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Protective antifungal effect of neem (Azadirachta indica) extracts on mango (Mangifera indica) and rain tree (Albizia saman) wood
International Biodeterioration & Biodegradation 63 (2009) 241–243
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Short communication
Protective antifungal effect of neem (Azadirachta indica) extracts on mango (Mangifera indica) and rain tree (Albizia saman) wood Md. Muktarul Islam a, Md. Iftekhar Shams a, b, *, G.N.M. Ilias c, Md. Obaidullah Hannan a a
Forestry and Wood Technology Discipline, Khulna University, Khulna-9208, Bangladesh Research Institute for Sustainable Humanosphere, Kyoto University, Kyoto 611-0011, Japan c Timber Products and Environment Monitoring Cell, Rural Electrification Board (REB), shiromoni, Khulna-9204, Bangladesh b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2008 Received in revised form 3 July 2008 Accepted 3 July 2008 Available online 20 November 2008
Neem (Azadirachta indica) extract (NE) and NE combined with copper sulfate and boric acid (NECB) were examined for their protective effect against fungal deterioration of mango (Mangifera indica) and rain tree (Albizia saman) wood. Growth of the white-rot fungus Schizophyllum commune was completely inhibited on solid medium containing 1.8% (w/w) NE or 5% (w/w) NECB. The average weight losses of NE and NECB treated wood blocks inoculated with S. commune were respectively 4.7% and 3.3% for M. indica and 4.1% and 3.0% for A. saman. These numbers were significantly lower compared to those obtained in the untreated condition. Similar observations were also noticed in the case of field tests. An average increase of life span of M. indica and A. saman due to NE treatment compared to untreated samples was about 6–7 times higher. Therefore, both NE and NECB treatments are promising preservation options for enhancing the durability of both M. indica and A. saman woods. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Neem extract NECB Weight loss Wood preservative
1. Introduction Degradation by microorganisms and termites causes significant damage to wood. Many investigations have examined the usefulness of natural and synthetic preservatives to extend the life of wood materials (Purushotham, 1970). However, most conventional preservatives cause environmental pollution and a few of them are hazardous to animals and human beings (Thompson, 1971; Onuorah, 2000). The toxicity of the conventional wood preservative chromated copper arsenate (CCA) is higher prior to impregnation, because this process results in the formation of insoluble complexes in the wood. Hexavalent chromium is reduced to the less toxic trivalent form and arsenic is converted to insoluble arsenates. CCA presents a high risk for workers exposed to liquid solutions (Eaton and Hale, 1993). Environmental and health concerns with the use of CCA, including possible arsenic exposure to humans have resulted in its use being significantly restricted or limited (Pohleven et al., 2002). Hence, there is now an increased awareness of the hazards associated with the production and application of treatment chemicals and the disposal of treated wood and unused solutions (Eaton and Hale, 1993). Neem (Azadirachta indica) is a tree that finds many applications in the production of medicines, building materials, fuel, lubricants, and pesticides. Neem extracts have many effects on insects. The
antifeedant and growth regulating effects are the most valued for pest management. Other effects include repellency, antioviposition, sterility, fecundity reduction, loss of flying ability, disruption of sexual communication, and reduction of guttural motility (Schmutterer, 1990; National Research Council, 1992). To date over 195 species of insects have shown some degree of sensitivity to neem extract, including insects that have become resistant to synthetic pesticides (Lindquist et al., 1990; Menn, 1990). Neem also produces a number of chemicals that are toxic against wood-decay microbes (Dhyani et al., 2004). Several reports have shown the usefulness of these plant products in combination with toxic metals in protecting wood against termites or fungi (Purushotham and Tewari, 1961; Jain et al., 1989; Jain and Narayan, 1991; Subbaraman and Brucker, 2001; Dev and Nautiyal, 2004). Mango (Mangifera indica) and rain tree (Albizia saman) are widely used woods in Bangladesh. Both woods are sensitive to attack by beetles and termites (Lahiry, 2001) as well as by decay and wood-staining fungi. In the present study, we have examined the efficiency of neem extracts (NE) alone and in combination with copper sulfate and boric acid (NECB) in protecting M. indica and A. saman woods against fungal bideterioration. 2. Material and methods 2.1. Collection and preparation of wood samples
