OR I G I N A L A R T I C L E
Changes in the prevalence of fungal species causing post-
harvest diseases of carrot in Finland
Satu Latvala1 | Minna Haapalainen2 | Petteri Karisto1 | Pirjo Kivijärvi3 |
Oona Jääskeläinen2 | Terhi Suojala-Ahlfors4
1Natural Resources, Natural Resources
Institute Finland (Luke), Jokioinen, Finland
2Department of Agricultural Sciences,
University of Helsinki, Helsinki, Finland
3Production Systems, Natural Resources
Institute Finland (Luke), Mikkeli, Finland
4Production Systems, Natural Resources
Institute Finland (Luke), Piikkiö, Finland
Correspondence
Satu Latvala, Natural Resources, Natural
Resources Institute Finland (Luke), Jokioinen,
Finland.
Email: satu.latvala@luke.fi
Present addresses
Minna Haapalainen, Natural Resources,
Natural Resources Institute Finland (Luke),
Helsinki, Finland; and Oona Jääskeläinen,
Verdera/Lallemand Plant Care, Espoo, Finland.
Funding information
the European Agricultural Fund for Rural
Development, Grant/Award Number: 15923
Abstract
Post-harvest diseases cause significant economic losses in the carrot production
chain. In this study, storage losses and fungal pathogens causing them were analysed
in the carrot yield from 52 different field plots in four areas in Finland in 3 years
(2016–2018). Over 30,000 carrots were sampled and analysed at three time points
during cold storage at 0–1
C. In March, after 5–6 months' storage, the average loss
due to diseases was 20%–21% every year. Decay of the root tip was the most com-
mon disease symptom, followed by pits on the side and black rot in the crown,
detected in 69.2%, 15.0% and 9.0% of the symptomatic samples, respectively. Both
intensive carrot cultivation practice and early timing of harvest increased storage
losses. Pathogens in 3057 symptomatic carrot tissue samples were isolated by cultur-
ing, and fungal species were identified. The most common fungal species detected
were Mycocentrospora acerina, Botrytis cinerea and Fusarium spp., especially
F. avenaceum. However, the frequency of different pathogens varied between the
different years and time points during storage. Species-specific PCR tests revealed
that M. acerina and F. avenaceum were present in many early time-point samples
where they could not yet be detected by the culturing method. In Finland, this study
on carrot post-harvest diseases is the first large-scale survey in which the fungal
pathogens were isolated and identified by laboratory tests. In comparison with the
previous studies, Fusarium spp. were detected more frequently in this study, while
grey mould and Sclerotinia rot were less frequent.
K E YWORD S
crop rotation, culturing method, Daucus carota, fungal diseases, Fusarium, harvest time, PCR
1 | INTRODUCTION
Carrot (Daucus carota) is the most important outdoor vegetable in
Finland with 76,000 tonnes production and 1726 ha cultivated area
in 2021 (OSF: Natural Resources Institute Finland, Horticultural Sta-
tistics). This production volume is approximately 10% of the
production in each of the major carrot producer countries in Europe:
Germany, Poland and the United Kingdom (EUROSTAT, online data
code APRO_CPSH1). However, in relation to the population in each
country, the carrot production volume in Finland is large. Because the
growing season is short in the Nordic countries, including Finland,
most of the carrot yield needs to be stored in the cold store
Received: 15 November 2023 Revised: 29 February 2024 Accepted: 12 March 2024
DOI: 10.1111/aab.12908
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2024 The Authors. Annals of Applied Biology published by John Wiley & Sons Ltd on behalf of Association of Applied Biologists.
Ann Appl Biol. 2024;1–13. wileyonlinelibrary.com/journal/aab 1
(temperature 0–1
C) for several months before sale. During the long
storage, post-harvest diseases cause significant losses, the level of
which differs between years and field plots (Suojala, 1999; Suojala-
Ahlfors & Laamanen, 2014). Hermansen and Amundsen (1995)
reported that in Norway typical losses due to diseases were 20%–
50% during 5–6 months' storage. In Finland, the average proportion
of diseased carrots in March, after 5–6 months' storage, was 36% and
25% in the years 2005 and 2006, respectively, while the proportion
of diseased carrots in different lots varied between 0 and 100%
(Vanhala et al., 2008).
Post-harvest diseases of carrot are mainly caused by different
soil-borne fungi and bacteria (Davis & Raid, 2002; Papoutsis &
Edelenbos, 2021; Snowdon, 1992). Infection can occur either during
the growing season in the field, during the harvesting process, or dur-
ing the storage. The pathogens present in the field soil, contaminated
surfaces, or diseased carrots in the same harvest lot can enter the car-
rot root through wounds caused by harvesting process and cause dis-
ease later during the storage (Davis & Raid, 2002; Kora et al., 2005a;
Papoutsis & Edelenbos, 2021).
Globally, the most damaging post-harvest diseases of carrot are
the fungal diseases Sclerotinia rot (Sclerotinia sclerotiorum (Lib.) de
Bary) and grey mould (Botrytis cinerea Pers. ex Fr.), which cause losses
especially after growing seasons with high precipitation (Davis &
Raid, 2002; Li et al., 2018; Kora et al., 2005b; Papoutsis &
Edelenbos, 2021). Both B. cinerea and S. sclerotiorum have a wide host
range and these fungi can overwinter as sclerotia in dead or living
plants or in the soil (Davis & Raid, 2002). Sclerotinia rot is a significant
problem in both production and storage, and it can spoil the carrots
already at the beginning of the storage period (Davis & Raid, 2002;
Papoutsis & Edelenbos, 2021). Botrytis cinerea can survive on various
surfaces in storage and packing sheds. In the modern refrigerated
packing and storage facilities, grey mould is generally a minor problem
(Davis & Raid, 2002). Losses due to grey mould can, however, increase
with the duration of storage, as desiccation makes the carrots more
susceptible to infections (Davis & Raid, 2002).
