Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN 968-878-105-3
MOLECULAR PHYLOGENY AND CULTIVATION
OF AGARICUS SPECIES
J. Geml 1,2 and D. J. Royse 1
1
Department of Plant Pathology, 211 Buckhout Lab
The Pennsylvania State University, University Park, 16802 PA, U.S.A.
2
Department of Botany, Szent István University,
Ménesi u. 44., Budapest, 1118, Hungary
<jig5@psu.edu>, <djr4@psu.edu>
ABSTRACT
A phylogenetic analysis was performed on 47 isolates of 34 Agaricus species using sequences from
the internal transcribed spacer-2 and the partial large subunit of ribosomal DNA. Our data confirm
the monophyly of the genus Agaricus, which is composed of three subgenera: Agaricus,
Lanagaricus and Coniogaricus. Within the subgenus Agaricus the following clades were found: 1)
Arvenses (A. abruptibulbus, A. albolutescens, A. arvensis, A. augustus, A. blazei, A. diminitivus, A.
excellens, A. fissuratus, A. macrocarpus, A. macrosporus, A. nivescens, A. osecanus, A. semotus, A.
sylvicola), 2) Campestres (A. californicus, A. campestris, A. cupreo-brunneus), 3) Fuscovelati (A.
fuscovelatus, A. lilaceps), 4) Hortenses (A. bisporus, A. bitorquis, A. devoniensis, A. impudicus, A.
spissicaulis, A. subfloccosus, A. subperonatus), 5) Subrutilescentes (A. lanipes, A. maskae, A.
subrutilescens). All four previously classified sections of the subgenus Agaricus (Agaricus,
Arvenses, Sanguinolenti and Xanthodermatei, sensu Heinemann 1978) represent non-monophyletic
groups with shared morphological and chemical features that may have evolved independently.
These species also were evaluated for cultural characteristics in small-scale pilot trials. While
isolates of A. bisporus, A. blazei, A. subfloccosus, and A. fissuratus showed the fastest growth rates
on agar media and in compost prepared for A. bisporus, significant intraspecies variation was found
in these values. Isolates of A. bisporus, A. bitorquis, and A. arvensis produced the highest yields,
while A. sylvicola, A. arvensis, and A. bitorquis showed the largest mean weight of basidiomes.
INTRODUCTION
Agaricus bisporus (Lange) Imbach is the most widely cultivated edible mushroom, accounting for
32% of the more than 6 million metric tons of mushrooms produced worldwide in 1997 (Chang
1999). While A. bisporus has maintained its leading role in the past decade, the mushroom industry
is undergoing considerable changes due to the rapid expansion of specialty mushroom (Lentinula,
Pleurotus, Flammulina etc.) production. In order to capitalize on the increasing demand for
specialty mushroom products worldwide, Agaricus spp. growers are seeking to diversify their
product line by offering a greater variety of species and products to consumers.
The number of commercial strains of A. bisporus available to meet specific demands for fresh and
processed products is limited, and these cultivars posses narrow genetic diversity (Royse and May
1982, Loftus et al. 1988, Sonnenberg 2000). Researchers (Kerrigan and Ross 1989, Kerrigan 1990,
Kerrigan et al. 1993, Rimóczi 1994ab, Rimóczi 2000, Xu et al. 1997, Callac et al. 2000) have
sought for unique germplasm of A. bisporus and have described geographical distribution, ecology,
lifecycles, genetic structure, and phylogeny of wild populations of this species. Some other species
of Agaricus, namely A. arvensis, A. bitorquis, A. macrosporus, A. subfloccosus and A. subrufescens
were investigated for potential commercial production and for use as breeding stocks (Elliott 1978,
Fritsche 1978, Fermor 1982, Kerrigan 1983, Martinez-Carrera et al. 1995, Noble et al. 1995, Geml
111
and Rimóczi 1999, Kerrigan et al. 1999, Calvo-Bado et al. 2000). Another species of Agaricus, A.
blazei, has gained popularity among consumers because of its purported medicinal value (Mizuno
1995, Iwade and Mizuno 1997). Other species of Agaricus, with unique culinary or medicinal value,
may soon be domesticatable as increasing knowledge of parameters necessary for their
fructification becomes available (Chang and Hayes 1978, Chang and Miles 1989, Miles and Chang
1997). Some of these species may be exploitable in breeding programs of A. bisporus, especially
considering the recent success in Agrobacterium-mediated transformation of this species (Challen et
al. 2000, Chen et al. 2000).