* Corresponding author: Research institute for Sustainable Humanosphere, Kyoto University, Kyoto 611-0011, Japan. Tel: þ81 774 38 3658; fax: þ81 774 38 3600. E-mail address: [email protected] (Md.I. Shams). 0964-8305/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2008.07.010
Mango (M. indica) and rain tree (A. saman) wood samples were collected from a sawmill. For each tree species, 30 samples
242
Md. M. Islam et al. / International Biodeterioration & Biodegradation 63 (2009) 241–243
containing sapwood and heartwood were collected for preservative treatments and 5 samples were used as controls. These samples were cut to 15 cm in length by 5 cm in width by 2.5 cm in thickness for field testing and to 1.9 cm in length by 1.9 cm in width by 1.9 cm in depth for laboratory tests. 2.2. Culture of Schizophyllum commune Schizophyllum commune is a white-rot fungus commonly found in almost all tropical countries. The strain used in this investigation was collected from a wooden fence at the Khulna University campus, in Bangladesh. It was isolated and identified in our laboratory. It was grown on 2% malt agar (MA) medium and the grown culture was maintained at 4 C. 2.3. Preparation of neem extract Leaves with small branches of neem (A. indica) were collected and they were crushed with a knife into smaller pieces. Two kilograms of this material was introduced into a 20-l aluminum container containing 10 l of distilled water. The suspension was kept for 1 h at room temperature. It was then boiled for 60 min with continuous stirring. After boiling, the mixture was cooled down and filtered through a cotton cloth to separate the extract from leaves and branches. The filtrate was collected and the solid phase was boiled again for about 30–40 min in 5 l of water and filtered. The filtrates from both extractions were combined, yielding finally 13 l of neem extract (NE). The concentration of dissolved solutes in NE preparation used in this work was measured with a hand refractometer by comparing the value of the refractive index of the solution to that of a pre-established standard curve. It was determined to be 1.8% (w/w). This preparation will be hereafter designated as 1.8% NE. 2.4. Preparation of NE mixed with copper sulfate and boric acid (NECB) In order to prepare the NECB solution 128 g of copper sulfate and 64 g of boric acid were added to 6 l of NE, making a 5% (w/w) preparation; this will be designated hereafter as 5% NECB.
direct contact with the medium. The linear radial growth of the fungus on the Petri plate was measured in millimeters in two directions at right angles to each other after 2, 4, 6, and 12 days of incubation at 25 2 C. Each assay was performed using five replicate cultures. When the mycelium of the fungus reached the edge of the control plate (containing no NE or NECB), the antifungal index (AI) was calculated according to the Amusant et al. (2005) equation:
Radialgrowthof thefungusonthetestmedium AI¼ ð%Þ¼ 1 Radialgrowthof thefungusonthecontrolmedium 100 The growth index varies from 0 to100 (it is 0 when there is no fungal inhibition, 100 when there is total fungal inhibition). 2.6.2. Laboratory test S. commune was grown in 250-ml glass bottles on 2% MA medium to establish an active mycelium. Five wood blocks of each species were used for each treatment. Wood blocks treated with NE or NECB and untreated control blocks were oven-dried at 103 C 2 C until constant weight to determine dry weight (initial weight, Mo). Then the wood blocks were sterilized in an autoclave for 20 min (15 psi at 121 C) and one wood block was introduced into each bottle containing actively growing S. commune cultures. The bottles were incubated for 12 weeks at 25 C and 70% RH. After the incubation period, the test blocks were withdrawn from the culture bottles. The fungal mats (mycelium) were carefully brushed off from the test blocks and were dried for 24 h at room temperature and then at 103 C 2 C overnight. After oven-drying the final weights (M) of the wood blocks were taken. The mass loss due to decay was calculated as the difference between dry mass of each wood block before and after incubation with the fungi and expressed as a percentage of dry mass loss. The efficiency of NE and NECB treatments was estimated from the differences in weight losses between untreated control wood blocks and NE- or NECBtreated blocks (Amusant et al., 2005):
% Loss of wood mass ¼ ½ðMo MÞ=Mo 100 where Mo ¼ oven dry weight of wood prior to the decay test, M ¼ oven dry weight of wood after the decay test.
