286

Mycol. Res. 104 (3) : 286292 (March 2000). Printed in the United Kingdom.

Genetic diversity of Alternaria alternata isolated from tomato

in California assessed using RAPDs

Paul F. MORRIS1, Mary S. CONNOLLY1 and Dina A. ST CLAIR2

" Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403, USA.
# Department of Vegetable Crops, University of California, Davis, California 956168746, USA.
Accepted 11 March 1999.

Black mould lesions were caused by Alternaria alternata in 76 % of 228 tomato fruit with characteristic sunken black lesions collected
from fields of processing tomatoes in California. Analysis of 29 RAPD primers revealed a high level of genetic diversity among the
69 isolates tested. Two major phenetic groups (Group 1 with 55 isolates and Group 2 with 14) were identified independently by
PCA and by UPGMA of Jaccard similarity coefficients. Only 34 of 137 RAPD markers were monomorphic for all isolates and the
genetic similarity between the two groups was 50 %. Co-infection of black mould lesions by genetically distinct strains of A. alternata
occurred in two of 10 isolates tested. There was no evidence for geographic clustering of isolates with high levels of genetic
similarity, suggesting that isolates are widely dispersed across California. Only one isolate was identified which also caused stem
canker disease on a susceptible tomato cv., suggesting that these strains play a minor role in causing black mould on processing
tomatoes in California. This isolate and two other known stem canker isolates were clustered together with 11 other isolates in
Group 2. Group 2-specific bands were also identified in a survey of seven additional isolates known to produce host-specific toxins.

Alternaria alternata (Fr.) Keissl. is the principal causative agent

of black mould, a disease characterized by sunken black lesions
on ripe tomato fruit (Pearson & Hall, 1975). A. alternata was
found to be the most prevalent fungus causing ripe fruit rot
in California processing tomatoes (Butler, 1959). A recent
survey indicated that 5 % of all fruit harvested from four
counties in California had visible symptoms of black mould
(Davis et al., 1997). Infection of ripe tomato fruit by this
airborne pathogen is promoted by high humidity and cool
temperatures, dew, or rainfall (Pearson & Hall, 1975).
Alternaria alternata f. sp. lycopersici Grogan, Kimble &
Misaghi (AAL) produces stem cankers, foliar symptoms and
lesions on green fruit of tomato cultivars which do not contain
the incompletely dominant allele Asc in the homozygous
condition (Grogan, Kimble & Misaghi, 1975 ; Gilchrist &
Grogan, 1976). These disease symptoms result from hostspecific toxins produced by AAL strains (Gilchrist & Grogan,
1976 ; Chen et al., 1992 ; Caldas et al., 1994). AAL isolates are
also capable of causing black mould on ripe tomatoes, but it
is not known whether they are a major cause of black mould
in the Central Valley of California. This region produces the
majority of tomatoes for the tomato processing industry in
the USA.
The potential for breeding resistance against A. alternata
isolates that cause black mould was examined using a sample
of California isolates on two tomato populations derived from
crosses of cultivated Lycopersicon esculentum with a related wild
species, L. cheesmanii (Cassol & St. Clair, 1994). Resistance to
black mould in L. cheesmanii f. sp. typicum accession LA 422 is

controlled by at least two genes and exhibits low (16 %)

heritability (Cassol & St. Clair, 1994). This study suggested
that breeding for resistance to A. alternata is possible.
Knowledge of pathogen diversity, however, and its relation to
aggressiveness, if any, is needed to determine how useful any
source of resistance would be in controlling black mould.
There is considerable morphological variation of spore size,
shape, and ornamentation among A. alternata isolates
(Simmons, 1978) and this is also reflected in estimates of
genetic variation. Petrunak & Christ (1992) identified 22
genotypes in a survey of 96 isolates collected from across the
United States which were clustered at a genetic distance of
0n35. Adachi et al. (1993) used a rDNA probe to assess genetic
variation of the Japanese pear pathotype in central and
western regions of Japan. Their results indicated that there
was also a high level of genetic variation in this pathotype.
Hybridization of DNA from several Alternaria spp. with a
rRNA probe indicated that isolates producing host-specific
toxins were genetically distinct from other Alternaria spp. but
not from isolates of A. alternata (Kusaba & Tsuge, 1994).
Sequence analysis of spacer regions of rRNA also supported
the conclusion that isolates which produce host-specific toxins
are variants of A. alternata (Kusaba & Tsuge, 1995).
We have found RAPD markers to be a reliable first step for
the analysis of genetic variation in plants (Williams & St Clair,
1993) and fungi (Francis & St Clair, 1993 ; Francis, Gehlen &
St Clair, 1994). RAPD markers are randomly distributed
throughout the genome and can be efficiently and reliably
sampled using established procedures (Williams et al., 1990 ;

