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The double life of Ceratobasidium: orchid mycorrhizal fungi and their potential for biocontrol of Rhizoctonia solani sheath blight of rice

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Ceratobasidium includes orchid mycorrhizal symbionts, plant pathogens and biocontrol agents of soilborne plant pathogens. It is not known to what extent members of the first guild also can participate in the others. Ceratobasidium spp. were isolated from roots of Colombian orchids and identified by phylogeny based on nrITS sequences. Phylogenetic grouping of Ceratobasidium spp. isolates corresponded to orchid host substrate (epiphytic vs. terrestrial). Isolates were tested for virulence on rice and for biocontrol of Rhizoctonia solani, causal agent of sheath blight of rice. All Ceratobasidium spp. isolates caused some signs of sheath blight but significantly less than a pathogenic R. solani used as a positive control. When Ceratobasidium spp. isolates were inoculated on rice seedlings 3 d before R. solani, they significantly reduced disease expression compared to controls inoculated with R. solani alone. The use of Ceratobasidium spp. from orchids for biological control is novel, and biodiverse countries such as Colombia are promising places to look for new biocontrol agents.
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The double life of
Ceratobasidium
: orchid mycorrhizal fungi and their potential
for biocontrol of
Rhizoctonia solani
sheath blight of rice
Ana Teresa Mosquera-Espinosa
1
Department of Agricultural Sciences, National
University of Colombia at Palmira
Research Group in Orchids, Ecology, and Plant
Systematics. National University of Colombia at
Palmira
Paul Bayman
Department of Biology, University of Puerto Rico at Rı
´o
Piedras, PO Box 23360, San Juan, Puerto Rico 00931-
3360
Gustavo A. Prado
Rice Pathology Program, CIAT-Colombia of Palmira
Arnulfo Go´mez-Carabalı´
Department of Animal Sciences, National University of
Colombia, Palmira
J. Tupac Otero
Department of Biological Sciences, National University
of Colombia, Palmira and Institute of Environmental
Studies, IDEA National University of Colombia at
Palmira
Research Group in Orchids, Ecology and Plant
Systematics. National University of Colombia at
Palmira
Abstract
:
Ceratobasidium
includes orchid mycorrhi-
zal symbionts, plant pathogens and biocontrol agents
of soilborne plant pathogens. It is not known to what
extent members of the first guild also can participate
in the others.
Ceratobasidium
spp. were isolated from
roots of Colombian orchids and identified by phylog-
eny based on nrITS sequences. Phylogenetic group-
ing of
Ceratobasidium
spp. isolates corresponded
to orchid host substrate (epiphytic vs. terrestrial).
Isolates were tested for virulence on rice and for
biocontrol of
Rhizoctonia solani
, causal agent of
sheath blight of rice. All
Ceratobasidium
spp. isolates
caused some signs of sheath blight but significantly
less than a pathogenic
R. solani
used as a positive
control. When
Ceratobasidium
spp. isolates were
inoculated on rice seedlings 3 d before
R. solani
,
they significantly reduced disease expression com-
pared to controls inoculated with
R. solani
alone. The
use of
Ceratobasidium
spp. from orchids for biological
control is novel, and biodiverse countries such as
Colombia are promising places to look for new
biocontrol agents.
Key words:
biological control, integrated pest
management, pathogenic fungi, soilborne pathogen
INTRODUCTION
The closely related genera
Ceratobasidium
and
Thanatephorus
(Agaricomycotina, Cantharellales) in-
clude fungi with a wide range of trophic strategies:
plant pathogens, saprotrophs and mycorrhizal symbi-
onts of orchids (Agrios 2002, Montalvo et al. 2006).
The best known pathogens are
Rhizoctonia solani
(5
Thanatephorus cucumeris
), which cause diverse diseas-
es of crops. Among these,
R. solani
causes sheath
blight of rice, considered the second most important
fungal disease of rice and the cause of large economic
losses worldwide (Correa et al. 2001, Chaudhary et al.
2003).
Rhizoctonia
also includes species with teleomorphs
in
Ceratobasidium,
which have characteristic binucle-
ate hyphal cells (often called ‘‘binucleate
Rhizocto-
nia
’’ or BNR, as opposed to the multinucleate cells
of
Thanatephorus
). Some
Ceratobasidium
species are
mycorrhizal symbionts of orchids, whose seeds re-
quire an association with a fungus to obtain sufficient
nutrition for germination (Arditti 1992, Otero et al.
2002, Rasmussen 2002). Orchid mycorrhizae are
unlike other types of mycorrhizae in that the fungus
probably receives little or nothing from the plant in
most cases (Rasmussen and Rasmussen 2009, Selosse
and Roy 2009). It is not clear how these fungi get the
carbon they pass to orchids; they could be parasites,
saprotrophs or mycorrhizal associates of other plants.
Understanding nutritional strategies of orchid my-
corrhizal fungi may lead to new strategies for
conservation of species of Orchidaceae, the largest
family of plants and one with many species that are
rare and becoming rarer (Flanagan et al. 2006,
Clements et al. 2011).
Other
Ceratobasidium
species are plant pathogens,
and some have been used for biocontrol of soilborne
plant pathogens (Burns and Benson 2000, Sneh et al.
2004).
Ceratobasidium
spp. isolates from soil also have
been used to control
R. solani
in crops such as cotton,
soybean, vegetable seedlings and grasses (Burpee and
Goulty 1984, Villajuan-Abgona et al. 1996, Jabaji-Hare
and Neate 2005, Khan et al. 2005, Wen et al. 2005),
but there are no reports of its use to control sheath
blight of rice. Sheath blight of rice is controlled
mainly with synthetic fungicides and resistant rice
Submitted 13 Mar 2012; accepted for publication 11 Jul 2012.
1
Corresponding author. E-mail: fitopatologia@hotmail.com
Mycologia,
105(1), 2013, pp. 141–150. DOI: 10.3852/12-079
#2013 by The Mycological Society of America, Lawrence, KS 66044-8897
141
varieties (Correa et al. 2001). Therefore, to help
develop an integrated pest management (IPM)
strategy for sheath blight of rice, we tested the
biocontrol ability of
Ceratobasidium
spp. isolates from
orchid roots.
Colombia is an ideal country in which to study
these issues; it has one of the richest orchid floras
in the world (Arditti 1992, Dressler 1993, Calderon
2006). However, its orchid mycorrhizal fungi are
almost entirely unexplored (Mosquera-Espinosa et al.
2010). Also, in some areas of Colombia
Rhizoctonia
sheath blight is a serious problem for rice production,
causing up to 40%yield loss in susceptible varieties
(Correa et al. 2001).
In this study we combined these themes: a
phylogenetic analysis of
Ceratobasidium
spp. isolates
from roots of orchids, assays of virulence of these
isolates on rice and their potential for biocontrol of
sheath blight. The objectives were: (i) Identify
Ceratobasidium
spp. isolates from roots of orchids in
different habitats in Colombia and use phylogenetic
analysis to determine whether habitat preferences
reflect phylogenetic relationships. (ii) Test their
virulence on rice plants. (iii) Test the potential of
Ceratobasidium
spp. isolates from orchids for biocon-
trol of
R. solani
on rice. We predicted that the least
virulent isolates of
Ceratobasidium
spp. would best
protect rice plants from
R. solani
, and that
Ceratoba-
sidium
spp. isolates from terrestrial orchids would
better protect rice plants from a soilborne pathogen
than isolates from epiphytic orchids. (iv) Determine
the effect of different doses of N and pasteurized vs.
unpasteurized soil on virulence and biocontrol by
Ceratobasidium
spp. High N was reported to increase
the incidence and severity of
R. solani
on rice (Savary
et al. 1995, Slaton et al. 2003, Cooke et al. 2006). We
expected pasteurization of soil would decrease the
biocontrol efficiency of
Ceratobasidium
spp. because
beneficial microorganisms might help in biocontrol
(Alexander 1981, Giri et al. 2005).
MATERIALS AND METHODS
Phylogenetic analysis of Ceratobasidium spp. isolated from
Colombian orchids.—
Collection of orchids from two regions
of Colombia, isolation of mycorrhizal fungi and amplifica-
tion and sequencing of the nuclear ribosomal ITS region
were reported by Mosquera-Espinosa et al. (2010). Sections
of root cortex with pelotons were surface-sterilized in 70%
EtOH, 2.5%NaClO and sterile distilled water 1 min each
and plated on potato dextrose agar (PDA) acidified with
0.2%lactic acid to inhibit growth of bacteria. Fungal
colonies were isolated in pure culture on PDA and isolates
of form-genus
Rhizoctonia
were selected for sequencing on
basis of hyphal morphology (Sneh et al. 1991). DNA was
isolated by a miniprep method and the nuclear ribosomal
ITS was amplified and sequenced (Otero et al. 2002).
Orchids sampled and fungi isolated are summarized
(TABLE I).
ITS sequences were aligned with the multiple sequence
alignment program Muscle (www.ebi.ac.uk/Tools/msa/
muscle/) and alignments were adjusted manually in BioEdit
5.0. The closest hits from BLAST queries in GenBank were
included in the dataset for phylogenetic analysis, along with
representative ITS sequences from several anastomosis
groups (AGs) of
Ceratobasidium
. Trees were rooted with
sequences of two
Tulasnella
isolates from the orchids
Spathoglottis plicata
(Singapore, AJ313458, Ma et al. 2003)
and
Vanilla poitaei
(Puerto Rico, GenBank DQ834391,
Porras-Alfaro and Bayman 2007).
