Crop Protection 44 (2013) 66e74

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Crop Protection
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Management of Cephaleuros parasiticaus Karst (Trentepohliales:

Trentepohliaceae), an algal pathogen of tea plant, Camellia sinsensis (L) (O. Kuntze)
Mohan Ramya, Ponnusamy Ponmurugan*, Devaraj Saravanan
Department of Biotechnology, K.S. Rangasamy College of Technology, K.S.R Kalvi Nagar, Tiruchengode 637 215, Namakkal District, Tamil Nadu, India

a r t i c l e i n f o a b s t r a c t

Article history: The efficacy of certain biocontrol agents were evaluated against Cephaleuros parasiticus Karst, a causal
Received 5 June 2012 organism of red rust disease in tea plants. Spraying of systemic fungicides was found to be superior to
Received in revised form biocontrol agents followed by algicides in controlling the disease under field condition. Dipping of shears
21 October 2012
in the solutions of fungicide, detergent or biocontrol agents failed to protect the disease spread. The
Accepted 27 October 2012
study on the impact of different harvesting practices on disease development showed the highest disease
incidence in continuously shear harvested fields and least in hand plucked fields. There was a reduction
Keywords:
in disease incidence in spraying of a solution containing urea and murate of potash mixture at 1% level.
Tea
Algae
The maximum green leaf yield and productivity index were recorded in biocontrol agents treated plots.
Pathogen Among the various group of biocontrol agents evaluated, Streptomyces sannanensis and Streptomyces
Red rust griseus belong to actinomycetes was better than bacterial and fungal antagonists in terms of disease
Cephaleuros parasiticus protection and increase in yield potential. Similarly, tea bush canopy architecture, physiological and
Biocontrol biochemical parameters were also increased in plants treated with biocontrol agents. In the case of
untreated control plots, the disease incidence was increased from 36% to 44.5% and the plants were
found unhealthy in terms of chlorosis, stunted growth and heavy flowering with banji buds. The shoots
collected from treated and untreated control plots were subjected to manufacture CTC black tea which
revealed almost all the tea quality parameters were significantly improved in the treated plot over the
untreated plots.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction diseases, red rust is an important disease prevalent in both young

Tea is the most popular and inexpensive beverage produced
from the young shoots comprising of three leaves and a bud of the
commercially cultivated tea plant (Camellia sinensis (L.) O. Kuntze).
It plays a major role in the economy of several under developed and
developing nations in Asia and Africa. In India, this crop is grown in
more than 500,000 ha, of which about 97,600 ha is cultivated by
123,000 small growers (one who owns less than 10 ha). As in any
other crop, many diseases affect the roots, stems and leaves of the
tea plant. Crop loss (loss in the yield) in tea due to pests, diseases
and weeds recorded as high as 43% (Baby, 2001). The majority of
diseases in tea are of fungal and bacterial origin except red rust
disease caused by an algal pathogen Cephaleuros parasiticus Karst.
Among the tea diseases, leaf diseases are very important due to the
fact that tea plants are cultivated for its young succulent leaves for
manufacturing of tea (Muraleedharan and Chen, 1997). Of the leaf
* Corresponding author. Tel.: þ91 4288 274741 44 (4 lines); fax: þ91 4288 274745.
E-mail address: drponmurugan@gmail.com (P. Ponmurugan).
and mature tea fields under adverse conditions of soil and climate.
According to the disease survey conducted by Ponmurugan et al.
(2010) red rust incidence was more severe in seedlings than
cultivar in certain pockets of southern Indian plantations in which
about 18 highly susceptible tea cultivars were identified.
The disease is a severe problem in India, Bangladesh, Sri Lanka,
China, Japan and Kenya (Muraleedharan and Chen, 1997; Islam and
Ali, 2011). The disease is sporadic in occurrence every year in most
of the tea plantations and usually varies in intensity within and
between localities (Prasanth et al., 2005). Crop loss due to this
disease is enormous when pathogen infects bare stalks and young
shoots. The disease has become a serious problem during the last
two decades due to extensive use of machines for harvesting tea
shoots (Gnanamangai and Ponmurugan, 2011). The disease is of
great importance, as the area under replanting and new clearings
with tea seedlings including biclonal seed stocks are increasing in
recent years. Moreover, capital loss (loss of entire bush) due to this
disease is substantial if continuous shear harvesting is practiced. It
has been recorded that due to red rust incidence, both physiological
and biochemical parameters were affected significantly in tea

0261-2194/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.cropro.2012.10.023
 M. Ramya et al. / Crop Protection 44 (2013) 66e74 67

