J Mycol Pl Pathol, Vol. 40, No.3, 2010
387
Bird’s Eye Spot Disease (Cercospora theae) in Tea Estates of South India
Balsubramanian Mythili Gnanamangai and Ponnusamy Ponmurugan
Department of Biotechnology, K.S.R. College of Technology, Tiruchengode 637 215, Namakkal District, Tamil Nadu, India.
E-mail: mythumithras@gmail.com
Abstract
Prevalence of the leaf spot disease in tea estates of South India among 38 clones and seedlings that are widely
cultivated for commercial purpose of manufacturing tea was studied. The disease was surveyed in 13344 hectares
using quadrates sampling on bushy canopy. The disease severity was more on UPASI clones and areas like Wynaad
and Central Travancore of Kerala were severely affected by the disease. UPASI clones such as UPASI-3, 17, 19 and
25-27, and Sri Lankan clones, like TRI-2024 and TRI-2025 showed less than 30% disease index. The popular
private estate clones such as SA-6, BSS-1, ATK-1 and BSB-1 were tolerant to the disease. The disease severity of
tea clones depended on the sandy loam soil type and severity of drought in the respective planting districts. The
causal agent of Bird’s eye spot disease in tea plants was isolated on PDA and identified as Cercospora theae Petch.
Three isolates were obtained from the infected tea leaves in Koppa area of Karnataka (KC10), Munnar of Kerala
(MC24) and Valparai of Tamil Nadu (VC38). All the isolates showed minor variations in terms of mycelial growth,
culture characteristics and in vitro life cycle. The pathogen could invade the leaf tissue by both inter-cellular and
intra-cellular invasions. C. theae produced erupting perithecium initially in the upper epidermis followed by lower
epidermis in late infection stage. Perithecia were orange red whose conidia were cylindrical, 1-3 septa and produced
by apical budding and its size varies from 38-68 x 4-5 μm. Pathogenicity of the three isolates were confirmed by
their virulence in potted plants. Tthe life cycle of the three isolates in detached leaves varied among isolates ranging
from 19 d in MC24 to 21d in KC10 and VC38.
Key words: Tea, Cercospora theae, bird’s eye spot disease, life cycle, growth pattern, spore germination
Citation: Gnanamangai BM and Ponmurugan P. 2010. Bird’s eye spot disease (Cercospora theae) in tea estates of
South India. J Mycol Pl Pathol 40(3):387-391.
Tea (Camellia sinensis (L.) O.Kuntze) is a perennial
woody plant. The average life span of a single tea plant
is more than 100 years; peculiar cultural condition and
warm humid climate of the tea growing areas are highly
conducive for disease development (Baby 2001). Tea
production is greatly hindered due to pests and diseases
and majority of the diseases are of fungal origin
(Muraleedharan and Chen 1997). Among these, fungal
attack is very important in terms of crop loss.
Cercospora theae Petch, is an Ascomycetes fungal
pathogen that attacks younger leaves initially and later,
it is confined to mature young leaves and to some extent
on bare stalk which bears a tri-bud of great economic
importance in tea manufacturing. This pathogen forms
typical necrotic spots of brown to black colour with a
red colored margin in the infected leaves.
Sporadic incidence of this disease was found
more in some of the estates in the Anamallais of
Southern zones of India during the monsoon season in
2007. Large scale occurrence of the disease in mature
tea leaves of South India was noticed for the first time
and continued to cause infection even now in many
estates (Ajay et al 2007). Although bird’s eye spot
disease of tea is known for over 40 years in South India,
the disease has become a major problem only in recent
years on the estates. This has made many to resort large
scale replanting, infilling and inter row planting to
increase production and productivity.
Unfortunately, majority of the clones and
seedlings used for planting are susceptible to the
disease. Further, any change in monsoon rainfall,
sunshine, temperature or relative humidity aggravates
the disease. Providing proper shade, drainage, soil
aeration and manuring especially with potassium for tea
plants can control the disease. Foliar spraying of copper
containing fungicides to bird’s eye spot affected plants
has been found useful for controlling the disease to
some extent. Despite its importance, no efforts have
been made to assess disease incidence in large areas in
South India and study the culture characteristics of the
pathogen.
