1. {5
3
J. Ecobiol. 11(4)299 - 305 (1999)
@ Palani Paramount Publications-Printed in lndia
PECTIC ENZYMES OF ALTERNARIA CEPULAE IN !.EAF
BLIGHT DISEASE OF ONION
B ANNADURAIN, P KARUNANIDHI** AND S MAHALINGAM**
*CENTRE FOR BIOTECHNOLOGY, DEPARTMENT OF BIOCHEMISTRY
C ABDUL HAKEEM COLLEGE MELVISHARAM - 632 509, TAMIL NADU, INDIA
* DEPARTMENT OFZOOLOGY, P.G. EXTENSION CENTRE, UNIVERSITY OF MADRAS
VELLORE 632 OO4, TAMIL NADU, INDIA
(Reived 20.'12.1996; revised 1 0' 1 0'99 ; accepted 1 9. 1 0'99)
ABSTRACT
A pure strain of Altern aria cepulae was isolated from leaf blight area of onion. The organism
was found io grow well in the Natural Onion medium, Czapeck and Lindberg medium, Bacons medium,
Ray's medium and Pectin medium as indicated by the growth rate. The production of endo PG enzyme
was maximum in Onion medium and in Pectin medium. The Nelson Somogyi's method was found to be
sensitive and accurate for the assay of endo PG activity.
Key words: Alterntaia cepulae,Pectic enzymes, Blight disease, Onion'
INTRODUGTION
Extensive studies on various diseases in onion (Altiurn cepa Linn.) have been
reported (Nolla 1927;Michail et a|1981; Maltitiz etal1984;Tanaka etal1985; Ramsey
et allSAd). t-eat btight disease due to Alternaria sp was reported as early as 1923 by
Ajrekar. ltwas also reported in Coimbatore (Ayyangar 1928) and Poona (Uppalef al
1g3S). A purple blotch disease in onion due to Alternaria porri (Ellis) was reported in
Bihar (Thirumalachar & Misra 1953) and Pubjab (Pandotra 1964)'
The leaf blight disease in onion occurred in and around Kaliyangadu of Periyar
district of Tamil Nadu since 1981. The epidemology of the disease was studied by
Khare and Nema (1981). The fungus Allium cepawas isolated from this disease and
used in this investigation. The pectic enzymes secreted by the pathogen degrade
the pectic constituents of the cell wall and middle lamella when the pathogen invades
the host in the leaf blight disease (Srinivasa ef a/ 1960; Bilgrami 1963). This paper
reports the isolates of the leaf blight causing fungus A.cepulae and culture
characteristics.
MATERIAI-S AND METHODS
lsolation of the fungus : The leaf blight causing fungus A. cepulae was isolated by adopting lhe
procedure of Blancher and Tatter (1981). The following media were experimented to find out the most
suitable medium forA. cepulae growth and pectic enzyme production. 1. Potato Dextrose Agar medium
1
l
ISSN: 0970-0937-001 1 - 299

2. 3OO BANNADURAI ETAL
(Boone & Keitt 1956) 2. Czapeck dox agar medium (Acha & Villaneva 1961) , 3. Naturalonion medium
(Bateman 1966) , 4. Pectin yeast extract medium iBootn 1971), 5. Glycerol medium (Collmer et a/
1982)' 6. Pectin medium (Strand et a|1982),7. Ray's medium lnay isso), 8. Czapeak dox and Linderberg
medium (Leonowicz et a/ 1985), 9. Glucose and Mineral salts medium (Rrngr.*r, i 1972),,10. Bacons
medium (Bacon ef a/ 1981).
Culture methods: The growth of the fungus and determination of mycelial dry weight was carried out
according to the method of cervone et al (1917) and Dube and Bordia (19g2).
Pectic enzyme preparation: The natural onion medium was prepared according to Bateman (1966)
Estimation of endo polygalacturonase (EPG EC 3.2.1.15): The viscometric method was carried out
according to the procedure of Ayers ef a/ (1966). Reducing sugar method was carried out according to
Nelson ('1944) and Somogyi (1952).
Estimation of Pectin Methyt Esterase (EC 3.1.1.11): PMe activity was estimated according to the
procedure of Kertesz (1951).
Estimation of Poly methyl Galacturonase (PMG EG 3.2.1.4,1): PMG was estimated by adopting the
method of Tallboys and Bush 91970).
Estimation of Pectin Trans Eliminase (PTE, EC4.2.2.1): PTE was estimated byfollowing the method
of Ayers ef a/ (1966) with stight modification of Besford et at (1972).