P. F. Morris, Mary S. Connolly and Dina A. St Clair

Skroch, Tivang & Nienhuis, 1993 ; Weeden et al., 1993).
Studies involving RAPDs have demonstrated that a relatively
small number of primers was sufficient to identify pathotypes
(Bayman & Cotty, 1993 ; Ouellet & Seifert, 1993) or vegetative
compatibility groups (Bayman & Cotty, 1993 ; Duncan, Barton
& OBrien, 1993) in various fungi. Genetic variation within
vegetative compatibility groups using RAPDs has also been
demonstrated (Jacobson, Miller & Turner, 1993).
For this survey, isolates were cultured from typical black
mould lesions on ripe field-grown fruit. In previous studies,
some single spore-derived isolates have been shown to be
avirulent (Adachi & Tsuge, 1994), hence we made no attempt
to ensure that isolates taken from a single lesion were clonal,
but instead reflected the original field lesion composition as
closely as possible. Our approach enabled us to address the
question of whether or not there were significant genetic
differences among lesions. The objectives of this study were :
(i) to determine what pathogens were primarily responsible
for black mould lesions on processing tomatoes from different
production areas in California, (ii) to determine the level of
genetic variation among A. alternata causing black mould in
different tomato production areas of California using RAPD
primers, and (iii) to determine if A. alternata and AAL isolates
could be distinguished with RAPDs.
MATERIALS AND METHODS
Collection, identification and maintenance of isolates
A total of 228 isolates was obtained from lesions characteristic
of black mould (Pearson & Hall, 1975) on 228 ripe fruit
collected from California processing tomato production areas
during Aug. to Oct. 1992. Of this total, 174 isolates were
identified as Alternaria alternata, 14 were Stemphylium spp., six
were Fusarium spp., three were Geotrichum spp. and 31 were
not identified. Isolates of A. alternata were characterized by
their abundant sporulation on V8 and potato dextrose agar
(PDA). The conidial chains were upright and branched with
dense ornamentation on the cell walls, typical of this species
Co1f, Co2a, Co3e, Co4c, Co5e
Co5h, Co6a, Co6b, Co6c, Co6d
Co6e, Co6g, Co6i

200 km

Co7c, Co8b, Co9c, Ro1d

Yo2d, Yo3c, Yo4a, Yo4f, Yo5b
Yo6a, Yo7c, Yo8c, Yo9a, Yo10d
Yo1d, D1, Aug1, Au31,
No30, Wd5, Sui2,
SL1c, SL2e, SL4a
STC, Wa1, Si4, EEEP
Male, Ma2a, Ma2c, Ma2e
Ma2f, Ma2g, Ma2h, Ma3g
Ma4b, Cr3c, Cr4a, Cr6e

V1b, V1c, V2a, V2b, V2c

V2d, V2e, V2f, V2g, V2h
V2i, V2j, V2k, V2h, V3j, V4d

AS27

Fig. 1. Map of California showing locations of processing tomato

production areas sampled for A. alternata isolates used in this survey.
Isolate codes which differ only by the last letter denote isolates
collected from the same microgeographical location.