In Northern Europe, liquorice rot is one of the major post-harvest
diseases of carrot (Hermansen et al., 2012). This disease is mainly
caused by Mycocentrospora acerina, although other fungal species in
the genera Fusarium and Alternaria can cause similar symptoms
(Mohamad, 2021). Plant roots usually get infected by M. acerina
already during the growing season via ascospores in the soil but can
also get infected during the harvesting process. During storage, the
disease can spread in the infected carrots, even at 0
C
(Snowdon, 1992). Yet the spread of M. acerina from diseased to
healthy roots is limited, since intact plant tissues are resistant to the
fungus (Davis & Raid, 2002).Mycocentrospora acerina is a polyphagous
fungus, having several weeds as alternate hosts that can maintain and
increase the fungus in the field soil, even in the years when carrot is
not grown (Hermansen, 1992). In Finland, liquorice rot became com-
mon in the 1980s (Tahvonen, 1989). Later, Suojala (1999) reported
that based on the disease symptoms liquorice rot and grey mould
were the most common storage diseases of carrot. Parikka (2008)
found that liquorice rot was the main disease detected in carrots
stored for more than 3 months, yet grey mould and Sclerotinia rot also
caused significant storage losses.
Fusarium spp. have also been associated with carrot storage dis-
eases. When occurring alone, they can cause brown leatherlike rot on
carrot surface and crown (Snowdon, 1992). Fusarium spp. are often
considered as secondary pathogens which utilize the tissue already
damaged by other pathogens, thus increasing the damage. Especially,
Fusarium avenaceum has been reported to cause spoilage of carrots
(e.g. Pascouau et al., 2023).
Various fungal species and oomycetes can cause pits and cavities
which spoil the appearance and marketability of carrots (Hiltunen &
White, 2002; Kastelein et al., 2007). Some pits and cavities can
already be seen at harvest in the field, for example, several Pythium
species can cause carrot cavity spot in the field (Hiltunen &
White, 2002). However, these oomycetes have a minor role in causing
post-harvest diseases that spoil carrots during the cold storage
(Davis & Raid, 2002). The majority of visible damages appearing dur-
ing a long cold storage are caused by fungal pathogens. In the
Netherlands Rhexocercosporidium carotae causing significant pit dis-
ease problems was reported (Kastelein et al., 2007), and in Norway
Athelia arachnoidea (synonyms Rhizoctonia carotae, Fibulorhizoctonia
carotae), causing crater rot, was reported as an important post-harvest
pathogen (Hermansen et al., 2012). Alternaria radicina is a seedborne
pathogen that occurs in most carrot production areas of the world
and causes black rot, appearing as dark pits and cavities (Davis &
Raid, 2002; Farrar et al., 2004; Kastelein et al., 2007; Papoutsis &
Edelenbos, 2021). Also, black root rot, caused by Berkeleyomyces basi-
cola (synonym Thielaviopsis basicola) and B. rouxiae, is an important
fungal disease affecting carrot quality worldwide (Nel et al., 2019;
Papoutsis & Edelenbos, 2021). B. basicola infection develops in stor-
age after handling of carrots in the packaging station, especially if the
storage temperature and humidity are too high, and symptoms lead to
vast decay of the carrots packaged in plastic bags (Papoutsis &
Edelenbos, 2021).
Bacterial soft rot can also cause spoilage of carrots, both in the
field, at warm temperatures and in case of waterlogging, and during
storage, under low oxygen (poorly ventilated) conditions (Farrar
et al., 2000; Lampert et al., 2017). Pectolytic bacteria identified as
causal agents of carrot soft rot include Pectobacterium, Erwinia and
Pseudomonas spp. (Kahala et al., 2012), and more recently Leuconos-
toc mesenteroides was found as a causal agent of bacterial oozing
(Lampert et al., 2017).
Carrot is a cool climate crop, sensitive to high temperatures, and
thus the global climate warming can affect the yield and quality of this
vegetable. Both thermal stress and elevated CO2 level have been
experimentally shown to reduce the yield and quality of carrot (Azam
et al., 2013; Ibrahim et al., 2006). As different fungal pathogens have
different optimal growth temperatures, the elevated temperatures
may also affect the pathogen populations, favouring some species and
suppressing others. The climate change has already altered the crop
growing conditions, which may affect the plants' susceptibility to dis-
eases and the occurrence of pathogens. Hence, knowledge of the cur-
rent pathogen incidence is needed for effective disease control. This
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study is the first large-scale survey on carrot post-harvest diseases in
Finland where the fungal pathogens were isolated and identified by
laboratory tests, in addition to identifying the disease symptoms. The
specific aims of this study were (i) to determine the level of post-
harvest losses due to the pathogens in the current climate conditions
and with the agronomical practices used in Finland, (ii) to identify the
main fungal pathogens spoiling carrots in cold storage at different
time points, and (iii) to compare two methods, fungal culturing and
PCR detection for identification of the main post-harvest pathogens.
2 | MATERIALS AND METHODS
2.1 | Carrot sampling
Carrot samples were collected manually from 52 field plots from four
regions in Finland—Southwest Finland, Häme, South Savo and North
Savo—at the end of the growing season in 2016, 2017 and 2018. The
samples were collected in September–October, as close to
the farmer's harvest time as possible, between 8 September and
4 October in 2016, from 14 September to 9 October in 2017 and
from 19 September to 9 October in 2018. Due to crop rotation, the
fields used for carrot growing were different in different years. From
each field, approximately 12 kg of carrots were collected as samples
from five different points, to form a 60-kg lot per field. The leaves
were manually removed on the field.
The number of collected lots was 16 in 2016, 15 in 2017 and
21 in 2018. In 2017, one lot was obtained from the farmer's storage,
while the other lots were collected directly from the field. The main
cultivars were Maestro (26 lots), Romance (eight lots) and Natalja
(seven lots). 21 lots were grown in organic soils and 31 lots in mineral
soils. Background information of farming practices and pre-plants
grown on the survey plots was collected by interviewing the farmers.