leaves (Ponmurugan et al., 2007). The predisposing factors of the
disease mainly are poor fertility of soil, lack of soil aeration, inad-
equate or complete absence of shade, extreme drought or frost,
improper fertilizer application, hard plucking, continuous shear
harvesting and water logging (Hajra, 2001). Due to attack by the
disease, plants become very weak and new shoots developed from
those are very thin with heavy banji shoots which in turn affect the
tea quality (Bore, 1996).
During the last several decades, control of diseases in tea fields is
predominantly done by the use of synthetic chemical fungicides
(Sanjay et al., 2008). Though broad-spectrum fungicides offer
powerful incentives in the form of good control, increased yield and
high economic returns, they have serious drawbacks such as
development of resistance to fungicides, resurgence of pathogens,
outbreak of secondary diseases, harmful effects on human health
and environment and presence of undesirable residues. In order to
control the disease, copper oxychloride, mancozeb and carbenda-
zim are being recommended to the tea planters (Baby, 2001). Early
days, red rust was controlled by manually removing the infected
leaves and burning them in the field.
Biocontrol strategy is extensively adopted to control various
stem and root diseases. The in vivo efficacy of Trichoderma and
Gliocladium bioformulations in controlling thorny stem blight and
Phomopsis canker (Ponmurugan and Baby, 2007) has been reported.
Recently blister blight caused by Exobasidum vexans, grey light
caused by Pestalotia theae and bird’s eye spot disease by Cercospora
theae were controlled very efficiently by spraying Trichoderma and
Pseudomonas bioformulations (Saravanakumar et al., 2007; Sanjay
et al., 2008; Gnanamangai and Ponmurugan, 2011). Since red rust
is very severe in southern India tea plantations, there is no suitable
recommendation made available to the tea planters. Moreover, no
attempt is made to control the disease using biocontrol agents as
the disease is caused by an algal pathogen, C. parasiticus. Studies
were undertaken to screen various bacterial, fungal and actino-
mycete antagonists along with a few algicides against the red rust
disease under field condition.
2. Materials and methods
2.1. Study site
Field trail was conducted in a naturally infected field in a tea
plantations in Valparai, Tamil Nadu State, India, lying at an eleva-
tion of 1035 m above mean sea level (10 300 , 77 00 E), with a highly
susceptible cultivar, UPASI-10 planted in 1991 at a spacing of
135  75  75 cm (Fig. 1a, b, c). Naturally infected field with an early

