Plant Pathology (1999) 48, 749–755
Identification and toxicity of Alternaria brassicicola, the
causal agent of dark leaf spot disease of Brassica species
grown in Thailand
P. Pattanamahakul and R. N. Strange*
Department of Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, UK
Fungi, with spores characteristic of the genus Alternaria, were isolated from necrotic lesions on leaves of cabbage,
cauliflower, Chinese kale and choi-sum growing in Thailand, and were proved by Koch’s postulates to be the causal
agents of a disease known as dark leaf spot. All isolates corresponded in morphology to descriptions of Alternaria
brassicicola and the identification was confirmed by analysis of the ITS1, 5·8S gene and ITS2 regions of rDNA, the
nucleotide sequences of isolates from all four plants being identical to each other and to the published sequence of a
known isolate of A. brassicicola. Culture filtrates of isolates of the fungus from each host, grown on a defined medium
consisting of Czapek–Dox nutrients supplemented with cations, were toxic to cells isolated from the four host plants.
Taking the data overall, filtrates from the cauliflower isolate were significantly less toxic than those from the other
isolates. Although the filtrate from the cabbage isolate was most toxic to cabbage cells and that of the choi-sum isolate
most toxic to choi-sum cells, filtrates of the Chinese kale and cauliflower isolates were most toxic to cells of plants
other than those from which they were isolated.
Keywords: Alternaria brassicicola, Brassica, dark leaf spot disease, toxicity, ITS regions, Thailand
Introduction
Species of the genus Brassica are important vegetable
crops grown in South east Asia, including Thailand, but
their production is often limited by diseases (Tsou &
Tsay, 1988). Diseases caused by the fungal genus
Alternaria are among the most serious, and they have
been reported from many countries including Australia
(Sivapalan & Browning, 1992), Canada (Petrie, 1974),
South Africa (Holtzhausen & Knox-Davies, 1974),
Taiwan (Wu, 1979), the UK (Maude & HumphersonJones, 1980) and the USA (Babadoost & Gabrielson,
1979). They are thought to be caused principally by
Alternaria brassicicola, although in some instances
Alternaria brassicae and Alternaria raphani are also
important. A. brassicicola is seed-borne, with high
incidences being reported from several countries. For
example, Maude & Humpherson-Jones (1980) found
that 86% of commercial brassica seed produced in the
UK between 1976 and 1978 was infected with A.
brassicicola and, more recently, in the state of Victoria,
Australia, Sivapalan & Browning (1992) showed that 26
*To whom correspondence should be addressed (e-mail:
[email protected]).
Accepted 29 July 1999.
Q 1999 BSPP
out of 44 samples of Brassica oleracea were infected
with the fungus.
Spread of the disease during the growing season is by
wind-blown or rain-splashed spores. Optimum conditions for sporulation are temperatures of 20–308C and a
minimum wet period of 13 h, epidemics occurring when
rainfall is frequent (Humpherson-Jones & Phelps, 1989;
Fontem et al., 1991). These conditions correspond to the
climate in some tropical countries such as Thailand,
where the average annual temperature is 26–288C and
there is daily rainfall during the growing season for
vegetables. A. brassicicola was first reported in Thailand
as the causal agent of dark leaf spot of brassicas in 1983
but its pathogenicity was not demonstrated (Sontirat et
al., 1983). Nevertheless, the disease appeared to cause
considerable damage to the crop in the country
(Visethsung & Saranak, 1988).
The taxonomy of Alternaria species has been based
principally on morphology and sometimes host plant
association. Each of the species occurring on brassicas
(A. brassicicola, A. brassicae and A. raphani) has a
distinct spore morphology. However, when the genus as
a whole is considered, species identification becomes
more difficult mainly owing to considerable variation in
spore sizes; some species, for example, having ranges of
spore dimensions that encompass those of several other
species (Rotem, 1994). Recently, significant advances in
749
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P. Pattanamahakul & R. N. Strange
fungal taxonomy and identification have come about
through DNA analysis. For example, restriction fragment length polymorphisms (RFLPs) have been used to
solve systematic problems in the genus Phythophthora
(Förster et al., 1990) and to measure genetic variability
in the Japanese pear pathotype of A. alternata (Adachi et
al., 1993). Random amplified polymorphic DNA
(RAPD) has been used to determine variation in
Rhizoctonia solani (Duncan et al., 1993) and Pseudocercosporella herpotrichoides (Nicholson & Rezanoor,
1994), and Sharma & Tewari (1998) and Cooke et al.
