Microbiology (2013), 159, 1946–1955
DOI 10.1099/mic.0.068205-0
Quantification of Alternaria brassicicola infection in
the Arabidopsis thaliana and Brassica rapa subsp.
pekinensis
Mukhamad Su’udi,1 Jong-Mi Park,1 Sang-Ryeol Park,1 Duk-Ju Hwang,1
Shin-Chul Bae,1 Soonok Kim2 and Il-Pyung Ahn1
Correspondence
Il-Pyung Ahn
[email protected]
Received 30 March 2013
Accepted 10 July 2013
1
National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707,
Korea
2
Wildlife Genetic Resources Centre, National Institute of Biological Resources, Incheon 404-708,
Korea
Black spot caused by Alternaria brassicicola is an important fungal disease affecting cruciferous
crops, including Korean cabbage (Brassica rapa subsp. pekinensis). The interaction between
Arabidopsis thaliana and Alt. brassicicola is a representative model system, and objective
estimation of disease progression is indispensable for accurate functional analyses. Five strains
caused black spot symptom progression on Korean cabbage and Ara. thaliana ecotype Col-0. In
particular, challenge with the strains Ab44877 and Ab44414 induced severe black spot
progression on Korean cabbage. Ab44877 was also highly infective on Col-0; however, the
virulence of Ab44414 and the remaining strains on Col-0 was lower. To unveil the relationship
between mycelial growth in the infected tissues and symptom progression, we have established a
reliable quantification method using real-time PCR that employs a primer pair and dual-labelled
probe specific to a unigene encoding A. brassicicola SCYTALONE DEHYDRATASE1
(AbSCD1), which is involved in fungal melanin biosynthesis. Plotting the crossing point values
from the infected tissue DNA on a standard curve revealed active fungal ramification of Ab44877
in both host species. In contrast, the proliferation rate of Ab44414 in Korean cabbage was 3.8
times lower than that of Ab44877. Massive infective mycelial growth of Ab44877 was evident in
Col-0; however, inoculation with Ab44414 triggered epiphytic growth rather than actual in planta
ramification. Mycelial growth did not always coincide with symptom development. Our quantitative
evaluation system is applicable and reliable for the objective estimation of black spot disease
severity.
INTRODUCTION
Alternaria brassicicola is a necrotrophic fungal pathogen
that causes black spot disease in many cruciferous
vegetables, such as broccoli, cabbage, cauliflower, radish
and turnip (Nowicki et al., 2012; Otani et al., 1995). A
recent survey reported that vegetable crop losses caused by
this fungus might be 20–80 % worldwide (Nowicki et al.,
2012). In addition to its economic importance, the
interaction between this pathogen and several ecotypes or
mutants of Arabidopsis thaliana has been used as a model
system to investigate functions of fungal virulence factors
and plant genes and signals modulating disease progression. Despite many accomplishments, no major gene for
resistance has been identified for black spot disease.
Abbreviations: CP, crossing point; ITS, internal transcribed spacer.
Two supplementary figures are available with the online version of this
paper.
1946
Accurate and objective disease evaluation is a critical step
in the evaluation of disease-resistant germplasm and effect
of fungicides. Traditionally, black spot progression has
been evaluated through scoring of visual symptoms.
Although this method is the easiest to perform and
remains useful for achieving some objectives, it does not
produce quantitative values. Macroscopic symptom assessment is not applicable in chronological estimation of
disease progression especially in the early period. More
seriously, this technique does not measure fungal proliferation but rather identifies symptoms on the host. To
avoid these problems, several alternative approaches have
been designed, such as microscopic observation (Jarnagin
& Harris, 1985), chemical detection and quantification of
pathogen-specific constituents (Martin et al., 1990), and
immunological detection (Harrison et al., 1990; Thornton
et al., 2010). However, these technical approaches are
usually laborious and not sensitive enough, and do not
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Alternaria brassicicola growth in planta
provide objective and quantitative results. During the past
three decades, a PCR-based strategy has been developed
and used for evaluation of fungal biomass within plant
tissue (Bretagne et al., 1995; Entz et al., 2005; Färber et al.,
1997; Jurado et al., 2006; Nicholson et al., 1997). Although
this technique has many advantages due to its broad
spectrum, simplicity, sensitivity and time- and labourefficiencies, it is basically qualitative and not quantitative
because the amount of amplicon does not reflect the copy
number of template DNA if the amount of each sample’s
amplified DNA has reached saturation level prior to the
checking point. More recently, crossing point (CP) valuedependent quantitative real-time PCR has been introduced
to solve this problem (Lievens et al., 2006; Selma et al.,
2008). As the CP value is able to correctly reflect the
logarithm of the input copy number or the amount of
template DNA, objective and quantitative measurement of
fungal DNA within a certain amount of sample DNA can
be performed by plotting the CP value of a sample on a
standard curve. Recently, a dual-labelled probe was used to
enhance the specificity of real-time PCR and is now widely
accepted as the most reliable quantitative technique for the
evaluation of fungal biomass within the host tissue (Kulik,
2011; Pavón et al., 2012; Su’udi et al., 2012; Zhao et al.,
2012).