However, all these data were not obtained for all the fields, and thus
only the frequency of carrot growing was utilized in the data analysis.
2.2 | Storage conditions and processing of carrot
samples
All carrot samples were taken to storage facilities (Luke Piikkiö, Kaarina,
Finland) where each 60-kg lot was divided into seven equal subsamples,
weighing approximately 8 kg each, and packed in woven polypropylene
bags. One of the subsamples was stored at 10
C for 6 weeks to predict
the storability of the lot (data not shown). Six subsamples were taken
to the cold store (temperature 0–1
C) for storage experiment. The stor-
age samples were inspected at three time points, with 6-week intervals,
in December, January, and March. Two parallel storage samples (a and
b) were analysed at each time point, and their weight was measured
before and after storage. The number of stored carrots in each year
and at each inspection time point is presented in Data S1.
The inspection was always started by separating the healthy look-
ing and diseased carrots. The number of carrots in both groups was
counted and the carrots were weighed. Storage loss due to diseases
was calculated both per number and per weight (the total weight of
carrots after storage).
2.3 | Analysis of the diseased carrots
The diseased carrots were washed and divided into four categories
based on visible (macroscopical) symptoms. If there was more than
one type of symptom present on a root, the same carrot was recorded
into several categories accordingly. The percentage of carrots in dif-
ferent symptom categories was counted per total number of carrots
in each lot. The symptom categories were:
1. Decay (starting) in the root tip.
2. Pits and cavities on the side of the root.
3. Black or dark brown rot in the crown of the root.
4. Carrots spoilt by grey mould or Sclerotinia rot, and the pathogen
identifiable by sclerotia. The disease was recognized as grey mould
if the split sclerotia were brown inside and as Sclerotinia rot when
they were white inside.
In addition to identifying the diseases based on the visible symp-
toms, samples were taken from maximum 30 carrots per lot in each
symptom category to identify the fungal pathogens by culturing
method. No samples from the carrots severely spoiled by grey mould
or Sclerotinia rot were plated on agar for fungal identification. In total,
3057 samples were taken for fungal identification tests (Data S1).
2.4 | Isolation and identification of fungi by
culturing method
Since pentachloronitrobenzene (PCNB) agar has been reported to be
semi-selective for Fusarium spp. (Nelson et al., 1983), we tested
whether it would be applicable for isolating the other fungal patho-
gens commonly affecting carrots. For comparison, different filamental
fungi isolated from symptomatic carrot roots were grown in parallel
on PCNB and potato dextrose agar (PDA) media. Two isolates of each
of the species F. avenaceum,M. acerina and B. cinerea, and two isolates
representing the Cylindrocarpon spp. complex (one Neonectria and one
Ilyonectria) were chosen for the experiment. Eight agar plugs (diameter
5 mm) were cut with a sterile cork bore from each 1-week-old fungal
culture on PDA. Four of these plugs were transferred onto PCNB
medium and the other four plugs onto PDA medium, one plug per
plate. These 90 mm plates were incubated at room temperature (20–
22
C) in darkness. The fungal cultures were photographed, and their
diameter was measured after 2, 5, and 7 days of incubation.
To isolate and identify fungal species from carrots classified in dif-
ferent symptom categories, a cubic piece with approximately 5 mm
sides was cut with a sterile scalpel at the edge between injured and
healthy-looking tissue. These tissue pieces were surface-sterilized by
soaking in 70% ethanol for 1 min, rinsed twice with deionized water
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and dried on sterile paper towel in laminar flow cabinet for 30 mins.
The pieces were then transferred onto PCNB agar medium (Nelson
et al., 1983), and incubated at room temperature (20–22
C) in dark-
ness for 5 days. Tips of the fungal hyphae growing out of the plant tis-
sue pieces on the PCNB agar were subsequently transferred onto
standard PDA medium (Difco™ Potato Dextrose Agar, Becton, Dickin-
son and Company). If different fungi grew from a single piece, they
were all transferred separately onto PDA. The pure cultures were
incubated in darkness at room temperature for 3–4 weeks. The fungal
species were tentatively identified by the culture features and spore
morphology according to the systematics by Gerlach and Niren-
berg (1982).
2.5 | Identification by molecular methods
To compare the results of the culturing method to PCR detection of
pathogens, we used both methods on a subset of samples. From the
2018 yield, 25 symptomatic carrots from the disease prediction sam-
ples, 10 from the December storage samples, 20 from the January
storage samples and 20 from the March storage samples were tested
by species-specific PCR in parallel with the culturing method (75 sam-
ples in total).
For this subset of samples, a piece of symptomatic tissue from
each carrot was split into two halves. One of them was surface-
sterilized and placed on a selective medium as described above. The
other half was frozen at 80
C prior to DNA extraction. Then 0.1 g
of tissue was ground using FastPrep-24 tissue and cell homogenizer
(MP Biomedicals) in tubes containing Lysing matrix A (MP Biomedi-
cals). The total DNA was extracted using DNeasy Plant Mini Kit
(Qiagen) according to the manufacturer's instructions.
The DNA samples were tested for the presence of M. acerina,
F. avenaceum and B. cinerea by single PCR reactions using species-
specific primers. The primers were KLO15F (CGGTCGGACTCCATT
CAAAC) and KLO20R (GCTCCGAAGCAAGTACGCCG) for M. acerina
(Hermansen et al., 2012), FA-F1 (AACATACCTTAATGTTGCCTCGG)
and FA-R1 (ATCCCCAACACCAAACCCGAG) for F. avenaceum
(Mishra et al., 2003), and Bc-F (CAGGAAACACTTTTGGGGATA)
and Bc-R (GAGGGACAAGAAAATCGACTAA) for B. cinerea (Fan
et al., 2015). The sizes of the expected PCR products were 314 bp,
314 bp and 354 bp, respectively.
