ABILITY of SELECTED BACTERIAL and FUNGAL BIOPROTECTANTS to LIMIT FUNGAL STAIN in PONDEROSA PINE SAPWOOD1 Bernhard Kreber Graduate Research Assistant

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ABILITY OF SELECTED BACTERIAL AND FUNGAL BIOPROTECTANTS TO LIMIT FUNGAL STAIN IN PONDEROSA PINE SAPWOOD1 Bernhard Kreber Graduate Research Assistant

and J. J. Morrell Assistant Professor Department of Forest Products Oregon State University Corvallis, OR 9733 1 (Received April 1992)

ABSTRACT The ability of 15 bacterial and fungal isolates to inhibit fungal stain of ponderosa pine sapwood was studied on small wood samples exposed in a moist environment. Several isolates including Bacillus subtilis were capable of inhibiting fungal stain although the protective effect was lost upon prolonged exposure. More detailed evaluations of two B. subtilis isolates revealed that performance was closely tied to the media on which the organisms were grown prior to wood application, with the optimal growth media appearing also to be associated with the highest degree of stain protection. Further evaluation of B. subtilis 733 A for evidence of production of water-soluble or volatile antibiotics suggested that antibiotics were not present on the wood, although further studies on bioprotectant mechanisms are suggested. Keywords: Bioprotection, Bacillus subtilus, fungal stain, antibiotics.

INTRODUCTION ability to prevent fungal degradation of wood Traditionally, chemical treatments have been products (Ricard 1966; Ricard and Bollen used to protect unseasoned lumber from bio- 1968; Bruce and King 1983, 1986a, b; Bruce logical discoloration during storage and trans- et al. 1990, 199 1; Bruce and Highley 199 1) or portation. However, potentially hazardous to eliminate incipient decay (Ricard et al. 1969; health and environmental effects have brought Morris and Dickinson 198 1; Highley 1989); the use of some antistain chemicals into ques- and has received much less attention as a pro- tion and indicate a need for new approaches tectant against wood discoloration. However, to biological stain prevention. several studies on the use of bacteria and fungi Biological protection or bioprotection is one (Benko 1986, 1988; Seifert et al. 1988) and alternative to chemical treatments. Bioprotec- other antimicrobial agents produced in a bio- tion uses one organism to inhibit the growth protectant culture fluid (Benko 1989; Croan or damaging effects of another. To date, bio- and Highley 199 la, b; Highley et al. 199 1) to protection has been studied primarily for its protect unseasoned lumber from biological discoloration have recently been published. In this study, 15 bacterial and fungal isolates I This is Paper 2819 of the Forest Research Laboratory, Oregon State University, Corvallis. This research was sup- were screened for their ability to prevent mi- ported by the USDA Center for Wood Utilization Re- crobial discoloration on unseasoned ponder- search. osa pine (Pinus ponderosa Laws.) sapwood. In