(1998) showed variation in RAPD patterns among
Alternaria species of diverse geographical origin. However, irreproducibility of RAPD profiles owing to factors
such as the quality of template DNA and polymerase,
differences in ramping times between temperatures and
reaction buffers have impaired the utility of the method
for comparative studies among laboratories (Schierwater
& Ender, 1993).
In contrast, analysis of DNA sequences, particularly
those of the ribosomal repeat unit, has proved to be a
definitive and rapid method for the identification and
taxonomic study of fungi as well as for studies of
evolution and speciation (White et al., 1990; Hillis &
Dixon, 1991). For example, Sherriff et al. (1994) used
rDNA sequences to demonstrate new species groupings
in the genus Colletotrichum and subsequently to
distinguish between Colletotrichum graminicola and
Colletotrichum sublineolum (Sherriff et al., 1995). The
phylogenetic relationship among several species of
Leptosphaeria has also been studied using the internal
transcribed spacers (ITS1 and ITS2) and 5·8S rDNA
regions (Morales et al., 1995). Kusaba & Tsuge (1995)
found that species of Alternaria, which had been
distinguished morphologically, were clearly separated
from each other by sequence variation of ITS1 and ITS2.
Similarly, Jasalavich et al. (1995) used the ITS regions
and 5·8S gene to distinguish four species of Alternaria:
A. brassicae, A. brassicicola, A. raphani and A. alternata
isolated from crucifers. Therefore, in this study this
region of the genome of Thai isolates of Alternaria was
sequenced in order to determine their identity.
Some organisms damage plants by secreting one or
more toxins. Species of Alternaria produce numerous
secondary metabolites, some of which are toxic to
plants. There are at least nine host-selective toxins
produced by Alternaria pathogens. These include three
pathotypes of A. alternata that are specific for Japanese
pear, strawberry and tangerine, and produce the hostselective AK–AF- and ACT-toxins, respectively
(reviewed in Otani et al., 1995). Other toxins produced
by species of Alternaria are nonhost-selective and
include tentoxin, tenuazonic acid, 3-epoxy radicinin
and deoxyradicinin (Nishimura & Kohmoto, 1983;
Robeson & Strobel, 1985; Ballio, 1991).
The objectives of this study were to identify the causal
agents of dark leaf spot diseases of the four brassica
crops grown in Thailand: cabbage (Brassica oleracea var.
capitata), cauliflower (B. oleracea var. botrytis), Chinese
kale (Brassica alboglabra) and choi-sum (Brassica
chinensis var. parachinensis), and to determine if they
produced toxic metabolites that might be involved in
the production of the necrotic lesions that characterize
the diseases.
Materials and methods
Isolation and storage of fungi
Fungi were isolated from disease lesions on cabbage
(seven isolates: designated CB001 to CB007), cauliflower (10 isolates: designated CF001 to CF010),
Chinese kale (nine isolates: designated CK001 to
CK009) and choi-sum (three isolates: designated
CS001 to CS003) growing in the Chiang Mai, Lamphun,
Kamphaengphet and Nakhon Sawan provinces of
northern Thailand. Spores were transferred from lesions
to sterile slides by means of a sterile needle. Single spores
were selected from the slide under a microscope and
transferred again by a sterile needle to acidified potato
dextrose agar (PDA). After incubation for 6–9 days at
258C in the dark, spores derived from these cultures were
subcultured onto the PDA.
Spores were released from the colonies by flooding
plates with sterile distilled water and agitating with a
sterile glass spreader. The resulting spore suspension was
freed from mycelia by filtration through four thicknesses
of sterile muslin, and the spores were washed three times
by centrifugation in sterile distilled water. They were
finally resuspended in sterile 10% glycerol at a density of
107 spores per mL and stored at ¹708C in a freezer or in
liquid nitrogen.