PCR was run using 0.4 μL Phire Hot Start II DNA polymerase with
its 1 reaction buffer (Thermo Scientific), 200 μM dNTPs, primers at
250 nM concentration, and 20 ng template DNA in 20 μL reaction
volume. The programme was as follows: 98
C for 30 s; 35 cycles of
98
C for 1\ 0 s, annealing for 10 s, extension at 72
C for 30 s; fol-
lowed by a final extension at 72
C for 5 min. Annealing temperatures
were 63
C for M. acerina and F. avenaceum, and 60
C for B. cinerea.
The PCR products were analysed by electrophoresis on 1.5% agarose
gels in Tris-borate-EDTA buffer. Gels were stained with ethidium bro-
mide and visualized under a UV-transilluminator (SCIE-PLAS).
A fragment of the translation elongation factor 1-alpha gene (TEF)
of a few Fusarium-positive fungal and carrot tissue samples was
amplified and sequenced as described in Haapalainen et al. (2016).
Primers ITS1 and ITS4 (White et al., 1990) were used to amplify a
fragment of ITS region of Pythium spp. by PCR, using the same proto-
col as described above, with the annealing temperature of 60
C. Prior
to Sanger sequencing, the PCR products of approximately 1 kb in size
were purified using QIAquick PCR Purification kit (Qiagen). The TEF
and ITS sequences obtained were compared to the sequences avail-
able in the GenBank, by using blastn tool (NCBI).
2.6 | Weather conditions
Weather data were obtained from the observation stations of Finnish
Meteorological Institute (https://fmi.fi/en). Mean temperature and
precipitation sum in May–September from four observation stations,
situated nearby the carrot fields, were compared to the long-term
averages in the period 1991–2020 (Data S2). In 2016, the mean tem-
perature in May–September was 0.7–1.3
C higher than the long-term
average, whereas in 2017 the mean temperature was 0.3–1.3
C lower
than the long-term average. The growing season in 2018 was the
warmest, and the mean temperature was 2.0–2.9
C higher than
the long-term average.
In Southwest Finland, the precipitation sum in the growing season
was lower than the long-term average in all the 3 years. In the other
regions the precipitation was close to the average in 2016 and 2017,
whereas in 2018 the precipitation sum was lower than the long-term
average.
2.7 | Statistical analyses
To compare the growth of different fungal species on solid PCNB and
PDA media, the measurement data of the two isolates representing
the same species or species complex (Cylindrocarpon) were combined
to obtain groups with N = 8 for each species. Comparisons between
these species groups were performed using Welch two sample t-test
in R (version 4.2.1). Difference between the species was considered
significant when the p-value of two-sided test was less than .05.
Significance of the effect of crop rotation and harvest time on
storage losses due to diseases was tested by Welch two sample t-test
in R. The harvest times of different carrot lots were classified into
three categories. The “average” harvest time (N = 14) was defined as
±1 week from the median harvest date in each year. Harvest dates
earlier than the “average” were classified to “early” harvest category
(N = 21) and harvest dates later than the “average” were classified to
“late” harvest category (N = 17). Significance of differences in the rel-
ative amounts of different fungal pathogens detected by culturing
method in the carrot lots with different crop rotation background or
different harvest time was tested by paired t-test in R. The signifi-
cance of differences in the frequency of different fungal pathogens
detected at different time points during the cold storage was tested
by Pearson's Chi-squared test in R. The p-values less than .05 were
considered significant.
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3 | RESULTS
3.1 | Storage losses due to diseases
Over 30,000 carrots collected from 52 different field plots in four
areas in Finland in 3 years (2016–2018) were sampled and analysed
at three time points during the cold storage at 0–1
C. The average
storage loss of carrots was roughly similar in all the 3 years. In
December the average proportion of diseased carrots was 4%–10%,
in January 11–12% and in March, in the end of the storage period,
20%–21% of the weight (Figure 1). However, in the individual lots the
storage losses varied between 0% and 76%. The carrot lots with
the best storability remained healthy up to the end of the storage
period, whereas in the poorest lots the storage loss was already high
in December. No significant differences were detected in the amount
of storage loss between the lots from different carrot cultivation
regions (data not shown).
3.2 | Disease symptoms
The most common symptom in carrots was softening and darkening
of the root tip (Figure 2a,b), comprising on average 69.2% of all the
visible symptoms developed during the cold storage. The appearance
of the root tip symptoms varied in different samples from slight dark-
ening of the end of the root tip to fully softened black tissue over 2–
3 cm from the tip and even beyond that. 45% of the root tip symp-
toms were severe, having darkened and softened tissue more than
1 cm up from the tip (Figure 2b), and 55% of the symptoms were
milder (Figure 2a). In the December samples the symptoms were still
mild, but later the disease progress was rapid in some lots. In March,
identifiable mycelium and sclerotia of B. cinerea were observed in
some symptoms started from the root tip (Figure 2c). In total, the pro-
portion of carrots visibly spoilt by grey mould or Sclerotinia rot was
6.8%. In March 2018, four sample lots from the parallel set b were dis-
carded due to an uncontrollable spread of Sclerotinia rot. Deep and/or
large pits and cavities, and small and clearly defined pits on the side of
the root were also detected (Figure 2d,e). On average, different pits
constituted 15.0% and black rot in the crown (Figure 2f) constituted
9.0% of the visible symptoms. During the cold storage all the symp-
toms caused by pathogens increased; however, the proportions of dif-
ferent symptom types increased at slightly different rates, and thus
the relative proportion of pit symptom decreased by time. The varia-
tion between individual carrot lots in the amount of diseased carrots
was large (Figure 1) and the proportions of different symptom types
were also different in different lots.