(Korea). Fungal inocula were prepared as follows: Alt. brassicicola
and Magnaporthe oryzae were grown on oatmeal agar (50 g oatmeal
flakes and 20 g agar per litre of distilled water); C. miyabeanus was
grown on sucrose proline agar (SPA) (Ahn et al., 2005); and Botrytis
cinerea, Fusarium and Rhizoctonia solani were cultivated on potato
dextrose agar (PDA, Difco). All strains were grown at 22 uC under
constant fluorescent light. Subsequently, conidial suspension or
actively growing mycelial block was transferred into complete liquid
medium and grown for 3 days at 25 uC, 150 r.p.m., in the dark. Prior
to sampling, the mycelial mass was filtered with Miracloth
(Calbiochem), subsequently frozen in liquid nitrogen and stored
at 280 uC.
Initially, short sequences of rDNA or internal transcribed
spacers (ITS) were the preferred target regions for primer
design owing to limited fungal genome data (Borneman &
Hartin, 2000; Gil-Serna et al., 2009; Martin & Rygiewicz,
2005). However, the copy number of both elements is not
fixed (Sone et al., 2000). Moreover, designing a primer pair
specific to the target organism is difficult because these
sequences are highly conserved and ubiquitous across all
filamentous fungi. Recently, the fungal genome project and
accumulated fungal sequences in the public database have
made an enormous amount of sequence information
available.
Mycological characteristics. For morphological characterization,
The objective of this study was to reveal the relationship
between symptom progression and Alt. brassicicola proliferation in two pathosystems: Ara. thaliana, the representative model plant, and Korean cabbage (Brassica rapa subsp.
pekinensis), one of the most important vegetables in Korea.
We have developed a convenient and reliable protocol to
produce objective and quantitative evaluation of Alt.
brassicicola within infected tissue DNA by employing a
primer pair and a dual-labelled probe specific to the unigene
AbSCD1, which encodes SCYTALONE DEHYDRATASE1.
An orthologue of AbSCD1 in Cochliobolus miyabeanus,
CmSCD1, has been used for quantification of brown leaf
spot disease in rice owing to its presence in the fungal
genome as a single copy (Su’udi et al., 2012).
METHODS
Fungal strains and pathogenicity assay. The fungal isolates used
in this study (Table 1) were obtained from the Korean Agricultural
Culture Collection (KACC), Rural Development Administration
http://mic.sgmjournals.org
Korean cabbage (Bra. rapa subsp. pekinensis) cv. ‘Seoul’ and Ara.
thaliana Col-0 ecotype were used for the inoculation assay.
Arabidopsis were germinated in pots containing a soil–vermiculate
(10 : 1) mixture and grown in a growth chamber at 22 uC, 70 %
relative humidity, with a light intensity of 250 mmol photons m22 s21
and a 16 h photoperiod. Inoculation was performed by spraying
conidial suspension of each Alt. brassicicola strain at a concentration
of 26105 conidia ml21 and 250 mg ml21 Tween 20 onto five Korean
cabbages or ten Arabidopsis plants until all the leaves were covered
with fine droplets. The infected plants were kept at 25 uC in a dark
chamber at almost 100 % humidity for 20 h and then transferred into
normal growth conditions (22 uC, 70 % relative humidity). The leaf
samples for DNA preparation were collected at the indicated times.
The images of symptom progression were taken at 4 days postinoculation (days p.i.). The pathogenicity assay was performed at least
three times and similar trends were obtained.
fungal conidia from each strain were retrieved from the medium with
sterilized distilled water, mounted on glass slides and observed under
a light microscope (Zeiss Axioplan 2). In each strain, the cell number
was estimated in more than 1000 conidia.
For the observation of conidial chain formation, one of the important
keys in Alt. brassicicola diagnosis, the surface of the colony was lightly
touched with transparent vinyl tape. Then, the tape was mounted on a
glass slide with a drop of sterilized distilled water, and the slide was
observed under a microscope. After taking pictures, the width and
length of each conidium were measured using the Java image
processing software ImageJ (http://rsbweb.nih.gov/ij/download.html).
DNA preparation and Southern blot analysis. Genomic DNA
from the fungal mass or infected plant materials was prepared using
the hexadecyl trimethyl ammonium bromide (CTAB) method
(Stewart & Via, 1993). DNA was precipitated using 2-propanol and
the concentration was quantified using a NanoDrop (Thermo
Scientific) and further confirmed by band brightness comparison
with the HindIII-digested lambda DNA marker (Invitrogen) using the
ImageJ software. The DNA concentrations from fungal and plant
samples were adjusted to 50 ng ml21 and fungal DNA was serially
diluted as indicated for standard curve construction by Taqman realtime PCR.
