BIOJOURNAL,VOL. ll NO.1 & 2,161.165 JUNE & DECEMBER, l999

GROWTH OF ALTERNARIA CEPULAE IN

LEAFBLIGHT DISEASE IN ONION

B. Annadurail and D. B. MotlagDepartment of Biochemistry, University of Madras, Chennai- 600 025

ABSTRACT

The activity of endopolygalactuonase (Endo PG) in infected Onion leaves and Pectinmedium by Atternaria cepulae, a leafblight causing f ungus was estimated. lt was found thatin both the cases, the activity of endo PG was maximum on the 1 6th day after the inocu lationof A. cepulae from Onion leaves.

INTRODUCTION

Endopolygalacturonase (Poly o, 1,4 galacturonide glycanohydrolase, EC 3.2.1.15) plays

a significant role in pathogenesis of many plant diseases (Cooper, 1980, Boothby, 1984,

Mills, 1985). Endopolygalacturonase is one of the prime macerating enzyme produced by

Alternaria ceputae during leafblight disease of Onion (Annadurai,1996, 1998,1999).

Endopolygalacturonase is produced both constitutively and adaptively by differentmicroorganisms (Annadurai, 1987).ln most instances, the enzymes are produced adaptivelyrather than constitutively (Keen & Horton, 1966). Fusarium monilitorme (Biehn, 1971) andScterotium rotfsii(Punja et. al., 1985) produce it adaptively in the presence of pecticsubstrates. A small number of pathogens like Prrcularia filamefosa (Ayers and Papavizas,1966) and Cotletorichum falcatum (Singh and Hussain, 1964) Verticillium alboatrum (Mussel

and Strouse, 1972) and Aphanomyces cutices (Ayers, 1965) are known to producepolygalacturonase in a constitutive manner.

pectin induces greater enzyme production in adaptive enzymes (Grant, 1985). Thepathogen in nature confronts with pectin substances not in isolation, but in combinationwith other carbohydrates (Annadurai, et al" 1998, 1999).These carbohydrates are reportedto control the productlon of Pectic enzymes (Patil and Diamond, 1968, Moran and Starr,

1969, Maldonado et. al. 1986). Catabolic repression of the endo PG levels in infected Onionleaves and in pectin medium were ascertained (Annadurai et. al., 1999). Systematicinvestigations were carried out on various nuitritional factors and culture conditionsinfluencing the production of endo PG of A. cepulae.

MATERIALS AND METHODS

Mycelial Dry Weight Deterrnination

Mycelial dry weight was determined by following the method of Annaduraiet al (1998).

After 16 days of inoculation ol A, cepulae in different physicochemical environment the

Head of the Department of Botany and Biochemistry, CAH College, Melvisharam-632509, Vellore District, Tamilnadu

1.

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ANNADUBAI AND MOTLAG .

contents of the erlenmeyer flask was filtered through a glassfunnel fitted with a coarsegrade sintered glass f ilter and washed thoroughly with water. The mat was pressed in f ilterpaper to remove the excess of moisture.This was transferred to a previously weighed f ilter

paper. lt was dried in an oven at 700C Overnight. lt was cooled to room temperature

lSZt t0C) in a desicator and weighed.

Estimation of EndopolygalacturonaseEndo PG activity was estimated by reducing sugar method according to Nelson (1944)

and Somogyi(1952).

The incubation mixture containing 1.0 ml of Sodium polypectate at pH 5.0 0.5 ml of

enzlrme were incubated at 3Z r 10C for t hour.The reaction was stopped by adding 2.0 mlofalkaline copper reagent. The tubes were kept in a boiling water bath for 30 minutes. Thecontrol treatments were carried out in the same manner except was added after adding thealkaline copper reagent.

The tubes were the.n cooled to room temperature (32 t 10C; and 1.0 ml ofArsenomolybdate reagent was added and tlren it was read at 530 mm in uv 260 Shimadzuspectrophotometer.

Estimation of ProteinProtein in the culture filtrate was estimated by employing the method of Lowry et. al.

(1951) using crystalline Bovine serum albumin as standard,

RESULTS AND DISCUSSION

Estimation of endopolygalacturonase activity in blight affected Onion leaves is showninTable 1.lt indicates that the lesion area and area around lesion in the inoculated leavesshows optimum endo PG activity. ln both the areas an endo PG activity starts from the 8thday of inoculation. The optimum endo PG activity was observed on the 16th day afterinoculation of A. cepulae.The difference in endo PG acitivty on 12th and 16th day is signif icantp < 0.01 in the lesion area and in the area around lesion.The endo PG activity is significantal0.1'/.level on 8th day and 2o/" level on 20th day.