Fig. 1. Biocontrol mechanism between Cephaleuros parasiticus and selected biocontrol agents. (a) Red rust infected tea field, (b) red rust lesions on mature tea leaves (arrows
indicate lesions), (c) formation of banji bud due to severe disease infection (arrow indicates a banji bud), (d) hyperparasitic potential of T. harzianum on C. parasiticus, (e) colo-
nization of T. harzianum on C. parasiticus filaments, (f) wrinkling and bursting of zoosporangium containing zoospores of C. parasiticus by S. sannanensis, (g) lysis of C. parasiticus
filaments by S. sannanensis (arrows indicate place of lysis). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)
68 M. Ramya et al. / Crop Protection 44 (2013) 66e74
history of red rust disease was selected based on the survey con-
ducted earlier by Ponmurugan et al. (2010). Experimental plots
were laid out in a randomized block design within the single
plantation. The study was conducted during disease seasons
consecutively from 2009 to 2011 and consisted of 25 bushes per
plot with five replicates. The newer chemicals and promising
biocontrol agents were evaluated as foliar sprays as per the
recommendations of UPASI Tea Research Institution, Valparai,
Tamil Nadu, India (Premkumar, 2008). Moreover, the standard
cultural operations like plucking, pruning, manuring and weeding
were adopted (Venkatesan et al., 2005) in the experimental plot as
per the UPASI-TRI recommendations.
2.2. Selection of fungicides and biocontrol agents
Contact fungicides such as mancozeb and copper oxychloride at
0.3% and systemic fungicides such as carbendazim and contaf at
0.05% were selected. In addition, algicides such as algaenozol and
algaenozine at 1000 ml ha1 were evaluated. The fungal biocontrol
agents such as Trichoderma harzianum (UP12) and Trichoderma
atroviride (MTCC 9641), Bacillus subtilis (KSRbs07) and Pseudo-
monas fluorescence (KSRpf11) belongs to bacterial biocontrol agents
and actinomycete antagonist like Streptomyces sannanensis
(KSRss11) and Streptomyces griseus (KSRsg02) were obtained from
native tea soils (Ponmurugan and Baby, 2007; Sanjay et al., 2008).
2.3. Mass production of biocontrol agents
Vermicompost was selected as the potential carrier material for
the mass multiplication of biocontrol agents. For vermicomposting
native earthworm species (Eisenia hortensis) are selected and the
setup is provided with optimal conditions for culturing. Feed
materials like cow dung, biogas slurry and other nitrogen rich
wastes like kitchen refusals were used. 50 ml of 7-day old Tricho-
derma liquid cultures (Potato Dextrose broth) was mixed with 100 g
of sterilized vermicompost and allowed to grow for 14 days. Simi-
larly, bacterial and actinomycete antagonists were grown on
nutrient and casein nitrate broth; respectively. About 25 ml of 3-
day old cultures further diluted to 1000 ml with water (1:4) to
which salicylic acid and ammonium sulphate were added at the
rate of 600 mg l1 as substrates to enhance the growth (Sanjay
et al., 2008). From this, 25 ml of grown culture were mixed with
100 g of vermicompost (Trillas et al., 2006). For field application,
the product was soaked in 2.0 L distilled water in a bucket and
allowed to stand overnight.
Spraying of fungicides, algicides and bioformulations was
carried out using a knapsack sprayer (30 L capacity) with a spray
volume of 300 l ha1 covering one row on either side of the tea
bush. Spore load in both the preparations was adjusted to 1  108
colony forming units per gram of sample (Gnanamangai and
Ponmurugan, 2011). Leaf samples were collected periodically for
the enumeration of biocontrol agents to evaluate the survival.
Enumeration of Trichoderma, Pseudomonas, Bacillus and Strepto-
myces species was carried out using Trichoderma selective medium,
nutrient and casein nitrate agar media respectively, following
dilution plate technique (Kuster and Williams, 1964; Elad and Chet,
1983). The circular green-grey areas that eventually form red rust as
the alga produces a profusion of rust-coloured microscopic spores
and filaments were collected and subjected to study periodically.
2.4. Impact of harvesting methods on red rust control
A study was conducted in a naturally infested field planted with
YK-7 cultivar (Sanjay et al., 2008). Treatments included hand
plucking with and without breaking back (removal of infected
leaves, cut leaves during mechanical shear harvesting and leaves
with chlorosis), harvesting using flat and step shears continuously.
In integrated system of harvesting, field was hand plucked in the
lean season (between January and March) and shear harvested
(flat/step) in the high cropping period (between April and
December). The total number of plucking rounds during the
experimental period was 82 in the hand plucked plots, 42 in the
continuous shear harvested ones and 55 in the integrated style of
harvesting. Disease assessments were made at each plucking
ground by picking 50 shoots (three leaves and a bud) at random
from the harvest of each replicate and examined them individually
for the presence of lesions.
2.5. Impact of dipping shears in different test solutions on red rust
control
The blades of shear harvesting machine were dipped into
biocontrol, fungicide, algaecide and 1% detergent (Teepol) solutions
before shearing. It was followed every time, after transferring the
plucked leaves to the basket. Sampling was done from the plucked
shoots (50 shoots/plot) at fortnightly interval and disease incidence
was recorded subsequently as per the method of Sanjay et al.
(2008).
2.6. Impact of urea and murate of potash foliar spray on red rust
control
The efficacy of a solution having urea and murate of potash
(MOP) was evaluated to maintain turgidity of cells, improve water
use efficiency, enhance plant growth and reduce leaf banji
(dormant shoot) formation which in turn to control red rust inci-
dence. Urea and MOP combination at 1% concentration was
prepared by dissolving it in tap water for field application. Urea and
MOP solution was sprayed by using a knapsack sprayer with a spray
volume of 200 L ha1 covering both the sides simultaneously as per
the recommendation of UPASI Tea Research Institution, Valparai,
Tamil Nadu, India. The treatments were applied two times per year
during AprileMay and the second were in OctobereNovember.
Data on green leaf yield and severity of red rust disease were
recorded (Huq et al., 2010).
2.7. Disease assessment and green leaf yield
The occurrence of red rust incidence was recorded directly on
the bush canopy using a quadrate method as per Sanjay et al.
(2008). A wooden frame of 30 cm2 size was placed over the
plucking table at random sites. The number of infected and unin-
fected intact leaves, cut leaves (damaged leaves), bare stalks, and
young shoots in 30 cm2 area were counted and the percentages
individually calculated. Percent disease incidence (PDI) was calcu-
lated using the formula PDI ¼ (IL þ CL þ BS þ YS)/4, where,
IL ¼ disease incidence on intact leaves; CL ¼ disease on cut leaves,
BS ¼ disease on bare stalk, YS ¼ disease on young shoot (all on
percentage basis). Green leaf yield and yield attributes, particularly
the percentage of banji occurrence was also recorded in the
experimental plots during every plucking round. The green leaf
yield potential was converted to made tea kg ha1 by the formula
i.e., yield ¼ green leaf yield in kg  19,000  0.225/number of
bushes in the experimental plot, where, 19,000 is the total bush
population in the hectare and 0.225 is the conversion factor for
green leaf (22.5% out turn i.e., average yield per year per hectare) to
made tea (Ponmurugan and Baby, 2007). Moreover, productivity
index (PI) in response to various treatments upon red rust control
and green leaf yield in trial plots was calculated according to the
method of Sharma and Sathyanarayana (1990).
 M. Ramya et al. / Crop Protection 44 (2013) 66e74
2.8. Tea bush canopy architectural analysis
Recovery of the treated bushes were monitored at every
plucking round (approximately 14e20 days interval) by assessing
various biometric parameters such as plucking surface of the bush,
plucking points containing three leaves and a bud, internodal
length, leaf moisture and dry matter contents using the method
adopted by Balasubramanian et al. (2010).
2.9. Physiological and biochemical variations
Since, the mother leaves of tea plants are a potent source
(Rajkumar et al., 1998), all the observations were restricted to the
mother leaves. Net photosynthetic rate (Pn), Transpiration rate (Tr)
and stomatal conductance (Sc) were measured using an infrared
gas analyzer (ADC LCA-3, UK) and an open type Parkinson leaf
chamber (ADC PLC-3). Water use efficiency was calculated from the
ratio between Pn and Tr. In order to analyse the biochemical
constituents, tea mother leaves (50 leaves) were collected from
different experimental plots and subjected to estimate total chlo-
rophyll (Harborne, 1973), sugar (Dubois et al., 1956), nitrogen
(AOAC, 1990), polyphenols (Bray and Thorpe, 1954), catechin
(Swain and Hillis, 1959) and caffeine (Egan et al., 1981) contents.
The sampling was done in each trail plots at every plucking round
for this study.
2.10. Tea quality constituents
Fresh tea shoots consisted of three leaves and a bud from treated
and untreated control blocks were collected separately during
plucking and subjected to miniature crush, tear, and curl (CTC)
manufacturing unit (UPASI-TRI, Valparai, India). The resultant
product was used to estimate various tea quality parameters such
as theaflavins, thearubigins (Ullah, 1972) and organoleptic evalua-
tion such as colour index, briskness index and flavour index was
done with the help of professional tea tasters and the scores for
various quality attributes were analyzed (Takeo and Oosawa, 1976).
Volatile flavour composition of tea was studied using simultaneous
distillation and extraction method (Hazarika et al., 1984). The
sampling was done for analysing tea quality parameters at monthly
intervals.
Fig. 2. Effect of various fungicides, algicides and biocontrol agents on red rust disease protection and green leaf yield potential. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
69
2.11. Statistical analysis
All the data were statistically evaluated using SPSS 14.0 statis-
tical package (SPSS, Inc. Chicago, IL). Moreover, the data obtained
were also subjected to analysis of variance (ANOVA) and treatment
means were compared by Duncan’s Multiple Range Test (DMRT).
The significant means were segregated by critical difference (CD) at
various levels of significance. The standard error (SE), standard
deviations (SD) and covariance analysis (CV) were also calculated
(Gomez and Gomez, 1984).
3. Results
3.1. Red rust disease control
The results on various fungicides, algicides and biocontrol
agents upon the control of red rust incidence in tea plantations
revealed that fungicides provided significant disease protection to
the tea plants when compared to biocontrol agents followed by
algicides (Figs. 2 and 3). Various treatments in trail plots protected
the disease to varying degrees and improved the plant health
considerably. Among the fungicides tested, systemic fungicides
registered the maximum disease control (61.5%) compared to
contact fungicides (53.5%). Of the systemic fungicides evaluated,
carbendazim (63.4%) was highly effective in inhibiting the disease
development followed by contaf (59.6%). Between the contact
fungicides, copper oxychloride (54.8%) gave the best disease
control. The algicides namely algaenozol and algaenozine were
ineffective in disease protection which accounted for by 28.6 and
29.2%; respectively. The biocontrol agents were effective moder-
ately in terms of disease protection (33.4e44.4%) but inferior to
fungicides. Among the different biocontrol agents tested, the acti-
nomycetes S. sannanensis (44.4%) was found to be better than
bacterial and fungal antagonists in protecting the plants against the
infection. Colonisation of the pathogen filaments by the antagonists
and wall lysis of C. parasiticus filaments were observed under
microscope. The zoosporangium containing zoospores of
C. parasiticus were wrinkled and ruptured due to the inhibitory
effect of all the selected antagonists (Fig. 1deg).
The disease incidence in the untreated control trail plots regis-
tered the highest as 44.5% and the bushes exhibited defoliation
70 M. Ramya et al. / Crop Protection 44 (2013) 66e74

