Plant Pathology (2010) 59, 712–720
Doi: 10.1111/j.1365-3059.2010.02306.x
Erysiphe trifolii – a newly recognized powdery mildew
pathogen of pea
R. N. Attanayakea, D. A. Glawea,b, K. E. McPheec, F. M. Dugand and W. Chend*
a
Department of Plant Pathology, Washington State University, Pullman WA 99164; bCollege of Forest Resources, University of
Washington, Seattle WA 98195; cDepartment of Plant Sciences, North Dakota State University, Fargo ND 58108; and dUSDA-ARS,
Washington State University, Pullman WA 99164, USA
Diversity of powdery mildew pathogens infecting pea (Pisum sativum) in the US Pacific Northwest was investigated using
both molecular and morphological techniques. Phylogenetic analyses based on rDNA ITS sequences, in combination with
assessment of morphological characters, defined two groups of powdery mildews infecting pea. Group I (five field samples
and three glasshouse samples) had ITS sequences 99% similar to those of Erysiphe pisi in GenBank and exhibited simple,
mycelioid type of chasmothecial appendages typical of E. pisi. Erysiphe pisi is normally considered as the powdery mildew
pathogen of pea. Group II (four glasshouse samples and two field samples) had ITS sequences 99% similar to those of
E. trifolii and produced chasmothecia with dichotomously branched appendages similar to those of E. trifolii. There are
fourteen nucleotide differences in the ITS region between the two groups. The correlation of rDNA ITS sequences with teleomorphic features for each of the two groups confirms their identity. Repeated samplings and artificial inoculations indicate
that both E. pisi and E. trifolii infect pea in the US Pacific Northwest. Erysiphe trifolii is not previously known as a pathogen
of pea. The existence of two distinct powdery mildew species infecting pea in both glasshouse and field environments may
interfere with the powdery mildew-resistance breeding programmes, and possibly explains putative instances of breakdown
of resistance in previously resistant pea breeding lines.
Keywords: chasmothecial appendages, ITS sequences, Pisum sativum, powdery mildew, single nucleotide polymorphism, taxonomy
Introduction
Powdery mildew of pea (Pisum sativum), caused by
Erysiphe pisi (in the past often reported as E. communis
auct. p.p. or E. polygoni auct. p.p.), is a serious disease
worldwide (Falloon & Viljanen-Rollinson, 2001). Two
different Erysiphe pisi varieties have been described in
Braun (1987), i.e. E. pisi var. pisi and E. pisi var. cruchetiana. In addition to species in Pisum, species in Medicago,
Vicia, Lupinus, Lens and a small number of other genera
are infected by E. pisi var. pisi, whereas Lathyrus and
Ononis species are hosts for E. pisi var. cruchetiana
(Braun, 1987).
Powdery mildew adversely affects total biomass,
number of pods per plant, number of seeds per pod,
plant height and number of nodes (Gritton & Ebert,
1975). Yield loss of 10–65% due to the disease has
been documented (Tiwari et al., 1997). Planting resistant cultivars is an economic and environmentally
friendly means to manage the disease. Resistance to
*E-mail: w-chen@wsu.edu
Published online 10 May 2010
712
powdery mildew in pea is controlled by two
recessive, independently inherited genes er-1 and er-2
(Heringa et al., 1969; Tiwari et al., 1997). Gene er-1
confers full resistance, whereas gene er-2 provides
only leaf resistance (Heringa et al., 1969). A third
gene, Er3, has been recently found in Pisum fulvum
and also contributes to genetic resistance to E. pisi
(Fondevilla et al., 2007). Although pea is always a
field crop (glasshouse production is not commercially
cost-effective), early generations of breeding materials
are often produced in the glasshouse, particularly in
temperate regions during winter months, in order to
obtain two generations per year. Even though powdery mildew symptoms are easily recognized, it can
be challenging to determine species assignment for a
given powdery mildew (Glawe, 2008). Determining
the pathogen to species level is very important for
pea breeding programmes because different resistance
genes may confer resistance to different species (Epinat et al., 1993). Taxonomic status of legume powdery mildews is incomplete in scope and not well
understood. For example, E. trifolii has been referred
to as a complex consisting of E. trifolii, E. baeumleri
and E. asteragali (Braun, 1987). The nature of this
No claim to original US government works
Journal compilation ª 2010 BSPP
Erysiphe trifolii on Pisum sativum
species complex remains to be determined (U. Braun,
Martin-Luther-Universität, Institut für Biologie, Germany, personal communication).
