Eur J Plant Pathol (2007) 117:293–305
DOI 10.1007/s10658-006-9098-0
FULL RESEARCH PAPER
Occurrence of Rhexocercosporidium carotae on cold
stored carrot roots in the Netherlands
Pieter Kastelein Æ Eveline S. C. Stilma Æ
Janneke Elderson Æ Jürgen Köhl
Received: 10 March 2006 / Accepted: 18 December 2006 / Published online: 24 January 2007
KNPV 2007
Abstract Winter carrot for the fresh market is
an important cash crop for many organic arable
farms in the Netherlands. In recent years carrot
roots from cold stores have been affected by
superficial dark brown to black spots. To gain
insight into the pathogens causing the blemish
and the effect of agronomic practices on their
occurrence, surveys were carried out among crops
harvested in 2001 and 2002. In addition carrots
harvested in 2003 were screened for root spotting
pathogens. Rhexocercosporidium carotae (syn.
Acrothecium carotae and Pseudocercosporidium
carotae) was the dominant pathogen in blackish
spots on carrots harvested in 2001. On carrots
harvested in 2002 and 2003 Alternaria radicina
was detected more frequently. Multiple regression analysis indicated that a higher occurrence of
the blemish may be linked with harvest conditions
and presence of umbelliferous plants. The effect
of the temperature on conidial germination,
mycelial growth and pathogenicity of R. carotae
was studied. The estimated optimum and maximum temperature for growth of R. carotae was 19
and 29C, respectively. Inoculation experiments
demonstrated that wounds are good invasion
P. Kastelein (&) E. S. C. Stilma J. Elderson
J. Köhl
Plant Research International BV, P.O. Box 16, 6700
AA Wageningen, The Netherlands
e-mail: pieter.kastelein@wur.nl
routes. Infection occurred at 3, 10 and 20C, but
not at 30C. Penetration into wounds was greatest
at 20C.
Keywords Acrothecium carotae Alternaria
radicina Organic farming Root spotting
Storage disease
Introduction
For many organic arable farms carrot for the
fresh market is an important cash crop. When
mature carrot roots are harvested before winter
and held in refrigerated storerooms for several
months, returns are more profitable. However, in
the last few years, superficial dark brown to black
spots have developed during storage of the roots
(Meier, 1998). When 5% or more of the roots are
affected, the whole consignment is rejected for
the fresh market. In organic farming application
of cultivation measures interfering with disease
cycles are needed to prevent the blemish. To
develop such preventive measures current knowledge of the pathogen(s) causing the spots and the
effect of agronomic practices on their occurrence
is required.
Several fungi may cause blackish lesions on
carrots during storage (Snowdon, 1991). Of these
Altenaria radicina, Chalaropsis thielavioides,
Mycocentrospora acerina, and Thielaviopsis basi-
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294
cola are considered of importance for carrot growing in the Netherlands. Infections by Rhexocercosporidium carotae are sporadically found (Meier,
1998). The last pathogen was first described by
Årsvoll (1965) as Acrothecium carotae. Subsequently, the fungus was successively classified in
the genera Pseudocercosporidium (De Hoog &
Van Oorschot, 1985) and Rhexocercosporidium
(Braun, 1994). The occurrence of R. carotae in rots
on stored carrots has also been reported in Norway
(Årsvoll, 1965), Denmark (Hobolth, 1983), Sweden
( Ewaldz, 1997; Jönsson, 2004; Pettersson, 1992)
and Canada (Shoemaker, Hambleton, Lacroix,
Tesolin, & Coulombe, 2002).
To gain an insight into the blemish on organically-grown carrots, blackish spots on cold-stored
roots were screened for fungi known to cause root
spotting, and information on cropping patterns,
crop husbandry and harvesting conditions was
analysed for relationships with disease occurrence.
This paper reports the widespread occurrence of
R. carotae on spotted carrots harvested in 2001 and
its subsequent diminution in the crops of the 2002
and 2003 growing seasons. For a better understanding of the pathogenicity of R. carotae, the
effect of temperature on conidial germination,
mycelial growth and invasiveness was studied. The
temperature range in which the fungus grew and
infected carrot roots is described.
Materials and methods
Carrot cultivation and sampling
During the 2001, 2002 and 2003 growing seasons,
a total of 60 crops of intermediate carrot varieties
from 26 different organic farms were sampled
(Table 1). Five farms participated during two
seasons, and five other farms all three seasons.
The farms were located on clay soils in the southwest, the centre and the north of the Netherlands.
All crops were grown to yield roots of 50–250 g
for long-term storage. The mean acreage of the
crops was 2 ha. The smallest crop covered .5 ha,
whereas the largest crop had an area of 6 ha. The
crops were sown in 5 cm strips on ridges between
the beginning of May and mid-June; 82% of the
crops were sown in May. The mean seed density
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Eur J Plant Pathol (2007) 117:293–305
Table 1 Number of carrot crops of the 2001, 2002 and
2003 growing seasons which were surveyed and numbers of
organic farms which participated in the study
Growing season
Total
2001 2002 2003
Number of carrot crops
19
Number of participating farms 15
23
14
18
12
60
26a
a
The total number of participating farms differs from the
result of the addition of the number of farms participating
in each of the three seasons, because several of these farms
participated during two or three seasons
was 2.0 million seeds ha–1. Minimum and maximum seed densities were 1.4 and 2.2 million seeds
ha–1, respectively. In all crops the distance
between ridges was 75 cm.
The weather during the 2001 and 2002 growing
seasons was warmer and wetter than normal
(Table 2). Of these two seasons 2001 was the
wettest. The 2003 growing season was also warm,
but relatively dry. By the end of the 2001 growing
season the foliage of 90% of the crops was
severely affected by leaf blight (Alternaria dauci).
