) indicate the direct utilizatin f disaccharides by sme fungi, but such recrds f direct assimilatin f cmplex sugars are rare Hawker and Chaudhari (1946) reprted that the rate f inversin f sucrse determined the amunt f grwth f Pdspra sp, Pyrnema cnfluens and Chatmium cchlides It is thus evident that nt nly the pathway f utilizatin f disaccharides may vary with different fungi, but even their rate f assimilatin may depend n the rate f hydrlysis f the sugar An attempt has, therefre, been made t undertake chrmatgraphic studies t establish the pathway f utilizatin (hydrlytic r nn-hydrlytic) f sme cmmn disaccharides (viz, sucrse, maltse and lactse) by three leaf spt causing fungi MATERIALS AND METHODS The three fungi, viz, Phyllsticta cycadina (Pass), Phyllsticta artcarpina (Syd et Buff) and Pestaltia mangiferce (Butl), which were respnsible 274

Assimilatin f Disaccharides by Sme Fungi 275 fr the leaf spt diseases f Cycas revluta, Artcarpus heterphyllus and Mangifera indica respectively, were islated frm the infected leaves f the abve

2 Assimilatin f Disaccharides by Sme Fungi 275 fr the leaf spt diseases f Cycas revluta, Artcarpus heterphyllus and Mangifera indica respectively, were islated frm the infected leaves f the abve mentined hsts The methds f islatin, purificatin and subculturing were similar t thse f Tandn and Bilgrami (1954) Asthana and Hawker's medium A* which has glucse as the nly surce f carbn was selected as the basal medium Glucse was replaced by different disaccharides and mixture f their hydrlytic prducts Care was taken t include the same amunt f carbn (ie, 2 gm per 1, ml in each case) Pyrex glass wares and purest available chemicals were used Fixed quantity f medium (viz, 25 ml) was taken in each 15 ml cnical flasks which were fractinallysterilizedbysteamingfrhalfanhur daily fr three successive days Slutins cntaining these sugars Were daily inculated fr 15 days with different rganisms by agar disc methd f Garrett (1936) The fungal mat frm each set was filtered separately n the sixteenth day when ne t fifteen days ld cultures were available The calculated dry weight was used as the quantitative measure fr daily grwth The filtrate frm each set was chrmatgraphically analysed t detect the presence f the sugars The methd described by Ranjan et al (1955) was used fr this purpse A circular piece f Whatman filter-paper N 1, diameter 27 cm having 12 radial sectrs separated by 12 radial cuts was used Drps f knwn vlume (5 ml) were taken frm the filtrate f each day and they were placed at the psitins lcated fr this purpse (just abve the numbers 1, 2, 3, 1, etc) Smetimes index slutins f knwn sugars were als kept n the same chrmatgram because this culd facilitate the identificatin and cmparisn f bands After placing the drps, the chrmatgrams Were run with n-butanlacetic acid and water (4: 1:5) Fr separatin f glucse and galactse (which gave a cmmn band with the abve slvent) n-butanl-pyridine and water (6: 4: 3) were used as the develping slvent The chrmatgrams had single paper wick (4 2 cm) and were run fr abut 7 hurs in the abve slvent They were then dried at rm temperature fr 2-3 hurs and were subsequently sprayed with a mixture f aniline-diphenyl amine phsphate (5 vlumes f 4% aniline, 5 vlumes f 4% diphenyl amine and 1 vlume f phsphric acid) After spraying, the chrmatgrams were left at rm temperature fr 3-4 hurs and were then placed in an electric ven at 11 C fr 9 secnds The average RI values f varius sugars were determined and they have been recrded in brackets at apprpriate places in the text * KNOB 35 gin, dextrse 5-grn, KH2PO gin, MgSO47HiO 75 gin, distilled water 1 litre

276 RN TANDON AND K S BILGRAMI The daily dry weight f these rganisms n different disaccharides has als been recrded and their grwth has becn cmpared with the grwth btained n a mixture f crrespnding

