Post-infection changes in sugars, ascorbic acid, phenols and protein of agarwood tree (Aquilaria malaccensis) were investigated after inoculation with fungi such as Chaetomium globosum and Fusarium oxysporum. In healthy trees, the biochemical constituents increased, whereas in infected trees they decreased after inoculation with fungi.
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Samanta, P Tamuli, P Boruah, R

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Journal of Spices and Aromatic Crops VoL 13 (2) : 87-91 (2004) 8) Indian Society for Spices Biochemical changes in agarwood tree (Aquilaria malaccensis Lamk.) during patho-genesis P Tamuli', P Boruah & R Samanta' Division of Plant Sciences and Ecology Regional Research Laboratory Jorhat - 785006, Assam, India Received 21 June 2003; Revised 08 June 2004; Accepted 16 July 2004 Abstract Post-infection changes in sugars, ascorbic acid, phenols and protein of agarwood tree (Aquilaria malaccensis) were investigated after inoculation with fungi such as Chaetomium globosum and Fusarium oxysporum. In healthy trees, the biochemical constituents increased, whereas in infected trees they decreased after inoculation with fungi. Key words: agarwood tree, Aquilaria malaccensis, ascorbic acid, biochemical changes, phenols, protein, sugars. Introduction The oil obtained from the agarwood tree, Aquilaria malaccensis Lamk. (Family : Thymelaeaceae) is used in cosmetic and phar-maceutical industries. Agar is considered to be a pathological product produced by fun-gal invasion of the host (Shu-Yuan et al. 1992). The agar bearing tree shows various symp-toms including a dark tinge on the wood compared to the pale brown buff colour of normal wood of healthy trees. A few work-ers have studied agar formation and reported that the agar zones are associated with mould and decay fungi (Bose 1938; Bhattacharyya et al. 1952; Jalaluddin 1970, 1977; Tamuli et al. 2000). Among the various fungal species associated with agar zones, a few could cause pathogenesis while others seem to be of saprophytic nature in different eco-geo-graphical conditions. The ultimate impact of host-pathogen interaction is manifested in alteration of composition of nutritional and structural metabolites in the host. Metabolic changes due to fungal infection have been reported in various plants (Reddy et al. 1984; Khatri et al. 1985; Prasad et al. 1988; Nema 1989). The present investigation was under-taken to study the changes in sugar, ascor-bic acid, phenol and protein contents dur-ing pathogenesis in A. malaccensis. Materials and methods Artificial inoculation with fungal isolates The most frequently isolated fungi (Chaetomium globosum and Fusarium oxysporum) were cultured on PDA plates. Ten day old cultures of C. globosum and F. oxysporum were inoculated alone and in com-bination to healthy plants by artificial bor-ing on to the plants (Tamuli et al. 2000). Ob-1Present address: Department of Botany, Darrang College, Tezpur - 784 001, Assam, India. 'Department of Life Sciences, Dibrugarh University, Dibrugarh - 786 004, Assam, India. 