3.3 | Applicability of the PCNB-PDA culturing
method for different fungal pathogens
The post-harvest fungal pathogens commonly found on carrot in
Finland—M. acerina, B. cinerea, F. avenaceum, Ilyonectria spp. and
Neonectria spp. (Cylindrocarpon spp. complex)—were all able to grow
well on PCNB medium (Table 1; Data S3; S4). However, some dif-
ferences in the growth rate were detected. During the first 2 days
on PCNB medium the B. cinerea isolates grew significantly less (t-
test, p < .05) than the other fungi, which did not significantly differ
from each other. In 5 days, M. acerina isolates had grown signifi-
cantly more than the other species (Table 1). The difference
between B. cinerea and F. avenaceum was also significant
(p = .0119), while neither of these fungi showed significant differ-
ence from the Cylindrocarpon species. In 7 days, M. acerina isolates
had grown significantly more than the other species, while there
were no significant differences between the other species. On PDA
medium, all the fungal species differed from each other significantly
in their growth during the first 2 days. In 5 days, there was no sig-
nificant difference between F. avenaceum and the Cylindrocarpon
isolates, while M. acerina and B. cinerea significantly differed from
them and from each other (Table 1). Both of the B. cinerea isolates
completely filled the PDA plates within 5 days. After 7 days,
M. acerina isolates had grown significantly more than F. avenaceum
and Cylindrocarpon isolates, which did not differ significantly from
each other (Data S3; S4).
F IGURE 1 Proportion of symptomatic carrots at three time points during the cold storage. N is the number of stored lots inspected at each
time point. In the box plots the line inside each box indicates the median value.
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3.4 | Pathogens in symptomatic carrots identified
by the culturing method
Altogether 3057 symptomatic samples taken from the cold-
stored carrots were tested by the culturing method for pathogen
identification by morphological characteristics (Data S1). Of
these sample pieces 72.6% were of the root tip symptoms,
17.1% of pits on the side of the root and 10.3% of the crown
black rot. The most common fungal species detected in the
symptomatic tissue samples were M. acerina, B. cinerea and
Fusarium spp., of which 65% were identified as F. avenaceum,
22% as F. sambucinum, and the rest as other species, including
F. culmorum and F. sporotrichioides.
Cylindrocarpon spp. were also frequently detected. Acremonium
spp., Mucor spp., and Phoma spp. were identified as minor species, as
well as some oomycetes, tentatively identified as Pythium spp. in 3%
of the tested samples, from all the symptom types. ITS sequencing
was performed for 13 of the isolates identified as Pythium, and based
on the sequence four isolates were confirmed as P. intermedium, one
as P. sylvaticum and another as P. irregulare, whereas the other seven
isolates remained as undefined Pythium isolates.
During the cold storage the frequency of different pathogens
detected in the symptomatic tissue samples changed in the course of
time. Fusarium spp. were typically detected early in the storage period,
whereas M. acerina and B. cinerea became more prevalent later in
January and March (Figure 3). Fusarium spp. and M. acerina were the
most frequent fungal species detected in all the three symptom types.
Acremonium spp., Mucor spp., and Phoma spp. and oomycetes that
were detected to a minor extent in the symptomatic tissues, are
included in the “other microbes” in Figure 3.
The proportions of different pathogens also varied between the
years. In 2016, B. cinerea was more common than in 2017 and 2018.
In 2017, M. acerina was found to be the most common fungus, and
Cylindrocarpon spp. were also detected from many of the symptomatic
root tip samples. In 2018, fungal isolates were only obtained from
30% of the symptomatic root tips and from 50% of the black crown
samples or large cavities on the side. Instead, bacteria were growing
from many samples on the culture medium (Figure 3).
F IGURE 2 Different symptoms observed in carrots. (a) and (b) softening and darkening of the root tip, (c) identifiable mycelium and sclerotia
of B. cinerea, (d) small and clearly defined pits on the side of the root, (e) deep and/or large pits and holes on the side of the root, and (f) black rot
in the crown.
6 LATVALA ET AL.
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/doi/10.1111/aab.12908 by Luonnonvarakeskus, W
iley O
nline Library on [26/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
3.5 | PCR detection in comparison to the culturing
method
In total, 75 symptomatic samples from different time points within the
storage period were tested by species-specific PCR in parallel with
the culturing method. Based on the identification by the culturing
method, samples having mainly one of the following fungi:
F. avenaceum, M. acerina or B. cinerea, or no culturable fungi at all,
were selected. Three of the chosen samples contained both
F. avenaceum and M. acerina. When DNA extracted from these sam-
ples was tested for F. avenaceum, M. acerina and B. cinerea by species-
specific PCR, more than one of the three fungi were detected in most
of the samples. Mycocentrospora acerina was detected in 66 samples,
F. avenaceum in 54 and B. cinerea in 20 samples.
In the disease prediction samples taken in November, M. acerina
and B. cinerea could already be detected by PCR, but not by the
culturing method. PCR tests also revealed that many storage samples
contained all the three fungi—B. cinerea, M. acerina and
F. avenaceum—even though only B. cinerea was growing on the culture
plates. While the two methods agreed quite well in the detection of
B. cinerea, many more positive results were obtained for M. acerina
and F. avenaceum by PCR than by culturing (Figure 4). In 30% of the
samples tested, none of these three fungal species was found by
the culturing method, whereas in species-specific PCR only one sam-
ple was negative for all three, suggesting that the detection limits of
the PCR methods were lower. By PCR, two different fungal species
were detected in 66.7% of the samples and all the three species
were detected in 10.7% of the samples.
Based on the TEF gene sequences, obtained from a few Fusarium-
positive samples from the culture plates and symptomatic carrot tis-
sue samples positive in FA-F1/FA-R1 PCR test, the main Fusarium
species was confirmed as F. avenaceum. Two of these sequences,
representing the two different sequence types detected, were depos-
ited to GenBank (accession numbers PP357886 and PP357887).