287
(Simmons, 1967, 1981). A randomly chosen subsample of the
1992 collection (65 A. alternata isolates out of 174) was used
to assess levels of genetic variation. Each isolate was derived
from a single lesion. In addition to these 65 isolates obtained
in 1992, four other isolates were added. Two AAL isolates
(AS27 and EEEP ; obtained from D. G. Gilchrist, University of
California, Davis) known to cause stem canker disease were
included to see if RAPDs could distinguish this pathotype.
Two other A. alternata isolates (No30, Aug1) were collected in
1990 from infected fruit of tomato plants grown in a field at
Davis, California (Fig. 1), and used in a study of inheritance of
resistance of A. alternata (Cassol & St Clair, 1994).
The aim of this study was to measure the diversity among
lesions since a lesion is a successful infection unit under field
conditions. Infected fruit were surface-sterilized in 0n8 %
sodium hypochlorite for 10 min, and rinsed in sterile distilled
water. A portion of infected inner carpel wall was dissected
out and transferred to acidified (pH 4n1) PDA plates amended
with streptomycin sulphate and chlortetracycline (each at
50 g ml") and grown under fluorescent lights at 25 mC.
Cultures that appeared to be infected with bacteria were plated
on acidified PDA media amended with chloramphenicol or
vancomycin (50 g ml"). Conidia from aseptic cultures were
transferred to V8 juice agar (Erwin & McCormick, 1971) and
to silica gel (Perkins, 1962) for long-term storage. Isolates
could be regrown after more than a year in storage by
sprinkling a few silica gel particles on V8 juice agar plates.
DNA extraction and RAPD analysis
Flasks containing 80 ml of 20 % V8 juice were inoculated with
conidia from a single isolate, placed in a shaker set at 150 rpm,
and grown in the dark at 25m for 68 d. Hyphae were
collected by filtration and stored at 80m. DNA was extracted
using potassium ethyl xanthogenate (Jhingan, 1992). After
extraction, DNA concentration was determined in a fluorometer (Hoeffer Instruments, model TKO-100, San Francisco)
according to the manufacturers directions. DNA stocks for
RAPDs were diluted in TE (0n1) buffer (10 m Tris pH 7n6,
0n1 m EDTA).
DNA was amplified by the RAPD technique (Williams et
al., 1990) in which random 10 bp oligonucleotide primers
(Operon Technologies, Alameda, California) were used to
produce amplified fragments ranging from 200900 bp. The
29 random primers used in this survey were OPA01, OPA02,
OPA09, OPA13, OPA16, OPA17, OPC05, OPC06, OPC09,
OPC10, OPC11, OPC14, OPC15, OPC18, OPC19, OPC20,
OPD01, OPD02, OPD03, OPD04, OPD05, OPD07, OPD08,
OPD11, OPD12, OPD13, OPD14, OPD15 and OPD20. Each
isolate was tested at a range of DNA concentrations, and the
highest concentration that resulted in the clearest amplification
of RAPD bands was used. This corresponded to 0n5 to 2n5 ng
DNA for all isolates in a reaction volume of 25 l. In addition,
each reaction contained 400 n 10 bp primer, 1n5 m MgCl
#
and 3n5 U Stoffel Taq (Applied Biosystems). DNA was
amplified in a PE Applied Biosystems model 480 DNA
thermal cycler with the following programme : 94m for 1 min,
initial denaturation cycle ; 1 min at 94m, 1 min at 35m, 2 min at
72m, 40 cycles ; 7 min at 72m, 1 cycle. The PCR products were

A survey of Alternaria alternata isolates

288

separated on 2n15 % agarose gels (1n08 % Nusieve GTG, FMC

BioProducts, Rockland, ME ; 1n07 % Ultrapure, BRL,
Gaithersburg, MD) in 1iTBE (0n45 Tris-borate,
0n001 EDTA, pH 8n3) buffer. Gels were run for 5 h at
3n5 V cm", stained with ethidium bromide and photographed
on a uv light box. In some experiments, the DNA fluorescence
patterns were acquired using the Foto\Eclipse image analysis
system, Fotodyne Inc, Hartland, WI.
DNA from two isolates with distinct RAPD patterns were
mixed in various ratios to assess the ability of the RAPDs
technique to detect rare alleles in DNA mixtures (Michelmore,
Paran & Kesseli, 1991). The DNA mixtures used were 5 %,
10 %, 15 %, 20 %, 25 %, 30 %, 40 % and 100 %. RAPD analysis
was performed as described above.
To test for co-infection of lesions by different isolates,
DNA from 4 to 5 single hyphal-derived cultures was analysed
in 10 parent isolates. RAPD analysis of the DNA was done
with five random primers using a PE Applied Biosystems
GeneAmp PCR System 2400 with the following program :
94m for 2 min, initial denaturation cycle ; 1 min at 94m, 1 min
at 35m, 2 min at 72m, 35 cycles ; 7 min at 72m, 1 cycle.

