Full text of DOI:10.1080/1065657X.2000.10701997

Compost Science & Utilization, (2000), Vol. 8, No. 3, 247-253
Identification of Bacteria in Mushroom Compost
Using Ribosomal RNA Sequence
K.L. Ivors, P.D. Collopy, D.M. Beyer and S. Kang
Department of Plant Pathology, The Pennsylvania State University,
University Park, Pennsylvania
Microorganisms in mushroom substrate play important roles in commercial mush-
room production. We have initiated the characterization of microorganisms in
Phase II compost using sequences of genes encoding small subunit ribosomal RNA
(rDNA). A specific area of rDNA was amplified from individually cultured organ-
isms as well as from total DNA extracted from the compost sample by polymerase
chain reaction. Thirteen rDNA fragments sequenced in this study corresponded to 12
different strains representing three divisions of bacteria. All of these strains, except
Bacillus licheniformis, have not previously been identified through rDNA sequencing,
suggesting that mushroom compost contains many novel organisms.
Introduction
It has been shown that both substrate preparation and mushroom (Agaricus bis-
porus Lange Imbach) cultivation are dynamic processes characterized by fluctuating
populations of microorganisms and that compost is colonized concurrently by dif-
ferent groups of microorganisms (Fergus 1964; Hayes 1969). Growth and develop-
ment of certain species are dependent upon others, not only for conversion of organic
material in compost to certain metabolites, but also for excretion of a wide array of
metabolites with growth-regulatory activities. Agaricus mycelium also interacts with
microbial communities in compost. It has been documented that mycelial spawn of
Agaricus growing in prepared compost produces volatile compounds that stimulate
Pseudomonas putida, and perhaps other microbes, to greater activity and can inhibit
other bacteria that are antagonistic to Agaricus (Hayes et al. 1969). Hence, the struc-
ture and dynamics of microbial communities in mushroom compost are believed to
significantly influence many facets of mushroom production, including the biocon-
version of ammonia and organic material in compost, mushroom fructification, and
the development of certain diseases. Given the importance of microorganisms in
mushroom production, there exists very limited information as to what types of mi-
croorganisms exist in compost and how they influence various stages of mushroom
production.
Considering the finding that only a very small portion of microorganisms pre-
sent in nature are cultivated by standard techniques (Amann et al. 1995), characteri-
zation of cultured microorganisms is inadequate for surveying the microbial diver-
sity in mushroom compost. Molecular phylogenetic approaches to the
characterization of the microbial diversity in environmental samples have made it
possible to identify specific types of microorganisms without cultivating them, al-
lowing us to overcome the limitations in culture-based surveys (Hugenholtz et al.
1
998; Hugenholtz and Pace 1996; Pace 1996; Wilson 1996). These approaches are
based primarily on the amplification of DNA extracted from environmental samples
by polymerase chain reaction (PCR) using universal primers targeted to genes en-
coding small subunit (16S or 18S) ribosomal RNA (rDNA). The amplified rDNAs are
individually cloned into a vector and sequenced. These rDNA sequences can then be
Compost Science & Utilization
Summer 2000 247

K.L. Ivors, P.D. Collopy, D.M. Beyer and S. Kang
used to identify the types of microorganisms in a given environmental sample by
comparison with the rDNA sequences of previously identified microorganisms in
public databases (http://www.ncbi.nlm.nih.gov/BLAST/; Maidak et al. 1997).
The long-term objective of this study is to determine the structure and dynamics
of microbial communities in mushroom compost through rDNA sequence-based sur-
veys, which will serve as a basis for addressing the question of how changes in the
structure of the microbial communities influence various aspects of mushroom pro-
duction. In this paper we report a method for extracting DNA from compost and also
present the identity of bacterial strains characterized in our preliminary analysis.