Microscopic examination of the fungi
The fungi were grown on PDA at 258C for 7–9 days and
conidia (100 per isolate) were examined under a
microscope. A slide culture technique was also used to
observe the morphology of the fungi (Booth, 1977).
Plant material
Seeds of cabbage and cauliflower were obtained from
Suttons Seeds Ltd, Torquay, UK and seeds of Chinese
kale and choi-sum were obtained from Horticultural
Research International, Wellesbourne, UK. They were
soaked in water overnight before sowing in Levington’s
multipurpose compost (Levington Horticulture Ltd,
Ipswich, UK) in 15-cm plastic pots (5–8 seeds per pot).
The plants were grown in a greenhouse at 20 6 28C with
a 16-h photoperiod and watered every other day.
Inoculation of plants
Spore suspensions of eight isolates removed from storage
(isolate CB001 and CB002 from cabbage, CF001 and
CF002 from cauliflower, CK001 and CK002 from
Chinese kale, and CS001 and CS002 from choi-sum)
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Plant Pathology (1999) 48, 749–755
Pathogenicity and toxicity of A. brassicicola
were diluted in sterile distilled water to obtain a
concentration of 105 spores per mL for inoculation of
plants. Droplets (5 mL) of the spore suspensions of each
fungal isolate were placed on healthy leaves (4–6
droplets per leaf) of 3-week-old cabbage, cauliflower,
Chinese kale and choi-sum plants (3 leaves per plant).
Control leaves were inoculated with 5 mL of sterile
distilled water. Inoculated plants were covered with
polythene bags for 3 days in order to maintain high
humidity, after which they were removed. Fungi were
re-isolated 10–14 days after inoculation from the
diseased leaves and compared with the original isolates.
Growth of fungi and extraction of DNA
Fungi (isolate CB001 and CB002 from cabbage, CF001
and CF002 from cauliflower, CK001 and CK002 from
Chinese kale, and CS001 and CS002 from choi-sum)
were grown in a medium consisting of Czapek–Dox
nutrients (45·5 g: Oxoid, Unipath Ltd, UK), mycological
peptone (1 g), yeast extract (1 g) and casein hydrolysate
(1 g), dissolved in 1 L water and supplemented with
200 mL of clarified V8 juice (Campbell Grocery Products
Ltd, UK) prepared by filtering the juice through four
thicknesses of muslin and centrifuging at 2000 g for
5 min. The medium was distributed to 250-mL conical
flasks (100 mL per flask) and autoclaved at 1218C for
20 min. Spores and mycelium were scraped from 7-dayold cultures growing on PDA and used to inoculate the
medium. After incubation for 2 days at 258C on an
orbital shaker (5-cm-diameter orbits, 150 r.p.m.) the
mycelia were harvested. DNA was extracted using a
commercial kit (Nucleon Phytopure Plant DNA Extraction Kit, Scotlab, UK) according to the manufacturer’s
instructions. Precipitated DNA was resuspended in
400 mL of TE buffer (10 mM Tris-HCl, 1 mM EDTA,
pH 8·0), RNase (1 mg mL¹1, 100 mL) was added and the
tube incubated at 378C for 2 h on a shaker.
PCR amplification
Primer pairs ITS1 (50 -TCC GTA GGT GAA CCT GCG30 ) and ITS4 (50 -TCC TCC GCT TAT TGA TAT GC-30 )
were used for PCR amplification of rDNA containing
the internal transcribed spacer (ITS) 1, the 5·8S gene and
the ITS2 regions (Jasalavich et al., 1995). Each DNA
sample (50 ng) and the two primers (10 pmol of each)
were added to a ‘Ready to Go’ PCR bead (Amersham
Pharmacia Biotech UK Limited, Buckinghamshire, UK)
which contained 1·5 units of Taq DNA Polymerase,
10 mM Tris-HCl (pH 9·0), 50 mM KCl, 1·5 mM MgCl2
and 200 mM of each dNTP when brought to a final
volume of 25 mL. The PCR reaction was performed for
35 cycles, with an initial 2 min at 948C for denaturation
and a final 7 min at 728C for extension in a Progene
Thermal cycler (Techne (Cambridge) Ltd, UK). Each
cycle consisted of 30 s at 948C, followed by 30 s at 588C
for annealing and 2 min at 728C for extension. Successful amplification was checked by electrophoresis in a
1·7% agarose gel.
