ASTM D4445-23

Standard Test Method for Fungicides for Controlling Sapstain and Mold on Unseasoned Lumber (Laboratory Method)

SIGNIFICANCE AND USE
5.1 This test method is useful as a screening procedure for selecting fungicides or formulations for more rigorous field evaluation.
SCOPE
1.1 This (laboratory) test method is used for determining the minimum concentration of fungicide, or formulation of fungicides, that is effective in preventing biodeterioration by sapstain fungi and molds in selected species of wood under optimum laboratory conditions.  
Note 1: From the results of this test, commercial treating solution concentrations cannot be estimated without further field tests.  
1.2 The requirements for test materials and procedures are discussed in the following order:    
Section  
Summary of Test Method  
4  
Apparatus  
6  
Reagents  
7  
Wood  
8  
Test Fungi  
9  
Culture Media  
10  
Preparation of Inoculum  
11  
Preparation of Test Chambers  
12  
Treatment of Samples  
13  
Inoculation and Incubation  
14  
Evaluation of the Test  
15  
Report  
16  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 14-Apr-2023
• ICS: 79.040 - Wood, sawlogs and sawn timber
• Technical Committee: D07 - Wood
• Drafting Committee: D07.06 - Treatments for Wood Products

Relations

• Refers: ASTM D9-12 - Standard Terminology Relating to Wood and Wood-Based Products
• Effective Date: 15-Oct-2012
• Refers: ASTM D5590-00(2010)e1 - Standard Test Method for Determining the Resistance of Paint Films and Related Coatings to Fungal Defacement by Accelerated Four-Week Agar Plate Assay
• Effective Date: 01-Jun-2010
• Refers: ASTM D5590-00(2010) - Standard Test Method for Determining the Resistance of Paint Films and Related Coatings to Fungal Defacement by Accelerated Four-Week Agar Plate Assay
• Effective Date: 01-Jun-2010
• Refers: ASTM D9-09a - Standard Terminology Relating to Wood and Wood-Based Products
• Effective Date: 15-Nov-2009
• Refers: ASTM D9-09ae1 - Standard Terminology Relating to Wood and Wood-Based Products
• Effective Date: 15-Nov-2009
• Refers: ASTM D9-09 - Standard Terminology Relating to Wood and Wood-Based Products
• Effective Date: 01-May-2009
• Refers: ASTM D1165-07 - Standard Nomenclature of Commercial Hardwoods and Softwoods
• Effective Date: 01-Sep-2007
• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006
• Refers: ASTM D5590-00(2005) - Standard Test Method for Determining the Resistance of Paint Films and Related Coatings to Fungal Defacement by Accelerated Four-Week Agar Plate Assay
• Effective Date: 01-Dec-2005
• Refers: ASTM D9-05 - Standard Terminology Relating to Wood and Wood-Based Products
• Effective Date: 01-Dec-2005
• Refers: ASTM D1165-03 - Standard Nonmenclature of Domestic Hardwoods and Softwoods
• Effective Date: 10-Jun-2003
• Refers: ASTM D5590-00 - Standard Test Method for Determining the Resistance of Paint Films and Related Coatings to Fungal Defacement by Accelerated Four-Week Agar Plate Assay
• Effective Date: 10-Nov-2000
• Refers: ASTM D5590-94 - Standard Test Method for Determining the Resistance of Paint Films and Related Coatings to Fungal Defacement by Accelerated Four-Week Agar Plate Assay
• Effective Date: 10-Nov-2000
• Refers: ASTM D9-87(1999) - Standard Terminology Relating to Wood
• Effective Date: 10-Apr-1999
• Refers: ASTM D9-87(1999)e1 - Standard Terminology Relating to Wood
• Effective Date: 10-Apr-1999

Overview

ASTM D4445-23: Standard Test Method for Fungicides for Controlling Sapstain and Mold on Unseasoned Lumber (Laboratory Method) provides a laboratory-based screening protocol for evaluating the effectiveness of fungicides in preventing the growth of sapstain fungi and mold on unseasoned wood. Developed by ASTM International, this standard plays a crucial role in wood protection, quality control, and treatment product development.

The method determines the minimum effective concentration of fungicides needed to inhibit biodeterioration by common wood-infecting molds and sapstain fungi under controlled laboratory conditions. While valuable for preliminary assessment, commercial treatment concentrations should be based on additional field trials.

