Developmental biology and infection cycle of Sclerotinia sclerotiorum causing stem rot of carnation in India

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Vol. 9(49), pp. 2328-2336, 14 December, 2015
DOI: 10.5897/AJMR2015.7690
Article Number: 8B784A656539
ISSN 1996-0808
Copyright © 2015
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJMR
African Journal of Microbiology
Research
Full Length Research Paper
Developmental biology and infection cycle of
Sclerotinia sclerotiorum
causing stem rot of carnation in India
VINOD KUMAR Selvaraj, RAJESHKUMAR Ponnusamy,
NAKKEERAN Sevugaperumal* and ERAIVAN Arutkani Aiyanathan K.
Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University,
Coimbatore – 641 003, India.
Received 2 August, 2015; Accepted 15 November, 2015
Carnation (Dianthus caryophyllus L.) is a cut flower with greater stipulation in the world cut flower
market. In India, carnations are cultivated under polyhouses in Nilgiris and Kodaikanal districts in the
state of Tamil Nadu. Carnations cultivation is impeded by various diseases, among them stem rot
caused by Sclerotinia sclerotiorum (Lib.) de Bary was found to be predominant in all varieties. Survey
among commercially cultivated varieties of carnation during 2013, revealed the occurrence of stem rot
incited by Sclerotinia sclerotiorum, for the first time in India. The pathogen was identified as Sclerotinia
sclerotiorum on the basis of phenotypic and genotypic characteristics. Carpogenic germination was
induced artificially, and life cycle of the fungi was studied. Microscopic studies of the apothecium
revealed the presence of spermatia, croziers, paraphyses as well as monomorphic ascospores specific
to S. sclerotiorum.
Key words: Apothecium, carnation, croziers, India, Sclerotinia, spermatia.
INTRODUCTION
Floriculture is a persuasive field in trade with high
potential returns per unit area. Because of this large
number of farmers are attracted towards cut flower
cultivation. In India, conditions prevailing in hilly regions
are highly favorable for the cultivation of various cut
flowers. Area under cut flower production is increasing
constantly (Indian Horticulture Database, 2015).
Carnations are one of the most preferred cut flower
varieties next to rose owing to their shelf life and greater
degree of available colours. Karnataka is the leading
state in India accounting for carnation production. Total
production of carnation all over India is 800 MT (Indian
Horticulture Database, 2015). Even though carnation
cultivation is increasing, it’s development is hampered by
various diseases. Among them, stem rot incited by S.
sclerotiorum was found to be highly destructive.
Sclerotinia has been previously reported as a
polyphagous fungi infecting more than 148 plant species
*Corresponding author. E-mail: nakkeeranayya@gmail.com. Tel: +91 7598489226.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License

(Saharan and Mehta, 2008). Sclerotinia is a facultative
parasitic fungus belonging to Ascomycota (Kirk et al.,
2008). It is a robust pathogen having greater rate of
survival even under adverse environmental conditions
and survives up to eight years in soil as sclerotial bodies
(Adams and Ayers, 1979). Yield losses have been
reported to be hundred per cent in susceptible crops
(Purdy, 1979). Three well documented species of
Sclerotinia (S. sclerotiorum, S. minor and S. trifoliorum)
are considered to be much important and likely to have
wide host range and reported with huge economic loss
(Saharan and Mehta, 2008). In India, stem rot of
carnation was identified for the first time in 2013. (Vinod
Kumar et al., 2015). The results of our study on survey,
morphological, molecular characterization and
apothecium induction in S. sclerotiorum are presented in
this communication.
MATERIALS AND METHODS
Survey
Commercially cultivated carnation varieties like Charmant pink,
Pudding yellow, Castor purple, Baltico white, White liberty, Golem
purple, Yellow liberty, Bizet, Gaudina red and Farida were surveyed
for the occurrence of stem rot in kothagiri, kodumudi, kunnur
regions of Nilgiris district in Tamil Nadu, India. The disease
incidence was calculated by using the formula proposed by
Wheeler (1969).