Estimation of Pectin Methyl Trans eliminate (PMTE, EC 4.2.2.31: PMTE activity was estimated
according to the method of Tattboys and Bush (1970)
Statistical analysis: The mean ( X ) anO standard deviations were calculated using the procedure of
Bailey (1984). The difference in results obtained was examined with 't'tests for small samples.
RESULTS AND DISGUSSTON
The growth of Alternaria cepulae was expressed as dry weight in grams in
different culture media (Fig.1). lt was observed that Czapeck and Lindeberg medium
was the most favourable for the growth. Becons medium and natural onion medium
also supported good growth.
The endo PG activity of A. cepulae in different culture media is shown in
Fig'2. lt is observed that A.cepulae produces maximum quantity of enzyme in natural
onion medium and in Pectin medium. The estimation of endo PG ictivity during
different periods of growth is shown in Fig.3. lt is also revealed that natuial onion
medium exhibits maximum EPG activity when compared to pectin medium. EpG
activity was found to be maximum on 16th day of incubation. These results are in
support of Bateman's (1966) observation of Bateman (1966). As the fungus grows
well in Ray's and in pectin medium, these two media were selected for tirge scale
production and purification studies.

3. PECTIC EI'IZYMES OF ALIERIVARIA CEPULAL 301
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Fig. 1. Potato Dextrose Agar, 2. Czapeck dox agar, 3. Natural onion leavbs,4. Natural onion bulbs, 5.
PJctin yeast extract, 6. Glycerol medium, 7. Pectin medium, 8. Ray's medium 9. Czapeaks and
Linderberg, l0.Baconsmedium. BarsarethemeahvalueofthegroMhofmyceliumofA.cepulae2S0
ml Erlenmeyer flask. Growth is expressed in mycelial dry weight in grams, Significance =
**
= P<0.05.
Fig. 2. Endo PG activity of A.cepulae after 16 days when grown in different culture media. 1 .Czapeck
medium, 2. Natural onion leaves medium, 3. Natural onion bulbs medium, 4. Pectin yeast extract
medium,5. Glycerol medium,6. Ray's medium, T.Czapeak and Linderberg medium9. Bacons medium.
Endo PG activity is expressed in relative in relative viscosity units i.e RVU=1000ff50 where T50 is the
time taken for 50% loss of viscosity. Bars are the mean value of endo PG activity in mg. Significance
**=P<0,05.
Fig. 3. Estimation of endo PG activity of A. cepulae grown in two media during different period of
incubation. Values displayed are the mean value +SD 16th day is significant P<0.05.
Fig.4. Estimation of endo PG acitivity of A. cepulae and the inhibitory effect of CaCl, during different
" period of incubation by viscometric method. Endo PG activity is expressed in relative in relative
viscosity units i.e RVU=1 000ff50 where Tro is the time taken for 50% loss of viscosity values given are
the mean value of 6 individual experiment+ SD
Fig. 5. Estimation of endo PG activity by viscometry at differeni pH Endo PG activity is expressed in
relative viscosity units RVU=1000/T50 where T50 is the time taken for 50% loss of viscosity. Values
given are the mean value of 6 individual experiment+ CaCI, inhibition is significant. P<0.05.
Flg. 6. Estimation of endo PG activity by TBA test at different pH values given are the mean + SD
Effect of CaCl, is significant. P<0.05).

4. 302 B ANNADURAI ET AL
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Fig. 7. Estimation of endo PG of A. cepulae by reducing sugar method at different pH Endo PG activity
is expressed in ug of galacturonic acid released per ml in half an hour at 32 + 1oC
Values given are the mean + SD
Effect of CaCl, is significant. P<0.05.
Fig. 8. Estimation of pectin methyl esterase of A.cepulae during period of incubation at different pH
PME activity is expressed in RVU with pectin as substrate (RVU=Relative viscosity units =1000ff50
where T5o is the time taken for 50% loss of viscosity. values given are the mean +sD
Activity is significant (P<0.05) at pH 6.0
Fig.9. Estimation of Poly methyl Galacturonase in different days of culture at different pH. AMG activity
is expressed in RVU with pectin as substrate (RVU=Relative viscosity units=1000/Tuo where Tro is the
time taken for 50% loss of viscosity. Values given are the mean+SD
Fig. 10. Absorption spectrum of the products from TBA test to estimate pectin Traseliminase (pTE) of
A.cepulae
Fig.l1. Estimation of Pectin Methyl Transeliminase with the without Cacl, at different pH.
PMTE activity was read directly at 235 nm with 4.0 ml of Pectin, 1ml of CaCl, or HrO and .1ml
of enzyme at 32+0C values given are the mean +SD.