3.6 | Effect of pre-crop and harvest time on
storage losses and pathogen community
On those fields in which carrot had also been grown during the previ-
ous 2 years, the proportion of symptomatic carrots was on average
twice as high as on the fields where carrot had not been grown. The
difference was statistically significant (p < .05) at all the three sam-
pling time points during the storage (Data S5). When classified by the
time of harvest, 21 carrot lots were harvested earlier and 17 later than
the average time and 14 lots were harvested at the average time
(median ± 1 week). The early harvested lots had significantly (p < .05)
higher proportion of symptomatic carrots than the lots harvested at
the average time or later (Data S5). However, when the proportions
of symptomatic carrots were compared between sample lots grouped
by both the harvest time and pre-crop (carrot or no carrot), it was the
combination of early harvest and intensive carrot culturing that signifi-
cantly increased the disease incidence (Figure 5). In December andT
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LATVALA ET AL. 7
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nloaded from
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iley.com
/doi/10.1111/aab.12908 by Luonnonvarakeskus, W
iley O
nline Library on [26/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
January, the group “Early harvest & Carrot grown in previous 2 years”
had significantly (p < .05) higher disease incidence than the other
groups, except for the group “Early harvest & No data on pre-crop”.
In March, the difference between the groups “Early harvest & Carrot
grown in previous 2 years” and “Average or late harvest &
Carrot grown in previous 2 years” was only marginally significant
(p = .0682).
The relative amounts of different fungal pathogens detected by
the culturing method were significantly different between the plots
where carrot had been or had not been grown during the last 2 years
(Chi-squared test, df = 3, in December χ2 = 15.4 and p = .0015, in
January χ2 = 34.6 and p < .001, in March χ2 = 24.7 and p < .001).
Especially, Fusarium spp. were significantly more frequent in the stor-
age lots with intensive carrot culture background (p < .05, paired t-
test), whereas Cylindrocarpon spp. were more frequently detected in
the samples from fields where carrot had not been grown during the
last 2 years (p = .0092; Figure 6a). In December and January,
B. cinerea was significantly more frequent in the samples with
intensive carrot cultivation background (p = .0136), whereas in March
there was no significant difference. The frequency of M. acerina was
higher in January and March in the samples from fields with intensive
carrot cultivation background, however, the difference was not statis-
tically significant. The effect of early harvest time on the pathogens
was similar to the effect of intensive carrot cultivation, except for
Fusarium spp. (Figure 6b). At all the time points, the proportions of
tests positive for M. acerina, B. cinerea, Fusarium spp. and Cylindrocar-
pon spp. were different between the early harvest samples and the
late or average harvest time samples (p < .001, Chi-squared test).
Especially, Cylindrocarpon spp. were more frequent in the late or aver-
age harvest time samples (p < .01, paired t-test). In contrast,
M. acerina was significantly more frequent in the early harvest sam-
ples than in the samples from lots harvested at late or average time in
2017 (p < .05, paired t-test), whereas for the samples harvested
in 2016 and 2018 the effect of harvest time was not significant.
When pre-crop and harvest time were viewed together, B. cinerea
was significantly more frequent in the early harvest samples when
F IGURE 3 Fungal pathogens and other microbes identified from symptomatic carrot samples by the culturing method. Samples were taken
from three different types of symptoms: root tip decay, pits on the side and crown black rot. The percentage of each sample type of all the tested
samples in each year is shown in a box inside each panel of the figure.
8 LATVALA ET AL.
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iley.com
/doi/10.1111/aab.12908 by Luonnonvarakeskus, W
iley O
nline Library on [26/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
carrot had been grown in the previous 2 years (p < .05, paired t-test).
In contrast, significantly more bacteria were detected in the early har-
vest samples when carrot had not been grown in the previous 2 years.
4 | DISCUSSION
In the carrot production chain different post-harvest diseases cause
significant economic losses and decline of quality (Davis & Raid, 2002;
Papoutsis & Edelenbos, 2021). In this study, the average loss due to
post-harvest diseases after 5–6 months' storage period was 20%–
21% each year (2016–2018), even though the weather conditions
were different during the growing season in the different years. This
is slightly less than the losses during storage reported in the earlier
Nordic studies (Hermansen & Amundsen, 1995; Vanhala et al., 2008).
However, there was a large variation in quality between the individual
storage lots. As the storage time increased, the number of diseased
carrots increased, and the symptoms became more severe. The most
common symptom was softening or decaying of the root tip, while
pits and cavities on the side of the carrot and black rot in the crown
were minor symptom types. The pit symptom increased at a lower
rate than the other symptom types and thus its relative proportion of
all the symptoms decreased during the storage.
To identify the major pathogens in the disease symptoms, we
chose to use two-step culturing method. Although the PCNB
medium was earlier reported to be semi-selective for Fusarium spp.
(Nelson et al., 1983), our culturing test results showed that the
most common fungal pathogens causing post-harvest diseases in
carrots in Finland were all able to grow well on the PCNB medium.
Especially, M. acerina, a common species causing carrot liquorice
rot, was found to grow faster on PCNB than F. avenaceum. As the
Cylindrocarpon species also grew on PCNB as well as F. avenaceum
and B. cinerea in 5 days, this medium did not seem to favour Fusar-
ium over the other main pathogens of carrot. The applicability of
PCNB medium for isolating other fungi than Fusarium spp. is sup-
ported by the observations of Castellá et al. (1997) who compared
PCNB medium and malachite green medium for their selectivity for
Fusarium spp. They reported that malachite green medium per-
formed as a potent selective medium for Fusarium spp., whereas
PCNB allowed the growth of many different fungal species, includ-
ing Zygomycetes and yeasts. Thus, this medium was chosen as the
first step medium for this study, to restrict the growth of soil bacte-
ria and enable the isolation of fungi from carrots, followed by culti-
vation of the fungi as pure cultures on PDA medium. In general,
when isolating fungi from plant roots, using a semi-selective
medium is necessary at the first step. Without this step, if placing
pieces of roots directly on a non-selective medium, bacteria would
overtake the fungi. Some fast-growing fungi—like B. cinerea—also
need to be restrained, to prevent them overtaking the other species
on the culture plate.
F IGURE 4 Comparison of two different methods in detection of
fungal pathogens. Symptomatic carrot samples from nine sample lots,
taken either before or during the cold storage, were tested for three
major pathogens by both the culturing method and by species-specific
PCR. For each sample lot, the proportion of positive culturing results
is shown on the x-axis and the proportion of positive species-specific
PCR tests is shown on the y-axis. The PCR tests were more sensitive,
yielding more positive results.