2.5. Determination of preservative retention All wood samples were oven-dried until constant weight. The samples for field testing were immersed in either 1.8% NE or 5% NECB solution for periods of 24 h, 48 h, or 72 h. The samples for laboratory testing were immersed in either 1.8% NE or 5% NECB solutions for a 72-h period. The retention of the preservative was expressed volumetrically using the following equation:
2.6.3. Field test Treated and untreated control samples were installed in a test yard as per Indian Standard method No. 4833 (Anon., 1968) for 12 weeks. Four samples were used from each treatment. After 12 weeks, the test samples were withdrawn from
40
Weight of salt ðpreservativeÞ Retention ¼ kg m3 Volume of sample
Mangifera indica
35
Albizia saman
2.6. Assays to determine the wood preservative efficacy of NE and NECB preparations 2.6.1. Agar plate assay Fungal sensitivity assays using S. commune were performed using 2% MA media which was prepared by dissolving 20 g of malt extract powder and 20 g of agar in 1 l of either 1.8% NE or 5% NECB solutions instead of water. After solidification of the medium, each Petri dish was inoculated with a 7-mm-diameter agar block taken from the periphery of a 20-day-old subculture of S. commune. The agar blocks were placed inverted so that the mycelial growth was in
Weight loss ( )
30 25 20 15 10 5 0 Control
NE
NECB
Fig. 1. Weight loss of M. indica and A. saman wood blocks after fungal exposure.
Md. M. Islam et al. / International Biodeterioration & Biodegradation 63 (2009) 241–243
Weight loss ( )
45 40
Mangifera indica
35
Albizia saman
30 25 20 15 10 5 0
24 Hours 48 Hours 72 Hours 24 Hours 48 Hours 72 Hours Control
NE
NECB
243
samples. Weight losses decreased slightly with increase of dipping duration. The overall weight loss in NECB-treated wood samples was slightly less than in NE-treated wood samples in both species. This is probably due to the contribution to wood protection of CB in the NECB mixture. Therefore, both NE and NECB treatment were efficient in protecting wood from wood-decay agents in soil. Their efficiency increased the lifetime of wood samples in field testing by a factor of 6 to 7 compared to the untreated controls. Together, these observation corroborate those of Venmalar and Nagaveni (2005), who showed that neem-oil- and copperizedneem-oil-treated test panels of rubber (Hevea brasiliensis) wood remained intact in field tests after 9 months whereas untreated samples were destroyed within 6 months of testing, and that the average life of neem-oil-treated cashew nut shells was increased by a factor of 4– 5.
Fig. 2. Weight loss of M. indica and A. saman wood blocks after field test.
References the field. They were carefully washed and then dried at room temperature and finally oven-dried at 103 C 2 C. From the initial and final weights, the percentage of weight loss and increase in durability were calculated with reference to the control samples. 3. Results and discussion The retention of neem extract (NE) was found to be 21.9 kg m3 for M. indica wood samples after 72 h and 15.5 kg m3 for A. saman samples after 24 h. The retention of NECB was 25.7 kg m3 for M. indica samples after 72 h and 17.9 kg m3 for A. saman after 24 h. Both NE and NECB inhibited S. commune growth completely in the agar plate assays where the calculated AI was still 100% for both preparations after 12 days of incubation. The average weight loss of NE- and NECB-treated wood blocks exposed to S. commune were 4.7% and 3.3%, and 4.1% and 3.0%, respectively, for M. indica and A. saman (Fig. 1). In control wood blocks, 32.4% and 30.5% weight losses were found for M. indica and A. saman, respectively, which is significantly (P < 0.05) higher than losses in treated wood blocks. White-rot fungi are generally more destructive than brown-rot fungi on hardwood species (Eaton and Hale, 1993); thus the deterioration caused in untreated M. indica and A. saman wood samples by S. commune was not exceptional. The extent of deterioration was drastically reduced in treated wood samples, where less than 5% weight loss was recorded for both treatments. Therefore NE and the combined NECB treatments resulted in significant protection against S. commune. Treatments resulting in less than 10% weight loss of test blocks are considered to be effective treatment and are grouped under the rubric ‘‘high resistance class’’; this classification of resistance is used only for comparison with natural resistance of durable species as per ASTM (Anon., 1981). In field tests, weight loss varied with wood species, preservative preparations, and period of treatment (Fig. 2). In the case of M. indica the average weight losses after 12 weeks of testing of samples treated with NE for 24, 48, and 72 h were 6.1%, 5.6%, and 5.0%, respectively, and for NECB the average weight losses were 4.6%, 3.8%, and 3.3%. In the case of A. saman wood, the average weight losses of samples treated with NE for 24, 48, and 72 h were 5.8%, 5.4%, and 4.8%, respectively, and for NECB they were 4.4%, 3.6%, and 3.0%. Weight losses of untreated control samples were found to be 37.0% and 36.4% for M. indica and A. saman, respectively, which is significantly (P < 0.05) higher compared to losses in treated