Key Topics

• Test Scope and Use

  • The method is designed for use as a screening tool to identify promising fungicide formulations for further, more rigorous field testing.
  • Results provide comparative data on the fungicidal efficacy, but do not substitute for field performance assessments.

• Test Method Outline

  • Sample Preparation: Unseasoned sapwood specimens, primarily from susceptible softwoods such as pine or spruce, as well as specific hardwoods, are used.
  • Fungicide Application: Lumber samples are treated with serial dilutions of fungicide solutions or dispersions.
  • Inoculation: Treated wood is inoculated with a range of standardized sapstain and mold fungi.
  • Incubation: Samples are incubated in controlled conditions, typically 25°C and high humidity, for 2-4 weeks.
  • Evaluation: Fungal growth is visually assessed and scored. The minimum concentration at which no growth (zero growth concentration, CGo) occurs is identified.

• Test Controls and Precision

  • Strict control specimens and consistent methodology are required to ensure valid results.
  • Variation may occur due to the biological nature of the test organisms; thus, repeatability is limited to within experimental groups, and broad reproducibility is not claimed.

Applications

• Fungicide Selection and Product Development

  • Used by chemical manufacturers, wood treaters, and research laboratories to screen and compare the efficacy of new or existing fungicidal treatments for unseasoned lumber.
  • Helps identify candidates for commercial wood protection formulations prior to costly and time-intensive field evaluations.

• Quality Assurance in Wood Processing

  • Enables wood processors to verify and compare the initial effectiveness of treatments intended to prevent sapstain and mold during storage, shipping, and processing of green lumber.

• Support for Regulatory Compliance

  • Provides standardized data for documentation, supporting the claims of fungicidal efficacy in product submittals and regulatory filings.

Related Standards

Practitioners applying ASTM D4445-23 may also consult these ASTM standards for consistency in terminology, materials, and related test methods:

• ASTM D9 – Terminology Relating to Wood and Wood-Based Products
• ASTM D1165 – Nomenclature of Commercial Hardwoods and Softwoods
• ASTM D1193 – Specification for Reagent Water (for ensuring sterile water in reagents)
• ASTM D5590 – Test Method for Determining the Resistance of Paint Films and Related Coatings to Fungal Defacement by Accelerated Four-Week Agar Plate Assay

Using ASTM D4445-23 ensures reliable laboratory evaluation and supports the development of effective strategies for controlling mold and sapstain on unseasoned lumber, contributing to longer-lasting, higher-value wood products in the marketplace.

Keywords: ASTM D4445, fungicides, sapstain, mold, unseasoned lumber, wood protection, test method, wood fungi, laboratory screening, lumber treatment