Tulasnella
was chosen as
an outgroup based on recent phylogenetic work on the
Cantharellales, which includes
Ceratobasidium
and
Thana-
tephorus
(Moncalvo et al. 2006).
Phylogenetic relationships were estimated with maximum
parsimony (MP) and maximum likelihood (ML) with
heuristic searches in PAUP 4.0b10 (Swofford 2001). The
ML tree was based on the HKY85 model of sequence
evolution as estimated by FindModel. Bootstrap support was
calculated for the MP tree using 1000 repetitions.
Virulence of Ceratobasidium spp. on rice plants.—
Nine of
the 12
Ceratobasidium
spp. isolates from Colombian orchids
were tested for virulence on rice in vitro and in vivo. Disease
incidence was tested on cut leaves (FAO 1985) and disease
severity on plants in the greenhouse (Liu et al. 2009).
Disease incidence on rice leaves in vitro.—
Mature leaves from
40 d old plants of
Oryza sativa
var.
japonica
cv. Koshihikari
(Kitazawa Seed Co., Oakland, California) were harvested.
Koshihikari is moderately susceptible to sheath blight
(Cartwright and Lee 2001). Seven centimeter-long pieces
were placed on filter paper in Petri dishes, with four pieces
per plate and 10 replicate plates per treatment. Each leaf
piece was inoculated with one 3 mm agar plug taken from
5 d old cultures on PDA. Plates were incubated at 25 C and
100%humidity, suitable for development of sheath blight
(Correa et al. 2001, Ferrero and Nguyen 2003). Percent
incidence was recorded 4 d after inoculation based on how
many of the four leaf pieces developed signs of sheath
blight (i.e. 0, 25, 50, 75 or 100%) (FAO 1985).
Rhizoctonia
sheath blight of rice first appears as irregular, elliptical, red-
brown lesions. In time the center of the lesions turns white-
gray, leaving a red-brown outer ring (Correa et al. 2001,
Agrios 2002). Sixteen isolates were tested: nine
Ceratobasi-
dium
spp. isolates described above, one
Ceratobasidium
sp.
and one
Tulasnella
sp. isolated from orchid mycorrhizae in
Puerto Rico, two
R. solani
potato pathogens (Rhs1AP and
BS69, both AG-3, Charlton et al. 2008), one
R. solani
rice
pathogen (AG1-1A) included as a positive control and two
negative controls (uninoculated leaf pieces and leaf pieces
inoculated with PDA only). The experiment had a
completely randomized design. Data were analyzed by
ANOVA. The large coefficient of variation was addressed
by transforming the data as !(X +0.5) (Go´ mez and Go´mez
1984). A post-hoc comparison of means was done with
Duncan’s multiple range test (
P
50.05) using SAS (2002).
142 MYCOLOGIA
Disease severity on rice plants in the greenhouse.—
Green-
house tests followed a standardized methodology (Liu et al.
2009): Twenty-one day-old plants of the variety Fedearroz
50, one of the most widely planted in Colombia, were used
(Jaramillo et al. 2003, Cuevas 2005). Fungi were inoculated
at the crown, simulating natural infection by the pathogen
in the soil (Agrios 2002). Each plant was inoculated with
one 3 mm diam plug taken from a 5 d old culture on PDA.
Once inoculated, each pot was covered with a microcham-
ber (made from a 2 L plastic soft drink bottle) to maintain
100%relative humidity and 30 C, conditions required for
infection by the fungus (Agrios 2002, Liu et al. 2009). To
maintain constant soil humidity, pots were partially sub-
merged in trays of water. Ten days after inoculation disease
severity was scored as percent leaf with lesions for each
plant, following the methodology of Liu et al. (2009); the
proportion of leaf area with lesions was estimated for the
lowest leaf affected, the two leaves above it, and the stem,
and the three leaves and stem were weighted 30%,25%,
25%and 20%respectively. For severity assays the
R. solani
AG-3 isolates from potato,
Ceratobasidium
spp. from Puerto
Rico and
Tulasnella
spp. were not included.
The experiment used five plants per pot, three pots per
treatment and a randomized block design in a factorial
experiment. A total of 72 combinations were tested: two soil
types (pasteurized vs. unpasteurized) 3three doses of N
(described below) 312 treatments (inoculation with nine
Ceratobasidium
spp. isolates and the three controls de-
scribed above). The experiment was repeated 1 mo after the
first run. Analyses by ANOVA combined data from the two
runs because no significant difference was observed
between them. Because the coefficient of variation was
TABLE I. Isolates of
Ceratobasidium
spp. from orchids
Isolate GenBank accession Orchid Habitat Locality %similarity Highest BLAST hit
Q1M13 GU206536
Notylia
sp. Epiphyte Valle del Cauca 93%
Ceratobasidium
AG-B(o),
AB219143
Q1.1 M14 GU206537
Habenaria
sp. 1 Terrestrial Valle del Cauca 94%
Ceratobasidium
YA
Cuevas, A. 2005,
DQ279043
Q1.2 M14 GU206538
Habenaria
sp. 2 Terrestrial Valle del Cauca 96%
Ceratobasidium
YA
Cuevas, A. 2005,
DQ279043
Q2.1 M14 GU206539
Cranichis
sp. 1 Terrestrial Valle del Cauca 100%
Ceratobasidium
AG-G, AB478783
100%
Ceratobasidium
,
EU605732
Q2.2 M14 GU206540
Cranichis
sp. 2 Terrestrial Valle del Cauca 100%
Ceratobasidium
,
DQ279043
Q1M15 GU206541
Vanilla
sp. Hemiepiphyte Valle del Cauca 99%Endophyte,
FJ613838
Q1 M16 1.1 GU206542
Epidendrum
melinanthum
1
Lithophyte Valle del Cauca 98%
Thanatephorus
cucumeris
,
AJ000202
97%
Ceratobasidium
AG-F, DQ102436
Q1 M16 1.2 GU206543
Epidendrum
melinanthum
2
Lithophyte Valle del Cauca 100%Uncultured
Ceratobasidium
,
EU002954
Q3 M17 1.1 GU206544
Trizeuxis falcata
1 Epiphyte Antioquia 97%
Ceratobasidium
JTO091,
AF472295
Q3 M17 1.2 GU206545
Trizeuxis falcata
2 Epiphyte Antioquia 97%
Ceratobasidium
JTO031,
AF503970
Q1M19 GU206547
Maxillaria
sp. Epiphyte Valle del Cauca 97%
Ceratobasidium
JTO031,
AF503970
Q2M19 GU206546
Dichaea
sp. Epiphyte Valle del Cauca 97%
Ceratobasidium
JTO075,
AF472291
R. solani
(Tolima
2399-1 oryzica 1)
GU206548
Oryza sativa
Pathogen CIAT, Colombia 100%
T. cucumeris,
FJ667267
MOSQUERA-ESPINOSA ET AL.: ORCHID MYCORRHIZAE AND DISEASE BIOCONTROL 143
high, data were transformed as !(X +0.5) (Go´ mez and
Go´ mez 1984). Post-hoc tests to compare means used
Duncan’s multiple range test with significance considered
P
50.05, using the statistical package SAS (2002).
Biocontrol of R. solani by Ceratobasidium spp. on rice plants
in greenhouse conditions.—
Experimental design and mea-
surement of disease severity were as described for the
virulence experiments described above. However, the time
of inoculation was modified as follows:
Ceratobasidium
spp.
isolates were inoculated 2 d before the virulent isolate of
R.
solani
. During these days the plants were incubated in a
60 m
2
chamber, with plastic curtains and three humidifiers
that ran 1 h twice a day to maintain an environment
conducive to development of disease. After inoculation,
plants were incubated in microchambers as described
above. The experiment was repeated 1 mo after the first
run.
Effects of soil N and pasteurization.—
In the greenhouse
experiments described above, the effect of N and of
pasteurized vs. unpasteurized soil on virulence of
Ceratoba-
sidium
spp. and biocontrol of
R. solani
was tested. N was
applied in three doses: 0, 150 and 250 kg N /ha, in the form
of ammonium sulfate (26%N) as reported Savary et al.
(1995), Slaton et al. (2003), Cooke et al. (2006). Soil was
collected from a rice field in Jamundı´, Valle del Cauca,
Colombia, to ensure that its physicochemical properties
were suitable for rice production (IGAC 2006).
RESULTS
Phylogeny of Ceratobasidium spp. isolates from
orchid roots.—
All isolates from orchids were assigned
to
Ceratobasidium
(in one case,
Thanatephorus
) based
on BLAST results and the binucleate condition of
young hyphae (in one case uninucleate: Q1M13 from
Notylia
sp., TABLE I) (Mosquera-Espinosa et al. 2010);
phylogenetic analysis agreed with these assignments.
Identification to species was not possible because the
isolates were divergent from named species and
because species-level taxonomy of
Ceratobasidium
is
poorly resolved. The isolate from a rice plant with
sheath blight (used here as a positive control) had
100%ITS homology with
R. solani
AG1-1A, the AG to
which the sheath blight pathogen belongs (Sneh et al.
1991, Agrios 2002, Bernardes de Assis et al. 2008).