Use of ribosomal DNA (rDNA) sequences to infer phylogenetic relationships among Agaric fungi
now is widely exploited (for example, see Bruns et al. 1991, Hillis and Dixon 1991, Hibbett et al.
1995, Lutzoni and Vilgalys 1995, Moncalvo et al. 1995, Nicholson 1995, Bunyard et al. 1996,
Hibbett et al. 1997, Hopple and Vilgalys 1999, Pine et al. 1999, Thon and Royse 1999, Moncalvo et
al. 2000ab). For phylogenetic analyses of Agaricus species, researchers have used sequence
variations found in rDNA (Mitchell and Bresinsky 1999, Calvo-Bado et al. 2000), mitochondrial
plasmid pEM (Robison and Horgen 1999) and the mitochondrial atp6 gene (Robison et al. 2001).
However, these investigations have used a limited number of species and the phylogenetic
relationships of many available Agaricus spp. remain unclear. The work reported herein was
designed to gain a better understanding of the evolutionary relationships among available species of
the genus Agaricus, and to observe mycelial growth and mushroom development of some species
on compost prepared for A. bisporus.
MATERIALS AND METHODS
Sequencing and phylogenetics
Forty-seven isolates of 34 Agaricus species were investigated including 32 isolates of 25 species
obtained from culture collections and research groups from France, Hungary and the United States.
Sequence data of an additional 15 Agaricus isolates were obtained from Genbank. Isolates were
grown in 50 ml potato dextrose yeast extract broth for 3 to 6 weeks depending on the growth rate of
the mycelium. The mycelium was filtered from the broth and DNA was extracted using the
PUREGENE DNA Isolation Kit (Gentra Systems, Minneapolis, MN). Internal transcribed spacer2 (ITS-2) and partial large subunit (LSU) regions were PCR amplified using four primers.
Amplification products were purified directly from reactions using the Wizard® PCR Prep system
(Promega, Madison, WI). Purified amplification products were sequenced using the Applied
Biosystems (ABI) BigDye terminator kit and an ABI 377 automated DNA sequencer (PerkinElmer, Foster City, CA). Each sample was sequenced in both directions with the same primers that
were used for PCR. Sequence ends were trimmed, manually edited and assembled into contigs
using the SeqMan II module in the Lasergene package (DNAStar Inc., Madison, WI). Sequences
were then aligned using the Clustal W algorithm (Higgins et al. 1991) of MegaAlign 4.03
(DNAStar Inc., Madison, WI) and manual editing. Sequence data of eight non-Agaricus species
were downloaded from Genbank and were included in the analysis. These species of reportedly
closely related genera (Moncalvo et al. 2000b) were chosen to investigate the phylogenetic origin of
the genus Agaricus. As a more distantly related species, Stropharia coronilla was chosen as
outgroup. Phylogenetic analysis was performed using PAUP version 4.0b4a (Swofford 2000). A
neighbor-joining (NJ) tree was constructed using the Kimura 2-parameter model. The stability of
clades was evaluated by bootstrap analysis with 1000 replications (Felsenstein 1985, Hills and Bull
1993).