388
Materials and Methods
Survey of bird’s eye spot disease. The bird’s eye spot
incidence was recorded directly on bush canopy using a
quadrate. Wooden frame of 30 cm2 size was placed over
the plucking table at random sites. The number of
infected and un-infected intact leaves, cut leaves, bare
stalks and young shoots in 30 cm2 area were counted.
Per cent disease incidence (PDI) was calculated using
the formula PDI = (IL+CL+BS+YS)/4; where, IL =
disease incidence on intact leaves; CL = disease on cut
leaves, BS = disease on bare stalk and YS = disease on
young shoot (all on per cent basis). A total of 13344 ha
were covered during the survey in which 38 clones and
seedlings were covered to estimate individual clonal
susceptibility to C. theae infection. After the survey, the
plants were classified into high, moderate and low to
disease susceptibility according to the PDI due to high
variability among planting districts. PDI less than 30
were classified under low susceptible clones, PDI
between 30 and 60 were placed under moderately
susceptible clones and PDI above 60 were categorized
as highly susceptible clones.
Isolation and pathogenicity of C. theae. From tea
estates at Koppa in Karnataka, Munnar in Kerala and
Valparai in Tamil Nadu belonging to various
agroclimatic zones of South India were selected for the
collection of diseased leaf samples to isolate the
pathogen. The leaf samples were examined by cross
sectioning the leaf to investigate the status of the cells in
diseased leaves. The diseased leaf samples were surface
sterilized with 0.1% mercuric chloride and infected
portions were cut carefully under aseptic condition
subsequently inoculated onto PDA medium. The
pathogen isolation was carried out three times using
samples from each place of collections. Pathogenicity of
the isolates was performed on 2-yr old potted plants of
susceptible clone UPASI-9 by inoculation on leaves
with C. theae after making wounds in the leaf portion
with a sterile scalpel. Mycelial discs were kept on the
wound portion, sprinkled with sterile water and wrapped
with polythene sheet. The plants were kept in a
greenhouse maintained by UPASI Tea Research
foundation at Valparai, to develop the disease. The
pathogen was re-isolated from the infected leaves. To
each isolate seven potted plants were subjected for
pathogenicity to confirm their virulence.
Cultural characteristics of C. theae. Cultural
characteristics of three isolates like colony colour,
colony margin, growth rate, perithicium formation,
number of spores per perithicium and its morphology
were studied by inoculating them individually into both
carrot extract agar (CEA) and PDA. The growth was
also studied by inoculating them into carrot extract
J Mycol Pl Pathol, Vol. 40, No.3, 2010
broth (CEB) and PDB where mycelial dry weight was
recorded. All the culture characteristics were studied
from three replicates in each isolates.
Life cycle of C. theae. Life cycle of the pathogen was
studied in vitro using fresh healthy tea leaves of most
widely used UPASI 9 clone in South India. The
detached healthy tea leaves were taken in a conical flask
containing moist cotton and were sterilized twice in
alternate days. The sterilized leaves were then wounded
with the help of a fine sterile needle and the spore
suspension was sprayed over the wounded ventral
surface of the leaves using a sterilized glass automizer.
The inoculated flasks were incubated at room temp with
sufficient moisture provided in the wet cotton. The
experiment was carried out with three replicates.
Various parameters like time taken to cover the entire
leaf and production of perithicium per leaf were
observed periodically.
Results and Discussion
Survey of Bird’s eye spot disease. In
phytopathological studies, distribution of the disease, its
host ranges, and varietal resistance are important
parameters for determining its economic importance.