Wood and Ffher Scrence, 25(1), 1993. pp. 23-34 O 1993 by the Society of Wood Sc~enceand Technology 24 WOOD AND FIBER SCIENCE, JANUARY 1993, V. 25(1) addition, one ofthese microorganisms, B. sub- Preparation of fungal stain suspensions. - tilis, was further examined for the effects of The mold and stain fungi listed in Table 2 were media composition on its bioprotective ca- added to 250-ml Erlenmeyer flasks containing pacities, its antibiotic production, and its effect 50 ml sterile (121 C, 20 min) 1°/o malt extract on spore germination. media and incubated at room temperature (23 MATERIALS AND METHODS C) on a rotary shaker at 80 RPM until an abundant dark pigmented mycelial biomass devel- Initial screening trials oped. The contents of each flask were then The bioprotectants evaluated in the initial vacuum filtered (1 bar) through Whatman #4 screening trials are listed in Table 1, and the filter paper with a Buchner funnel, and the mold and stain fungi used for testing (selected collected spores and hyphal fragments were from the Forest Research Laboratory culture rinsed with distilled water off the filter paper collection, Oregon State University, Corvallis) and into a single 500 ml beaker. The combined are recorded in Table 2. The bioprotectant cul- biomass in the beaker was then blended for tures were maintained on agar slant tubes with about 10 seconds, and enough distilled water the appropriate growth media (Table I), and was added to the beaker to obtain 1 liter of the mold and stain fungi were maintained on suspension. This suspension was then drained malt agar (MA) (1.5% malt extract, 1% agar) into a squeeze bottle and kept at room tem- plates. All cultures were stored in a cold room perature until needed. Additional fungal bio- (0 C) until needed. Chemicals used for media mass, prepared as described above, was added composition were purchased form Difco Lab- to the fungal stain suspension at approximately oratories (Detroit, MI) or Sigma Chemical 2-week intervals to maintain fungal viability. Company (St. Louis, MO). Bioprotectant testing. -For each bioprotec- Preparation of bioprotectant suspensions. - tant tested, 20 steam sterilized (100 C, 15 min) Bacterial bioprotectant suspensions were pre- ponderosa pine specimens (0.5 x 1.0 x 3.0 pared by transferring the bacterial inocula into cm) were placed in a 400-ml aluminum con- 250-ml Erlenmeyer flasks containing 50 ml tainer, covered with weighted wire to keep them sterile (1 2 1 C) liquid growth media of the ap- submerged during treatment, and flooded for propriate type (Table 1) and incubating the about 8 minutes with 250 ml of the biopro- flask contents at room temperature (23 C) on tectant suspension. While all replicates were a rotary shaker (manufactured by Lab Line prepared at the same time, inoculum density Instr. Inc., Melrose Park, IL) at 80 RPM for was not quantified. An additional set of pon- approximately 65 hours. The resulting cultures derosa pine specimens, used as the control, was were aseptically harvested by draining the flask flooded with 250 ml distilled water for the same contents into 400-ml aluminum trays and add- time period. The containers were then drained ing 200 ml sterile (121 C, 45 min) distilled and the specimens were removed, allowed to water to produce 250-ml suspensions. To pre- surface dry for approximately 2-3 minutes, and pare the fungal bioprotectant suspensions, the sprayed on both wide faces to the point of test fungi were incubated at 27 C on MA plates runoff with the prepared fungal stain suspen- (or, for S. cerevisiae, at room temperature on sion. The specimens were then incubated em- malt extract in a flask) until abundant sporu- ploying a method described by Seifert et al. lation developed. Each plate was then flooded (1988). For each bioprotectant, five replicate with 4 ml distilled water, and the spores and tests were conducted. For each test, four wood hyphal fragments that rose to the water's sur- specimens treated with both bioprotectant and face were harvested and placed in 400-ml alu- stain fungi, and one control specimen treated minum trays, and enough distilled water was with stain fungi only, were placed on glass rods added to each tray to produce 250-ml suspen- in a sterile glass petri dish containing three sions. filter paper discs, and 5 ml distilled water was Krt,ber and MorreN-BIOPROTECTION AGAINST FUNGAL STAIN 2 5

TABLE1. Bioprotectants evaluated on ponderosa pine.

