Biological Efficiency Study


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Abstract The effects of various combinations of wheat
bran, rye and millet (at 20% and 30% of total dry sub-strate
wt) on crop cycle time, biological efficiency (BE)
and mushroom quality were evaluated for a commercial-ly
used isolate of Grifola frondosa (maitake). Supple-ments
were combined with a basal ingredient of mixed
oak (primarily red oak) sawdust, and the resulting mix-ture
was pasteurized, cooled, inoculated and bagged with
an autoclaving mixer. Times to mushroom primordial
formation and mushroom harvest were recorded, and
mushroom quality was rated on a scale of 14, where 1
was the highest quality and 4 was the lowest quality. The
combinations of 10% wheat bran, 10% millet and 10%
rye (BE 47.1%, quality 1.8 and crop cycle 12 weeks) and
10% wheat bran plus 20% rye (BE 44%, quality 1.7 and
crop cycle 10 weeks) gave the most consistent yields and
best basidiome quality over time.
Introduction
Strong consumer demand has stimulated increased world-wide
production of maitake (Grifola frondosa). The in-creased
demand for maitake is due to the unique culinary
and medicinal properties associated with this choice
mushroom. Annual commercial production has increased
41-fold (to 33,100 t in 1997; Chang 1999) since 1981, the
year when commercial production of maitake first began
in Japan (Takama et al. 1981). Maitake production and
consumption is also increasing rapidly in the United
States (up 38% in 19992000; USDA 2000). Presently,
most maitake is marketed as food. Powdered basidiomes
also are used in the production of many health foods such
as maitake tea, whole powder, granules, drinks, and tab-lets
(Royse 1997; Mizuno 1999).
Commercial production of most maitake is on
synthetic substrate contained in polypropylene bags. A
common substrate used for commercial production of
maitake is supplemented sawdust. Oak (Lee 1994; D.J.
Royse, unpublished data) is the most popular choice in
the United States and Japan, while beech (Kirchhoff
1996; Yoshizawa et al. 1997) and larch (Stamets 2000)
are also preferred to a lesser extent in Japan. In China,
cottonseed hulls have been successfully used as a
substitute for sawdust (Zhao et al. 1983). Brans, derived
from cereal grains, such as rice (Takama et al. 1981),
wheat (Mayuzumi and Mizuno 1997), oats and corn,
are widely used as nutrient supplements. Other nutrient
supplements used for maitake cultivation include
millet (D.J. Royse, unpublished data), corn meal
(Kirchhoff 1996), and soybean cake (Mizuno and
Zhuang 1995).
There are not many reference texts available for use
in producing maitake. The techniques currently used to
grow maitake are mostly adapted from those used to pro-duce
other specialty mushrooms, such as shiitake. Exten-sive
research has been carried out on the most efficient
methods, genotypes and nutritional formulation of spe-cialty
mushrooms other than maitake (Diehle and Royse
1986; Royse and Bahler 1988; Royse et al. 1990;
Stamets 2000). The rapid increase of maitake production
in Japan and the United States has focused the need to
develop more efficient substrate formulas to improve
yield and quality and to shorten the crop cycle. In this
study, two experiments were conducted to determine the
effects of selected nutrient supplements at various levels
on maitake crop cycle time, biological efficiency (BE),
yield and quality. Significant differences among different
formulations were found and the best combinations of
nutrient supplements among those tested were identified.
For continued growth of the commercial industry, efforts
directed toward improving BE, yield, quality, and re-duced
time to primordium formation and harvest are de-sirable.
Q. Shen D.J. Royse ( .)
Department of Plant Pathology,
The Pennsylvania State University, University Park, PA 16802,
USA
e-mail: [email protected]
Tel.: +814-8657322, Fax: +814 8637217
Q. Shen D. J. Royse
Effects of nutrient supplements on biological efficiency,
quality and crop cycle time of maitake ( Grifola frondosa)
Received: 9 April 2001 / Received revision: 18 May 2001 / Accepted: 2 June 2001 / Published online: 11 August 2001
Springer-Verlag 2001


Materials and methods
Substrates and preparation
The major substrate ingredient mixed oak sawdust (mostly
Quercus rubra L.) with approximately 30% moisture was ob-tained
from a local sawmill in Centre County, Pennsylvania. The
general substrate formulation consisted of sawdust, nutrient sup-plements
and 0.2% gypsum (CaSO 4 ). The nutrient supplements
used in the study included white millet (Panicum miliaceum L.),
wheat bran (Triticum aestivum L.) and rye (Secale cereale L).
Moisture contents of the substrates were adjusted to 5558% of
the fresh weight. All ingredients were combined, mixed, pasteur-ized
(20 min at 111C), cooled, inoculated, and bagged with an
autoclaving paddle mixer as previously described by Royse
(1985). Dry matter contents of the processed substrates were de-termined
by drying 100 g of the processed substrates in an oven
for 24 h at 105C.
