A STUDY OF ROUGH BARK DISEASE ON CINNAMON

(Cinnamomum zeylanicum Blume); DISEASE SYMPTOMS,

DEVELOPMENT AND THE CAUSAL AGENT WITH SPECIAL
REFERENCE TO ITS MORPHOLOGY, HISTOPATHOLOGY AND
NUTRITIONAL STATUTES OF AFFECTED PLANTS

G. G. JAYASINGHE1*, K. L. WASANTHA KUMARA2,

M. W. G. C. WIJAYAWARDANA2, K. G. G. Wijesinghe1, D. N.
SAMARAWEERA1 and K. M. M. PRIYANGIKA1
1National Cinnamon Research and Training Center, Department Export

Agriculture, Palolpitiya, Thihagoda, Sri Lanka

2Faculty of Agriculture, University of Ruhuna, Mapalana, Sri Lanka

ggjaya2005@yahoo.co.in
 Contents
 Introduction
 Research problems
 Objectives
 Methodology
 Result and discussion
 Conclusions
 References
 Introduction
 Rough bark disease (RBD) is the most
important disease of cinnamon
cultivation in Sri Lanka

 RBD is responsible for considerable

bark yield reduction in cinnamon which
is the most important part in the
harvest

 The disease is found in almost all the

cinnamon growing areas in Sri Lanka
 .…Introduction

RBD is initiated as minute black lesions at the bark of

semi hard wood shoots and those black lesions turn into
large brown spots with a black margin spreading
gradually throughout the bark

Leaves of the infected plants show interveinal

chlorosis under severe conditions, leading to the death
of infected immature plants. Diseased bark cannot be
peeled properly affecting cinnamon processing
 .…Introduction

• Kularatne (1994) reported that the disease

was caused by algae considering its slow
spreading nature and other symptoms.

• Kumara (1999) reported scab-like condition

in cinnamon

• Identification and control practices were

studied (Jayasinghe and Rathnasoma, 2013)
 Research Problems
 RBD is the most common disease of cinnamon and
also it is a major problem in many cinnamon growing
areas in Sri Lanka
 No detailed information available on the etiology and
histopathology of the disease
 Therefore, it is crucial to confirm pathogen and
study the penetration pattern of the causal organism
inside the plant tissues in order to understand
disease development and to manage the disease
effectively
 Objectives
• To confirm of the causal organism using
Koch’s postulate
• To study the morphological characteristics
of the pathogen
• To study the penetration pattern of causal
organism/s inside the tissues of host plant
• To find out the relationship between
nutritional status of the plant with disease
intensity
 Methodology
1. Isolation of Pathogen
• Pathogen was isolated from the diseased
plants and cultured on PDA media

• Sub cultures of isolated pathogen/s was

established for further experiments (Spore
preparation for inoculation)

• Slide culture technique was performed to

observe the colony characters,
morphology of the mycelium and spores
under optical microscope
 …..Methodology
2. Pathogenicity test

Koch's postulation was performed by

introducing isolated pathogens into
healthy plants using three inoculation
methods
– Injuring by clean sand paper
– wrapping the cotton plug with
inocula on the stem
– Spraying the spore solution into
the stem
 …..Methodology

3. Histopathological Study

• Different types of cross and

longitudinal sections were prepared
using microtome from diseased and
healthy areas of the stem

• Compare of healthy and diseased

tissues of host plant through
microscope in order to understand
the penetration pattern of
pathogen/s
 …..Methodology

4. Nutrient Analysis of the Infected Cinnamon Leaves

• Healthy and infected leaves of different severity
levels of disease were tested for major plant
nutrients N, P and K