Phylogenetic analyses grouped the isolates into
three clades, labeled ‘‘epiphytic’’, ‘‘
Thanatephorus
’’
and ‘‘terrestrial’’ (FIG. 1), which corresponded to the
habitats of the host orchids, but also to some extent
with host phylogeny. Isolates from the epiphytic
orchids
Dichaea
,
Maxillaria
,
Notylia
and
Trizeuxis
(all in subfamily Epidendroideae) formed a clade
with 73%bootstrap support. This epiphytic clade also
included reference sequences from
Ceratobasidium
AG-Q and mycorrhizal fungi of epiphytic orchids in
Puerto Rico (clades A, B and C, Otero et al. 2002;
Otero et al. 2007), as well as from an epiphytic orchid
from Singapore (AJ318430) and an isolate from
strawberry (Japan, AB219143, pathogenicity not stated).
The ‘‘
Thanatephorus
’’ clade, with 69%bootstrap
support, included a
Ceratobasidium
spp. isolate from
the epiphytic orchid
Epidendrum melinanthum
1 (also
in subfamily Epidendroideae) and a reference se-
quence from an orchid from Puerto Rico (JTO048).
It also included reference sequences from patho-
gens listed as
Rhizoctonia
,
Thanatephorus
and
Cerato-
basidium
with a global distribution:
Thanatephorus
FIG. 1. Phylogeny of
Ceratobasidium
isolates from
orchids and related fungi based on ITS sequences. The
maximum likelihood tree was based on a 580 bp alignment
with 195 informative characters. Two ITS sequences from
Tulasnella
were defined as the outgroup. Dotted lines mark
clades discussed in the text. Numbers at the nodes show
bootstrap values from the corresponding maximum parsi-
mony (MP) tree. The asterisk marks the only branch not
shared with the MP tree. 2Ln likelihood 53773.74826. The
corresponding maximum parsimony analysis generated
32 equally parsimonious trees, 606 steps long, CI 50.650,
RI 50.788.
144 MYCOLOGIA
AG1-1A (Benin, AJ000202) and AG-3 (USA,
AF354064), and
Ceratobasidium
AG-F (Israel,
DQ102436). A subclade with 100%bootstrap support
included sequences from rice pathogens: the isolate
of
R. solani
from Colombia (GU206548) and
Thanatephorus
from Japan (FJ667267) and from
China (EF429316).
The ‘‘terrestrial’’ clade, with 98%bootstrap sup-
port, included two mycorrhizal isolates each from the
terrestrial orchids
Cranichis
sp. and
Habenaria
sp.
(both subfamily Orchidoideae) and one from the
lithophytic orchid
Epidendrum melinanthum
(Epiden-
droideae). It also included reference sequences of
Rhizoctonia
sp. AG-G (Italy, AY927346); sequences of
a
Ceratobasidium
spp. endophyte from the epiphytic
orchid
Rhynchostylis retusa
(India, EU605732) and
from apple seedling (USA, EU002954), pathogens of
Viburnum
(Italy, AB478783) and another from an
unnamed host (the Netherlands, DQ279043). The
remaining mycorrhizal isolate, from a terrestrial root
of
Vanilla
sp. (subfamily Vanilloideae), grouped with
90%bootstrap support with two reference sequences:
Ceratobasidium
AG-O from soil (Japan, AF354094)
and a root endophyte (China, FJ613838). The root
from which this
Ceratobasidium
spp. was isolated was
terrestrial (
Vanilla
is hemiepiphytic, or simultaneous-
ly terrestrial and epiphytic, Porras-Alfaro and Bayman
2007).
Virulence of Ceratobasidium on rice.— Ceratobasi-
dium
isolates from orchids induced signs characteris-
tic of sheath blight on detached rice leaves, but at low
incidence (FIG. 2A). The isolate of
R. solani
used as a
positive control caused significantly higher disease
incidence than all
Ceratobasidium
spp. isolates (85%,
P
#0.0001) and the two isolates of
R. solani
from
potato (RS1AP and BS69, both belonging to AG-3)
(FIG. 2A). There were no significant differences in
disease incidence among the nine
Ceratobasidium
spp. isolates and a
Tulasnella
spp. isolate from orchids
(
P
#0.0001). Uninoculated controls had no lesions.
Severity on rice plants in greenhouse tests.—Ceratobasi-
dium
spp. isolates from orchids varied in severity of
sheath blight, 0.3–1.4%, with only one isolate signif-
icantly greater than zero (Q1.1M14, FIG. 2B). All
isolates from orchids caused significantly less severe
disease than the
R. solani
rice pathogen, which had a
mean 34.5%severity (
P
#0.0001, FIG. 2B). There
were significant differences among the
Ceratobasi-
dium
spp. isolates from orchids, with lowest disease
severity in isolates Q1M19 (from the epiphytic orchid
Maxillaria
sp.), Q1M13 (from the epiphytic orchid
Notylia
sp.) and Q1M161.2 (from the lithophytic
orchid
Epidendrum melinantum
2). The last two
isolates mentioned also caused low disease incidence
in detached leaves (FIG.2A). The isolate from
Epidendrum melinanthum
1 in the
Thanatephorus
clade, which might be expected to be pathogenic, was
relatively low in both severity and disease incidence.
Biocontrol of R. solani on rice plants by Ceratobasi-
dium spp. isolates from orchids.—Ceratobasidium
spp.
isolates protected rice plants from
R. solani
when the
disease severity was significantly less than that on
plants inoculated with
R. solani
alone (the positive
control) (Sneh et al. 2004). In general, disease
severity was low: 16.5%in the positive control
FIG. 2. Sheath blight incidence and severity caused by
isolates of
Ceratobasidium
and
R. solani
on rice leaves and
plants. A. Disease incidence. B. Disease severity. Column 1:
R. solani
isolate from rice in Colombia (positive control)
(TABLE I). Columns 2–10:
Ceratobasidium
isolate from
orchids. Columns 11–12: negative controls without fungi
(with and without sterile agar plug). Soil was not pasteur-
ized. Column 13–15:
Ceratobasidium
from orchid in Puerto
Rico (Otero et al. 2002);
R. solani
RS1AP and BS69 are from
potato in USA, AG-3;
Tulasnella
from
Vanilla
in Puerto Rico
(Porras-Alfaro and Bayman 2007). Bars with the same letter
were not significantly different (
P
#0.05) according to
Duncan’s multiple range tests. Vertical lines: standard
deviation (s.d). nd: not determined.
MOSQUERA-ESPINOSA ET AL.: ORCHID MYCORRHIZAE AND DISEASE BIOCONTROL 145
(FIG. 3) vs. 34.5%(FIG. 2B) in the virulence experi-
ment (above). This difference probably was due to
environmental differences between the times of the
two experiments, although they were run in the same
greenhouse and both experiments were repeated. In
all treatments with
Ceratobasidium
spp. there was
significant reduction in disease severity compared to
the positive control; severity was 5.4–10.6%(
P
#
0.0001, FIG. 3). Differences among
Ceratobasidium
isolates were significant, with plants inoculated with
isolates Q2.2M14 (from the terrestrial orchid
Cranchis
sp. 2) and Q1M19 (from the epiphytic orchid
Maxillaria
sp.) showing lowest severity, 5.4%and
7.2%respectively. A few uninoculated control plants
developed lesions, perhaps as a result of native
Rhizoctonia
in the soil, but disease severity on control
plants was not significantly greater than zero.
Effect of N and soil type on virulence and biocontrol by
Ceratobasidium spp.—
In both virulence and bio-
control experiments, high N (250 K N/ha) signifi-
cantly increased severity of sheath blight (
P
50.02
and
P
#0.0001 respectively; TABLE II). Disease
severity was slightly greater when pasteurized soil
was added compared to unpasteurized soil; this
difference was significant in the biocontrol experi-
ment (
P
50.03; 7.8%vs. 7.4%) but was not significant
in the virulence experiment.
DISCUSSION
Ceratobasidium spp. as mycorrhizal symbionts of
orchids.—
Orchids live in diverse habits and associate
with diverse mycorrhizal fungi (Kottke et al. 2008,
Valadares et al. 2012). Many tropical, epiphytic
orchids, which include the majority of species, are
associated with
Ceratobasidium
spp. (Otero et al.
2002). Tropical, terrestrial orchids are largely unstud-
ied in terms of mycorrhizal associations, but several of
those studied also form associations with
Ceratobasi-
dium
spp. (Athipunyakom et al. 2004, Porras-Alfaro
and Bayman 2007). Although some orchid species are
highly specific for particular groups within
Ceratoba-
sidium
spp. (Otero et al. 2004, 2007, 2011) overall
patterns of specificity are not clear. This is partly
because
Ceratobasidium
spp. is slow to form tele-
omorphs in culture (Sneh et al. 1991) and thus
difficult to identify without DNA sequencing and
partly because mycorrhizae of relatively few tropical
orchid species have been studied.
The present study makes three novel contributions
to our knowledge of orchid mycorrhizal relationships:
relating groups of mycorrhizal fungi with orchid
habitat identifying orchid mycorrhizal fungi from
Colombia, which has more than 3000 species of
orchids (Calderon 2006), none of whose mycorrhizal
fungi have been identified except for our preliminary
report (Mosquera-Espinosa et al. 2010); testing
pathogenicity and biocontrol of
Ceratobasidium
spp.
from orchids on other plants.
