Australasian Plant Pathol.

(2013) 42:449–459
DOI 10.1007/s13313-013-0204-4

Infection pathway of Botrytis cinerea in capsicum fruit

(Capsicum annuum L.)
Thong D. Le & Glenn McDonald & Eileen S. Scott &
Amanda J. Able

Received: 30 October 2012 / Accepted: 19 February 2013 / Published online: 22 March 2013
# Australasian Plant Pathology Society Inc. 2013

Abstract Botrytis cinerea, which causes grey mould, in- Keywords Preharvest and postharvest inoculation .
fects fruit of a number of horticultural crops during their Flowering . Fruit ripening . Latent infection . Grey mould
development but then remains latent until the ripening pro-
cess when the disease manifests. However, how B. cinerea
grows in capsicum fruit after harvest has not been fully Introduction
characterised. The present research has examined the
growth of B. cinerea in fruit of two cultivars of capsicum Botrytis cinerea Pers. Fr. [teleomorph Botryotinia
(cv. Aries and cv. Papri Queen) that were inoculated either fuckeliana (de Barry) Whetzel], which causes grey mould
before or after harvest. Three concentrations of conidial of fruit in a number of horticultural crops, can infect during
suspensions (104, 105 and 106 conidia mL−1) were used to flowering and/or the early stages of fruit development as
inoculate flowers at three preharvest stages – anthesis, 3 days well as mature fruit often via wounds. Following infection
after anthesis (DAA) and 6 DAA, and fruit at three of flowers, B. cinerea may remain latent until reactivated
postharvest ripening stages – deep green (DG), breaker red when ripening causes physiochemical and biochemical
(BR) and red (R). Inoculation with water served as a control. changes in the fruit, leading to fruit rot and the development
Rot development was then monitored daily during of grey mould if environmental conditions are suitable
postharvest storage at 10 °C by measuring the length and (Droby and Lichter 2004). The processes of infection by
width of lesions. Cv. Aries was more susceptible to B. B. cinerea and development of grey mould are well
cinerea than cv. Papri Queen regardless of whether inocu- established for some fruit, such as red raspberry
lation occurred preharvest or postharvest. Flowers often (Dashwood and Fox 1988), strawberry (Jarvis and Borecka
died when inoculated at anthesis. Regardless of cultivar, as 1968), grape (Keller et al. 2003) and tomato (Lavy-Meir et
inoculum concentration increased the number of flowers al. 1989). Inoculation of crops such as strawberry, raspberry
that died also increased. However, disease development on (Jarvis and Borecka 1968; Dashwood and Fox 1988) and
fruit was not affected by inoculum concentration or the grape (Keller et al. 2003) at flowering leads to a greater
timing of inoculation before harvest. When fruit were inoc- incidence of grey mould in ripe fruit after harvest than at
ulated after harvest, grey mould developed most rapidly in other stages of development. When tomato fruit were
BR fruit of cv. Papri Queen and in R fruit of cv. Aries. The infected by B. cinerea during flowering, grey mould only
understanding of infection of B. cinerea revealed by this developed at the stem-end when fruit were stored (Lavy-
research and its implications for disease management are Meir et al. 1989). However, no relationship between flower
discussed. infection by B. cinerea and postharvest decay was found in
nectarine and plum (Fourie and Holz 1994). Infection by B.
cinerea occurs via the flower parts, after which the fungus
T. D. Le : G. McDonald : E. S. Scott : A. J. Able (*) remains latent in plant tissue. Disease then develops after
School of Agriculture, Food and Wine, The University
harvest or through wounding of fruit.
of Adelaide, Waite Research Institute, PMB 1,
Glen Osmond, SA 5064, Australia Infection by B. cinerea and the timing of grey mould
e-mail: [email protected] development are therefore likely to be related to a number of
450 T.D. Le et al.