With this objective, an extensive survey was conducted
in various planting districts of South India. The result on
the survey showed that different clones and seedlings
have different levels of disease severity (Table 1) and
their susceptibility varied in different agroclimatic zones
(Table 2). It has been noted that disease incidence was
relatively high in Wynaad and Central Travancore
followed by Nilgiris districts. The UPASI clones such
as UPASI-1, 2, 4-8, 10-14, 16 and 20-24 were found
highly susceptible to bird’s eye spot disease. Clones of
other estates such as Yellapatty (YK-7) and
Singampatty (SMP-1) were also found highly
susceptible to the disease. Other clones such as UPASI9, 15, 18, CR-6017, W-35 and BSS-2 were moderately
susceptible. There were about 12 clones which
expressed low susceptibility (Table 2).
UPASI clones such as UPASI-3, 17, 19 and
25-27, and Sri Lankan clones, like TRI-2024 and TRI2025 were placed in low susceptible categories. The
popular private estate clones such as SA-6, BSS-1,
ATK-1 and BSB-1 were tolerant to the disease. The
disease severity of tea clones depended on the sandy
loam soil type and severity of drought in the respective
planting districts. Moreover, the disease susceptible and
tolerant nature of tea plants was reported to be due to
water stress, biotic and abiotic factors including cultural
operations like tipping, pruning, mechanical shear
harvesting and careless plucking (Baby et al 2001).
J Mycol Pl Pathol, Vol. 40, No.3, 2010
389
Table 1. Incidence of Cercospora theae on tea plantations of South India
Clones
or
seedling
Anamallais
Central
Travancore
(Kerala)
(Tamil Nadu)
2200 m*
900 m
UPASI-1
11.4
32.5
UPASI-2
10.3
UPASI-3
4.3
UPASI-4
31.4
20
UPASI-5
36
UPASI-6
31.4
20
UPASI-7
UPASI-8
12
13.7
UPASI-9
UPASI-10
3.4
40
UPASI-11
16.3
UPASI-12
16.6
UPASI-13
10.3
10.3
UPASI-14
0.6
32
UPASI-15
9.2
42.9
UPASI-16
5.7
20.2
UPASI-17
2.9
UPASI-18
UPASI-19
8
UPASI-20
UPASI-21
21.7
49.7
UPASI-22
8.8
28
UPASI-23
8.8
28
UPASI-24
UPASI-25
1.7
UPASI-26
9.1
UPASI-27
20
TRI-2024
5.1
3.4
TRI-2025
12.7
ATK-1
SMP-1
8.3
29.8
CR-6017
3.4
5.1
W-35
9.7
SA-6
7.4
YK-7
23.4
BSB-1
BSS-1
BSS-2
*Altitude m above mean sea level
High Range
(Kerala)
2050 m
18.7
23.1
9.7
23.1
3.7
21
15.6
4.9
9.7
4.8
10
-
Leaf infection. The cross sectioned leaf infected with
C. theae exhibited necrotized cells in the affected region
and normal cells in the healthy region. The necrotized
regions slowly invaded the healthy cells. The pathogen
could invade the leaf tissue by both inter-cellular and
intra-cellular invasions. Changes caused by Septoria
apiicola during leaf spot disease in celery was reported
to show no effect on the epidermis or stomata until late
in to infection cycle when pycnidia erupted through
Chickmagalur
(Karnataka)
1100 m
4.3
6.3
3.4
15.4
3.4
4.6
5.1
5.7
4.7
4.7
2.3
3.4
12.9
-
Nilgiris
(Tamil Nadu)
1050 m
20.3
7.4
9.1
10.3
7.4
32
32
22.3
10.7
15.6
7.4
6.3
20.4
6.9
20
4.7
21.3
3.4
9.1
15.6
6.3
4.6
4.6
9.1
10.7
12.6
6.3
4
10.7
15.6
4.6
22.9
NilgirisWynaad
(Kerala)
1858 m
47.6
45.1
27.4
27.4
16.9
7.4
15.4
45.1
16.3
16
12.8
21.6
10.7
6.6
16.3
14.1
25.1
6.6
7.4
Wynaad
(Kerala)
974 m
44.6
35.4
51.4
21.4
12
16.6
30.3
27.4
29.7
35.1
32
16.6
42.9
35.4
29.7
37.2
17.4
39.4
37.1
12
epidermis of the upper and lower leaf surfaces (Suzane
et al 1999). C. theae revealed a character of erupting
perithecium initially in the upper epidermis followed by
lower epidermis in late infection stage. All the three
isolates confirmed their virulence in potted plants in
green house by exhibiting the necrotic spot in both
mature and young leaves (Table 3). The isolates were
comparable on their pathogenicity and hence all the
three isolates were studied further.