Powdery mildew occurs on pea in both glasshouse and
field conditions, and inconsistencies in resistance have
been noted between the two. For example, some pea
breeding lines were resistant to powdery mildew in the
glasshouse, but were susceptible in the field (K.E.
McPhee, unpublished data). Such inconsistent results
could be due to different disease pressures in different
environments, or glasshouse environments may alter the
susceptibility of host plants to powdery mildew strains
(Braun, 1987; Cunnington et al., 2005). Another possible
explanation is the presence of powdery mildew strains
with different host ranges as reported in Cook & Fox
(1992) or even different pathogen species in different
environments.
The objective of this study was to determine if more
than one species of powdery mildew infects pea in the US
713
Pacific Northwest, and if so, whether infections from a
given species are present in both glasshouse and the field.
Materials and methods
Powdery mildew samples
A total of 18 powdery mildew samples (eight from glasshouses and 10 from fields or natural areas) were collected
from pea and other legume plants (Table 1). The eight
glasshouse samples (seven from pea and one from lentil)
were from four disconnected glasshouses located at least
500 m apart from each other. Three of the four glasshouses (all with entrances facing north) are located on the
Pullman campus of Washington State University and
form a triangle. The fourth glasshouse is located 16 km
away in Moscow, Idaho. There was minimum chance of
transferring inoculum from one glasshouse to another
because the glasshouses were usually not planted with
Table 1 Sample designation, host plant, location and sampling date, appendage morphology, ITS sequence group (number of nucleotides considered),
GenBank accession and species determination of powdery mildew (Erysiphe spp.) samples used in this study
Sample
designation
Host
plant
Location
Samples from glasshouses
GH 04
Pisum sativum
Collection
date
Greenhouse 112, 2004
Pullman, WA
GH 05
P. sativum
Greenhouse 112, 2005
Pullman, WA
GH 06
P. sativum
Greenhouse 112, 2006
Pullman, WA
GH 07-119 P. sativum
Greenhouse 119, 2007
Pullman, WA
GH M 07
P. sativum
Greenhouse,
2007
Moscow, ID
GH N 07
P. sativum
Plant Growth
2007
Facility Rm 134,
Pullman, WA
Lif 07
P. sativum
Plant Growth
2007
Facility Rm 134,
Pullman, WA
LGHN 07 Lens culinaris
Greenhouse 119, 2007
Pullman, WA
Samples from agricultural fields or uncultivated areas
EI 08-1
P. sativum
Pullman, WA
2008
EI 08-2
P. sativum
Pullman, WA
2008
FF 06
P. sativum
Fairfield, WA
2006
GE 07
P. sativum
Genesee, ID
2007
LI 08-1
P. sativum
Pullman, WA
2008
LI 08-2
P. sativum
Pullman, WA
2008
SP 07
P. sativum
Pullman, WA
2007
Lathyrus
Lathyrus sp.
Pullman, WA
2007
Medicago Medicago lupulina Pullman, WA
2007
Melilotus
Melilotus albus
Colfax, WA
2006
a
Not available.
Not determined.
b
Plant Pathology (2010) 59, 712–720
Ratio of
appendage
length to
ITS sequence
chasmothecial Appendage group (number GenBank Species
diameter
type
of nucleotides) accession determination
December 1Æ5–2Æ5
Mycelioid
I (646)
FJ378870
E. pisi
November N ⁄ Aa
N⁄A
II (646)
FJ378874
E. trifolii
November N ⁄ A
N⁄A
II (644)
FJ378873
E. trifolii
N⁄A
N⁄A
I (646)
FJ378872
E. pisi
December N ⁄ A
N⁄A
I (646)
FJ378871
E. pisi
December 4–6Æ5
Branched
II (646)
FJ378875
E. trifolii
December 4Æ5–7
Branched
II (280)
N⁄A
E. trifolii
August
4–7
Branched
II (568)
FJ378882
E. trifolii
July
July
August
July
July
July
July
September
September
October
N⁄A
N⁄A
N ⁄ Db
N⁄D
N⁄A
N⁄A
N⁄D
2–3
N⁄A
N⁄A
N⁄A
N⁄A
Mycelioid
Mycelioid
N⁄A
N⁄A
Mycelioid
Mycelioid
N⁄A
N⁄A
II (549)
II (533)
I (646)
I (645)
I (505)
I (549)
I (602)
I (646)
II (580)
II (646)
GU361633
GU361634
FJ378867
FJ378869
GU361635
GU361636
FJ378868
FJ378879
FJ378877
FJ378878
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
April
trifolii
trifolii
pisi
pisi
pisi
pisi
pisi
pisi
trifolii
trifolii
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R. N. Attanayake et al.
peas at the same season except one case in 2007 (see location and collection data in Table 1). At each sampling,
specimens were taken from four to five well-separated
plants in a given glasshouse and processed separately. If
the specimens were identical in ITS sequences (usually the
case), they were considered as one biological sample
because they were likely asexually propagated from one
source. Seven of the 18 samples were from pea plants
from either commercial fields or experimental field plots.