Strain number Source Growth medium Bacteria Forintek Canada Corp., Ottawa, Nutrient broth (NB) Bacillus subtilis Cohn 733 A Canada (0.3%) Bacillus subtilis Cohn Quantum 4000 HB J. Loper, USDA Agric. Re- NB search Service (ARS), Corval- lis, OR Pseudomonas cepacia T. Highley, Forest Products Kings B Media (KB) (2% Burkholder Laboratory, Madison, WI proteose peptone, 1Yo glycerol, 0.15% potassi- um phosphate monoba- sic, 0.1 5% magnesium sulfate) Pseudomonas jluorescens Pf-5 (#3832) J. Loper, ARS, Corvallis, OR KB Migula Pseudomonas putida A-12 J. Loper, ARS, Corvallis, OR KB Migula Pseudomonas putida N-1R J. Loper, ARS, Corvallis, OR KB Migula Streptomyces tendae 695A Forintek, Ottawa, Canada Potato-Dextrose-Broth (PDB) Streptomyces sp. C 248 Forintek, Ottawa, Canada PDB Streptomyces sp. C 254 Forintek, Ottawa, Canada PDB Fungi Gliocladium aurifilurn 675 A Forintek, Ottawa, Canada Malt agar (MA) (1.5% malt extract, 1% agar) Gliocladium virens H 3 J. Loper, ARS, Corvallis, OR MA J. H. Miller, J. E. Giddens, & A. A Foster Penicillurn thomii Maire 655 A Forintek, Ottawa, Canada MA Saccharomyces cerevisiae 591 A Fonntek, Ottawa, Canada MA Meyen ex Hansen Trichoderma harzianum Forest Research Lab (FRL), Or- MA Rifai egon State University, Cor- vallis Trichoderma harzianum FRL, Oregon State University, MA Rifai Corvallis

added. For additional controls, specimens samples were examined weekly and, after 4 treated with stain fungi or bioprotectant only weeks, were evaluated with methods described were placed in separate petri dishes. in the next section. The dishes were coded independently to Evaluation of bioprotectants. -At the end of minimize evaluation bias and were then in- the 4-week incubation period, average degree cubated in the dark at 27 C for 4 weeks; deion- of surface stain (ADS) was determined, and ized water was added as necessary to maintain percentage of fungal stain inhibition (FSI) was a moist environment. The incubated wood calculated for each of the bioprotectants tested. 26 WOOD AND FIBER SCIENCE, JANUARY 1993, V. 25(1)

TABLE2. Mold and stain fungi used to evaluate biopro- 3 = heavily stained tectant capabilities on ponderosa pine sapwood. (more than 50% of the wood surface Strain stained) Species number Alternaria alternata (FR : FR) Keissl. ED 13 Percentage of FSI was calculated with the following equation: Aspergillus niger van Tieghea BOB

Aureobasidium pullulans [de Bary] [1 - (ADS/ADSCO~~IO,)Ix 100 (1) G. Amauda Zm 3 ADS and ADS,,,,, are the average degree of Bispora betulina (Corda) S. J. Hughes 1 stain on treated and control specimens, re- (leratocystis pillfera (Fries) C. Moreaub 55 A spectively. Clladosporum elatum (C. Harz) Nannf. 563-1- 1 Efects of growth media on Hormoconis resinae (Lindau) Arx & G. A. De Vries P 1600 bioprotectant performance ~enicil~iurnfrequentans WestIinga 43 Bacillus subtilis #733 A and B. subtilis ATCC Phialophora fastigata (Lagerberg & Melin) 6633 were selected to quantify the effects of Conant 14 different growth media on bioprotectant per- Rhinocladiella atrovirens Nannf. in Melin formance against stain fungi. Most of these & Nannf. 68 growth media had been used in prior studies, Ulocladium chartarum (G. Preuss) E. Sim- for example, for antibiotic production (Kugler mon 100 et al. 1990). The two bioprotectants were cul- 'Not tncluded tn suspension prepared for antibiotic production test. tured in l -5 liters each test medium ', Not included in fungal stain suspension used for inltial screening trials. 3) for 0.30/o nutrient broth; for this medium, the bioprotectants were cultured in six 250-ml Erlenmeyer flasks containing 50 ml The following modified grading system pro- sterile nutrient broth, and the resulting 300 ml 988) was used posed by BenkO of culture were added to 1.2 liters distilled wa- quantify ADS: ter to produce 1.5 liters of fluid. 0 = unstained wood Treatment of wood with bioprotectant culture (no visible signs of stain) jluids and fungal stain suspension. -The same 1 = incipient establishment of microbial wood-treatment procedures were used for this growth experiment as for the initial screening trials, (evidence of microbial growth on sur- except that wood specimens for this test mea- face) sured 0.5 x 1.5 x 3.0 cm, an 8-week incu- 2 = moderate stain bation period was used, and one half of the (at least 25% of the wood surface stained) treated specimens were resprayed with the fun-

TABLE3. Culture conditions of growth media used to evaluate the efect of dzfferent media on bioprotectant performance against wood-staining fungi.

