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In vitro evaluation of an integrated approach using Trichoderma harzianum and chitosan for the control of sapstain
2008 - IRG/WP 08-10659
In vitro assays were undertaken to evaluate the control of two sapstain fungi, Leptographium procerum and Sphaeropsis sapinea by a combination of chitosan or chitosan oligomer (CODP-14) and a naturally mutated strain of Trichoderma harzianum. Spore germination and hyphal growth of the test fungi were assessed on media amended with chitosan or chitosan oligomer with and without T. harzianum using either simultaneous inoculation or various time intervals between inoculation of the media with sapstain fungi and inoculation with T. harzianum. There was no mycelium growth of the test fungi regardless of chitosan concentrations used when either L. procerum or S. sapinea were simultaneously inoculated with T. harzianum. However, the dose response of chitosan or chitosan oligomer on the test fungi was apparent when there was a time interval before the introduction of T. harzianum. There was a greater growth reduction at higher concentrations (0.075-0.1%v/v) and overall, chitosan oligomer was more effective than chitosan aqueous solution. Furthermore, the results also suggest that generally, either chitosan or T. harzianum alone was able to restrict or delay the germination of spores but the combination of chitosan and T. harzianum inhibited spore germination and hence colony formation of test fungi regardless of time delay.
C Chittenden, T Singh

Growth of two selected sapstain fungi and one mould on chitosan amended nutrient medium
2003 - IRG/WP 03-10466
In vitro studies were undertaken to investigate the effect of chitosan on growth of Leptographium procerum, Sphaeropsis sapinea and Trichoderma harzianum. Chitosan was tested at three molecular weight (MW) ranges and different concentrations formulated as either a powdered suspension or as a solution. The results generally showed that low MW chitosan produced a greater inhibitory effect on growth of test fungi than medium and high MW, irrespective of the chitosan formulation used. However, chitosan was more effective when applied in solution with much lower concentrations exerting inhibition of test fungi than chitosan suspensions. Furthermore, susceptibility of test fungi to chitosan differed, with T. harzianum being the most tolerant and S. sapinea the most sensitive species irrespective of chitosan formulation used.
C Chittenden, R N Wakeling, B Kreber

In vitro studies on the effect of chitosan on mycelial growth and spore germination of decay fungi, moulds and staining fungi
2004 - IRG/WP 04-10507
The effect of solubilised, low molecular weight chitosan on established mycelial growth of a range of decay fungi, moulds and staining fungi was investigated using nutrient medium amended with different concentrations of chitosan that ranged from 0.1 to 0.4% weight per volume (%w/v). Also, spore germination of Trichoderma harzianum and Leptographium procerum was examined on chitosan amended nutrient medium using visual and microscopic assessment. The results showed that chitosan affected mycelial growth of a wide range of test fungi which generally showed lower growth rates as the chitosan concentration increased. However, the degree of inhibition exhibited by chitosan varied with fungal species. Under the present test conditions, chitosan was fungistatic, but not fungitoxic, against established mycelium of the staining fungi tested and also two moulds, Botrytis cinera and Cladosporium herbarum, but not against any of the decay fungi tested. Spores of T. harzianum germinated on chitosan amended nutrient medium at all chitosan concentrations tested, except for the highest level (0.2%w/v) used, while L. procerum failed to germinate on 0.15 and 0.2%. Also, T. harzianum and L. procerum spores incubated on 0.15% and 0.2% chitosan failed to germinate when placed onto fresh malt extract agar suggesting fungitoxic activity of chitosan at these higher concentration levels.
C Chittenden, B Kreber, N McDowell, T Singh

Remediation of CCA-treated wood by chitin and chitosan
2005 - IRG/WP 05-50229
Chitin and chitosan are naturally abundant biopolymers which are interest of to research concerning the sorption of metal ions since the amine and hydroxyl groups on their chemical structures act as chelation sites for metal ions. This study evaluated the removal of copper, chromium, and arsenic elements from chromated copper arsenate (CCA)-treated wood via biosorption by chitin and chitosan. Exposing CCA-treated sawdust to various amounts of chitin and chitosan for 1, 5, and 10 days enhanced removal of CCA components compared to remediation by deionized water, only. Remediation process with 2.5 g chitin-containing solution for 10 days removed 74% copper, 62% chromium, and 63% arsenic from treated sawdust. Remediation of treated sawdust samples using same amount of chitosan resulted in 57% copper, 43% chromium, and 30% arsenic removal. The results suggest that chitin and chitosan are able to remove copper from CCA-treated wood. Thus, these most abundant natural amino polysaccharides could be important in the remediation of waste wood treated with the newest formulations of organometallic copper compounds and other water-borne wood preservatives containing copper.
S N Kartal, Y Imamura