The remaining three samples were from other legume species found in uncultivated areas (roadsides and parks):
black medick (Medicago lupulina), sweet pea (Lathyrus
sp.) and sweet clover (Melilotus albus).
Some of the samples were taken from the same glasshouse over the years (samples GH 04, GH 05 and GH 06,
Table 1). However, there was little chance of carry-over
of powdery mildew inoculum from the previous year’s
glasshouse crop. Each year in August (the hottest month
of the year), all glasshouses in the cool season grain
legume breeding programme are routinely completely
vacated, cleaned, and disinfected with GreenShield
(Whitmire Micro-Gen Research Laboratories). Following disinfestations, they are allowed to naturally heat by
solar radiation (with the cooling systems shut off) for
2 weeks to sanitize the glasshouse for insects and pathogens. It is assumed that each year the powdery mildew
inoculum was from unidentified sources outside of the
glasshouses.
sequences of Erysiphe species found on fabaceous hosts
from the NCBI GenBank database were included in the
analysis. Sequences were aligned using BioEdit (Hall,
1999) and ambiguously aligned sites were removed. Phylogenetic analyses were conducted using PAUP* 4.0b8
(Swofford, 2002). Trees were obtained from maximum
likelihood (ML) and parsimony (MP) methods. MP analysis was performed using the heuristic search option with
1000 random addition sequences to increase the
likelihood of finding the most parsimonious tree. The
branch-swapping algorithm used was tree-bisectionreconnection (TBR) with ‘MulTrees’ option in effect.
Branches collapsed (creating polytomies) if branch length
was zero. Gaps were treated as missing data. Strength of
internal branches of the resulting trees was tested with
Bootstrap analysis using 1000 replications (Felsenstein,
1985). In the ML method, the most appropriate evolution
model was determined for a given data set using PAUP*
4.0b8 (Swofford, 2002) and DT-ModSel (Minin et al.,
2003). A starting tree was obtained with the neighbourjoining (NJ) (Saitou & Nei, 1987) method with the JC69
model of evolution. With this tree, likelihood scores were
calculated for 56 alternative models of evolution by PAUP.
Once the model of evolution was chosen, it was used to
construct a phylogenetic tree with the Minimum Evolution (ME) method from the heuristic search option in
PAUP* 4.0b8. Starting branch lengths were obtained using
the Rogers-Swofford approximation method.
ITS sequencing
Morphological characterization
Total genomic DNA was isolated from about 100 mg of
powdery mildew (conidia and mycelia) using the FastDNA Kit (BIO 101 Inc.), and ITS sequences were
obtained from all samples as described in Attanayake
et al. (2009). Polymerase Chain Reaction (PCR) was performed with total genomic DNA using the ITS1 and ITS4
primer pair (White et al., 1990) or Erysiphe-specific ITS
primer pair, EryF (5¢-TACAGAGTGCGAGGCTCA
GTCG-3¢) and EryR (5¢-GGTCAACCTGTGATCCA
TGTGACTGG-3¢) (Attanayake et al., 2009). Amplified
DNA fragments were first cloned and transformed into
competent Escherichia coli cells (Invitrogen Crop).
Following blue-white colony selection, plasmids were
isolated from positive colonies and at least five clones from
each sample were sequenced using one of the six primers:
EryF, EryR, ITS1, ITS4, M13F and M13R (Attanayake
et al., 2009). Nucleotide sequences were determined from
both strands using an ABI PRISM 377 automatic sequencer (Applied Biosystems) at the Sequencing Core Facility
of Washington State University. Sequences were used as
queries in BLAST (http://www.ncbi.nlm.nih.gov/BLAST)
searches to identify most similar sequences available in
the GenBank databases.