In 2002, 39% of the crops were severely affected
by this disease, whereas in 2003 it caused hardly
any damage. Powdery mildew was the most
important foliar disease of the 2003 growing
season, with 24% of the crops severely affected.
The crops were harvested between the end of
September and mid-November; 84% of the crops
were harvested in October. The time between
sowing and harvest averaged 138 days; the shortest and longest growing times were 115 and
168 days, respectively. Harvesting was done with
‘top-lift’-type carrot harvesters equipped with
topping bars and an elevator system that enables
direct filling of storage boxes in the field. The
topped roots were transported to a cold-storage
building on the harvest day and stored unwashed.
On the harvest day of each crop the soil and the
carrot roots were sampled. After the harvester
had passed by, 250 ml topsoil of three arbitrarily
chosen carrot ridges was collected to determine
the water content of the soil. From at least ten
arbitrarily chosen storage boxes 20–30 roots were
collected until two 15 l crates were filled. Dependent on the size of the roots, 200–400 roots were
collected from each crop. A sub-sample of 100
roots was used to assess the pre-storage sanitary
Eur J Plant Pathol (2007) 117:293–305
295
Table 2 Weather conditions during the 2001, 2002 and 2003 growing seasons
Month
April
May
June
July
August
September
October
November
Season
Temperaturea
Global radiationb
Precipitationc
2001
2002
2003
Normald
2001
2002
2003
Normald
2001
2002
2003
Normald
8.0
13.7
14.9
18.5
18.7
13.6
14.4
7.5
13.7
9.3
13.2
16.5
17.4
18.8
15.1
10.0
8.0
13.5
9.9
13.2
17.8
18.8
19.3
13.9
7.5
8.0
13.6
8.3
12.7
15.2
17.4
17.2
14.2
10.3
6.2
12.7
37.7
64.0
59.6
58.9
49.7
27.0
19.7
8.7
325.4
42.4
51.5
56.4
52.3
44.6
34.0
18.5
8.9
308.5
47.9
54.2
63.4
60.0
43.7
38.0
22.0
9.2
338.4
40.3
54.7
54.2
54.7
47.8
30.8
18.7
8.7
309.9
75
34
50
71
108
177
55
90
660
52
41
67
88
112
39
82
87
568
46
85
40
57
22
53
74
49
426
44
57
71
70
62
75
78
82
539
a
Country means of the monthly and seasonal mean aerial temperatures in C. The country means are based on the
measurements of the five main weather stations of the Royal Netherlands Meteorological Institute
b
Country means of the monthly and seasonal sums of the global radiation in kJ cm–2
c
Country means of the monthly and seasonal sums of the precipitation in l m–2
d
Long-term mean over the period 1971–2000
To assess the sanitary condition of the samples, the
roots were sprayed clean with tap water and allowed
to dry. Then the roots were visually inspected for
pests and diseases, which were identified by the
appearance of lesions or damage. For each sample
the number of roots affected by the various blemishes was recorded to calculate incidences. Amounts
of root surface affected by the pests or diseases
(severities) were not assessed. The decimal notation
of the fraction of roots affected by blackish spots was
used for data analysis.
106 and 263 lesions were examined. For each
lesion size, shape and colour were recorded. Then
the lesion with surrounding tissue was excised
from the carrot root, placed on water-soaked
filter paper in a Petri dish and incubated for
2 weeks at 15C under NUV-illumination (Philips
‘TL’D 18W/08) with a daily photoperiod of 12 h.
Lesions were inspected with a stereomicroscope
for presence of conidia or chlamydospores of
A. dauci, A. radicina, C. thielavioides, M. acerina,
T. basicola and R. carotae, which are known to
cause blackish spots (Snowdon, 1991). Fungi were
identified by spore morphology (Årsvoll, 1965;
Ellis, 1971). The method used to screen for root
spotting pathogens does not allow for the
detection of species with mycelia sterilia (e.g.
Rhizoctonia solani).
For each sample the number of lesions infected
by the various pathogens was recorded to calculate incidences. The decimal notation of the
fraction of lesions infected by R. carotae was
used for data analysis.
Screening for root spotting pathogens
Data collection
Only carrot roots affected by superficial dark
brown to black spots were screened for presence
of root spotting pathogens. From each sample all
spots up to a maximum of 25 arbitrarily chosen
spots were processed. From the roots sampled in
2001, 2002 and 2003, respectively, a total of 224,
Data on the crops harvested in 2001 and 2002 on
farmland, cropping patterns, crop health, crop
husbandry and harvesting conditions were gathered by means of field observations, crop analysis
and questionnaires. Two weeks before the
planned harvest date the crops were surveyed
condition. The other roots were crated unwashed
and stored in a cold room under conditions similar
to those during commercial cold-storage. The
samples of the 2001 and 2002 crops were stored
for 4–5 months, whereas those of 2003 were stored
for 3 months. After storage the post-harvest
sanitary condition of the samples was assessed.