Materials and Methods
Culture Conditions
Bulk compost samples were taken after pasteurization during Phase II compost-
ing, two days prior to spawning, from the Mushroom Test Demonstration Facility
(MTDF) at The Pennsylvania State University (University Park, Pennsylvania). The
compost formulation consisted of a standard wheat straw bedded-horse manure mix-
ture containing poultry manure, gypsum, and brewers grain. The compost was pre-
pared according to conventional Phase I and Phase II processes. Phase I composting
was followed by a controlled conditioning stage (Phase II) in which the compost was
pasteurized at 60°C for two hours and cooled to 50°C before taking the sample. In or-
der to culture microorganisms from the compost, 5 g compost was added to 45 ml of
sterile water in a 250 ml flask and mixed in a shaker (250 rpm for 30 min). This com-
post extract was serially diluted with sterile water and then plated onto three differ-
ent media: Bacto nutrient agar (Difco, Detroit, Michigan), compost agar, and compost
agar amended with antibiotics (50 mg nystatin and 50 mg cycloheximide per liter of
agar). One half of the plates inoculated was incubated at 37°C and the other half was
incubated at 28°C. Compost agar was prepared in the following way: 100 g wet com-
post was added to 1000 ml distilled water and autoclaved for 1 hour. This sterilized
mixture was filtered twice through three layers of cheesecloth, centrifuged at 2000 rpm
for 10 minutes to remove the remaining debris, and its volume was adjusted back to
1000 ml by adding distilled water. One hundred ml of this compost extract was added
per liter of compost agar.
Isolation of DNA
Both ground (by mortar and pestle with the addition of liquid nitrogen) and in-
tact compost samples (250 mg) were subjected to DNA extraction using FastDNA
Spin Kit for soil (BIO101, Vista, California) according to the manufacturer’s protocol
except that constant vortexing (15 min or 30 min), instead of a special bead beater,
termed FastPrep System (Savant, Farmingdale, New York), was used. The DNA re-
leased from lysed cells was purified using DNA binding matrix and spin columns
included in the kit. The amount of purified DNA was quantified using a spec-
trophotometer, and purified DNA (5 µl) was digested with 5U of MboI in 20 µl of re-
action at 37°C for two hours. To release DNA from colonies on the agar plates, a small
fraction of individual colonies was transferred to 20 µl of water in a 1.5 ml tube us-
ing a toothpick. This tube was incubated in boiling water for 5 min, chilled on ice for
5
min, and centrifuged for 5 min before collecting the DNA solution.
248 Compost Science & Utilization
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Identification of Bacteria in Mushroom Compost Using Ribosomal RNA Sequence
DNA Analyses
Total DNA extracted from mushroom compost (100-140 ng) or from individual
colonies (5 µl) was amplified in a 50 µl PCR mixture (10 mM Tris-HCl [pH 8.3], 50
mM KCl, 1.5 mM MgCl2, 0.001% gelatin, each dNTP at 0.25 mM, 0.2 µM of each
primer, and 2.5 units of Taq polymerase) using a PTC-100 thermal cycler (MJ Re-
search, Watertown, MA). The primers, 530F and 1494R, were designed as previous-
ly described (Borneman et al. 1996). This mixture was subjected to 30 cycles of am-
plification (one cycle of 1 min at 96°C, 30 sec at 50°C, and 1 min at 72°C followed by
2
9 cycles of 30 sec at 94°C, 20 sec at 55°C, and 1 min at 72°C) followed by a 2 min ex-
tension at 72°C. The amplified DNA was separated on a 1 % agarose gel. A DNA frag-
ment of approximately 1 kb was cut from the gel and purified using the QIAquick
gel extraction kit (QIAGENE, Valencia, California), which was then ligated to the
pGEM-T vector (Promega, Madison, WI). Competent cells of Escherichia coli were
transformed with the ligated DNA and then plated on LB plates containing ampi-
cillin and X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside). White E. coli
colonies, presumably carrying pGEM-T with an rDNA insert, were transferred to 96-
well microtiter plates containing LB-ampicillin and cultured, and the plates were
stored at –80°C after adding 1/3 vol of 80% glycerol. Plasmid DNA was prepared
from clones in this rDNA library using the QIAprep kit (QIAGEN, Valencia, Cali-
fornia) and sequenced using the T7 primer and the Big Dye terminator sequencing
kit (Perkin-Elmer, Foster City, California). The sequencing reactions were analyzed
using an automated DNA sequencer, ABI377 (PE Applied Biosystems, Foster City,
California). The PCR fragments from the cultured cells were directly sequenced us-
ing 530FS (5’-GAGTGCCAGCMGCCGCGG-3’) primer. The sequence of rDNA was
compared with the sequences in the nonredundant nucleotide database available
through the National Center for Biotechnology Information (NCBI, Bethesda, Mary-
land) using the BLAST algorithm (Altschul et al. 1997).