Amusant, N., Moretti, C., Richard, B., Prost, E., Nuzillard, J.M., Thevenon, M., 2005. Chemical compounds from Eperua falcata and Eperua grandiflora heartwood and their biological activities against wood destroying fungus (Coriolus versicolor). The International Research Group on Wood Preservation. IRG/WP 05–30373. Anon, 1968. Methods for field testing of preservatives. Indian standard 4833. Anon.,1981. Standard method of accelerated laboratory test of natural decay resistance of woods. American society for testing and material Designation: D, 2017–2081. Dev, I., Nautiyal, S.N., 2004. An eco-friendly wood preservative formulation (CRCNSL). Journal of Timber Development Association of India 50 (1-2), 19–25. Dhyani, S., Tripathi, S., Indra, D., 2004. Preliminary screening of neem (Azadirachta indica) leaf extractives against Poria monticolada wood destroying fungus. Journal of Indian Academy of Wood Science 1-2, 103–112. Eaton, R.A., Hale, M.D.C., 1993. Wood: Decay, Pests and Protection. Chapman and Hall, London. Jain, J.K., Narayan, V., 1991. Arsenated and borated cashew nut shell liquid: low cost environmentally safe preservatives. Journal of Timber Development Association of India 37, 25–27. Jain, J.K., Narayan, V., Gazwal, A.K., 1989. Studies on the efficacy of cashew nut shell liquid as compared to normal creosote against termites in the termite mound. Journal of Timber Development Association of India 35, 51–54. Lahiry, A.K., 2001. Applied Wood Preservation. Magnum Opus, Dhaka, Bangladesh, 428 pp. Lindquist, R.K., Adams, A.J., Hall, F.R., Adams, I.H.H., 1990. Laboratory and greenhouse evaluations of Margosan-O against bifenthrin-resistant and -susceptible greenhouse whiteflies, Trialeurodes vaporariorum (Homoptera: Aleyrodidae). In: Locke, J.C., Lawson, R.H. (Eds.), Proceedings of a Workshop on Neem’s Potential in Pest Management Programs. USDA-ARS, Beltsville, MD, pp. 91–99. ARS-86. Menn, J.J., 1990. USDA interest in neem research. In: Locke, J.C., Lawson, R.H. (Eds.), Proceedings of a Workshop on Neem’s Potential in Pest Management Programs. USDA-ARS, Beltsville, MD, pp. 1–3. ARS-86. National Research Council, 1992. Neem: a tree for solving global problems. National Academy Press, Washington, DC. Onuorah, E.O., 2000. The wood preservative potential of heartwood extracts of Milicia excelsa and Erythrophleum suaveolens. Bioresource Technology 75, 171–173. Pohleven, F., Humar, M., Amartey, S., Benedik, J., 2002. Tolerance of wood decay fungi to commercial copper based wood preservatives. The International Research Group on Wood Preservation IRG/WP 02–30291. Purushotham, A., 1970. Protection of pulpwood (timber, bamboo) from deterioration due to biological agencies (fungi and insects, etc.) during transit and storage. Journal of Timber Development Association of India 16, 51–53. Purushotham, A., Tewari, M.C., 1961. A preliminary note on the preparation of copper and zinc preservatives from cashew nut shell liquid. Journal of Timber Preservation Association of India 7, 8–10. Schmutterer, H., 1990. Future tasks of neem research in relation to agricultural needs worldwide. In: Locke, J.C., Lawson, R.H. (Eds.), Proceedings of a Workshop on Neem’s Potential in Pest Management Programs. USDA-ARS, Beltsville, MD, pp. 15–22. ARS-86. Subbaraman, R.B., Brucker, B.R., 2001. Method for using neem extracts and derivatives for protecting wood and other cellulosic composites. United States Patent 6294571. Thompson, W.S., 1971. Contribution of the wood preserving industry to water pollution. In: Thompson, W.S. (Ed.), Proceeding of conference on pollution abetment and control in the wood industry. Mississippi Forest Products Laboratory, Mississippi State College, pp. 50–75. Venmalar, D., Nagaveni, H.C., 2005. Evaluation of copperised cashew nut shell liquid and neem oil as wood preservatives. The International Research Group on Wood Preservation IRG/WP 05–30368.