F IGURE 5 Effect of harvest time and pre-crop on the proportion
of symptomatic carrots at different time points during storage. At all
the time points the group “Early harvest & Carrot grown in previous
2 years” showed a higher proportion of symptomatic carrots than
most other groups. The groups significantly (p < .05) different from
each other are marked with different letters. Group means are shown,
and the error bars indicate standard deviation. The numbers on the
right side of the legend denote the number of sample lots in each
group.
LATVALA ET AL. 9
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nloaded from
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iley.com
/doi/10.1111/aab.12908 by Luonnonvarakeskus, W
iley O
nline Library on [26/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
By using this culturing method, M. acerina, Fusarium spp. and
B. cinerea were the fungal pathogens most frequently detected from
the symptomatic carrot tissues. Compared to the earlier studies in
Finland (Mukula, 1957; Tahvonen, 1985), the proportion of Fusarium
spp. was higher and the proportion of B. cinerea (grey mould) was
lower in the symptomatic carrots in this study. Most strikingly,
S. sclerotiorum that had earlier been a devastating post-harvest patho-
gen was only found in some single storage lots. This may be, at least
partially, due to the improved storage conditions, as previously sug-
gested (Geeson et al., 1988). Proper crop rotation and good knowl-
edge of the host plants have also been found important in control of
Sclerotinia rot: cultivation of oil seed rape or other hosts of
S. sclerotiorum prior to carrot can cause quick spoilage of the crop
already during the early stage of storage (Parikka, 2008).
When species-specific PCR methods were compared with the
culturing method in identifying the three main fungal pathogens,
B. cinerea was detected equally well by both methods, whereas more
samples were found positive for M. acerina and F. avenaceum by PCR
than by culturing. By PCR M. acerina could even be detected in the
beginning of the storage period, when the amount of this fungus was
still low, and it could be occluded on the culture medium by other
more abundant fungi. The growth rate of different fungal species and
different strains of the same species also differ, and thus the ones
with a higher growth rate can supersede the others, even when they
were equally abundant in the original sample. On the other hand, the
culturing method allows identification of new pathogens and the main
pathogen(s) in each sample and distinguishing between the viable and
non-viable microbes in the plant tissue.
Cylindrocarpon spp. were also frequently detected from the symp-
tomatic tissue in the root tip, pits and cavities. Few reports are avail-
able of the importance of Cylindrocarpon spp. as causal agents of
carrot diseases (Mohamad, 2021; Mukula, 1957; Sweetingham, 1983).
However, as Cylindrocarpon spp. are considered to cause pits and cavi-
ties on parsnip (Channon & Thomson, 1981) and potato (Choiseul
et al., 2006), they may also have a role in the symptom development
on carrot. Alternaria radicina, Athelia arachnoidea or Rhexocercospori-
dium carotae species, found causing pit symptoms in carrot in other
countries in Europe (Årsvoll, 1969; Hermansen et al., 2012; Kastelein
et al., 2007; Snowdon, 1992), were not detected in the samples from
Finland. No symptoms characteristic of carrot infection by Berkeleyo-
myces spp. were detected in this study, neither were these fungi
detected by culturing. Our results thus confirm the previous reports in
that Berkeleyomyces spp. have not been found in Finland (Nel
et al., 2019).
Although the focus of this study was on the fungal pathogens,
the culture method used for detection also revealed the presence of
some Pythium spp. in 3% of the carrots tested. Different Pythium spp.
have been reported to cause carrot cavity spot, and species involved
in the disease appear to vary according to geographical areas (Allain-
Boulé et al., 2004; Howard et al., 1978). P. irregulare and P. sylvaticum,
detected in this study, are fast-growing species which are often iso-
lated from the older cavities, and Hiltunen and White (2002) sug-
gested them to be secondary invaders that utilize the moisture and
decaying organic matter. Although Pythium spp. are major pathogens
affecting carrot during the growing season, they have a minor role in
post-harvest diseases spoiling carrots in storage (Davis & Raid, 2002).
For the carrots grown in 2018, in a warm growing season, no fun-
gal pathogens could be isolated from the majority of the symptomatic
tissues but instead, bacteria were growing in these samples. The bac-
terial species were not identified in this study, however, Kahala et al.
(2012) previously reported that different strains of the genera Pseudo-
monas, Pectobacterium and Erwinia were the primary bacteria causing
decay of carrots in Finland during storage and marketing. These pec-
tolytic bacteria can cause considerable financial losses, because they
account for a large proportion of bacterial rot of fruits and vegetables
during storage, transit, and marketing (Kahala et al., 2012).
The incidence of the main fungal pathogens also varied between
the 3 years of this study. During storage time, water loss from the car-
rot root makes it susceptible to fungal pathogens, especially to grey
mould infection. The infection often starts from the delicate root tip,
F IGURE 6 Effect of pre-crop (a) and harvest time (b) on the
occurrence of different fungal pathogens and bacteria in the disease
symptoms detected during storage. For each time point, the bars
show how much more (or less) each type of microbe was detected in
the symptomatic carrots in (a) samples from fields where carrot had
been grown in the previous 2 years (N = 802) in comparison to
samples from fields where carrot had not been grown in the previous
2 years (N = 820), or (b) samples from early harvest lots (N = 1341) in
comparison to lots harvested late or at the average time (N = 1714).
pp, percentage points.
10 LATVALA ET AL.
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nloaded from
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iley.com
/doi/10.1111/aab.12908 by Luonnonvarakeskus, W
iley O
nline Library on [26/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
since it desiccates easily (Snowdon, 1992). The biggest weight loss
during the storage was observed in the 2016 harvest, and this could,
at least partially, explain the high incidence of grey mould that year.