Fig. 3. Effect of various fungicides, algicides and biocontrol agents on red rust disease protection and productivity index.

with heavy flowering and yellowing of leaves (chlorosis). Moreover, 3.4. Physiological and biochemical response of red rust infected
the diseased leaves appeared as circular green-grey areas that leaves
eventually form rust red as the alga produced a profusely rust-
coloured sporangia. Physiological and biochemical parameters were found to be
superior in biocontrol agents treated plant leaves followed by
3.2. Green leaf yield improvement fungicides and algicides (Table 2). Physiological parameters such as
Net photosynthetic rate (Pn), transpiration rate (Tr), water use
There was a strong correlation between green leaf yield, efficiency (WUE), and stomatal conductance (SC) achieved the
productivity index and disease protection after imposing various maximum in biocontrol agents treated plants. Same trend was seen
treatments in the trail plots. The yield attributes were maximum in in biochemical parameters too. Biochemical parameters such as
the plots treated with biocontrol agents (Figs. 2 and 3). Among the total sugars, nitrogen, protein, amino acids, polyphenols, catechin
different biocontrol agent tested, S. sannanensis showed the highest and caffeine were found to be superior with the treatment of
yield about 3583 kg ha1 made tea along with productivity index biocontrol formulation compared to fungicides (Table 2). Among
about 1.7 followed by S. griseus about 3542 kg ha1 made tea. In the various biocontrol agents tested, S. sannanensis and S. griseus
addition the bushes were found healthy plucking surface of the were better than fungal and bacterial biocontrol agents. Pn and Tr
bush with good flushing in these treatments. When compared with rates recorded were 8.42 per mol min2 m2 and 3.89 mol s2 m2;
the untreated control plots, algicides showed a moderate response respectively in S. sannanensis treated bushes. Similarly, polyphenols
in terms of yield potential. A significant yield increase was recorded
in plots treated with carbendazim (3418 kg ha1 made tea) and Table 1
contaf (3403) fungicides. The yield and productivity index were Effect of various fungicides, algicides and biocontrol agents on canopy architectural
very least in untreated control plots which accounted as analysis of tea bush.
2727 kg ha1 made tea. S. no Treatment Plucking Plucking Leaf Dry Banji
details surface points moisture matter (%)
3.3. Tea bush canopy architectural analysis (cm2) (per sq.ft) (%) (%)
1. Mancozeb 7219def 120.0bcde 67.33defgh 19.05cd 42.27gh
2. Copper 7297def 121.1bcde 68.15defgh 19.09cd 41.80gh
Biocontrol agents treated bushes showed an increase in various
oxychloride
biometric parameters such as plucking surface, number of plucking 3. Carbendazim 7400ghi 127.3fghi 69.00defgh 21.38efg 35.55def
points, internodal length, leaf moisture and dry matter contents 4. Contaf 7332def 125.8fghi 70.55hijk 21.38efg 35.55def
significantly (Table 1). These results were greatly in line with 5. Algaenozol 6589bc 120.5bcde 63.23bc 17.08bc 51.74hi
the observation of green leaf yield and productivity index. 6. Algaenozine 6418bc 122.2bcde 64.12bc 18.18bc 52.83hi
7. P. fluorescence 7412ghi 130.5jklm 69.24defgh 21.48efg 30.53def
Plucking surface of the bush ranged between 7412 and 8695 cm2 8. B. subtilis 7489ij 130.8jklm 70.80hijk 22.88hijk 31.70def
in biocontrol agents treated bushes followed by 7332e7400 cm2 9. T. atroviride 7500ij 125.5fghi 70.33hijk 22.08hijk 28.77bcd
in systemic fungicides sprayed plants. The plucking surface 10. T. harzianum 7389ghi 127.7fghi 69.50defgh 23.18kl 27.78bcd
of biocontrol treated plots exhibited fairly dark green, mildly 11. S. griseus 8035jk 135.5jklm 70.27hijk 22.44hijk 23.89ab
12. S. sannanensis 8695jk 135.5jklm 72.31kl 22.44hijk 22.59ab
pubescent, slender and moderate easiness at harvest. The results of
13. Untreated 2021a 111.1a 57.57a 13.13a 75.53j
plucking points per unit area were in line with the result of leaf control
moisture, dry matter and banji contents. The banji content was CD at P ¼ 0.05 144.15 13.11 11.11 10.14 8.89
reduced in all the treatments compared to that of control bushes, Values are the mean of five replications.
however, the reduction was highest in biocontrol treated bushes Values followed by similar alphabet denote they are not significantly different at
ranged between 22.6 and 31.7% (Table 1). P < 0.05 and vice versa according to DMRT.
 M. Ramya et al. / Crop Protection 44 (2013) 66e74 71