Culture conditions Medium Time (hr) Temp ("C) RPM Reference Nutrient broth (NB) (0.3 or 1.5%) 60 2 5 80 Claus and Fritz 1989 PA media 18 3 7 225 Ozcengiz et al. 1990 Penassay broth (PB) 48 30 250 Bernier et al. 1986 PL media 48 25 120 Kugler et al. 1990 Tryptic soy-broth (TSB) 72 37 250 Anker et al. 1948 Yeast-dextrose-carbonate (YDC) 7 2 25 80 Wilson et al. 1967 Yeast-malt-dextrose broth (YMBD) 25 25 80 Seifert et al. 1987 Kreber and Morrell-BIOPROTECTION AGAINST FUNGAL STAIN 2 7 gal stain suspension at 2-week intervals to sim- shaker at 120 RPM for 10 hours at room tem- ulate repeated influx of staining organisms. perature. The resulting extracts in the two flasks After 8 weeks, the effect of different media were combined and filtered through a sequence on the two bioprotectants' performance was of 0.8-, 0.45, and 0.22-p membranes. The fil- assessed. Average degree of surface stain (ADS) trate was then concentrated in an Amicon and percentage of fungal stain inhibition (FSI) Stirred Ultra-Filtration Cell with Amicon Di- were determined as for the initial screening aflo YM 3 and YM 10 membranes (all man- trials. Wood specimens were then split longi- ufactured by Amicon Inc., Danvers, MA), to tudinally, and the average degree of internal produce concentrated extracts with molecular stain (ADIS) was determined based on the fol- weight cutoffs of <3,000, 3,000-10,000, and lowing grading system: > 10,000. The concentrated extracts were then stored at 4 C until needed. unstained wood (no visible signs of Antibiotic production in each extract was internal stain) tested by pipetting a small amount (0.05 ml) small (1- or 2-mm diameter) stained of the extract into a well (0.4 cm in diameter, spots visible near the end grain 0.3 cm deep) cut into the center of an MA petri thin (0.5 mm) stained shell around no dish. The petri dishes were then abundantly more than 25% of the circumference seeded with a fungal stain suspension prepared stained shell (0.5 mm) around no more as for the initial screening trials but containing than 50% of the circumference a slightly different combination of fungi (see stained shell (0.5 mm) around more Table 2). The seeded MA dishes were incu- than 50% of the circumference bated for 7 days in the dark at 26 C and then stained shell (0.5 mm) around the en- were examined for evidence of zones of inhi- tire circumference bition around the well. All tests were per- more than 50% of the internal wood formed in two replicates. In addition, as con- surface area stained trols for each test, one MA dish received only Percentage of internal fungal stain inhibition the 0.05 ml of concentrated extract and an- (IFSI) was calculated as for FSI [Eq. (I)] but other was seeded with the fungal stain suspen- using the ADIS values. sion only. Antibiotic production Bioprotectant efect on spore germination Antibiotic production represents one poten- Inhibition of spore germination is critical for tial bioprotection mechanism and was inves- successful protection against wood-staining tigated for B. subtilis #733 A with the following fungi. This test compared fungal spore ger- procedure. Ponderosa pine specimens treated mination in the presence and absence of Ba- for 8 minutes with the 0.3 NB B. subtilis #733 cillus subtilis #733 A. Steam sterilized pon- A suspension and sprayed with the fungal stain derosa pine specimens (0.45 x 1.4 x 1.5 cm) suspension, and control specimens treated only were dipped into 250-ml Bacillus subtilis #733 with the bioprotectant, fungal stain suspen- A suspension (prepared as described for the sion, or 300 ml distilled water, were incubated initial screening trials) or, for the controls, into at 27 C as for the initial screening trials. 250 ml sterile distilled water, for approxi- After 1, 2, 3, or 4 weeks of incubation, a mately 8 minutes. Samples were then allowed series of concentrated extracts was prepared to surface dry for 2-3 minutes prior to being from the treated and control specimens as fol- sprayed to runoff with A. niger spores that had lows. Ten specimens were removed from the been cultured on PDA and flooded from the incubator, placed in two 250-ml Erlenmeyer mycelial mat with 0.001% Tween 80. The flasks, each containing 50 ml 0.001% Tween treated wood specimens were then placed in 80 (Manooleate), and incubated on a rotary an 8.2- x 3.0- x I .O-cm aluminum incubation 28 WOOD AND FIBER SCIENCE, JANUARY 1993, V. 25(1) chamber with a 6.6- x 1.4- x 0.5-cm molded depression and covered with a glass slide. The (a) incubation environment was sealed with min- 2- uBsubhb733A eral oil, which allowed oxygen to diffuse into g control the chamber but prevented moisture loss from < 1 - the chamber and restricted entry of competing microbes.