112
Cultivation experiments
The maximum radial growth rates of isolates on agar were determined following the inoculation of
2% malt extract agar, supplemented with 1‰ yeast extract (MYA), with a single 2 mm agar plug
taken from the leading edge of a colony. Four replicate plates were prepared per culture and
incubated at 25 °C for 3 to 10 weeks. Mycelial growth (maximum radius) was measured at 3 to 10
days intervals (depending on the growth rate). Isolates were examined for growth rate on
conventional phase II wheat straw-based compost prepared at the Mushroom Test Demonstration
Facility of the Pennsylvania State University according to the short method of composting by
Sinden and Hauser (1953). One spawn grain was placed in the center of a sterile 50-ml Petri dish
containing 20 g of phase II compost. Four replicate plates were prepared per culture and the
inoculated substrate was incubated at 25 °C (2 wk). Mycelial growth (radius; maximum length) was
measured at 3 to 7 day intervals (depending on growth rate). Mushrooms were grown in small-scale
trials conducted in controlled environmental chambers at the Mushroom Research Center of The
Pennsylvania State University. The substrate (2.5 kg) was filled into plastic containers (three
replicates per strain), and grain spawn was mixed into the substrate at 1.5% (v/w). Standard
cultivation methods for A. bisporus were used for all strains (Wuest and Bengston 1982). Fruit
bodies were harvested at a slightly open, “portobello” stage. The lower part of the stipe was
trimmed removing the adherent casing material. The number and weight of basidiomes harvested
from each container were recorded and average yield per 100 kg wet substrate, average basidiome
weight, and average number of days to fruiting were calculated. In all production experiments a
commercial hybrid strain of A. bisporus (Korona 2) served as a control.
RESULTS
Phylogeny
Amplification of the ITS-2 and partial LSU yielded fragments of approximately 500 and 550 bp,
respectively. The assembled sequences ranged in size from 910 to 1020 bp. An alignment of 928
base pairs, including gaps, was generated for phylogenetic analysis. The neighbor-joining analysis
resulted in a single tree (Figure 1). Several clades were found as follows: 1) Arvenses group bootstrap value of 56%, 2) Campestres group - bootstrap value of 52%, 3) Fuscovelati group –
bootstrap value of 69% 4) Hortenses group - bootstrap value of 83%, and 5) Subrutilescentes group
- bootstrap value of 88%.
113
97
58
97
64
97
A abruptibulbus
A albolutescens
A arvensis1
A arvensis 2
A augustus 1
A augustus 2
A. blazei
A diminitivus
56
A semotus
A excellens
A fissuratus
A sylvicola 2
A macrocarpus
A macrosporus 1
A macrosporus 2
A nivescens
A macrosporus 3
A osecanus
A sylvicola 1
91
A bernardii 1
A bernardii 2
A bisporus 1
A bisporus 2
A bisporus 3
A subfloccosus
A bitorquis 1
96
83 A bitorquis 2
A impudicus
A bitorquis 3
A devoniensis
A subperonatus
A spissicaulis
52
A californicus
A campestris 1
A campestris 2
A cupreobrunneus
A fuscofibrillosus
69
A fuscovelatus
A lilaceps
A hondensis
A lanipes
88
A maskae
A subrutilescens
A silvaticus
A 95
xanthoderma 1
A xanthoderma 2
A placomyces
Chlorophyllum molybdites
100
Leucoagaricus naucinus
Leucocoprinus caepestipes
Macrolepiota procera
Cystolepiota cystidiosa
Lepiota felina
Entoloma nitidum
Stropharia coronilla
Arvenses
Hortenses
Campestres
Fuscovelati
Subrutilescentes
0.01 substitutions/site
Figure 1. Phylogram generated by neighbor-joining analysis of sequences from ITS-2 and partial
LSU rDNA showing recognized clades of Agaricus spp.
Bootstrap values are based on 1000 replications (only values of main groups are shown).
114
Mycelial growth and mushroom production
Isolates of A. blazei, A. bisporus, and A. fissuratus showed the fastest growth on MYA, with
average values of 1.29, 1.18, and 0.61 mm/day, respectively (Figure 3). However, the highest
growth rate means on compost were of A. bisporus, A. blazei, and A. subfloccosus, 5.09, 4.29, and
3.16 mm/day, respectively (Figure 4). The highest yields were obtained from A. bisporus, A.
bitorquis, and A. arvensis; 36, 35, and 23 kg mushroom per 100 kg wet compost, respectively.