Table 2
Physiological and biochemical variations in the mother leaves of treated and untreated red rust infected tea bushes.

S. no Treatment details Physiological parameters Biochemical parameters (% by mass)

Pn Tr WUE SC Chlorophyll Sugar Nitrogen Polyphenols Catechin

1. Mancozeb 3.23bc 2.44cd 2.08bc 0.17b 1.32cd 4.22bc 2.32bc 15.56bc 4.12a
2. Copper oxychloride 3.43bc 2.23bc 2.02bc 0.21bc 1.23bc 4.23bc 2.23bc 15.41bc 4.40ab
3. Carbendazim 4.20de 3.48ef 2.48cd 0.20bc 1.48de 4.45cd 3.14cd 17.47fg 5.89de
4. Thiophanate methyl 4.00de 2.97cd 2.87cd 0.20bc 1.51de 4.58cd 3.20cd 17.56fg 5.45d
5. Algaenozol 3.08bc 2.08a 2.04bc 0.21bc 1.20bc 4.10bc 3.00cd 16.22de 4.00a
6. Algaenozine 3.01bc 2.03a 2.14bc 0.23bc 1.23bc 4.21bc 2.88cd 16.11de 4.08a
7. P. fluorescence 7.24fg 3.33e 3.27ef 0.27bc 1.84d 4.69de 4.17fg 17.14fg 5.94de
8. B. subtilis 8.38h 3.33ef 3.23ef 0.25bc 1.83d 4.64de 4.18fg 17.00cd 6.24f
9. T. atroviride 7.75fg 3.47ef 3.21ef 0.26bc 1.82d 4.80ef 3.97e 17.28cd 5.90de
10. T. harzianum 7.56fg 3.41ef 3.12ef 0.28bc 1.90de 4.90ef 4.00f 17.38cd 6.00de
11. S. griseus 8.37h 3.88fg 3.81fg 0.30cd 1.93de 5.12fg 4.00f 18.00h 6.37fg
12. S. sannanensis 8.42h 3.89fg 3.80fg 0.31cd 1.90 5.12fg 4.23g 18.88i 6.23f
13. Untreated control 2.86a 2.23bc 1.17a 0.10a 1.01 3.02a 1.14a 13.31a 5.01c
CD at P ¼ 0.05 2.11 1.08 2.08 1.12 2.13 2.78 2.32 02.21 2.00

Values are the mean of five replications. Values followed by similar alphabet denote they are not significantly different at P < 0.05 and vice versa according to DMRT.
Pn: net photosynthetic rate (per mol min2 m2), Tr: net transpiration rate (mol s2 m2), WUE: water use efficiency (Ratio of Pn/Tr rate), SC: stomatal conductance
(mol s2 m2), Chlorophyll: total chlorophyll (mg/g).