Assessment of the sanitary condition of root
samples
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296
for symptoms of foliar diseases. The adjoining
plots were examined for presence of umbelliferous crops and field margins were searched for
umbelliferous plants. The 100-root sub-samples
used for assessment of pre-storage sanitary conditions were also used for assessing dirt tare,
topping efficiency, harvest damage (abrasions and
other injuries) and plant analysis. The weight loss
after washing was used to assess dirt tare. To
assess topping efficiency the roots were grouped in
four classes (0 = roots without remainders of
petiole bases on the crown, 1 = length of the
remainders of the petiole bases 0–1 cm, 2 = length
of the remainders of the petiole bases 1–2 cm,
3 = remainders of petiole bases longer than 2 cm
or one or more dead leaves attached to the
crown). Then the roots in each class were counted
and an index for topping efficiency was calculated
using the formula:
Index ¼ 100 ð0 N0 þ :33 N1 þ :67 N2 þ N3 Þ=Ntotal
in which N0, N1, N2 and N3 is the number of
carrots grouped in the classes 0, 1, 2 and 3,
respectively and Ntotal is the total number of
carrots in the sample. An index for topping
efficiency of 0 indicates that the crowns of all
roots were without remainders of petiole bases or
leaves; 100 indicates that all roots had long
remainders of petiole bases or dead leaves
attached to the crown, i.e. removal of the foliage
was unsatisfactory. The same formula was also
used to calculate an index for mechanical damage
caused during harvest. For assessing crop damage,
the roots were grouped in the classes 0 (no visible
damage), 1 (1–10% of the periderm surface
showing abrasions and wounds), 2 (11–25% of
the periderm surface showing abrasions and
wounds) and 3 (>25% of the periderm surface
showing abrasions and incisions). An index for
mechanical damage of 0 indicates that none of the
roots showed visible damage; whereas an index of
100 indicates that all roots were severally damaged (class 3). From carrots without blemishes, a
random sample of 25 roots was sorted to determine dry matter content and contents of calcium
(Ca), magnesium (Mg), potassium (K) and sodium
(Na). The carrots were shredded and a fraction of
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Eur J Plant Pathol (2007) 117:293–305
the chips was weighed and dried at 105C to
determine the dry matter content (Houba, Van
der Lee, & Novozamsky, 1997). The bulk of chips
was dried at 70C and pulverized in a laboratory
mill (Peppink Engineering Works, Amsterdam,
The Netherlands) with a 1 mm mash sieve. The
resulting powder was digested with H2SO4–salicylic acid–H2O2 and selenium (Temminghoff,
Houba, Van Vark, & Gaikhorst, 2000). The
amounts of Ca, K and Na in the digest were
determined by flame atomic emission spectrometry, whereas the amount of Mg was determined by
flame atomic absorption spectrometry. To assess
soil wetness during harvest of the carrot crops the
water content of the soil samples, obtained from
carrot ridges on the day of harvest, was determined (Houba et al., 1997).
Questionnaires were used to obtain information on acreage, soil conditions (e.g. soil type, %
particles <16 lm, pH, % organic matter and
nutrient levels) and cropping history of the plot
(e.g. carrot-cropping frequency, crops and green
manure crops grown in the ten preceding seasons
and occurrence of blackish spots in previous
carrot crops), primary and secondary tillage
practices (e.g. management of crop residues, seed
bed preparation and building of ridges), carrot
variety, seed quality (e.g. seed size, hot water
treatment), sowing (e.g. sowing time, drill width,
seeding rate), time and uniformity of emergence,
crop husbandry (e.g. weed and pest management,
manuring and irrigation), harvest (e.g. harvesting
equipment, harvesting time, weather conditions,
wetness of the soil and the foliage) and the
bordering plots (crops grown during current and
preceding season, management of crop residues).
Isolation and cultivation of R. carotae
Isolations were made on Potato Carrot Agar (PCA;
20 g l–1 potatoes, 20 g l–1 carrots and 15 g l–1 agar
(Oxoid, nr. 3)) amended with 25 mg l–1 tetracycline. Peeled potatoes and scraped carrots were
shredded and stored in 20 g portions at –20C. To
prepare the medium the potato and carrot shreds
were soaked in water for 2 h, cooked for 5 min and
filtered through cheesecloth. The filtrate was
adjusted to 1 l and the agar was added. PCA was
autoclaved for 15 min at 120C.
Eur J Plant Pathol (2007) 117:293–305
Monoconidial isolate 840 of R. carotae was
obtained from a black lesion on cold stored carrots
of a 2001 crop grown in Flevoland (the Netherlands). The isolate was stored on PCA-slants at
3C. Mycelial inoculum was produced on Carrot
Juice Agar (CJA). CJA contained 200 ml l–1
Akwarius carrot juice (Natuproducts BV, Harderwijk, The Netherlands) and 20 g l–1 agar. CJA
was autoclaved the same way as PCA. Cultures
were incubated in the dark for 4 weeks at 18C.
Agar discs (5 mm diam) with mycelium from the
edge of the colonies was used as mycelial inoculum. Conidial inoculum was produced on carrot
leaf extract enriched CJA. Fresh carrot leaves
were crushed using a Pollähne leaf press (Meku,
Wennigsen, Germany); 80 g pressed leaves were
then mixed with 400 ml water in a blender (Waring, New Hartford, Conn., USA). The fibre was
separated from the leaf mousse by pressing the
fluid through a colander. The leaf extract was
stored in 100 ml portions at –20C. Enriched CJA
contained 200 ml l–1 Akwarius carrot juice,
400 ml l–1 leaf extract and 20 g l–1 agar. The
medium was autoclaved the same way as PCA.
Cultures were incubated at 18C, first for 3 weeks
in the dark and thereafter for 7–10 days under
NUV-illumination with a daily photoperiod of
12 h. Conidial suspensions were obtained by
flooding the cultures with sterile tap water, releasing the conidia from the mycelium by streaking the
cultures with a glass Drigalski spatula, and straining the fluid through sterile nylon gauze with a
mesh of 200 lm. Densities of conidial suspensions
were determined by means of a haemocytometer
and adjusted with sterile tap water. Conidial
suspensions were kept on ice and used within 4 h.
Effect of temperature on conidial germination
in vitro
Conidial germination over time at different temperatures was determined on Petri dishes (50 mm
diam) containing 6 ml Malt extract Agar (MA).