The specimens were incubated at room tem- O 1 2 3 4 perature for 6,8, 10, 12,24, or 36 hours. Then, a 20-p-thick section was cut from each specimen with a Spencer microtome (manufactured by Spencer Lens Co., Buffalo, NY), stained with 1°/o safranin 0 followed by steaming pico- aniline blue according to the procedure described by Wilcox (1964), and mounted in water. A Leitz microscope at 10 x magnification was then used to examine each section for spore germination as evidenced by production of a Time (weeks) 2- to 3-p-long germ tube. Percentage of spore FIG. 1. Bioprotection associated with application of B. germination per square centimeter of wood subtilis #733 A as (a) average degree of surface stain (ADS) surface area was calculated with the following and (b) percentage of fungal stain inhibition (FSI). ADS for sprayed specimens at 32 C was 0. equation:

wood, possibly because of incomplete sub- where: strate colonization (Seifert et al. 1987). Competition for nutrients and space, and/ A = mean of three replicate conidia germi- or antifungal compounds produced in B. sub- nation counts at a selected time tilis culture fluids, have been suggested as B = potential germination based on mean probable modes of action of B. subtilis isolates number of colony-forming units pro- against wood-staining microorganisms (Ber- duced on PDA nier et al. 1986; Florence and Sharma 1990). C = surface area of ponderosa pine speci- However, these hypotheses were drawn either mens from culture analysis or from short-term (2-

RESULTS AND DISCUSSION week) stain tests on rubbenvood, and so their relevance to longer protective periods is ques- Initial screening trials tionable. To accurately assess the microbial The most effective bioprotectant was Bacil- activity of bioprotectants in regard to wood- lus subtilis isolate 733 A, which completely staining microorganisms, the activity should prevented fungal discoloration on ponderosa be evaluated exclusively on wood substrates pine sapwood over a 4-week period (Fig. la), and for longer time periods. producing 89% fungal stain inhibition in com- Although Gliocladium virens #H-3 reduced parison with the control (Table 4). However, fungal stain by 62.96% in comparison with the after an additional 4 weeks, B. subtilis #733 A control, it also produced an extensive greenish showed a gradual decrease in antagonistic ac- aerial mycelium and spores on the wood sur- tivity against mold and stain fungi (Fig. lb). faces that were detrimental aesthetically. Sei- This result suggests that B. subtilis #733 A de- fert et al. (1988) reported similar effects with layed but could not completely prevent fungal G. roseum when evaluating it as a potential invasion of unseasoned ponderosa pine sap- bioprotectant. Unless G. virens is found to pro- Kreber and MorreN-BIOPROTECTION AGAINST FUNGAL STAIN 29

TABLE4. Average degree of surface stain (ADS) and percentage of fungal stain inhibition (FSI) for bioprotectants 4 weeks after wood treatment.