On the other hand, the highest incidence of M. acerina (liquorice rot)
was detected in the 2017 harvest, after the growing season with the
highest amount of precipitation. Previously, Hermansen et al. (2012)
reported that in Norway the incidence of carrot crown rot and tip rot
caused by M. acerina showed significant positive correlation with pre-
cipitation in July and September. In the presence of free water, the
optimal germination temperature of M. acerina was reported to be
from 14 to 22
C in vitro, however, the fungus did germinate even at
4
C (Yang et al., 2022). Thus, the weather during the growing season
can significantly affect the build-up of M. acerina inoculum. Other fac-
tors, such as pre-crop, soil type, cropping systems and fertilization, as
well as competition with other organisms can affect the development
of disease (Ghorbani et al., 2008; Hermansen et al., 1997; Kastelein
et al., 2007).
The prevalence of different fungal species detected in the symp-
tomatic carrots also changed during the time in storage. Fusarium spp.,
especially F. avenaceum, which was a minor fungus in the old report
(Tahvonen, 1985), were prevalent fungi in the December samples,
whereas M. acerina was more prevalent in the January and March
samples. This suggests that M. acerina infection develops slowly,
which agrees with those earlier reports stating that the amount of
M. acerina increased during the prolonged storage at low temperature
(Årsvoll, 1969; Snowdon, 1992). In addition toM. acerina and Fusarium
spp., B. cinerea was often detected in the wide shallow pits. Although
B. cinerea has been earlier considered a major pathogen spoiling the
crown of the carrot (Snowdon, 1992), in this study it was very rarely
detected in the crown rot samples.
Fusarium species have not been commonly known as carrot
post-harvest pathogens, however, recently carrot dry rot caused by
Fusarium spp. has been reported in Europe (Le Moullec-Rieu
et al., 2020; Stankovic et al., 2015). The pathogen was identified as
F. avenaceum in both Serbia and France, and in France also
F. tricinctum was identified as a carrot pathogen (Le Moullec-Rieu
et al., 2020). In this study the proportion of Fusarium fungi detected
in the carrot pit symptoms was high compared to the previous
reports in Nordic countries (Årsvoll, 1969; Hermansen et al., 2012;
Kastelein et al., 2007; Mukula, 1957; Tahvonen, 1985), and espe-
cially F. avenaceum was frequently found, suggesting it has become
an important pathogen of carrots in Finland. As the optimal growth
temperatures for Fusarium spp. are relatively high, 20–25
C (Li
et al., 2014; Moore, 1945), it is possible that these fungi could have
benefited from the recent elevated temperatures during the growing
season (Aalto et al., 2022).
The fungal pathogens detected from the carrot storage lots in this
study differ from each other regarding the applicable disease control
measures. While M. acerina is a soil-borne pathogen hosted by many
weed plants, controlling B. cinerea with agronomical practices is gener-
ally challenging, as the airborne spores are abundant in the autumn
(Snowdon, 1992). However, in this study grey mould was more com-
mon in those fields where carrot had been grown in the previous
years, suggesting harmful effect of poor crop rotation on this disease
as well. Fusarium spp. were also more common in the carrots from the
plots with poorer crop rotation. An early harvest and/or carrot inten-
sive crop rotation seemed to increase the incidence of both B. cinerea
and M. acerina. Although the pre-crop (carrot or no carrot) alone was
not a strong explanatory factor of the disease incidence level,
together with the early harvest time it resulted in a significantly higher
disease incidence. This result agrees with the previous studies in
which poor crop rotation (Hermansen et al., 1997) and early harvest
(Suojala, 1999) were reported to increase the risk of storage losses of
carrot.
5 | CONCLUSIONS
In the 3 years of this study, the total amounts of symptomatic carrots
were similar after 5 months in storage. However, the proportions of
different pathogens detected from the symptoms were different each
year. Of the most common fungal pathogens detected, Fusarium spe-
cies were prevalent after 2 months of storage, whereasM. acerina and
B. cinerea became more abundant later. From a large proportion of the
symptoms no fungal pathogens could be isolated, and instead bacteria
were detected. Thus, bacteria may also have an important role in
spoiling carrots during storage, especially after a warm growing sea-
son like in the year 2018. Sclerotinia rot (S. sclerotiorum), which earlier
was a major problem, was rarely detected in this study. Some of the
fungal pathogens causing serious storage losses in other countries in
Europe, like Alternaria radicina, Athelia arachnoidea or Rhexocercospori-
dium carotae were not detected in this study in Finland.
Altogether, controlling storage diseases is a complex challenge for
which no single solution can be found. Our results imply the impor-
tance of optimal harvest time and proper crop rotation, which con-
firms the results of earlier studies. Information on the harvest time of
each carrot lot could be more systematically used for planning the
order in which the lots are sold to the customers, to avoid long stor-
age of the early harvest lots. In addition, good storage conditions, with
low temperature and high relative humidity, as well as avoiding
mechanical damage are key factors in improved storability of carrot.
ACKNOWLEDGEMENTS
This work was funded by the European Agricultural Fund for Rural
Development (project 15923). We are very grateful to our colleague
Dr. Asko Hannukkala, who died in May 2020, for his precious contri-
bution to this work. We thank Sirkka Jaakkola and Paula Rannikko
from Häme University of Applied Sciences and Heikki Inkeroinen from
ProAgria Eastern Finland for their help in collecting the samples. The
technical assistance of Auli Kedonperä, Sanna Kulmala, Johanna Rih-
tilä, Senja Räsänen, Aila Siren and Senja Tuominen from Natural
Resources Institute Finland (Luke) is greatly acknowledged. We also
thank Anneli Virta from Luke Jokioinen for sequencing.
CONFLICT OF INTEREST STATEMENT
The authors declare that they have no conflict of interest.
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ORCID
Satu Latvala https://orcid.org/0000-0002-6433-2798
Minna Haapalainen https://orcid.org/0000-0002-2497-4059
Petteri Karisto https://orcid.org/0000-0003-4807-0190
Terhi Suojala-Ahlfors https://orcid.org/0000-0001-7543-870X
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How to cite this article: Latvala, S., Haapalainen, M., Karisto,
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Changes in the prevalence of fungal species causing
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