MA contained 1 g l–1 malt extract (Oxoid) and
15 g l–1 agar. The day before use, the required
Petri dishes with MA were placed in incubators at
0, 5, 10, 15, 20, 25 and 30C. The temperature of
0C was obtained by placing the Petri dishes in a
box surrounded on all sides with melting ice. On
297
the day of inoculation two conidial suspensions
were prepared at a 12 h interval. The suspensions
containing 5 · 105 conidia ml–1 were atomized
onto the medium to obtain approximately 75
conidia mm–2 on the agar surface. Immediately
after inoculation the Petri dishes were replaced in
the appropriate incubator. The total incubation
period of the conidia depended on the temperature. The incubation periods (0C: 72 h; 5C: 24 h;
10C: 20 h; 15C: 10 h; 20C: 10 h; 25C: 20 h;
30C: 16 h) were divided into 2 h (incubation for
10 h), 4 h (incubation for 16–24 h) or 24 h (incubation for 72 h) intervals and after each time
interval the fungal growth in two Petri dishes was
stopped by placing the plates over filter paper
saturated with household ammonia water (4.8%
NH4OH). The use of two conidial suspensions
made it possible to avoid late night sampling
during the course of the experiment.
Conidial germination was quantified by microscopic examination of 100 arbitrary, free-lying
conidia per plate. A conidium was assessed as
germinated whenever the length of the germ tube
was at least one third of the length of the
conidium. For each incubation period/incubation
temperature combination, the average was taken
of the % germinated conidia found in the
duplicate plates to describe conidial germination
over time. The experiment was carried out twice.
Effect of temperature on mycelial growth
in vitro
Mycelial growth rates of R. carotae were determined at 0, 5, 10, 15, 20, 25 and 30C. Mycelial
inoculum was placed in the centre of Petri dishes
(90 mm diam) containing 17 ml half strength
CJA. At each temperature level four plates were
incubated in the dark for 28 days. Radial growth
of colonies along two perpendicular lines was
recorded at 7-day intervals. The weekly growth
rates were measured and for each Petri dish the
mean growth rate was used for data analysis. The
experiment was carried out twice.
Inoculation of carrots
Unwashed mature carrot roots (cv. Nerac) were
obtained from an organic farm in Flevoland and
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298
stored at 3C for up to 6 months. Before use the
carrots were subsequently sprayed clean with tap
water, surface-sterilized for 2 min in .5% sodium
hypochlorite, rinsed three times in water and
dried for 1 h. Shortly before inoculation with
R. carotae, carrots were wounded as described
below at four spots at one side of the root. The
wounded areas were 1–2 cm long and about 1 cm
wide. After inoculation of the fresh wounds with
20 ll suspension containing 1 · 105 conidia ml–1,
the carrots were incubated in moist chambers as
specified below for 3 weeks. Wounds treated with
sterile tap water were used as the non-inoculated
control. At the end of the incubation period the
inoculated patches of skin were examined for
colonization and development of blackish spots.
A crosscut was made through the lesions to
measure the penetration depth.
In one experiment three types of wounding
were applied. Wounding type 1 (no wounds), type
2 (abrasions) was wounding by a graze with
medium-coarse sandpaper and type 3 (open
wounds) was by superficially removing a piece
of the periderm with a parer. The experiment was
set up in a randomized complete block design.
Each of the ten repetitions contained two inoculated carrots and one non-inoculated control
carrot per wounding type. The carrots were
incubated at 18C. For each repetition the decimal notation of the fraction of blackish spots and
the mean of the depths of the eight lesions per
wounding type was used for statistical analysis.
The experiment was carried out once.
Another experiment assessed the effect of the
temperature on the ability of R. carotae to infect
open wounds. The temperatures were 0, 3, 10, 20,
or 30C. At each temperature 20 inoculated
carrots and ten non-inoculated control carrots
were incubated. The decimal notation of the
fraction of colonized patches and the mean of the
depths of the four lesions per carrot was used for
statistical analysis. The experiment was carried
out once.
Statistical analysis
To interrelate disease occurrence with the information collected on the crops grown in 2001 and
in 2002 a method was used similar to that
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Eur J Plant Pathol (2007) 117:293–305
described by Den Belder, Elderson, Van den
Brink, and Schelling (2002). For each season a
separate multiple linear regression analysis was
performed with the RSEARCH procedure in the
Genstat 6.1 programme (Payne et al., 2002). The
information analysed included in total 14 categorical variables and 21 quantitative variables,
which were grouped into six categories (Table 3).
Using arcsin-transformed values of the dependent
variables ‘fraction of roots affected by blackish
spots’ or ‘fraction of spots with R. carotae’ the
analysis was performed in two steps. In step 1 the
independent terms were presented in four groups:
(1) the quantitative variables of the categories
‘soil conditions’, ‘crop rotation’, ‘surrounding
area’ and ‘crop husbandry’; (2) the quantitative
variables of the categories ‘harvest conditions’,
and ‘ composition of the carrots’; (3) categorical
variables of the categories ‘soil conditions’ and
‘crop husbandry’; (4) categorical variables of the
categories ‘crop rotation’ and ‘surrounding area’.
For each group the terms best explaining the
dependent variables were selected based on
Mallow’s Cp-values close to the number of independent terms (p) and the largest adjusted R2. In
step 2 of the analysis the terms selected in step 1
were combined and presented for a second round
of selection and elimination of independent terms
to provide models with numbers of p as small as
possible.