Chasmothecia (when available) and conidia were
removed from leaves with an insect needle, mounted in
water and examined at 100–1000· using bright field
microscopy (Carl Zeiss Model Axioskop 40). Taxonomic characters such as chasmothecial appendages
and diameters, number of asci per chasmothecium,
number of ascospores per ascus, lengths and widths of
asci, ascospores, conidia and conidiophore foot cells
were examined and recorded. Five plants from each
glasshouse (one from each of the four corners and one
from the middle) were observed. At least 50 measurements were made for each character from each sample
and results were compared with the species descriptions in Braun (1987).
Phylogenetic analyses
Thirteen of the 18 ITS sequences determined in this study
that were at least 568 bp long (Table 1) along with 15 ITS
Pathogenicity assays
A detached leaf assay was carried out to confirm pathogenicity of E. trifolii on pea. Fresh inoculum (conidia) was
obtained from Lens culinaris, M. albus and P. sativum,
and used in cross inoculation of the same three host species as described below. The ITS sequences of the three
inoculum sources were determined before the inoculation
experiment. Powdery mildew-infected M. albus plants
were obtained from the Boyer Park, Washington, and
subsequently transplanted to a separate glasshouse
to maintain fresh powdery mildew inoculum. Powdery
mildew conidia from P. sativum and Lens culinaris were
Plant Pathology (2010) 59, 712–720
Erysiphe trifolii on Pisum sativum
obtained from naturally infected glasshouse-grown
plants.
Fresh leaves of Lens culinaris cv. Crimson, M. albus (PI
90186) and P. sativum cv. Dark Skin Perfection, grown in
a separate glasshouse where no powdery mildew was
observed, were surface disinfected with 70% ethanol for
30 s followed by three serial washings with sterilized distilled water (Spurr, 1979). Leaves of a given plant were
then placed in three replicate Petri dishes (moist chambers) as described in Attanayake et al. (2009). Moist
chambers were made with 9 cm diameter Petri dishes and
sterilized wet filter papers. Sterilized 200 lm metal mesh
(4 · 4 cm) was kept between filter paper and the leaf to
prevent the leaf from directly contacting water. Each leaf
petiole was inserted into a 200 lL pipette tip (narrow end
flame sealed) filled with 1% sucrose solution, thereby
prolonging greenness of the leaves. Conidia from P. sativum were used to inoculate detached leaves of M. albus
and Lens culinaris, and conidia from M. albus and Lens
culinaris were used separately to inoculate detached
leaves of P. sativum. Conidia were applied on the abaxial
leaf surfaces using a fine paint brush (Lim, 1973) until a
white powdery appearance was visible. The paint brush
was disinfested by rinsing in 95% ethanol, followed by
air-drying, between each treatment. Aseptic techniques
were used during the inoculation procedure to minimize
cross contamination, and mock-inoculated leaves (a
paint brush without conidia was applied to the leaf surface) served as controls to monitor potential contamination. All inoculation work was conducted in a biological
safety cabinet. Inoculated leaves were incubated at room
temperature under white fluorescence light with a 12 h
photoperiod (Warkentin et al., 1995). Symptom development was monitored using a dissecting microscope (Carl
Zeiss Model Stemi 2000 C) and recorded as presence or
absence of powdery mildew colonies at 2-day intervals
until leaves become senescent (usually 20 days). DNA
from freshly developed powdery mildew colonies was
isolated using the microwave method as described in
Attanayake et al. (2009), and used in PCR and DNA
sequencing as described above. The experiment was performed twice.
An observational study was carried out in a glasshouse to see if the pea powdery mildew pathogen can
infect soybean. Two soybean genotypes L84-2237 (PI
547870) and Harosoy (PI548573) were planted along
with four pea cultivars: Dark Skin Perfection, Lifter,
Medora and Radly. The two soybean genotypes are
known to be susceptible to E. diffusa (Dunleavy, 1978;
Lohnes & Nickell, 1994). Twelve seeds of each genotype (four seeds per 15-cm pot and three pots per genotype) were arranged randomly, and maintained at 16 h
photoperiod, 19–24C day temperature and 15C night
temperature. Upon first appearance of symptoms,
development of powdery mildew, revealed by the presence of white powdery colonies, was observed and
recorded at 2-day intervals for 7 weeks, then at weekly
intervals until plants matured (in about 10 weeks). The
experiment was repeated once.
