New Zealand Journal of Agricultural Research

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Detection of Alternaria radicina and A. dauci from

imported carrot seed in New Zealand

J.J. Soteros

To cite this article: J.J. Soteros (1979) Detection of Alternaria radicina and A.�dauci from imported
carrot seed in New Zealand, New Zealand Journal of Agricultural Research, 22:1, 185-190, DOI:
10.1080/00288233.1979.10420859

To link to this article: https://doi.org/10.1080/00288233.1979.10420859

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 N.Z. Journal of Agricultural Research 22 (1979): 185-90

Detection of Alternaria radicina and

A. dauci from imported carrot seed
in New Zealand
J. J. SOTEROS,"

Plant Diseases Division, DSIR, P.B., Auckland, New Zealand

Seven laboratory methods and a glasshouse test used to determine the incidence
of two seed-borne pathogens, Alternaria radicina and A. dauci, are compared and
discussed. Incidence ranged from 0-26% and 0-1 %, respectively. The two most
efficient methods of detection were a heat treatment and the Copenhagen
germinator test. The heat treatment was the only method that detected internal
infections of the seed. Disease symptoms were expressed in the filter paper,
Copenhagen germinator, and pot tests. In glasshouse tests there was no correlation
between seed infection and seedling emergence. Hyphae of A. radicina were
carried in the inner pericarp layers of the seed and in the testa. It is suggested that
seed infection by A. radicina develops from infection of umbel parts through the
pedicel and ovary wall.

INTRODUCTION tamination and internal infections of the seed;

Black root rot of carrots incited by Alternaria (ii) determine the effects of seed-borne inoculum
radicina (Meier et al.) Neerg. and leaf blight of on germination and seedling emergence, and (iii)
carrots caused by A. dauci (Kuhn) Groves & determine the location of the pathogens in the
Skolko are both known to be seed-borne patho- seed.
gens of carrot (de Tempe 1962; Noble & Richard- METHODS
son 1968).
Seven imported carrot seed cultivars, purchased
In New Zealand little was known of these two locally in packets of 113 g, were examined. Four
pathogens until recently; Scott & Wenham (1973) hundred seeds per cultivar were used for each
found that, of a total of 86 imported carrot seed test in accordance with the International Seed
lines, 19 were infected with A. radicina and 7 Testing Association (1966). In all plate tests
carried A. dauci. (tests 1-6),20 seeds were placed equidistant from
Seed-borne inoculum is important in initiating each other on 9 ern petri dishes. The dishes were
disease in carrot crops, and A. radicina has been left at room temperature under a near ultra violet
reported to affect seed germination (Neergaard (NUV) tube and (4 + 40 W) white daylight
1945) and to cause considerable losses of seed- tubes in a 12 h light, 12 h dark period, and
lings by damping-off (Mouce & Bosher 1943). examined at 5, 7, and 10 days using a low power
A. dauci has also been known to cause consider- stereoscopic microscope. Each cultivar was
able reduction in germination (Hewett 1964). examined by all the following methods:
Efficient and reliable methods for detection of Laboratory tests
seed-borne pathogens are essential for the assess- 1. Seed washings
ment of control treatments in infected seed lines.
This involved three tests:
This investigation was undertaken to: (i) (a) Microscopic examination - seed was
compare different methods used to measure the shaken in 20 ml sterile water and the seed
incidence of these two seed-borne pathogens in washings examined microscopically for spores.
imported carrot seed, in order to determine (b) Potato dextrose plates - the washings
whether they distinguish between external con- were then centrifuged and each pellet was
" Present address: Plant Health Diagnostic Station, resuspended in 5 ml sterile water. Five 1 ml
Ministry of Agriculture and Fisheries, P.B.. Levin, aliquots were pipetted and streaked on 5
New Zealand potato dextrose agar (PDA) plates.
Received 13 April 1978: revision 13 July 1978
186 N.Z. JOURNAL OF AGRICULTURAL RESEARCH. VOL. 22, 1979