To analyse the effect of the temperature on
mycelial growth of R. carotae a Logan curve
(Logan, Wollkind, Hoyt, & Tanigoshi, 1976) was
fitted through the mycelial growth rates at the
different temperatures. Logan curves are described
by:
8
>
< YðTÞ ¼ w½expðqðT TbÞÞexpðqðTM
TbÞÞðTMTÞ=DT for T TM
>
:
YðTÞ ¼ 0 for T TM
in which Y is mycelial growth rate, T is temperature, Tb is the lower threshold temperature and
TM is the upper lethal temperature. Values of w,
q, Tb, TM and DT were estimated by non-linear
regression analysis. The equation of the Logan
curve and the estimates of the parameters were
used to calculate the optimum temperature (the
Eur J Plant Pathol (2007) 117:293–305
299
Table 3 Numbers of independent variables per category
of information on the carrot crops harvested in 2001 and
2002
Category
Quantitative
variables
Categorical
variables
Soil conditionsa
Crop rotationb
Surrounding areac
Crop husbandryd
Harvest conditionse
Chemical composition
of carrot rootsf
2
5
0
3
6
5
1
4
4
5
0
0
Table 4 Occurrence of blackish spots on cold stored carrot roots collected in 2001, 2002 and 2003
Growing season
2001
2002
2003
a
Prevalencea
100
83
100
Incidenceb
Mean
Range
9
2
31
2–21
0–10
7–66
Percentage samples in which blackish spots were found
b
Mean and range of the percentages of affected roots in
the 19, 23 and 18 samples collected in 2001, 2002 and 2003,
respectively
a
Quantitative variables: % particles <16 lm, % organic
matter; Categorical variable: soil type
b
Quantitative variables: No. of gramineous crops in the
three and five preceding seasons, No. of leguminous crops
in the three and five preceding seasons, carrot-cropping
frequency; Categorical variables: preceding crop, second
last crop, green manure crop preceding carrot, green
manure crop after second last crop
c
Categorical variables: presence of umbelliferous plants
in the field margins, presence of a carrot crop on a
bordering plot, cultivation of carrots on a bordering plot
during the preceding season, presence of trees or shrubs in
the near environment
d
Quantitative variables: crop length, cultivated area,
seeding rate; Categorical variables: variety, optimal
seedling emergence (yes or no); irrigation during
emergence (yes or no), application of N and K during
growing season (yes or no)
e
Quantitative variables: aerial temperature, soil
temperature, soil moisture, tare weight, index for topping
efficiency, index for mechanical damages
f
Quantitative variables: dry matter content, Ca-, K-, Mgand Na-content
temperature with the highest Y(T) value) for
growth.
Analysis of variance (ANOVA) was used to
analyse the data of the inoculation experiments.
Least significant difference (LSD) tests (a = .05)
were used for evaluating the significance of
differences between pairs of treatment means.
Results
Occurrence of blackish spots
At harvest time of 2001 no blackish spots were
found on carrot roots. However, in 2002 and 2003
the blemish was already found at harvest in 26
and 11% of the samples. In these samples .4–7%
of the roots showed blackish spots. At the end of
storage all samples collected in 2001 and 2003
contained carrots showing blackish spots
(Table 4). The blemish affected 2–21% and
7–66% of the roots in the samples of 2001 and
2003, respectively. In 83% of the samples of the
2002 growing season only 1–10% of the roots
showed blackish spots, whereas the other samples
did not contain spotted roots. The mean incidence
of the blemish was 9, 2 and 31% for the samples
of 2001, 2002 and 2003, respectively. The diversity
in appearance of the lesions, even within samples,
was considerable. Some spots were dark brown or
black in colour, others had a brown centre with a
black edge. In some cases the centre of the spot
was sunken. Most lesions were irregularly shaped,
although also round, oval and bar like spots
occurred. Lesions were superficial, up to 2 mm
deep. In 2001 and 2002 the majority of the lesions
were black coloured, whereas in 2003 dark brown,
slightly sunken lesions with a black edge were
predominant.
Occurrence of root spotting pathogens
In the scarce blackish spots that were already
present at harvest, either A. dauci, A. radicina, or
C. thielavioides was detected. From blackish spots
which had developed during cold storage of the
carrots harvested in 2001, 2002 and 2003, respectively, 66, 53 and 32% revealed spores of fungi
known to cause root spotting. In 50% of the
lesions on roots harvested in 2001 R. carotae was
detected (Table 5). This pathogen was found to
occur in samples from all three regions surveyed.
The prevalence of R. carotae for the 2001 growing
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Table 5 Occurrence of pathogens in blackish spots on
cold stored carrot roots and their distribution among the
samples collected in 2001, 2002 and 2003
Root spotting
pathogena
% lesions with
pathogen
% samples with
pathogenb
2001 2002 2003 2001 2002 2003
Rhexocercosporidium
carotae
Alternaria radicina
Alternaria dauci
Chalaropsis
thielavioides
Mycocentrospora
acerina
No sporulation of
root spotting
pathogens
50
11
1
74
22
6
8
6
1
23
14
5
25
2
0
26
47
5
35
13
4
61
17
0
1
0
4
11
0
22
34
47
68
16
39
28
Number of lesions or 224
samples examined
106
263
19
23
18
a
The identity of the pathogens was established by spore
morphology
b
For each year the sum of the percentages of samples
with the various root spotting pathogens differs from 100
because in most samples more than one pathogen was
detected
season was 74%. On carrots harvested in the
following seasons R. carotae was, with a prevalence of 22 and 6% for 2002 and 2003, respectively, of lesser importance. In the samples of
these seasons A. radicina was detected most
frequently. Furthermore, A. dauci, C. thielavioides and M. acerina were detected in blackish
spots. In most samples more than one root
spotting pathogen occurred. Mostly one pathogen
was detected in the lesions, however, in ten out of
the 593 lesions screened R. carotae and another
root spotting pathogen (A. dauci: 5·; A. radicina:
3·; C. thielavioides: 2·) was present. Other
pathogen combinations were not found. No relationships were found between lesion type (size,
shape or colour) and root spotting pathogen
detected in the lesions.
