IRG/WP 01-10407

THE INTERNATIONAL RESEARCH GROUP ON WOOD PRESERVATION

Section 1 Biology

Wood Extractive Concentration and SEM Examination of Pretreated Southern Yellow Pine Wood Chips with Blue-Stain Fungi for Mushroom

Production

Suki C. Croan and John Haight

U.S. Department ofAgriculture, Forest Service Forest Products LaboratoryOne Gifford Pinchot Drive

Madison, Wisconsin, USA 53705-2398

Paper prepared for the 32nd Annual Meeting Nara, Japn

May 20-25th, 2001

IRG SECRETARIAT SE-100 44 Stockholm

Sweden

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Wood Extractive Concentration and SEM Examination of Pretreated

Southern Yellow Pine Wood Chips with Blue-Stain Fungi for Mushroom Production

Suki C. Croan and John Haight

Mushroom-producing white-rotting basidiomycetes either do not colonize or else

colonize very poorly on freshly prepared southern yellow pine wood chips. This study

evaluates the resinous extractive content of southern yellow pine before and after

treatment with colorless mutant blue-stain fungi. The blue-stain fungi penetrate into the

sapwood of southern yellow pine and utilize nonstructural resinous extractives,

simultaneously reducing the total resinous extractive content. Scanning electron

microscopic examination showed that heavy mycelial growth with good sporulation

occurred on the surface of wood chips within 2 days and in parenchyma cells within 6

days. Ophiostoma spp. removed 61.1% to 99.9% of the extractives from the southern

yellow pine wood within a period of 4 to 5 days. We conclude that white-rot

basidiomycetes can easily colonize and produce fruiting bodies on treated southern

yellow pine wood wastes.

Keywords: Fruiting bodies, blue stain fungi, Cartapip™ 97 (colorless mutant isolate,

Ophisostoma piliferum), conidia, Coremia, synnemata, white-rot, mushroom-producing

basidiomycete, wood decay, southern yellow pine, wood extractives.

INTRODUCTION

Four of the eleven species of southern yellow pine [loblolly pine (P. taedua), short leaf

pine (P. echinata), long leafpine (Pinus palustris), and slash pine (P. elliottii) ] account

for about 93% of all standing timber. These trees generally grow in the southeast and

south central United States (Hoadley, 1990). Southern yellow pine usually contains of a

high concentration of wood extractives, ranging from 3% to 9% (Koch, 1972) and 1% to

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5% (Zabel and Morrell, 1992) of the total dry weight of wood. In exceptional cases, the

extractives may represent up to 40%, depending upon the season of the year when they

were harvested (Zabel and Morrell, 1992). These extractives can be divided into three

subgroups: terpenes and terpenoid, aliphatic compounds (mainly fats and waxes), and

phenolic compounds (Sjostrom, 1981, pp. 87) : these are primarily low molecular weight

compounds that are soluble in neutral, nonpolar, organic solvents and cold water (Gao

and Breuil, 1995; Zabel and Morrell, 1992). They are usually found in resin canals,

epithelial cells, and ray parenchyma cells (Sjostrom, 198 1, pp. 83-103).

Some extractives are toxic to bacteria, fungi, and termites, but other extractives give color

and odor to wood. The mushroom-producing ligninolytic white-rot basidiomycetes

mainly attack hardwood but they grow only rarely or poorly on softwood such as

southern yellow pine. Wood extractives may be responsible for the mycelia growth of

white-rotting fungi.

The blue stain fungi Ophiostoma spp. are initial wood colonizers and attack wood chips,

logs, sawn timber, veneer, sawdust, and wood products; they penetrate rapidly and deeply

into the sapwood of conifers and have the ability to reduce wood extractive concentration

of sterilized southern yellow pine chips (Blanchette et al., 1992). They invade resin

canals, epithelial cells, and ray parenchyma cells. Growth of these fungi occurs primarily

in ray parenchyma cells, where the fungi assimilate available nutrients, mainly the

nonstructural wood components, wood extractives. As the fungi grow on wood and

penetrate into the wood, their pigmented hyphae discolor it. Although the fungi do not

degrade the major components of wood cell walls, lignin, and cellulose, the metabolic

action substantially reduces wood extractives (Blanchette et al., 1992).