Spawn, spawn run, primordial development
and basidiome development
Isolate WC828 (apparently of Asian decent and available from the
authors) was selected for this study because it is a commercially-used
cultivar in the United States and is also an isolate that consis-tently
produces high yield and quality at the Mushroom Research
Center (MRC), The Pennsylvania State University. Spawn was
prepared in 500 ml flasks with a spawn formula as follows:
100 ml beaker level full of Hesco (Watertown, S.D.) mushroom
rye grain, 50 ml beaker of hardwood sawdust, one-half teaspoon
CaSO 4 , and 120 ml of warm tap water. After inoculation with
spawn, virgin polyethylene bags were used to contain moist
(5558%) substrates (2,650 g per bag) for incubation. Spawn run
temperatures were maintained at 201C. The bags were sealed
with a twist tie and, after the spawned substrate was incubated for
1 week, 20 slits (5 mm long) were made at the top of each bag
with a sharp scalpel to allow for gas exchange. Spawn run is de-fined
as the period from the beginning of inoculation to primordia
formation. After primordia formation, two holes were cut in the
polyethylene bags, exposing the developing primordia. The top of
the bag was folded over, exposing only the developing primordia
to the fruiting environment. Taped bags then were moved to a pro-duction
room for basidiome development (Fig. 1). The period of
basidiome development was initiated when the primordia began to
grow and differentiate to form small pilei and stipes. A crop cycle
of 12 weeks or less was considered short, both on the basis of our
experience and compared to the 15-week crop cycle reported by
Stamets (2000).
Harvesting and determination of BE and quality
Mushrooms were harvested from the substrate when the caps were
fully mature. The substrate clinging to the main stipe was removed
and the clusters of mushrooms were weighed. BE was determined
as the ratio of the weight of fresh mushrooms harvested per kg dry
substrate, and was expressed as a percentage (Royse 1992). The
shape and color of the basidiome was used to evaluate the quality
of maitake [rated 14 (Table 1) based on the description of
Kunitomo (1992) and our observations].
Experimental design
Two experiments (two crops per experiment; four crops in total)
were conducted to evaluate the effects of two levels of total nutrient
supplements (20% and 30%) on BE, mushroom quality and crop
cycle time. Ten combinations (simplex lattice mixture design; SAS
Institute 1996) of wheat bran, millet and rye were tested for each of
the two experiments. Experiments were conducted as completely
randomized designs (10 replicates per treatment) and carried out at
the MRC. Environmental conditions were as described by Royse
(1985). Briefly, relative humidity (90 to 95%) was maintained by
water atomizers placed in air handling ducts, 4 h of light were pro-vided
daily by eight (1.22 m, 40 W) cool-white fluorescent bulbs,
and temperatures were maintained at 172C. Sufficient air changes
were maintained to hold CO 2 concentrations below 700 ppm (l/l).
The SAS program JMP (SAS Institute 1996) was used to analyze
data. The general linear models procedure was used to perform an
Fig. 1 Maitake (Grifola frondosa; WC828) fruiting from oak saw-dust
supplemented with wheat bran (10%) and rye (20%) 63 days
(top) and 68 days (bottom) after inoculation.
Table 1 Rating scale (14) for
evaluating basidiome quality
for Grifola frondosa (maitake)
grown on sawdust substrate
supplemented with various
combinations and levels of
nutrients
Rating Description
1 The best quality mushrooms with black to dark gray color, uniform and no misshapen pilei
2 Mushrooms with gray to light gray color and mostly uniform shape
3 Mushrooms with more than one half of the pilei misshapen
4 Mushrooms with misshapen, immature and undeveloped pilei

76
analysis of variance. Treatments with zero values were excluded
from the data analysis. Tukey-Kramer Honestly Significant Differ-ence
(HSD) was used to separate treatment means (SAS Institute
1996). To test for uniformity between crops within the same experi-ment,
a two-way analysis of variance (ANOVA) was used with crop
as a source of variation. If significant differences were found be-tween
the two crops for BE and quality, then both crops within an
experiment were analyzed separately. If no significant difference was
found for the F-test for crops as a source of variation within an ex-periment,
then data was combined for the two crops for the ANOVA.
Results
Effects on crop cycle time
Total nutrient levels (20% and 30% of total dry substrate
weight) and various combinations of wheat bran, millet
and rye significantly influenced mushroom crop cycle
time (Fig. 2). All treatments resulted in completion of
the crop cycle, except treatment 4 (millet only) at both
levels, and treatments 2, 3 and 7 at the 20% level. At the
20% level, crop cycle times for treatments 8 [wheat bran
(13.3%) : millet (0%) : rye (6.7%)] and 5 [wheat bran
(6.7%): millet (0%): rye (13.3%)]were the shortest
(10 weeks). At the 30% level, 9 weeks was the shortest
crop cycle achieved (treatments 8 and 10).