When the leaf blight causing fungus A. cepulae is grown in synthetic pectin medium(Table 2), the mycelial growth as indicated by dry weight slowly increases and it is signif icantupto 20 days (p < 0.001).The difference is not significant after 20 days.The protein contentalso increases from 548 pg/ml.The difference is significant on 8th day and 12th day. lt issignificant at 5% Ievelfrom 16th days to 30th day. Endo PG activity slowly decreases to B0

to 564 units on the 16th day.Thereafter the activity slowly decreases.The difference of endoPG acitivity is significant (p < 0.001).

The results presented inTable 1, indicates that the EPG activity in lesion area andaround lesion area began on the 16th day in synthetic pectin medium also (Table 2).TheEPG activity is more in the area around the lesion than in the lesion itself.This suggests thatendo PG enzymes initiate the pathogenesis by the cell wall and pave way for the entry of thehyphae (Bateman, DF and Basham, H.G.1976). After the 16th day of growth of the fungus,the EPG activity slowly decreases.

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BIOJOURNAL, JIJNE & DECEMBER, lggg

Table - 1

Estimation of endo PG of in blight affected onion leaves caused by A. cepulae

sl.No.

Days after d.f.inoculation

Control treatments lnoculated leaves

Wounded Healthyarea area

Lesion area Around lesion AwayfromLesionarea

UnitsRVU/ml

UnitRVU/ml

1. 4 days

2. I days

3. 12 days

4. 16 days

5. 20

5

5

5

5

5

NIL

NIL

NIL

NIL

NIL

NIL

NIL

NIL

NIL

NIL

NIL NIL

17 = 3.27 25 + 1-7

29 = 2.45 34 t 2.1

45 t 1.8 ++ 40 x. 1.4

23 t 1.41 NS 46 x.2.62

++

+

+

NIL

NIL

NIL

NIL

NILValues given are the mean value of EpG activity in RVU unit * SD.

EPG activity is expressed in Belative viscosity units (RVU - 100/Ts') whereTuo is the timetaken in seconds tor 5oo/o loss of viscosity. The procedure is explai-nld in the iJxt.d.f. - Degrees of freedom =.number of observation - 1.

Significance = ++ P < 0.001, + = P < 0.001 (N = 1)

Table - 2

Estimation of endo PG of A. cepulae in synthetic pectin medium

Sl. Culture d.f.No. Age

Mycelialdry wt. Protein Endo PG activity

ln gms/ Signi-flask t SD ficance

Sd Significance

Units t SD!g,I Signi-ficance

1. 4 days 5 0.35 t 0.01 549.00 * 9.70 80.00 t 12.002- 8 days 5 0.58 t 0.02 ++ s64.00 + 4.10 ++ 144.00

= 1.76 ++3. 12 days 5 0.85 r 0.03 ++ 604.00 * 5.73 ++ 2}4.oo r 10.6 ++4. 16 days 5 1.40 t0.03 ++ 612.00 t 4.52 + 364.00 t 1.85 ++5. 20 days 5 1.90 t 0.07 ++ 620.00 x. 4.s2 + 300.00 r 1.85 ++

6. 25 days 5 1.92 r 0.07 NS 624.00 t 1.85 + 284.00 t 8.98 ++

7. 30 days 5 1.94 t 0.08 NS 628.00 t 1.54 + 188.00 t 8.58 ++

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ANNADUNAI AND MOTLAG

Protein content is expressed in pg/ml by adopting the method of Lowry et aL, withBSA as standard.

EFG activity is expressed in units as pg of galacturonic acid released from one ml in30 minutes from galacturonic acid standard.

Mycelial dry weight is in grams t Sd

Allvalues expressed are the mean value (X) of e individualexperiments * SD

Significance : ++ = p < 0.001, * = p < 0.05, NS = Not significant

REFERENCES

Annadurai, B, (1987), Studies on endopolygalacturonase in leafblifght disease of Onion(Allium cepa Linn) caused by Alternaria cepulae (Ponnappa) and its interaction withphytohormones, Ph. D.Thesis, Department of Biochemistry, University of Madras.

Annaduari, B, Palani,8., Mahalingam, S., and Singaravetu G., (1998). Effect of aflatoxin orRBC,WBC and hemoglobin of Rattus rattus narvegicus, Biojournal, 10.165 - 172.

Annadurai, 8., Prabhakaran. V., Md., Faruk, S and Arulkumaran. P (1998). Production ofamylase in Aspergillus oryzae Biojournal 10,179-185.

Annadurai, 8., Mahalingam, S., Palani, 8., and Singaravelu, G., ('1999). Production of af latoxinI in contaminated stored grains, J. Ecotoxicol, Environ, Monit. g (1) 13-17.