5. PECTIC ENZYMES OF ALTERNARIA CEPIJLAE 303
Effects of Cacl, on endo PG as inhibitor is shown in Fig.4. lt was observed
that the endo pG actiriity was inhibited by 0.025 M CaClr. Bateman and Lumsden
if gOS) have reporteA tnat high calcium content in the nost is often associated with
dis"ase resistance. High acctrmmulation of calcium (Ca..) is observed in infected
region. This Ca** rendeied resistance to further action of fungal endo PG (Goodman
it"it rca7;. lt is well known that the availability of calcium.and the influence of auxin
affects the pectin enzymes by the formation of calcium bridge (Ediginton ef a/ 1958;
Corden ef al 1959).
The endo pG activity was estimated by viscometric method (Fig.6) and by
Reducing sugar method (Fig.7). The optimum pH in all the methods is pH 5.0. A
second iealiat pH 7.0 wal also observed. These activities were suppressed by
0-025 M CaClr. but of the three methods, the estimation of reducing sugar method
was found to 5e more sensitive and accurate for the experimental studies.
The,estimation of EPG activity with sodium polyetate as substrate two peaks
at pH 5.0 and,7.0 Batemin (1966) ind Rombouts and Pilnik (1972) have reported
tni.O.OZS rtr Cacl, inhibition at pH 7.0 indicates the presence of endo PG. But trans
,eliminases are reported to be activated by Cacl, at alkaline pH (Batem-an 1966;
no*uoutr & pilnik 1972). Therefore, it is likely thiat tvvo forms of endo PG may be
excreted by A. cepu/ae in the natural onion medium as well as in pectin medium'
similar observations were noted in other methods of estimation of EPG activity'
pectin methyl esterase activity during different period of incubation is shown
in Fig.g. .lt is observed that the PME ictivity is maximum on 12th day of incubation at
pH dO; poly Methyl Gatacuronase activity during different periods of incubation at
different pH-is shown in Fig.9.
'ltjndicates that the enzyme did not prefer pegtin as its
substrate. Hence, PMG activity may be absent'
Absorption spectrum of the products from TBA test to estimate pectin trans
eliminases activity ort A. ceputae is shown Fig.10. It indicates that the absorption is
not equivalent to b.t at 55&nm. Hence, the activity is not significant'
pectin Methyl Transeliminase activity and the effect to CaCl, suppresses the
activity. lt is not aciivated at alkaline pH. Hence, PMTE activity is absent.
The activities of PTE, PMTE and PMG were very low. EPG which has high
activity seems to be the major enzyme involved in cell *.11^!:go9ation of oni-on'
in"r" r".rlts are in support of the observation of Sandu (1982) and'Carrol (1985)'
I REFERENGE
Acha I G and Villaneuva J R 1g61 A selective medium for the formation of Ascospores by Aspergil/us

6. 304 B ANNADURAI ET AL
nidulans. Nature 199: 329.
Ajrekar S L 1923 Annual report of the workdone under the plant pathologist to'the Government of
- Bombay, Poona for the year 1921-22, Bombay Department. Agr. nnn. Rept.p. 1oz-104.
Ayers W A, Papvizas G e and piem A F 1 966 Polygaiactuionate transJiminase and'potygrtr"rron"="
. production by Rhrzoctonia sorani. phytopathorogy s6: 1006-1011.
Ayyangar C R 1928 A-Leaf spot and blight disease of onion caused by Alternaria palanduisp. Nov.
Agr. Res lnst. pusa Bull.ll:1-14.
Bacon C W, Porter J K and Robbins J D 1981 ln vitro production of auxin by Balancia epichse.
Phytochemis try 24: 1 429-1 431 .
Bailey T J 1984 staticar methods in biorogy; Hodder and stoughton, London.
Bateman D F and Lumsden R D 1965 Retation of calcium conient and nature of the pectic substances
in bean hypocotyls of different ages of susceptibility to an isolate of Rhizoctonia sotani.
Phytopathotogy 55: 734-tSB.
Bateman D F 1966 Hydrolytic and transeliminative degradation of pectic substances,by extracellular
_ enzymes of Fusarium sorani phaseo/i. phytopathorogy 56i23g-244.
Bateman D F and Millar R L 1966 Pectic enzyme in tissue o"egration. Ann. Rev. of phytioathol,4:
1 1 9-146.
Besford R T and Hobson G E 1972 Pectic enzyme associated with the softening of tomato fruit.
Phytochemistry 1 1 : 2201-Z2OS.
Boone D M and Keitt G W 1956 Fhytotoxins in plant diseases. Academic press, New york.
Blancher R o and Tattar T A 1981. ln field and laboratory guide to tree pathology, Academic press, New
York.
Booth C '1971 Culture media: Methods in microbiology 4: 4}-g4.Academic press, New york.