Factors affecting the occurrence of blackish
spots and R. carotae
A high index for mechanical damage (Table 6,
crops harvested in 2001, P = .006) or a high
temperature (Table 6, crops harvested in 2002,
P = .030) during harvest was associated with a
high incidence of blackish spots. In the carrot
samples of 2001 from plots where umbelliferous
plants were present in the field margin, the
incidences of the blemish were higher (Table 6,
P = .014) than in the samples from plots with
margins in which umbelliferous plants were not
found. The occurrence of R. carotae in blackish
spots was linked with the presence of umbelliferous plants in the field margin and the calcium
content of roots (Table 7, crops harvested in 2001,
P = .004 and P = .005, respectively) or the cultivation of carrots on one of the bordering plots
during the 2001 growing season and topping
efficiency during harvest (Table 7, crops harvested in 2002, P = .006 and P = .023, respectively). A high calcium content of roots (2001) or
a high index for topping efficiency, i.e. more roots
Table 6 Accumulated analysis of variance, the variable being the incidence of black spotted carrots after cold storage of
root samples from 19 and 23 carrot crops harvested in 2001 and 2002, respectively
Source of variation
Carrot crops harvested in 2001, adjusted R2 = 40.1%
Index for mechanical damages caused during harvesta
Presence of umbelliferous plants in the field marginsb
Residual
Total
Carrot crops harvested in 2002, adjusted R2 = 16.8%
Aerial temperature during harvestc
Residual
Total
a
d.f.
SS
F-value
P>F
Coefficient
1
1
16
18
.075
.057
.119
.224
9.99
7.62
.006
.014
.006
.155
1
21
22
.033
.127
.160
5.46
.030
.013
The index for mechanical damages caused during the harvest in 2001 ranged from 8.7 (few damaged roots) to 46.7
b
Umbelliferous plants were found in the field margins of 16% of the crops grown in 2001
c
For the crops harvested in 2002 the mean aerial temperature on the harvest day ranged from 6.1 to 15.6C
123
Eur J Plant Pathol (2007) 117:293–305
301
Table 7 Accumulated analysis of variance, the variable being the incidence of R. carotae infected blackish spots on cold
stored roots from 19 and 23 carrot crops harvested in 2001 and 2002, respectively
Source of variation
Carrot crops harvested in 2001, adjusted R2 = 46.9%
Presence of umbelliferous plants in the field marginsa
Calcium content of roots at harvest timeb
Residual
Total
Carrot crops harvested in 2002, adjusted R2 = 28.6%
Cultivation of carrots on a bordering plot during the preceding seasonc
Index for topping efficiency during harvestd
Residual
Total
d.f.
SS
F-value
P>F
Coefficient
1
1
16
18
.999
.931
.119
.224
11.18
10.42
.004
.005
.643
–1.239
1
1
20
22
.013
.008
.028
.043
9.32
6.11
.006
.023
.071
–.003
a
Umbelliferous plants were found in the field margins of 16% of the crops grown in 2001
b
The calcium content of the crops grown in 2001 ranged from 3.9 to 4.6 g kg–1 fresh carrots
c
In 2002, 4% of the crops bordered on a plot on which carrots were grown in 2001
d
For the crops harvested in 2002 the index for topping efficiency ranged from 7.0 (few roots with remainders of petiole
bases attached to the crown) to 42.8
with long remainders of petiole bases (2002), was
associated with a low incidence of R. carotae in
the blackish lesions.
Effect of temperature on germination
of R. carotae conidia
Germination of conidia was slowest at 0C
(Table 8). At this temperature the first germinated conidia were found 48 h after inoculation,
whereas at higher incubation temperatures the
first germ tubes appeared within 4 h. After 72 h
Table 8 The effect of temperature on the germination of
R. carotae conidia
Temperature (C)
0
5
10 15
20
25 30
Total incubation period (h) 72 24 20 10 10 20 16
48 4 4
2
4 4 4
Time (h) of initial conidial
germinationa
Maximum percentage of
60 94 99 100 100 63 35
germinated conidia at the
end of the total incubation
period
– 20 16 10 10 16 8
Time (h) of maximum
conidial germinationb
a
Time of initial conidial germination is the time of the
first sampling after inoculation in which germinated
conidia occurred
b
Time of maximum conidial germination is the time of
the first sampling in which the maximum percentage of
germinated conidia was reached
incubation at 0C an average of 60% conidia had
germinated. At 5–20C more than 90% conidia
had germinated within 24 h. At 25 and 30C the
increase in % germinated conidia had stopped
before the end of the incubation period at
average levels of 63 and 35%, respectively.
Effect of temperature on mycelial growth
of R. carotae
At temperatures between 0 and 20C the mycelial
growth rates ranged from .6 ± .6 mm per week at
0C to 15.8 ± .5 mm per week at 20C (Fig. 1). At
25C the growth rate was 5.1 ± .4 mm per week
and at 30C the fungus did not grow at all. The
Logan curve fitted through the observed growth
rates, matched the data of temperatures above
5C more accurately than those of 0C. At 0C
the actual growth rate was much lower than the
estimated rate. On the basis of the Logan equation the optimum and maximum temperatures for
growth of R. carotae were 19 and 29C, respectively.
Pathogenicity of R. carotae
In the inoculation experiment at 18C with three
types of wounding (no wounds, abrasions and
open wounds) R. carotae developed dark brown
lesions in all types of wounds. This discolouration
did not develop in water controls. Periderm
123
302
Eur J Plant Pathol (2007) 117:293–305
Radial growth rate
(mm week-1)
20
Table 10 Effect of temperature on the ability of R. carotae to infect patches of periderm wounded with a parer
(open wounds)
15
10
5
0
0
5
10
15
20
25
30
Temperature (°C)
Fig. 1 Mycelial growth rates of R. carotae at different
temperatures. The line represents the fitted Logan curve
Y(T) = 107 · [exp(.096 · (T – 5)) – exp(.096 · (29 – 5) –
(29 – T)/10)]; R2 = 90.6%. Each point and error bar
represents the mean and corresponding standard deviation
of the eight growth rates measured at that temperature
without mechanical wounding was most resistant
to the pathogen: only an average of 36% of the
inoculated patches showed small lesions
(Table 9). All open wounds and an average of
96% of the abrasions were penetrated by the
fungus. In all cases lesion depths did not exceed
2 mm. Depths of lesions in patches with abrasions
did not differ from those in patches with open
wounds.