The primary objective of this study was to use the colorless isollate Cartapip™ 97 of the

blue-stain fungi, to remove the wood extractives in southern yellow pine wood waste. A

second objective was to facilitate the mycelial growth of the mushroom-producing,

white-rot basidiomycetes and mushroom production on pretreated southern yellow pine

chips.

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METHODS AND MATERIALS

Fungi

Dikaryotic isolates of mushroom-producing white-rot basidiomycetes Hericium

erinaceus (Bulliard: Fries) Persoon (M-13), Pleurotus citriopileatus (Singer) (FP-

102361) (LM-17), Pleurotus cystidiosus O.K. Miller (FP-140088) (M19), Pleurotus

ostreatus (Jacquin: Fries) Kummer (FP-101509),(HHB-9790)(M-23), and Pleurotus

sujor-caju (Fr.) Sing. (FP-140078) (M-29). The colorless isolate Cartapip™ 97 of blue-

stain fungus Ophiostoma pilferum was obtained from Clariant Co. (Charlotte, NC). The

fungi were maintained on 1.5% (w/v) malt extract (Bacto, Difco, Detroit, MI) and 2%

(w/v) agar (Bacto, Difco).

The mycelial isolates of white-rot and blue stain fungi were plated on 1.5% (w/v) malt

extract agar (MEA). Malt extract agar 90-mm-diameter plates were inoculated with a

mycelium/agar plug (6 mm diameter) of a young, actively growing margin of the colony

at the center of the plate and incubated at 24°C in the dark for 1 to 2 weeks or until

mycelial growth had covered the entire surface of the MEA plates.

Southern yellow pine chips treatment–Southern yellow pine wood chips were

obtained from Bienville National Forest, Mississippi. Four kilograms (dry weight 49%)

of frozen southern yellow pine (Pinus spp.) chips of various sizes (0.5 to 3.5 cm by 0.2 to

0.25 cm) and 4 L distilled water were added to an air-permeable polyethylene bag

(24X36”, VWR Scientific, ORD# 801648450000). The bag was autoclaved at 121°C for

45 min. The autoclaved wood chips were inoculated with 2 × 109 spores of Cartapip™

97 per kg wood chips. They were mixed and incubated at 24°C in the dark for 4-1/2

days. At the end of the incubation period, all wood chips were autoclaved.

Resinous extractives determination–After treatment, the southern yellow pine chips

were oven dried at 50°C and ground into 30-mesh sawdust with a Wiley mill (Authur H

Thomas Co., Scientific Apparatus, Philadelphia, PA, USA). The oven-dried sawdust (1 g

dry weight) was extracted in a Soxhlet extractor with diethyl ether overnight (Brush et al.,

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1994). The data constitute the percentage of weight loss on the basis of dry weight in a

vacuum oven at 50oC.

Scanning electron microscopy–The treated southern yellow pine chips were embedded in

O.C.T compound (Electron Microscopy Sciences) and sectioned radially at -20°C on a

cryostat(International-HarrisModel,CTD). Digital images were taken oftreated wood chip

surfaces with a JEOL 845 Scanning electron microscope.

GrainSpawnProduction–A mixture of 500 g barley, 5 g gypsum (calcium sulfate),

and 600 mL water was utilized for spawn production. Calcium sulfate was used to loosen

the substrate for aeration and to adjust the pH. Each ingredient was individually weighed

in a polypropylene autoclavable bag (20.2 by 42 cm) with a microporous filter patch

(Sunbag, Santomi Sangyo, LTD, Japan). The procedure followed was the one described

in Croan (2000). The bags were loosely tied to allow air exchange and incubated at 24°C

in total darkness for 2 to 4 weeks or until mycelial growth had covered the surface of all

the grain.