The relationships among
Ceratobasidium
isolates
reflected host substrate: Fungi from epiphytic orchids
formed a clade apart from those from terrestrial
orchids (the sample of lithophytic orchids was too
small to be conclusive) (FIG. 1). This association
between habit and phylogeny was expected for two
reasons. First, many groups of orchids have switched
mycorrhizal partners, especially mycoheterotrophic
orchids (Selosse and Roy 2009, Otero et al. 2011);
second, soil and tree bark are different habitats, and
FIG. 3. Effect of isolates of
Ceratobasidium
on disease
severity of
R. solani
on rice. Column 1:
R. solani
alone
(positive control). Columns 2–10:
Ceratobasidium
isolate
from orchids inoculated 2 d before pathogen. Columns 11–
12: negative controls without either pathogen or biocontrol
strain (with and without sterile agar plug). Soil was not
pasteurized. Bars with the same letter were not significantly
different (
P
#0.05) according to Duncan’s multiple range
tests. Vertical lines: standard deviation (s.d).
TABLE II. Effect of nitrogen on severity of sheath blight
disease in pathogenicity and biocontrol experiments
a
Nitrogen dose
added
%disease severity in
pathogenicity tests
%disease severity in
biocontrol tests
250 kg N/ha 3.8 a 9.3 a
150 kg N/ha 3.3 b 7.4 b
0 kg N/ha 2.9 b 6.0 c
a
Plants in the greenhouse were inoculated with
Ceratoba-
sidium
(pathogenicity) or
Ceratobasidium
+
R. solani
(biocontrol). Each value is the mean of all
Ceratobasidium
treatments. Within each column, values followed by the
same letter were not significantly different (
P
#0.05)
according to Duncan’s test.
146 MYCOLOGIA
different groups of fungi are likely to dominate. For
example, fungi in roots of
Vanilla
differed significantly
between substrates, with
Ceratobasidium
spp. more
common in roots in soil and
Tulasnella
spp. more
common in roots on tree bark (Porras-Alfaro and
Bayman 2007). However, the phylogenetic relation-
ships among
Ceratobasidium
spp. isolates also reflected
host phylogeny, at least at subfamily rank (FIG. 1). This
was due to the association between orchid habitat and
systematics; all the terrestrial orchids sampled be-
longed to the relatively primitive subfamily Orchidoi-
deae, whereas the epiphytic orchids belonged to the
derived subfamily Epidendroideae.
Thanatephorus
formed a clade within
Ceratobasi-
dium
(FIG. 1) implying that
Ceratobasidium
as cur-
rently defined is paraphyletic, as has been reported in
Gonza´lez et al. (2001), Moncalvo et al. (2006), Porras-
Alfaro and Bayman (2007). This clade was composed
mostly of pathogens, including a rice pathogen from
Colombia but also included mycorrhizal fungi of
orchids in Colombia and Puerto Rico.
The ability of
Ceratobasidium
spp. isolates to
stimulate germination and growth of orchid seeds
was predicted by phylogenetic relationships, suggest-
ing a genetic basis for this behavior (Otero et al. 2004,
2005). We therefore expected that closely related
isolates would be very similar in disease severity and
biocontrol ability on rice, but there was no evidence
of this (FIG. 2). The fact that most of the isolates
showed pathogenicity on rice (FIG.2), although
slight, suggests that they might be at least partly
parasitic in nutritional mode. Although a number of
Ceratobasidium
species are plant pathogens, the
nutrition of orchid mycorrhizal species is unknown.
Understanding their nutrition might be useful in
conservation of the orchid species which depend on
them for seed germination (Otero et al. 2007).
The
Ceratobasidium
spp. isolates described here
came from the cortical cells of orchid roots with
visible pelotons (Mosquera-Espinosa et al. 2010),
which suggests that the fungi had a functional
mycorrhizal relationship with their orchid hosts
(Hadley and Williamson 1972, Sua´rez et al. 2006).
However, not all fungi in orchid roots are mycorrhizal
andwedidnottestfunctionalaspectsofthe
association. Symbiotic seed germination experiments
demonstrated a functional mycorrhizal relationship
with fungi closely related to the ‘‘epiphytic’’ clade
(FIG. 1) (Otero et al. 2004, 2005).
Ceratobasidium species as plant pathogens and
biocontrol agents.—
In this study
Ceratobasidium
spp.
isolated from orchids caused lower incidence and
severity of disease on rice than
R. solani
AG1-1A
isolated from rice (FIG. 2). Studies have shown that
some
Rhizoctonia
AGs are more virulent to the host
from which they were isolated than on other hosts
(Bolkan and Ribeiro 1985, Bernardes de Assis et al.
2008). Some authors have recommended that viru-
lence tests should score disease from the lowest level
to the highest to identify hypovirulent
Ceratobasidium
spp. isolates with biocontrol potential against soil-
borne pathogens (Sneh et al. 2004), as we do here.
Nonpathogenic
Ceratobasidium
spp. have been
isolated from soil and some show biocontrol activity
against soilborne plant pathogens. They have been
used against
R. solani
causing damping-off in a variety
of crops: beans (Xue et al. 1998, Wen et al. 2005),
corn (Pascual et al. 2000), soybeans (Khan et al.
2005), cotton and radish (Ichielevich-Auster et al.
1985, Sneh et al. 2004, Jabaji-Hare and Neate, 2005),
cucumber (Villajuan-Abgona et al. 1996) and grasses
(Burpee and Goulty 1984). The relationship of these
groups to the
Ceratobasidium
species that form
mycorrhizae with orchids is not clear, but in some
cases they are closely related (FIG. 1; Otero et al. 2002,
2004, 2007; Porras-Alfaro and Bayman 2007). Biocon-
trol is sometimes more effective when the biocontrol
agent is related to the pathogen, because their
requirements are more likely to be similar.
However, as with any potential biocontrol agent,
the potential pathogenicity of these isolates on the
hostplantmustbeconsideredcarefully.Some
anastomosis groups (AGs) of
Thanatephorus
and
Ceratobasidium
are pathogenic on a narrow range of
hosts (e.g. AG-3 on potato) whereas others attack a
range of hosts (e.g. AG4 in different crops) (Bolkan
and Ribeiro 1985, Agrios 2002). Although all the
Ceratobasidium
spp. isolates tested were significantly
less virulent on rice than an isolate of
R. solani
that
grouped with AG1-1A, they were still capable of
causing lesions typical of sheath blight (FIG. 4). Many
biocontrol agents have a certain level of virulence to
the host (Burns and Benson 2000). Low infection
does not necessarily imply a decrease in yield, and in
some cases may increase yield and plant success,
mainly by the production of plant growth-promoting
substances (Sneh 1998, Sneh et al. 2004).
Some
Ceratobasidium
spp. isolates produced abun-
dant mycelium on rice plant tissues and surrounding
soil (FIG.5A,B),aswellassclerotia(FIG.5C);
extensive colonization is usually considered a favor-
able sign for biocontrol. Studies have not described
this colonization in BNR used for biocontrol; on the
contrary, they have been described as slow-growing
compared to soilborne pathogens (Sneh et al. 2004).
Relative efficacy of Ceratobasidium spp. isolates.—
Differences in biocontrol among
Ceratobasidium
spp.
isolates were significant in some cases (FIG. 3). It is
MOSQUERA-ESPINOSA ET AL.: ORCHID MYCORRHIZAE AND DISEASE BIOCONTROL 147
interesting that those most effective at biocontrol
were not those that were least virulent (FIGS. 2, 3).
The most effective biocontrol occurred with a
Ceratobasidium
spp. isolate from the terrestrial orchid
Cranichis
sp. 2 (Q2.2M14) (FIG. 3). This isolate had
low disease severity in pathogenicity tests, although
not as low as some other isolates (FIG. 2). The
combination of high protection in the biocontrol
experiment and low severity in the pathogenicity
experiments suggests that this isolate is the most
promising candidate for biocontrol of rice sheath
blight. A further advantage of Q2.2M14 is that it is a
fungus isolated from roots of a terrestrial orchid and
is more likely to survive and provide effective
biocontrol in the soil environment than an isolate
from an epiphytic orchid adapted to live on tree bark.
Effect of doses of N and soil on virulence and biocontrol
by Ceratobasidium spp.—
The relationship between
high N and increased disease severity agreed with Savary
et al. (1995), Slaton et al. (2003), Cooke et al. (2006).
Increased severity in pasteurized soil also was expected,
because pasteurization (as well as killing pathogens)
destroys beneficial microorganisms that compete with
pathogens (Alexander 1981, Giri et al. 2005). Chemical
analysis showed that K and Zn occurred at low levels (K
50.34 cmol/kg, Zn 53.6 mg/kg), which could have
had a negative effect on the response of plants to
infection by
R. solani
. These two elements affect rice’s
resistance to soilborne pathogens (Guerrero 1991,
Marschner 1995). However, disease severity in this
study never reached 40%, the threshold at which sheath
blight begins to cause economically significant losses in
rice (Correa et al. 2001, Liu et al. 2009).