factors such as the genetic background of the host, devel- Osmocote® (Scotts Australia Pty Ltd, Australia), hydrated
opmental stages of plants or fruit and physiological changes lime (0.7 kg m −3 ) and Aglime (1 kg m −3 ) (Aglime
in the fruit during ripening (Droby and Lichter 2004; Fertilisers, Australia)].
Williamson et al. 2007). As fruit mature, accumulation of Capsicum plants were grown in a greenhouse at 25±
sugars and organic acids during ripening provides appropri- 5 °C under a natural photoperiod cycle at the Waite
ate nutrients for B. cinerea to germinate and develop quickly Campus of the University of Adelaide (N-34° 58″ 13″
(Williamson et al. 2007). On the other hand, the accumula- W-138° 37″ 51″).
tion of antifungal proteins, including chitinases and
glucanases, that occur in grapes during ripening has been Isolation, maintenance and culture of B. cinerea
linked to resistance to B. cinerea (Derckel et al. 1998;
Salzman et al. 1998) while a decline in phytoalexins, such Botrytis cinerea was isolated from a naturally-infected cap-
as stilbene and resveratrol, has been shown to contribute to sicum fruit (bell pepper). Briefly, a symptomatic tissue piece
increased susceptibility to B. cinerea (Bais et al. 2000). (5 mm2) was placed on potato dextrose agar (PDA, Difco,
Changes to cell wall structure and composition as USA) amended with 100 mg L−1 streptomycin sulphate
well as ethylene production may also have an effect (Sigma, St Louis, MO) and incubated at 21 – 24 °C under
on infection by B. cinerea and grey mould development a 12 h light/12 h dark cycle for 2 weeks. A pure culture was
in fruit. Ethylene produced by tomato fruit during rip- then established by placing a 5 mm2 disc cut from the edge
ening has been shown to promote growth of B. cinerea of the culture on to new PDA plates. These were then
(Barkai-Golan and Lavy-Meir 1989), while B. cinerea incubated as described earlier. Pure cultures were then
has also been shown to produce ethylene and stored using a swab technique (Sue Pederick, South
polygalacturonase to allow greater access to nutrients Australia Research and Development Institute, personal
within the cell wall (Cantu et al. 2009). Although cap- communication). The end of a sterile cotton-tipped plastic
sicum belongs to the Solanaceae, it is non-climacteric, swab (Copan Italia, Brescia Italy) was rolled over a sporu-
whereas tomato is climacteric (Villavicencio et al. lating 3-week-old culture to coat it with conidia and the
1999). As such, the process involved in infection of swab was then inserted into a plastic tube and stored at 4 °C.
capsicum by B. cinerea may differ from that in tomato. A culture of B. cinerea was prepared by touching a stored
The aim of this study therefore was to determine the swab to the surface of PDA and incubating as described
incidence of grey mould development in capsicum fruit previously. Conidia were prepared by placing a disc from
during storage when plants or fruit were inoculated with the edge of a culture of B. cinerea in the centre of an 8.5-cm
B. cinerea at various flowering (preharvest) or ripening Petri dish containing V-8 juice [Campbell’s V-8 juice
(postharvest) stages. (900 mL L−1), dextrose (15 g L−1) and agar (20 g L−1)].
Plates were then incubated at 21–24 °C under a 12 h
light/12 h dark cycle for 2 weeks. Cultures were flooded
Materials and methods with 20 mL of Tween-20 solution (three drops of Tween-20
in 1 L sterile nanopure water) and agitated with a spreader.
Plant materials and conditions The conidial suspension was filtered through two layers of
sterile cheesecloth to remove mycelia. Conidia were washed
Plants of the Capsicum annuum cultivars Aries (bell, twice by centrifugation at 1,000 rpm for 5 min (Volpin and
large-fruit pepper) and Papri Queen (sweet, long, small- Elad 1991) and re-suspended in fresh Tween-20 solution.
fruit pepper), previously characterised for their Conidia were counted using a haemocytometer and the
postharvest physiology and ripening behaviour (Pham concentration adjusted as required using sterile nanopure
2007), were used. Pham (2007) determined that fruit water.
from cv. Papri Queen turned red more quickly and to
a greater extent than cv. Aries. However, their suscep- Inoculation by B. cinerea
tibility to B. cinerea had not been documented.
Seeds for cv. Aries and cv. Papri Queen were supplied by Flowers and young fruit
Monsanto Seeds and Fairbanks Seeds (South Australia),
respectively. Seeds were sown in 15-cm diameter plastic Flowers were inoculated with B. cinerea at three stages:
pots containing approximately 2.5 kg of UC potting mix anthesis (flowers fully open), 3 days after anthesis (DAA)
(University of California at Davis) (Baker 1957), and seed- (when petals had started to turn brown and young fruit ap-
lings thinned to one plant per pot. The UC mix was prepared proximately 0.3 – 0.5 cm in length had appeared) and 6 DAA
by combining two-thirds washed Waikerie sand and one (when fruit had set and were ~1 cm in length). Flowers and
third peat moss with the addition of fertiliser [2.25 kg m−3 fruit on two to four flowering nodes on each plant were
Infection of capsicum fruit by Botrytis cinerea 451