Plant Pathology (2010) 59, 712–720
715
Results
ITS sequences
PCR using primers ITS1 and ITS4 generated products of
about 650 bp from most samples. Full length (646 bp) or
partial sequence of the ITS region was obtained from all
the samples and representative sequences were used in
BLAST searches (Table 1). All ITS sequences obtained from
a single glasshouse at a given time were virtually identical
(similarity more than 99Æ4%) and therefore considered as
one biological sample. Sequences of samples obtained
from pea fell into two discrete groups (referred to as
Group I and Group II in Table 1 and hereafter). Fourteen
single nucleotide polymorphisms (SNPs) were found
between these two groups when 602 nucleotides of the
ITS region were compared. Group I included five field
samples (FF 06, SP 07, GE 07, LI 08-1 and LI 08-2) and
three glasshouse samples (GH 04, GH 07-119 and GH M
07). Group II included four glasshouse samples (GH 05,
GH 06, GH N 07 and Lif 07) and two field samples (EI
08-1 and EI 08-2). Although sequences for a few samples
(e.g. Lif 07, Table 1) were incomplete, assignment to one
of the two groups was easily accomplished based on some
of the 14 unique SNPs. BLAST search using the FF 06 sample (Group I) as query had 99% similarity (one nucleotide
difference) to E. pisi (Accession AF011306 from Lathyrus latifolius deposited by Saenz & Taylor (1999)). The
sequences in GenBank showing the next highest similarities (fourteen base pair differences) were eleven identical
sequences (EF196666 to EF196675 and AY739112) for
E. diffusa deposited by Almeida et al. (2008), and several
‘Oidium sp.’ (AB078800 etc.) deposited by Takamatsu
et al. (2002). In the BLAST search using the GH N 07
sequence (Group II) as a query, the sequences in GenBank
that showed the highest similarity (one base-pair difference) were three identical sequences (AB079853 to
AB079855) of Oidium sp. from three different hosts from
Japan which were in an E. bauemleri ⁄ E. trifolii clade
(Fig. 1 in Okamoto et al., 2002). The sequences in GenBank that showed the next highest similarity (three base
pair differences) were five identical sequences
(AB015913, AB163926, AB167523, AB167524 and
AF298542) of E. trifolii (Takamatsu et al., 1999; Cunnington et al., 2003; Matsuda et al., 2005) and another
sequence (AB015933) of E. baeumleri (Takamatsu et al.,
1999).
Phylogenetic analyses
Of 570 total characters (nucleotides), 93 characters were
phylogenetically informative for parsimony analysis. Parsimony analysis using PAUP* 4.0b10 generated 24 equally
most parsimonious trees. Tree topologies were almost
consistent among all the trees except for branch lengths
and branching orders of the terminal branches. The
majority rule consensus tree is shown in Fig. 1. For ML
analysis, DT-ModSel selected shape parameter alpha,
0Æ295144, HKY85 + G model, transition ⁄ transversion
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R. N. Attanayake et al.
Figure 1 Majority rule consensus tree (unrooted) based on the internal transcribed spacer (ITS) sequences from 28 taxa of Erysiphe spp.
showing the relationship among strains of several powdery mildew species found on legumes. Bootstrap values based on 1000 replications
are shown above the branches. Roman numerals at the right of taxa indicate the ITS sequence grouping in Table 1. Taxa in bold are the
sequences determined in this study.
ratio 1Æ30921 (kappa = 2Æ6403874) and nucleotide frequencies as A = 0Æ19757, C = 0Æ28096, G = 0Æ27493,
T = 0Æ24653. Tree topology of the ML tree was similar to
that of the MP tree (not shown). Thirteen sequences
obtained in this study fell into two major clades (Fig. 1).
Clade I (97% bootstrap support) consisted of E. pisi GenBank sequences obtained from Lathyrus latifolius
(AF011306) and P. sativum (AF073348) hosts from USA
and Australia, respectively, and most of the pea field samples, some glasshouse samples and a sample from Lathyrus sp. obtained in this study. Clade II had 93% bootstrap
support and comprised the rest of the glasshouse and field
samples obtained in this study and GenBank sequences
E. trifolii and E. baeumleri obtained from Vicia
(AB015919 and AB015933) and E. trifolii (AB015913
and AF298542) from Japan and Switzerland, respectively. Erysiphe diffusa formed a separate distinct clade
with 100% bootstrap support. Surprisingly, the ITS
sequence (AB104519) of a fungus deposited as E. pisi
from Melilotus sativa from Iran is distinct from all the
other E. pisi sequences.
