1、Designation: D4445 09Standard Test Method forFungicides for Controlling Sapstain and Mold onUnseasoned Lumber (Laboratory Method)1This standard is issued under the fixed designation D4445; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revi
2、sion, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This (laboratory) test method is used for determining theminimum concentration of fungicide, or form
3、ulation of fungi-cides, that is effective in preventing biodeterioration by sap-stain fungi and molds in selected species of wood underoptimum laboratory conditions.NOTE 1From the results of this test, commercial treating solutionconcentrations cannot be estimated without further field tests.1.2 The
4、 requirements for test materials and procedures arediscussed in the following order:SectionSummary of Test Method 4Apparatus 6Reagents 7Wood 8Test Fungi 9Culture Media 10Preparation of Inoculum 11Preparation of Test Chambers 12Treatment of Samples 13Inoculation and Incubation 14Evaluation of the Tes
5、t 15Report 161.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to e
6、stablish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D9 Terminology Relating to Wood and Wood-Based Prod-uctsD1165 Nomenclature of Commercial Hardwoods and Soft-woodsD1193 Specification fo
7、r Reagent Water3. Terminology3.1 DefinitionsFor definitions of terms used in this testmethod, refer to Terminologies D9 and D1165.4. Summary of Test Method4.1 Unseasoned sapwood specimens are treated either byspraying with, or by immersing in, solutions or dispersions ofa fungicide formulation prepa
8、red at five or more concentrationlevels. The specimens are exposed to sapstain fungi and molds.Options for testing the toxicity of fungicides include testingagainst individual fungi or against several fungi by using amixed spore suspension for the inoculation of the specimens.4.2 The intensity of su
9、rface fungal growth is estimated afterincubation and the results used to determine the minimumchemical treatment concentration giving zero growth (CGo).5. Significance and Use5.1 This test method is useful as a screening procedure forselecting fungicides or formulations for more rigorous fieldevalua
10、tion.1This test method is under the jurisdiction of ASTM Committee D07 on Woodand is the direct responsibility of Subcommittee D07.06 on Treatments for WoodProducts.Current edition approved Sept. 15, 2009. Published October 2009. Originallyapproved in 1984. Last previous edition approved in 2003 as
11、D4445 03. DOI:10.1520/D4445-09.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International,
12、 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.6. Apparatus6.1 Incubation Room (or Incubation Cabinet), maintained ata temperature of 25 6 1C, and relative humidity between 70and 80 %.6.2 Steam Sterilizer.6.3 Containers:6.3.1 Sterile Petri Dishes, with minimum s
13、ize of 150(diameter) by 25 mm (height) with lid or,6.3.2 Aluminum Pans, with minimum size of 240 by 100 by20 mm (height) with aluminum foil cover.6.4 Spacers:6.4.1 U-Shaped Glass Rod, with 3 mm diameter or,6.4.2 Polyethylene Mesh, cut to cover the bottom of theselected container(s).7. Reagents7.1 Pu
14、rity of WaterReference to water shall be understoodto mean sterile reagent water conforming to Type IV ofSpecification D1193.8. Wood8.1 General PropertiesThe wood species to be testedshall be selected on the basis of their susceptibility to stainingfungi (pine or spruce species are preferred). Sapwo
15、od of theselected wood species, unseasoned (moisture content higherthan 40 %), free of knots, visible decay, sapstain, and mold,shall be used (Note 2). If the fungicide is to be used to protecthardwood, the inclusion of sapwood from a hardwood speciesis recommended.NOTE 2If wood for the test is coll
16、ected in a sawmill where logs arestored in water, it is necessary to collect lumber from at least threedifferent logs since depletion of nutrients during water storage maystrongly affect the growth of molds and staining fungi. Ensure that thelumber collected in a sawmill has not been treated with a
17、sapstain andmold preventive, and if there is any doubt, at least 10 mm of surface woodmust be removed and discarded.8.2 Size of SpecimensSpecimens shall be 7 by 20 mm incross section and 70 mm long.8.3 Preparation of SpecimensWithin two days of collect-ing, the samples shall be cut from the wood usi
18、ng a sharp sawblade. To prevent drying, the specimens shall be stored inpolyethylene bags. For storage longer than one day, but lessthan one year, tightly packed specimens shall be frozen (20Cor lower) in polyethylene bags . For these longer storage cases,the contents of one bag shall be limited to