• Content of major plant nutrients (N, P and K) were

analyzed using stranded procedures and tests

• Leaf samples of four different disease intensities

and control (healthy leaves) were compared
 …..Methodology

Treatments:
• T1 - No any symptoms of disease in stem
• T2 - Initial stage of disease (only black tiny lesions on the stem)
• T3 – Low intensity of disease (lesion size on stem is less than 25 mm2)
• T4 – Moderate intensity of disease (lesion size in stem is more
than 25mm2, but no interveinal chlorosis in leaves)
• T5 – Higher intensity of disease (interveinal chlorosis in leaves)
 …..Methodology

Analysis
• ANOVA was performed using SAS
statistical package for N,P and K

• Regression analysis was done to find

out the relationship between the
nutritional status of plant with disease
intensity
 Results & Discussion
1. Isolation and identification
• Colonies of RBD isolates range from woolly to cottony, white or
whitish, pale to pale greyish
• The fast growing mycelium is initially white and gradually turns
to a light gray color on potato-dextrose agar (PDA)
• Normally Phomopsis sp. produce two types of hyaline, non
septate conidia- namely alpha and beta (Rehner and Uecker,
1994)
• Some spp. produces only α-conidia on the agar medium
(Banihashemi and Javadi, 2009)
• RBD of cinnamon , Phomopsis sp. produce only the alpha
conidia in PDA. which are aseptate, generally hyaline,
fusiform and usually biguttulate
• Mature hyphae of Phomopsis sp. are septate

Alpha
conidia

Septate hypae
 .…Results & Discussion

Pycnidia
Alpha
conidia

Cross section of the pycnidia

• All isolates produced pycnidia on PDA at 29 0C.

It contains alpha conidia
 .…Results & Discussion

Spore formation on cinnamon stem Conidiophore formation on PDA

• Morphology of the spores of Phomopsis sp. which

were formed naturally in host are very similar to
those the spores formed on PDA
 2. Confirmation of Causal Organism

 Six weeks after inoculation, minute black color lesions were

appeared as the first signs of disease in some cinnamon plants
when they are artificially inoculated with isolated spores (106 - 107
per ml).
 The symptoms developed were similar to the natural infection of
Phomopsis sp.
The pathogen that was re-isolated from inoculated plants
and cultured on PDA. It very similar to the original isolate in
morphologically

Original isolate of Phomopsis sp. Re-isolated of Phomopsis sp.

20
3. Studying the penetrate pattern of causal organism/s
inside the tissues of host plant.

Epidermis
Cortex
Phloem
Cambium
Xylem
Pith
Xylem Ray

Cross section of healthy stem (×40) Longitudinal section of healthy

stem (×40)

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 As a defense mechanism of plants, plant cells die
immediately when contact with the fungal cells. It is a
hypersensitivity reaction
 Sapwood under the bark (including xylem tissues) of
infected stem is usually turn into dark brown or black,
indicating tissue death due to the fungal invasion

Diseased area
Cross section of the infected stem Longitudinal section of the
(x40) infected stem (x40)

22
Dark brown or black color area of Penetration of the pathogenic fungi inside
the Xylem (x40) the xylem tissues of the plant (x100)

Microscopic observations showed that RBD fungi is associated with

the xylem tissues at the final stage
They might be blocked xylems so as to interrupt the mineral and
water conduction showing symptoms of interveinal chlorosis in
cinnamon leaves before dying of the plants.

23
 Most vascular pathogens colonize the nutrient-
poor xylem vessels because the xylem is
composed of dead tracheary elements with
relatively low osmotic pressure (Yadeta and
Thomma, 2013)
 P. vaccinii develops numerous tyloses in the
xylem at advance stage of disease in blueberry
plant (Daykin and Milholland, 1990).

 Final destination of P. helianthi was the vessel

elements after destruction of phloem, adjacent
mesophyll and cambium in leaves of sunflower
(Heller and Gierth, 2001).
4. Relationship between nutritional status of the plant with disease
intensity.