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: D4445 − 23
Standard Test Method for
Fungicides for Controlling Sapstain and Mold on
Unseasoned Lumber (Laboratory Method)
This standard is issued under the fixed designation D4445; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
2.1 ASTM Standards:
1.1 This (laboratory) test method is used for determining the
minimum concentration of fungicide, or formulation of D9 Terminology Relating to Wood and Wood-Based Prod-
ucts
fungicides, that is effective in preventing biodeterioration by
sapstain fungi and molds in selected species of wood under D1165 Nomenclature of Commercial Hardwoods and Soft-
woods
optimum laboratory conditions.
D1193 Specification for Reagent Water
NOTE 1—From the results of this test, commercial treating solution
D5590 Test Method for Determining the Resistance of Paint
concentrations cannot be estimated without further field tests.
Films and Related Coatings to Fungal Defacement by
1.2 The requirements for test materials and procedures are
Accelerated Four-Week Agar Plate Assay
discussed in the following order:
3. Terminology
Section
Summary of Test Method 4
3.1 Definitions—For definitions of terms used in this test
Apparatus 6
Reagents 7
method, refer to Terminologies D9 and D1165.
Wood 8
Test Fungi 9
4. Summary of Test Method
Culture Media 10
Preparation of Inoculum 11
4.1 Unseasoned sapwood specimens are treated either by
Preparation of Test Chambers 12
spraying with, or by immersing in, solutions or dispersions of
Treatment of Samples 13
a fungicide formulation prepared at five or more concentration
Inoculation and Incubation 14
Evaluation of the Test 15
levels. The specimens are exposed to sapstain fungi and molds.
Report 16
Options for testing the toxicity of fungicides include testing
1.3 The values stated in SI units are to be regarded as
against individual fungi or against several fungi by using a
standard. No other units of measurement are included in this
mixed spore suspension for the inoculation of the specimens.
standard.
4.2 The intensity of surface fungal growth is estimated after
1.4 This standard does not purport to address all of the
incubation and the results used to determine the minimum
safety concerns, if any, associated with its use. It is the
chemical treatment concentration giving zero growth (CGo).
responsibility of the user of this standard to establish appro-
5. Significance and Use
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
5.1 This test method is useful as a screening procedure for
1.5 This international standard was developed in accor-
selecting fungicides or formulations for more rigorous field
dance with internationally recognized principles on standard-
evaluation.
ization established in the Decision on Principles for the
6. Apparatus
Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical
6.1 Incubation Room (or Incubation Cabinet), maintained at
Barriers to Trade (TBT) Committee.
a temperature of 25 °C 6 1 °C, and relative humidity between
70 % and 80 %.
6.2 Steam Sterilizer.
This test method is under the jurisdiction of ASTM Committee D07 on Wood
and is the direct responsibility of Subcommittee D07.06 on Treatments for Wood
Products. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved April 15, 2023. Published May 2023. Originally contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
approved in 1984. Last previous edition approved in 2019 as D4445 – 10 (2019). Standards volume information, refer to the standard’s Document Summary page on
DOI: 10.1520/D4445-23. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D4445 − 23
6.3 Containers: 9.1.2.1 Trichoderma pseudokoningii Rifai (ATCC 26801).
6.3.1 Sterile Petri Dishes, with minimum size of 140 mm 9.1.2.2 Cephaloascus fragrans Hanawa (ATCC 12091).
(diameter) by 20 mm (height) with lid or,
9.1.2.3 Clonostachys rosea (Link:Fries) (ATCC 10521).
6.3.2 Aluminum Pans, with minimum size of 240 mm by
9.2 Softwoods:
100 mm by 20 mm (height) with aluminum foil cover.
9.2.1 Sapstain Fungi:
6.4 Spacers:
9.2.1.1 Diplodia gossypina Cooke (Pole-Evans, anamorph)
6.4.1 U-Shaped or Straight Glass Rods, with 3 mm diameter
(ATCC 34643).
or,
9.2.1.2 Ceratocystis pilifera (Fr.) C. Moreau (ATCC
6.4.2 Polyethylene Mesh, cut to cover the bottom of the
15457).
selected container(s).
9.2.1.3 Aureobasidium pullulans (de Bary) Arnaud (ATCC
16624).
7. Reagents
9.2.2 Mold Fungi:
7.1 Purity of Water—Reference to water shall be understood
9.2.2.1 Trichoderma pseudokoningii (Rifai) (ATCC 26801).
to mean sterile reagent water conforming to Type IV of
9.2.2.2 Cephaloascus fragrans Hanawa (ATCC 12091).
Specification D1193.
9.2.2.3 Clonostachys rosea (Link:Fries) (ATCC 10521).
9.3 General Consideration—In addition to the above fungi,
8. Wood