For Southern blot analysis, fungal genomic DNA (5 mg) from four
Alt. brassicicola strains (Ab44877, Ab44415, Ab43923 and Ab44414)
was digested with EcoRI or SphI, incubated at 37 uC for 4 h and
loaded onto 0.7 % agarose gel. The gel was blotted onto a HybondN+ nylon membrane (Amersham Pharmacia Biotech), and the
membrane was further processed according to standard DNA blot
procedure (Southern, 1975). A 268 bp fragment of AbSCD1 was
used to make a radiolabelled probe. The primers used for probe
amplification were AbSCD1_7F (59-GAG AAG AAC GAA TTG AAG
CC-39) and AbSCD1_275R (59-ATT GCT TCC CAG ATC TTG TC39) (Fig. 1). The probe was labelled with [a-32P]dCTP by random
priming (Feinberg & Vogelstein, 1983)) using the Rediprime II DNA
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M. Su’udi and others
Table 1. Fungal strains used in the evaluation of primer pair specificity
Fungal strain
Alternaria brassicicola
Alternaria brassicicola
Alternaria brassicicola
Alternaria brassicicola
Alternaria brassicicola
Magnaporthe oryzae
Cochliobolus miyabeanus
Botrytis cinerea
Fusarium moniliforme
Fusarium graminearum
Rhizoctonia solani
Strain/collection no.
PCR result*
HostD
KACC42464
KACC44877
KACC44415
KACC43923
KACC44414
KI197
Cm85
KACC40574
KACC41032
KACC46434
KACC40101
+
+
+
+
+
2
2
2
2
2
2
Armoracia rusticana
Brassica rapa
Raphanus sativus
Armoracia rusticana
Brassica oleracea
Oryza sativa
Oryza sativa
Cucumis sativus
Oryza sativa
Hordeum vulgare
Solanum tuberosum
*+, Strain detected; 2, strain not detected.
DOriginal host harbouring each strain.
600
9416
6557
675
4361
4
3
441
Ab4
5
392
7
487
441
Ab4
4
5
392
3
441
Ab4
23 130
525
Ab4
450
Ab4
7
bp
441
487
375
Ab4
300
SphI
EcoRI
Ab4
(b)
75
150
225
Ab4
(a)
4304
3181
750
825
2501
2322
2027
900
975
1050
1057
Probe
*
SphI
(c)
*
SphI
*
SphI
SphI
6000
6500
7000
7500
EcoRI
EcoRI
8000
8500
9000
EcoRI AbSCD1
SphI
9500
SphI
10 000 10 500 11 000 11 500 12 000 12 500 13 000
13 500
3181
4304
bp
2501
Fig. 1. Genomic DNA sequence of AbSCD1 and Southern blot analysis. (a) Three exon regions are indicated with capital
letters and the translated amino acid sequences are shown. Primer pairs (AbSCD1_123F and AbSCD1_219R) and probe
sequence for Taqman real-time PCR are designated with dotted boxes and an arrow. The primer pair AbSCD1_7F and
AbSCD1_275R is indicated with dashed boxes and the probe was amplified using this pair. The single SphI restriction site is
indicated with a solid box. (b) Southern blot analysis of AbSCD1. After digestion of genomic DNA with EcoRI and SphI, the
membrane was hybridized with the 269 bp amplicon as the probe. (c) Restriction map of the ATCC 96866 genomic DNA
spanning 8 000 bp around AbSCD1 (arrow). Asterisks indicate the three SphI sites that might be responsible for the difference
in Southern blot patterns between Ab43923 and Ab44877, Ab44415 and Ab44414. There were no EcoRI sites and only a
single SphI site within the probe region of AbSCD1.
1948
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Microbiology 159
Alternaria brassicicola growth in planta
Labelling system kit (GE Healthcare). After hybridization, membranes
were washed with 26SSPE, 0.1 % SDS, exposed on BAS film
(Fujifilm) and developed using the Personal Molecular Imager (PMI)
system (Bio-Rad).
RESULTS
Plasmid construction and PCR amplification. A 97 bp fragment
To establish a Taqman real-time PCR-based Alt. brassicicola quantification system, a primer pair and dual-labelled
probe specific to the AbSCD1 gene encoding SCYTALONE
DEHYDRATASE1 were designed. In addition to its
presence as single copy within the fungal genome, this
gene might be involved in melanin biosynthesis. In fungi,
melanin is indispensable for the survival and longevity of
fungal hyphae and spores (Wheeler, 1983). Melanin is also
important for appressorium formation and for prepenetration development. Initially, the genomic sequence of
AbSCD1 was retrieved from the Alt. brassicicola strain
ATCC 96866 genome database (http://genome.jgi.doe.gov/
Altbr1/Altbr1.home.html). This strain was isolated from
cabbage (Brassica oleracea) seeds with Alternaria black spot
symptoms. This gene spans three exons which are
separated by two short intronic sequences (Fig. 1a). The
open reading frame (ORF) consists of 558 nt encoding 185
amino acids. From the whole genomic sequence, a 97 bp
region was selected for primer and probe design (Fig. 1a).