A Q-type PCA was also performed using NTSYS-pc as an

independent test of the clustering of isolates by UPGMA
(Rohlf, 1992 ; Francis et al., 1994). The data matrix of RAPD
bands was standardized (0 mean, unit variance), productmoment correlations were calculated among the variables, and
eigenvectors were extracted from the resulting matrix. The
standardized data were projected onto the eigenvectors to
obtain a plot of the first three principal components. The
Mantel test (Sokal, 1979) was used to test the hypothesis that
the genetic variation of isolates was associated with
geographic origin ; non-significance indicates no association.
To test the hypothesis that phenetic grouping was
associated with toxin production, six isolates from ATCC and
one isolate (PetoS) obtained from Dr B. Gabor (PetoSeed,
Woodland, CA) were analysed with RAPDs. RAPD analysis
of these seven isolates was performed with five primers
(OPA01, OPA13, OPC10, OPC11, OPD05) for the presence
of RAPD bands that distinguished two major phenetic groups
of isolates.

Southern analysis

All isolates that were tested with RAPDs (except three lost
during culturing : Yo7c, Yo3c, Ma4b) were also assayed for
the ability to cause stem canker on a susceptible cv. Each
isolate was tested at least twice on susceptible (L. esculentum
cv. Earlypak 7) and resistant (L. esculentum cv. Cal Ace) plants
using either the whole plant method of Gilchrist & Grogan
(1976) or the petiole assay of Adachi et al. (1993). Lesion
development was evaluated 5 d after inoculation.

RAPD gels were blotted onto Hybond-N (Amersham,

Arlington Heights, IL) by alkaline transfer (Southern, 1975).
Selected RAPD-generated bands used as probes (RAPD bands
OPC11330 and OPC11350 : bands were denoted by the
Operon primer number, followed by the band size in bp) were
excised from gels, purified with Gene Clean II (Bio101, La
Jolla, CA) and subcloned using the TA cloning kit (Invitrogen,
San Diego, CA). White colonies were checked for the presence
of the RAPD insert by PCR amplification using vector-specific
primers according to the manufacturers directions. The
amplified insert was purified by gel filtration (Ultrafree-MC,
Millipore Corporation) and labelled with [-$#P]dCTP using
the Prime-It II random primer labelling kit (Stratagene, La
Jolla, CA). Hybridization of the membranes was done as
previously described (Francis & St. Clair, 1993). Only very
short exposures (530 min) were necessary when Southern
blots of RAPD gels were probed with a cloned RAPD band
from one isolate because these blots contained selectively
amplified DNA fragments.
RAPD data analysis
Only bright bands that could be clearly distinguished as
present or absent for all isolates were scored. Bands that were
amplified in only one isolate were not scored. A total of 137
bands generated by 29 oligonucleotide primers was used to
assess levels of genetic variation. Statistical analysis of the
data was performed using the NTSYS-pc program (Rohlf,
1992). The degree of genetic relatedness or similarity was
estimated using the Jaccard coefficient [a\(nd)] in which the
data are defined by a two-way contingency table such that for
any pairwise comparison of isolates, a l (1, 1), b l (1, 0), c l
(0, 1), d l (0, 0), with 1 denoting presence of a band and 0
denoting absence of a band, and n l (ajbjcjd). Clustering
of similarity matrices was by UPGMA and the projection of
phenograms was done using the TREE program of NTSYS-pc.

Stem canker disease evaluation

RESULTS AND DISCUSSION

Lesion identification and analysis of genetic variation
Black mould symptoms can be produced by several plant
pathogens, and for this reason, we first set out to confirm that
A. alternata remains the primary cause of black mould on
processing tomatoes. In this survey, 76 % (174 out of a total
of 228 infected fruit) of all isolates identified from typical black
mould lesions were A. alternata. These results are comparable
with a much earlier survey completed by Butler (1959) who
reported that 65 % and 51 % of all ripe fruit rot lesions in
tomatoes were caused by A. tenuis (now A. alternata) in
surveys from 1956 and 1957, respectively.
Amplification of A. alternata DNA produced reproducible,
clear RAPD bands (Fig. 2). In this survey, 34 of 137 bands
scored were monomorphic in all 69 isolates tested. Two bands
(OPC11350, OPC11330) were tested as hybridization
probes on Southern blots of RAPD gels to confirm that bands
of the same molecular weight were homologous. Each probe
hybridized strongly to a single band (Figs 34) in all of the
isolates in which that size fragment was amplified (Fig. 2). In
addition, probe OPC11330 hybridized to a second band of
350 bp (Fig. 4).
Computation of the Jaccard coefficient and clustering by
UPGMA produced a single tree (Fig. 5) for the 69 isolates
(cophenetic correlation r l 0n90). The 69 isolates were
clustered into two major phenetic groups (Gp1 with 55