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Plant Pathology (1999) 48, 749–755
751
DNA sequencing and analysis
DNA sequencing of PCR products was performed using
the ABI PRISM Dye Terminator Cycle Sequencing
Ready Reaction Kit and AmpliTaq DNA Polymerase,
FS (Perkin-Elmer Corporation, California, USA),
according to the manufacturer’s instructions, with
primers ITS1, ITS2, ITS3 and ITS4 (White et al., 1990;
Jasalavich et al., 1995).
The sequences of the PCR products were aligned using
the Clustal method in conjuction with the programme
LASERGENE NAVIGATOR (DNASTAR Inc., Wisconsin, USA).
Homologies to the sequence data (ITS1, 5·8S and ITS2
regions) were searched using remote BLAST (Basic Local
Alignment Search Tool) services of the National Center
for Biotechnology Information over the WWW interface.
Preparation of isolated cells
The third or fourth true leaf from 2–3-week-old plants
grown in the greenhouse as described earlier was
harvested and cut into 2–4 mm2 pieces, and the pieces
were placed in a digestion solution (Pectolyase Y-23
(ICN Biomedicals Inc., Ohio, USA) 50 mg mL¹1;
Macerozyme (Yakult Honsha Co. Ltd, Tokyo, Japan)
15 mg mL¹1; and bovine serum albumin (BSA: Sigma,
USA), 500 mg mL¹1, dissolved in a holding buffer (HB)
consisting of 50 mM citric acid, 1 mM MgSO4, 1 mM
KH2PO4, NaOH 5·8 g L¹1, glucose 100 g L¹1 adjusted
to pH 5·8). After vacuum infiltration of the mixture and
stirring for 15–20 min, the disintegrating leaf pieces
were filtered through muslin and the cells in the filtrate
were washed three times by centrifuging at 80 g in HB
for 5 min. They were finally resuspended in ice-cold HB
at a density of 0·2 A units at l ¼ 620 nm (7·5–9·0 × 104
cells per mL).
Phytotoxicity test
Fungal isolate CB001 from cabbage, CF001 from
cauliflower, CK001 from Chinese kale and CS001
from choi-sum were grown in Czapex–Dox liquid
medium supplemented with cations (ZnSO4?7H2O
50 mg L¹1, LiCl 5 mg L¹1, CuCl2?2H2O 20 mg L¹1,
MnCl2?4H2O 20 mg L¹1, CaCl2?2H2O 100 mg L¹1,
CoCl2?6H2O 20 mg L¹1, Na2MoO4?2H2O 20 mg L¹1
and H3BO3 10 mg L¹1). The medium was distributed in
30-mL aliquots in 250-mL conical flasks and, after
autoclaving at 1218C for 20 min and cooling, inoculated
with 1 mL of spore suspension (105 spores per mL) of the
fungal isolates. Cultures were incubated at 258C without
shaking and filtrates were harvested in triplicate at 3, 6,
9, 12 and 15 days by filtering through three thicknesses
of Whatman no. 1 filter paper on a Buchner funnel.
Filtrates were freeze-dried and reconstituted at 10 times
the original concentration by dissolving in HB. Uninoculated medium was treated in the same way. The
concentrates from all three replicates for all incubation
periods and for all fungal isolates, as well as uninoculated media to serve as controls (72 preparations in
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P. Pattanamahakul & R. N. Strange
total), were tested for toxicity on cells isolated from
leaves of cabbage, cauliflower, Chinese kale and choisum by the method used by Latif et al. (1993), with
fluorescein diacetate as the live/dead stain (Widholm,
1972). Probit percentage cell death was plotted against
log2 dilution factor in order to determine the dilution
that killed 50% of the cells, which was designated as one
unit of activity. Maximum toxic activity values for each
combination of fungal isolate and plant cell were
subjected to analysis of variance using a two factor
completely randomized design. Significance was determined at P < 0·05, using the least significant difference
test. All calculations were performed using the MSTAT
statistical analysis programme (Freed & Eisensmith,
version 1·3).