Nucleotide sequencing also indicated that AbSCD1 in our
strains is identical with that in ATCC 96866 (data not
shown).
was amplified from Alt. brassicicola genomic DNA using Pfu
polymerase (Nanohelix). The primers used in the PCR were
AbSCD1_123F (59-GCA GAC AGC TAC GAT AGC AA-39) and
AbSCD1_219R (59-GAT GCA TTT GCG GAG AC-39) (Fig. 1). The
amplicon was purified, A-tailed and ligated into the pGEM-T Easy
vector (Promega) at 4 uC for 16 h. The ligation product was
transformed into Escherichia coli strain DH5a, plated on Luria–
Bertani (LB) agar medium supplemented with ampicillin (Amp) and
incubated at 37 uC for 16 h. Several colonies were picked and grown
in LBAmp broth for plasmid preparation. The resulting plasmid
construct (pGEM-T Easy : : AbSCD1) was confirmed by EcoRI
digestion and DNA sequencing.
For the primer specificity test, PCR was performed with primer sets
AbSCD1_123F and AbSCD1_219R using fungal genomic DNA
(83.3 ng) as a template. PCR conditions were as follows: 95 uC for
5 min, followed by 30 cycles of 95 uC for 15 s, 55 uC for 15 s and
72 uC for 30 s, with a final extension at 72 uC for 5 min. PCR
products were loaded onto a 2 % (w/v) agarose gel containing
ethidium bromide. The pictures were taken using a gel documentation system (Bio-Rad).
Cloning of the ITS regions. For the molecular identification of the
fungal strain, the 582 bp ITS regions were amplified from the
genomic DNAs of five Alt. brassicicola strains using the primers
AbITS1 (59-TCC GTA GGT GAA CCT GCG-39) and AbITS4 (59-TCC
TCC GCT TAT TGA TAT GC-39). After cloning the product into the
pGEM-T Easy vector, the nucleotide sequences of the cloned ITS
regions were identified using M13F and M13R primers.
Taqman real-time PCR. The fluorescent dyes 6-carboxyfluorescein
(FAM) and matching Black Hole quencher 1 (BHQ1) were added to
the 59 and 39 ends to generate dual-labelled probe. The probe
sequence for Taqman real-time PCR was 59-tca cac atg gca gGA
CTG GGA CC-39. A 10-fold dilution series (1010 to 1020.1 gene
copies) of pGEM-T Easy : : AbSCD1 plasmid was used as a template
to generate a standard for gene copy number. Similarly, Alt.
brassicicola genomic DNA was diluted threefold, ranging from
83.3 ng to 1 pg, to generate a standard for fungal DNA
concentration. Real-time PCR was performed in triplicate on the
LightCycler 480 II (Roche) with triplicates for 40 cycles (15 s at
95 uC and 15 s at 60 uC), starting with an initial incubation at 95 uC
for 4.5 min. The CP values obtained from this reaction were set
automatically by the system. For the evaluation of pathogen
ramification in planta, the leaves from five Korean cabbages or ten
whole plants of infected Arabidopsis were combined and roughly
ground. DNA preparation, quantification and Taqman real-time
PCR were performed as described above. The amount of pathogen
DNA (in nanograms) within 1 mg of plant DNA was determined by
plotting CP values on a standard curve.
Fungal ramification in planta. To observe fungal growth within
the plants, the leaves were recovered at 3 days p.i. The leaves were
stained with 2.5 mg ml21 aniline blue staining solution and
resuspended in lactophenol (equal volumes of glycerol, lactic acid
and phenol). After boiling gently for 1 min, the samples were further
stained in the same solution for 1 day. Excess aniline blue was
removed with several lactophenol washes. Then, the mycelial growth
within the infected tissue was observed under a light microscope
(Peng et al., 1986).
http://mic.sgmjournals.org
Validation of AbSCD1 for evaluation of
Alt. brassicicola proliferation
One of the most important requirements of a gene as a
detection target for Taqman real-time PCR is that the gene
should exist in a fixed copy number in the genome (Salvioli
et al., 2008; Baumgartner et al., 2010). A Southern blot was
performed to determine the copy number of AbSCD1 within
the Alt. brassicicola genome. A 269 bp fragment of this gene
was used as a probe (Fig. 1a, c). In the probe region and the
whole genomic DNA sequence of AbSCD1, no EcoRI site and
only a single SphI site were present. As shown in Fig. 1b,
single- and double-bands were evident in the genomic DNA
digested with EcoRI and SphI, respectively. These data
indicated that AbSCD1 exists as a single copy within the Alt.
brassicicola genome. Therefore, AbSCD1 is an appropriate
target in the establishment of a Taqman real-time PCR.
AbSCD1 was also present as a single copy in Ab43923, and its
Southern blot pattern was different from those of other
strains (Fig. 1b). The 2 675 697 bp contig 2 of ATCC 96866
contains AbSCD1 (Fig. 1c).