19、as many specimensas are used for a single experiment.9. Test Fungi39.1 Hardwoods:9.1.1 Sapstain Fungi:9.1.1.1 Diplodia natalensis P. Evans (ATCC 34643).9.1.1.2 Ceratocystis virescens (Davidson) C. Moreau(ATCC 11066) a form of C. coerulescens found on Americanhardwoods.9.1.1.3 Aureobasidium pullulans
20、 (d. By) Arnaud. (ATCC16624).9.1.2 Mold Fungi:9.1.2.1 Trichoderma pseudokoningii Rifai (ATCC 26801).9.1.2.2 Cephaloascus fragrans Hanawa (ATCC 12091).9.1.2.3 Gliocladium roseum (Link) Bainier (ATCC 10521).9.2 Softwoods:9.2.1 Sapstain Fungi:9.2.1.1 Diplodia natalensis P. Evans (ATCC 34643).9.2.1.2 Ce
21、ratocystis pilifera (Fr.) C. Moreau (ATCC15457).9.2.1.3 Aureobasidium pullulans (d By) Arnaud (ATCC16624).9.2.2 Mold Fungi:9.2.2.1 Trichoderma pseudokoningii (Rifai (ATCC 26801).9.2.2.2 Cephaloascus fragrans Hanawa (ATCC 12091).9.2.2.3 Gliocladium roseum (Link) Bainier (ATCC 10521).9.3 General Consi
22、derationIn addition to the above fungi,others that are known to cause discoloration on wood speciesused in test include, for example, Cytospora sp. (Pine);Phialophora sp.; Graphium sp.; Ceratocystis sp.; Alternariasp.; Penicillium sp.; Aspergillis sp.; Trichoderma sp.10. Culture Media10.1 Agar Subst
23、rateFor both stock culture tube and petridish cultures of the test fungi, use a nutrient medium: that is,malt extract agar (MEA, 2 % malt extract plus 2 % agar),potato dextrose agar (PDA, 0.4 % potato starch, 2 % dextroseplus 2 % agar), or similar commercial mixtures of MEA orPDA prepared in accorda
24、nce with manufacturer instructions.PDA stimulates sporulation in some sapstain fungi (for ex-ample, Aureobasidium pullulans). Sterilize the medium at121C, 0.1 MPa, for 20 min.11. Preparation for Inoculum11.1 If the toxicity of a fungicide is being tested againstindividual fungi, maintain aseptic con
25、ditions when preparingthe spore suspension; if the general effectiveness of a fungicideis being tested using a mixed spore supension, aseptic condi-tions are unnecessary. For laboratory experiments requiring arelatively small volume (about 100 mL) of inoculum, prepara-tion using only the stock test
26、tube cultures is an option. Forlarger volumes of inoculum, prepare from cultures grown onpetri dishes.NOTE 3Before using any stock test tube culture, reinoculate newtubes for future use.11.2 For the preparation of a spore suspension, add 5 mL ofsterile water to each culture tube or 10 mL to petri di
27、shes, andrub the surface of the MEA or PDA culture with a blunt glassrod to loosen the spores. After collecting the spores andcombining them with other similarly collected spores, ifdesired, adjust the water volume to that required.Although it isa good practice to prepare fresh spore suspensions jus
28、t beforeuse, their storage, even without refrigeration, for 2 to 3 weeksis permissible.11.3 For nonsporulating cultures, obtain a mycelial suspen-sion for use by aseptically scraping the surface mycelium offand blending it with sterile water.3The following numbers refer to standard strains of test f
29、ungi maintained in theAmerican Type Collection (ATCC), P.O. Box 1549, Manassas, VA20108, www.atc-c.org.D4445 09211.4 To evaluate a fungicide use at least six test fungi (threesapstain and three mold) individually, as well as one mixedspore suspension of selected fungi.12. Preparation of Test Chamber
30、s12.1 To maintain high humidity in the petri dishes duringthe test period, place eight to ten layers of absorbent paper onthe bottom of each dish. Wet the papers with water until freewater appears, and press out any air bubbles trapped under andbetween the paper disks. Place a U-shaped glass rod (3
31、mm indiameter) (Fig. 1) or polyethylene mesh spacer (Fig. 2)ontopof the saturated papers in a sterile petri dish.12.2 Aluminum ContainersTo maintain high humidity inthe containers, treat as with the petri dishes. Instead of aU-shaped glass rod however, place two straight rods (3 mm indiameter by 200
32、 mm long) or a polyethylene mesh spacer ontop of the saturated papers. Sterilize if required.13. Treatment of Specimens13.1 SpecimensIf the wood samples were stored frozen,allow them to thaw in the polyethylene bags. Because of thevariation in the susceptibility of wood to fungi, distribute anequal
33、number of specimens from each log, into each treatmentper fungus. If specimens were taken from lumber where logidentity is not available, select the specimens randomly fortesting. Autoclave the specimens before treatment at 121C,0.1 MPa, for 20 min.13.2 Number of SpecimensUse a minimum of ten speci-
34、mens per concentration of a fungicide for each fungus tested.Also, use a minimum of ten untreated control specimens foreach fungus tested.13.3 Preparation of Treating SolutionEvaluate each fun-gicide using at least five concentrations. Select the lowestconcentration of a fungicide or formulation to