(c) Filter paper - the washed seed from each for 40 days and the number of plants which
cultivar was placed on sterile dampened filter emerged and showed disease symptoms were
papers in petri dishes. recorded.
2. Filter paper method
Untreated seed was placed on 2 sterile damp RESUI.TS
filter papers in a petri dish. In this work the organisms from 7 tests (Table
3. Agar method 1) are listed in order of their frequency of
Untreated seed was plated on 2% malt agar occurrence.
(MA). Laboratory test methods
4. Modified agar method In the preliminary tests using 7 cultivars, a low
Untreated seed was plated on MA containing incidence of A. radicina was found in 3 cultivars
benomyl at 10 ppm active ingredient. while A. dauci was found in only one cultivar. In
2 cultivars neither pathogen was detected.
5. Surface sterilisation method
Seed was treated with 1% NaGCl for 5 min, The results of the seven detection methods
then rinsed in 2 changes of sterile distilled used on the same day are given in Table 2.
water and allowed to dry before plating on 1. Seed washings
MA. (a) Microscopic examination - This method
Other surface sterilisation methods - Seed which would be expected to detect loose,
was treated with formalin or 0.1 % HgClz for sur~ace-borne spores and mycelium, was
5 min and different concentrations of ethyl tedious and time consuming. Spores and
alcohol (10-90%) for 1 min. After treatment mycelium were not observed by this method.
the seed was rinsed in 2 changes of sterile The results do not, however, exclude the pos-
distilled water and dried before plating on MA. sibility that pathogens are carried on the
surface of the seed.
6. Heat treatment method (b) Potato dextrose plates - No colonies of
Seed was placed in preheated petri dishes and A. radicina or A. dauci were obtained from
heated at 100°c for 1 h in a hot-air oven and streaking PDA plates with the seed washings.
allowed to cool at room temperautre before
plating on MA. (Modification of Malone's (c) Filter paper - Washed seed had a low
method 1962). incidence of both pathogens. The fungi were
seen as a dark mycelial growth and conidia
7. Copenhagen germinator on infected seedlings or ungerminated seed.
Untreated seed was placed on seed test filter
paper on glass strips over which perspex TABLE 1 - Micro-organisms isolated from untreated
funnels were placed. These were set up in a carrot seed
germinator and the glass lid was closed to
maintain a high relative humidity. The tem- Organisms Frequency of occurrence
perature was maintained at 30°c for 8 h under
Range
light (2 X 40 W daylight tubes) and 22°c (Numbers Mean (%)
for 16 h darkness. The test was inspected per test (For 7 tests)
after 7 days and ended at 14 days.
All the laboratory methods except the seed Alternaria alternata iS5-220 44.92
washing method (La) and the Copenhagen germ- Bacteria 68-150 40.00
inator test are plate tests, whereby untreated or Ulocladium consortiale 20- 76 32.79
treated seeds are placed on filter paper or an Epicoccum purpurascens 19- 72 29.79
agar medium for the detection of the pathogens. Cladosporium herbarum 23- 67 10.89
The Copenhagen germinator test is similar to the Yeasts 20- 46 10.12
filter paper method in which symptoms of infec- Rhizopus sp, 12- 28 7.71
tion are expressed. Fusarium spp, 7- 28 5.93
Mucor sp, 9- 27 4.29
Glasshouse tests Pleospora herbarum 8- 24 3.82
Four hundred untreated seeds per cultivar were Alternaria radicina 0- 26 3.71
sown in sterilised soil in 12 em diameter pots, Hormodendron sp. 0- 8 1.07
each pot containing 20 seeds. The pots were Phoma sp. 1- 7 0.85
placed in trays in the glasshouse under a day Phomopsis sp. 0- 5 0.66
temperature of 20-30°c and a night temperature Alternaria dauci 0- 1 0.04
of 12·-18°c. Seedlings were kept in the glasshouse
 SOTEROS: DETECTION OF Alternaria 187

TABLE 2 - Incidence (% infected seed) of A. radicina and A. dauci from carrot seed cultivars using
different methods of detection

ASC ABH EUS

Sample no. ADF Homes Egmont AGB Spring
Cultivar Dividend Improved Gold Topweight Market