Annadurai, B., B, Karunanidhi, P. and Mahalingam, S., (1999). Effect of sugars on amylaseactivity ol Aspergillus oryzae, J. Ecotoxicol. Environ. Monit I (3),2A9-212.

Annadurai, B., Karunanidhi, P. and Mahalingam, S., (1999). Pectic enzymes ol Alternaria.cepulae in leafblight disease of Onion J. Ecobiol, 11 (4) 299-305.

Annadurai, 8., and Motlag, B. D.,1996. Extracellular enzymes ol Alternaria cepulaein leafblightdisease of onion, Biojournal, 4105-109.

Annaduari, 8., Gopinath, D., and Palani, R., 1998. Studies on the role of the cetl wall degradingenzymes in leafblight disease of onion (Allium cepa). caused by Alternaria cepulaeBiojournal, 1 0, 173-178.

Ayers, W. A. & Papavisas, G: C. (1965), An exocellular pectolytic enzymes ol Aphanomyceseute i ch es. Phyto Patho logy, 55. 249-253.

Ayers, W. A, Papavizas G. C. and Diem, A.F. (1966), Polygalacturonase transeliminase andPolygalacturonase production by Rhizoctonia solani. Phytopathology,56,1006-1011.

Bateman D.F. & Basham HG (1976) Degradation of Plant cellwall and memberanes bymicrobial enzymes, in physiological plant pathology (R Heitfuss and PH Williamseditors). Berlin, Heidelberg, New york, Springer Varlag, 316-355.

Biehn W.L. & Dimond A.E. (1971a), Effect of galactose on Polygalacturonase production andpathogenesis by Fusarium oxysporum f sp lycopersici. Phytopathology,6l, 242-243.

Biehn W.L. & Dimond A.E. (1971b), Effect of Pectin source and sugars on polygalacturonaseproduction by Ceratocystis ulmi. Phytophathotogy, 61, 745-746.

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BIOJOURNAL, JUNE & DECEMBER, 1999

Boothby D. & Magreola N.O. (1984) Production of Polysaccharide degrading enzymes ofcochliobolus sativus and Fusarium culmorum grown in liquid culture.Trans. Br. Mycolsoc.,85, 275-280.

Cooper R.M. & Wood R.K.S. ('1980), Cell wall degrading enzymes of Vascular wilt Fungi lll,possible involvement of endopectin lyase in Verticillium wilt of Tomato. Physiol Pl Pathol,16,285-300

Grant (1985),The effect of Pectin and Related Compounds on Encystment and Germination. of Phytopthora palmivora zoospores J. Gen. Microbiol,131.669-676.

Keen N.T. & Horton J.C. (1966), lnduction and repression of endo polygalacturonase synthesisby Pyrenochaeta terrestris. Canad J. Microbiol, 12. 443-483.

Lowry O.H., Rosebrough N.J. Farr. A.L.O. & Randall R.J. (1951) Protein measurement withthe Folin phenol reagent J. Biol Chem., 193,265-295.

Maldonado M. C., Navarro, A. & Calliori D.A.S. (1986), Production of Pectinasesby Aspergillussp using differently pretreated lemon peel as the carbon source. Biotechnol Lett, 8,501 -504.

Mills P.R. &Wood R.K.S. (1985), Degfadation of Cellwall material from unprotected andsystemically protected cucumber plants by extracellular enzlymes of Colletotrichumlagenarium. trans. Brit Mycol soc, 85, 291-298.

Moran F. & Starr M.P. (1969), Metabolic regulation of Polygalacturonic acid trans. eliminasein Eriwinia. Eur. J. Bio.chem, 11,291-295.

Mussel H.W. & Strouse B.(1972), Characterization of two polygalacturonase produced byVerticiltium alboatrum Canad J. of Biochemistry, 50, 625-632.

Nelson N. (1944) A Photometric adaptation of the Somogyi method for the determination ofGlucose. J. Biol Chem., 155,375-380.

Patil S.S. & Diamond A.E. (1968), Repression of Polygalacturonasesynthesis in Fusariumoxysporum sp. lycopersici by sugars, Phytopathology, 58, 676-682.

PuniaZ. K. Huang J.S. & Jenkinks S.F. (1985), Relationship of mycelialgrowth and productionof Oxalic acid and cell wall degrading enzymes to virulence in Sclerotium rotfsii.CanadJ of Plant Pathol,7,109-117.

Somogyi M. (1952) Notes on Sugar determination J. Biol Chem. 195, 19.

Singh, G. P. and Hussain, A (1964) Relation of hydrolytic enzyme activity with virulence ofstrains of Colletotrichum falcatum. Phytopathology, 54, 1 1 00-1 1 01 .

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