Specificity and sensitivity are important for most quantification strategies (Francis et al., 2006; Pfaffl, 2004). In
our work, the specificity of the primer pair was tested by
comparing the amplification patterns of five Alt. brassicicola and several other fungal species (Table 1). PCR was
performed using the same primer set used for cloning as
described in the Methods section. Fig. 2 shows that the
primers used in the PCR specifically and successfully
amplified the 97 bp AbSCD1 fragments. The primer pair
did not react with DNA of other fungal species. Owing to
its specificity, the primer pair can be used in the evaluation
of Alt. brassicicola in biological materials.
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M. Su’udi and others
2
3
4
5
6
7
8
9
10 11 12
35
Fig. 2. Specific PCR amplification of AbSCD1. Lanes: M, 100 bp
DNA ladder; 1, Alt. brassicicola KACC42464; 2, Alt. brassicicola
KACC43923; 3, Alt. brassicicola KACC44414; 4, Alt. brassicicola KACC44415; 5, Alt. brassicicola KACC444877; 6, M.
oryzae KI197; 7, Cochliobolus miyabeanus Cm94; 8, Bot. cinerea
KACC40574; 9, Fusarium moniliforme KACC41032; 10, F.
graminearum KACC46434; 11, C. miyabeanus; 12, Ara. thaliana
(Col-0 ecotype) genomic DNA.
Relationships among CP value, copy number, and
amount of genomic DNA
With respect to sensitivity, Taqman probe real-time PCR
promises more accurate assessment than real-time PCR
using SYBR Green (Gunel et al., 2011; Matsenko et al.,
2008; Yin et al., 2001). We prepared two standard curves
dealing with the relationships among CP value, copy
number, and amount of fungal genomic DNA (Fig. 3).
Taqman probe and primers were designed as shown in Fig.
1(a). The standard curve for the relationship between CP
values and gene copy number was generated by using
pGEM-T Easy : : AbSCD1 plasmid DNA as the template in a
Taqman probe real-time PCR. The templates were serially
diluted 10-fold from 1010 to 1020.1. The slope obtained
from this standard curve was 23.5072, with a high linear
correlation (R251) (Fig. 3a). The standard curve for the
relationship between CP values and the amount of fungal
DNA was generated using a similar strategy; known
concentrations of fungal genomic DNA were serially
diluted threefold from 83.3 ng to 1 pg. The linear
correlation between DNA concentrations and CP values
was high (R2 .0.998), with a regression slope of 23.0491
and 23.1494 for Ab44877 and Ab44415, respectively (Fig.
3b). Hence, we estimated the genome copy number and
fungal DNA concentration by plotting the CP values from
the infected samples on the above linear regressions.
Mycological and molecular identification of
Alt. brassicicola
The colony morphology on oatmeal agar was typical of
growth of Alt. brassicicola. Approximately 5 to 6 days p.i.,
the colony colour changed to dark black, and active
sporulation was evident. To confirm the species diagnosis,
we analysed other mycological characteristics and molecular evidence, such as the shape/size of the conidia, the
formation of conidial chains during sporulation, and the
nucleotide sequences of the ITS regions (Kirk et al., 2008).
1950
(a) 40
Crossing point value
1
30
25
20
15
10
y = –3.507x + 44.77
R2 = 1
5
0
0
2
4
6
8
log10(plasmid copy number)
10
12
(b) 40
35
Crossing point value
bp M
1 517
1 000
700
500
300
200
100
30
25
20
15
10
5
0
–4
Ab44877, y = –3.0494x + 26.846
R2 = 0.9983
Ab44415, y = –3.1494x + 26.547
R2 = 0.9992
–3
–2
–1
0
1
log10(ng fungal genomic DNA)
2
3
Fig. 3. Relationships among CP value, copy number, and amount
of genomic DNA. (a) Relationship between CP value and copy
number. A 10-fold dilution series of the AbSCD1 clone was used
as a template. (b) Relationship between CP value and DNA
in threefold dilution series of two genomic DNAs from Alt.
brassicicola strain Ab44877 (filled circles) and Ab44415 (empty
circles).
The conidium was divided into two to seven cells by the
transepta and longisepta. The percentages of three- and
four-celled conidia from Ab44415, Ab44877, Ab42464,
Ab43923 and Ab44414 were 27, 28, 44, 52, 28 % and 18, 36,
32, 23, 18 %, respectively (Fig. S1, available in Microbiology
Online). The percentage of three- and four-celled conidia
reached up to 61 %. Although Ab44415 was slightly larger
than the remaining strains, no significant differences were
found in conidial length and width. The larger conidia
usually contained more cells. The mean values of the
width6length in three- and four-celled conidia were
8.82±0.93 mm618.23±1.96 mm and 10.04±1.25 mm6
23.72±2.61 mm, respectively. Conidial chain formation
during sporulation has been recognized as one of the
important keys in the diagnosis of the Alt. brassicicola
species, and our strains showed abundant conidial chain
formation (Fig. 4a). ITS region sequencing of the five
strains and subsequent comparative analyses with that of
Alt. brassicicola confirmed that our strains were all Alt.
brassicicola (Fig. S2).