The temperature had a marked effect on the
ability of R. carotae to penetrate open wounds. At
0C an average of 51% of the wounds were
colonized by the pathogen. However, at 0C the
colonized tissues did not develop into brown
Table 9 Ability of R. carotae to infect different types of
wounds. Percentage patches infected by the pathogen and
lesion depths after incubation of inoculated patches of
periderm during 3 weeks at 18C
Wounding
typeA
Infected patches
(%)B
No wounds
36 ± 28 a
Abrasions
96 ± 12 b
Open wounds 100 ± 0 b
Penetration depth
(mm)B
.05 ± .06 a
.81 ± .35 b
.92 ± .26 b
A
No wounds: periderm without mechanical wounding;
Abrasions: periderm wounded by a graze with sandpaper;
Open wounds: periderm removed with a parer
B
Values presented are the means and standard deviations
for the percentage of infected patches and the mean lesion
depth of eight patches on carrot roots with ten replicates.
Values followed by different letters are significantly
different (LSD-test; a = .05)
123
Temperature
(C)
Colonized patches
(%)A
Penetration depth
(mm)A
0
3
10
20
30
51
90
98
85
0
.05
.17
.62
.80
.00
±
±
±
±
±
5b
17 c
8c
29 c
0a
±
±
±
±
±
.03
.10
.19
.38
.00
a
b
c
d
a
Percentage patches colonized by the pathogen and lesion
depths after incubation of inoculated wounds during
3 weeks at 0, 3, 10, 20 and 30C
A
Values presented are the means and standard deviations
for the % of colonized patches and the mean lesion depth
of four patches per carrot root with 20 replicates. Values
followed by different letters are significantly different
(LSD-test; a = .05)
lesions (Table 10). After incubation at 3, 10 and
20C brown lesions appeared in an average of 90,
98 and 85% of the wounds, respectively. The
colour of the lesions ranged from pale brown at
3C to dark brown at 20C. At 30C no signs of
infection were found. The lesion depth was
greatest at 20C.
Discussion
In the Netherlands A. radicina is considered to be
the main cause of blackish spots in stored carrots.
In addition C. thielavioides, T. basicola and
M. acerina are increasingly detected (Meier,
1998). Except for T. basicola, these root spotting
pathogens were found to occur in lesions on
carrots collected during present surveys. Furthermore A. dauci and R. carotae were detected in
lesions. So far, R. carotae was occasionally
isolated by the Dutch Plant Protection Service
in the years 1986–1988. Extent of disease occurrence and geographical distribution were poorly
documented. Therefore the frequent occurrence
of R. carotae in organic carrot roots grown in 2001
was unexpected.
Except for the reports of Årsvoll (1965) and
Ewaldz (1997), most reports (Hobolth, 1983;
Jönsson, 2004; Pettersson, 1992; Shoemaker et al.,
2002) on loss of stored carrots due to R. carotae do
not elaborate on the extent of disease occurrence.
Eur J Plant Pathol (2007) 117:293–305
In surveys of stored carrots made during 1962–
1965 in Norway the pathogen was found to be
common in the county Rogaland but occurred
sporadically in other parts of the country. During
winter of 1963/1964 R. carotae rot affected about
20% of the storage stock of carrots grown in
Rogaland (Årsvoll, 1965). In field experiments
conducted in 1991–1992 in Sweden R. carotae
affected on average 77.5% of the stored carrots
from the two experiments of 1991, but was not
found in 1992 (Ewaldz, 1997). Also during the
present study incidences of R. carotae in stored
carrots were substantial in one season and minor
in the two subsequent seasons.
The fungus R. carotae has been described by
Årsvoll (1965) and Shoemaker et al. (2002). By
laboratory experiments Årsvoll (1965, 1971)
obtained information on the ecophysiology and
pathogenicity of the fungus. However, knowledge
on the effect of agricultural practices and environmental factors on disease occurrence and
disease severity is lacking. In a first effort to fill
this hiatus, information on cropping patterns,
crop husbandry and harvesting conditions was
analysed for relationships with disease occurrence. The results of the multiple regression
analysis point to a possible importance of issues
associated with harvesting (mechanical damages,
topping efficiency and aerial temperature), the
calcium content of roots and the presence of
umbelliferous plants in the field margin or carrot
crops on a bordering plot during the preceding
season. Although the number of carrot crops was
relatively small in proportion to the number of
independent variables, the following factors best
explain the occurrence of the blemish or the
presence of R. carotae in blackish spots: (1) Many
pathogenic fungi enter plants through wounds, i.e.
mechanical damages, formed during harvest (Agrios, 1997). (2) The topping efficiency represents
the harvestability of the crop and is determined
by the vigour of the foliage, the uniformity of the
ridges and adjustments of the harvester. Healthy
and strong foliage, for example, allows the gripper belts of top-lifting harvesters to go at a lower
speed, thus reducing the chance of mechanical
damage. (3) The temperature has an effect on
processes involved in host defence (Bostock &
Stermer, 1989) as well as in fungal pathogenesis
303
(Årsvoll, 1971). (4) Calcium reduces the severity
of several diseases because of its effect on the
composition of cell walls and their resistance to
penetration by pathogens (Moerschbacher &
Mendgen, 2000) and its role in early signal
transduction in host–pathogen interactions (Boller & Keen, 2000). (5) In many diseases of annual
crops the inoculum survives in perennial weeds or
alternate hosts, and every season it is carried from
them to the annual crop and other plants. Some
fungi affecting annual plants overwinter as mycelium, resting or other spores or as sclerotia in
infected plant debris (Agrios, 1997; Krupinsky,
Bailey, McMullen, Gossen, & Turkington, 2002).