Fruiting Body Production

Wood waste–Seven hundred grams of pretreated southern yellow pine chips of various

sizes (0.5 to 3.5 cm by 0.2 to 0.25 cm), with or without 20% wheat bran and 650 mL

distilled water, were placed in autoclavable bags with a microporous filter patch. Each

bag was mixed manually, loosely tied, and autoclaved at 121°C for 45 min. After the

bags were cooled, 50 mL of 40% glucose was added to each bag to give a final

concentration of 1.5% glucose. The bags were then inoculated with grain spawn at a level

of approximately 10 to 15% (wet weight basis). The contents were manually mixed, and

the bags were loosely tied and incubated at 24°C in the dark for 3 to 5 weeks or until the

mycelium had completely colonized the substrate.

The bags were placed in a refrigerator for 3 to 5 days, cut open, exposing the colonized

substrate to the air, and placed in an incubator. The temperature was maintained at 22°C

to 26°C under a standardized light cycle (approximately 8-10 h light, 14-16 h dark)

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using a fluorescent ceiling light (General Electric, 2 to 15 W, standard, cool white).

Humidity and moisture were maintained at 90 to 95% with a constant vapor-like spray of

distilled water, using a Herri-michifier (part# L122-74, trio model #707). Fruiting bodies

were harvested when the caps reached reached 5 to 10 cm in diameter. Fruiting bodies

were harvested for up to 3 flushes.

RESULTS AND DISCUSSION

The wood extractives are mostly low molecular weight compounds that are easily

extracted from wood by solvents such as water, alcohol, ether, or benzene. The

percentage of the extractives account for 4.05% to 9.02% (with 0.04 standard deviation)

of the dry weight of southern yellow pine wood chips depending upon the batch of wood

chips received and the season of the year and amount of rainfall (Terry Conners,

Mississippi Forest Products Laboratory, personal communication) when they were

harvested. The colorless isolate Cartapip™ 97 of sapstain fungus Ophiostoma piliferum

attacked the southern yellow pine chips. The wood chips were treated with 2 billion

spores of colorless isolate of Cartapip™ 97 per kilograms of wood chips. The Cartapip™

97 removed 61.1% to 99.8% of the extractives (diethyl ether extractible) of dry weight of

wood chips within 4 to 5 days. SEM examination showed that Ophiostoma piliferum

were colonized on the entire surface of the wood chips within 2 days, producing

filamentous heavy mycelial growth (Fig. 1) with sporulation (Fig. 2). The Cartapip™ 97

removed 25.9% of the extractives within 2 days. The mycelium of Ophiostoma pilferum

passed from one cell to another through border pits and tracheids (Fig. 3). The mycelia

appeared in the ray parachyma cells within 6 days (Fig. 4).

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Figure 1-4. The colonization of Ophiostoma piliferum (1) with sporulation (2); border

pits and tracheids (3); ray parenchyma cells (4) on southern yellow pine chips.

The fungi that cause the decay of wood are the brown-rot (red-rot) and white-rot in wood,

which are found in forests, in stored logs, on standing trees, on stumps, in woodlands, and

elsewhere. Brown-rot basidiomycetes attack mainly softwoods and occasionally also

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hardwood. Mushroom-producing white-rot basidiomycetes attack mainly hardwood but

only rarely softwood. Our study demonstrated that mushroom-producing white-rot fungi,

did not grow or else grew very poorly on untreated fresh southern yellow pine chips. All

these mushroom-producing white-rot basidiomycetes, Hericium erinaceus, and Pleurotus

citriopileatus, P. cystidiosus, P. ostreatus and P. sajor-caju did not grow on untreated

fresh pine chips but dense, filamentous heavy mycelial growth occurred on the entire

surface of treated southern yellow pine chips and fruiting bodies were formed all around

wood chips (Fig. 5 to 9).