These results suggest that where rice is fertilized with N
sheath blight is more likely to cause a significant
reduction in yield. N affects physiological processes
involved in rice yield (leaf growth, photosynthesis,
tillering etc., Jaramillo et al. 2003). Growers who apply
N fertilizers to increase yield may not realize that they are
predisposing the plant to attack by fungal pathogens
such as
R. solani
(Fedearroz 2000, Slaton et al. 2003,
Cooke et al. 2006). Rice growers who fertilize are
therefore those most in need of effective biocontrol
strategies. Inducing suppressive soils by inoculation with
biocontrol organisms is an established strategy for
control of pathogens (Sneh 1998). Such a strategy could
be used with
Ceratobasidium
to protect rice fields from
attack by
R. solani
, minimizing its adverse effects on yield.
This study provides evidence that
Ceratobasidium
isolates from orchids may be effective at biocontrol
of
Rhizoctonia
sheath blight of rice and have low
virulence on rice (under greenhouse conditions) with
potential for application as part of an IPM program.
However, pathogenicity on other hosts, particularly
on crop plants that are grown with (or in rotation
with) rice, and mode of action should be tested
before field trials can be considered.
ACKNOWLEDGMENTS
We thank the Rice Pathology Group at CIAT, Palmira,
Colombia, for assistance, the NSF-sponsored CREST-
CATEC program at UPR Rı´o Piedras (HRD 0734826) for
support, and Marc Cubeta for the
R. solani
AG-3 isolates.
The first author was supported by a fellowship from
COLCIENCIAS Doctorados Nacionales 2006. The authors
declare no conflicts of interest.
LITERATURE CITED
Agrios GN. 2002.
Fitopatologı
´a
.2nded.Me´xico, DF:
Editorial Limusa S.A. 830 p.
FIG. 5. Growth and colonization by
Ceratobasidium
isolates in biocontrol experiments on rice. A. growth on
the surface of the plant (black arrows); B. on soil surface
around the plant (black and white arrows); C. production of
sclerotia on soil (red arrows). Bars 51 cm.
FIG. 4. Differences in severity of sheath blight on rice plants
inoculated with: A.
R. solani
isolate from rice in Colombia
(positive control) (TABLE I) (35%); B.
Ceratobasidium
Q1M16
1.2, from the orchid
Epidendrum melinanthum
2(1%); C.
Ceratobasidium
Q1M13, from the orchid
Notylia
sp. (2%).
148 MYCOLOGIA
Alexander M. 1981.
Introduccio´n a la microbiologı
´a del suelo
.
1st ed. Me´ xico, DF: AGT Editor S.A. 491 p.
Arditti J. 1992. Fundamentals of orchid biology. New York:
John Wiley & Sons. 483 p.
Athipunyakom P, Manoch L, Pileuk C. 2004. Isolation and
identification of mycorrhizal fungi from 11 terrestrial
orchids. Kasetsart J Nat Sci 36:216–228.
Bernardes de Assis J, Peyer P, Rush MC, Zala M, McDonald
BA, Ceresini P. 2008. Divergence between sympatric
rice and soybean-infecting populations of
Rhizoctonia
solani
anastomosis group-1 IA. Phytopathology 98:
1326–1333, doi:10.1094/PHYTO-98-12-1326
Bolkan HA, Ribeiro WRC. 1985. Anastomosis groups and
pathogenicity of
Rhizoctonia solani
isolates from Brazil.
Plant Dis 69:599–601, doi:10.1094/PD-69-599
Burns J, Benson M. 2000. Biocontrol of damping-off of
Catharanthus roseus
caused by
Pythium ultimum
with
Trichoderma virens
and binucleate
Rhizoctonia
fungi.
Plant Dis 84:644–648, doi:10.1094/PDIS.2000.84.6.644
Burpee L, Goulty G. 1984. Suppression of brown patch
disease of creeping bentgrass by isolates of nonpatho-
genic
Rhizoctonia
spp. Phythopathology 74:692–694,
doi:10.1094/Phyto-74-692
Caldero´n Sa´enz E. 2006. Libro rojo de plantas de Colombia.
Vol. 6. Orquı´deas, primera parte. Bogota´: Instituto von
Humboldt. 828 p.
Cartwright R, Lee F. 2001. Management of rice diseases. In:
Slaton NA, ed. Rice production handbook. Misc. Publ.
192. Little Rock, Arkansas Coop. Ext. Serv. p 87–100.
Clements MA, Otero JT, Miller JT. 2011. Phylogenetic
relationships in Pterostylidinae (Cranichideae: Orchi-
daceae): combined evidence from nuclear and plastid
DNA sequences. Aust J Bot 59:99–117, doi:10.1071/
BT10190
Cooke B, Gareth D, Kaye B. 2006. The epidemiology of
plant diseases, 2nd ed. The Netherlands: Springer.
576 p.
Correa F, Meneses R, Gutie´ rrez A, Garcı´a A, Antigua G,
Go´ mez J, Calvert L. 2001. Guı´a para el trabajo de
campo en el manejo integrado de plagas del arroz. Cali,
Colombia: CIAT, IIA, FLAR. 72 p.
Cuevas A. 2005. Cincuenta razones para sembrar Fedearroz
50. Fedearroz 53:32–39.
Charlton ND, Carbone I, Tavantzis SM, Cubeta MA. 2008.
Phylogenetic relatedness of the M2 double-stranded
RNA in
Rhizoctonia
fungi. Mycologia 100:555–564,
doi:10.3852/07-108R
Chaudhary RC, Nanda JS, Tran DV. 2003. Guı´a para
identificar las limitaciones de campo en la produccio´n
de arroz. Published online at: ftp://ftp.fao.org/
docrep/fao/006/y2778s/y2778s00.pdf. [last accessed
20 Jun 2012].
Dressler RL. 1993. Phylogeny and classification of the
orchid family. London: Cambridge Univ. Press. 313 p.
FAO. 1985. Manual para pato´ logos vegetales. 2nd ed. Chile:
FAO. 438 p.
FEDEARROZ. 2000. Manejo y conservacio´n de suelos para la
produccio´ n de arroz en Colombia. Bogota´: Fedearoz. 78p.
Ferrero A, Nguyen N. 2003. The sustainable development of
rice-based production systems in the world. FAO, Italy.
Published on line at: http://www.fao.org/docrep/008/
y5682e/y5682e0g.htm. [last accessed 20 Jun 2012].
Flanagan NS, Peakall R, Clements MA, Otero JT. 2006.
Conservation of difficult taxonomic groups: the case of
the Australian orchid,
Microtis angusii
. Conserv Genet
7:847–859, doi:10.1007/s10592-006-9119-8
Giri B, Giang PH, Kumari R, Prasad R, Varma A. 2005.
Microorganisms in soils: roles in genesis and functions.
In: Buscot F, Varma A, eds. Soil biology. Vol. 3. Berlin:
Springer-Verlag. 419 p.
Go´mez K, Go´mez A. 1984. Statistical procedures for
agricultural research. 2nd ed. New York: John Wiley
& Sons. 608 p.
Gonza´lez D, Carling DE, Kuninaga S, Vilgalys R, Cubeta MA.
2001. Ribosomal DNA systematics of
Ceratobasidium
and
Thanatephorus
with
Rhizoctonia
anamorphs. Myco-
logia 93:1138–1150, doi:10.2307/3761674
Guerrero R. 1991. Fertilizacio´ n de cultivos en clima ca´lido.
2nd ed. Bogota´: Mono´ meros Colombo-Venezolanos.
312 p.
Hadley G, Williamson B. 1972. Features of mycorrhizal
infection in some Malayan orchids. New Phytol 71:
1111–1118, doi:10.1111/j.1469-8137.1972.tb01989.x
Ichielevich-Auster M, Sneh B, Koltin Y, Barash I. 1985.
Suppression of damping-off caused by
Rhizoctonia
species
by a nonpathogenic isolate of
R. solani
. Phytopathology
75:1080–1084, doi:10.1094/Phyto-75-1080
Jabaji-Hare S, Neate SM. 2005. Nonpathogenic binucleate
Rhizoctonia
spp. and benzothiadiazole protect cotton
seedlings against
Rhizoctonia
damping-off and
Alter-
naria
leaf spot in cotton. Phytopathology 95:1030–
1036, doi:10.1094/PHYTO-95-1030
Jaramillo S, Pulver E, Duque MC. 2003. Efecto del manejo
de la fertilizacio´ n nitrogenada en arroz de riego, sobre
la expresio´ n del potencial de rendimiento de lı´neas
e´ lites y cultivares comerciales. Palmira, Colombia:
Fondo Latinoamericano de Arroz de Riego (FLAR) y
Centro Internacional de Agricultura Tropical (CIAT).
12 p.
Khan FU, Nelson BD, Helms TC. 2005. Greenhouse
evaluation of binucleate
Rhizoctonia
for control of
R.
solani
in soybean. Plant Dis 89:373–379, doi:10.1094/
PD-89-0373
Kottke I, Haug I, Setaro S, Suarez JP, Weiß M, Preusing M,
Nebelb M, Oberwinkler F. 2008. Guilds of mycorrhizal
fungi and their relation to trees, ericads, orchids and
liverworts in a Neotropical mountain rain forest. Basic
Appl Ecol 9:13–23, doi:10.1016/j.baae.2007.03.007
Liu G, Jia Y, Correa-Victoria F, Prado G, Yeater K, McClung A,
Correll J. 2009. Mapping quantitative trait loci respon-
sible for resistance to sheath blight in rice. Phytopathol-
ogy 99:1078–1084, doi:10.1094/PHYTO-99-9-1078
Marschner H. 1995. Mineral nutrition of higher plants. 2nd
ed. New York: Academic Press. 889 p.