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Microbiology 159
Alternaria brassicicola growth in planta
(a)
Ab44877
Ab44415
Ab43923
Ab44414
Pathogen growth
in planta
Symptoms
Formation of
conidial chain
Conidia
Mock
(×10 000)
35
30
25
20
15
10
5
0
50
5
41
4
Ab
44
3
41
44
Ab
7
92
87
43
Ab
7
92
Ab
3
44
41
Ab
5
44
41
4
Ab
43
87
44
Ab
M
oc
k
0
44
10
oc
20
Ab
30
k
Gene copies
40
M
Fungal DNA (ng)
(b)
Pathogenicity assay of Alt. brassicicola in the two
pathosystems and microscopic observation
Among the six strains of Alt. brassicicola tested, Ab44877
and Ab44414 showed the highest virulence on Bra. rapa
subsp. pekinensis and the remaining three strains also
induced some degree of disease symptoms on the same
host species (Fig. 5a). No visible alteration was evident on
the plants at 1 day p.i. At 2 days p.i., almost all the
infected leaves showed abundant small, water-soaked
spots, and some part of the leaves had turned yellow.
The spots developed into big lesions at 3 days p.i. At
4 days p.i., almost all the infected leaves had turned
yellow, and some leaves were severely withered. All tested
strains induced severe chlorosis and withering on the
outer leaves; however, Ab44877 and Ab44414 provoked
severe yellowing and black spot symptoms on the inner
leaves. In the other host, Ara. thaliana ecotype Col-0,
strain Ab44877 was also highly virulent and the remaining
strains also induced disease symptoms (Fig. 4a). Massive
water-soaked lesions were evident on the infected leaves at
1 day p.i., and the yellowing and withering occurred at
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Fig. 4. Alternaria black spot progression on
the Ara. thaliana ecotype Col-0 and quantification of pathogen growth in planta using
Taqman real-time PCR. (a) Conidial shape
(top row) and formation (second row) of the
conidial chain during sporulation observed
10 days p.i. on oatmeal agar. Bars, 20 mm.
After spraying conidial suspension of each
strain onto the ten plants and maintaining them
in a 100 % relative humidity chamber for 1 day
and subsequently in a growth chamber, the
plants showing representative symptom development were photographed at 4 days p.i.
(third row). Red arrowheads indicate typical
Alternaria black spot symptoms on the
Arabidopsis. Bars, 3 cm. At 3 days p.i.,
infected leaves with representative symptom
development were harvested and stained with
aniline blue. After removal of excess dye,
conidial germination/appressorium formation
and invasive/epiphytic mycelial growth of each
strain were observed under a microscope
(bottom row). c, Conidia; g, germ tube; a,
appressoria; im, invasive mycelia; eg, epiphytic
growth. Mock indicates treatment with 250 mg
ml”1 Tween 20 only. Bars, 20 mm. (b)
Quantification of pathogen DNA in plant DNA
from the Arabidopsis inoculated with Alt.
brassicicola at 3 days p.i. Five whole plants
inoculated with each fungal strain were harvested and combined. The amount of fungal
DNA (ng) in 1 mg of infected leaf DNA was
assessed by plotting the CP value on a
standard curve. The pathogenicity assay was
performed at least three times and representative results are shown.
4 days p.i. In contrast with the results from Bra. rapa
subsp. pekinensis, Ab44414 did not cause severe
symptoms; the symptoms were almost identical to those
induced by Ab43923 and Ab44415.
To observe the pathogen’s mycelial growth, we harvested
infected Arabidopsis leaves at 4 days p.i. and analysed them
under a microscope after aniline blue staining (Fig. 4a).
Active conidial germination, appressorium formation, and
massive invasive mycelial growth were evident on the
Arabidopsis leaves inoculated with strain Ab44877; however, invasive mycelial growth within the leaves challenged
with Ab43923, Ab44415 and Ab44414 was relatively
restricted. In addition, some degrees of epiphytic growth
were observed on the Arabidopsis leaves infected with Alt.
brassicicola.
Quantification of Alt. brassicicola in Arabidopsis
and Korean cabbage
Two host species, Ara. thaliana Col-0 and Korean cabbage,
Bra. brassica rapa subsp. pekinensis, were spray-inoculated
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M. Su’udi and others
(a)
Mock
Ab44877
Ab44414
was estimated chronologically (Fig. 5b). At 1 day p.i., there
were no distinct differences among the strains and the
mean amount of fungal DNA within 1 mg of infected
Korean cabbage DNA was 236 pg. Ab44415, Ab4393 and
Ab42464 DNA did not increase noticeably by the next day;
however, Ab44877 and Ab44414 fungal DNA increased and
reached 2376 and 1241 pg by 2 days p.i. The amounts of
DNA of the remaining three strains were less than 1 ng at
3 days p.i., but those of Ab44877 and Ab44414 sharply
increased to 15 ng and 4 ng, respectively.