Isolates of A. sylvicola, A. arvensis, and A. bitorquis produced mushrooms with the greatest average
basidiome weight, 98.4, 73.2, and 47.2 g; while the longest time periods before fruiting were
recorded for A. sylvicola, A. bitorquis, and A. blazei, i.e. 55, 53, and 48 days, respectively (Figure
2). Although a small number primordia of A. fissuratus were observed on compost, they did not
develop into mature mushrooms.
110
98
100
90
Yield (kg/100 kg)
80
Basidiome w eight
(g)
73
Days to fruiting
70
60
55
53
48
47
50
45
45
41
40
39
36
35
33
30
23
25
35
24
21
19
20
23
10
7
0
ARV
BIS
BIS-C
BIT
BLA
SUF
SYL
Figure 2. Mean yield, basidiome weight and number of days to fruiting (rounded to nearest whole
number).
ARV= A. arvensis, BIS= A. bisporus, BIS-C= A. bisporus Control, BIT= A. bitorquis, BLA= A. blazei, SUF=
A. subfloccosus, SYL= A. sylvicola.
115
Agar (mm/day) By Species
Agar
(mm
/day)
1
0
ALB ARV AUG BER BIS BIS-C BIT
BLA DIM EXC FIS
FUF FUV
LIL MAC MAS
NIV
SUF SUR SYL
FUF FUV
LIL MAC MAS
NIV
SUF SUR SYL
Species
Compost (mm/day) By Species
7
6
Com
post 5
(mm
/day)
4
3
2
1
0
ALB ARV AUG BER BIS BIS-C BIT
BLA DIM EXC FIS
Species
Figures 3-4. Maximum radial growth rate of Agaricus species on malt-yeast extract agar (MYA)
and mushroom compost.
ALB= A. albolutescens, ARV= A. arvensis, AUG= A. augustus, BER= A. bernardii, BIS= A. bisporus, BISC= A. bisporus Control, BIT= A. bitorquis, BLA= A. blazei, DIM= A. diminitivus, EXC= A. excellens, FIS=
A. fissuratus, FUF= A. fusco-fibrillosus, FUV= A. fuscovelatus, LIL= A. lilaceps, MAC= A. macrocarpus,
MAS= A. macrosporus, NIV= A. nivescens, SUF= A. subfloccosus, SUR= A. subrutilescens, SYL= A.
sylvicola. Means and standard errors are shown as lines and bars (within diamonds). Vertical end points of
diamonds form the 95% confidence interval for the mean. Dotted line shows overall mean for each
experiment.
116
DISCUSSION
Our data support the theory of monophyletic evolution of the genus Agaricus - bootstrap value of
97% - as previously proposed by others (Heinemann 1978, Cappelli 1984, Kerrigan 1986, Singer
1986, Bohus 1995, Mitchell and Bresinsky 1999). However, four previously classified sections of
the subgenus Agaricus (Agaricus, Arvenses, Sanguinolenti and Xanthodermatei, sensu Heinemann
1978) likely represent paraphyletic groups with shared morphological and chemical features that
may have evolved independently. Furthermore, our results suggest possible evolutionary groups
within the genus.
The largest of these groups is the Arvenses clade, that contained 14 species (A. abruptibulbus, A.
albolutescens, A. arvensis, A. augustus, A. blazei, A. diminitivus, A. excellens, A. fissuratus, A.
macrocarpus, A. macrosporus, A. nivescens, A. osecanus, A. semotus, A. sylvicola). These species
were previously recognized as members of the section Arvenses (Heinemann 1978), sections
Arvenses and Minores (Cappelli 1984) and the group Arvenses (Kerrigan 1986). Despite the
observed morphological and genetic differences, many recognized taxa of the section Arvenses are
able to interbreed (Calvo-Bado et al. 2000). Therefore, the appropriate taxonomic level of this
section is yet to be clarified based on the concordance of morphological, biological and
phylogenetic species concepts as proposed by (Taylor et al. 2000).