The inconsistency in the results of the analysis for
the crops harvested in 2001 and 2002 may be
connected with the low number of crops involved.
Because of this low number of observations it is
possible that a single observation strongly influenced the outcome of the analyses. Furthermore,
the inconsistency may be connected with the
annual differences in the abundance of the
various root spotting pathogens in general (occurrence of blackish spots) and of R. carotae in
particular (presence of R. carotae in lesions).
Therefore, further observational and experimental research is needed to verify the importance of
above-mentioned contributing factors.
The use of mechanical harvesting equipment
always incurs damage to carrot roots (Tucker,
1974). The inoculation experiment with different
wounding types (Table 9) has demonstrated that
R. carotae can effectively infect damaged areas of
the skin of carrot roots. So, avoidance of mechanical damage during harvest is a possible way to
prevent infections by this pathogen. Quite often
foliar diseases (e.g. leaf blight) and the current
logistics of carrot root harvesting make this
impracticable. Since curing has been found to
reduce losses in crops infested by M. acerina
(Hoftun, 1993), pre-storage treatments to harden
the periderm and heal wounds may also be useful
to control R. carotae. But measures that prevent
crops from becoming contaminated will be more
effective. For this purpose inoculum sources of R.
carotae need to be known. The relationships with
umbelliferous plants and the cultivation of carrot
crops during the preceding season indicate that
infected umbelliferous plants or their remains
123
304
may be a source of inoculum which is vectored by
wind or otherwise to susceptible carrot crops.
However, a small scale screening of umbelliferous
plants in field margins of the crops surveyed in
2002 did not lead to the identification of hosts of
R. carotae (data not presented). Furthermore, the
use of the carrot disk soil assay described by
Tabachnik, DeVay, Garber, and Wakeman
(1979) to detect T. basicola in soil, did not yield
positive results for R. carotae with soil samples
taken during spring 2003 from four fields on
which affected crops had been grown in 2002
(data not presented). A more systematic survey of
the presence of the pathogen on wild and cultivated umbelliferous plants, as well as on crop
remains or in soil is therefore needed to identify
inoculum sources.
At harvest time blackish spots associated with
R. carotae were not found, whereas such lesions
were indeed present after cold storage, thus
indicating the blemish had developed during
storage. Furthermore, the occurrence of R. carotae
in spots was linked with harvest damage. To
explore at what temperatures contaminated
wounds can develop into lesions, the effect of
temperature on aspects of the pathogenicity of
R. carotae, i.e. conidial germination, mycelial
growth and the penetration of carrot roots was
studied. The responses of the germination rate,
growth rate and lesion depth to the different
temperatures showed similar trends. The temperature growth curve for R. carotae isolate 840
(Fig. 1) resembles that described by Årsvoll
(1965) for a Norwegian isolate. For the latter
isolate a minimum temperature for growth was
found under –3C. Temperatures close to 18C
and a little higher than 25C were the estimates of
the optimum and maximum temperature for
growth, respectively. By fitting a Logan curve
through the weekly mycelial growth rates at
different temperatures, accurate estimates were
obtained for two cardinal temperatures of the
temperature growth curve for R. carotae isolate
840, i.e. an optimum of 19C and a maximum of
29C. With its ability to grow at 0–5C, its
optimum growth temperature >15C and its
upper limit for growth >20C, R. carotae meets
the specifications of psychrotrophic (psychrotolerant) microorganisms (Morita, 1975; Singleton &
123
Eur J Plant Pathol (2007) 117:293–305
Sainsbury, 1987). Despite its cold tolerating
nature, the pathogen failed to penetrate wounded
root tissues at 0C. The rate of development of
defence barriers in the periderm tissues was
probably faster than the rate of germination and
growth of R. carotae. The development of brown
lesions at 3–20C (Table 10), on the other hand,
demonstrates that at these temperatures the
pathogen could outgrow the host’s defence mechanisms. The absence of infections at 30C is
certainly connected with the inability of
R. carotae to grow at this temperature. Because
R. carotae, as is the case with the black spot
pathogens M. acerina (Lewis, Davies, & Garrod,
1981) and T. basicola (Punja, Chittaranjan, &
Gaye, 1992), is able to infect open wounds at
temperatures used in curing procedures, it
remains to be seen whether pre-storage temperature treatments can prevent wounds, already
contaminated at harvest, from becoming infected.
Only wounds which are already healed are no
longer amenable to infection by M. acerina and
T. basicola. For that reason the positive effect of
pre-storage temperature treatments described by
Hoftun (1993) might not be the result of speeding
up wound healing, but could just as well be
connected with the colonization of the wounded
tissue by antagonists amongst the resident microflora of the carrot rhizoplane. Treating wounds
with antagonists might even be more effective in
controlling root decay by R. carotae than temperature treatments supporting wound healing.
The availability of the roots on the elevator of
‘top-lift’-type carrot harvesters offers possibilities
to apply such biological control agents.
The present study demonstrates that R. carotae
can infect wounds and thrives at the low temperatures occurring during cold storage of carrot
roots. There are indications that umbelliferous
plants or their remains may be sources of inoculum. However, the life cycle of R. carotae is
unknown. Wild and cultivated umbelliferous
plants and crop remains or soil need to be tested
for the presence of the pathogen. With conventional mycological methods the slow growth of
R. carotae brings about the risk of the pathogen
remaining undetected because it is overgrown by
other fungi. These disadvantages can be circumvented by the use of molecular detection methods.
Eur J Plant Pathol (2007) 117:293–305
Molecular tools, such as real-time PCR, may also
be usable to assess effects of control measures
such as curing.
Acknowledgements This study was supported by the
Dutch Ministry of Agriculture, Nature and Food Quality
LNV programme 342 and by the Dutch Product Board
Horticulture.
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