randomly selected and tagged at each of these stages before air-dried and then placed on a sterile, moist cloth in
direct application of 100 μL suspension of 104, 105 or 106 clear plastic containers and stored as described earlier.
conidia mL−1 (Halfon-Meiri and Rylski 1983; Elad and Volpin The inoculated ripe fruit and fruit derived from inocu-
1993; Fallik et al. 1996) onto the stigma in the flower or the lated flowers and young fruit were monitored daily and
stem-end of young fruit by using a pipette (Utkhede and the number of fruit exhibiting rot was recorded and
Mathur 2005). Controls were treated with sterile nanopure expressed as a percentage. Rot development was quanti-
water in the same way. After inoculation, flowers and fruit fied by determining the approximate lesion area on indi-
were covered with plastic bags for at least 8 – 12 h to maintain vidual fruit. Lesion area was calculated by multiplying the
humidity (Volpin and Elad 1991). There were 15 or more length and width of decayed areas as measured using
replicate flowers, one per plant, on 15 or more plants per digital calipers (digiMax, Switzerland). Polynomial re-
treatment to ensure that at least 10 fruit were obtained for each gressions were fitted to data using GenStat (14th Edition,
treatment for assessment of grey mould development VSN International Limited, UK) and time to reach 50 % of
postharvest. Plants were assigned at random to locations on final lesion size, as an approximation of disease rate
the greenhouse bench and the experiment was conducted (Horowitz et al. 2004), was determined by using the clos-
twice between October 2010 and February 2011. est fit polynomial regression in GenStat.

Fruit after harvest Presence of B. cinerea at harvest in fruit derived

from inoculated flowers
Fruit at three ripening stages, as described by Pham (2007),
were harvested for inoculation: Deep Green (DG) – Microscopy and reisolation on PDA were used to determine
completely green and intense colour; Breaker Red (BR) – if B. cinerea was present in fruit derived from inoculated
40 to 50 % red surface colouration and Red (R) – 100 % red flowers. Two surface-sterilised red fruit derived from inoc-
with low intensity. Postharvest inoculation was conducted as ulated flowers treated with each conidial concentration were
described by Fallik et al. (1996). Fruit were surface- collected randomly to assess the extent of hyphal growth in
sterilised by dipping in 2 % sodium hypochlorite for 1 min fruit at harvest. Each fruit was cut into three pieces: top
and rinsed briefly with sterile reverse osmosis water then (a 5 mm wide piece at the stem-end of fruit), centre (a
air-dried. The fruit were wounded on opposite sides to a 5 mm wide piece around the equator of the fruit) and
depth of 1.5 mm by puncturing them with the point of a bottom (5 mm at the blossom-end). Six replicate seg-
sterile 1.5 mm diameter nail. Each wound site was inocu- ments of 5 mm2 were cut from each. Segments random-
lated with 40 μL of suspension containing 104, 105 or 106 ly selected from each piece were surface sterilised by
conidia mL−1. Controls were treated with sterile nanopure dipping in 2 % sodium hypochlorite for 2 min, rinsed
water. Inoculated fruit were placed randomly on a sterile, briefly with sterile water and then placed on PDA plates
moist cloth in clear plastic containers (30 cm length × 20 cm so that each plate contained a segment from the top,
width × 10 cm height) and stored at 10 °C and a relative centre and bottom of fruit. The plates were then incu-
humidity (RH) of>90 %. The containers contained calcium bated at ambient temperature (22±2 °C) in natural light
carbonate and potassium permanganate to absorb excess and observed daily. Isolation of B. cinerea was con-
carbon dioxide and ethylene released from fruit, respective- firmed by microscopic examination (× 400) of conidia
ly (Ozdemir and Floros 2004). Each treatment had 20 fruit and conidiophores (Maas 1998).
and the experiment was conducted twice.
Statistical analysis
Assessment of grey mould development
Experiments were conducted twice and designed randomly
For the 0 DAA treatment, dead flowers were counted using the factors of cultivar, time of inoculation and conidial
and expressed as a percentage of total flowers inoculat- concentration. Data were subjected to analysis of variance
ed per treatment. A total of 45 flowers was examined (ANOVA) using GenStat. Since the repeated experiments
per treatment. were statistically similar they were combined in a second
Fruit derived from inoculated flowers (a total of 20 ANOVA. Means of the treatments were compared using the
across two experiments for each treatment) were Least Significance Differences (LSDs) at P<0.05. Means
harvested at the R stage for assessment of grey mould and standard errors were determined using Microsoft Excel.
development during 28 days of postharvest storage at Time series ANOVA was performed for each time of inoc-
10 °C. Fruit were surface-sterilised by dipping in 2 % ulation or ripening stage with different conidial concentra-
sodium hypochlorite for 1 min and rinsed briefly with tions. Multifactorial analyses also accounted for treatment
reverse osmosis (RO) water. Surface-sterilised fruit were interactions.
452 T.D. Le et al.