Isolation
The pathogen was isolated from both infected stem tissue and
sclerotial bodies. Infected stem tissues and sclerotial bodies were
surface sterlized with 0.1% mercuric chloride (HgCl2) for 60 s and
then the chemical traces were removed by rinsing thrice in sterile
distilled water. Surface sterlized tissues and sclerotia were plated
on sterile Petri plates containing potato dextrose agar (PDA)
medium, amended with 100 µg/ml of streptomycin sulphate and
incubated at 20 ± 2°C for 7 days. After emergence of fungal growth,
the pathogen was pure cultured by single hyphal tip technique
(Tutte, 1969).
Pathogenicity
Pathogenicity experiments were conducted on 30 days old potted
cuttings of carnation variety, Charmant pink as per method
described by Kim and Cho (2003). The cuttings were grown in pots
with sterilized pot mixture. Mycelial discs and sclerotial bodies of
the pathogen were inoculated to cuttings separately. Mycelial discs
(9 mm) were placed in stem portion, 2 cm above ground and
covered with moist cotton. Sclerotial bodies (3-5) covered in muslin
cloth were buried in soil in proximity with the collar region of the
healthy cuttings. Both the tests were replicated thrice with three
cuttings per replication. After inoculation the pots were covered with
polybags and incubated, ambient inside the polyhouse maintained
at Elkhil Agrotech Pvt Ltd at Udhagamandalam. Healthy control was
also maintained subsequently. After expression of symptoms the
pathogen was re- isolated to confirm pathogenicity.
Vinod Kumar et al. 2329
Identification
The pathogen was subjected to both morphological and molecular
characterization. The study fungus was compared with well
documented species of Sclerotinia viz., S. sclerotiorum, S. minor
and S. trifoliorum. Morphological discrimination was based on
ascospore morphology and size of sclerotia. Molecular identification
was carried out by sequencing the 18S-28S rRNA gene.
Morphological characterization
Species differentiation was possibly made between them by
studying the colony character, sclerotia and ascospore morphology
(Ekins et al., 2005). Apothecium was induced as per the procedure
proposed by Cobb and Dillard (2004).
Induction of apothecium
Before induction of apothecium the sclerotial bodies were subjected
for a conditioning process. Medium to large sclerotial bodies (7.4 to
15.5 mm) were selected for better per cent rate of carpogenic
germination. Sclerotial bodies were tied in cheese cloth bags and
immersed in a container filled with tap water and incubated at 60 C.
Fresh air was continuously circulated by using an aquarium pump.
In this conditioning process, water was replenished once in a week
till stipe initiation.
After conditioning process, sclerotia with stipe initials were
transferred to Petri plates containing clean, sterilized, dry sand.
Sand was moistened and plates were overlaid with lids in order to
prevent desiccation. Moisture was maintained with sterile distilled
water. Plates were incubated at 20°C in alternate dark and light
periods with Bright Boost cool white fluorescent light-18W/840
(Phillips, China make), till apothecia were produced. The
conditioning treatment was replicated thrice and a control was also
maintained. Apothecium was subjected for ultramicrotome, and
observed under microscope to study the ascus and ascospore
morphology.
Microtome sectioning
In order to study the ultra-structures, apotheium was subjected to
ultra microtome studies as demonstrated by Johanson (1940). The
specimen was soaked for a minimum of 12 h in Formalin: Alcohol:
Acetic acid: Water in the ratio of 10:50:5:35. Later the specimen
was washed in a series of 60, 70, 80, 90 and 100 per cent Teritary
Butly Alcohol. Then the specimens were embedded in molten wax
at 52-54°C. Then after drying 12 micron thick sections were made
with Spencers rotary microtome. After sectioning the specimens
were subjected to de waxing with xylene: ethanol (1:1) for 30 min.
After de waxing, the specimens were stained with safranin. Excess
stains were washed with series of 50, 70 and 90 per cent ethanol.
Later the specimens were dried and then mounted on glass slides
with DPX mount.