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Short communication
Protective antifungal effect of neem (Azadirachta indica) extracts on mango (Mangifera indica) and rain tree (Albizia saman) wood Md. Muktarul Islam a, Md. Iftekhar Shams a, b, *, G.N.M. Ilias c, Md. Obaidullah Hannan a a
Forestry and Wood Technology Discipline, Khulna University, Khulna-9208, Bangladesh Research Institute for Sustainable Humanosphere, Kyoto University, Kyoto 611-0011, Japan c Timber Products and Environment Monitoring Cell, Rural Electrification Board (REB), shiromoni, Khulna-9204, Bangladesh b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2008 Received in revised form 3 July 2008 Accepted 3 July 2008 Available online 20 November 2008
Neem (Azadirachta indica) extract (NE) and NE combined with copper sulfate and boric acid (NECB) were examined for their protective effect against fungal deterioration of mango (Mangifera indica) and rain tree (Albizia saman) wood. Growth of the white-rot fungus Schizophyllum commune was completely inhibited on solid medium containing 1.8% (w/w) NE or 5% (w/w) NECB. The average weight losses of NE and NECB treated wood blocks inoculated with S. commune were respectively 4.7% and 3.3% for M. indica and 4.1% and 3.0% for A. saman. These numbers were significantly lower compared to those obtained in the untreated condition. Similar observations were also noticed in the case of field tests. An average increase of life span of M. indica and A. saman due to NE treatment compared to untreated samples was about 6–7 times higher. Therefore, both NE and NECB treatments are promising preservation options for enhancing the durability of both M. indica and A. saman woods. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Neem extract NECB Weight loss Wood preservative
1. Introduction Degradation by microorganisms and termites causes significant damage to wood. Many investigations have examined the usefulness of natural and synthetic preservatives to extend the life of wood materials (Purushotham, 1970). However, most conventional preservatives cause environmental pollution and a few of them are hazardous to animals and human beings (Thompson, 1971; Onuorah, 2000). The toxicity of the conventional wood preservative chromated copper arsenate (CCA) is higher prior to impregnation, because this process results in the formation of insoluble complexes in the wood. Hexavalent chromium is reduced to the less toxic trivalent form and arsenic is converted to insoluble arsenates. CCA presents a high risk for workers exposed to liquid solutions (Eaton and Hale, 1993). Environmental and health concerns with the use of CCA, including possible arsenic exposure to humans have resulted in its use being significantly restricted or limited (Pohleven et al., 2002). Hence, there is now an increased awareness of the hazards associated with the production and application of treatment chemicals and the disposal of treated wood and unused solutions (Eaton and Hale, 1993). Neem (Azadirachta indica) is a tree that finds many applications in the production of medicines, building materials, fuel, lubricants, and pesticides. Neem extracts have many effects on insects. The
antifeedant and growth regulating effects are the most valued for pest management. Other effects include repellency, antioviposition, sterility, fecundity reduction, loss of flying ability, disruption of sexual communication, and reduction of guttural motility (Schmutterer, 1990; National Research Council, 1992). To date over 195 species of insects have shown some degree of sensitivity to neem extract, including insects that have become resistant to synthetic pesticides (Lindquist et al., 1990; Menn, 1990). Neem also produces a number of chemicals that are toxic against wood-decay microbes (Dhyani et al., 2004). Several reports have shown the usefulness of these plant products in combination with toxic metals in protecting wood against termites or fungi (Purushotham and Tewari, 1961; Jain et al., 1989; Jain and Narayan, 1991; Subbaraman and Brucker, 2001; Dev and Nautiyal, 2004). Mango (Mangifera indica) and rain tree (Albizia saman) are widely used woods in Bangladesh. Both woods are sensitive to attack by beetles and termites (Lahiry, 2001) as well as by decay and wood-staining fungi. In the present study, we have examined the efficiency of neem extracts (NE) alone and in combination with copper sulfate and boric acid (NECB) in protecting M. indica and A. saman woods against fungal bideterioration. 2. Material and methods 2.1. Collection and preparation of wood samples