and catechin contents estimated were 18.88 and 6.23%; respec-
tively in the same bushes. In the case of systemic fungicides, car-
bendazim was the best in enhancing metabolic activities like
physiological and biochemical attributes.
3.5. Analysis of tea quality parameters in treated shoots
Quality parameters of CTC black tea such as theaflavins, thear-
ubigin, caffeine, colour, briskness and flavour indexes were increased
significantly irrespective of treatments over the untreated on red rust
infected bushes. Similar to that of the physiological and biochemical
parameters, quality parameters followed the same trend in bushes
treated with biocontrol agents. The untreated bushes registered
significantly low values when compared to all the other treatments
tested. The contents of theaflavin and thearubigin were less due to
decrease in polyphenols and catechin in untreated control bushes
followed by shoots treated with contact fungicides and algicides. The
highest theaflavins, thearubigin and caffeine contents registered
were 8.05, 0.89 and 3.63%; respectively in treatments applied with
S. sannanensis. Similarly, the highest quantitative estimation of
colour (4.88), briskness (17.54) and flavour (3.78%) indices was
recorded in the same treatment (Table 3).
3.6. Impact of harvesting methods on red rust disease control
Hard plucking without breaking back method was found to be
the best in terms of reducing disease incidence (10.57%) followed
by bare stalk plucking (12.77%). The disease incidence was highest
in continuously harvested plots with both flat and step shears and it
was comparatively low when an integrated schedule was followed
(Table 4). Among the different harvesting schedules, an integrated
schedule with both flat and step shears was the best in reducing
disease resistance and also increasing green leaf yield significantly.
Among the two integrated schedules adopted, integrated schedule
with flat shears was found to be better than the integrated schedule
with step shears reducing in terms of green leaf yield. The green
leaf yield was 3535 and 3496 kg ha1 made tea, respectively in the
in integrated schedule with flat shears and the integrated with step
shears. The yield was comparatively low in continuous shearing
which accounted between 3018 and 3087 kg ha1 made tea,
respectively in flat and step shears (Table 4).
3.7. Effect of dipping shears in different test solutions on red rust
control
Among different treatments tested to control the disease
incidence upon dipping the shears in solution containing deter-
gent, fungicide, algaecide or biocontrol agents before shearing in
field, dipping shears in carbendazim solution was found to be the
best (Table 5). The incidence was 14.5% in the plots where the
shears were dipped into a carbendazim solution. However, it was
19.3% in the plots where the shears were dipped into a contact
fungicide, copper oxychloride. Dipping of shears with teapol
resulted in 40% disease incidence followed by algaenozol (34.5%).

Table 3
Analysis of quality parameters of CTC black teas manufactured from treated and untreated red rust infected leaves (% by mass).

S. no Treatment details Theaflavin Thearubigin Caffeine Colour index Briskness index Flavour index
1. Mancozeb 6.11de 0.51bc 3.00bcde 4.03bc 14.22bc 2.18de
2. Copper oxychloride 6.23de 0.54bc 3.08bcde 4.08bc 14.89bc 2.21de
3. Carbendazim 7.14fghi 0.62de 3.20fg 4.32bde 15.11c 3.00fg
4. Thiophanate methyl 7.74fghi 0.60de 3.22fg 4.42bdef 15.00c 3.01fg
5. Nimbicidine 5.34bc 0.50bc 3.00bcde 4.00bc 14.20bc 2.00bc
6. Nimbidoxin 5.47bc 0.52bc 3.00bcde 4.03bc 14.21bc 2.00bc
7. P. fluorescence 8.00ij 0.80fghi 3.61ghi 4.20bc 16.88de 3.85hij
8. B. subtilis 7.88fghi 0.80fghi 3.57ghi 4.33bde 17.04ef 3.68hij
9. T. atroviride 8.00ij 0.84fghi 3.60ghi 4.31bde 16.18d 3.71h
10. T. harzianum 7.91fghi 0.86fghi 3.50ghi 4.04bc 16.18d 3.70h
11. S. griseus 8.01ij 0.82fghi 3.64ij 4.88b 17.54fg 3.81hij
12. S. sannanensis 8.05ij 0.89fghij 3.63ij 4.88b 17.54fg 3.78hij
13. Untreated control 1.55a 0.46ab 2.00a 2.02a 11.18a 1.00a
CD at P ¼ 0.05 1.43 0.51 1.04 2.21 1.23 1.37

Values are the mean of five replications.

Values followed by similar alphabet denote they are not significantly different at P < 0.05 and vice versa according to DMRT.
72 M. Ramya et al. / Crop Protection 44 (2013) 66e74

Table 4 4000 100

3687
Effect of harvesting methods on red rust disease incidence. Yeild (kg ha-1 made tea) Disease incidence (%)

Treatment details Disease Green leaf yield Productivity

incidence (%) (kg ha1 made tea) index 80
3000
f c c
Hand plucking with 10.57 3384 1.5  0.2 2523
breaking back
60
Bare stalk plucking 12.77e 3213de 1.4  0.1d
Continuous shearing 23.50ab 3018f 1.2  0.2ef 2000 47
(flat shears)
Continuous shearing 23.00 ab
3087 de
1.2  0.2 ef 40
(step shears)
25
Integrated schedule 17.45cd 3535ab 1.7  0.1a 1000
(flat shears) 20
Integrated schedule 18.23cd 3496ab 1.6  0.1b
(step shears)
CD at P ¼ 0.05 02.48 408.34 0.12 0 0
Values are the mean of five replications. Urea + MOP Treated Untreated control
Values followed by similar alphabet denote they are not significantly different at
P < 0.05 and vice versa according to DMRT. Fig. 4. Effect of urea and murate of potash on red rust disease control and green leaf
Disease incidence and green leaf yield were calculated at every plucking round (14e yield. Values are the mean of five replications. The line on each bar represents SE and
20 days intervals). the values in the bars followed by the common letters(s) are not significantly different
Productivity index was calculated in response to various treatments upon red rust at the 5% level according to DMRT.
control and green leaf yield in each trail plot. The green leaf yield potential was
converted into made tea kg ha1 (Refer 2.7. Materials and Methods section).
3.9. Enumeration of biocontrol agents