Results
None of the control plants inoculated with sterile
distilled water developed symptoms. Koch’s postulates
were completed by re-isolating from the inoculated
plants fungi that had the same morphological features as
those originally isolated.
Sequence data
DNA was successfully extracted from the fungal isolates
using the commercial kit and yielded the PCR products
of about 600 bp. Sequences of the PCR products
obtained from all four primers were aligned and the
maximum homology of the nucleotides was ascertained.
This region contained the ITS1 region (179 bp), the 5·8S
gene (159 bp) and ITS2 region (157 bp). All isolates had
the same sequence and they were identical to a fungus
previously identified as A. brassicicola (Jasalavich et al.,
1995).
Morphology of the fungal isolates and completion of
Koch’s postulates
Fungal cultures obtained from leaf spot lesions on
cabbage, cauliflower, Chinese kale and choi-sum plants
growing in northern Thailand corresponded in morphology to A. brassicicola (Wiltshire, 1947). On PDA, spores
were produced in rarely branched chains of up to 15 and
had no beak. They were dark olivaceous brown, nearly
cylindrical (24·88 6 7·38 × 10·93 6 1·71 mm; range 13–
44 × 8–21) with 1–6 transverse septa and up to 2
longitudinal septa. Colonies on PDA were velvety and
dark olivaceous brown. They grew at an average rate of
7·8–8·8 mm per day and sporulated copiously.
Healthy cabbage, cauliflower, Chinese kale and choisum plants developed disease symptoms when inoculated with droplets of spore suspension. Within 2 days,
leaf tissue under an inoculum droplet became necrotic
and the area of necrosis was surrounded by a halo of
chlorosis. On further incubation, lesions increased in
size and became darker as the fungus sporulated and
eventually the infected area became dry and paper thin.
Phytotoxicity of isolates
Culture filtrates of all four fungal isolates (i.e. CB001,
CF001, CK001 and CS001) were toxic to cells of the
four plant types (cabbage, cauliflower, Chinese kale and
choi-sum) but not to the uninoculated medium. Analysis
of variance of the maximum toxicity values showed
highly significant differences among isolates and among
cells. Overall the cabbage isolate was the most toxic and
the cauliflower the least (Table 1). Cells of the four plant
types were of similar sensitivity to culture filtrates of the
Chinese kale isolate and also reacted similarly but less
sensitively to culture filtrates of the cauliflower isolate.
In contrast, cells of cabbage and choi-sum were sensitive
to culture filtrates of the choi-sum isolate but the cells of
the other two plants were considerably less sensitive.
The isolate from choi-sum was distinct in that maximum
activity peaked at 12 days whereas in most interactions
activity continued to rise throughout the period of
culture (Fig. 1).
Table 1 Maximum toxic activity (units per mL) of culture filtrates of four isolates of A. brassicicola when tested on plant cells of the four brassicas. Maximum activity was attained in filtrates from 12- to 15-day-old cultures. Analysis of variance was performed using a two factor completely randomized design
Plant cell type
Isolate
name*
Cabbage
Choi-sum
Cauliflower
Chinese kale
Mean
sensitivity of all
plant cell types
CB001
516·88 6 176·82 a
262·12 6 10·61 d
467·05 6 18·47 ab
381·85 6 56·63 bc
406·80
CK001
342·68 6 70·88 cd
438·63 6 65·05 abc
373·02 6 52·00 bc
356·28 6 52·83 cd
377·65
CS001
341·97 6 93·28 cd
402·20 6 75·37 bc
129·74 6 55·38 ef
152·28 6 42·06 e
256·55
CF001
95·42 6 19·43 ef
23·57 6 5·23 f
45·48
Mean toxicity
of all isolates
324·24
26·99 6 5·50 f
282·49
35·94 6 1·42 f
251·44
228·50
Values designated by different letters are significantly different at P < 0·05 (least significant difference test).
*CB, cabbage isolate; CK, Chinese kale isolate; CS, choi-sum isolate; CF, cauliflower isolate.