Chitosan for wood protection - state of the art
2005 - IRG/WP 05-30378
The aim of this paper was to give a state of the art description of chitosan as a wood protecting agent. Chitosan is a metal free natural compound derived from crustacean shells and is under evaluation as an environmentally benign wood protecting agent. Information from journals states that chitosan may act both fungistatically and at higher concentrations, as fungitoxic, but the mode of action is not yet fully understood. The hypothesis with most support in the literature is that chitosan interacts with the cell membrane and causes alterations in permeability. It is not proven that chitosan is more effective against a particular class of fungi, but morphological changes and reduction in growth rate is reported from a range of fungi. Results from agar plate growth rate tests are not conclusive with respect to whether high or low molecular weights are most effective against micro-organisms. Other factors than the molecular weights may influence microbial activity of the species studied, i.e. FA, pH, and internal distribution of the monomers, concentration and additives. In results available in the literature it is obvious that there is a dose-response relationship between chitosan and antimicrobial activity. In agar plates a lethal/totally inhibiting concentration is usually between 0.1 and 1 %. Chitosans in solution are more effective against antimicrobial growth than chitosans in suspension (i.e. solid chitosan particles). This is further reflected in that higher concentrations of chitosan are needed in wood than in agar amended plates. If the treated wood is subjected to leaching, around 5 % (w/v) chitosan solutions seems to be needed for good efficacy against fungal decay. Some tests where chitosan fails in decay tests are probably because of the use of to low concentrations, or to low penetration of chitosan solution due to high molecular weight.
M Eikenes, G Alfredsen, E Larnøy, H Militz, B Kreber, C Chittenden

Solution stability and Mechanical properties of Chitosan treated Pine
2005 - IRG/WP 05-30377
During the last years the research on chitosan as a wood preservative has been enhanced. Up to now, most of the research has been applied to the anti-fungal properties of chitosan, and no research was conducted on the solution stability of chitosan solutions in repeated trials by impregnation of wood, or on the mechanical properties of chitosan-treated wood. In this paper, the stability of high- and low-molecular weight chitosan solutions (2,4% (w/v) concentration) was investigated in 15 repeated impregnation cycles using pine (Pinus sylvestris) samples. Changes in the following parameters of the treatment solution were examined: uptake of chitosan, viscosity, pH, molecular weight and concentration. In addition, the following mechanical properties of chitosan-treated pine were determined using a paired experimental design: modulus of elasticity (MOE), static bending, impact bending strength, static hardness, shear strength, tensile strength, compression strength and adhesion of paint to wood. Overall, an average chitosan uptake by the pine samples in the order of 15 to 16 kg/m³ was obtained. In the trials for determination of solution stability, the uptake, viscosity and concentration remained unchanged. However, the pH of the solutions increased, and the average chitosan molecular weight decreased, with the high molecular weight chitosan displaying larger changes than low molecular weight chitosan. No significant changes in the mechanical properties of pine treated with high-and low molecular weight chitosan (2,4% (w/v) concentration) were determined as compared to untreated wood samples.
E Larnøy, M Eikenes, H Militz

PXTS; A Metal Free Oligomer Wood Preserving System - A Summary of Data To Date
2004 - IRG/WP 04-30350
The world is continually looking for new wood preservative technology, especially to address environmental concerns. Recent changes in the USA have limited the use of arsenical containing formulations to industrial use through a negotiated voluntary cancellation of most residential label uses. PXTS (Polymeric Alkylphenol Polysulfide) offers a brand new technology that is an all organic system, like creosote or pentachlorophenol, but without the associated human toxicity. PXTS has been extensively tested and evaluated over the last 6+ years in both laboratory and field efficacy tests, and has undergone extensive physical and chemical property tests on both the active ingredient and on the PXTS treated wood . This paper summarizes the results of the testing on PXTS and PXTS treated wood through 2003. Lengthy field trials have now proven the efficacy of PXTS in both harsh and very severe test sites where attack is rampant from insects and decay organisms. Additionally, laboratory efficacy tests have shown that the PXTS performance profile is superior to creosote in many applications, extending the life of wood treated with PXTS many fold over that of untreated controls. Laboratory soft rot test indicate that PXTS may be as much as 6 times more effective than AWPA P-1/P-13 creosote. Tests in marine waters, although not presented here, have also proven PXTS superior to creosote in harsh Florida environments riddled with Limnoria. Although work is continuing on the evaluation of this new preservative, this document represents the most complete portfolio of information on PXTS presented to an international audience.
M H Freeman, D D Nicholas, D Renz, R Buff