88 servations on inoculated plants were made at an interval of 10 days up to 40 days after inoculation. Estimation of biochemical constituents Sugars The soluble sugars were extracted by boil-ing 5 g of sample with 80% alcohol (Dayal & Joshi 1968). Total and reducing sugars were estimated by Lane and Eynon's method us-ing methylene blue as internal indicator (AOAC 1945). Non-reducing sugars (sucrose) were calculated by subtracting the value of reducing sugar from the total acid hydrolysable sugars and these were multi-plied by 0.95 to correct for the water taken up during hydrolysis. Ascorbic acid Wood chips (5 g) were extracted with 5% metaphosphoric acid solution and made up to 50 m!. Ascorbic acid content was estimated volumetrically using 2, 6 - dichlorophenol in-dophenol as indicator (Bessey & King 1933). Phenols Phenolic compounds were extracted by boil-ing 5 g plant material with 80% alcohol for 5 min (Chandramohan et al. 1967). Total phenols in the alcohol extract wi's estimated by employing Folin-Ciocalteau reagent (Bray & Thorpe 1955) an~ Catechol was used as standard. Protein Protein content of plant samples was esti-mated by the method of Lowry et al. (1951) and Wildman & J ogendorf (1952) using bo-vine albumin as standard. Results and discussion Sugar content, The reducing and non-reducing sugar con-tents increased from 10 days (18.80 and 10.71 mg go, dry wt., respectively) to 40 days of incubation (20.23 and 11.43 mg go, dry wt., respectively) in healthy plants (Table 1). However,in naturally infected and inocu-lated wood, reducing sugar contents de-ramuli et al. creased with increase in days of incubation. In naturally infected wood, it decreased from 10.92 mg go, (10 days of incubation) to 9.67 mg go, (40 days of incubation). In wood samples inoculated with C. globosum and F. oxysporum, reducing sugar contents decreased from 19.46 to 17.41 mg go, and 18.74 to 17.63. mg go" respectively. In the wood infected with dual culture of C. globosum and F. oxysporum, it decreased from 18.43 to 17.220 mg go, during the same period. Similarly, non-reducing sugars in infected wood also de-creased with increase in days of incubation. The total sugar content was maximum (31.66 mg go,) in healthy wood and minimum in natu-rally infected wood (17.20 mg go,) at 40 days after inoculation. Artificially inoculated plants contained 26.89, 26.97 and 26.59 mg go, of to-tal sugars when inoculated with C. globosum and F. oxysporum alone and as dual cultures, respectively. A similar trend of decreased sugar levels in diseased plants was observed by Prasad et al. (1988) and Nema (1989). The depletion of sugars during host-parasite interaction might be due to increased respiration or utilization of sugars by the fungi which depends on the capability of fungi to secrete carbohydrate de-grading enzyme (Prasad et al. 1988). Nema (1989) suggested that reduction in sugars dur-ing disease development might be due to uti-lization of sugars probably for energy and synthetic reactions involved in multiplication of the pathogen. Ascorbic acid content Ascorbic acid content in healthy wood in-creased during the test period (10 to 40 days). In healthy wood, ascorbic acid content in-creased from 9.21 to 10.04 mg go, during the test period (Table 2). In infected samples ascorbic acid content decreased with inocu-lation period. The decline in ascorbic acid con-tent was maximum in naturally infected wood which was followed by the wood inoculated with F. oxysporum, C.globosum and F. oxysporum in combination and C. globosum alone. The decreased level of ascorbic acid in infected plants might be due to ascorbic acid Biochemical changes in agarwood tree 89 Table 1. Effect of fungal inoculation on sugar content of Aquilaria malaccensis plants (stem) during different periods of incubation Sugar Treatment Sugar content (mg g! dry wt.) 10 days 20 days 30 