Moncalvo JM, Nilsson RH, Koster B, Dunham SM, Bernauer
T, Matheny PB, Porter TM, Margaritescu S, Weiß M,
Garnica S, Danell E, Langer G, Langer E, Larsson E,
Larsson KH, Vilgalys R. 2006. The cantharelloid clade:
dealing with incongruent gene trees and phylogenetic
MOSQUERA-ESPINOSA ET AL.: ORCHID MYCORRHIZAE AND DISEASE BIOCONTROL 149
reconstruction methods. Mycologia 98:937–948, doi:10.
3852/mycologia.98.6.937
Mosquera-Espinosa AT, Bayman P, Otero JT. 2010.
Cerato-
basidium
como hongo micorrı´zico de orquı´deas en
Colombia. Acta Agron 59:316–326.
Otero JT, Ackerman JD, Bayman P. 2002. Diversity and host
specificity of endophytic
Rhizoctonia
-like fungi from
tropical orchids. Am J Bot 89:1852–1858, doi:10.3732/
ajb.89.11.1852
———, ———, ———. 2004. Differences in mycorrhizal
preferences between two tropical orchids. Molec Ecol
10:207–212.
———, Bayman P, Ackerman JD. 2005. Variation in
mycorrhizal performance in the epiphytic orchid
Tolum-
nia variegata
in vitro: the potential for natural selection.
Evol Ecol 19:29–43, doi:10.1007/s10682-004-5441-0
———, Flanagan N, Herre A, Ackerman JD, Bayman P.
2007. Widespread mycorrhizal specificity correlates to
mycorrhizal function in the Neotropical, epiphytic
orchid
Ionopsis utricularioides
(Orchidaceae). Am J
Bot 94:1944–1950, doi:10.3732/ajb.94.12.1944
———, Thrall PH, Clements M, Burdon JJ, Miller JT. 2011.
Codiversification of orchids (Pterostylidinae) and their
associated mycorrhizal fungi. Aust J Bot 59:480–497,
doi:10.1071/BT11053
Pascual C, Raymundo A, Hyakumachi M. 2000. Efficacy of
hipovirulent binucleate
Rhizoctonia
spp. to control
banded leaf and sheath blight in corn. J Gen Plant
Pathol 66:95–102, doi:10.1007/PL00012928
Porras-Alfaro A, Bayman P. 2007. Mycorrhizal fungi of
Vanilla
: diversity, specificity and effects on seed
germination and plant growth. Mycologia 99:510–525,
doi:10.3852/mycologia.99.4.510
Rasmussen HN. 2002. Recent developments in the study
of orchid mycorrhiza. Plant Soil 244:149–163, doi:10.
1023/A:1020246715436
———, Rasmussen FN. 2009. Orchid mycorrhiza: implica-
tions of a mycophagous life style. Oikos 118:334–345,
doi:10.1111/j.1600-0706.2008.17116.x
Savary S, Castilla P, Elazegui A, McLaren G, Ynalvez M, Teng
S. 1995. Direct and indirect effects of nitrogen supply
and disease source structure on rice sheath blight spread.
Phytopathology 85:959–965, doi:10.1094/Phyto-85-959
Selosse MA, Roy M. 2009. Green plants that feed on fungi:
facts and questions about mixotrophy. Trends Plant Sci
14:64–70, doi:10.1016/j.tplants.2008.11.004
Slaton N, Cartwright R, Meng J, Gbur E, Norman R. 2003.
Sheath blight severity and rice yield as affected by
nitrogen fertilizer rate, application method and fungi-
cide. Agron J 95:1489–1496, doi:10.2134/agronj2003.
1489
Sneh B. 1998. Use of non-pathogenic or hypovirulent fungal
strains to protect plants against closely related fungal
pathogens. Biotechnol Adv 16:1–2, doi:10.1016/S0734-9750
(97)00044-X
———, Burpee L, Ogoshi A. 1991. Identification of
Rhizoctonia
species. St Paul, Minnesota: American
Phytopathological Society. 133 p.
———, Yamoah E, Stewart A. 2004. Hypovirulent
Rhizocto-
nia
spp. isolates from New Zealand soils protect radish
seedlings against Damping-off caused by
R. solani
.NZ
Plant Protect 57:54–58.
Sua´ rez JP, Michael W, Abele A, Garnica S, Oberwinkler F,
Kottke I. 2006. Diverse tulasnelloid fungi form mycor-
rhizas with epiphytic orchids in an Andean cloud
forest. Mycol Res 110:1257–1270, doi:10.1016/j.mycres.
2006.08.004
Valadares RB, Pereira MC, Otero JT, Cardoso EJ. 2012.
Narrow fungal mycorrhizal diversity in a population of
the orchid
Coppensia doniana
. Biotropica 44:114–122,
doi:10.1111/j.1744-7429.2011.00769.x
Villajuan-Abgona R, Kageyama K, Hyakurnachi M. 1996.
Biocontrol
of Rhizoctonia
damping-off of cucumber by
non-pathogenic binucleate
Rhizoctonia
. Eur J Plant
Pathol 102:227–235, doi:10.1007/BF01877961
Wen K, Seguin P, St-Arnaud M, Jabaji-Hare S. 2005. Real-
time quantitative RT-PCR of defense-associated gene
transcripts of
Rhizoctonia solani
-infected bean seedlings
in response to inoculation with a nonpathogenic
binucleate
Rhizoctonia
isolate. Phytopathology 95:345–
353, doi:10.1094/PHYTO-95-0345
Xue L, Charest PM, Jabaji-Hare SH. 1998. Systemic
induction of peroxidases, 1,3-b-glucanases, chitinases
and resistance in bean plants by binucleate
Rhizoctonia
species. Phytopathology 88:359–365, doi:10.1094/
PHYTO.1998.88.4.359
150 MYCOLOGIA
... W. circinata mycelia grown on PDA were scraped, weighed and diluted in autoclaved distilled water to obtain 2 suspensions with concentrations of 5 and 10 g L -1 . The leaves and sheaths were sprayed at 55 DAS and the plants kept under high humidity (95-100 %), with temperatures of 27-30 ºC during the day and 22-25 ºC at night (Prabhu et al. 2002, Mosquera-Espinosa et al. 2013). The plants were assessed every 2 days, for 10 days, observing the presence or absence of symptoms on the roots, leaves and sheaths. ...
... For all 3 assays, the plants were kept in a greenhouse under high humidity (95-100 %), daytime temperatures of 27-30 ºC and 22-25 ºC at night (Prabhu et al. 2002, Mosquera-Espinosa et al. 2013. ...
... The present study showed that the two W. circinata application methods (via spraying or mycelial disks applied directly to the soil) did not damage the BRS Tropical rice plants at 20 and 55 DAS. Carvalho et al. (2015) inoculated BRS Primavera rice plants with W. circinata and also found that the fungus is not pathogenic to rice, confirming our results of no pathogenicity during the vegetative or reproductive stages and corroborating Mosquera-Espinosa et al. (2013). ...
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The BRS Tropical rice cultivar was developed for tropical foodplains, but is susceptible to sheath blight, making the Waitea circinata bioagent an important tool in managing the disease. This study aimed to assess the W. circinata mechanisms involved in the parasitism and resistance induction to sheath blight. The in vitro antagonism was assessed by optical microscopy and scanning electron microscopy, followed by quantification of the lytic enzymes involved in parasitism, such as chitinase, glucanase and protease. An effect of the W. circinata mycoparasite against Rhizoctonia solani was observed. The W. circinata application suppressed the sheath blight by up to 65 % and increased the chitinase, glucanase and lipoxygenase activity 72 h after the inoculation and that of the peroxidase 96 h after the inoculation. Thus, W. circinata showed to be efficient in suppressing sheath blight by parasitism and induced resistance via the activation of biochemical mechanisms. KEYWORDS: Oryza sativa ; Rhizoctonia solani ; controle biológico
... Besides forming endomycorrhizal associations with orchids, rhizoctonian fungi can occupy various ecological niches [5]. They have been detected as endophytes of other plant families [6,7], as ectomycorrhizal fungi on tree roots [7,8] or as plant pathogens [9,10]. Their predominant nutritional mode is, however, saprotrophy via the decomposition of dead organic matter in the soil [2,11,12]. ...
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Orchid mycorrhizal fungi (OMF) from the rhizoctonia aggregate are generally considered to be soil saprotrophs, but their ability to utilize various nutrient sources has been studied in a limited number of isolates cultivated predominantly in liquid media, although rhizoctonia typically grow on the surface of solid substrates. Nine isolates representing the key OMF families (Ceratobasidiaceae, Tulasnellaceae and Serendipitaceae), sampled in Southern France and the Czech Republic, were tested for their ability to utilize carbon (C), nitrogen (N) and phosphorus (P) sources in vitro in both liquid and solid media. The isolates showed significant inter-and intra-familiar variability in nutrient utilization, most notably in N sources. Isolates produced generally larger amounts of dry biomass on solid medium than in liquid one, but some isolates showed no or limited biomass production on solid medium with particular nutrient sources. The largest amount of biomass was produced by isolates from the family Ceratobasidiaceae on most sources in both medium types. The biomass production of Tulasnellaceae isolates was affected by their phylogenetic relatedness on all sources and medium types. The ability of isolates to utilize particular nutrients in a liquid medium but not a solid one should be considered when optimizing solid media for symbiotic orchid seed germination and in understanding of OMF functional traits under in situ conditions.