3 276 RN TANDON AND K S BILGRAMI The daily dry weight f these rganisms n different disaccharides has als been recrded and their grwth has becn cmpared with the grwth btained n a mixture f crrespnding mnsaccharides, which wcrc frmed by the hydrlysis f varius disaccharides OBSERVATIONS 1 Assimilatin f Sucrse (R 61)--Chrmatgraphic analysis f all the three hsts shwed the presence f this sugar as well as f its hydrphytic prducts (viz, glucse and fructse) Mst f the fungi hydrlyse this sugar befre assimilatin Dally chrmatgraphic analysis f the medium revealed that the present rganisms als utilized sucrse thrugh a hydrlytic pathway The rate f cnversin f sucrse, hwever, varied with the rganisms It was bserved that between 2nd and 3rd day, the tw species f Phyllsticta cnverted the entire sucrse int a mixture f glucse (RI 66) and fructse (RI -69) and wlthin hat perid it (sucrse) ttally lst its identity in the medium The same chrmatgrams als established that bth these fungi fully assimilated glucse, three days earlier than frvctse The hydrlysis rate f this sugar (viz, sucrse) by Pestaltia mangiferce was much slwer because it culd nt be hydrlysed cmpletely even upt 7th day It was als nticed that bth the hydrlytic prducts f sucrse were simultaneusly cnsumed n the 15th day The daily dry weight grwth rate f these fungi n sucrse as well as a mixture f its hydrlytic prducts is summarized in Table I It is evident frm the table that grwth rate f bth the species f Phyltsticta increased upt 5th day but in each case there was a prgressive fall frm the 6th day The dry weight f P cycadina increased upt 15th day while that f P artcarpina increased upt 16th day Pestaltia mangiferae, hwever, did nt shw any grwth ult 3rd day Chrmatgraphic analysi s f the medium had indicated that sucrse was nt hydrlysed by Pestaltia mcngiferce upt that day (3rd day) Table I als shws that the grwtb rate f Pestaltia mangiferce increased upt 6th day and then it decreased The dry weight f the rganism, hwever, cntinued t increase upt the 16th day These rganisms were als grwn n a mixture f glucse and fructse and it was bserved that all the three fungi exhibited almst similar behaviur n this mixture as n sucrse except fr the fact that Pestaltia mangiferte which did nt shw any grwth n sucrse upt 3rd day was capable f prdueing mycelium frm the 2nd day 2 Assimilatin f Maltse (R -55)mMaltse n hydrlysis yields glucse nly Chrmatgraphic analysis f the medium cntaining this

4 Assimilatin f Disaccharides by Sme Fungi 277 O CO L c c c e I Q L O LO O, O O O * * O ' O ' - J, I ' " ' 33 ' " "-- = " " =,"n I3#,

278 R N TANDON AND K S BILGRAMI sugar revealed the frmatin f glucse during its assimilatin The rate f hydrlysis f maltse als varied with the rganism Culture medium inculated with P cycadina shwed the

5 278 R N TANDON AND K S BILGRAMI sugar revealed the frmatin f glucse during its assimilatin The rate f hydrlysis f maltse als varied with the rganism Culture medium inculated with P cycadina shwed the presence f glucse (Rf 66) n the 3rd day but it disappeared n the 8th day Traces f maltse were als bserved upt 8th day In the medium n which P artcarpina was grwing, the frmatin f glucse was bserved n the secnd day In this ease presence f glucse was recrded upt 9th day, while that f maltse upt 7th day nly The rate f hydrlysis f maltse by Pestaltia mangiferte was very slw Glucse appeared n the 6th day, and persisted upt 11h day while maltse was present nly upt 1th day Dtu'ing the prcess f assimilatin f maltse by these fungi, it was bserved that besidesrglucse ne mre sugar was als frmed in the medium The R value f this band (band III) was -18 which indicated it t be malttrise (an ligsaccharide) and it was suspected that this sugar was synthesized during the hydrlysis f maltse Fr further cnfirmatin f this band the chrmatgram was sent t Prfessr K V Giri* at Bangalre Giri (1955) in a persnal cmmunicatin als agreed that the particular band (band HI) was f malttriset The daily dry weight f these rganisms n maltse and its hydrlytic prduct (glucse) is recrded in Table II Table II indicates that grwth rate f bth the species f Phyllsticta increased upt 5th day, while that f Pestaltia mangiferte cntinued t increase upt 7th day The dry weight f Phyllsticta cycadina and Pestaltia mangiferae increased upt 15th day Phyllsticta artcarpina, hwever, shwed an increase in the dry weight upt 16th day N grwth f Pestaltia mangifem was evident upt 3rd day This appears t be cnnected with delayed and slw hydrlysis f maltse by this fungus A cmparisn f grwth f these rganisms n maltse with that f glucse shwed that all the three rganisms attained better mycelial grwth n maltse than n glucse Chrmatgraphic analysis f the medium cntaining glucse nly revealed that it was cnsumed in 9, 12, and 14 days by Phyllsticta cycadina, p artcarpina and Pestaltia mangiferte respectively 3 Assimilatin f Lactse (RI 51)--Lactse has nt been reprted t be present in plants Hydrlysis f lactse with acids r lactase yields a mlecule each f glucse and galactse Daily chrmatgraphic analysis f the medium shwed that this sugar was nt hydrlysed by any f the three * Prfessr f Bichemistry, Indian Iastitute f Science, Bangalre-3 f In spite f best effrts, this sugar culd nt be btained in synthetic frm and it was, therefre, nt pssible t include it fr detailed studies r t cmpare the band btained with the band f malttrise,

