Thyme oil completely inhibited Aspergillus, Penicillium and
Trichoderma on Southern Pine
http://www.fpl.fs.fed.us/documnts/pdf2006/fpl_2006_yang001.pdf
AMERICAN WOOD PROTECTION ASSOCIATION
Multi-component Biocide Protects Wood from Fungi and Insects in UC2 Applications
Carol A. Clausen Vina W. Yang
US Department of Agriculture, Forest Service Forest Products Laboratory Madison, Wisconsin
ABSTRACT
Results that demonstrate wood protection from mold, decay
and termites in laboratory and above ground field tests are presented for an experimental synergistic biocide, called Durazol.
Keywords: biocide, moldicide, termiticide, decav/iingi,
termite, synergy
INTRODUCTION
Development of synergistic biocides to protect wood in
interior applications has been of particular interest since the recent increase of indoor mold infestations. Many products have been developed to address the recent influx of indoor mold infestations.
Researchers must emphasize the use of environmentally benign chemicals due to the need for safety of human occupants. Some typical strategies employed include one or more of the following: naturally occurring antimicrobial or insecticidal chemicals, synergistic combinations of chemicals, chemicals with known performance (and previous EPA registration). Unfortunately, safely controlling fungal growth in the same environment as human occupants is difficult to accomplish. Since fungi and humans are both eukaryotic, (i.e. higher multi-cellular organisms with organized nuclei), metabolic inhibitors of fungi are likely to be toxic to humans (Clausen and Yang 2005a). Use of naturally occurring antimicrobials needs to be approached with caution, since many but not all naturally occurring compounds are toxic in tissue culture. Chemicals with previous EPA approval are limited, but certainly, new, unique combinations of such chemicals can create new formulations withantimicrobial or insecticidal properties (Clausen and Yang
2003). Occasionally, combinations of known fungal or insect inhibitors are synergistic, i.e. combined chemicals are more effective than the individual components at higher concentrations. One such synergistic combination,
called Durazol, incorporates some known antimicrobials and insecticides, namely boric acid and propionic acid with a quaternary amine compound and an azole to provide protection against mold fungi, stain fungi, decay fungi, and termites at lower concentrations than any of the individual components alone (Clausen and Yang 2004; 2005b; 2005c; 2007). A patent application was filed with the US Patent and Trade Office (USPTO) on 3/21/07 (Clausen et al. 2007).
MATERIALS AND METHODS
Organisms
Mold fungi: Three mold fungi, Aspergi/lus niger 2.242,
Penicilliu,n chrysogenum PH02 and Trichoderma viride ATCC 20476 were grown and maintained on 2% malt agar (Difco, Detroit, MI, USA) at 27°C, and 80% relative humidity (RH). Aureobasidium
pullulans MDX- 18 was grown and maintained on 2% potato dextrose agar at 27°C, and 80% relative humidity.
Spore suspensions of test fungi were prepared by washing the surface of individual 2-wk old Petri plate cultures with 10-15 ml of sterile deionized water (Dl) according to ASTM standard D4445-91 (ASTM 1998). The wash water was transferred to a spray bottle and diluted to approximately 100 mL with DI water to yield -3 x spores/ml. The spray bottle was adjusted to deliver 1 niL inoculum per spray. Individual spore suspensions were used to inoculate
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monoculture Petri plate tests or the mold test chamber,
while Aureobasidium pullulans was used exclusively for inoculation of the mold chamber test.
Decay fungi: Two brown-rot fungi, Gloeophyllum traheuin
Mad-563 and Postia placenta Mad-698 and one white-rot fungus, Trametess versicolor Mad-697 were grown and maintained on 2% malt extract agar at 27°C and 70% RH.
Termites: Subterranean termites, Reticulitermesfiavipes were
collected in Janesville, WI and maintained at the Forest Products Laboratory, Madison, WI.
Coptotermesformosanus were collected and maintained at the Fonnosan Termite Research Facility in McNeill, MS.
Chemical formulation
Durazol’s chemical formulation consists of 0.5% propionic acid, 0.1% boric acid, 1.1% dimethyl cocoamine, 0.1% thiabendazole and 0.3% propylene glycol in
an aqueous solution, however, due to low solubility, thiabendazole must be solubilized in 70% ethanol prior to addition to the treatment solution.
Treatment method and chemical retention
Unseasoned southern pine specimens were brushed, sprayed, or
immersed (15 sec) in treatment solution.
Chemical treatment retentions were determined from the
average difference in specimen weight before and after dipping and reported based on the volume of the specimen. Specimeds were held in a closed container for 24 hr at 25°C prior to challenge with test organisms.
Additionally, chemical retention in unseasoned pine was compared with kiln-dried pine. Retention rates were also compared for unseasoned pine, aspen, and Douglas-fir.