J Mycol Pl Pathol, Vol. 40, No.3, 2010
390
Table 2. Susceptibility of tea clones and seedlings to C. theae infection on tea plantations of South India
Planting districts
State
Area
(ha)
High
Anamallais
Tamil Nadu 2090
UPASI-1, 2,
Central Travancore Kerala
1045 4-8, 10-14, 16,
High Range
Kerala
2090
20-24, YK-7,
Chickmagalur
Karnataka
950
SMP-1
Nilgiris
Tamil Nadu 3115
Nilgiris-Wynaad
Kerala
2129
Wynaad
Kerala
1925
#
High > 60 PDI, Moderate 30-60 PDI, Low < 30 PDI
Table 3. Pathogenicity of C. theae isolates on a tea
clone UPASI-9 in pot culture
Isolates
Mature
leaves
infected
23
27
Young
leaves
infected
29
32
Spots
/leaf
VC38
CD (P=0.05)
24
4.8
31
4.81
59.4
0.17
CV (%)
38.1
42.3
1.5
KC10
MC24
61.5
63.1
Growth of C. theae. Among the three isolates, MC24
isolate showed a better growth than KC10 and VC38 in
both CEA and PDA. The isolate MC24 covered the
entire plate in 8 d in CEA and 12 d in PDA. Similarly,
the isolate VC38 took 12 d. KC10 took merely 12 d to
cover the plate in CEA but could not do so even after 16
d in PDA (Table 4). Kilpatric and Johnson (1956) had
also reported that carrot leaf extract and CEA are the
best media for the growth and sporulation of
Cercospora species.
Among the two liquid media tested, CEB was
significantly superior to PDB. The growth of MC24
isolate was more in CEB than the other two isolates.
The morphological features varied among the isolates
where KC10 exhibited dull white, cottony thick,
mycelium with spore measuring 15.1 x 4.1 μm; MC24
showed dull orange white, cottony thick mycelium with
spore measuring 19.4 x 4.9 μm; and VC38 had white
cottony sparse mycelium with spore measuring 15.3 x
4.7 μm. The disparity in nutritional sources, physical
conditions like altitude (763 m at Koppa in Karnataka,
2400 m at Munnar in Kerala and 1050 m at Valparai in
Tamil Nadu, and age of tea plants from 30, 46 and 71
yrs, respectively at the three locations apparently
influenced the variation among the three isolates.
Clonal susceptibility#
Moderate
UPASI-9, 15, 18,
CR-6017, W-35, BSS-2
Low
UPASI-3, 17-19,
25-27,TRI-2024, 2025
SA-6, BSS-1,
ATK-1, BSB-1
Conidial features of pathogen. The isolated cultures
were stained with lactophenol cotton blue stain and its
conidial size, morphology and mycelial features were
determined. The size of conidia ranged from 15-20 μm
in length and 4-6 μm in width. The spores were uniform
in width with slightly elevated curved ends and 1-3
septate in nature. Crous et al (1993) described that
Calonectria ilicicola causing black rot in soyabean and
peanut is an anamorph of Cercospora theae which
produces orange red perithecia whose conidia were
cylindrical, 1-3 septa and produced by apical budding
and its size varies from 38-68 x 4-5 μm. The mycelial
features of C. theae indicated aerial mycelium with an
orange imprint underneath the Petri dish, the mycelial
width was 1-2 µm and exhibited multiseptate branched
hyphae. The pathogen produced an orange coloured
droplet that later turned into dark reddish-black masses
which were the fruiting bodies of the pathogen,
perithecia. Janice and Chris (1997) showed similar
perithecium formation in leather leaf infected by
Calonectria and Cylindrocladium species. The isolates
exhibited similar features with minor differences in their
size. Deepadavis and Beena (2007) reported that in leaf
spot of Ivy gourd, the pathogen C. cocciniae was unable
to sporulate in culture media but showed branched and
septate hyphae that were 3.8-5.8 μm in size.