Results inoculation and/or conidial concentration. The interactions

of cultivar x time of inoculation (P=0.241), cultivar x co-
Effect of inoculum concentration of B. cinerea nidial concentration (P=0.244), and cultivar x time of inoc-
on the incidence of dead flowers ulation x conidial concentration (P = 0.885) had no
significant effect during storage.
As the concentration of conidia applied to flowers (at When flowers of cv. Papri Queen were inoculated with
anthesis) increased, the numbers of flowers that died 105 and 106 conidia mL¯1 at anthesis, time to 50 % maxi-
also significantly increased, regardless of cultivar (Table 1). mum lesion area on fruit was significantly less (P=0.021)
However, significantly more flowers of cv. Aries than of than for those derived from flowers inoculated with 104
cv. Papri Queen died at each inoculum concentration conidia mL−1 (Fig. 3a). However, there was no significant
(P=0.001). difference in time to 50 % maximum lesion area among
conidial concentrations when flowers were inoculated at 3
Rot development in fruit derived from inoculated flowers or 6 DAA. When flowers of cv. Papri Queen were inoculat-
and young fruit ed with 105 or 106 conidia mL−1 at anthesis or 3 DAA, time
to 50 % maximum lesion area on fruit was significantly
Regardless of when flowers or young fruit were inoculated shorter (P=0.021) than that for flowers inoculated at 6
with B. cinerea (0, 3 or 6 DAA), symptoms of grey mould DAA. For cv. Aries, there was no significant difference (P
were first observed on fruit developing from flowers inocu- =0.17) in time to 50 % lesion area among flowering stages.
lated with B. cinerea at 12 days after harvest (DAH) and When flowers were inoculated with 105 conidia mL−1 at
then increased steadily during storage of both cultivars anthesis or 6 DAA, time to 50 % maximum lesion area on
(Figs. 1 and 2). Generally, for both cultivars, by 28 DAH fruit was significantly longer (P=0.013) than for fruit from
the highest conidial concentration (106 conidia mL−1) flowers inoculated with 106 conidia mL−1 (Fig. 3b).
appeared to cause more decay and more fruit exhibited rot
than those inoculated with the two lower concentrations. Presence of B. cinerea at harvest in fruit derived
However, regardless of stage, lesion area on fruit and the from inoculated flowers
percentage of fruit exhibiting rot where flowers or young
fruit had been inoculated with 106 conidia mL−1 were not Botrytis cinerea was isolated infrequently at harvest from
significantly different (P=0.052), at any time during stor- fruit derived from inoculated flowers. The number of seg-
age, from those where flowers had been inoculated with the ments with hyphal growth was not significantly different
two lower concentrations. Lesion area and the percentage of (P=0.12) among conidial concentrations or flowering stages
fruit exhibiting rot where flowers had been inoculated with for both cultivars. Nevertheless, the pathogen was isolated
the same conidial concentration were not significantly dif- more often from the stem-end of the fruit regardless of
ferent (P= 0.09) over time of storage among different conidial concentration. Generally, more of these segments
flowering stages for both cultivars. showed hyphal growth when inoculated with 106 conidia
Data analysis to examine treatment interaction showed mL−1 (Table 2). Botrytis cinerea appeared to be isolated
that only cultivar had a significant effect (P<0.001) on more frequently from segments cut from fruit of cv. Aries
lesion size from 14 DAH to the end of storage. The two than from cv. Papri Queen. However, when flowers were
cultivars responded in the same way with change of time of inoculated with the same conidial concentration at the same
flowering stage, the number of segments with hyphal
Table 1 Effect of concentration of conidia of Botrytis cinerea on the growth was not significantly different (P=0.095) between
incidence of dead flowers (%) in two cultivars of capsicum. Flowers cv. Aries and cv. Papri Queen.
were inoculated at anthesis. Means for n=90 across two experiments
are shown Grey mould development in fruit inoculated postharvest
−1
Conidial concentration (mL ) Dead flowers (%)
Symptoms of grey mould on inoculated fruit of both
cv. Aries cv. Papri Queen cultivars were observed after 4 days post-inoculation
(DPI) and increased steadily for the next 8 days
104 45.3a 10.6a
(Figs. 4 and 5). All inoculated fruit had developed grey
105 66.8b 15.7a
mould by 8 DPI and B. cinerea was isolated from all
106 80.2b 35.8b
lesions. Lesions were significantly larger (P<0.001) on
LSD (P value) 20.1 (P=0.037) 7.6 (P=0.045)
fruit of cv. Aries than those of cv. Papri Queen when
Means with the same letters in each column were not significantly they were inoculated with the same conidial concentra-
different as determined using the LSD (P<0.05) tion at the same ripening stage.
Infection of capsicum fruit by Botrytis cinerea 453