Species ADS of treated specimens ADS of control specimens FSIa (%) B. subtilis 733 A 0.30b (0.46p B. subtilis Q 4000 HB 2.60 (0.74) P. cepacia 2.70 (0.64) P. jluorescens Pf-5 3.00 (0.00) P. putida A- 12 1.70 (0.84) P. putida N- 1R 2.70 (0.64) Strept. tendae 695 A 2.30 (0.95) Strept. sp. C 248 3.00 (0.00) Strept. sp. C 254 2.75 (0.62) G. uurifilium 675 A 2.75 (0.62) G. virens H 3 1.00 (0.77) P. thomii 655 A 1.80 (0.92) S. cerevisiue 59 1 A 2.15 (0.85) T. harzianum 25 2.45 (0.86) T. harzianum 142 2.70 (0.64)

., % FSI = [I - (ADS/ADS,,,,,,,,, )I x 100. %ADS values represent mean of 20 replicates. Values in parentheses represent standard deviations. " ADS,,,.,,, values represent mean of 10 replicates. ' Negat~veFSI values denote increased staining compared to controls. duce antifungal compounds that are particu- the yellow stain. Previous studies have re- larly effective against the mold and stain fungi ported Trichoderma spp. production of vola- prevalent on unseasoned sapwood, this species tile and nonvolatile antibiotics that inhibit se- is probably not a viable bioprotectant against lected decay fungi (Dennis and Webster 197 1a, fungal stain. b; Bruce et al. 1984). With a 34.62% reduction in fungal stain The failure of the remaining tested biopro- compared to the control, Pseudomonas putida tectants to prevent microbial stain on pon- #A- 12 also exhibited some potential for bio- derosa pine specimens may reflect inappro- protection of unseasoned lumber, probably by priate growth media, adverse environmental competing with stain fungi for nutrients and conditions, and/or the inability of these species space or by antibiosis. Fluorescent Pseudo- to compete with microbial competitors once monas spp. are able to control pythopathogens they became established on the wood sub- through both antibiosis (Thomashow et al. strate. Klingstrom and Johanssen (1973) noted 1990) and iron competition (Loper 1988), and, that different microbial strains within the same with the latter mechanism, to reduce chla- species can produce markedly different results. mydospore germination in the rhizosphere of This observation was borne out in our study plants (Elad and Baker 1985). by the different results for B. subtilis isolates Ponderosa pine specimens treated with 733 A and Q 4000 HB. Trichoderma harzianum #25 contained a yellow stain and conspicuous green spores start- Efect of growth media on ing at 2 weeks after treatment, a point at which bioprotectant performance most control specimens had already exhibited Incubation ofthe two B. subtilis isolates (733 progressive stain patterns. Although the yellow A and ATCC 6633) on eight types of media stain was aesthetically detrimental, mold and prior to wood application resulted in drarnat- stain fungi growth was not observed on the ically different degrees of stain prevention. wood, suggesting the presence of antimicrobial Generally, the best results were obtained when secondary metabolites in or associated with the isolates were incubated on their preferred 30 WOOD AND FIBER SCIENCE, JANUARY 1993, V. 25(1)

TABLE5. Percentages of surface and internal fungal stain inhibition (FSI and IFSIP achieved with B. subtilis #733 A after 8 weeks of incubation on various media.

Temperature (OC) 32 26 20 Medium Resprayed" Not resprayed' Resprayed Not resprayed Resprayed Not resprayed 1.5 NB 11.66 (68.18) 0.3 NB 55.76 (86.84) PB 1.66 (N.D.) YMDB 0.00 (9.37) YDC 0.00 (3.92) TSB 0.00 (-40.0) PA 0.00 (20.60) PL 0.00 (-11.11)

' FSI and IFSI values calculated as [I -(ADS/ADS,,.,.,,)] x LOO. IFSI values are in parentheses. Negative values denote increased staining compared to Controls. "After initial treatment, specimens were resprayed at 2, 4, and 6 weeks with fungal stain suspension. Specimens were sprayed with fungal stain suspension during initial treatment only. Wot determined because treated and control specimens exhibited no signs of internal stain.