Cervone F, Scala A, Forest M, Cacace M G and Noviello 1977 Endopoly galacurono sefrom Rhizoctonia
fragariae purification and characterization two isoenzymes.' gGnemica et biophysica lcta482:379-385.
Collmer A and Bateman D F 1982 Regulation of extracellular pectate lyase in Erwinia chrysanthmi:
evidence that reaction products of pectate lyase and exopoiy D galacturosidase mediate induction
on D-galacuronan. Physiol. pl.pathol. 21 : 1 27 -1 39.
Dube H C and Bordia S 1982 lmpact of sugars on pectic acid lyase production by Hetminthaspoium
sacchari. lndian Phytopath. 3i:434-432.
Edging L W, Cordon M E and Diamond A E 1958 The Role of pectic substances in chemically induced
resistance of Fusarium wilt of romato. phytopathology 51: 179-182.
Goodman R N, Kiraly Z and Zaitlin M 1967 The biochemistry anJ pnysiology of infectious plant diseases.
D Van Nostrand Company inc. London.
Kertesz Z l, 1951 The pectic substances Interscience publications, New york p.628.
Khare U K and Nema K G 1981 slud]es on purple blodtch of onion sporulation on host and dispersalof
conidia. lndian phytopath. 34: 214-21g,
Leonowicz A, Szklaz G and wasolewska M w 1985 The effect of fungal laccase on fractional
Lignosulphonates (peritan Na.). phytochemistry 24: 393_3g6.
Maltitz P A and Broembasen S.L 1984 Phytopthoia porri on onions in sourth Africa. ptant
disease 68:732-
Mc' Canol D R and Thor E 1984 Phytolytic cellulolytic and proteolytic activities expressed by culture of
Endothia parasitica and inhibition of these activities by iomponents extracted from Chinese
and American chestnut inner bar. physior. pr. pathor. io: gozgze.
Michail S H and Salem M A 1981 Reaction ofonion cultivars to scald disease incited by A/fe rnaria poyi.
Acta phytopafh. Aca Sci. Hung. i6:299-305.
NelsonN 1941 A photometric adaption of the somogyi method for the determination of glucose. J.
Biol. Ghem. 153: 375-380
Nolla J AB 1927 A new A/fernarla disease of onions (Attium c"pa L1. phytopathol ogy. 17t1s-1i2.
Pandotra V R 1s64 Purple Blotch disease of onion of Punjab: its occurence, prir,rg'.,'rl.ity ,-nj r,o"r
range. Proc. lnd. Acad. Sci. Sect. B. 60: 599_603.

7. PECTIC ENZYMES OF ALTERNARIA CEPULAE 305
Ramsey G R and Lorbeer J W 1986 Flower blight and scape girdling of onion grown for seed production
in New York. Phytopathology 76: 599-605.
Ramsey G R and Lorbeer J W 1986 Pathogenicity of sp on onion umbels and scape under controlled
conditions. Phytopathology 76: 606-615.
Ramsey G R and Lorbeer J W 1986 The role of temperature and free moisture in onion flower blight.
Phytopathology 76: 61 2-61 6.
Rangaswami G 1972 Disease of crop plants in lndia. Prentice Hall of lndia Pvt. Ltd.,New Delhi'
Ray p M 1956 The destruction of lndole Acetic Acid ll Spectrophotometric study of the enzymatic
reaction. Arch. Biochem. Biophys. 64: 193-216.
Rombouts F M and Pilnik W 1972 Research on Pectin depolymerases in the sixties: a literature review.
Crit. Rev. Fd. Technol. 3-26.
Sandu D K and Kalra M K 1982 Production of Cellulose, Xylene and Pectinase by Trichoderma
longibrachiagum on different substrates. Trans. Brit. Mycol. Soc 79: 409-413.
Somogyi M 1952 Notes on sugar determination. J. Biol' Chem. 19: 19-23.
Sriniviian K V and Viiayalakshmi N 1960 Thiamine and biotin requirements of two strains of
Gtomerella tucumanesis (speg) ARX and Mueller. Gurr. Sci. (lnd) 29: ',l03=104.
Stand L L, Corden M E and Mc Donald D L 1976 Characterization of two endopolygalacturnase isozymes
produced by Fusarium oxysporumf .sp. Lycopersici. Biochimicaet biophysiczaacta429:870-
883.
Tallboys p W and Bush Ln V 1970 Pectic enzymes produced by Verticillium species. Trans. Brit.
Mycol. Soc. 55: 367-381.
Tanaka tyt, Cnee K and Komochis 1985 Cultivation of onion Res. Bull of the Hokkaido Natl. Agric.
expt. Stn. 141:17-28.
Thirumalchar M J and Misra J N 1953 Some disease of economic plants in Bihar. lndia. I and ll (Abst)
RAM.33:(6).