D1
Co9c
Male
Sui2
Au31
Cr6e
Yold
Yo9a
Co7c
Yo8c
SL4a
V3j
Wa1
SL1c
No30
Co3e
Wd5
Rold
SL2e
STC
Aug1
Yo4f
AS27
EEEP

P. F. Morris, Mary S. Connolly and Dina A. St Clair

800
600
400

200
3

D1
Co9c
Male
Sui2
Au31
Cr6e
Yold
Yo9a
Co7c
Yo8c
SL4a
V3j
Wa1
SL1c
No30
Co3e
Wd5
Rold
SL2e
STC
Aug1
Yo4f
AS27
EEEP

Figs 24. PCR amplification products and Southern analysis of a

RAPD gel. Fig. 2. PCR amplification of DNA from 24 isolates
representing both Gp1 and Gp2. A mol. wt marker consisting of a
100 bp ladder is shown in the first and last lanes of the gel. Single and
double arrows denote bands (350 and 330 bp, respectively) cloned
for Southern hybridizations shown in Fig. 3 and Fig. 4. All isolates
with the 330 bp band were classified as Gp2. Fig. 3. Southern
hybridization of OPC11 amplified A. alternata DNA using PCR
amplified plasmid probes of OPC11350. Fig. 4. Southern
hybridization of the Gp2-specific band, OPC11330. Two bands,
330 and 350 bp, were detected in all Gp2 isolates (Yo9a, SL4a, SL1c,
Co3e, Rold, SL2e, Aug1, AS27, and EEEP). Isolate Wa1 contains a
band at 320 bp which does not hybridize with the probe.

isolates and Gp2 with 14). The genetic similarity within

groups (73 % for Gp1, 80 % for Gp2) was much higher than
the 50 % similarity between the two phenetic groups. When
the genetic variation of the isolates was analyzed by scoring
only the first 40 bands, the same two major phenetic groups
were identified. Using this limited data set, 65 of the 69
isolates could be distinguished from each other, but clustering
by UPGMA produced more than six trees (data not shown).
Principal component analysis was performed to provide a
method independent of UPGMA clustering to evaluate
variation among the isolates. The first three components
explained 38 %, 5n5 % and 5 % of the total variation,
respectively. The three dimensional plot in Fig. 6, therefore,
summarized 48n5 % of the total variation in the collection. The
two major phenetic groups were found to be projected in
distinct areas of space. Interestingly, isolate Wa1, which had
the least degree of genetic similarity relative to other
members of Gp1 (Fig. 5), was projected in space remote from
both Gp1 and Gp2.
Although the level of similarity among isolates clustered
within the same major group was high, RAPDs identified
bands that could be used to distinguish uniquely all 69 isolates
included in this study. The RAPD data presented here and
other molecular studies (Adachi et al., 1993) confirm that even
among isolates collected from a single species, the level of
variation among isolates is high. One possible explanation for
this diversity is that there exists an unknown sexual stage :

289
04

06

08

10
V1b
Co2a
Yo3c
Si4
D1
Yo4f
Male
Co1f
Yold
Yo6a
Wd5
V2d
Au31
Yo5b
Co4c
Co5e
V3j
Co6i
Co8b
No30
Co6g
Cr3c
Ma2g
V2h
V2a
V2g
V2b
V2c
V2k
Ma2a
Ma4b
Co5h
Ma2e
Ma3g
Yo10d
Cr4a
Yo2d
SL4a
Sui2
STC
V2i
Co6e
Co7c
Cr6e
V2j
Ma2f
Ma2c
Yo7c
Co9c
Co6a
V1c
V4d
V2e
V2f
Wa1
Co3e
Co6d
Yo4a
Ma2d
Yo8c
SL1c
SL2e
Ma2h
Rold
Aug1
AS27
EEEP
Co6c
Yo9a

Fig. 5. Phenetic tree of 69 A. alternata isolates. UPGMA cluster

analysis was based on the Jaccard similarity coefficient. AS27, EEEP,
and Aug1 are stem canker (AAL) isolates in Gp2. Isolate codes which
differ only by the last letter denote isolates that were collected from
the same microgeographical location.