The effect of chitosan on the growth and physiology of two wood inhabiting fungi
2006 - IRG/WP 06-10590
The effect of chitosan on the wood inhabiting fungi Sphaeropsis sapinea and Trichoderma harzianum was evaluated at cellular level. Increasing concentrations of chitosan caused an increase in the amount of peroxide in cultures of S. sapinea, which was accompanied by a simultaneous decrease in superoxide. The same effect was not observed in T. harzianum. The growth of both fungi was inhibited when hydrogen peroxide was added to the culture medium, but the inhibitory effect of hydrogen peroxide was alleviated by addition of catalase. Furthermore, addition of catalase to the chitosan-amended medium also lead to improved growth for both species, suggesting that oxidative stress might act concurrently to chitosan toxicity under these conditions. Chitosan affected membrane permeability in both species and lead to increased leakage of potassium ions. The results are discussed with a view to the potential mechanisms mediating chitosan toxicity in these two fungi.
D Vesentini, T Singh

Effect of chitosan on the morphology and ultrastructure of two wood inhabiting fungi
2007 - IRG/WP 07-10606
An investigation was undertaken to compare the antifungal effects caused by chitosan against two selected wood degrading fungi Sphaeropsis sapinea and Trichoderma harzianum on their growth, morphological and ultra-structural characteristics, using differential interference microscopy, epifluorescence microscopy and transmission electron microscopy. The effect of chitosan on fungal morphology was pronounced against both test fungi but was more severe on S. sapinea compared to T. harzianum. Increasing concentrations of chitosan induced excessive branching, vacuolation and a reduction in hyphal diameter. Transmission electron microscopy (TEM), which provided information on cell ultrastructure showed more sever changes in S. sapinea hyphae from chitosan treatment as compared to T. harzianum. The information provided suggests that growth inhibitory effect of chitosan is related to significant alterations in the organisation of vital cell components, and indicated that plasma membrane may be the primary target of chitosan action, and that the two fungi differ in the extent to which they are affected. Fluorescence microscopy provided further indication that the effect of chitosan on fungal hyphae is mediated through alterations in the plasma membrane properties.
T Singh, A P Singh, G Daniel

Efficacy of chitosan in combination with GRAS (Generally Recognised As Safe) compounds as a potential wood preservative
2007 - IRG/WP 07-30414
In vitro studies were undertaken to evaluate the synergy between chitosan and selected GRAS (Generally Recognised As Safe) compounds against two sapstain fungi Sphaeropsis sapinea and Leptographium procerum. Bioassays performed on nutrient medium suggested that some of the GRAS compounds tested including sodium benzoate, potassium sorbate and ascorbic acid had a synergistic effect when combined with chitosan against both test fungi. However the degree of activity varied depending on concentration used and species tested. Synergy between chitosan and sodium benzoate/or potassium sorbate was also apparent when bioassays were performed on sterile wood discs. After 3 weeks of incubation, discs dip-treated with either 1.5% chitosan or 0.5% sodium benzoate/or potassium sorbate had 60%-100% coverage by the test fungi. This was reduced to 5-10% fungal coverage on discs treated with the combination of chitosan and sodium benzoate and virtually no growth was observed on discs dip-treated with chitosan and potassium sorbate at the concentrations tested. Furthermore, fungicidal activity against S. sapinea was confirmed after re-culturing wood pieces taken from these discs on fresh malt-agar medium.
C Chittenden, K Thämelt, T Singh

Antifungal activity of different molecular weights of a biopolymer chitosan against wood decay fungi
2008 - IRG/WP 08-30456
In recent years chitosan has been investigated as a natural polymer for wood preservation against fungal decay. From an environmental point of view, chitosan seems to possess a potential approaches as wood protecting agent. In this study, three different molecular weights of chitosan compounds (with approximately the same degree of deacetylation 83 ± 2 %) were evaluated as in vitro and in vivo assessments against two wood decay fungi (Coriolus versicolor and Poria placenta). Average molecular weights of chitosan samples were determined by measurements of intrinsic viscosity and were found to be 3.60×105 Dalton for low molecular weight (LMW), 6.11×105 Dalton for medium molecular weight (MMW) and 9.53×105 Dalton for high molecular weight (HMW). In vitro antifungal assay was carried out using of a mycelial radial growth inhibition technique. The results showed that the antifungal activity was increased with a decreasing of the molecular weight and a LMW chitosan was exhibited a high antifungal potency against Coriolus versicolor and Poria placenta with EC50 of 1876 and 1744 mg.L-1, respectively. In addition, the results revealed that Poria placenta was more sensitive to these compounds than Coriolus versicolor. Preliminary in vivo biological test was carried out on malt agar using a miniblock technique for beech and Scots pine sapwood with Coriolus versicolor and Poria placenta, respectively. After six weeks of exposure to fungal attack all chitosan protective systems tested proved their relative effectiveness when compared to the control specimens. Furthermore, chitosan LMW was the most effective compound among all treatments at the higher concentration against the tested fungi.
A S O Mohareb, M E I Badawy