Morphological observations
All samples used in this study displayed typical powdery
mildew symptoms. Mycelia of all samples were mainly
epiphyllous, in white, effuse patches often covering the
entire adaxial and abaxial surfaces of leaves, stems and
sometimes pods. Hyphae were branched, septate, hyaline, thin-walled; lobed appressoria were solitary or in
opposite pairs. Single conidia formed terminally on conidiophores. In all samples conidiophore foot cells were
erect, straight to sometimes flexuous, and cylindrical.
After detecting ITS sequence identity among the several
samples obtained from a given glasshouse, one set of morphological measurements was taken into account. Conidial dimensions were graphically depicted using box plots
in Fig. 2. Conidial lengths and widths for E. pisi ranged
from 23Æ5–60 · 7Æ5–20Æ5 lm, whereas those for E. trifolii were 23–60Æ5 · 9Æ5–19 lm (Fig. 2). Similarly, the
conidial dimensions for the two species in Braun (1987)
also overlapped (24–55 · 13Æ5–22 lm for E. pisi and 30–
45 · 16–21 lm for E. trifolii). Conidiophore foot cell
measurements also varied considerably amongst the samples (data not shown). Chasmothecia were observed in
six P. sativum samples (FF 06, GH 04, GE 07, GH N 07,
Lif 07 and SP 07). Chasmothecia were scattered or gregarious, with irregularly polygonal peridial cells. Mature
dark brown chasmothecia enclosed several sessile or
short-stalked asci. Ascospores were ellipsoid to ovoid.
Two kinds of chasmothecial appendages were observed
(Table 1). Chasmothecial appendages of the samples FF
06, GH 04, GE 07 and SP 07 (from Group I) were short,
mycelioid, simple, septate, brown coloured at the base,
becoming pale towards the tip and hyaline at the upper
half and often interwoven with each other and with other
mycelia (Fig. 3a). Chasmothecia of samples GH N 07 and
Lif 07 (from Group II) displayed long, flexuous, dichotomously branched appendages (Fig. 3b). Appendage apices were straight, 3–5 times loosely branched, diffuse,
and often deeply cleft (Fig. 3c). Removal of Group II
chasmothecia from the leaf surface was accomplished
more easily than was the case for Group I samples because
the latter had appendages interwoven with the surrounding
Plant Pathology (2010) 59, 712–720
Erysiphe trifolii on Pisum sativum
717
and for Group II was 4–7, whereas in Braun (1987) it is
0Æ5–3Æ5 and 2–6 for E. pisi and E. trifolii, respectively.
The highly branched chasmothecial appendage apices
observed in Group II samples differed from descriptions
of E. trifolii in Braun (1987, 1995) but agreed with the
descriptions of E. trifolii on lentils in Attanayake et al.
(2009) and with apices on an authentic specimen of E. trifolii as discussed below.
Pathogenicity assays
Figure 2 Box plots showing variations of conidial lengths and widths
among 14 samples of Erysiphe pisi (hatched boxes) and E. trifolii
(open boxes) used in this study. The boxes and middle lines
represent the middle 50 percentiles and medians, respectively. The
whiskers represent upper and lower limits and asterisks represent
outliers.
hyphae. Most of the other characters measured (such as
length and width of conidiophore foot cells) displayed
overlapping values between the two groups as well as
amongst the individual samples (data not shown). However, the ratio of chasmothecial appendage length to
chasmothecial diameter is clearly different between the
two groups. Ratios for Group I and Group II agreed with
the descriptions for E. pisi and E. trifolii, respectively, in
Braun (1987). The ratio of chasmothecial appendage
length to chasmothecial diameter for Group I was 1–3
(a)
(b)
The ITS sequences of the three samples that were used for
inoculation belonged to Group II (E. trifolii). In the
detached leaf assay, signs of powdery mildew were visible
on leaves of M. albus and Lens culinaris about 10 days
after inoculation with conidia obtained from P. sativum.
Similarly, signs of powdery mildew were visible after the
same time interval on P. sativum leaves when conidia
from M. albus or Lens culinaris were used. Mock-inoculated controls remained free of symptoms during the
entire period of the experiment. Repeating the experiment gave the same results. ITS sequences of conidia from
the infected leaves were identical to those of the inoculum
used.
In the observational study in the glasshouse all four pea
cultivars were heavily infected by powdery mildew,
exhibited chasmothecial production, and the ITS
sequence of the samples from the pea plants were of
Group II (E. trifolii). However, the two soybean genotypes (L84-2237 and Harosoy, susceptible to E. diffusa)
remained symptomless during the entire period of the
experiment. The same results were obtained when the
experiment was repeated.