10 10 10
2500 100

A 90 D
2000 80

70

1500 60

50

1000 40

30

500 20

10

0 0
0 10 12 14 16 18 20 22 24 26 28 10 10 10

2500 100

Percentage of fruit exhibiting rot

B 90 E
Lesion area (mm /fruit)

2000 80

70
2

1500 60

50

1000 40

30

500 20

10

0 0
10 12 14 16 18 20 22 24 26 28 10 10 10

2500 100

C 90 F
2000 80

70

1500 60

50

1000 40

30

500 20

10

0 0
10 12 14 16 18 20 22 24 26 28 10 10 10
-1
Time after harvest (days) Conidial concentration (conidia mL )

Fig. 1 Grey mould development and the percentage of fruit of capsi- (b and e) and 6 DAA (c and f). Data are means±SE from n=20 across
cum cv. Papri Queen exhibiting rot where the flowers had been inoc- two experiments. LSDs (P<0.05) for comparisons among individual
ulated with 104, 105 or 106 conidia mL−1 at anthesis (a and d), 3 DAA conidial concentrations at each time point after harvest are shown

Multifactorial analysis showed that, regardless of conidial DG (Figs. 4 and 5). Similarly, cultivars responded differently
concentration, fruit of different ripening stages from different to conidial concentration from day 6 to day 8 post-inoculation.
cultivars responded differently only from day 6 to day 8 post- In cv. Papri Queen (Fig. 4), lesion area on fruit inoculated with
inoculation. In cv. Papri Queen, lesions on BR fruit were 106 conidia mL−1 was significantly larger (P=0.007) than on
significantly larger (P<0.001) than those on fruit inoculated fruit inoculated with 105 and 104 conidia mL−1. However,
at DG or R, and lesions on R fruit of cv. Aries were signifi- lesion area on fruit inoculated with 105 conidia mL−1 was
cantly larger (P<0.001) than those on fruit inoculated at BR or not significantly different (P=0.007) from lesion area on fruit
454 T.D. Le et al.

10 10 10
7000 100
A D
90
6000
80
5000 70

60
4000
50
3000
40

2000 30

20
1000
10

0 0
10 12 14 16 18 20 22 24 26 28 10 10 10

7000 100
B E

Percentage of fruit exhibiting rot

90
6000
Lesion area (mm /fruit)

80
5000 70
2

60
4000
50
3000
40

2000 30

20
1000
10

0 0
10 12 14 16 18 20 22 24 26 28 10 10 10

7000 C 100 F
90
6000
80
5000 70

60
4000
50
3000
40

2000 30

20
1000
10

0 0
10 10 10
10 12 14 16 18 20 22 24 26 28
-1
Time after harvest (days) Conidial concentration (conidia mL )
Fig. 2 Grey mould development and the percentage of fruit of capsi- e) and 6 DAA (c and f). Data are means±SE from n=20 across two
cum cv. Aries exhibiting rot where the flowers had been inoculated experiments. LSDs (P<0.05) for comparisons among individual conid-
with 104, 105 or 106 conidia mL−1 at anthesis (a and d), 3 DAA (b and ial concentrations at each time point after harvest are shown

inoculated with 104 conidia mL−1. In cv. Aries (Fig. 5), lesion 007) than that on fruit inoculated with 104 conidia mL−1.
area on fruit inoculated with 106 conidia mL−1 was signifi- Interactions between ripening stage x conidial concentration
cantly larger (P=0.007) than on fruit inoculated with the two (P=0.052) or cultivar x ripening stage x conidial concentra-
lower conidial concentrations and lesion area on fruit inocu- tion (P=0.69) were not significantly different at any time
lated with 105 conidia mL−1 was significantly greater (P=0. during storage post-inoculation.
Infection of capsicum fruit by Botrytis cinerea 455

10 10 10 humidity and the presence of nutrients in flowers (Bristow et

30 A al. 1986; Williamson et al. 2007), leading to death of grape
(McClellan and Hewitt 1973) and tomato flowers (Eden et
25 al. 1996). The observation that more flowers of cv. Aries
than of cv. Papri Queen died following inoculation with
20 conidia may reflect a more plentiful supply of nectar and
pollen in the larger flowers of cv. Aries. Indeed, when
Time (days) to 50 % maximum lesion size