A survey of Alternaria alternata isolates

290

Gp1

Gp2
Yo10d
Ma3g

08

V4d

Aug1
SL1c
Yo9a

V1c
V2h

Co6d

Wa1
12

08

04

00

04

08

Fig. 6. Principal component analysis of 69 A. alternata isolates.

Selected isolates are labelled to illustrate that this analysis separated
the isolates into the same two groups as shown in Fig. 5.

Alternaria anamorphs of several Lewia spp. have already been

described (Simmons, 1986). Significant genetic variation in
asexually reproducing fungi is not atypical, however. High
levels of genetic variability have been found in Fusarium
oxysporum (Gordon & Martyn, 1997) and in asexual
populations of Uromyces appendiculatus (Groth, McCain &
Roelfs, 1995).
The Mantel statistic, Z, revealed no significant geographic
population substructures. Isolates collected from the same
geographic area were no more similar than isolates collected
from widely separated locations. The lack of geographic
substructures suggests that spore dispersal by wind and rain
is effective in scattering isolates over a wide geographic area.
Analysis of the data indicated that some of the bands could
be used to indicate which major phenetic group the isolates
belonged. In Gp2 isolates, 21 bands (OPA01410, OPA09
600, OPA09450, OPA13230, OPA13700, OPC09730,
OPC09500, OPC10650, OPC11330, OPC15380,
OPD01470, OPD03690, OPD04380, OPD05490,
OPD08260, OPD121300, OPD121200, OPD12180,
OPD13750, OPD15530, OPD20280) were present in the
majority of cases, but were always absent in Gp1 isolates. In
addition, eight Gp1-specific bands (OPC15520, OPC18430,
OPD01480, OPD03300, OPD04520, OPD07350,
OPD15510, and OPD20280) were identified. Thus the
presence of one of these bands could be used to reliably place
an isolate into one of the two phenetic groups.
When two genetically distinct isolates were mixed together
and subjected to RAPD analysis, a faint band characteristic of
the genotype in lowest abundance was detectable when the
DNA from that isolate constituted 10 % of the DNA mixture
(data not shown). The same band was much more prominent
when that isolate constituted 20 % or more of the mixture.
Thus RAPD bands did not represent an averaging of all
possible bands in a mixture, but rather the more abundant
alleles were preferentially amplified and detected. The aim of
this study was to measure the genetic variation among lesions,
since a lesion is a successful infection unit under field

conditions. Therefore, each isolate for this survey was derived

from a single lesion and was not a single spore-derived
culture. RAPD analysis of DNA bulks by Michelmore et al.
(1991), and our own DNA mixing experiments using two
genetically distinguishable isolates, support the conclusion
that RAPDs detect the predominant alleles in a DNA mixture
and not the rarer alleles. Thus even if isolates are composed
of more than one strain, with RAPD analysis it is still possible
to detect major differences between them.
The aetiology of black mould lesions on ripe fruit is still
unclear. Entry into the fruit may be an active process but
opportunistic infections may also occur as a result of sunburns,
bruising or cracking of ripe fruit. Given the abundance of aerial
spores, both active and opportunistic infections could result in
lesions being composed of genetic mixtures. If most infections
occur by hyphal growth through tiny cracks on the surface,
the genetic diversity of lesions might be expected to be
primarily clonal or mixtures of one or two isolates. Such a
finding would suggest that control of this disease might be
achieved by selecting for a more elastic epidermis, capable of
fruit expansion without loss of integrity under the environmental conditions present in the Central Valley of
California.
The genetic variability among spores within 10 lesions was
assessed by RAPD analysis using 5 primers and scoring 40
bands. All of the isolates from one lesion could clearly be
distinguished from isolates from other lesions by scoring
bands amplified by these primers. In eight of the 10 lesions,
however, all the isolates appeared to be homozygous. The
RAPD patterns produced by three primers on four single
hyphal-derived cultures from two of the lesions in this survey
are shown (Figs. 78). Each of the three primers amplified at
least one band that could be used to distinguish isolates from
the two lesions. No unique bands were, however, identified
from isolates within the same lesion.
Co-infection by different isolates of single lesions of
Japanese pear leaves by the Japanese pear pathotype of
A. alternata was first confirmed by RFLP analysis of rDNA
variants of single spore-derived cultures (Adachi & Tsuge,
1994). RAPD analysis of single spore-derived cultures may be
a more sensitive method for identifying genetic variants, since
several RAPD bands can be scored for every primer used.
Our tentative conclusion from a small survey is that lesions
on tomato fruit have minimal genetic variation and are
predominantly clonal. In the laboratory, we have frequently
observed that A. alternata spores germinated profusely on the
surface of a tomato fruit but failed to cause disease. When the
skin of the fruit was broken, however, black mould always
developed. Given that the variability of the pathogen is high
in tomato fields, a finding that many infections are clonal or
mixtures of only a few isolates may suggest that wounds or
cracks are the primary source of entry of this pathogen. These
findings certainly justify a more extensive survey of field
isolates to determine the typical genetic makeup of lesions.
Stem canker disease evaluation
Only one isolate, Aug1, in addition to the stem canker isolates
AS27 and EEEP, produced characteristic symptoms on the