Results
DNA Extraction From Mushroom Compost
We have modified a previously reported method (Borneman et al. 1996) to extract
DNA from a bulk mushroom compost sample taken after pasteurization during Phase
II composting. This modified method utilizes a vortex instead of a special bead beater
to mechanically lyse microorganisms. The extracted DNA was subsequently purified.
Yields of DNA per gram of the compost ranged from 55 µg to 74 µg (Table 1). Grind-
ing the compost samples prior to DNA extraction significantly increased yield (21 to
3
5%) relative to unground samples. In
the absence of grinding, increased vor-
texing (30 min vs. 15 min) appeared to
have increased the yield slightly (11%).
However, when the compost was ground
before DNA extraction, we observed no
increase in yield with 30 minutes of vor-
texing, suggesting that 15 minutes of vor-
texing is sufficient for an efficient DNA
extraction. Gel electrophoresis showed
that the size of the purified DNA frag-
TABLE 1.
DNA yields from four different extraction
conditions
No
Grinding
Grinding
Length of Vortexing
DNA Yielda
15 min 30 min 15 min 30 min
55 61 74 74
aµg of DNA per 1 g of compost. The data were from a
single experiment. We assumed that 1 O.D.
corresponds to 50 µg DNA per ml.
260
Compost Science & Utilization
Summer 2000 249

K.L. Ivors, P.D. Collopy, D.M. Beyer and S. Kang
ments ranged from less than 1
kb to larger than 12 kb (Figure
A). Increased vortexing time
A
3
B
3
1
2
4
5
1
2
4
5
1
and grinding of the sample
appear to slightly decrease
the average size of purified
DNA fragments. We tested
the quality of the purified
DNA by restriction analysis
using a 4 base cutter, MboI
(
Figure 1B). Four different
DNA samples appear to have
been completely digested by
the enzyme, suggesting that
these DNA samples contain
very few, if any, impurities
that could inhibit subsequent
enzymatic reactions. Success-
ful amplification of a segment
of rDNA by PCR from these
samples also supports the
statement that this DNA ex-
traction method produces
high quality DNA.
Figure 1. Gel electrophoresis of DNA extracted from Phase II mushroom
compost. Samples of 10 µl purified DNA were separated on a 0.7%
agarose gel (Figure 1A). DNA samples (5 µl) were also digested with
MboI and subsequently separated on a 1.0% agarose gel (Figure 1B). In
both figures: Lanes 1 and 3 correspond to DNA extracted from ground
compost, lanes 2 and 4 contain DNA extracted from the compost without
grinding. The length of vortexing the samples was either 30 min (lanes 1
and 2) or 15 min (lanes 3 and 4). Lane 5 contains a 1 kb DNA ladder
(GIBCO BRL, Rockville, Maryland) as size markers.
Construction And Characterization Of An rDNA Library
The DNA was amplified by PCR using a pair of universal primers designed for
amplifying most 16S rDNA. To construct a clone library of rDNA, the amplified
rDNA was cloned into pGEM-T vector. The library consisted of 192 E. coli colonies
stored in two 96-well microtiter plates. PCR analysis showed that 89% of the clones
in the library contained an insert of approximately 1 kb. Eight clones in the library
were sequenced (Table 2). The identity between these sequences and the known
rDNA sequence in the database ranged from 90% to 96%, suggesting that these se-
quences correspond to previously unidentified bacteria. These bacteria belong to
TABLE 2.
