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J. Ecobiol. 11(4)299 - 305 (1999)@ Palani Paramount Publications-Printed in lndia

PECTIC ENZYMES OF ALTERNARIA CEPULAE IN !.EAFBLIGHT 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 lheprocedure 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

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ISSN: 0970-0937-001 1 - 299

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 Linderbergmedium (Leonowicz et a/ 1985), 9. Glucose and Mineral salts medium (Rrngr.*r, i 1972),,10. Baconsmedium (Bacon ef a/ 1981).

Culture methods: The growth of the fungus and determination of mycelial dry weight was carried outaccording 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 outaccording to the procedure of Ayers ef a/ (1966). Reducing sugar method was carried out according toNelson ('1944) and Somogyi (1952).

Estimation of Pectin Methyt Esterase (EC 3.1.1.11): PMe activity was estimated according to theprocedure of Kertesz (1951).

Estimation of Poly methyl Galacturonase (PMG EG 3.2.1.4,1): PMG was estimated by adopting themethod of Tallboys and Bush 91970).

Estimation of Pectin Trans Eliminase (PTE, EC4.2.2.1): PTE was estimated byfollowing the methodof 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 estimatedaccording to the method of Tattboys and Bush (1970)

Statistical analysis: The mean ( X ) anO standard deviations were calculated using the procedure ofBailey (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 indifferent culture media (Fig.1). lt was observed that Czapeck and Lindeberg mediumwas the most favourable for the growth. Becons medium and natural onion mediumalso supported good growth.

The endo PG activity of A. cepulae in different culture media is shown inFig'2. lt is observed that A.cepulae produces maximum quantity of enzyme in naturalonion medium and in Pectin medium. The estimation of endo PG ictivity duringdifferent periods of growth is shown in Fig.3. lt is also revealed that natuial onionmedium exhibits maximum EPG activity when compared to pectin medium. EpGactivity was found to be maximum on 16th day of incubation. These results are insupport of Bateman's (1966) observation of Bateman (1966). As the fungus growswell in Ray's and in pectin medium, these two media were selected for tirge scaleproduction and purification studies.

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 extractmedium,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 ofincubation. 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 arethe 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. Valuesgiven 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).

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 activityis expressed in ug of galacturonic acid released per ml in half an hour at 32 + 1oC

Values given are the mean + SDEffect of CaCl, is significant. P<0.05.

Fig. 8. Estimation of pectin methyl esterase of A.cepulae during period of incubation at different pHPME activity is expressed in RVU with pectin as substrate (RVU=Relative viscosity units =1000ff50where 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 activityis expressed in RVU with pectin as substrate (RVU=Relative viscosity units=1000/Tuo where Tro is thetime 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) ofA.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.

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)'

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304 B ANNADURAI ET AL

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