Discussion
Powdery mildew is a recurring problem in glasshousegrown pea in the US Pacific Northwest. Such frequent
occurrence of powdery mildew in glasshouses has facilitated selection of resistance materials in breeding programmes. However, some of the resistant materials
(c)
0.1 mm
50 µm
50 µm
Figure 3 Comparison of chasmothecial appendages of Erysiphe pisi and E. trifolii. (a) Short myceliod chasmothecial appendages of E. pisi.
(b) Long flexuous chasmothecial appendages (arrow) of E. trifolii. (c) Highly branched appendage apex of E. trifolii (arrow).
Plant Pathology (2010) 59, 712–720
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R. N. Attanayake et al.
selected in the glasshouse are susceptible to powdery mildew in the fields (K.E. McPhee, unpublished data). The
inoculum sources in the glasshouse are often unknown,
but because of disinfestation procedures they must originate from outside the glasshouse.
Both teleomorphic characters and ITS sequences were
used to distinguish pathogen species found on pea in both
glasshouse and field in the US Pacific Northwest. Based
on ITS sequences, two distinct groups of powdery mildew
pathogens were found. The two groups differed in 14
nucleotide positions in the ITS region and also exhibited
readily distinguishable chasmothecial appendage morphology. The correlation of morphological differences
with ITS sequence differences confirms that the two
groups belong to different species of the genus Erysiphe.
Group I had ITS sequences most similar to those of previously deposited sequences of E. pisi (Saenz & Taylor,
1999; Cunnington et al., 2003), and produced chasmothecia with short mycelioid, simple appendages conforming to the descriptions of E. pisi, a well-documented
pathogen of pea. However, anamorphic characters could
not be used to differentiate the two species due to intraspecific variations (Fig. 2). The variations could be due to
genetic differences and environmental conditions such as
relative humidity. For example, relatively larger conidia
were consistently observed in detached leaf assays conducted in moist chambers (high relative humidity) than
those on the field samples (R.N.Attanayake, unpublished
data).
Determination of the species identity of the Group II
samples was more complicated. ITS sequences of Group
II were most similar to those of the E. trifolii complex
(Takamatsu et al., 1999; Okamoto et al., 2002; Cunnington et al., 2003; Khodaparast et al., 2003). However,
members of this group produced branched chasmothecial
appendages, resembling those described for E. diffusa or
E. pisi var. cruchetiana in Braun (1987). Neither E. diffusa, E. pisi var. cruchetiana nor E. trifolii has previously
been reported to infect pea. Erysiphe diffusa produces
dichotomously branched, rigid chasmothecial appendages (Braun, 1987), whereas E. trifolii was not reported
to produce dichotomously branched chasmothecial
appendages in Braun (1987). A recent study (Attanayake
et al., 2009) employing ITS sequences, plus morphological observation of an authentic specimen of E. trifolii
(WSP 70928) originating from GZU Dupla Fungorum
that was determined by U. Braun (Scheuer, 2003), demonstrated that E. trifolii can produce long, flexuous and
dichotomously branched chasmothecial appendages.
Erysiphe trifolii differs from E. pisi var. cruchetiana by
producing frequently dichotomously branched long, flexuous appendages, whereas the latter has irregularly
branched, short appendages.
These findings help differentiate E. trifolii from E. pisi
on pea. Erysiphe trifolii can also be differentiated from
E. diffusa, in spite of the fact that both species may
produce highly dichotomously branched appendage
apices, on the basis of the flexuous nature of appendages
in E. trifolii (Fig. 3b). Erysiphe diffusa is a well docu-
mented pathogen of soybean (Dunleavy, 1978; Lohnes &
Nickell, 1994) and was also tentatively identified (on the
basis of appendage morphology) as a causal agent of
powdery mildew on lentil in Canada (Banniza et al.,
2004). The powdery mildew pathogen from pea did not
cause any visible disease symptoms on soybean genotypes
known to be susceptible to E. diffusa, and ITS sequence
of E. diffusa from wild soybean was distinct from those of
the pea powdery mildew pathogens (Attanayake et al.,
2009). These findings indicate that the pea powdery mildew samples constituting Group II are not referable to
E. pisi or E. diffusa, but are in fact E. trifolii.
Phylogenetic analysis further supported this conclusion. The phylogenetic analysis in this study revealed two
groups of powdery mildews: Group I was in congruence
with the morphological characters of E. pisi, and formed
a clade with all previously identified E. pisi sequences
except the Iranian sample (AB104519) from Melilotus sativa, whereas Group II was in congruence with E. trifolii.