* Corresponding author: Research institute for Sustainable Humanosphere, Kyoto University, Kyoto 611-0011, Japan. Tel: þ81 774 38 3658; fax: þ81 774 38 3600. E-mail address: [email protected] (Md.I. Shams). 0964-8305/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2008.07.010
Mango (M. indica) and rain tree (A. saman) wood samples were collected from a sawmill. For each tree species, 30 samples
242
Md. M. Islam et al. / International Biodeterioration & Biodegradation 63 (2009) 241–243
containing sapwood and heartwood were collected for preservative treatments and 5 samples were used as controls. These samples were cut to 15 cm in length by 5 cm in width by 2.5 cm in thickness for field testing and to 1.9 cm in length by 1.9 cm in width by 1.9 cm in depth for laboratory tests. 2.2. Culture of Schizophyllum commune Schizophyllum commune is a white-rot fungus commonly found in almost all tropical countries. The strain used in this investigation was collected from a wooden fence at the Khulna University campus, in Bangladesh. It was isolated and identified in our laboratory. It was grown on 2% malt agar (MA) medium and the grown culture was maintained at 4 C. 2.3. Preparation of neem extract Leaves with small branches of neem (A. indica) were collected and they were crushed with a knife into smaller pieces. Two kilograms of this material was introduced into a 20-l aluminum container containing 10 l of distilled water. The suspension was kept for 1 h at room temperature. It was then boiled for 60 min with continuous stirring. After boiling, the mixture was cooled down and filtered through a cotton cloth to separate the extract from leaves and branches. The filtrate was collected and the solid phase was boiled again for about 30–40 min in 5 l of water and filtered. The filtrates from both extractions were combined, yielding finally 13 l of neem extract (NE). The concentration of dissolved solutes in NE preparation used in this work was measured with a hand refractometer by comparing the value of the refractive index of the solution to that of a pre-established standard curve. It was determined to be 1.8% (w/w). This preparation will be hereafter designated as 1.8% NE. 2.4. Preparation of NE mixed with copper sulfate and boric acid (NECB) In order to prepare the NECB solution 128 g of copper sulfate and 64 g of boric acid were added to 6 l of NE, making a 5% (w/w) preparation; this will be designated hereafter as 5% NECB.
direct contact with the medium. The linear radial growth of the fungus on the Petri plate was measured in millimeters in two directions at right angles to each other after 2, 4, 6, and 12 days of incubation at 25 2 C. Each assay was performed using five replicate cultures. When the mycelium of the fungus reached the edge of the control plate (containing no NE or NECB), the antifungal index (AI) was calculated according to the Amusant et al. (2005) equation:
Radialgrowthof thefungusonthetestmedium AI¼ ð%Þ¼ 1 Radialgrowthof thefungusonthecontrolmedium 100 The growth index varies from 0 to100 (it is 0 when there is no fungal inhibition, 100 when there is total fungal inhibition). 2.6.2. Laboratory test S. commune was grown in 250-ml glass bottles on 2% MA medium to establish an active mycelium. Five wood blocks of each species were used for each treatment. Wood blocks treated with NE or NECB and untreated control blocks were oven-dried at 103 C 2 C until constant weight to determine dry weight (initial weight, Mo). Then the wood blocks were sterilized in an autoclave for 20 min (15 psi at 121 C) and one wood block was introduced into each bottle containing actively growing S. commune cultures. The bottles were incubated for 12 weeks at 25 C and 70% RH. After the incubation period, the test blocks were withdrawn from the culture bottles. The fungal mats (mycelium) were carefully brushed off from the test blocks and were dried for 24 h at room temperature and then at 103 C 2 C overnight. After oven-drying the final weights (M) of the wood blocks were taken. The mass loss due to decay was calculated as the difference between dry mass of each wood block before and after incubation with the fungi and expressed as a percentage of dry mass loss. The efficiency of NE and NECB treatments was estimated from the differences in weight losses between untreated control wood blocks and NE- or NECBtreated blocks (Amusant et al., 2005):
% Loss of wood mass ¼ ½ðMo MÞ=Mo 100 where Mo ¼ oven dry weight of wood prior to the decay test, M ¼ oven dry weight of wood after the decay test.