Country of origin Australia Europe

A.r. A.d. Ax. A.d. A.r. A.d. A.r. A.d. A.r. A.d,

la.Microscopic examination 0 d 0 d 0 d 0 d 0 d 0 d
b.PDA plates 0 d 0 d 0 d 0 d 0 d 0 d
c.Filter paper, washed seed 0.8 d 7.2 c 8.5 c 3.2 d 1.0 d 0.5 d
2. Filter paper, untreated seed 0.2 d 6.0 c 7.5 c 2.0 d 0.2 d 0.2 d
3. Agar 1.5 d 12.2 b 13.7 b 3.0 d 1.0 d 1.0 d
4. Modified agar 1.5 d 14.5 b 17.2 b 3.5 d 1.0 d 1.0 d
5. Surface sterilisationt 0 d 3.5 cd 5.8 cd 1.5 d 0 d 0 d
6. Heat treatment 2.0 d 19.5 a 22.0 a 3.5 d 1.0 d 1.0 d
7. Copenhagen gerrninator 2.2 d 24.0 a 25.5 a 4.5 d I.2d 1.0 d

Figures flanked by a letter in common do not differ significantly (P < 0.01). Comparison is by columns
A.r. = A. radicina
Axl. c= A. dauci
t Treated with 1% NaOCI for 5 min.

2. Filter paper method Effect of surface sterilisation on spores of

A similar infection level to method lc was
found with untreated seed. Sporulation was
sparse when plates were kept in continuous
darkness, but under NUV and daylight conidia
of both pathogens were abundant on infected
seed and infected seedlings.
3. Agar method
This method indicated a high incidence of
A. radicina but did not distinguish between
external or internal infections of the seed. nor
did it provide an accurate assessment of the
incidence of the pathogens since saprophytic
fungi can suppress and mask internally borne
pathogens.
4 Modified agar methods
Higher numbers for the pathogens were
obtained with this method than the previous
methods. It allowed the expression of the
pathogens without affecting them; at the same
time many of the saprophytic fungi were
eliminated except those that tolerate benomyl.
5. Surface sterilisation methods
Tests using different surface sterilants or dis-
infectants affected the incidence of the
pathogens. All these methods gave lower
incidences of A. radicina and A. dauci than
untreated seed.
A. radicina and A. dauci - To determine the
effects of NaGCI on the pathogens, spores were
exposed to treatment of 0.1-1 % NaGCI for 5,
to, 15, and 20 min. Three hundred spores
were assessed for germination for each treat-
ment. Concentrations of 0.1-0.4% NaGCI
did not affect the viability of the spores signi-
ficantly compared with the untreated controls.
The viability of the spores was affected by
concentrations of NaGCI above 0.4% and no
spores remained viable after treatments of
0.5% or at higher concentrations of NaGCI for
15 min. Only 13% of the spores remained
viable after treatment of 1 % NaGCl for 5 min
and 6% after 10 min. Spores were not viable
after longer treatment periods.
6, Heat treatment method
This method revealed a higher incidence than
the other agar plate methods. This method
can be used to detect low levels of seed
infection. Although the treatment kills the
seed it eliminates the difficulty of examining
germinated seeds in petri dishes.
Effect of heat treatment on spores 0/
A. radicina and A. dauci - To determine the
effects of heat treatment on A. radicina and
A dauci, spores of both fungi were exposed
to dry air heat at temperatures of 10-100° c
188 N.2. JOURNAL OF AGRICULTURAL RESEARCH, VOL. 22, 1979