Effects on BE and quality
Twenty percent level of supplements
The BEs and quality of two crops for the 20% level
of combined wheat bran, millet and rye are shown in
Table 2. BE ranged from 42% (treatment 8) to zero
(treatments 2, 3, 4 and 7). Treatments 8 (13.3% wheat
bran and 6.7% rye), 6 (6.7% each of wheat bran, millet
and rye), 5 (6.7% wheat bran and 13.3% rye) and 9
(13.3% wheat bran and 6.7% millet) had the highest
BEs. A significant difference for BE was found between
crops I and II. However, the results from the two crops
were not in conflict. Results for crop II were similar to
crop I except the overall values for BE and quality were
lower. Total means for BE for crop I and II were 33.9%
and 31.2%, respectively. There was no significant differ-ence
in mushroom quality for any of the treatments in
both crops I and II. When wheat bran and rye were used,
both combinations of 6.7%:13.3% (treatment 5) and
13.3%:6.7% (treatment 8) produced high BEs. When
wheat bran and millet were used, only the combination
of 13.3%:6.7% (treatment 9) produced high BEs. This
suggests that a higher level of wheat bran might provide
additional yield increases. The combination of wheat
bran, millet and rye was also effective in stimulating
mushroom yield.
Thirty percent (30%) level of supplements
The BEs and quality for two crops for the 30% level of
wheat bran, millet and rye are shown in Table 3. Signifi-cant
differences in BEs and quality were found in both
crops. In crop I, BEs ranged from 48.9% (treatment 6) to
zero (treatment 4), and quality ranged from 1.6 (treat-ment
3) to 2.5 (treatment 10). The BEs for treatments 6
(10% each of wheat bran, millet and rye) (48.9%) and 5
(10% wheat bran and 20% rye) (44.1%) were signifi-cantly
higher than the other treatments. Treatment 10
(30% wheat bran only) resulted in lower mushroom
Fig. 2 Graphic summary (10
treatments) of crop cycle time
of Grifola frondosa (WC828)
as influenced by 20% (top) and
30% (below) levels of wheat
bran, millet and rye used alone
or in various combinations.
Ratios shown below each treat-ment
number indicate percent-ages
of wheat bran: millet: rye

quality. There was no significant difference in quality for
the other treatments. In crop II, BEs ranged from 45.2%
(treatment 6) to zero (treatment 4), and quality ranged
from 1.4 (treatment 3) to 2.3 (treatment 10). Similarly to
crop I, a combination of 10% each of wheat bran, millet
and rye (treatment 6) and 10% wheat bran plus 20% rye
(treatment 5) had the highest BEs. In this crop, combina-tions
of 20% wheat bran plus 10% rye (treatment 8) and
20% wheat bran plus 10% millet (treatment 9) also were
significantly higher. Quality results for crop II were the
same as crop I with treatment 10 (30% wheat bran only)
significantly lower than the others. Significant differ-ences
for BE were found between crops I and II. The
overall BE for crop I (36.6%) was higher than that for
crop II (33.4%). There was no significant difference in
quality between the two crops. Overall results for com-bined
data were the same as that for each individual
crop.
Comparison of the 20% and 30% levels of nutrient
supplement showed that, in general, increasing the nutri-ent
level increased yield, although this was not always
the case. For example, BEs for treatments 7, 8 and 10
were higher for the 20% level than for the 30% level. In
addition, some treatments did not produce mushrooms at
the 20% level of supplementation, while at the 30%
level, relatively high yields were obtained (treatments 2,
3 and 7). The BEs of treatments 6 (wheat bran: millet:
rye = 10%:10%:10%) and 5 (wheat bran: rye =
10%:20%) were significantly higher than the other treat-ments
at the 30% and 20% levels.