Mycelial growth of mushroom-producing basidiomycetes covered the entire surface of

wood chips pretreated with colorless isolates (Cartapip™ 97). The mycelial growth of

Hericium erinaceus on the entire surface of southern yellow pine chips was white,

filamentous, and dense or heavy. The basidiomycetes produced a white to pale yellowish,

oval to round, solid mass of beardlike spine decurrent fruiting bodies on treated wood

chips, composed of beardlike mass 30 to 50 mm in diameter and 25 to 40 mm high

(Figure 2) (called Lion's Mane). The color of the mushrooms changed to yellowish

brown or dark brown with age. These mushrooms could be flushed 2 times under

optimum conditions, producing a harvest of 280 g and 30 g.

The cap clusters (fruiting bodies) of Pleurotus citrinopileatus were golden to bright

yellow in color, 20 to 70 mm in size, and convex to plane and had white gills. White

stem, 5-10 mm were centrally attached to the caps. The mushrooms formed on the

surface of wood chips supplemented with 20% wheat bran (Fig 6) (called the Golden

Oyster). These mushrooms were flushed up to 3 times, producing 356 g - 3 1 g.

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Figure 5. Fruiting of

Hericium erinaceous

on southern yellow

pine chips.

Figure 3. Fruiting bodies

formation of Pleurotus

citriopileatus

Pleurotus cystidiosus (M 19) produced the Abalone mushrooms or Miller's Oyster

mushrooms, convex to hollow dark grayish brown in the center, with smooth brown to

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light brown margins. White thick stems, 5-15mm, with white gills were centrally attached

to the caps. Numerous sterile cells on black caps, conidiophores bearing conidia at the

apex [termed synnemata or coremia by Miller (1969)] grew on treated pine chips

supplemented with 20% bran ((fig 7). The fungi did not produce these black synnemata

on the MEA plates but other species of Pleurotus cystidiosus (M18) produced numerous

synnemata around the colony and agar plug of inoculum area on MEA plates. They grew

poorly on the wood chips and did not form synnemata.

Figure 7. Fruiting bodies

formation of Pleurotus

cystidiosus with numerous

black minute synnemata on

treated pine chips

supplemented with 20% wheat

bran.

Pleurotus ostreatus produced blue oyster mushrooms, grayish-dark blue at the center,

with lighter grayish blue smooth margins and white gills on pretreated southern yellow

pine chips. The caps of the fruiting bodies were initially hollow with a curved inner

surface (Fig 8). This species could be flushed up to 3-5 times 366 g - 50 g.

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Figure 8. Fruiting bodies

formation of Pleurotus

ostreaus (blue oyster) on

treated southern

yellow pine chips.

Pleurotus sajor-caju produced “Pheoenix” mushrooms that were initially dark grayish

blue, small, hollow caps with long stem on barley as a substrate. The mushrooms were

flushed three times, producing 425,300, and 160 g (Fig 9). When 100% pretreated

southern yellow pine chips were used, the mushrooms were relatively flat and fan-shaped

with wavy margins and shorter stems.

Figure 9-10. Pleurotus sajor-caju on barley (Fig 9),

and on 100% treated pine chips (Fig 10).

In conclusion, the results of this study demonstrate that the colorless isolates Cartapip™

97 of the blue-stain fungus Ophiostoma piliferum can remove 6 1.1% to 99.9% of the

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extractives from southern yellow pine. The mushroom-producing white-rot fungi

Hericium erinaceus, Pleurotus citriopileatus, P. cystidiosus, P. ostreatus and P. sajor­

caju can be easily colonized and can successfully produce mushrooms on treated

southern yellow pine chips.

ACKNOWLEDGMENT

I wish to thank Dr. Roger C. Pettersen, Analytical Chemist at the Forest Products

Laboratory, for analysis of wood extractives and Scott I. Gamb, student assistant from

Madison Area Technical College, for scanning electron microscopic examination.

REFERENCES

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