Cellulase, pectinase, peroxidase and polyphenol oxidase activities were determined in healthy,naturally infected and inoculated agarwood (Aquilaria malaccensis) plant parts at varioustime intervals to study the changes in activities of these enzymes during pathogenesis. &nbsp;Samplesinfected naturally with Chaetomium globosum and &nbsp;Fusarium oxysporum exhibited higheractivity of all the enzymes compared to healthy samples
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R Samanta, P Tamuli, P Baruah,

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240Aquilaria malaccensis  Lamk.  (Family:Thymalaeaceae) bears resinous patches offragrant wood known as 'agar'.  Agar isconsidered to be a pathological productproduced by fungal invasion of the host.Many workers have studied agar formationand reported that the agar zones areassociated with mould and decay fungi(Bhattacharyya et al. 1952; Tamuli et al. 2000).Among the different fungal species associatedwith agar zones, a few could exhibitpathogenesis while others seem to besaprophytic.  One of the most commonsymptoms in the fungal disease is thedisorganization of host tissues brought aboutby enzymes secreted by the pathogens whichattack the host walls.Enzyme activities of agarwood (Aquilaria malaccensis Lamk.) stem under pathogenesisP Tamuli1, P Baruah & R Samanta2Division of Medicinal, Aromatic and Economic PlantsNorth East Institute of Science and TechnologyJorhat-785 006, Assam, India.E-mail:pbaruah@rrljorhat.res.inReceived 18 July 2006; Revised 15 February 2008; Accepted 31 July 2008AbstractCellulase, pectinase, peroxidase and polyphenol oxidase activities were determined in healthy,naturally infected and inoculated agarwood (Aquilaria malaccensis) plant parts at varioustime intervals to study the changes in activities of these enzymes during pathogenesis.  Samplesinfected naturally with Chaetomium globosum and Fusarium oxysporum exhibited higheractivity of all the enzymes compared to healthy samples.Keywords: agarwood, Aquilaria malaccensis, Chaetomium globosum, enzyme activity,Fusarium oxysporum.Changes in cellulolytic and pectinolyticenzyme activity during pathogenesis havebeen reported by various workers (Prasad etal. 1988; Reddy & Reddy 1988).  Increase inperoxidase and polyphenol oxidase activityin tissues of various plants following infectionby various pathogens have been reported(Loebenstein & Linsey 1961; Addy &Goodman 1972).  The present investigationwas undertaken to study the changes incellulase, pectinase, peroxidase andpolyphenol oxidase activity duringpathogenesis in agarwood.Artificial inoculation of fungal isolatesThe most frequently isolated fungi(Chaetomium globosum  Kunze.  and1Present address: Department of Botany, Darang College, Tezpur-784 001, Assam, India.2Department of Life Sciences, Dibrugarh University, Dibrugarh-786 004, Assam, India.Journal of Spices and Aromatic CropsVolume 17 (3) :  240–243(2008) Indian Society for Spices241Fusarium oxysporum  Schlecht.) wereinoculated (in combination also) to thehealthy plant by artificial boring on to theplants (Tamuli et al. 2000).  Inoculation wasmade with these two fungi and theircombination.Enzyme activities were measured in healthy,naturally infected and artificially inoculatedplant samples at an interval of 10, 20, 30 and40 days after inoculation.  For estimation ofcellulase activity, wood chips were ground in15 ml of 0.5 M NaCl solution, and activitywas determined according to the methoddescribed by Verma & Singh (1975) andspecific activity was calculated. Pectinaseactivity was determined according to Dubey& Mathur (1975).  The reaction mixtureconsisted of 1% pectin and 1 ml of enymeextract.  One unit of enzyme activity wasexpressed as 1% loss of viscosity after 30 min.Peroxidase was extracted following themethod of Purohit et al .  (1979) and theactivity determined by the method ofChakravarti & Nandi (1978).  The activity ofpolyphenol oxidase was determined by themethod of Purohit et al. (1979); catechol wasused as substrate.Cellulase and pectinase activityMaximum activity of these enzymes wererecorded in plants 20 days after inoculationwhich, however, reduced at 40 days (Tables 1& 2).  Specific activity of celluase and pectinasewas highest in naturally infected wood, andin healthy wood.Peroxidase activityProxidase activity in healthy samplesremained more or less unchanged up to 40days of incubation (Table 3).  The highestspecific activity (0.077) was recorded innaturally infected wood and the lowest (0.052)in healthy wood.Polyphenol oxidase activityIn healthy samples, polyphenol oxidaseactivity did not increase considerably withprolongation of incubation (Table 4).However, considerable increase in polyphenoloxidase activity was observed in infectedsamples with increase in days of incubation.Specific activity was maximum (0.119) innaturally infected wood and minimum (0.054)in healthy wood.The investigation indicated that all thetreatments showed maximum activities ofboth the enzymes on the 20th day ofincubation, while on the 30th and 40th dayof incubation, activities of both the enzymesdeclined (Tables 1 & 2).  Thus, higher cellulaseand pectinase activities observed in infectedwood might be responsible fordisorganization of host tissues leading to thecolonization of pathogens.  Prasad et al.(1988) reported alteration in the enzymeactivities of seeds in finger millet due toTable 1.  Effect of fungal inoculation on cellulolytic enzyme activity of Aquilaria malaccensisduring different periods of incubationTreatment 10 days 20 days 30 days 40 daysH 2.42 ± 0.53 3.23 ± 0.47 2.74 ± 0.38 2.65 ± 0.31NI 4.40 ± 0.57 6.97 ± 0.45 6.07 ± 0.51 6.06 ± 0.29CG 3.08 ± 0.44 4.84 ± 0.49 3.93 ± 0.46 3.61 ± 0.32FO 3.35 ±  0.72 5.01 ± 0.62 4.24 ± 0.48 4.08 ± 0.24CG + FO 3.49 ± 0.61 5.45 ± 0.53 4.79 ± 0.54 4.77 ± 0.32Time (t) Treatment (tr) t x trSEd (Specific activity) 0.118 0.132 0.217CD P=0.05 0.239 0.267 0.440       P=0.01 0.320 0.358 0.588Values indicate specific activityH=Healthy; NI=Naturally infected; CG=Chaetomimum globosum; FO=Fusarium oxysporumEnzyme activities of agarwood242 Tamuli et al.Aspergillus flavus.  They found the activitiesof pectolytic and cellulolytic enzymes wereenhanced due to the influence of A. flavus.They suggested that higher per cent ofpectolytic and cellulolytic enzymes are usedas a tool for penetration of fungi as theseenzymes are responsible for permeabilitydamage of the cell.  Similar results were alsoobtained by Reddy & Reddy (1988) in fruitrot of coccinia.  They suggested thatincreased activities of these enzymes ininfected tissues compared to healthy tissuesmay be attributed to pathogen activity.The activities of peroxidase and polyphenoloxidase increased along with the incubationperiod in all the treatments (Tables 3 & 4).However, maximum activity of these enzymesTable 2.  Effect of fungal inoculation on pectinolytic enzyme activity of Aquilaria malaccensisduring  different periods of incubationTreatment 10 days 20 days 30 days 40 daysH 3.35 ± 0.41 5.18 ± 0.34 4.38 ± 0.27 4.24 ± 0.44NI 4.66 ± 0.48 7.92 ± 0.48 6.71 ± 0.31 6.55 ± 0.43CG 3.49 ± 0.34 5.61 ± 0.21 4.95 ± 0.24 4.86 ± 0.36FO 3.67 ± 0.38 5.40 ± 0.38 4.78 ± 0.52 4.65 ± 0.43CG + FO 3.71 ± 0.37 6.02 ± 0.43 5.18 ± 0.33 4.96 ± 0.41Time (t) Treatment (tr) t x trSEd (Specific activity) 0.118 0.132 0.217CD P=0.05 0.239 0.267 0.440       P=0.01 0.320 0.358 0.588Values indicate specific activityH=Healthy; NI=Naturally infected; CG=Chaetomimum globosum; FO=Fusarium oxysporumwere recorded in naturally infected samplesand minimum in healthy samples.Peroxidase and polyphenol oxidase activitiesin apple leaves inoculated with Erwiniaamylovora Burril was studied by Addy &Goodman (1972).  