others that are known to cause growth/discoloration on wood
8.1 General Properties—The wood species to be tested shall
which may also be used for testing include, Alternaria sp.;
be selected on the basis of their susceptibility to staining/mold
Aspergillus brasiliensis Varga et al. (ATCC 9642); Aspergillus
fungi (pine or spruce species are preferred when evaluating
niger van Tieghem (ATCC 6275); Aspergillis sp.; Ceratocystis
softwoods). Sapwood of the selected wood species, unseasoned
sp.; Cytospora sp. (Pine); Graphium sp.; Penicillium citrinum
(moisture content higher than 40 %), free of knots, visible
Thom (ATCC 9849); Penicillium sp.; Phialophora sp.; Tricho-
decay, sapstain, and mold, shall be used (Note 2). If the
derma virens (Miller et al.) von Arx, anamorph (ATCC 9645);
fungicide is to be used to protect hardwood, the inclusion of
Trichoderma sp.
sapwood from a susceptible hardwood species, such as red oak
or hickory, is recommended.
10. Culture Media
NOTE 2—If wood for the test is collected in a sawmill where logs are
10.1 Agar Substrate—For both stock culture tube and Petri
stored in water, it is necessary to collect lumber from at least three
dish cultures of the test fungi, use a nutrient medium: that is,
different logs since depletion of nutrients during water storage may
malt extract agar (MEA, 2 % malt extract plus 2 % agar),
strongly affect the growth of molds and staining fungi. Ensure that the
lumber collected in a sawmill has not been treated with a sapstain and
potato dextrose agar (PDA, 0.4 % potato starch, 2 % dextrose
mold preventive, and if there is any doubt, at least 10 mm of surface wood
plus 2 % agar), or similar commercial mixtures of MEA or
must be removed and discarded.
PDA prepared in accordance with manufacturer instructions
8.2 Size of Specimens—Specimens shall be 7 mm by 20 mm
(for example, culturing substrate and growth conditions as
in cross section and 70 mm long.
defined by the ATCC). PDA stimulates sporulation in some
sapstain fungi (for example, Aureobasidium pullulans). Steril-
8.3 Preparation of Specimens—Within two days of
ize the medium at 121 °C, 0.1 MPa, for 20 min.
collecting, the samples shall be cut from the wood using a
sharp saw blade. To prevent drying, the specimens shall be
11. Preparation of Inoculum
stored in polyethylene bags. For storage longer than one day,
but less than one year, tightly packed specimens shall be frozen
11.1 Maintain aseptic conditions when preparing individual
(–20 °C or lower) in polyethylene bags. For these longer
and mixed spore suspensions. For laboratory experiments
storage cases, the contents of one bag shall be limited to as
requiring a relatively small volume (about 100 mL) of
many specimens as are used for a single experiment.
inoculum, preparation using only the stock test tube cultures is
an option. For larger volumes of inoculum, prepare from
9. Test Fungi
cultures grown on Petri dishes. Use only freshly prepared
9.1 Hardwoods:
cultures to ensure vigor of the fungi.
9.1.1 Sapstain Fungi:
NOTE 3—Before using any stock test tube culture, reinoculate new tubes
9.1.1.1 Diplodia gossypina Cooke (Pole-Evans, anamorph)
to confirm growth and for future use.
(ATCC 34643).
11.2 For the preparation of a spore suspension, add 5 mL of
9.1.1.2 Davidsoniella virescens (Davison) de Beer et al., a
sterile water to each culture tube or 10 mL to Petri dishes, and
form of C. coerulescens found on American hardwoods.
gently rub the surface of the MEA or PDA culture with a blunt
9.1.1.3 Aureobasidium pullulans (de Bary) Arnaud (ATCC
glass rod to loosen the spores. Carefully aspirate the spore
16624).
suspension from plate using a sterile pipet, avoiding collection
9.1.2 Mold Fungi:
of mycelial fragments or agar.
11.3 For nonsporulating cultures, obtain a mycelial suspen-
The following numbers refer to standard strains of test fungi maintained in the
sion for use by aseptically scraping the surface mycelium off
American Type Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, www.atc-
c.org. and blending it with sterile water.
D4445 − 23
11.4 After collecting the spores, shake or gently vortex to polyethylene mesh spacer over saturated papers. Maintain
liberate spores and break up spore clumps. Filter spore suspen- aseptic conditions throughout.
sion through a sterile funnel containing a thin layer of glass
13. Treatment of Specimens
wool (see methods in Test Method D5590).
13.1 Specimens—If the wood samples were stored frozen,
11.5 Using a counting chamber, adjust the water volume
allow them to thaw in the polyethylene bags. Because of the
such that the spore suspension contains 0.8 to 1.2 × 10
variation in the susceptibility of wood to fungi, distribute an
spores/mL. For mixed spore suspensions, combine equal vol-