Classification of clones in the rDNA library
Identity
(%)
Accession
Sizea
Closest relativeb
no
c
Putative Division
Clone
T2G6
T2G7
T2G11
T2H6
T2H8
T2H9
T2H11
T2H12
515
587
610
588
570
431
518
589
Thermobacillus xylanolyticus
Chrondromyces crocatus
95
91
90
96
96
93
90
90
AJ005795
M94275
Low G+C gram positive
Proteobacteria delta subdivision
Proteobacteria beta subdivision
Actinobacteria
Leptothrix sp.
AB008801
X89939
Streptosporangium cinnabarium
Polyangium cellulosum
M94282
Proteobacteria delta subdivision
Proteobacteria gamma subdivision
Low G+C gram positive
Actinobacteria
Iron-oxidizing lithotroph ES-1
Unidentified rumen bacterium RFN 82
Microthrix parvicella
AF012541
AB009230
X82546
a
The number of nucleotides obtained by sequencing.
b
c
Organism with rDNA sequences showing the highest homology to the clone based on BLASTN search.
NCBI database accession number of the closest relative.
250 Compost Science & Utilization
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Identification of Bacteria in Mushroom Compost Using Ribosomal RNA Sequence
three bacterial divisions, the Proteobacteria (4 clones), the low G+C gram-positive
bacteria (2 clones), and the Actinobacteria (2 clones).
Characterization Of Cultured Microorganisms
Bacteria in this compost sample have also been cultured and identified to compare
results with the sequence-based survey. We cultured microorganisms on three differ-
ent media (nutrient agar, compost agar, and compost agar amended with antibiotics)
at two different temperatures (37°C and 28°C). Sixty colonies, ten from each of the six
growth conditions, were randomly picked and subjected to rDNA amplification by
PCR. Although a PCR product of expected size (~1 kb) was obtained from 90% of
colonies grown on nutrient agar, only 20% of colonies from compost agar produced a
PCR product. A PCR product was obtained from 50% of the colonies picked from com-
post agar amended with antibiotics.
Five PCR products, three (labeled N1-N3) from colonies on nutrient agar and two
labeled C1 and C2) from colonies on compost agar amended with antibiotics, were se-
(
quenced (Table 3). Taxonomic classification of these sequences was performed by com-
paring them with the DNA database sequences using BLAST (Altschul et al. 1997). These
sequences represent at least four different members of the low G+C gram-positive divi-
sion of bacteria (Hugenholtz et al. 1998). The sequence of N1 showed 100% identity to
1
6S rDNA of Bacillus licheniformis. The remaining strains appear to correspond to previ-
ously unidentified members of the low G+C gram-positive bacteria. Although both N2
and N3 showed the highest degree of homology to Caryophanon latum, there existed 9
nucleotide differences between the sequences of N2 and N3, indicating that they repre-
sent different members of the division. The sequence of C1 was identical to that of C2,
suggesting that they had derived from identical, or highly related, species.
TABLE 3.
Classification of cultured compost bacteria based on their rDNA sequences
Identity
(%)
Accession
No
Sizea
Closest relativeb
c
Putative Division
Clone
N1
N2
N3
C1
C2
a
528
523
524
391
554
Bacillus licheniformis
Caryophanon latum
100
97
97
97
96
X68416
Low G+C gram positive
Low G+C gram positive
Low G+C gram positive
Low G+C gram positive
Low G+C gram positive
X70314
Caryophanon latum
X70314
Unidentified bacterium
Unidentified bacterium
AJ000981
AJ000981
The number of nucleotides obtained by sequencing.
b
c
Organism with rDNA sequences showing the highest homology to the clone based on BLAST search.
NCBI database accession number of the closest relative.