for 1 h. Viability of the spores was not affected Comparison of laboratory detection methods
after heating at lO-60 e for 1 h compared
0
The 4 methods revealing the highest incidence
with the untreated controls. Few spores of A. radicina and A. dauci were further tested
remained viable after treatments at 90°c and to determine their reliability. Differences in the
no spores remained viable after IOOoe for 1 h. incidence of the pathogens were approximately
Effect of surface sterilisation and heat treat- the same when the tests were repeated. There
ment on the microjlora of carrot seed - The were significant differences between the methods
effect of surface sterilisation (l % NaGCI for (Table 2). The Copenhagen germinator test and
5 min) and heat treatment (lOOoe for 1 h) on the heat treatment method were confirmed to be
the major microflora of 4 of the carrot samples, the most efficient and reliable methods in detect-
ADF, ASC, ABH, and AGB, are shown in ing the pathogens from carrot seed, and in all
Table 3. Surface sterilisation did not eliminate instances the former test gave a higher incidence
surface-borne contaminants and had an adverse of the pathogens.
effect on A. radicina. The heat treatment Tests carried out over 1 year to determine the
always reduced the saprophytic contaminants longevity of the pathogens with age of the seed
and did not appear to affect A. radicina. showed that A. radicina was not affected after
7. Copenhagen germinator 1 year from time of harvest (samples ADF, ASC,
This method was the most efficient and detec- ABH, and AGB) and A. dauci after 3 years from
ted the highest incidence of seed infection. time of harvest (samples EUS). The incidence of
The germ ina tor provides an environment for disease in the seed became higher with increasing
disease development which can be readily age of the seed (Table 4). This can be explained
observed from symptoms on the seedlings. if competitive saprophytic micro-organisms on the
i.e., infection of the hypocotyl, radicle, and seed do not remain viable as long as the patho-
cotyledons. and from sporulation on infected gens. Six-year-old carrot seed was tested and
seed and diseased seedlings. The test provides found to have viable A. radicina infection.
a reliable method for detecting pathogens and Glasshouse tests
detects high levels of infection. Seedling emergence was relatively unaffected
even in cultivars which showed a fairly high
TABLE 3 - Effect of surface sterilisation and heat incidence of seed infection with A. radicina.
treatment on the microflora of carrot seed It appears that soil conditions were not conducive
for the initiation of seedling infections. Glass-
Incidence - (')0 house tests and the filter paper method and
infected seed) Copenhagen germinator test (Table 5) showed
Fungi Surface
sterilisation Heat that there was no correlation between seed
(1 % treatment infection and seedling blight or between seed
NaGCl for (lOO°c for infection and seedling emergence.
Untreated 5 min) for 1 h)
Nature of seed transmission
Alternaria a!ternata 38.7 19.1 3.2 Sections of infected seed were made with a
Ulocladium freeze microtome, then cleared and stained with
consortiale 23.5 13.2 1.2 lactophenol cotton blue. Hyphae were prevalent
Epicoccum in the pedicel of the seed, iR, the endocarp (inner)
purpurascens 16.5 9.9 2.9 layers of the pericarp, and were occasionally
Cladosporium found in the testa, but were not found in the
herbarum 10.2 7.5 1.5
Alternaria radicina 7.6 2.7 12.2
endosperm or embryo. Sections of the infected
Pleosoora herbarum 6.8 4.2 1.9
seed were plated on PDA and the fungus was
A. radicina.

TABLE 4 - Longevity of A. radicina in carrot seed

Incidence (%) by direct plating

Months
Sample no. 4 7 10 12

ASC 8.2 10.2 11.7 14.5

AGB 10.5 12.7 15.2 17.8
 SOTEROS: DETECTION OF Alternaria 189

TABLE 5 - Effect of A. radicina and A. dauci on seed germination and seedling emergence

Sample no. Filter paper method Copenhagen germination Glasshouse testst

Seed Seed Seedling

infection Germination infection Germination blight Emergence
(%) (%) (%) (%) (%) (%)

ADF 0.5 89.7 2.5 83.3 0.2 84.5

ASC 8.0 73.2 23.5 85.0 1.5 84.5
ABH 6.5 79.2 22.2 84.5 3.0 85.5
AGB 3.0 87.7 5.2 72.5 0.5 86.5
EUS 0.7 81.5 3.0 79.2 0 84.0

No significant correlation (1' = 0.32) between seed infection and germination or between seed infection
and emergence
t Percentage means of 4 replications