Discussion
Knowledge is currently very limited as to how various
nutrient types and levels influence maitake crop cycle
Table 2 Percentage biological efficiency (%BE) and quality rating for Grifola frondosa (WC828) grown on substrate supplemented
with various combinations of selected nutrients (wheat bran, millet and rye at 20% total)
Treatment Selected nutrient supplements (%) Crop I Crop II Mean (Crop I and II)
Wheat Bran Millet Rye BE (%) Quality a BE (%) Quality BE (%) Quality
1 0 0 20 29.2 b 1.8 NS d 25.3 b 1.9 NS 27.3 1.9
2 0 6.7 13.3 0 c 00
3 0 13.3 6.7 0 0 0
4 02000 0 0
5 6.7 0 13.3 36.1 a 1.6 NS 36.6 a 1.7 NS 36.4 1.7
6 6.7 6.7 6.7 38.5 a 1.4 NS 36.1 a 1.4 NS 37.3 1.4
7 6.7 13.3 0 0 0 0
8 13.3 0 6.7 42.0 a 1.3 NS 38.3 a 1.2 NS 40.1 1.3
9 13.3 6.7 0 32.9 ab 1.8 NS 30.2 ab 1.7 NS 31.5 1.8
10 20 0 0 24.4 b 1.6 NS 20.7 b 1.9 NS 22.6 1.8
Crop Means 33.9 31.2 32.5
a Quality rating based on scale of 14 where 1 is highest quality
b Means in the same experiment in the same column followed by
the same letter are not significantly different at the P=0.05 level
according to Tukey-Kramer HSD
c Treatments where no fruiting occurred (0.0) were eliminated
from the analysis of variance
d Not significant
Table 3 Percentage biological efficiency (%BE) and quality rating for Grifola frondosa (WC828) grown on substrate supplemented
with various combinations of selected nutrients (wheat bran, millet and rye at 30% total)
Treatment Selected nutrient supplements (%) Crop I Crop II Mean (Crop I and II)
Wheat Bran Millet Rye BE (%) Quality a BE (%) Quality BE (%) Quality
1 0 0 30 39.0 b b 2.0 ab 30.8 bc 2.0 ab 34.9 2.0 a
2 0 10 20 31.6 b 1.9 ab 28.5 bc 1.6 ab 30.1 1.8 a
3 0 20 10 33.4 b 1.6 a 27.9 bc 1.4 a 30.7 1.5 a
4 03000 c 00
5 10 0 20 44.1 ab 1.7 a 43.8 a 1.6 ab 44.0 1.7 a
6 10 10 10 48.9 a 1.7 a 45.2 a 1.8 ab 47.1 1.8 a
7 10 20 0 34.1 b 2.0 ab 30.9 bc 1.9 ab 32.5 2.0 a
8 20 0 10 39.2 b 1.7 a 34.2 ab 1.8 ab 36.7 1.8 a
9 20 10 0 36.9 b 2.0 ab 36.1 ab 1.8 ab 36.5 1.9 a
10 30 0 0 22.2 c 2.5 b 22.8 c 2.3 b 22.5 2.4 b
Crop means 36.6 33.4 35.0
a Quality rating based on scale of 14 where 1 is highest quality
b Means in the same experiment in the same column followed by
the same letter are not significantly different at the P=0.05 level
according to Tukey-Kramer HSD
c Treatments where no fruiting occurred (0) were eliminated from
the analysis of variance

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time, mushroom yield and quality due to the short histo-ry
of its commercial cultivation. Our results clearly indi-cate
that type and quality of nutrient supplements influ-ence
crop cycle time, yield and basidiome quality. Wheat
bran is one of the most important factors for reducing
crop cycle time. Formulations with only rye produced
mushrooms, but were significantly lower in BEs than
formulations with combinations of wheat bran and rye.
Comparison of the supplement levels of 20% and 30%
showed that, in most cases, as the nutrient levels in-creased
BEs also increased. In fact, no mushrooms were
produced when total nutrient levels were at a 10% level,
regardless of nutrient combination (Shen and Royse, un-published
data). The combination of 10% wheat bran,
10% millet and 10% rye and the combination of 10%
wheat bran plus 20% rye were the best overall formula-tions
for isolate WC828.
We found that better quality mushrooms and more
consistent yields were produced from a more nutrition-ally
balanced substrate. Combinations of two or three
nutrients selected from wheat bran, rye or millet were
the most desirable formulations found. For example,
higher levels of wheat bran alone significantly short-ened
the crop cycle, but produced poorer quality mush-rooms
and lowered BEs. On the other hand, increasing
wheat bran levels in sawdust substrates containing mil-let
and rye, or both, increased productivity and often im-proved
mushroom quality. Millet also resulted in poor
yield and quality when used alone. However, when it
was used together with wheat bran and rye, significantly
higher BEs and quality were achieved. Royse (1985)
noted similar trends in nutritional work on shiitake
(Lentinula edodes). Additional work evaluating the
effects of other types and quantities of nutrients on
mushroom BE and quality might reveal more productive
combinations than we found in this study. We also sug-gest
that additional nutritional investigations be coupled
with the use of two or more strains of diverse genetic
origin. This would help minimize the potential indepen-dent
effect of germplasm on crop cycle time, yield and
mushroom quality.
Acknowledgements The authors thank Tom Rhodes, Doug Keith,
Henry Shawley and Vija Wilkinson for technical assistance.

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