In addition, E. baeumleri was also grouped with E. trifolii.
There are taxonomic ambiguities in the placement of
E. baeumleri. It is not clear whether E. trifolii and E. baeumleri are conspecific or two distinct species (U. Braun,
Martin-Luther-Universität, Institut für Biologie, Germany, personal communication). Erysiphe trifolii has
been regarded as a complex of similar species consisting
of E. trifolii, E. baeumleri and E. asteragali (Braun,
1987). However, in this analysis the bootstrap support
to separate the E. baeumleri clade was only 71%. The
species identity of the Iranian sample (AB104519) from
M. sativa needs reassessment because the ITS sequence
was drastically different from those of the other E. pisi
sequences.
Results of this study demonstrated that both E. pisi and
E. trifolii are present and cause disease on pea in both field
and glasshouse conditions. The sample Lif 07 was from
pea cv. Lifter which is resistant to E. pisi (McPhee &
Muehlbauer, 2002), but severely infected by E. trifolii in
the glasshouse. Ondřej et al. (2005) reported a similar situation: Pisum sativum germplasm lines resistant to E. pisi
were susceptible to E. baeumleri in fields in the Czech
Republic. Therefore, powdery mildew fungi infecting pea
are more diverse than previously assumed (Braun, 1987;
Falloon & Viljanen-Rollinson, 2001). These findings
may explain some inconsistent responses of individual
pea breeding lines to powdery mildew infection. Glasshouse infections may not adequately reflect host ranges in
natural or field situations. Cook & Fox (1992) observed
that Vicia faba (faba bean) grown in glasshouses were
infected with E. pisi var. pisi while such infection is not
reported in fields in Britain. Okamoto et al. (2002) also
found Oidium subgenus Pseudoidium causing powdery
mildew on its non-natural host, Eustoma grandiflorum
(prairie gentian), under glasshouse conditions. Taylor
(2008) suggested that climatic change may have favoured
increased aggressiveness of existing pathogen races, causing the breakdown of red clover resistance to powdery
mildew. Nevertheless, in the present work E. trifolii was
observed on pea in both fields and glasshouses and
Plant Pathology (2010) 59, 712–720
Erysiphe trifolii on Pisum sativum
formed the teleomorph on pea (Table 1), indicating that
infection of pea by E. trifolii is not a glasshouse artifact.
During winter months only a small acreage of winter
pea is grown in the field in the US Pacific Northwest and is
at a significant distance from the glasshouse facilities,
suggesting that powdery mildew inoculum for the glasshouse plants likely comes from volunteer pea plants or
alternative hosts, or from resting states (chasmothecia)
on plant debris. Both E. pisi and E. trifolii were found on
other legumes (Lathyrus sp., Medicago lupulina, Melilotus albus and Lens culinaris) commonly found in the US
Pacific Northwest. These legume species could be inoculum sources for glasshouse grown peas during winter
months. Although both E. pisi and E. trifolii were
detected in glasshouses, at a given time only one of the
species (based on ITS sequences) was detected in a given
glasshouse. This suggests that the winter inoculum, originating external to glasshouses, was very limited during
the colder months but propagated rapidly upon gaining
entry to a suitable internal environment.
This report is the first to document E. trifolii causing
powdery mildew on pea, and documents the disease in
both field and glasshouse conditions. It is likely that
E. trifolii has been a pathogen of pea for a long time,
but has not been recognized until now. Recognition of
E. trifolii as a pea pathogen is significant for pea breeding programmes. Pathogen species identity is important
because different species may interact with pea genotypes differently. Although it may be difficult to completely exclude a given powdery mildew species from a
glasshouse, attempts can be made to introduce the
desired powdery mildew species into glasshouse screenings or at least determine which species is being
screened at a given time.
Acknowledgements
The authors want to thank Professor Uwe Braun, MartinLuther-Universität, Institut für Biologie, Bereich Geobotanik, Herbarium, Neuwerk 21, D-06099 Halle ⁄ S.
Germany for advice concerning the taxonomic status of
Erysiphe trifolii, Dr Eric H. Roalson, Department of Biology, Washington State University, Pullman, WA for helping in phylogenetic analysis, and Shari Lupien, Plant
Introduction, USDA ARS, Pullman for maintaining
Melilotus albus plants infected with powdery mildew.
The research was funded in part by the USDA-CSREES
Cool Season Food Legume Research Program.
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