TABLE 6- Isolation of A. radicina from infected the present study). In the present study spores of
carrot umbels A. radicina and A. dauci were also found to be
affected by surface sterilising. Since the modified
Infected with agar and heat treatment methods detected a high
Number of A. radicina
Infected parts platings (%) level of seed infection, it appears these methods
have a minimal effect on the pathogens and at
Bract leaves 66 100 the same time they reduce the masking effects of
Bractlets 52 48 the seed microflora. Differences in the incidence
Pedicels 120 80 of A. radicina, as detected by the filter paper
method and the Copenhagen germinator, cannot
Floral segments and
development ovules 240 69 be explained easily. The principal differences
Endosperm 80 0 were temperature and light which may have had
an effect on the detection of the pathogen.
Embryos 80 0
It is desirable that methods used for the
detection of pathogens detect both external con-
Infected carrot plants were left to go to seed taminants and internal infections of the seed.
and developing umbels (inflorescences) were Although the seed washings test did not show
examined. Infected parts of umbels were surface surface-borne spores of the pathogens, both
sterilised and plated on PDA. A. radicina was A. radicina (de Tempe 1962) and A. dauci
isolated from umbel parts and from developing (Hewett 1964) have been found to be carried on
seed (Table 6). No deep seated infections in the seed in heavily contaminated seed lines. Any
endospermic and embryonic tissues were found. real, although not statistical, differences between
the Copenhagen germinator and the heat treat-
DISCUSSION ment method would explain the higher incidence
An efficient method of detection is important if of A. radicina from external-borne spores. The
the levels of seed infection are to be determined. heat treatment was found to affect externally-
The efficiency of methods for the detection of borne saprophytic fungi (Table 3) as well as
A. radicina and A. dauci from carrot seed is pre- spores of both A. dauci and A. radicina. Spores
sumably influenced by the position of the patho- of A. radicina were not detected from seed wash-
gens in the seed, and by the presence of other ings and internal infections of the seed were
micro-organisms. The latter appear to be found to be from hyphae. As external contamina-
antagonistic to A. radicina, inhibiting the growth tion of the seed came from surface-borne spores,
of the pathogen. Suppression or elimination of cells, or hyphae and most of the micro-organisms
of these micro-organisms should provide a more were unable to survive high temperatures, the
accurate assessment of the incidence of the heat treatment enabled internal hyphal infections
pathogens. Unfortunately surface sterilisation, of the seed by A. radicina to be detected. Internal
used to suppress the other micro-organisms, also seed infections appeared to be less susceptible to
greatly reduces the incidence of the pathogens heat treatment so that this method presumably
(Malone 1962; Hewett 1964; also confirmed in detects internal infections of the seed.
190 NZ. JOURNAL OF AGRICUI.TURAL RESEARCH,
Significant differences were found between the
detection methods used, the four best methods
being in order of increasing efficiency, the agar,
modified agar, heat treatment, and the Copen-
hagen germinator methods. . The ~openhagen
germinator test detected the highest incidence of
A. radicina and was also efficient in revealing
symptoms of infection which can be readily
asessed. The test was confirmed to be an efficient
and reliable method (de Tempe 1962).
In glasshouse tests the level of seed infection
was not correlated with seedling blight. Al-
though the primary source of infection comes
from seed-borne inoculum, the present study
showed no serious losses of carrot seedlings. This
is in agreement with Maude (1966) who showed
that seed quality and germination were not greatly
affected, and that pre- and post-emergence
seedling damping-off were not significant.
From the location of hyphae in the seed it is
suggested that seed infection of A. radicina pro-
gresses from. infections of the pedicel and ovary
wall.
Acknowledgments
Applied Mathematics Division, DSIR, for assis-
tance with statistical analysis; Miss Gillian Laundon
and Dr A. F. Rainbow, Plant Health and Diagnostic
Station, MAF, for helpful suggestions with the manu-
script.
VOL. 22, 1979
REFERENCES
de Tempe, J. 1962: Comparison of methods for seed
health testing. Proceedings of the International
Seed Testing Association 27: 819-28.
Hewett, P. D. 1964: Testing carrot seed infection
with Alternaria porri f. sp. dauci. Proceeding,
of the International Seed Testing Association
29: 463-71.
International Seed Testing Association 1966: Inter-
national Rules for Seed Testing 1966: Ibid.
31 (1): 1-152.
Malone, J. P. 1962: IV. The application of heat to
seed oats as an aid in the detection of Pyrena-
phora avenae by the Ulster method. Ibid. 27:
856-61.
Maude, R. B. 1966: Studies on the etiology of black
rot, Stemphvlium radicinum (Meier, Dreschler
and Eddy) Neerg., and leaf blight, Alternaria
dauci (Kuhn) Groves and Skolko, on carrot
crops; and on fungicide control of their seed-
borne phases. Annals of Applied Biology 57:
83-93.
Mounce, 1.; Bosher, J. E. 1943: Seedling blight of
carrots caused by Alternaria radicina. Scientific
Agriculture 23: 421-3.
Neergaard, P. 1954: "Danish Species of Alternaria
and Stemphylium" (Translated by H. Ander-
son). Oxford University Press, London 560 p.
Noble, M.; Richardson, M. r. 1968: An annotated
list of seed-borne diseases. Proceedings of the
International Seed Testing Association 33: 1-191.
Scott, D. T.; Wenham, H. T. 1973: Occurrence of
two seed-borne pathogens, Alternaria radicina
and Alternaria dauci, on imported carrot seed
in New Zealand. N.Z. Journal of Agricultural
Research 16: 247-50.