Disease intensity N% P% K%
T1 0.616a 0.078a 1.032a
T2 0.609a 0.059ab 0.816ab
T3 0.588a 0.060ab 0.658bc
T4 0.630a 0.042b 0.634bc
T5 0.537a 0.046b 0.429c
P-value 0.7575 0.062 0.007
CV(%) 15.491 23.072 21.455
LSD (α=0.05) 0.168 0.025 0.279

• T1 - No any symptoms of disease in stem

• T2 - Initial stage of disease (only black tiny lesions on the stem)
• T3 – Low intensity of disease (lesion size on stem is less than 25 mm2)
• T4 – Moderate intensity of disease (lesion size in stem is more than 25mm2, but no
interveinal chlorosis in leaves)
• T5 – Higher intensity of disease (interveinal chlorosis in leaves)

2
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CONCLUSIONS
 Pathogenicity tests confirmed the responsible
causal organism of rough bark disease in cinnamon
as Phomopsis species.

 The disease reaches critical stages when the fungus

invades the xylem tissues of stem showing
interveinal chlorosis of leaves as the pathogen
blocks the water & mineral nutrient translocation.

27
Further studies such as molecular based
techniques to identify the pathogen at the
species level

Comprehensive analysis of soil & plant is

crucial to investigate relationship between
disease severity and plant nutrients

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 Acknowledgements
Authors acknowledge the suggestions and
comments made by

– Mr. W. D. L. Gunaratne, Executives Director, International

Pepper Community, Indonesia

– Dr. Danushka Udayanaga, Visiting Scientist, USDA-ARS

Systematic Mycology and Microbiology Lab, West Beltsville,
USA to improve the quality of this paper.
 References
• Daykin, M. E. and Milholland, R. D. (1990) Histopathology of blueberry twig blight caused by Phomopsis
vaccinii. Phytopathology, 80: 736 - 740
• Heller. A., and Gierth, K. (2001) Cytological observations of the infection process by Phomopsis helianthi
(Munt. –Cvet) in leaves of sunflower. J Phytopathology 149: 347-357
• Jayasinghe, G. G. and Rathnasoma, H. A. (2013) Efficacy of fungicides for managing rough bark disease
(Phomopsis sp.) in cinnamon (Cinnamomum zeylanicum Blume), Proceedings - Part 1 – Abstracts,
SLAAS, Colombo, Sri Lanka. 2nd – 6th December, p.35
• Kularatne, R. S. (1994) Studies on rough bark disease on cinnamon. Annual Report, Research Division,
Department of Export Agriculture, Sri Lanka, p. 23
• Kumara, K. L. W. (1999) Study of leaf galls, leaf spots and scab-like condition of cinnamon (Cinnamomum
zeylanicum Blume) in Matara District, Proc. SLAAS, Colombo, Sri Lanka. 29th November to 3rd December,
p. 65.
• Mahadevakumar, S. and Janardhana, G. R. (2016) Leaf and fruit rot disease of brinjal caused by Diaporthe
vexans (Phomopsis vexans) in six agro-ecological regions of South West India. Plant Pathology &
Quarantine 6 (1): 5 -12
• Moster, L., Crous, P. W., Kang, J. and Phillips, A. J. L. (2001) Species of Phomopsis and Libertella sp.
occurring on grapevines with specific references to South Africa: morphological, cultural, molecular and
pathological characterization. Mycologia, 93 (1): 146 - 167
• Rosskopf, E. N., Charudattan, R., Shabana, Y. M. and Benny, G. L. (2000) Phomopsis amaranthicola, a new
species from Amaranthus sp. Mycologia, 92: 114 – 122
• Udayanga, D., Liu, X., McKenzie, E. H. C., Chukeatirote, E., Bahkali. A. H. A. and Hyde, K. D. (2011) The
genus Phomopsis: biology, applications, species concepts and name of common phyto-pathogens.
Fungal Diversity, 50: 189 – 225
• Yadeta, K.A. & Thomma, B.P.H.J. (2013) The xylem as battleground for plant hosts and vascular wilt
pathogens. Front plant Sci., 4: 97

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