days 40 days Reducing sugar H 18.80 ± 1.03 19.20 ± 0.97 19.63 ± 0.58 20.23 ± 1.12 NI 10.92 ± 0.92 10.71 ± 0.72 9.83 ± 0.84 9.67 ± 0.63 CG 19.46 ± 0.74 18.22 ± 0.57 17.84 ± 1.20 17.41 ± 0.86 FO 18.74 ± 0.58 18.55 ± 0.73 18.02 ± 0.68 17.63 ± 0.92 CG+FO 18.43 ± 0.72 17.96 ± 0.66 17.74 ± 0.91 17.22 ± 0.83 Time (t) Treatment (tr) t x tr SEd 0.173 0.192 0.387 CD (P=0.05) 0.300 0.389 0.784 CD (P=O.Ol) 0.469 0.521 1.050 Non-reducing sugar H 10.71± 0.88 10.85 ± 1.04 11.19 ± 0.67 11.43 ± 0.82 NI 8.19 ± 1.12 7.72 ± 0.77 7.64 ± 0.81 7.53 ± 0.47 CG 10.76 ± 0.87 10.51 ± 0.67 10.23 ± 0.62 9.48 ± 0.74 FO 10.72 ± 0.66 10.54 ± 0.72 9.63 ± 0.67 9.34 ± 0.53 CG+FO 10.53 ± 0.93 10.38 ± 0.76 9.74 ± 0.82 9.37 ± 0.65 Time (t) Treatment (tr) t x tr SEd 0.164 0.188 0.382 CD (P=0.05) 0.332 0.381 0.774 CD (P=O.OI) 0.445 0.510 1.036 Total sugar H 29.53 ± 0.57 30.06 ± 0.93 30.82 ± 1.14 31.66 ± 1.02 NI 19.11 ± 0.73 18.43 ± 0.67 17.47 ± 1.06 17.20 ± 0.84 CG 30.22 ± 0.76 28.73 ± 0.89 28.07 ± 0.52 26.89 ± 0.71 FO 29.46 ± 0.92 29.09 ± 1.12 27.65 ± 0.47 26.97 ± 0.85 CG+FO 28.96 ± 0.81 28.34 ± 0.73 27.48 ± 0.83 26.59 ± 0.87 Time (t) Treatment (tr) t x tr SEd 0.178 0.197 0.392 CD (P=0.05) 0.361 0.399 0.794 CD (P=O.OI) 0.483 0.534 1.063 H-Healthy; NI=NaturaIly infected; CG=Chaetomium globosum; FO=Fusarium oxysporum Table 2. Effect of fungal inoculation on ascorbic acid content of Aquilaria malaccensis plants (stem) during different periods of incubation Treatment Ascorbic acid content(mg g'! dry wt.) 10 days 20 days 30 days 40 days H 9.21 ± 0.30 9.35 ± 0.32 9.67 ± 0.24 9.80 ± 0.22 NI 8.49 ± 0.42 7.45 ± 0.49 7.37 ± 0.83 6.96 ± 0.44 CG 9.19 ± 0.20 9.06 ± 0.29 8.98 ± 0.14 8.70 ± 0.45 FO 9.25 ± 0.37 8.90 ± 0.24 8.80 ± 0.39 8.04 ± 0.54 CG+FO 9.23 ± 0.48 8.78 ± 0.36 8.74 ± 0.42 8.13 ± 0.62 Time (t) Treatment (tr) t x tr SEd 0.178 0.199 0.398 CD (P=0.05) 0.360 0.403 0.806 CD (P=0.01) 0.482 . 0.539 1.078 H=Healthy; NI=Naturally infected; CG=Chaetomium globosum; FO=Fusarium oxysporum 90 ramuli et al. Table 3. Effect of fungal inoculation on phenol content of Aquilaria malaccensis plants (stem) during different periods of incubation Treatment Phenol content (rg g~! dry wt.) 10 days 20 days 30 days 40 days H 32.12 ± 1.63 32.45 ± 1.70 35.04 ± 1.70 35.22 ± 2.87 NI 30.14 ± 8.16 29.23 ± 1.25 29.26 ± 1.70 27.31 ± 9.43 CG 31.06 ± 1.41 30.31 ± 9.42 29.33 ± 8.16 29.31 ± 1.25 FO 30.22 ± 4.71 29.41 ± 8.16 28.14 ± 9.43 27.22 ± 4.71 CG+FO 31.04 ± 4.71 29.24 ± 4.71 28.11 ± 1.41 27.34 ± 4.71 Time (t) Treatment (tr) t x tr SEd 0.154 0.168 0.312 CD (P=0.05) 0.312 0.340 0.632 CD (P=O.OI) 0.418 0.456 0.846 H=Healthy; NI=Naturally infected; CG=Chaetomium globosum; FO=Fusarium oxysporum degenerating enzymes either by the fungus alone or by the activity of the host-pathogen complex. Reddy et al. (1984) observed a gradual loss in ascorbic acid content in in-fected fruits of acid lime (Citrus aurantifolia). Phenol content Phenol content increased from 10 days (32.12 >Lg g'!) to 40 days (35.22 >Lg g'!) of incubation in healthy wood. But a reverse trend was no-ticed in naturally infected and inoculated wood (Table 3). Khatri et al. (1985) observed that the amount of total phenols declined in rice leaves due to infection by. Entyloma oryzae. He suggested that the reducti"n in concen-tration of phenolic compounds might be as-sociated with susceptibility of the host to the invading pathogen. Nema (1989) suggested that in betel vine, the leaf spot pathogen is inhibited by the phenolic compounds but when the