Molecular characterization
The fungus was cultured on potato dextrose broth at 20 ± 20C for 4-
5 days. Then the mycelium was collected, dried and powdered by
freezing in liquid nitrogen. The genomic DNA was extracted by
Cetyl Trimethyl Ammonium Bromide (CTAB) method as described
by Chakraborty et al. (2010).
PCR amplification of 18S-28S rRNA gene
The genomic DNA was used as a template and subjected for the
Percent Disease Incidence = No .of infected plants
Total No .of plants x 100

2330 Afr. J. Microbiol. Res.
Table 1. Prevalence of stem rot in different varieties of carnation in Nilgiris district of Tamil
Nadu.
Variety
Colour
Percentage of stem rot incidence
Kothagiri
Kodumudi
Kunnur
Yellow liberty
Yellow
12.60
3.60
4.3
Gaudina red
Red
8.60
2.90
3.70
Farida
Pink
11.5
3.80
3.50
Bizet
Light pink
14.70
4.40
4.70
Castor purple
Purple
24.60
6.60
6.80
Golem purple
Purple
18.30
5.50
5.60
White liberty
White
13.50
4.50
5.00
Charmant pink
Light Pink
38.50
9.60
10.4
Pudding yellow
Yellow
22.20
7.60
7.20
Baltico white
White
28.40
8.60
8.30
Mean incidence
18.29
5.81
5.95
PCR amplification of 18S-28S rRNA gene using the primer pairs,
ITS 1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS 4 (5’-
TCCTCCGCTTATTGATATGC-3’) (White et al., 1990). The PCR
reaction was performed with ready to use Taq DNA Polymerase 2x
master mix supplied from Ampliqon, comprising Tris-HCl pH 8.5,
(NH4)2So4, 3mM MgCl2, 0.4mM dNTPs,
0.2units/μl Ampliqon Taq DNA polymerase and inert red dye. The
programming cycle was accomplished as follows: initial
denaturation at 940C for 2 min, 30 cycles of denaturation at 940C for
30 s, annealing at 570C for 30 s, extension at 720C for 1 min and
final extension at 720C for 7 min (Saitoh et al., 2006).
Life cycle assessment
Different stages of the pathogen associated with its life cycle were
studied by inoculating mycelium and sclerotia of the pathogen on
seedlings. Subsequently, the developmental biology of apothecium
and their components were studied.
RESULTS AND DISCUSSION
Survey
This is the first record of sclerotinia rot of carnations in India.
Survey results revealed that the variety Charmant pink was
highly susceptible to stem rot. In survey the stem rot
incidence was relatively greater in Kothagiri compared to
that of Kunnur and Kodumudi. Among the varieties
surveyed, pink coloured variety Charmant pink was highly
susceptible with 38.50 per cent disease incidence
followed by Baltico white (28.40%) and Castor purple
(24.60%). Least incidence was observed in the variety
Gaudina red (8.60%). However the same was
severely susceptible to fairy ring spot and blossom
blight. Mean stem rot incidence in Kothagiri was
18.29 per cent whereas in kunnur and kodumudi stem
rot incidence was comparatively less viz., 5.81 and
5.94, respectively (Table 1). Consistent relative
humidity and relatively lower temperature, prevailing
in Kothagiri region have resulted in greater loss
compared to other regions.
Symptomatology
Symptoms were found to be associated from seedling to
maturity stage. However, the plants are highly
susceptible at seedling stage. Initial symptoms include
paleness of the plant accompanied with drooping leaves
(Figure 1a). However, the most typical symptom is the
presence of cottony white mycelial growth on root zone
(Figure 1b), as well as collar region of the plant.
Subsequently, the plant dries to straw yellow and finally
dies. Besides, longitudinal splitting of the infected stems
revealed plenty of dark, black, sclerotial bodies (12-20) of
varying shapes and sizes (Figure 1c). Saharan and
Mehta (2008) described that symptom includes, cottony
white mycelium on the root zone and collar region
accompanied with dark, black, irregular sclerotia inside
the stem, followed by, complete drying of the plant to
straw yellow. Similar descriptions were made by Purdy
(1979).