equal number of specimens from each log, into ea
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: D4445 − 10 (Reapproved 2019) D4445 − 23
Standard Test Method for
Fungicides for Controlling Sapstain and Mold on
Unseasoned Lumber (Laboratory Method)
This standard is issued under the fixed designation D4445; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This (laboratory) test method is used for determining the minimum concentration of fungicide, or formulation of fungicides,
that is effective in preventing biodeterioration by sapstain fungi and molds in selected species of wood under optimum laboratory
conditions.
NOTE 1—From the results of this test, commercial treating solution concentrations cannot be estimated without further field tests.
1.2 The requirements for test materials and procedures are discussed in the following order:
Section
Summary of Test Method 4
Apparatus 6
Reagents 7
Wood 8
Test Fungi 9
Culture Media 10
Preparation of Inoculum 11
Preparation of Test Chambers 12
Treatment of Samples 13
Inoculation and Incubation 14
Evaluation of the Test 15
Report 16
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of
regulatory limitations prior to use.
1.5 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
This test method is under the jurisdiction of ASTM Committee D07 on Wood and is the direct responsibility of Subcommittee D07.06 on Treatments for Wood Products.
Current edition approved July 1, 2019April 15, 2023. Published August 2019May 2023. Originally approved in 1984. Last previous edition approved in 20152019 as
D4445–10(2015).D4445 – 10 (2019). DOI: 10.1520/D4445-10R19.10.1520/D4445-23.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D4445 − 23
2. Referenced Documents
2.1 ASTM Standards:
D9 Terminology Relating to Wood and Wood-Based Products
D1165 Nomenclature of Commercial Hardwoods and Softwoods
D1193 Specification for Reagent Water
D5590 Test Method for Determining the Resistance of Paint Films and Related Coatings to Fungal Defacement by Accelerated
Four-Week Agar Plate Assay
3. Terminology
3.1 Definitions—For definitions of terms used in this test method, refer to Terminologies D9 and D1165.
4. Summary of Test Method
4.1 Unseasoned sapwood specimens are treated either by spraying with, or by immersing in, solutions or dispersions of a fungicide
formulation prepared at five or more concentration levels. The specimens are exposed to sapstain fungi and molds. Options for
testing the toxicity of fungicides include testing against individual fungi or against several fungi by using a mixed spore suspension
for the inoculation of the specimens.
4.2 The intensity of surface fungal growth is estimated after incubation and the results used to determine the minimum chemical
treatment concentration giving zero growth (CGo).
5. Significance and Use
5.1 This test method is useful as a screening procedure for selecting fungicides or formulations for more rigorous field evaluation.
6. Apparatus
6.1 Incubation Room (or Incubation Cabinet), maintained at a temperature of 25 6 1°C,25 °C 6 1 °C, and relative humidity
between 7070 % and 80 %.
6.2 Steam Sterilizer.
6.3 Containers:
6.3.1 Sterile Petri Dishes, with minimum size of 140140 mm (diameter) by 20 mm (height) with lid or,
6.3.2 Aluminum Pans, with minimum size of 240 by 100240 mm by 100 mm by 20 mm (height) with aluminum foil cover.
6.4 Spacers:
6.4.1 U-Shaped Glass Rod,or Straight Glass Rods, with 3 mm diameter or,
6.4.2 Polyethylene Mesh, cut to cover the bottom of the selected container(s).
7. Reagents
7.1 Purity of Water—Reference to water shall be understood to mean sterile reagent water conforming to Type IV of Specification
D1193.
8. Wood
8.1 General Properties—The wood species to be tested shall be selected on the basis of their susceptibility to stainingstaining/
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
D4445 − 23
mold fungi (pine or spruce species are preferred). preferred when evaluating softwoods). Sapwood of the selected wood species,
unseasoned (moisture content higher than 40 %), free of knots, visible decay, sapstain, and mold, shall be used (Note 2). If the
fungicide is to be used to protect hardwood, the inclusion of sapwood from a hardwood species susceptible hardwood species, such
as red oak or hickory, is recommended.
NOTE 2—If wood for the test is collected in a sawmill where logs are stored in water, it is necessary to collect lumber from at least three different logs
since depletion of nutrients during water storage may strongly affect the growth of molds and staining fungi. Ensure that the lumber collected in a sawmill
has not been treated with a sapstain and mold preventive, and if there is any doubt, at least 10 mm of surface wood must be removed and discarded.
8.2 Size of Specimens—Specimens shall be 77 mm by 20 mm in cross section and 70 mm long.