The observations on the enumeration of biocontrol agents

Dipping of shears with a solution containing biocontrol
showed that they could survive well in tea leaf samples for more
agents showed a moderate results which accounted in the range
than one year; however, there was a drastic decline in population
of 26.4e33.0%. The green leaf yield potential was high in
density with the age of the sample (Fig. 5). The survival rate of
biocontrol agents treated plants where it was between 3330 and
biocontrol agents after application on 12th month revealed the
3538 kg ha1 made tea.
population level of all the biocontrol agents was in the range 1.0e
2.5 cfu  104 g1 leaf dry weight.
3.8. Impact of urea and murate of potash foliar spray on red rust
control
4. Discussion
The efficacy of a solution containing urea and MOP upon red
Red rust is a predominant disease in southern Indian tea plan-
rust incidence and yield potential showed that there was a reduc-
tations affecting the yield potential significantly. The affected leaves
tion in red rust incidence and increase in green leaf yield in the
become variegated (greenish yellow or greenish pale white) which
field after imposing the treatments (Fig. 4). The disease incidence
in turn influence the bush health and tea quality parameters. The
recorded was 25.5% and yield was 3687 kg ha1 made tea. It
percentage of red rust incidence was reduced considerably after
was 47.3% and 2523 kg ha1 made tea in plots treated with Urea
imposing the treatments under field condition. Among the fungi-
and MOP while in disease incidence and yield; respectively in
cides and biocontrol agents, systemic fungicides gave a better
untreated control plots.
protection than that of biocontrol agents (Figs. 2 and 3). It might be
due to immediate action of fungicides upon the pathogen. Among
Table 5 the systemic fungicides, carbendazim (63.4%) was better than
Effect of dipping shears in different fungicides, algicides and biocontrol agents on contaf (59.6%) in terms of disease protection. According to Kalim
red rust disease incidence. et al. (2000), carbendazim is well known to have direct action on
Treatments Disease Green leaf yield Productivity
incidence (%) (kg ha1 made tea) index
12
Dipping of shears in 19.3gh 3304bcde 1.5  0.1bcde
copper oxychloride
cfu x 104 g-1 leaf dry weight