medium (0.3 NB for B. subtilis #733 A, and gradual loss of antibiotic from the wood sur- Penassay broth for B. subtilis ATCC 6633) face, which enabled the resprayed spores to (Tables 5 and 6). Between the two isolates germinate more easily. grown on their preferred media, the combined Most of the media evaluated had no signif- FSI and IFSI data showed B. subtilis #733 A icant effect on bioprotectant results, perhaps to be the superior bioprotectant of unseasoned due to inappropriate culture conditions. Benko ponderosa pine sapwood. and Highley (1990a, b) reported that media As well as growth media, the incubation composition significantly affects the activity of temperature of the treated wood specimens and fungitoxic bacterial compounds used for wood the number of times the specimens were treatment. However, their studies tested these sprayed with fungal stain suspension also af- compounds against only two wood-staining fected the performance of B. subtilis #733 A fungi, Ceratocystis coerulescens (Miinch) Bak- (Fig. 2). Favorable incubation temperatures (26 shi and Trichoderma harzianum Rifai. These and 32 C) may have enabled the bioprotectant two fungi might behave differently in com- rapidly to exploit readily available nutrients, petition with other sapwood-staining fungi such thereby excluding microbial competitors. An- as those used in the current study. other possibility is that any temperature-sen- sitive antifungal compounds produced in the Antibiotic production culture broth of the biological agent may have None of the extracts prepared from the treat- remained active for a longer time period at ed wood specimens inhibited growth of mold these temperatures. The consistent decrease in and stain fun@ in comparison to the controls. protection with respraying may be due to a Either antibiotics were not produced, an in- Kreber and Morrell-BIOPROTECTION AGAINST FUNGAL STAIN 3 1

TABLE6. Percentages of surface and internal fungal stain inhibition (FSZ and ZFSZ). achieved with B. subtilis ATCC 6633 after 8 weeks of incubation on various media.

Temperature (T) - - 32 26 20 Medium Resprayedb Not resprayed' Resprayed Not resprayed Resprayed Not resprayed 1.5 NB 5.00 0.00 6.66 0.00 5.00 0.00 (70.73) (20.00) (16.84) (58.06) (59.58) (14.76) 0.3 NB

YMDB

YDC

TSB

" FSI and IFSl values calculated as [(I - (ADS/ADS,,.,,,,)] x 100. IFSI are in parentheses. Negative values denote increased staining compared to controls. "After initial treatment, specimens were resprayed at 2, 4, and 6 weeks with fungal slain suspension. ' Specimens were sprayed with fungal stain suspension during initial treatment only. Not determined because treated and control specimens exhibited no signs of internal stain.

appropriate extraction procedure deactivated them (for example, through pH changes or presence of proteolytic enzymes), or the sol- vent used for the extraction was unable to re- lease the antibiotics from the wood substrate (Dawson-Andoh and Morrell 199 1). Our results suggest that competition for nutrients and space are the dominant modes of action in this bioprotectant system although assaying for specific antifungal compounds known to be produced by particular isolates of B. subtilis (Kugler et al. 1990) or refining the extraction procedure may have produced more precise results. Bioprotectant eflect on spore germination In our test conditions, B. subtilis #733 A did not inhibit A. niger spore germination (Table 7). This result is consistent with that of Seifert Temperature ("c)

et 987)7 found' as we did' that FIG. 2. ERects of optimal media on the ability ofbio- the biOprOtectant ''ionized the protectants to protect ponderosa pine from microbial stain: (a) B. subtilis #733 A with 0.3 NB: (b) B. subtilis ATCC wood substrate in clusters. In some instances, . . ,., however, close spatial relationships such as 6633 with PB. 3 2 WOOD AND FIBER SCIEN(3E, JANUARY 1993, V. 25(1)