15 conidial suspension was amended with 0.1 M glucose, the

percentage of conidia that germinated in vitro was much
10 higher than that without glucose (data not shown). The
lower death rate of cv. Papri Queen may also be linked to
5
the presence of antifungal compounds, as reported for resis-
tant flower tissues of strawberry (Terry et al. 2004).
When infection of capsicum fruit by B. cinerea occurred
0
0 DAA 3 DAA 6 DAA early in their development, grey mould appeared to remain
quiescent until after harvest of the fruit. Grey mould devel-
30
oped quickly in ripening fruit during incubation in a
B favourable environment. Inoculation with B. cinerea at an-
25
thesis was expected to result in more grey mould develop-
ment on capsicum fruit than inoculation at 3 and 6 DAA as
20 observed for raspberry (Dashwood and Fox 1988) and grape
(Keller et al. 2003). However, the present research showed
15 that inoculum concentration had a greater influence than
developmental stage of fruit at which inoculation occurred
10 on the severity of grey mould. Lesions were generally
largest and developed most quickly on fruit derived from
5 flowers inoculated with the highest conidial concentration
(10 6 conidia mL −1 ) than with lower concentrations.
0 Development of grey mould in red raspberry also did not
0 DAA 3 DAA 6 DAA differ among fruit that had been inoculated with B.cinerea at
Fig. 3 Time after harvest for lesions to reach 50 % of maximum size
five different growth stages from flowering to red fruit
on fruit of cv. Papri Queen (a) and cv. Aries (b) derived from flowers (Williamson et al. 1987). Similarly, in plum and nectarine,
inoculated with three conidial concentrations (104, 105 or 106 conidia there was no relationship between infection of flowers by B.
mL−1). Data are means±SE from n=20 across two experiments. LSDs cinerea and postharvest decay of fruit although fungal in-
(P<0.05) for comparisons among individual conidial concentrations
and flowering stage of each cultivar are shown
fection was observed in sepals and floral tubes. However,
sepals and floral tubes did not attach to the young fruit
(Fourie and Holz 1994). In contrast, we observed that petals
Discussion remained attached to capsicum fruit for at least 7 days after
flowering. Petals might therefore have served as a source of
Fruit of capsicum cv. Aries were much more susceptible to nutrients for B. cinerea during the preharvest period, regard-
infection by B. cinerea than those of cv. Papri Queen regard- less of the stage at which inoculation occurred. Moreover,
less of whether inoculation occurred before or after harvest. withered attached styles on young fruit were more important
Botrytis cinerea can infect capsicum fruit during flowering for penetration and growth of B. cinerea on raspberry than
and the early development stages, causing grey mould devel- were the living floral parts (Williamson et al. 1987). In the
opment in fruit during storage. Degree of rot increased with present research, conidia placed on the stem-end of young
increasing inoculum concentration. Development of grey fruit at 3 DAA and 6 DAA may have become lodged in the
mould on fruit inoculated with B. cinerea after harvest was groove, which is assumed to retain more moisture than other
affected by the inoculum concentration and the ripening stage parts of fruit. In addition, shed pollen was observed on the
of fruit. tiny fruit at 3 DAA and 6 DAA which, along with withered
Capsicum flowers (10.6 – 80.2 %) died following inoc- floral parts including the petals, would provide nutrients for
ulation with conidia at all concentrations tested, but partic- subsequent growth of B. cinerea. In support of this sugges-
ularly at 106 conidia mL−1. Germination of conidia and the tion, B. cinerea was isolated most frequently from the stem-
subsequent penetration of host cells is promoted by high end of fruit. Further research to examine the role of withered
456 T.D. Le et al.

Table 2 Number of segments

from which Botrytis cinerea was Position on fruit cv. Papri Queen cv. Aries
isolated on PDA from tissues
from three positions on fruit Anthesis 3 DAA 6 DAA Anthesis 3 DAA 6 DAA
derived from inoculated flowers
of capsicum cv. Aries and cv. 104 conidia mL¯1
Papri Queen (n=6) Top 0 1 0 0 0 1
Centre 0 0 0 0 0 0
Bottom 0 0 0 1 1 0
105 conidia mL¯1
Top 1 0 0 2 0 1
Centre 0 0 0 0 1 0
Bottom 0 0 0 0 0 1
106 conidia mL¯1
Top 1 2 2 2 2 2
Centre 0 0 0 0 2 0
Bottom 0 0 0 0 0 1
Control
Top 0 0 0 0 0 1
Centre 0 0 0 0 1 0
Bottom 0 0 0 0 0 0