Life cycle of C. theae. Inoculation of C. theae into the
sterilized leaf materials showed that the pathogen
completed its life cycle by 19-21 d in vitro. Spores of all
the isolates germinated on the leaves and visible growth
was observed on 3 d after incubation. The mycelial
growth in KC10 was initiated in 4 d, covered the leaves
in 8 d, showed orange colored pigment in 19 d, and
black colored fruiting bodies (perithecium) appeared in
21 d. In MC24, the mycelium was initiated in 3 d,
covered the leaves in 6 d, and fruiting bodies appeared
by 19 d. In case of VC38, the mycelial growth was
initiated in 4 d, covered the leaves by 10 d and
perithecium started to develop on 21 d.
J Mycol Pl Pathol, Vol. 40, No.3, 2010
391
Table 4. Growth of C. theae isolates
Days
KC10
MC24
Growth of isolates in PDA (mm)
1
0
0
2
13.3
13.3
4
14.3
18.3
8
33.3
36.7
10
45.7
47.3
12
79.3
88.7
CD (P=0.05)
6
6.7
CV (%)
8.7
8.7
Growth of isolates in CEA (mm)
1
0
0
2
12.3
19.7
4
21.7
31.7
8
53.3
79.3
10
74.7
86.3
12
86.7
89.7
CD (P=0.05)
5.2
5.3
CV (%)
5.7
4.4
Dry weight of C. theae in PDB (mg)
3
83.7
61
6
87.3
187.7
9
106.7
275.3
12
126.3
346.7
15
161.7
778.3
18
136.3
352.3
21
121
317.7
24
109.3
285.7
27
96.7
93
CD (P=0.05)
0.04
0.06
CV (%)
2.22
3.6
Dry weight of C. theae in CEB (mg)
3
88.7
62.7
6
115.3
279
9
147
558.3
12
193.3
1406.7
15
131.7
1246
18
128
463
21
111.7
458. 7
24
91.7
442.7
27
87.7
175
CD (P=0.05)
0.11
0.05
CV (%)
6.2
3.1
VC38
0
14.3
15.3
34.7
45.7
86.3
5.4
7.3
0
21.3
37.3
86.7
89.7
90
3
2.6
72.7
134.3
341
629
847
607.7
578.3
449.3
80.3
0.05
2.9
68.3
176
649.7
1132.3
1032
830.3
762.3
339.7
168
0.06
3.5
The number of perithecia counted on 26 d was
more with MC24 (236) with that of KC10 (126) and
VC38 (180). Life cycle of other tea pathogen, Tunstallia
aculeate causing thorny stem blight was reported to be
57 d in vitro on sterilized tea stem bits (Chandramouli
and Parthiban 1992).
Similarly, life cycle of Phomopsis theae causing stem
canker was reported as 10-13 d (Ponmurugan and Baby
2005).
Thus the present study revealed the prevalence
and disease severity among estates of South India and
provided a basis for more extensive studies on C. theae
for its control. It is apparent from the results that all the
three isolates are virulent to most of the commercial tea
clones and needs suitable integrated management.
Acknowledgements
The authors are thankful to the Director, Department of
Biotechnology, Principal and Chairman of KSR
Educational Institutions, Tiruchengode, Tamil Nadu,
India for providing necessary facilities and
encouragement.
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Received: May 5, 2010
Accepted: Aug 26, 2010