6 Assimilatin f Disaccharides by Sme Fungi + 279,, O N N N

7 O ICO ",, 28 RN TANDON ANO K S Bn,GgAVH 1 to COO u C C'I O C,,I,1 L 1 O c,i 9,, L - O " O C) O c a j aj - x -,,, K M O I cj :z ' I

Assimilatin f Disaccharides by Sme Fungi 281 rgar/isms and glucse r galactse were, therefre, nt frmed in the culture medium The abve results als established that lactse was finished n 9th and 12th

8 Assimilatin f Disaccharides by Sme Fungi 281 rgar/isms and glucse r galactse were, therefre, nt frmed in the culture medium The abve results als established that lactse was finished n 9th and 12th day by Phyllstieta cyeadina and P artearpina respectively, while Pestaltia mang(ferce culd nt utilize all the lactse f the medium even in 15 days The daily dry weight and grwth rate f these rganisms n lactse and a mixture f its hydrlytic prducts is recrded in Table III It is evident frm Table III that the grwth rate f P eycadina and P artearpina increased upt 5th and 7th day respectively, while the rate f grwth f Pestaltia mangferce was never very high and was nt significantly different between 2nd and the 8th day These fungi shwed much better grwth n a mixture f gluce and galactse (hydrlytic prducts f lactse) than n lactse Chrmatgraphic analysis f the medium cntaining this mixture revealed that all the three fungi assimilated glucse (Rf 67) ne day earlier than galactse (R 65) Glucse was assimilated in 9, 11 and 13 days by P eyeadina, P artearpina and P mangiferce respectively DISCUSSION Present investigatins clearly shwed that sucrse and maltse were gd carbn surces fr all the three rganisms Similar results with these tw sugars were als btained by Herrick (194), Blank and Talley (1941), Cantin (1949), Fergus (1952),, Wlf (1953) and Agarwal (1955) fr the fungi investigated by them Daily chrmatgraphic analysis f the medium indicated that all the present fungi assimilated sucrse and maltse thrugh a hydrlytic pathway The results further shwed that ut f the tw hydrlytic prducts f sucrse, glucse was assimilated three days earlier than fructse "by the tw species f Phyllstieta, thugh bth glucse and fructse were present in their hsts Separate investigatins by Bilgrami (1956) als cnfirmed these results and it was bserved that bth the species f Phyllstieta gave better mycelial grwth n glucse than n fructse Pestaltia mangiferte differed frm the tw species f Phyllsticta, because it was a slw hydrlysing fungus, and it cnsumed bth the hydrlytic prduets f sucrse simultaneusly Hawker (1947) reprted that the amunt f mycelium prduced by Melanspra destruens was different when this fungus was grwn n sucrse r n a mixture f glucse and fructse Her results indicated that it was nt always essential that a cmplex carbhydrate (eg, sucrse) r its hydrlytic prducts (eg, mixture f glucse and fructse) shuld always be equally gd surces The results btained with the present rganisms differed frm thse f Hawker (1947) It was bserved that there was n significant difference in dry weight f these fungi n sucrse r tt mixture f its hydrlytic prducts Recrds n daily rate f assimila-

282 RN TAX'DON AND K S BILGRAM] tin f sucrse had indicated that Pestaltia mangiferte shwed slightly delayed respnse n sucrse than n a mixture f glucse and fructse It appears that this may be due t

9 282 RN TAX'DON AND K S BILGRAM] tin f sucrse had indicated that Pestaltia mangiferte shwed slightly delayed respnse n sucrse than n a mixture f glucse and fructse It appears that this may be due t delayed and slw activity f the enzyme invertase Chrmatgraphic analysis f the medium n which P mangifer was grwing als cnfirmed that glucse and fructse were nt frmed in the culture medium till 3rd day and grwth appeared after that perid Thus:it is als clear that sucrse is nt assimilated by these rganisms till it is hydrlysed Pestaltia mangiferte shwed delayed respnse n maltse als, as the grwth f this fungus started n the 4th day, thugh glucse (hydrlytic prduct f maltse) was detected in the medium n the 6th day It appears that whatever glucse was frmed n the 4th and 5th day was entirely taken up by this rganism, s that n trace was left in the medium and hence it culd nt be detected chrmatgraphically The behaviur f lactse was different frm that f sucrse r maltse as it was nt nly a pr surce f carbn, but was assimilated thrugh a nn-hydrlytic pathway and in this respect the present results seem t cnfirm the bservatins f Marglin (1942) The present results als established that the different disaccharldes may differ in their behaviur, because it was dearly bserved that the grwth n sucrse was similar, n maltse it was better and n lactse it was prer than n their hydrlytic prducts Chrmatgraphic studies shwed the presence f malttrise (an lig 9- saccharide) which appears t have been synthesized during the prcessf assimilatin and which seems t play an imprtant rle in making maltse a better carbn surce than its hydrlytic prduct It may be mentined that recently varius wrkers, including Gift, Nigam and Srinivasan (1954) as well as Bustn and Jabbar (1954), have reprted that varius fungi culd play an imprtant part in enzymic synthesis f ]igsaccharides frm disaccharides The exact rle f malttrise in assimilatin f maltse is, hwever, nt dearly understd by any investigatr The present investigatins indicate that these fungi were capable f synthesizing ligsaccharide nly frm maltse and nt frm the ther tw disaccharides, viz, sucrse and lactse SUMMARY Phyllsticta cycadina, P artcarpina and Pestaltia mangifer were islated frm the leaf spt diseases f Cycas revluta, Artcarpus heterphyllus and Mangifera indica respectively A detailed study f the assimilatin Of sucrse, maltse and lactse by these rganisms was undertaken Sucrse and maltse were assimilated thrugh a hydrlytic pathway Pestalta mangifer utilized bth lucse and fructse snultaneusl),, but the tv