Mold-resistance Effect of Bamboo Wood Treated with CCC-organic Complexes
2009 - IRG/WP 09-30514
Mold resistant effect of CCA, ACQ, CuAz, CCC and the compound of CCC and propiconazole were researched on bamboo wood of Phyllostachys pubescens were reported in this paper. Results showed that all of the test fungicides could protect bamboo wood better from Penicillam citrinum than from Trichoderma viride and Aspergillus niger. The complex of CCC and propiconazole had the best resisting effect on all of the test mold fungi. ACQ had better resisting effect on Penicillam citrinum, but not so effective on Trichoderma viride and Aspergillus niger, especially on Aspergillus niger. CuAz had better effect only on Penicillam citrinum. CCA and CCC had better resisting effect on Penicillam citrinum only at higher retentions, but had poor effect on the other two test fungi.
Sun Fangli, Yang Le, Chen Anliang, Bao Binfu, Li Qiao

Characterisation and evaluation of various chitosan oligomers, and decay resistance
2011 - IRG/WP 11-30562
Chitosan, a polymer of D-glucosamine, is known for its antimicrobial activity. However, the physicochemical properties of chitosan depend upon three principal factors, i.e. source of raw material, molecular weight and degree of deacetylation. Here, we report synthesis and characterization of chitosan oligomers prepared by deaminative depolymerization of chitosan (s) obtained from Sigma Aldrich and China (industrial grade). Subsequently their antifungal activies were evaluated using both agar nutrient medium and wood decay bioassays. The nutrient medium bioassay results showed that chitosan(s) and chitosan oligomers affected mycelial growth to different magnitudes. Nevertheless, two of the low-MW chitosan oligomers completely inhibited the growth of all the tested fungi as compared to high-MW chitosan oligomers. However, the work presented in this paper, depicts that antifungal activity increases by decreasing the degree of polymerization of chitosan oligomers, which is a contrary to the reported literature. The wood decay trial confirmed the antifungal activity of chitosan oligmers against basidiomycetes but highlighted the leachability of chitosan when exposed to water.
I Hussain, C Chittenden, T Singh

Novel microscopic approaches to visualise chitosan within impregnated wood
2012 - IRG/WP 12-20485
Chitosan, a deacetylated product of an abundant naturally occurring biopolymer chitin, has been used in a range of applications, particularly in food and health areas, as an antimicrobial agent. In the work reported here Pinus radiata wood was impregnated with chitosan as an environmentally compatible organic biocide. It is important to understand micro-distribution of bioprotectants in impregnated wood products to properly evaluate their performance. We developed a novel microscopic techniques to visualise impregnated chitosan within wood tissues using light microscope and field emission scanning electron microscope (FE-SEM) in combination with a dye that specifically stained wood cell walls and a heavy metal stain that chemically react with chitosan. Sections were viewed with the light microscope without staining with a dye as well as after staining with the dye toluidine blue. Light microscopy was also undertaken on sections that had been stained with 1% aqueous osmium tetroxide (OsO4). For SEM observations, the sections were treated with OsO4 and then examined with the FE-SEM, first in the secondary electron imaging mode (SEI) and then in the backscattered electron imaging (BEI) mode, imaging the same areas of a section in both SEI and BEI modes. The preparation techniques employed and the combined use of light and scanning electron microscopy provided valuable complementary information, revealing that chitosan had penetrated into the cavities (cell lumens, intercellular spaces) of all sizes present within wood tissues and had also impregnated early wood cell walls.
A Singh, T Singh

Analysis of hindgut bacterial phyla frequency and diversity in subterranean termites exposed to chitosan-treated wood
2017 - IRG/WP 17-10882
The termite hindgut contains a microbial community that symbiotically aids in digestion of lignocellulosic materials. For better understanding of the dynamics of the bacteria-termite relationship, a species survey of bacterial hindgut microbes in subterranean termites (Reticulitermes flavipes: Kollar) collected from Louisville, Mississippi was performed after exposure to chitosan-treated and control (water-treated) wood samples. Total genomic DNA was isolated from termite hindguts, amplified and 16S ribosomal RNA (rRNA) gene fragments were analyzed using next-generation sequencing techniques. Twenty-seven bacteria phyla were identified in both treatment groups, with six bacteria phyla showing significantly differences in abundance between the chitosan-treated and control groups. These results suggest that there was a treatment driven effect on the hindgut bacteria diversity.
O Raji, J D Tang, T Telmadarrehei, D Jeremic