floral parts and shed pollen on young fruit in latent infection Lavy-Meir 1989). Although ethylene production by cap-
of capsicum is therefore required. sicum during storage is low due to its non-climacteric
When fruit were inoculated after harvest, grey mould nature (Pham 2007), ethylene production by mature red
development was affected by the ripening stage, in that fruit of cv. Aries was higher than mature green fruit
grey mould developed most rapidly on BR fruit of cv. (Villavicencio et al. 1999) and by mature BR fruit of
Papri Queen and on R fruit for cv. Aries. That obser- cv. Papri Queen was higher than mature DG and red
vation may be related to differences in biochemical fruit (Pham 2007) suggesting that grey mould growth
compounds due to the physiological changes that occur should be greater on mature red than mature green fruit
during ripening. One such biochemical change may be a of cv. Aries and BR fruit of cv. Papri Queen. Similarly,
greater phenolic content in capsicum fruit at the green reduction of disease incidence caused by B. cinerea
than the red stage (Navarro et al. 2006; Conforti et al. associated with low ethylene production was found in
2007). Tao et al. (2010) reported that six polyphenolic strawberry (Babalar et al. 2007) and kiwi fruit (Niklis et
compounds, which act as antioxidants, inhibited conidial al. 1995) both of which are also non-climacteric.
germination and germ tube elongation of B. cinerea in The response to inoculation of detached, surface-
vitro. Increasing antioxidant content also decreased the sterilised R fruit of the two cultivars with B. cinerea
susceptibility of grape (Pezet et al. 2003) and apple fruit was similar to preharvest inoculation, in that lesions on
(Davey et al. 2007) to infection by B. cinerea. fruit of cv. Aries were significantly larger than those on
Capsaicin, a phytoalexin known to be antifungal cv. Papri Queen for all inoculum concentrations.
(Lopez-Malo et al. 1998), has also been shown to be Biochemical factors in different cultivars might contrib-
more abundant in green than in red fruit of the cv. ute to differences in susceptibility to grey mould as
Paprika (Eissa et al. 2007; Kim et al. 2011). Similarly, mentioned previously. Antioxidant content (primarily
the decrease in resveratrol (a phytoalexin) observed from phenolics) of fruit of cayenne pepper (cv. Yellow
during grape ripening was linked to susceptibility to Bell) was significantly greater than that of bell pepper
infection by B. cinerea (Adrian et al. 1997). Thus, (cv. Mesilla) (Howard et al. 2000). Similarly, higher
larger quantities of polyphenolic compounds in green concentrations of phenolic compounds were found in a
capsicum fruit may restrict hyphal growth of B. cinerea resistant grape cultivar than in a susceptible cultivar
in green fruit compared with red fruit. Additionally, (Goetz et al. 1999; Pezet et al. 2003). Pham (2007)
pathogenesis-related (PR) proteins have been detected showed that cv. Papri Queen has much more intense
in green fruit but not in red fruit (Aizat et al., spice colour than cv. Aries, suggesting it contains more
unpublished data). Furthermore, internal ethylene may capsaicinoids (Deli et al. 2001). As such, a greater
induce grey mould development (Barkai-Golan and phenolic content of fruit from cv. Papri Queen may play
Infection of capsicum fruit by Botrytis cinerea 457

10 10 10 10 10 10
2500
4000
A
A 3500

2000 3000

2500
1500
2000

1000 1500

1000
500
500

0
0
3 4 5 6 7 8
3 4 5 6 7 8

4000
2500 B B
3500

Lesion area (mm /fruit)

2000
Lesion area (mm2/fruit)

3000

2
2500
1500
2000

1000 1500

1000
500
500

0 0
3 4 5 6 7 8 3 4 5 6 7 8

2500 4000
C C
3500
2000
3000

1500 2500

2000
1000
1500

1000
500

500

0
0
3 4 5 6 7 8
3 4 5 6 7 8
Time post inoculation (days)
Time post inoculation (days)
Fig. 4 Grey mould development on fruit of cv. Papri Queen inoculated Fig. 5 Grey mould development on fruit of cv. Aries inoculated with
with 104, 105 or 106 conidia mL−1 at Deep Green (a), Breaker Red (b) 104, 105 and 106 conidia mL−1 at Deep Green (a), Breaker Red (b) and
and Red (c). Data are means±SE from n=20 across two experiments. Red (c). Data are means±SE from n=20 across two experiments. LSDs
LSDs (P<0.05) for comparisons among individual conidial concentra- (P<0.05) for comparisons among individual conidia concentrations at
tions at each time point post inoculation are shown each time point post inoculation are shown

a role in restricting development of B. cinerea. glucanases) may have an effect on grey mould develop-
Furthermore, PR proteins (such as chitanase and ment in fruit of different cultivars. Wurms (2005)
458 T.D. Le et al.