10 Assimilatin f Disaccharides by Sme Fungi 283 species f Phyllsticta behaved differently and cnsumed glucse three days befre the ther hydrlytic prduct f sucrse (viz, fructse) Synthesis f an ligsaccharide (malttrise) was recrded during the assimilatin f maltse Lactse was used thrugh a nn-hydrlytic pathway and was fund t be a pr surce but a mixture f its hydrlytic prducts supprted gd grwth ACKNOWLEDGMENTS The authrs are indebted t Prfessr Shri Ranjan, Head f the Btany Department, AUahabad University, fr prviding the necessary labratry facilities They als wish t express their thanks t Prf Giri fr sending his pinin n the chrmatgrams shwing the presence f malttrise 1 Agarwal, GP 2 Bilgrami, KS 3 Blank, L M and Talley, P J 4 Bustn, H W and Jabbar, A ' 5 Cantin, E C 6 Fergus, C L 7 Garrett, S D 8 Giri, K V 9, Nigam, V N and Srinivasan, K S 1 Hawker, L E 11: 12 Herrick, J A 13 Mandels, G R REFERENCES "Cultural and pathlgical studies f sme Fungi lmper fectii,'" D Phil Thesis, University fallahabad, 1955 "Pathlgical and physilgical studies f sm fungicausing leaf spt diseases," 1bid, 1956 "The carbn utilizatin and carbhydrate activity f Phymattrichum mnivrum," Amer J Bt, 1941, 28, "Synthesis f ligsaccharides by Chaetmium glbsum," Chem and Indust (Lndn), 1954, 2, 48 "The physilgy f aquatic Phycmycete Blastcladia pringsheimii," Amer J /Rt, 1949, 36, "The nutritin f Penicillium digitatum," Myclgia, 1952, 44, "Sil cnditin and take all disease f wheat," Ann appl BiL, 1936, 93, Persnal Cmmunicatin, 1955 "Enzymic synthesis f ligsaccharides frm sucrse and lactse by Aspergillus flavus," J Indian Inst Sci, 1954, 36, "Effect f temperature f strage n the rate f lss f fertility f stck cultures f Melanspera destruens," Nature (Lndn), 1947, 159, 136 "Grwth and fruiting f certain Ascmycetus fungi," Ann Bt (NS), 1946, 1, "The carbn and nitrgen metablism f Stereum gausapatum," Obe J Sci, 194, 4, "Metablism f sucrse and related ligsaccharides by spres f fungus Myrthecium verrucaria," 'Plant Physil, 1954,

11 284 R N TANDON AND K S BII_GRAMI 14 Marglin, A S 15 Ranjan, S, Gvindj and Lalriya, M M 16 Smith, V M 17 Tandn, R N and Bilgrarni, KS 18 Wlf, F T PhD Thesis, University f West Virginia, 1942 "Chrmatgraphic studies n amin acid metablism f healthy and diseased leaves f Crtn sparsiflrus," Prc Nat Inst Sci, 1955, 21, "On mechanism f enzyme actin by Marasmius chrdalis,'" Arch Bichem, 1949, 23, "Effect f sme nitrgen cmpunds n grwth and sprulatin f Phyllsticta cycadina," Prc Nat Acad Sci, (India), 1954, 24 B, "Utilizatin f carbn and nitrgen cmpunds by Ustilag zew,'" Myclgia, 1953, 45,

* Manuscript received August 25, t Horticultural Research Section, CSIRO, Glen Osmond, South Australia.

* Manuscript received August 25, t Horticultural Research Section, CSIRO, Glen Osmond, South Australia. SUCROSE BREAKDOWN AND SYNTHESIS IN THE RIPENING GRAPE BERRY* By P. J. HARDyt Sucrse is the majr translcated sugar in the grapevine (Swansn and EIShishiny 1958), but in the berry glucse and fructse tgether