DISCUSSION
Ab44415
Ab43923
Control
Ab44414
Ab44415
Ab43923
Ab42464
Ab44877
Fungal DNA (ng)
(b) 16
14
12
10
8
6
4
2
0
Ab42464
0
1
2
3
Days p.i.
Fig. 5. Alternaria black spot progression on the Bra. rapa subsp.
pekinensis (Korean cabbage) and quantification of pathogen
growth in planta using Taqman real-time PCR. (a) After spraying
conidial suspension of each strain onto five plants and maintaining
the plants in a 100 % relative humidity chamber for 1 day and
subsequently in the greenhouse, plants showing representative
symptom development were photographed at 3 days p.i. Mock
indicates treatment with 250 mg ml”1 Tween 20 only. White-lined
boxes indicate active symptom progression on the inner leaves of
Korean cabbage. Bars, 15 cm. (b) Chronological quantification of
pathogen DNA (ng) per microgram of DNA of plants inoculated
with Alt. brassicicola. Outer leaf samples were harvested from the
five plants daily. Each sample was 4 cm ¾ 4 cm and all replicates
were combined. The amount of fungal DNA in 1 mg of infected leaf
DNA was quantified by plotting the CP value on a standard curve.
The pathogenicity assay was performed at least three times and
representative results are shown.
with conidial suspensions from the five strains of Alt.
brassicicola and the pathogens’ growth within infected
tissues harvested at 4 and 3 days p.i. was evaluated by
Taqman real-time PCR. By plotting the CP values on the
standard curve, the amount of fungal DNA and the
number of genomes within 1 mg of infected tissue DNA
were calculated (Figs 4b and 5b). As expected, the amount
of Ab44877 DNA within and on the Arabidopsis tissues was
40 950 pg or 3 256 033 genomes per microgram of infected
tissue DNA. In contrast, the amount of Ab44414 DNA was
only 5870 pg or 658 516 genomes (Fig. 4b). The amount of
Alt. brassicicola within the infected Korean cabbage tissues
1952
Real-time PCR-based strategy is a powerful tool for
molecular detection and quantification. This method is
widely used not only in human disease-related clinical
detection (Rodu et al., 1991; Taylor et al., 2010), but also
in the evaluation of microbial population (Fierer et al.,
2005; Kolb et al., 2003; Mühling et al., 2008). PCR-based
detection and quantification are also convenient, fast
and reliable methods for many other biological applications (Mandal et al., 2011; van Brouwershaven et al.,
2010).
Previously, the quantification of Alt. brassicicola was
performed using SYBR Green I in combination with
5.8S rDNA-specific primers (Brouwer et al., 2003).
Subsequently, another SYBR Green fluorescence-dependent evaluation was presented (Gachon & Saindrenan,
2004). The significant development in the latter experiment
was the introduction of a primer pair specific to
CUTINASE A, a gene that exists within the Alt. brassicicola
genome in a fixed copy number. Although we have
attempted the same experiment several times employing
the same primer pair, the standard curves obtained were
not reliable and the R2 values were less than 0.96 (data not
shown). In this investigation, we present relationships
among the CP value, copy number, and amount of fungal
genomic DNA with high reliability (Fig. 3b). To further
confirm the reliability of our evaluation system, we
constructed two standard curves using two series of CP
values from two serial dilutions of genomic DNA from two
different strains, Ab44877 and Ab44415. Southern blot
analysis revealed that AbSCD1 exists as a fixed, single copy
within their genome. If all of the experimental conditions,
such as template DNA quantification, are ideal, then the
above two standard curves should be identical regardless of
the strain differences. As expected, the two standard curves
almost completely merged with a high degree of reliability.
These results indicate that our DNA evaluation is
preferable and reliable for fungal quantification of Alt.
brassicicola biomass. Our method was able to meet all of
the requirements with respect to specificity (Francis et al.,
2006; Pfaffl, 2004) and sensitivity (Gunel et al., 2011;
Matsenko et al., 2008; Yin et al., 2001). In addition, this
method is relatively free from rDNA contamination during
the PCR because the target of this system is fungal
AbSCD1, which exists as a single copy within the fungal
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Microbiology 159
Alternaria brassicicola growth in planta
genome. Fixed gene copy number is one of the most
important requirements for the proportional relationship
between the amount of fungal DNA and fungal biomass
within infected tissue (Baumgartner et al., 2010; Salvioli
et al., 2008). Therefore, this method should be preferred
over previously reported methods (Brouwer et al., 2003;
Gachon & Saindrenan, 2004).