2.5. Determination of preservative retention All wood samples were oven-dried until constant weight. The samples for field testing were immersed in either 1.8% NE or 5% NECB solution for periods of 24 h, 48 h, or 72 h. The samples for laboratory testing were immersed in either 1.8% NE or 5% NECB solutions for a 72-h period. The retention of the preservative was expressed volumetrically using the following equation:
2.6.3. Field test Treated and untreated control samples were installed in a test yard as per Indian Standard method No. 4833 (Anon., 1968) for 12 weeks. Four samples were used from each treatment. After 12 weeks, the test samples were withdrawn from
40
Weight of salt ðpreservativeÞ Retention ¼ kg m3 Volume of sample
Mangifera indica
35
Albizia saman
2.6. Assays to determine the wood preservative efficacy of NE and NECB preparations 2.6.1. Agar plate assay Fungal sensitivity assays using S. commune were performed using 2% MA media which was prepared by dissolving 20 g of malt extract powder and 20 g of agar in 1 l of either 1.8% NE or 5% NECB solutions instead of water. After solidification of the medium, each Petri dish was inoculated with a 7-mm-diameter agar block taken from the periphery of a 20-day-old subculture of S. commune. The agar blocks were placed inverted so that the mycelial growth was in
Weight loss ( )
30 25 20 15 10 5 0 Control
NE
NECB
Fig. 1. Weight loss of M. indica and A. saman wood blocks after fungal exposure.
Md. M. Islam et al. / International Biodeterioration & Biodegradation 63 (2009) 241–243
Weight loss ( )
45 40
Mangifera indica
35
Albizia saman
30 25 20 15 10 5 0
24 Hours 48 Hours 72 Hours 24 Hours 48 Hours 72 Hours Control
NE
NECB
243
samples. Weight losses decreased slightly with increase of dipping duration. The overall weight loss in NECB-treated wood samples was slightly less than in NE-treated wood samples in both species. This is probably due to the contribution to wood protection of CB in the NECB mixture. Therefore, both NE and NECB treatment were efficient in protecting wood from wood-decay agents in soil. Their efficiency increased the lifetime of wood samples in field testing by a factor of 6 to 7 compared to the untreated controls. Together, these observation corroborate those of Venmalar and Nagaveni (2005), who showed that neem-oil- and copperizedneem-oil-treated test panels of rubber (Hevea brasiliensis) wood remained intact in field tests after 9 months whereas untreated samples were destroyed within 6 months of testing, and that the average life of neem-oil-treated cashew nut shells was increased by a factor of 4– 5.
Fig. 2. Weight loss of M. indica and A. saman wood blocks after field test.
References the field. They were carefully washed and then dried at room temperature and finally oven-dried at 103 C 2 C. From the initial and final weights, the percentage of weight loss and increase in durability were calculated with reference to the control samples. 3. Results and discussion The retention of neem extract (NE) was found to be 21.9 kg m3 for M. indica wood samples after 72 h and 15.5 kg m3 for A. saman samples after 24 h. The retention of NECB was 25.7 kg m3 for M. indica samples after 72 h and 17.9 kg m3 for A. saman after 24 h. Both NE and NECB inhibited S. commune growth completely in the agar plate assays where the calculated AI was still 100% for both preparations after 12 days of incubation. The average weight loss of NE- and NECB-treated wood blocks exposed to S. commune were 4.7% and 3.3%, and 4.1% and 3.0%, respectively, for M. indica and A. saman (Fig. 1). In control wood blocks, 32.4% and 30.5% weight losses were found for M. indica and A. saman, respectively, which is significantly (P < 0.05) higher than losses in treated wood blocks. White-rot fungi are generally more destructive than brown-rot fungi on hardwood species (Eaton and Hale, 1993); thus the deterioration caused in untreated M. indica and A. saman wood samples by S. commune was not exceptional. The extent of deterioration was drastically reduced in treated wood samples, where less than 5% weight loss was recorded for both treatments. Therefore NE and the combined NECB treatments resulted in significant protection against S. commune. Treatments resulting in less than 10% weight loss of test blocks are considered to be effective treatment and are grouped under the rubric ‘‘high resistance class’’; this classification of resistance is used only for comparison with natural resistance of durable species as per ASTM (Anon., 1981). In field tests, weight loss varied with wood species, preservative preparations, and period of treatment (Fig. 2). In the case of M. indica the average weight losses after 12 weeks of testing of samples treated with NE for 24, 48, and 72 h were 6.1%, 5.6%, and 5.0%, respectively, and for NECB the average weight losses were 4.6%, 3.8%, and 3.3%. In the case of A. saman wood, the average weight losses of samples treated with NE for 24, 48, and 72 h were 5.8%, 5.4%, and 4.8%, respectively, and for NECB they were 4.4%, 3.6%, and 3.0%. Weight losses of untreated control samples were found to be 37.0% and 36.4% for M. indica and A. saman, respectively, which is significantly (P < 0.05) higher compared to losses in treated
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