... Moreover, this fungus was found to inhabit soils as saprophyte and roots as endophyte by exerting growth promotion on olive trees. Growth promotion of BNR has already been observed in Scot pine and apple (Kaparakis and Sen 2006;Grönberg et al. 2011;Manici and Caputo 2020); while in orchids BNR has been reported to exert growth promotion through mycorrhizal-like mechanisms (Mosquera-Espinosa et al. 2013). Other beneficial effects of BNR, such as antagonistic and defence-inducing towards root pathogens, in both woody and herbaceous crops have been largely reported in literature (Sneh 1996;Villajuan-Abgona et al. 1996;Poromarto et al. 1998;Muslim (Sneh et al. 1996). ...
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Purpose Binucleate Rhizoctonia sp. (BNR), indigenous to old olive groves of the Apulia region (Southern Italy), and its relationship with olive trees were investigated to search for beneficial soil microorganisms suitable for supporting plant growth and crop productions in degraded semi-arid soils. Methods Binucleate Rhizoctonia sp. was first isolated from five ancient olive groves using olive tree plantlets as "living baits". The functional relationship of BNR with olive trees was estimated with in-pot growth assays, using native soil amended with an artificial inoculum from the native BNR isolates. BNR was then quantified in the olive groves by digital PCR using a TaqMan assay specifically designed on the ITS regions of the native AG-A isolates. Results Digital PCR was able to discriminate BNR (Ceratobasidium sp.) from the phylogenetically close Basidiomycetes such as Rhizoctonia solani (Thanatephorus cucumeris). This assay detected an amount of BNR in five olive groves ranging from traces up to 105 target gene copies per µL of DNA. When artificially added to native soil samples, BNR AG-A increased olive tree growth by 50% and 25% respectively under constant optimal water availability and repeatedly induced water stress. Indole-3-acetic acid production was demonstrated for the BNR AG-A isolates, thus, in part, explaining growth promotion. Conclusion Thanks to the absolute quantification of fungal DNA fragments in soil with digital PCR, findings suggest that new soil functional indicators, such as the growth promoting Rhizoctonia AG-A, may be identified with this research approach to be investigated as a biotic resource for enhancing soil ecosystem services.
... A plant pathogenic fungus, Diaporthae spp., and an organic matter decomposer, Petriella spp., were more represented at medium densities of P. goodeyi (151-533 nematodes × 100 cc soil −1 ). The latter was also more represented, with Wallemia and the endophytes Phialocephala and Ceratobasidium spp., an orchid mycorrhyza and biocontrol agent, respectively (Mosquera-Espinosa et al., 2013), in samples with higher Helicotylenchus spp. numbers (Supplementary Table 6). ...
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Agriculture affects soil and root microbial communities. However, detailed knowledge is needed on the effects of cropping on rhizosphere, including biological control agents (BCA) of nematodes. A metabarcoding study was carried out on the microbiota associated with plant parasitic and other nematode functional groups present in banana farms in Tenerife (Canary Islands, Spain). Samples included rhizosphere soil from cv Pequeña Enana or Gruesa and controls collected from adjacent sites, with the same agroecological conditions, without banana roots. To characterize the bacterial communities, the V3 and V4 variable regions of the 16S rRNA ribosomal gene were amplified, whereas the internal transcribed spacer (ITS) region was used for the fungi present in the same samples. Libraries were sequenced with an Illumina MiSeq TM in paired ends with a 300-bp read length. For each sample, plant parasitic nematodes (PPN) and other nematodes were extracted from the soil, counted, and identified. Phytoparasitic nematodes were mostly found in banana rhizosphere. They included Pratylenchus goodeyi, present in northern farms, and Helicotylenchus spp., including H. multicinctus, found in both northern and southern farms. Metabarcoding data showed a direct effect of cropping on microbial communities, and latitude-related factors that separated northern and southern controls from banana rizosphere samples. Several fungal taxa known as nematode BCA were identified, with endophytes, mycorrhizal species, and obligate Rozellomycota endoparasites, almost only present in the banana samples. The dominant bacterial phyla were Proteobacteria, Actinobacteria, Planctomycetes, Bacteroidetes, Chloroflexi, and Acidobacteria. The ITS data showed several operational taxonomic units (OTUs) belonging to Sordariomycetes, including biocontrol agents, such as Beauveria spp., Arthrobotrys spp., Pochonia chlamydosporia, and Metarhizium anisopliae. Other taxa included Trichoderma harzianum, Trichoderma longibrachiatum, Trichoderma virens, and Fusarium spp., together with mycoparasites such as Acrostalagmus luteoalbus. However, only one Dactylella spp. showed a correlation with predatory nematodes. Differences among the nematode guilds were found, as phytoparasitic, free-living, and predatory nematode groups were correlated with specific subsets of other bacteria and fungi. Crop cultivation method and soil texture showed differences in taxa Ciancio et al. Banana Rhizosphere Metabarcoding representations when considering other farm and soil variables. The data showed changes in the rhizosphere and soil microbiota related to trophic specialization and specific adaptations, affecting decomposers, beneficial endophytes, mycorrhizae, or BCA, and plant pathogens.
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Mycorrhizal symbiosis has been related to the coexistence and community assembly of coexisting orchids in few studies despite their obligate dependence on mycorrhizal partners to establish and survive. In hyper-diverse environments like tropical rain forests, coexistence of epiphytic orchids may be facilitated through mycorrhizal fungal specialization (i.e., sets of unique and dominant mycorrhizal fungi associated with a particular host species). However, information on the role of orchid mycorrhizal fungi (OMF) in niche differentiation and coexistence of epiphytic orchids is still scarce. In this study, we sought to identify the variation in fungal preferences of four co-occurring epiphytic orchids in a tropical rainforest in Costa Rica by addressing the identity and composition of their endophytic fungal and OMF communities across species and life stages. We show that the endophytic fungal communities are formed mainly of previously recognized OMF taxa, and that the four coexisting orchid species have both a set of shared mycorrhizal fungi and a group of fungi unique to an orchid species. We also found that adult plants keep the OMF of the juvenile stage while adding new mycobionts over time. This study provides evidence for the utilization of specific OMF that may be involved in niche segregation, and for an aggregation mechanism where adult orchids keep initial fungal mycobionts of the juvenile stage while adding others.
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Bacterial resistance to antibiotics is a serious public health problem that needs new antibacterial compounds for control. Fungi, including resupinated fungi, are a potential source to discover new bioactive compounds efficient again to bacteria resistant to antibiotics. The inhibitory capacity against the bacterial species was statistically evaluated. All the species (basidiomata and strains) were molecularly characterized with the ITS1-5.8S-ITS2 barcoding marker. The strains Ceraceomyces sp., Fuscoporia sp., Gloeocystidiellum sp., Oliveonia sp., Phanerochaete sp., and Xenasmatella sp. correspond to resupinate Basidiomycetes, and only the strain Hypocrea sp. is an Ascomycete, suggesting contamination to the basidiome of Tulasnella sp. According to the antagonistic test, only the Gloeocystidiellum sp. strain had antibacterial activity against the bacterial species Escherichia coli of clinical interest. Statistically, Gloeocystidiellum sp. was significantly (<0.001) active against two E. coli pathotypes (O157:H7 and ATCC 25922). Contrarily, the antibacterial activity of fungi against other pathotypes of E. coli and other strains such as Serratia sp. was not significant. The antibacterial activity between 48 and 72 h increased according to the measurement of the inhibition halos. Because of this antibacterial activity, Gloeocystidiellum sp. was taxonomically studied in deep combined morphological and molecular characterization (ITS1-5.8S-ITS2; partial LSU D1/D2 of nrDNA). A new species Gloeocystidiellum lojanense, a resupinate and corticioid fungus from a tropical montane rainforest of southern Ecuador, with antibacterial potential against E. coli, is proposed to the science.
Chapter
South America is undoubtedly the cradle of orchid diversity. However, few aspects of the biology of this plant group have been explored in the region. Orchids establish an important relationship with fungi that supply them with nutrients in the early stages of development to stimulate the germination of their tiny seeds. These fungi are called orchid mycorrhizal fungi (OMF), and their interaction with orchids forms a particular group of mycorrhizae: the orchid mycorrhiza (OM). In this chapter, we present the advances of the research conducted in South America, which explores some aspects of this interesting interaction. We have noticed that most studies on OM are academic documents deposited in university library repositories or published in local scientific journals. About half of the studies have focused on determining the diversity of OMF associated with a few orchid species of interest. Studies of phylogeny, morphological, and symbiotic seed germination are other of the main topics addressed. Research on ultrastructure and community ecology is hardly representative, while evolutionary implications, mutualistic networks, and metabolic aspects are the least explored topics. We encourage collaborations with the international scientific community to continue investigating complex questions that allow us to understand the role of mycorrhizas in the evolutionary success of tropical orchids. Moreover, we believe that it is important to propose attractive research that generates interest not only in the academic community but also in ordinary people who have traditionally been related to orchids (e.g., growers) in order to develop the enormous potential of the region in this field.KeywordsMycorrhizal fungi Seed germination Orchidaceae Symbiosis Rhizoctonia-like fungi
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Mycorrhizal symbiosis has been related to the coexistence and community assembly of coexisting orchids in few studies despite their obligate dependence on mycorrhizal partners to establish and survive. In hyper-diverse environments like tropical rain forests, coexistence of epiphytic orchids may be facilitated through mycorrhizal fungal specialization. However, information on the role of orchid mycorrhizal fungi (OMF) in niche differentiation and coexistence of epiphytic orchids is still scarce. In this study, we sought to identify the variation in fungal preferences of four co-occurring epiphytic orchids in a tropical rainforest in Costa Rica by addressing the identity and structure of their endophytic fungal and OMF communities across species and life stages. We show that the endophytic fungal communities are formed mainly of previously-recognized OMF taxa, and that the coexisting orchid species display distinct (OMF) communities while keeping a base of shared fungi. We also found that adult plants keep the OMF of the juvenile stage while adding new mycobionts over time, a strategy that may serve as a complementary mechanism to fulfill the nutritional needs associated with reproduction. This study provides evidence for niche partitioning in coexisting tropical epiphytic orchids through the utilization of specific OMF, and for an aggregation mechanism where adult orchids keep initial fungal mycobionts of the juvenile stage while adding others.