pathogen is successful in causing the disease, the phenolic compounds in highly susceptible cultivar depleted the most. It is also probable that the infected host is unable to produce phenolic compounds in the pres-ence of the pathogen. Protein content In healthY' plants, slight increase in protein content (8.23 to 8.40 mg g'!) was observed with increase in days of incubation (Table 4). All infected samples exhibited a decreasing trend in protein content with incubation. Among the infected samples, naturally in-fected wood contained minimum protein con-tent (6.13 mg g'!). Decrease in protein con-tent might be due to degradation of the host proteins by the proteolytic enzymes secreted Table 4. Effect of fungal inoculation on protein content of Aquilaria malaccensis plants (stem) during different periods of incubation Treatment Protein content (mg g'! dry wt.) 10 days 20 days 30 days 40 days H 8.23 ± 0.12 8.27 ± 0.33 8.37 ± 0.21 8.40 ± 0.24 NI 6.83 ± 0.29 6.30 ± 0.08 6.23 ± 0.12 6.13 ± 0.25 CG 8.20 ± 0;29 7.83 ± 0.21 7.67 ± 0.12 7.57 ± 0.17 FO 8.13 ± 0.31 7.93 ± 0.12 7.70 ± 0.29 7.63 ± 0.19 CG+FO 8.07 ± 0.31 7.77 ± 0.33 7.50 ± 0.43 7.37 ± 0.31 Time (t) Trea tmen t (Ir) t x tr SEd 0.119 0.133 0.267 CD (P=0;05) 0.242 0.270 0.541 CD (P=O.OI) 0.324 0.362 0.724 H=Healthy; NI=Naturally infected; c;C=Chaetomium globosum; FO=Fusarium oxysporum Biochemical changes in agarwood tree by the pathogens. Similar results were also reported by Prasad et al. (1988) in muskmelon fruits infected with fruit-rot fungi. References A 0 A C 1945 Official Methods of Analysis of the Association of Official Analytical Chemists. 6th Edn. Association of Official Analytical Chemists, Washing-ton, DC. 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Ghosh H K, Bhargava S N & Tandon R N 1966 Studies on fungal disease of some tropi-cal fruits. VI. Post infection changes in 91 ascorbic acid contents of mango and 'pa-paya. Indian Phytopathol. 19 : 262-268. Jalaluddin M 1970 Disease of forest tree of Paki-stan. Agric. Pakistan 21 : 363-370. Jalaluddin M 1977 A useful pathological condi-tion of wood. Econ. Bot. 31 : 222 - 224. Khatri R K, Shastry R P, Reddy P N & Nema K G 1985 Metabolic changes in rice leaves in-fected by EntylDma Dryzae. Indian Phytopathol. 38 : 769-771. Lowry 0 A, Rosenburg M J, Lewis F A & Randall R J 1951 Protein measurement with Falin phenol reagent. J. BioI. Chern. 193 : 263-275. Nema A G 1989 Sugar and phenol contents of betel vine leaves after inoculation with leaf spot bacterium. Indian Phytopathol. 42 : 31-37. Prasad M M, Roy A K & Krishna A 1988 Bio-chemical changes in muskmelon fruits by fruit-rot fungi. Indian Phytopathol. 41: 641-643. Reddy B C. Reddy P V & Gopala Raju D 1984 Post-infection changes in acid lime fruits caused by Aspergillus niger. Indian Phytopathol. 37 : 185-187. Shu-Yuan Q, Lu Bi-Yao, Zhu Liang-Feng & Li Bao-Ling 1992 Formation of oxo-agarospirol in Aquilaria sinensis. Plant Physiol. Comm. 28 : 336~339. Tamuli P, Boruah P, Nath S C & Samanta R 2000 Fungi from diseased agarwood tree (Aquilaria agallDcha Roxb.) : Two new records. Adv. For. Res. India 22: 182-187. Wildman S & Jogendorf A 1952 Leaf proteins. Ann. Rev. Plant Physiol. 3: 131-147. 

Biochemical changes in agarwood tree (Aquilaria malaccensis Lamk.) during pathogenesis

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Biochemical changes in agarwood tree (Aquilaria malaccensis Lamk.) during pathogenesis

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