TABLE7. 17ffect of B. subtilis #733 A on germination of protectant is incapable of completely protect- A. niger conidiospore germination on ponderosa pine sap- ing ponderosa pine sapwood. However, spatial wood at selected times. relationships between biological agents and Spore germmation on wood specimens (%p other more important wood staining fungi need Tlme" Control B. subtilrs-treated more detailed investigation over longer time periods than were used in this study. Our results indicate that bioprotection against mold and stain fungi is a viable pros- pect. However, considerable basic research in ,' As a percent of A. niger spores initially applied. ', Hours after spore application. After 24 hours of incubation spore gener- areas such as optimizing growth of potential ation was too dense to be quantified. bioprotectants and determining the effect of environmental factors (temperature, wood those described by Campbell (1989) between moisture content) on bioprotection still needs B. subtilis cells and swollen conidiospores were to be done. Furthermore, a better understand- noted. In these cases, competition for nutrients ing of microbial interactions on wood surfaces may have been occurring, but the destructive is essential before large-scale use of bioprotec- sampling procedure we used prevented us from tion is attempted. verifying this possibility. REFERENCES Although A. niger may not be representative of all mold and stain fungi, its rapid growth ANKER,S. H., A. J. BALBINA,J. GOLDBERG,AND F. L. rate is typical of these organisms and highlights MELENEY.1948. Bacitracin: Methods of production, concentration, and partial purification, with a summary the need for bioprotectants to be highly com- of the chemical properties of crude bacitracin. J. Bac- petitive for both nutrients and space. Modi- teriol. 55:249-255. fication of the wood substrate in favor of the BENKO,R. 1986. Protection of wood against the blue bioprotectant, to promote uniform substrate stain. Ref. no. 18. International Congress of IUFRO, colonization by the bioprotectant, may also be Ljubljana, Yugoslavia. . 1988. Bacteria as possible organisms for biolog- necessary (Dawson and Morrell 1990; Morrell ical control of blue-stain. IRG/WP/I 339. International 1990). Research Group on Wood Preservation, Stockholm, Sweden. CONCLUSIONS . 1989. Bacterial control of blue-stain on wood with Pseudomonas cepacia 6253. Laboratory and field Of the bioprotectants we tested, Bacillus test. IRG/WP/1380. International Research Group on subtilis #733 A provided superior protection Wood Preservation, Stockholm, Sweden. against wood-staining microorganisms on , AND T. L. HIGHLEY. 1990a. Selection of media ponderosa pine sapwood, but only on opti- on screening interaction of wood attacking fungi and mized media at higher temperatures. Strategies antagonistic bacteria. 1. Interaction on agar. Mater. Org. 25:161-171. such as nutritional selection (Benko and High- , AND -. 1990b. Selection of media on ley 1990a, b) or modification of the wood sub- screening interaction of wood attacking fungi and an- strate in favor of B. subtilis (Dawson-Andoh tagonistic bacteria. 2. Interaction on wood. Mater. Org. and Morrell 1990; Morrell 1990) may further 25: 173-1 80. improve the organism's bioprotective capa- BERNIER,R., J. M. DESROCHERS,AND L. JURASEK.1986. Antagonistic effect between Bacillus subtilis and wood bilities. staining fungi. J. Inst. Wood Sci. 10:214-216. Although we found no evidence of antibiotic BRUCE,A., W. J. AUSTIN,AND B. KING. 1984. Control production with B. subtilis, it is possible that of growth of Lentinus lepideus by volatiles from Trich- these compounds either were produced in very oderma. Trans. Br. Mycol. Soc. 82:423428. small quantities or were volatile, and thus were , A. FAIRNINGTON,AND B. KING. 1990. Biological control of decay in creosote treated distribution poles. not detectable with our test methods. 3. Control of decay in poles by immunizing commensal The fact that B. subtilis #733 A did not in- fungi after extended incubation period. Mater. Org. 25: hibit spore germination suggests that this bio- 15-28. Kreber and Morrell-BIOPROTECTION AGAINST FUNGAL STAIN 33

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