P. F. Morris, Mary S. Connolly and Dina A. St Clair

7

OPC-05

OPC-10

OPC-19

291
8

OPC-05

OPC-10

bp

OPC-19
bp

1600

1600

800

800

400

400

200

200

Figs 78. RAPD patterns for 2 selected isolates using 3 random primers (OPDC05, OPC10, and OPC19). Lanes 1 and 14 are 100 bp
markers. Fig. 7. RAPD profiles of four single hyphal-derived cultures of isolate Au31. Fig. 8. RAPD profiles of four single hyphalderived cultures of isolate Sui2.
Table 1. Strains of Alternaria alternata producing host-specific toxins and
associated with Gp2 strains.
Origin of isolate

Isolate

Host

Source

California, U.S.A.
California, U.S.A.
California, U.S.A.
California, U.S.A.
California, U.S.A.
Greece
Kansas, U.S.A.
England
England
Italy

Aug1*
AS27*
EEEP*
PetoS
28329
60647
34956
56835
56836
66868

Tomato
Tomato
Tomato
Tomato
Tomato
Tomato
Sorghum
Wheat
Wheat
Sunflower

This study
D. Gilchrist
D. Gilchrist
B. Gabor
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC

* Isolates included in RAPD survey of genetic diversity.

leaves of the susceptible cv. Earlypak 7. The separation of all

isolates tested into two phenetic groups, Gp1 and Gp2 (Figs
78), and the clustering of the three stem canker isolates in
Gp2 suggested that the ability to produce toxins may be
associated only with Gp2 isolates. To test this hypothesis,
seven additional isolates which produced host-specific toxins
on a variety of crop plants (Table 1) were obtained and
subjected to RAPD analysis with five primers that amplified
bands specific to Gp2 isolates. All seven isolates amplified the
majority of Gp2-specific bands (data not shown).
The evolutionary relationship between A. alternata pathotypes which produce host-specific toxins, and other Alternaria
isolates is still uncertain. Phylogenetic analysis of rDNA RFLPs
and sequence data of spacer regions of ribosomal DNA
support the inclusion of these pathotypes within A. alternata
(Kusaba & Tsuge, 1994, 1995). All isolates produced a large
number of secondary compounds, however, many of which
have not been characterized (Andersen & Thrane, 1996).
HPLC profiles of culture media from a collection of 36 isolates
from Danish malting barley indicate that there was significant
variation in both the quantity and identity of compounds that

are produced by different isolates (Anderson & Thrane, 1996).

Some compounds, such as alternariol and alternariol monomethyl ether were diagnostic of all A. alternata isolates.
Several other unidentified compounds appeared to be the
most useful diagnostic characters for associating an isolate
with a specific cluster group. If further research on A. alternata
suggests that patterns of secondary metabolites are characteristic of phenetic or phylogenetic groups, then molecular
markers such as the Gp2-specific bands could be used for rapid
characterization of field isolates.
A C K N O W L E D G E M E N TS
We thank D. Francis, E. Jones, P. Gepts, and D. Shaw for helpful comments on
the manuscript, and D. Gilchrist and B. Gabor for isolates. This work was
supported in part by Van Den Bergh Foods Company, California Tomato
Research Institute, the California League of Food Processors and the Ohio
Board of Regents.

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