8.3 Preparation of Specimens—Within two days of collecting, the samples shall be cut from the wood using a sharp saw blade.
To prevent drying, the specimens shall be stored in polyethylene bags. For storage longer than one day, but less than one year,
tightly packed specimens shall be frozen (–20°C(–20 °C or lower) in polyethylene bags . bags. For these longer storage cases, the
contents of one bag shall be limited to as many specimens as are used for a single experiment.
9. Test Fungi
9.1 Hardwoods:
9.1.1 Sapstain Fungi:
9.1.1.1 Diplodia natalensisgossypina P. Evans Cooke (Pole-Evans, anamorph) (ATCC 34643).
9.1.1.2 Davidsoniella virescens (Davison) de Beer et al,al., a form of C. coerulescens found on American hardwoods.
9.1.1.3 Aureobasidium pullulans (d. By) Arnaud.(de Bary) Arnaud (ATCC 16624).
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 roseumClonostachys rosea (Link) Bainier (Link:Fries) (ATCC 10521).
9.2 Softwoods:
9.2.1 Sapstain Fungi:
9.2.1.1 Diplodia natalensisgossypina P. Evans Cooke (Pole-Evans, anamorph) (ATCC 34643).
9.2.1.2 Ceratocystis pilifera (Fr.) C. Moreau (ATCC 15457).
9.2.1.3 Aureobasidium pullulans (d By)(de Bary) Arnaud (ATCC 16624).
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 roseumClonostachys rosea (Link) Bainier (Link:Fries) (ATCC 10521).
9.3 General Consideration—In addition to the above fungi, others that are known to cause discoloration on wood species used
The following numbers refer to standard strains of test fungi maintained in the American Type Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, www.atcc.org.
D4445 − 23
in test include, for example, growth/discoloration on wood which may also be used for testing include, CytosporaAlternaria sp.
(Pine); sp.; PhialophoraAspergillus brasiliensis sp.; Varga et al. (ATCC 9642); GraphiumAspergillus niger van Tieghem (ATCC
6275); Aspergillis sp.; Ceratocystis sp.; AlternariaCytospora sp. (Pine); Graphium sp.; Penicillium citrinum Thom (ATCC 9849);
Penicillium sp.; AspergillisPhialophora sp.; Trichoderma virens (Miller et al.) von Arx, anamorph (ATCC 9645); Trichoderma sp.
10. Culture Media
10.1 Agar Substrate—For both stock culture tube and petriPetri dish 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 % dextrose plus 2 % agar),
or similar commercial mixtures of MEA or PDA prepared in accordance with manufacturer instructions. instructions (for example,
culturing substrate and growth conditions as defined by the ATCC). PDA stimulates sporulation in some sapstain fungi (for
example, Aureobasidium pullulans). Sterilize the medium at 121°C,121 °C, 0.1 MPa, for 20 min.
11. Preparation forof Inoculum
11.1 If the toxicity of a fungicide is being tested against individual fungi, maintain Maintain aseptic conditions when preparing
the spore suspension; if the general effectiveness of a fungicide is being tested using a mixed spore supension, aseptic conditions
are unnecessary. individual and mixed spore suspensions. For laboratory experiments requiring a relatively small volume (about
100 mL) 100 mL) of inoculum, preparation using only the stock test tube cultures is an option. For larger volumes of inoculum,
prepare from cultures grown on petri dishes.Petri dishes. Use only freshly prepared cultures to ensure vigor of the fungi.
NOTE 3—Before using any stock test tube culture, reinoculate new tubes to confirm growth and for future use.
11.2 For the preparation of a spore suspension, add 5 mL of sterile water to each culture tube or 10 mL to petriPetri dishes, and
gently rub the surface of the MEA or PDA culture with a blunt glass rod to loosen the spores. After collecting the spores and
combining them with other similarly collected spores, if desired, adjust the water volume to that required. Although it is a good
practice to prepare fresh spore suspensions just before use, their storage for up to one week with refrigeration is permissible-
.Carefully aspirate the spore suspension from plate using a sterile pipet, avoiding collection of mycelial fragments or agar.
11.3 For nonsporulating cultures, obtain a mycelial suspension for use by aseptically scraping the surface mycelium off and
blending it with sterile water.
11.4 After collecting the spores, shake or gently vortex to liberate spores and break up spore clumps. Filter spore suspension
through a sterile funnel containing a thin layer of glass wool (see methods in Test Method D5590).
11.5 Using a counting chamber, adjust the water volume such that the spore suspension contains 0.8 to 1.2 × 10 spores/mL. For
mixed spore suspensions, combine equal volumes such that the final mixture contains equivalent spore concentrations from each
of the test fungi selected.
NOTE 4—Certain test fungi may not be ame
...