wood extractive concentration and sem examination of
TRANSCRIPT
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
1
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
2
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.
3
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.,
4
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)
5
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).
6
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
7
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.
8
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
9
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.
10
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
11
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
12