Discussion
A better understanding of how the microbial communities change in mushroom
compost and how these changes influence various aspects of mushroom production
could open up the possibility of targeted manipulation of specific organisms during dif-
ferent phases of mushroom production. Such manipulation could be used to alleviate
composting odors, to better manage mushroom disease epidemics, or to increase avail-
able nutrients for mushroom growth. As an initial step toward characterizing the dy-
namics of the microbial communities in mushroom compost, we have constructed and
Compost Science & Utilization
Summer 2000 251

K.L. Ivors, P.D. Collopy, D.M. Beyer and S. Kang
characterized an rDNA library that contains rDNA fragments amplified from the ge-
nomic DNA of microorganisms in a Phase II compost sample. The sequences of rDNA
provide information about the identity of organisms and can also be used for inferring
genetic and physiological properties of these organisms through comparison with well-
studied relatives. Strain-specific probes can also be developed based on these se-
quences, and such probes can offer rapid and comprehensive methods of surveying the
structure and dynamics of microbial communities in mushroom compost.
Considering the successful amplification of many bacterial rDNAs, as well as fun-
gal rDNAs, from the purified DNA sample (Ivors, Beyer and Kang, unpublished re-
sults), the method employed in this study appears highly efficient. The resulting DNA
samples were pure enough for efficient restriction digestion and PCR. We replaced the
use of expensive equipment for mechanical lysis (Borneman et al. 1996) with a short
vortexing step. In comparison to a previous reported protocol for extracting DNA from
compost (Malik et al. 1997), our method is much less time consuming and does not in-
volve the use of harmful chemicals. In contrast, the method employed to release ge-
nomic DNA from cultured bacteria appears inefficient. Nearly 50% of the colonies sub-
jected to PCR failed to produce a rDNA fragment, which could have been caused by
one or a combination of factors. It is possible that the primer set was inadequate for ef-
ficiently amplifying rDNA from certain groups of bacteria. However, considering a
previous work showing that the same primer set was successfully used to amplify
rDNA from highly diverse groups of bacteria as well as fungi (Borneman et al. 1996),
this possibility seems unlikely. An alternative is that the boiling of cells suspended in
water was not sufficient to release genomic DNA from certain groups of bacteria. It is
also possible that inhibitors of PCR could have been released along with genomic
DNA, preventing the amplification of rDNA.
The number of unique rDNA sequences detected in our survey (12 of 13) suggests
that the compost sample analyzed contained a large number of unique microorganisms,
many of which probably correspond to novel organisms. For instance, all eight rDNA
clones from the rDNA library represented three bacterial divisions and corresponded to
species that had not previously been identified through sequencing of rDNA. Sequenc-
ing of rDNA from five cultured organisms revealed a similar pattern: all of the rDNA
sequences, except the one corresponding to Bacillus licheniformis, have not previously
been identified. These five cultured bacteria belong to the same division (low G+C gram
positive). Assuming that all of the colonies that failed to produce an rDNA fragment by
PCR belonged to other bacterial divisions, this result suggests that members of the low
G+C gram positive division predominate the cultured bacteria under the growth con-
ditions used in our study. In addition, members of this division have been shown to be
prevalent in other compost and self-heating organic materials (Storm 1985). For exam-
ple, of the 652 colonies cultured from various samples of solid-waste compost, Storm
identified 87% of them as Bacillus spp.. However, considering the results obtained from
characterizing the rDNA library, the prevalent presence of this division in cultured bac-
teria from Phase II mushroom compost does not seem to accurately reflect its relative
biomass within the microbial community. Only two of the eight rDNA clones belonged
to members of this division. In contrast, Proteobacteria were represented by four clones,
and the remaining two clones corresponded to Actinobacteria.
Acknowledgments
We want to thank anonymous reviewers of the manuscript for many helpful sug-
gestions. We also want to thank Dr. Ewen Mullins for critical reading of the manu-
252 Compost Science & Utilization
Summer 2000

Identification of Bacteria in Mushroom Compost Using Ribosomal RNA Sequence
script. Kelly Ivors's assistantship is being funded by MIFBAR (Mushroom Industry
Farmer Based Applied Research). Contribution number 2066 from the Department of
Plant Pathology, The Pennsylvania Agricultural Experiment Station.
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