Isolation
On PDA, pure cultured study fungus produced fluffy white
mycelial growth. The hyphae was hyaline, septate and
metamorphosed into irregular, black sclerotial bodies of
various size following mycelia aggregation as per the
previous reports (Colotelo, 1974; Purdy, 1979). In this
study, the pathogen produced dense cottony white
mycelium on PDA and fungal mass covered the entire
Petriplate within 5 days. After 8 days large, dark, black,
irregular sclerotial bodies appeared in a circular fashion
along the corners of the Petri plate (Figure 2a). The
hyphae were hyaline and septate. Sclerotia were
produced, superseding the accumulation of nutrients in

Vinod Kumar et al. 2331
Figure 1. (a) Pale drooping infected plant, (b) Root zone colonized by mycelium of S. sclerotiorum,
(c) Sclerotial bodies inside the infected stem.
Figure 2 . (a) Colony morphology of S. sclerotiorum, (b) Dew drops on
developing sclerotia.
the form of dew drops (Figure 2b). The morphology and
development of our isolate resembled S. sclerotiorum as
described by Kohn (1979) and Wang et al. (2008).
Sclerotial size and number were greater in Petri plates
exposed to light, compared to that incubated under
complete darkness.
Pathogenicity
Inoculation of mustard plants with 6 mm mycelial disc of
the pathogen S. sclerotiorum expressed the symptom of
stem rot disease after 10 days when incubated at 100%
relative humidity at 22oC (Kim and Cho, 2003).
Pathogenicity in Brassica sp, Canola, peanut and Bell
pepper has been established successfully (Young et al.,
2012; Khangura and Macleod, 2013; Faske et al., 2014;
Gonzalez et al., 1998). Similarly, in our study
pathogenicity was proved by inoculating mycelial discs
and sclerotial bodies. The typical symptoms were
expressed by the plants after 25 days of inoculation. The
plant turned pale and showed drooping symptom (Figure
Figure 3. Pathogenicity with typical drooping and sclerotial bodies
in stem.
3a) and, typical rot was observed in the stem portion
accompanied with small, black sclerotial bodies (Figure
3b). Hundred per cent infection was observed in the
plants inoculated with mycelia disc and sclerotial bodies.
The pathogen was re-isolated several times and
morphological characters were akin to that of the
previously isolated pathogen. Thus Koch postulates were
established.
Developmental biology of S. sclerotiorum and
morphological characterization
Identity of the pathogen was confirmed by morphological
characterization mainly based on sclerotial size and
ascospore morphology. Morphology of study fungus was
compared with three well documented commonly
occurring species of Sclerotinia. Species differentiation
was confirmed among three plant pathogenic species
viz., S. sclerotiorum, S. minor and S. trifoliorum by
morphologically characterization as proposed by Ekins et
al. (2005). The most distinguishing parameter was the

2332 Afr. J. Microbiol. Res.
Figure 4. (a) Sclerotia with stipe initials, (b) Melanization and elongation of stipe initials, (c) Initiation of disc differentiation, (d)
Growing disc, (e) Completely matured apothecium, (f) Apothecial disc loaded with ascospores.
sclerotial size that differentiated S. minor (1-2 mm dia)
from other two species viz., S. trifoliorum and S.
sclerotiorum that produced larger sclerotial bodies (3-100
mm dia). S. minor with comparatively smaller sclerotia
enables separation from S. trifoliorum and S. sclerotiorum
(Jagger, 1920; Willets and Wong, 1971; Wong, 1979;
Tariq et al., 1985). Ascospore morphology enabled
further separation between the other two species viz., S.
sclerotiorum and S. trifoliorum. Monomorphic
ascospores were observed in S. sclerotiorum, whereas S.
trifoliorum is known to produce dimorphic ascospores
(Kohn, 1979; Uhm and Fujii, 1983; Ekins et al., 2005).