reported that higher chitinase activity in kiwi fruit cv. Barkai-Golan R, Lavy-Meir G (1989) Effects of ethylene on the
susceptibility to Botrytis cinerea infection of different tomato
Hayward resulted in reduction of grey mould incidence
genotypes. Ann Appl Biol 114:391–396
compared with cv. Hort16A. High levels of chitinase Bristow P, McNicol R, Williamson B (1986) Infection of strawberry
were also related to low disease incidence in the resis- flowers by Botrytis cinerea and its relevance to grey mould
tant cultivar of tomato cv. Geronimo (Cota et al. 2007). development. Ann Appl Biol 109:545–554
Cantu D, Blanco-Ulate B, Yang L, Labavitch JM, Bennett AB, Powell
As such, more research is required to examine the
ALT (2009) Ripening-regulated susceptibility of tomato fruit to
relationship between antioxidants or biochemical sub- Botrytis cinerea requires NOR but not RIN or ethylene. Plant
stances and grey mould in a range of cultivars of Physiol 150:1434–1449
capsicum. Conforti F, Statti GA, Menichini F (2007) Chemical and biological
variability of hot pepper fruits (Capsicum annuum var.
Grey mould developed faster on wounded fruit inoculat-
acuminatum L.) in relation to maturity stage. Food Chem
ed with B. cinerea after harvest than on unwounded fruit 102:1096–1104
when B. cinerea was present but latent before harvest. Cota I, Troncoso-Rojas R, Sotelo-Mundo R, Sánchez-Estrada A,
Wounds made during harvest were also found to be the main Tiznado-Hernández M (2007) Chitinase and β-1, 3-glucanase
enzymatic activities in response to infection by Alternaria
infection pathway of B. cinerea in kiwi fruit (Michailides
alternata evaluated in two stages of development in different
and Elmer 2000). Conidia of B. cinerea germinated in juice tomato fruit varieties. Sci Hortic 112:42–50
around wounds on red raspberry and lesions were visible Dashwood E, Fox R (1988) Infection of flowers and fruits of red
3 days later (McNicol et al. 1990). The moisture and release raspberry by Botrytis cinerea. Plant Pathol 37:423–430
Davey MW, Auwerkerken A, Keulemans J (2007) Relationship of
of nutrients, such as sugars and organic acids, probably
apple vitamin C and antioxidant contents to harvest date and
promoted germination and penetration of wounded tissue postharvest pathogen infection. J Sci Food Agric 87:802–813
by B. cinerea (Williamson et al. 2007). Wounding probably Deli J, Molnár P, Matus Z, Tóth G (2001) Carotenoid composition in
also caused cell death allowing B. cinerea, a necrotroph the fruits of red paprika (Capsicum annuum var. lycopersiciforme
(Staats et al. 2005), to colonise tissue rapidly. rubrum) during ripening: biosynthesis of carotenoids in red pa-
prika. J Agric Food Chem 49:1517–1523
The activity of B. cinerea on capsicum flowers and young Derckel JP, Audran JC, Haye B, Lambert B, Legendre L (1998)
fruit in this study suggests that minimising exposure to inoc- Characterization, induction by wounding and salicylic acid, and
ulum can reduce loss of fruit preharvest. Furthermore, activity against Botrytis cinerea of chitinases and β-1,3-
avoiding sources of inoculum during harvest and careful glucanases of ripening grape berries. Physiol Plant 104:56–64
Droby A, Lichter A (2004) Post-harvest Botrytis infection: etiology,
postharvest handling may help to prevent loss during subse- development and management. In: Elad Y, Williamson B,
quent storage and marketing of fruit. Tudzynski P, Delen N (eds) Botrytis: biology, pathology and
control. Kluwer Academic Press, Dordrecht, pp 349–367
Acknowledgments The authors thank Monsanto Seeds and Fair- Eden M, Hill R, Beresford R, Stewart A (1996) The influence of
banks Seeds for supplying capsicum seeds. The first author is inoculum concentration, relative humidity, and temperature on
grateful to the Agricultural Science and Technology Project of infection of greenhouse tomatoes by Botrytis cinerea. Plant
Vietnam for awarding a PhD scholarship. We thank Sue Pederick Pathol 45:795–806
(South Australian Research and Development Institute) for sharing Eissa HA, Mostafa В, Hussein A (2007) Capsaicin content and quality
her techniques for long-term storage of B. cinerea and Dr Olena characteristics in different local pepper varieties (Capsicum
Kravchuk (the University of Adelaide) for her advice about mul- annum) and acid-brine pasteurized puree. J Food Technol
tifactorial analysis. 5:246–255
Elad Y, Volpin H (1993) Reduced development of grey mould (Botrytis
cinerea) in bean and tomato plants by calcium nutrition. J
Phytopathol 139:146–156
Fallik E, Grinberg S, Alkalai S, Lurie S (1996) The effectiveness of
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