The Campestres group (A. californicus, A. campestris, A. cupreo-brunneus) included
morphologically similar species. Although A. californicus often mistaken in the wild for A.
campestris, it was placed earlier in the section Xanthodermatei, based on its inedibility, phenolic
odor and some other features (Kerrigan 1986).
Another well-supported group found in our work is Hortenses (A. bisporus, A. bitorquis, A.
devoniensis A. impudicus, A. spissicaulis, A. subfloccosus and A. subperonatus). Our results, in
agreement with Heinemann (1978), Kerrigan (1986) and Robison et al. (2001), but in disagreement
with Cappelli (1984), confirms the close relatedness of A. bisporus, A. subfloccosus and A.
subperonatus. The placement of A. impudicus could not be made with confidence until additional
isolates of this species are investigated.
Although species of the Subrutilescentes clade (A. lanipes, A. maskae and A. subrutilescens) were
placed in different sections/groups previously by Heinemann (1978) and Kerrigan (1986), the
grouping of these species is well-supported. In addition, several similar morphological and
ecological characteristics have been reported for A. lanipes and A. subrutilescens (Cappelli 1984,
Kerrigan 1986).
Classification of A. bernardii, A. fusco-fibrillosus, A. hondensis, A. placomyces, A. silvaticus and A.
xanthoderma still is not clear. Sequence data from additional Agaricus species of the section
Sanguinolenti (A. annae, A. benesii, A. bohusii, A. depauperatus, A. haemorrhoidarius, A.
mediofuscus, A. squamulifer etc.) are needed to further elucidate the evolutionary relationships of
this diverse group. Also, future studies should include species of other subgenera (Conioagaricus
Hein. and Lanagaricus Hein.) in order to further elucidate the evolution of the genus Agaricus.
Knowledge currently is very limited about the growth and production requirements of all but a few
species of Agaricus. Our research clearly indicates, there are several species in the genus Agaricus,
that can be cultivated with the commercial method developed primarily for A. bisporus. Some
isolates of these species showed values of the same level or even superior to A. bisporus in some
aspects, i.e., growth on agar media, basidiome weight etc.; and the full potential of these species is
117
yet to be determined by adapting production methods to their ecological requirements. Others,
showing slow growth or producing no basidiomes in these experiments (A. albolutescens, A.
augustus, A. bernardii, A. diminitivus, A. excellens, A. fusco-fibrillosus, A. fuscovelatus, A. lilaceps,
A. macrocarpus, A. macrosporus, A. nivescens, and A. subrutilescens), could still possess yet-to-bediscovered valuable features that might be exploited in mushroom breeding.
Because the species of Agaricus we examined in this study are distributed predominantly in the
temperate regions, future phylogenetic studies should include tropical species from the subgenus
Agaricus, as well as from the subgenera Conioagaricus and Lanagaricus. A better understanding of
the phylogenetic relationships and cultivation requirements of species of Agaricus may help the
selection and breeding of commercial lines and help to improve commercial production of these
mushrooms.
ACKNOWLEDGEMENTS
This work is part of a Ph.D. program of J. Geml, lead by Dr. Imre Rimóczi at the Department of
Botany of the Szent István University (Hungary), and was supported by a joint scholarship of the
Hungarian Fulbright Commission and the Soros Foundation. Special thanks go to Dr. Philippe
Callac (INRA, France), Dr. Richard W. Kerrigan (Sylvan Inc., USA), Dr. Mark G. Loftus (Amycel–
Spawn Mate, USA), Vija Wilkinson (PSU Mushroom Culture Collection, USA), the Korona Spawn
Plant (Hungary), and the Hungarian Museum of Natural History for providing cultures. Thanks go
to Dr. David M. Geiser for assistance with the phylogenetic analysis, and to Patrick Collopy and Dr.
Qing Shen for technical assistance and advice.
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