According to previous studies (Saharan and Mehta, 2008;
Pellegrini et al., 1989) S. sclerotiorum produced larger
sclerotia compared to other species and produced
monomorphic ascospores. In our study, the pathogen
produced, large, dark, black irregular, sclerotial bodies
ranging from 7x4 to 15x5 mm under in vitro. This
confirmed the identity between S. sclerotiorum and S.
trifoliorum eliminating the chance to be S. minor.
In order to study the ascospore morphology, sclerotia
were subjected for carpogenic germination. Sanogo and
Puppala (2007) reported that S. sclerotiorum produced
tan to beige coloured apothecial discs. In our study,
conditioned sclerotia produced stipe initials after 2
months. After fifteen days of incubation, the black
stipe initials got melanised, grew in length and cup
shaped; ochraceous apothecium were produced from the
stipe initials at the rate of 10-20 per sclerotia. The
apothecia formed were of varying sizes ranging from 1.0
to 1.5 cm. The developmental stages of apothecium viz.,
stipe initiation, melanisation and elongation of stipe
initials, disc differentiation and maturation were observed
and recorded (Figure 4). Disc differentiation of
apothecium was observed only under the exposure of
stipe initials to light. Under darkness, length of the stipe
increased without disc differentiation. This revealed the
importance of cool white fluorescent light in the
development of apothecium. All the three replications
subjected to conditioning process produced apothecium
@ 10-15 apothecium/sclerotia; however, control
remained the same.
The ascocarp was made up of four layers viz.,
hymenium, subhymenium, medullary excipulum and ectal
excipulum. A row of vertically arranged asci constituted
the fertile hymenial layer (Figure5a) which is the outer
most and open part of apothecial disc. The subhymenium
was composed of closely packed longitudinal cells
immediately below hymenium (Figure 5b). Balloon like
elongated parenchyma formed the medullary excipulum
(Figure 5c). The ectal excipulum was composed of
compactly arranged globose pseudoparenchymatous

Vinod Kumar et al. 2333
Figure 5a. Hymenial layer with ascus arranged in a row (450X magnification), b.
Subhymenium (1000X magnification), c. Medullary excipulum (1000X magnification), d. Ectal
excipulum (1000X magnification).
cells that fabricate the stroma (Figure 5d).
Ascus and ascospore
Ascospore morphology enables the separation between
S. sclerotiorum and S.trifoliorum.
S. sclerotiorum and S. trifoliorum are reported to produce
monomorphic and dimorphic ascospores, respectively
(Evans et al., 2008; Ekins et al., 2005). In the present
study, asci were cylindrical, sac like, elongated, with
truncated apex and measured 179.39 µm x 8.65 µm at
1000X magnification (Figure 6a). The lateral wall was thin
(1.89 µm) and, apex region was thick (5.28 µm) when
measured under 1000x magnification (Figure 6b). Each
ascus contained eight, ellipsoidal, hyaline, monomorphic
ascospores measuring 16.84 µm x 8.65 µm at 1000x
magnification. Monomorphic ascospores confirmed the
identity as S. sclerotiorum distinguishing from S.
trifoliorum. These results confirmed the identity of the
pathogen as S. sclerotiorum. Asci were intercepted with
supporting sterile hyphae called paraphyses (Figure 6c).
The paraphyses were hyaline, filliform with clavate apex.
Ascospores were monomorphic and uniform as per the
distinctive character of S. sclerotiorum (Figure 6a).
Ascospores measured 16-19 µ in length and 8-9 µm in
width. Ascospores that germinated at both ends were
also observed (Figure 6d). Developmental stages of
ascospores like empty ascus and immature ascus with
under developed ascospores were also observed and
recorded (Figures 6e and 6f).
Spermatia and croziers
Besides ascospores, spermatia or microconidia were also
observed. Small, hyaline, globose spermatia were
singly attached to phialides borne laterally on the
hyphae (Figure 6g). Even though spermatia were
observed, their exact role in reproduction remains unclear.
Small hook like projections with bulged apex resembling
croziers were observed in the apothecium (Figure 6h).