Mold test
Petri plate test: Five treated specimens and untreated
controls were placed in a Petri dish (150x25mm)
(B-D Falcon, Los Angeles, Calif.) containing four layers of
blotting paper that was saturated with 30 ml.DI water. A polyethylene mesh spacer was used to elevate specimens and prevent chemical leaching.
Specimens were sprayed with I mL of individual spore
suspension 24 hr post-treatment. Petri dishes weresealed in polyethylene bags to prevent drying, and incubated
at 27°C, 70% RH. Specimens wereindividually rated for mold growth at 4 and 8 wk on a scale
of 0 to 5 with 0 indicating no growth and
5 indicating heavy growth.
Environmental chamber test: A polypropylene mold chamber
fabricated according to ASTM 3273-00 (ASTM 1986) was placed in a room maintained at 30°C and 70% RH. The mold chamber relies on a circulating fan above water to provide 100% humidity and non-sterile soil for a supplemental source of circulating mold spores. The soil was additionally inoculated with mold spores from three test fungi,Aureobasidium pullulans, Aspergillus niger and Penicilliu,n
chrvsogenum, two weeks before placing the test specimens in the chamber. Five southern pine test specimens (75 x 100 mm by 12.5 mm thick) were conditioned at 27°C, 70% RH and weighed prior to immersion in Durazol for —15 sec. Treated specimens were reweighed and held for 24 hr in a closed container.
Treated specimens and untreated controls were vertically suspended across the width of the environmental chamber over inoculated soil. Specimens were sprayed with spore suspensions of A. niger, P. chrysogenum and T viride and incubated at 30°C. After 8 wk incubation, specimens were individually rated for mold growth on a scale of 0 to 5 with 0 indicating no growth and 5 indicating 100% coverage.
Soil block decay test
Soil block culture bottles were prepared according to AWPA
El 0 (2006) with a modification of wood block size to 1 x 1 x 1 cm. In soil block bottles, southern yellow pine feeders were inoculated with brownrot fungi, G. trabeum and P. placenta and maple feeders were inoculated with the white-rot fungus, T. versicolor. Bottles were incubated at 27°C and 70% RH for 3 weeks until the fungus completely colonized each feeder. Pre-weighed southern yellow pine or maple blocks, conditioned at 27°C and 70% RH, were vacuum treated with 2 % Durazol for 2 x 20 min at 25 in Hg.
Based on solution uptake, the boron retention in the blocks was 0.048 pcf (as B 2 03 ). The total Durazol retention was 0.97 pcf. Treated blocks were conditioned for 2 wk at 27°C, 70% RH and reweighed before adding them to pre-grown soil block bottles and incubating at 27°C, 70% RH for 12 weeks. Untreated blocks served as controls. Following incubation, surface mycelia was removed and blocks were dried at 60°C overnight before reconditioning at 27°C, 70% RH for 2 wk. Blocks were reweighed and the average percent weight loss was calculated.
Termite test
A laboratory no-choice test was conducted according to AWPA
El (2006) with Reticu/itermesjlavipes at USDA Forest Products Laboratory in Madison, WI and on Coploterines formosanus at the Formosan Termite Research Facility at McNeill. MS.
Above ground test methods
Ground proximity test: A standard test for evaluation of
preservative treatments for lumber and timbers against subterranean termites in above-ground, protected applications (UCI and UC2) (AWPA E21) was initiated in July 2006 on southern yellow pine pressure-treated with 2% or 4% Durazol (0.97 and 1.93 pcf, respectively), disodium octaborate tetrahydrate (0.37 pet),
and untreated controls (N=I0). Specimens willbe evaluated annually for termite and fungal decay.
Above ground deck test: Five 2″ x 6″ x 30″
long southern pine deck boards were pressure treated with
2% Durazol and installed in a moderate decay hazard zone in
Madison. WI. After 2 years, there are nosigns of decay, mold, or stain on any surface of the treated specimens. They are rated as sound with no evidence of weakening, softening, or discoloration from deteriorating organisms.
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RESULTS AND DISCUSSION
Chemical Retention
Comparative retention rates for Durazol applied by spray,
brush and dip are shown in Table 1. Brushing
and immersion provide roughly twice the treatment that
spraying does. Retention rates for different wood
types dip-treated with Durazol and the comparison of
kiln-dried with unseasoned SYP are shown in Table
2. Pine retains the most treatment whether it is kiln-dried
or unseasoned. Unseasoned Douglas-fir and aspen
demonstrate similar retention rates, although they both
retained approximately 3.4 times less than kiln-dried
pine and 1.5 times less than unseasoned pine.
Table I. Comparison of Durazol retention rate for brush,
spray and dip application on southern
yellow pine.
Ave.
Application
retention
method
(kg/rn)
Spray 0.54
Brush 0.95
Dip 0.83
33AMERICAN WOOD PROTECTION ASSOCIATION
Table 2. Comparative retention of different wood types and
seasoning conditions dip-treated with