Chapter
Biofertilizers and biological products are increasingly being used to enlarge the productivity of crops. Of these, microbes known as Plant Growth-Promoting Microorganisms (PGPM) are the most valuable as biofertilizers, having the capacity to directly impact the growth and development of plants. Plant Growth-Promoting Fungi (PGPF) and Plant Growth-Promoting Bacteria (PGPB) help crops to face biotic and abiotic stresses by enhancing the defense system and several other parameters related to plant growth. This chapter is focused on explaining the function and positive influence of the PGPF and PGPB on several crops, and also to provide a general view of the application of microorganisms in modern agriculture
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Most of the microbes present in soils are beneficial to the plant and the environment. Soil microbes assist plants in their development and growth and vice versa plants provide nutrition and shelter to the microbes for their development. Plant and microbe interaction enrich the soil in their texture and quality. Soil improvement reduces the dependency of plant on chemical fertilizers and provides many benefits to the plants. Microbes are natural organisms; their processes are slow. Genetic engineering and biotechnology tools may hasten the microbial process and could convert less utilized microbes into more utilization. In today’s scenario, utilizing the microbial approaches in enhancing the productivity of plant is more progressive movement in the direction of sustainable agriculture and clean environment.KeywordsSoil improvementEnvironmentSustainable agricultureMicrobial approachesPGPRsMycorrhiza
Article
Mycorrhizal fungi are essential for the germination of orchid seeds. However, the specificity of orchids for their mycorrhizal fungi and the effects of the fungi on orchid growth are controversial. Mycorrhizal fungi have been studied in some temperate and tropical, epiphytic orchids, but the symbionts of tropical, terrestrial orchids are still unknown. Here we study diversity, specificity and function of mycorrhizal fungi in Vanilla, a pantropical genus that is both terrestrial and epiphytic. Mycorrhizal roots were collected from four Vanilla species in Puerto Rico, Costa Rica and Cuba. Cultured and uncultured mycorrhizal fungi were identified by sequencing the internal transcribed spacer region of nuclear rDNA (nrITS) and part of the mitochondrial ribosomal large subunit (mtLSU), and by counting number of nuclei in hyphae. Vanilla spp. were associated with a wide range of mycorrhizal fungi: Ceratobasidium, Thanatephorus and Tulasnella. Related fungi were found in different species of Vanilla, although at different relative frequencies. Ceratobasidium was more common in roots in soil and Tulasnella was more common in roots on tree bark, but several clades of fungi included strains from both substrates. Relative frequencies of genera of mycorrhizal fungi differed significantly between cultured fungi and those detected by direct amplification. Ceratobasidium and Tulasnella were tested for effects on seed germination of Vanilla and effects on growth of Vanilla and Dendrobium plants. We found significant differences among fungi in effects on seed germination and plant growth. Effects of mycorrhizal fungi on Vanilla and Dendrobium were similar: a clade of Ceratobasidium had a consistently positive effect on plant growth and seed germination. This clade has potential use in germination and propagation of orchids. Results confirmed that a single orchid species can be associated with several mycorrhizal fungi with different functional consequences for the plant.
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We reassessed the circumscription of the cantharelloid clade and identified monophyletic groups by using nLSU, nSSU, mtSSU and RPB2 sequence data. Results agreed with earlier studies that placed the genera Cantharellus, Craterellus, Hydnum, Clavulina, Membranomyces, Multiclavula, Sistotrema, Botryobasidium and the family Ceratobasidiaceae in that clade. Phylogenetic analyses support monophyly of all genera except Sistotrema, which was highly polyphyletic. Strongly supported monophyletic groups were: (i) Cantharellus-Craterellus, Hydnum, and the Sistotrema confluens group; (ii) Clavulina-Membranomyces and the S. brinkmannii-oblongisporum group, with Multiclavula being possibly sister of that clade; (iii) the Sistotrema eximum-octosporum group; (iv) Sistotrema adnatum and S. coronilla. Positions of Sistotrema raduloides and S. athelioides were unresolved, as were basal relationships. Botryobasidium was well supported as the sister taxon of all the above taxa, while Ceratobasidiaceae was the most basal lineage. The relationship between Tulasnella and members of the cantharelloid clade will require further scrutiny, although there is cumulative evidence that they are probably sister groups. The rates of molecular evolution of both the large and small nuclear ribosomal RNA genes (nuc-rDNA) are much higher in Cantharellus, Craterellus and Tulasnella than in the other cantharelloid taxa, and analyses of nuc-rDNA sequences strongly placed Tulasnella close to Cantharellus-Craterellus. In contrast analyses with RPB2 and mtSSU sequences placed Tulasnella at the base of the cantharelloid clade. Our attempt to reconstruct a “supertree” from tree topologies resulting from separate analyses that avoided phylogenetic reconstruction problems associated with missing data and/or unalignable sequences proved unsuccessful.
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The phylogenetic relationships of anastomosis groups (AG) of Rhizoctonia associated with Ceratobasidium and Thanatephorus teleomorphs were determined by cladistic analyses of internal transcribed spacer (ITS) and 28S large subunit (LSU) regions of nuclear-encoded ribosomal DNA (rDNA). Combined analyses of ITS and LSU rDNA sequences from 41 isolates representing 28 AG of Ceratobasidium and Thanatephorus supported at least 12 monophyletic groupings within Ceratobasidium and Thanatephorus. There was strong support for separation of Ceratobasidium and Thanatephorus, however, six sequences representing different AG of Ceratobasidium grouped with certain sequences within the Thanatephorus clade. Phylogenetic analysis of ITS sequence data from 122 isolates revealed 31 genetically distinct groups from Thanatephorus (21 groups) and Ceratobasidium (10 groups) that corresponded well with previously recognized AG or AG subgroups. Although phylogenetic analysis of ITS sequences provided evidence that several AG of Ceratobasidium may be more closely related with some AG from Thanatephorus, these relationships were not as strongly supported by bootstrap analysis.
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Plant disease epidemiology is a dynamic science that forms an essential part of the study of plant pathology. This book brings together a team of 35 international experts. Each chapter deals with an essential component of the subject and allows the reader to fully understand how each exerts its influence on the progress of pathogen populations in plant populations over a defined time scale. Since the first edition of the text was published in 1998, many new developments have occurred in the subjects covered, particularly molecular diagnostics, modelling, fungicide resistance and information technology. The second edition of the book is a comprehensive text on all aspects of plant disease epidemiology that should serve as an invaluable reference work for those involved in this fascinating science of crop plants.
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An understanding of the mineral nutrition of plants is of fundamental importance in both basic and applied plant sciences. The Second Edition of this book retains the aim of the first in presenting the principles of mineral nutrition in the light of current advances. This volume retains the structure of the first edition, being divided into two parts: Nutritional Physiology and Soil-Plant Relationships. In Part I, more emphasis has been placed on root-shoot interactions, stress physiology, water relations, and functions of micronutrients. In view of the worldwide increasing interest in plant-soil interactions, Part II has been considerably altered and extended, particularly on the effects of external and interal factors on root growth and chapter 15 on the root-soil interface. The second edition will be invaluable to both advanced students and researchers.
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This is a large and expensive book. Free copies are not available for distribution. Please do not ask. It can be purchased from Zip Publishing (info@zippublishing.com). This illustrated reference work provided a detailed scientific approach to orchid biology. There are 15 chapters: history (in Asia, Africa, Europe, New Guinea and Australia), including the history of the discovery of orchid reproduction; classification and naming of orchids; evolution of the Orchidaceae, and of plant parts individually; cytology; physiology; phytochemistry; morphology; anatomy; mycorrhiza (including orchid-fungus specificity, seed germination and root characteristics); pollination (with attention to attractants and pollinators); embryology; reproduction (including reproduction through seeds, germination, and sexual and asexual propagation); heredity and breeding; ecology (with an account of the habitats in which orchids exist, as well as notes on climate, carbon fixation, seed dispersal and conservation); and commercial and ethnobotanical uses. Each chapter has a bibliography. -J.W.Cooper This a book. The author cannot send copies. It is available for purchase at www.amazon.com. -Joseph Arditti