The croziers later developed into ascus through meiosis
and mitosis.

2334 Afr. J. Microbiol. Res.
Figure 6. (a) Ascus with ascospore (1000X magnification), (b) Ascus wall (1000X magnification), (c) Paraphyses with
bulged apex, (d)Germinating ascospore (1000X magnification), (e) Immature empty ascus, (f) Underdevloped
ascospores inside ascus, (g) Spermatia (1000X magnification), (h) Croziers (1000X magnification).
The phenotypic characterization of apothecium revealed
the components of apothecium identical to previous
reports viz., ascospore (Saharan and Mehta, 2008),
germinating ascospores (Saharan and Mehta, 2008),
paraphyses (Saharan and Mehta, 2008), spermatia
(Saharan and Mehta, 2008; Rollins, 2007) and crozier
formation (Pellegrini et al., 1989). Thus developmental
biology of the pathogen was comparatively studied.
Molecular characterization
The ITS region encompassing 18S rRNA, ITS1, 5.8S
rRNA, ITS 2 and 28S rRNA can be regarded as an
environmental barcodes for the identification of fungi
(Bellemain, 2010). The 18S-28S rRNA gene has been
reported to have approximately 600 bp (Wang et al.,
2008; Jeon et al., 2006). In this study, the PCR product
produced an amplicon length of ~600 bp (Figure 7) and
the same was sequenced by Sanger dideoxy sequencing
method at Excelris genomics, Ahmedabad. Sequencing
was carried out using ABI 3730xl Genetic Analyzer
(Applied Biosystems, USA). After sequencing the
sequence was analysed with BLAST in NCBI
(KP676452). The nucleotide sequence of 18S-28S rRNA
gene acquired from the study fungus had 99% match with
all the three species viz., S. sclerotiorum (KM272350), S.
Figure 7. PCR amplification of 18S - 28S rRNA gene of S.
sclerotiorum.
trifoliorum (JQ743329) and S. minor (KC836493). Under
this predicament, morphological characterization gave a
strong distinctive identification of the pathogen. This
confirmed the identity of the pathogen as S. sclerotiorum.

Vinod Kumar et al. 2335
Figure 8. Life cycle of S. sclerotiorum in carnation.
Life cycle assessment
The life cycle and different stages of the pathogen were
studied. The soil was rich in inoculum due to off season
cultivation of leguminous plants. The pathogen resides in
soil in the form of sclerotial bodies. The sclerotial bodies
are hard and resistant to adverse environmental
conditions. Sclerotia play a major role in the disease
cycle, since they resist adverse environmental condition
and provide required inoculum potential for disease
establishment (Willets and Wong, 1980). Conducive
environment favours stem rot in carnation, superseding
myceliogenic/carpogenic germination of sclerotia (Bolton
et al., 2006).
In the present study sclerotial bodies served as primary
source of inoculum. Carpogenic germination of sclerotia
and airborne dissemination of ascospores have been
previously well documented (Heffer Link and Johnson,
2007; Saharan and Mehta, 2008; Purdy, 1979). Under
carpogenic germination, apothecia were produced, inside
which fertilization occurs and leads to the production of
ascospores. The airborne ascospores gets drifted in wind
and fall over the healthy flowers. Germinated ascospores
gain access to colonize the root zone when the flowers
fall off to ground following senescence. Carpogenic
germination of sclerotia into apothecium was not
observed till now in the field. However, an assumption
was made and a lifecycle was designed based on
previous studies (Figure 8).
Conclusion
Survey on the distribution of the pathogen S. sclerotiorum
revealed that, stem rot disease is highly prevalent in
Kothagri, Tamil Nadu. Developmental biology of the
pathogen was studied in detail. Life cycle assessment
shows the mode of infection of the pathogen as mycelium

2336 Afr. J. Microbiol. Res.
colonizing root zone and advancing collar region. This
provides basic information for future studies to control the
disease by breaking developmental biology and infection
cycle of the fungus.
Conflict of Interests
The authors have not declared any conflict of interests.
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