Based on the above system, we have evaluated the in planta
growth of each strain in the two different pathosystems
Ara. thaliana ecotype Col-0 and Korean cabbage, Bra. rapa
subsp. pekinensis. Strain Ab44877 caused the most severe
black spot symptoms. The quantification of growth of
Ab44877 based on CP values and the microscopic
observation of the infected tissues indicated the active
proliferation of this strain in both host species. Although
Ab44414 also induced intensive symptoms on Korean
cabbage, the development of symptoms on Arabidopsis by
this strain was low and similar to those on Arabidopsis
caused by Ab43923 and Ab44415. A comparison of CP
values revealed 3.8-fold lower mycelial growth of Ab44414
than of Ab44877 in Korean cabbage. Similar to symptom
development, strains Ab44414, Ab43923 and Ab44415 did
not show significant differences with respect to fungal
growth within and on the Arabidopsis plants. Uncoupling
strong symptom development from relatively lower in
planta mycelial growth of Ab44414 on Korean cabbage
indicated the additional contribution of other unknown
virulence factor(s) such as enzymes and/or toxic secondary
metabolites that specifically affect symptom progression
by Ab44414 in Korean cabbage. A similar disagreement
between symptom development and fungal growth within
the host tissue has been described in the interactions
between necrotrophic Septoria glycines and soybean and
between necrotrophic Bot. cinerea and Arabidopsis
(Hoffman et al., 1999; Thomma et al., 1999). Alt.
brassicicola is also a representative necrotrophic pathogen
and several studies have focused on the effects of molecules
synthesized and secreted during prepenetration morphogenesis and symptom development (Fan & Köller, 1998;
Wight et al., 2009). The results for symptom development
and mycelial growth of the strains Ab44877 and Ab44414
indicate the importance of fungal strains in the evaluation of
resistant germplasms or genetic resources. Furthermore,
black spot evaluation methods should be chosen carefully
and appropriately according to the aims of the investigation.
Although invasive mycelial growth of Ab44877 was superior
to that of the other strains, this strain and the remaining
three strains (Ab43923, Ab44414 and Ab44415) also grew
epiphytically on Arabidopsis (Fig. 4a). Therefore, some
portions of the evaluated fungal DNA originated from these
mycelia. Owing to the lack of methods that are able to
distinguish real invasive mycelial growth from epiphytic
growth, results of in planta Alt. brassicicola growth should be
analysed with consideration of epiphytic growth.
Investigation of the 8000 bp region containing AbSCD1
from Alt. brassicicola strain ATCC 96866 revealed the
http://mic.sgmjournals.org
presence of six SphI sites (Fig. 1c). One of the sites is
located within the second exon of the AbSCD1 and the
probe regions for Southern blot analysis in this research.
The expected Southern blot result is identical to the pattern
for Ab43923 which comprises a 3181 bp band containing
the 39 region of AbSCD1 and a 2501 bp band harbouring
the 59 region of AbSCD1 (Fig. 1b). Interestingly, Southern
analyses of the remaining Alt. brassicicola strains revealed a
shift of the smaller band from 2.5 to 4.3 kbp. These results
strongly imply the absence of the three SphI sites (indicated
with asterisks in Fig. 1c) that are located upstream of
AbSCD1 in the genomes of Ab44877, Ab44415 and
Ab44414. Cloning via PCR and sequencing of these
600 bp regions of Ab44877, Ab44415 and Ab44414 further
confirmed the absence of these three SphI sites (data not
shown). Nucleotide sequence analysis indicated that the
EcoRI fragment of ATCC 96866 that contains AbSCD1 is
12 119 bp long and this result coincided with the Southern
blot results from the EcoRI-digested Ab43923. In contrast,
other strains’ EcoRI fragments harbouring AbSCD1 are
larger by as much as 1.8 kbp.
In sum, we have described a reliable method to evaluate
Alt. brassicicola’s mycelial growth based on CP values
inversely reflecting the amount of fungal DNA. Our
method is dependent on a specific primer pair and an
additional dual-labelled probe and guarantees the accurate
and objective estimation of fungal biomass within infected
tissues. Dependent on the results presented here and
several observations not described here, we can make
some suggestions for the establishment of a fungal mass
estimation system by real-time PCR. Prior to all, the
confirmation of the copy number and nucleotide sequence
of a target gene is indispensable. After evaluation of the
specificity of the primer pair, sequencing of the resulting
amplicon and Southern analyses using the amplicon as the
probe are recommended. Exact quantification of fungal
template DNA and infected tissue DNA is a critical step for
the reliable standard curve construction and estimation of
fungal DNA among tissue DNA. Several trials based on
absorbance at 260 nm did not yield consistent values, but
band brightness-based measurements were reproducible.
Although the aim of this investigation was to establish a
fungal biomass quantification system, our data here also
imply the value of symptom development results and the
exact characterization of the tested fungal strains. These
attributes are especially important in the design of
experiments using necrotrophic pathogens and also in
the analysis of the results. Finally, the CP values from the
infected tissue DNA are composed of two kinds of DNA
from actual invasive mycelial growth and epiphytic growth.
This fact should not be ignored in the quantitative data
analyses.
ACKNOWLEDGEMENTS
This research was supported by grants from the Research Program
for Agricultural Science and Technology Development (project no.
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M. Su’udi and others
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