35、be below theexpected effective strength and each of the following concen-trations shall be twice the previous concentration. Start thepreparation of the set of concentrations of each fungicide bypreparing the highest concentration in an amount equal to twicethe volume required for treatment of the s
36、amples. Then dilutehalf of this preparation with an equal volume of water to obtainthe next preparation. Therefore, a serial set of concentrations isprepared by continuing the dilutions in this way.13.4 Treating ProcedureCarry out the treatment in a600-mL beaker (Fig. 3). Place two unused test piece
37、s edgewiseon the bottom of the beaker, and the specimens, four or five ina layer, also on edge, crosswise on the previous layers untilthey reach the top, but not extending above the rim of thebeaker. Holding down the specimens with a finger bearingdown on a watch glass, pour the prepared solution in
38、to thebeaker. After 15 s, pour the solution out, still holding thespecimens down so that they cannot move. Similarly, treatuntreated control specimens with water. After the treatment,tightly cover the beaker with a piece of plastic sheet to preventdrying, and store overnight. This allows draining of
39、 excesssolution and time for the fungicide to be deposited or fixed inthe wood before inoculation.13.5 After overnight storage, place the samples into theprepared petri dishes or aluminum pans for inoculation.14. Inoculation and Incubation14.1 Inoculation of the SpecimensStir the spore suspen-sion f
40、requently during inoculation. Perform inoculation using atransfer pipet fitted with a rubber bulb; streak about 0.25 mLofspore suspension along the length of one flat side of eachspecimen in the culture vessels. Application may also beaccomplished by spraying. Allow a small amount of the sporesuspen
41、sion to run down on at least one of the crosscut ends.14.2 Place the petri dishes in polyethylene bags to preventdrying and incubate at 25C in an incubator preferably in thedark. Incubation time is between 2 and 4 weeks. Rewet thepaper pads with sterile water as necessary during the incuba-tion peri
42、od to maintain a “damp condition.”14.3 Incubate the aluminum pans at 25C for a period ofbetween 2 and 4 weeks.15. Evaluation of the Test15.1 After 2 or 4 weeks, or both, estimate the growth offungi visually and score using a scale of 0 to 5, the 5 beingmaximum intensity (Table 1). Base the estimate
43、on the inten-sity of growth and discoloration, and not only on the surfacearea covered by the fungi, since the latter may be correlatedonly to the distribution of the original inoculum and notnecessarily to the subsequent growth activity of the fungi.15.2 Determine the effective concentration, or co
44、ncentrationfor zero growth (CGo), as follows:15.2.1 At each concentration, average the scores given foreach fungus or for the mixed fungi, or both.15.2.2 If the toxicity was tested with individual fungi orwith more than one mixture of fungi, sum the average scoresfor each concentration (as shown und
45、er “Total” in Table 1).FIG. 1 Arrangement of Treated Wood Specimens on Glass RodWithin the Petri Dishes Before IncubationD4445 09315.2.3 Express fungal growth for each concentration as apercentage of the fungal growth in the controls (for example, inTable 1 at a concentration of 0.011 % for fungicid
46、e “A”),percent of total 511.514.73 100 5 7815.2.4 Plot the “percentage of total(s)” against the logarithmof treating solution concentration and draw the best-fit, straightline to these points (Fig. 4).15.2.5 The concentration where the line crosses the axis oftreating solution concentration is the e
47、stimated CGo. Forexample, the line for Fungicide A crosses the x-axis atapproximately 0.88. The anti-log of 0.88 is 0.13, so theestimated CGo is 0.13 %.FIG. 2 Arrangement of Treated Wood Specimens on Polyethylene Spacing Within the Petri Dishes Before IncubationFIG. 3 Arrangement of Test Material fo
48、r Treatment in a 600-mLBeakerTABLE 1 Fungicide Scoring After IncubationAFungicideConcentrationin TreatingSolutionC.f. T.p. MTotal Scoresfor Stain andMoldPercent ofStain andMold(Based onControl)A 0 (control) 5.0 4.7 5.0 14.7 0.011 2.2 4.3 5.0 11.5 780.022 1.5 3.3 4.7 9.5 670.045 0.9 1.6 2.0 4.5 310.0
49、9 0.0 0.5 0.9 10.0 100.18 0.0 0.1 0.1 0.1 0NaTCP 0 (control) 5.0 4.3 5.0 14.3 0.12 5.0 3.0 5.0 13.0 910.24 5.0 1.5 5.0 11.5 800.48 3.8 0.2 2.2 6.2 430.96 2.5 0.5 1.5 4.5 311.9 0.1 0.0 0.0 0.1 0AScoring assessed after three weeks incubation, for two fungicides, “A” andsodium tetrachlorophenate (NaTCP) at five concentrations, using Cephaloascusfragrans (C.f.), Trichoderma pseudokoningii (T.p.) and a mixture (M) containing thespores of two Penicillium sp., Aspergillus niger and Ceratocystis pilifera. Eachscore is an average of eight samples.NOTE 1CGo f