Dipping of shears in 14.5gh 3371bcde 1.5  0.2bcde 10

carbendazim
P. fluorescence
Dipping of shears in 34.5cde 3285fg 1.4  0.2fg 8 B. subtilis
algaenozol T.atroviride
cde bcde bcde
Dipping of shears in 33.0 3392 1.5  0.1
T. atroviride 6 T. harzianum
cde bcde
Dipping of shears in 32.7 3330 1.5  0.2bcde S. griseus
P. fluorescence 4 S. sannanensis
Dipping of shears in 26.4f 3538a 1.7  0.1a
S. scannensis
Dipping of shears in 40.0ab 3228fg 1.4  0.2fg 2
teepol
Untreated control 45.5ab 2524h 0.7  0.2h 0
CD at P ¼ 0.05 04.88 260.5 0.12 Initial 3 6 9 12
Values are the mean of five replications. Months after treatment
Values followed by similar alphabet denote they are not significantly different at
P < 0.05 and vice versa according to DMRT. Fig. 5. Survival of selected biocontrol agents in tea leaf samples. Values are the mean
Productivity index was calculated in response to various treatments upon red rust of five replications. The line on each bar represents SE and the values in the bars
control and green leaf yield in each trail plot. The green leaf yield potential was followed by the common letters(s) are not significantly different at the 5% level
converted into made tea kg ha1 (Refer 2.7. Materials and Methods section). according to DMRT.
 M. Ramya et al. / Crop Protection 44 (2013) 66e74
the pathogen through inhibition of spindle formation during
mitosis.
The reduction of red rust disease incidence was achieved
moderately with treatments where other biocontrol agents like
P. fluorescence, B. subtilis, T. atroviride and T. harzianum were used.
This clearly showed the inhibitory effect of antagonists on the
growth and reproduction of the pathogen C. parasiticus (Rozeena
and Ahmad, 2007). The filaments of C. parasiticus were
completely colonized by the antagonists and thereby the wall lysis
of filaments was observed. Moreover, the zoosporangium con-
taining zoospores of C. parasiticus were wrinkled and ruptured
subsequently due to the action of antagonists (Fig. 1). This clearly
indicated the potential of selected antagonists in parasitising the
pathogen. Further, the microscopic observations confirmed this
view. Studies have shown that Trichoderma spp. is known to
produce antibiotic substances (Papavizas, 1985) which would have
helped to suppress the development of the pathogen in situ.
The yield potential and attributes (productivity index) were
found the highest in the bushes amended with biocontrol agents,
which accounted in the range of 3088e3583 kg ha1 made tea.
The highest green leaf yield recoded was 3583 kg ha1 made tea in
the bushes sprayed with S. sannanensis (Figs. 2 and 3). From Figs. 2
and 3 observed that there was an increase in plucking surface,
plucking points, leaf moisture and dry matter contents and
reduction in banji content after imposing various treatments.
However, all these parameters were highest again in treatments
where biocontrol agents were applied. It may be due to the
recovery of plants from red rust infection and thereby increasing of
plant metabolism as well. It is also established that biocontrol
agents enhances growth by producing growth stimulating
hormones such as auxins, cytokinins and gibberellins (Windham
et al., 1986). A moderate yield was recorded in the plots treated
with systemic fungicides which are not only due to the disease
control but also due to their phytotonic effect (Sundaramoorthy
et al., 2012).
In order to control red rust disease in tea plantations, different
types of harvesting methods were adopted. The results were pre-
sented in the Table 4. The hard plucking without breaking back
method was found to be the best in terms of disease incidence
(10.57%) followed by bare stalk plucking (12.77%) method. The
highest disease incidence was observed in continuously harvested
plots. Similar observations were recorded by Chandramouli and
Baby (2002) in his studies on the relation of harvesting methods
and thorny stem blight disease of tea. The highest yield was
recorded in the treatments were integrated schedule was followed.
Continuous hard form of shearing will reduce the health of the bush
and make them susceptible to many pathogens (Sanjay et al., 2008).
Dipping the shears in detergent/fungicide or biocontrol agents’
solutions before shearing failed to protect the plants from the
disease. The treatments were imposed to prevent the spread of the
disease through shears. Failure of these treatments can be
explained in the light of endophytic nature of the pathogen.
Further, the superior performance of systemic fungicides to contact
fungicides also substantiated this view.
Data on the reduction of red rust incidence and production of
green leaf yield were recorded upon foliar spray of urea and MOP
solution. The disease incidence recorded was 25.5% and yield was
3687 kg ha1 made tea after imposing treatments (Fig. 4). Studies
have shown that potash fertilizer plays an important role in
developing bush frame as well as providing rigidity to the bushes
against pathogen vulnerability. Potassium enhances disease resis-
tance in plants by strengthening stalks and stems contributes to
a thicker cuticle which guards against disease and water loss,
controls the turgor pressure within plants to prevent wilting and
curling (Cakmak, 2005).
73
After application of treatments, physiological and biochemical
parameters were improved in red rust infected plants (Table 2).
Moreover, the biocontrol treated plants are physiologically more
active compared to that of the fungicides treated and untreated
control plants. In general, when the plants are physiologically
active, biochemical constituents are synthesized in larger amount
which resulted an increase in green leaf yield. Important
biochemical constituents, which are responsible for the formation
of quality attributes of black tea in the shoots ready for harvest.
Among the important biochemical constituents, polyphenols and
catechin contents directly influence the quality of made tea
(Bagyalakshmi et al., 2012). These parameters were found to be
high in biocontrol treated plants which ultimately produce the high
content of theaflavin, thearubigin and caffeine contents (Table 3).
It has been reported that the chemical basis of quality in black tea
indicated that theaflavin and thearubigin are two important factors
determining the liquoring characteristics of black tea (Hazarika
et al., 1984). The liquor characteristics are reflected in the flavour,
colour and briskness indexes in made tea samples manufactured
from biocontrol treated bushes (Table 3). Most of the quality
constituents in made tea manufactured from blister blight infected
shoots treated with ergosterol biosynthesis inhibitors (EBI’s) have
been shown to increase considerably (Baby et al., 2004).
The results on the enumeration of various biocontrol agents
covering bacterial, fungal and actinomycetes group revealed that all
the antagonists could survive well in the tea leaf samples for more
than a year (Fig. 5). The Survival of biocontrol agents in leaf samples
after using bioformulations were well reported earlier in grey
blight, blister blight and bird’s eye spot diseases of tea (Ajay et al.,
2004; Sanjay et al., 2008; Gnanamangai and Ponmurugan, 2011).
Competitive saprophytic ability of Trichoderma, Pseudomonas,
Bacillus and Streptomyces species has been reported which confirms
this view (Ahmad and Baker, 1987). Due to their saprophytic nature,
they could survive well in tea leaves and thus act as effective barrier
to fresh entry of the pathogen.
From the study, it may be concluded that biocontrol agents
could be effectively incorporated in the red rust disease manage-
ment in tea. The bioformulation containing actinomycetes could be
applied in the red rust infected fields in the rhizosphere soil at the
dosage of 2.5 kg/ha to reduce the disease incidence and to increase
the green leaf yield. Though red rust disease is caused by an algal
pathogen, biocontrol agents could be used extensively as an eco-
friendly approach to control the incidence in tea field. The usage of
algicides were incompetent in tea field due to the inefficient
binding (interaction) with tea leaves and the biotic and abiotic
factors involved. Tea harvesting practices like continuous plucking
also makes the usage of algicides ineffective and the selected
algicides may also be host (tea plants) specific.
Acknowledgements
The authors wish to express their sincere thanks to the
Chairman and Principal of K.S. Rangasamy College of Technology,
Tiruchengode, Tamil Nadu, India for their support and constant
encouragement. Dr. P. Mohan kumar, Director, UPASI Tea Research
Institute, Valparai and Dr. S. Marimuthu, Assistant General
Manager, Parry Agro Tea Industries Limited, Valparai, Tamil Nadu,
India are gratefully acknowledged for their excellent support in
conducting field trails.
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