They noted an increasedactivity of both the enzymes in inoculatedleaves as compared to control.  Loebenstein& Linsey (1960) suggested that the higherperoxidase activities in infected plants mightbe the consequence of greater breakdown ofcarbohydrates through monophosphateshunt due to which precursors of phenoliccompounds are produced which are oxidisedby peroxidase to quinones in the presence ofH2O2 to overcome the pathogen.Table 3.  Effect of fungal inoculation on peroxidase activity of Aquilaria  malaccensis during   dif-ferent periods of incubationTreatment 10 days 20 days 30 days 40 daysH 0.050 ± 0.002 0.051 ± 0.003 0.050 ± 0.002 0.052 ± 0.003NI 0.064 ± 0.003 0.073 ± 0.002 0.075 ± 0.004 0.077± 0.004CG 0.052 ± 0.002 0.057 ± 0.002 0.059 ± 0.002 0.062 ± 0.003FO 0.054 ± 0.004 0.055 ± 0.002 0.058 ± 0.002 0.060 ± 0.003CG + FO 0.053 ± 0.002 0..057 ± 0.002 0.060 ± 0.003 0.062 ± 0.002Time (t) Treatment (tr) t x trSEd (Specific activity) 0.002 0.003 0.007CD P=0.05 0.004 0.006 0.014       P=0.01 0.005 0.008 0.019Values indicate specific activityH=Healthy; NI=Naturally infected; CG=Chaetomimum globosum; FO=Fusarium oxysporum243The changes in activity of various enzymesin naturally infected agarwood indicated thatthese enzymes were involved in the infectionprocess and development of symptoms of thedisease in agarwood plants.ReferencesAddy S K & Goodman R N 1972 Polyphenol oxi-dase and peroxidase activity in appleleaves inoculated with a virulent oravirulent strain of Erwinia amylovora.Indian Phytopathol. 25: 575-579.Bhattacharyya B, Datta A & Baruah H K 1952On the formation and development ofagar in Aquilaria agollocha. Sci. Cult. 18:240-241.Chakravarti N & Nandi B 1978 Enzyme activityin banana fruits rotted byBotryodiplodia theobromae. Pat. ActaAgrobot. 31: 41-46.Dubey H C & Mathur S 1975 Hydrolytic andtrans-eliminative degradation of pecticsubstances by extra-cellular enzymes ofVerticillum dahlia.  Indian Phytopathol.28: 346.Table 4.  Effect of fungal inoculation on polyphenol oxidase activity of Aquilaria malaccensisduring different periods of incubationTreatment 10 days 20 days 30 days 40 daysH 0.052 ± 0.003 0.052 ± 0.003 0.054 ± 0.002 0.054 ± 0.002NI 0.064 ± 0.003 0.084 ± 0.003 0.095 ± 0.004 0.119± 0.008CG 0.055 ± 0.002 0.066 ± 0.003 0.074 ± 0.004 0.085 ± 0.003FO 0.054 ± 0.003 0.068 ± 0.002 0.079 ± 0.002 0.083 ± 0.003CG + FO 0.055 ± 0.002 0.067 ± 0.002 0.077 ± 0.004 0.090 ± 0.002Time (t) Treatment (tr) t x trSEd(Specific activity) 0.003 0.003 0.007CD P=0.05 0.006 0.006 0.014       P=0.01 0.008 0.008 0.019Values indicate specific activityH=Healthy; NI=Naturally infected; CG=Chaetomimum globosum; FO=Fusarium oxysporumLoebenstein G & Linsey N 1961 Peroxidase ac-tivity in virus infected sweet potatoes.Phytopathology 51: 533-537.Prasan B K, Shanker U, Narayan N, Kishor A &Dayal S 1988 Alteration in the enzymeactivities of seeds of finger millet due toAspergillus flavus.  Indian Phytopathol.41: 578-580.Purohit S S, Ramawat K G & Arya H C 1979Polyphenols and some oxidative en-zymes in rust infected leaves of Cyperusrotandus.  Indian Phytopatol. 32 : 255-259.Reddy P B & Reddy S N 1988 Role of oxidasesand cell degrading enzymes in the fruitrot of coccinia caused by Rhizoctoniasolani.  Indian Phytopathol. 41: 138-141.Tamuli P, Boruah P, Nath S C & Samanta R 2000Fungi from diseased agarwood tree(Aquilaria agallocha Roxb) : Two newrecods. Adv. For. Res. India 22: 182-187.Verma J P & Sing M P 1975 Cellulases, pectinasesand proteases of Indian isolates ofXanthomonas malvacearum .  IndianPhytopathol. 28: 379-383.Enzyme activities of agarwood

Enzyme activities of agarwood (Aquilaria malaccensis Lamk.) stem under pathogenesis

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Enzyme activities of agarwood (Aquilaria malaccensis Lamk.) stem under pathogenesis

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