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Pathogenicity and toxicity of A. brassicicola
753
Figure 1 Toxic activity (units per mL) of culture filtrates of the four isolates of A. brassicicola – (a) isolate CB001 from cabbage, (b) CF001 from
cauliflower, (c) CK001 from Chinese kale and (d) CS001 from choi-sum – when tested on cells of cabbage (V), cauliflower (B), Chinese kale
(O) and choi-sum (X). Each point is the mean of three replicates.
Discussion
In Thailand, Alternaria sp. has been considered to be the
cause of leaf spot disease on brassicas since the 1960s
(Puckdeedindan, 1966), but A. brassicicola was only
recognized as the possible causal agent in 1983 when
Sontirat et al. (1983) isolated the fungus from lesions on
the four brassicas used in this study. However, he did not
perform pathogenicity tests.
In the present work, lesions on the leaves of cabbage,
cauliflower, Chinese kale and choi-sum consisted of
dark, central necrotic spots 1–10 mm in diameter
surrounded by a chlorotic halo that accorded with the
descriptions given by Weimer (1924) and Wiltshire
(1947) for Alternaria leaf spot diseases of brassicas.
Spores isolated from the lesions and cultured on PDA
gave fungal colonies with spore morphologies that were
consistent with those described for A. brassicicola by
Wiltshire (1947) but were smaller (13–44 × 8–21 mm)
compared with 18–130 × 8–30 mm. The fungus was
proved to be the cause of the disease by the completion
of Koch’s postulates.
Confirmation of the identity of the pathogen was
sought by sequence analysis of rDNA. These experiments showed that the sequences of the internal
transcribed spacers and 5·8S gene of the Thai isolates,
which were identical to each other, were also identical to
that of a Canadian isolate of A. brassicicola (Jasalavich
et al., 1995). Thus, the causal agent of dark leaf spot of
brassicas in Thailand was confirmed as A. brassicicola at
the molecular level. Two interpretations of the perfect
match of the DNA sequences of the Thai and the
Canadian isolates are that either these regions are
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Plant Pathology (1999) 48, 749–755
particularly conserved within A. brassicicola or that
the Canadian and Thai isolates are of common origin.
The latter seems more likely, because the fungus is seedborne and infected seed may have provided the means of
transport from one side of the world to the other (Petrie,
1974).
Profuse production of chains of the dark spores of the
fungus is one reason for the dark appearance of the
lesions caused by the pathogen. They serve not only to
spread the disease in the current crop but also may
contaminate seed, thus ensuring the pathogen’s survival
between crops (Babadoost & Gabrielson, 1979; Wu
1979; Sivapalan & Browning, 1992). Another reason for
the dark central area of the lesions and necrotic
symptoms may be the production of toxins that kill
host cells (Bains & Tewari, 1987). Although there are
numerous precedents for the production of toxins by
species of Alternaria, little work has been reported on
phytotoxic metabolites produced by A. brassicicola.
MacDonald & Ingram (1986) reported that methanol
extracts of the culture filtrates of the fungus were
phytotoxic. Cooke et al. (1997) also found that spore
germination fluids of the fungus were phytotoxic. More
recently, Otani et al. (1998) reported that spore germination fluids of the fungus contained a host-specific high
molecular weight phytotoxin.
In the present study, the Thai isolates of A. brassicicola also produced toxic culture filtrates, the isolates
varying in toxigenicity and the cells of different brassica
species varying in sensitivity to them. However, there
was no consistent relationship between the plant from
which the isolate originated and the sensitivity of its cells
to the culture filtrate.
754
P. Pattanamahakul & R. N. Strange
Acknowledgements
We thank the Development and Promotion of Science
and Technology Talent Project of the Thai Government
for providing financial assistance throughout this study,
Dr Vicha Sardsud and his staff at Chiang Mai University
for help in isolating fungi from infected brassica species,
Horticultural Research International, Wellesbourne, for
seed of Chinese kale and choi-sum, the Central Research
Fund of London University for a grant to enable the
purchase of a thermocycler and Miss Laura Winskill for
sequencing the PCR products.
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