Biological control of coffee leaf Journal of Biopesticides, 2(1): 94-98 (2009) 94

Biological control of cof fee leaf r ust pathogen, Hemileia

vastatrix Berkeley and Broome using Bacillus subtilis and
Pseudomonas fluorescens
S. Daivasikamani1 and Rajanaika2

ABSTRACT
Bacterial antagonists isolated from coffee rhizosphere soils were evaluated at different concentrations alone
and in combination against Hemileia vastatrix, the causal organism of leaf rust disease of coffee under in
vitro and in vivo conditions. Under in vitro conditions, the rhizobacterium, Bacillus subtilis inhibited the
growth of urediospores to an extent of 68.20% at a test dosage of 1 x 109 cfu ml-1 followed by Pseudomonas
fluorescens to an extent of 64.50% at the same test dose. Combination of P. fluorescens and B. subtilis at 1 x
108 cfu ml -1 inhibited the growth of urediospores of the CLR pathogen to an extent of 61.46%. The natural
inhibition of germination in the check treatment was 37.79%. Among fungicides used for comparison, the per
cent inhibition over control was maximum in Bayleton (89.03%) followed by Bordeaux mixture (80.64%). Under
in vivo conditions, during the period of season-I, maximum reduction in disease index was recorded in
treatment with Bayleton (71.84%) followed by Bordeaux mixture (53.37%). Among the bioagents, B. subtilis
recorded maximum disease reduction (42.98%) at a test dosage of 1 x 109 cfu ml-1 followed by P. fluorescens
(33.65 %) at the same test dose. P. fluorescens and B. subtilis in combination at 1 x 108 cfu ml-1 reduced the
disease incidence to an extent of 26.45%. The season-II (post-monsoon period) has recorded less disease
reduction compared to season-I (pre-monsoon period).

Key words: Hemileia vastatrix, arabica coffee, bacterial antagonists.

INTRODUCTION are the infection propagules disseminated by wind, water,

Coffee trees belong to the genus Coffea L. of the family insects etc. Severe rust incidence may lead to a foliage
Rubiaceae. Coffea arabica L. (arabica coffee) and Coffea loss up to 50% and berries up to 70% (Bhat et al., 2000).
canephora Pierre ex Froehner (robusta coffee) are the two Hence, effective management of this major disease is of
species of Coffea now commercially cultivated throughout most important in sustained production and productivity
the coffee growing countries. Arabica coffee is highly of coffee.
susceptible to many major diseases compared to robusta At present, adopting cultural practices, planting resistant
coffee (Anonymous, 2003). In India, the cultivated area varieties and application of contact and systemic
under arabica coffee is about 1.79 lakh ha. with a produ fungicides are the control measures recommended
ction of 0.99 lakh MT. The average productivity was 657 (Anonymous, 1998). Though satisfactory control of the
kg ha -1 clean coffee in the year 2006-07 (Anonymous, disease by using various fungicides has been documented
2008). (Mayne et al., 1933; Muthappa and Nirmala Kumari, 1976;
Coffee leaf rust (CLR) caused by the fungus Hemileia Muthappa, 1981; Daivasikamani and Govindarajan, 1989;
vastatrix Berkeley and Broome is an obligate parasite and Hanumantha et al., 1989), continuous use of fungicides
is host specific. It is a major disease of arabica coffee may pose many problems like toxicity to non-target
causing economic loss and is reported from over fifty organisms, development of resistance in the populations
coffee growing countries. The fungus was first observed of the pathogen, fungicide residues in the final product
on coffee in India during 1869 and infects only the foliage and environment and ground water pollution.
and very rarely the young branches. The fungus exists in In the recent years there is a shift in the control of plant
different physiological forms (races). The uredinial stage diseases from the regular use of pesticides to an alternate
is the only viable perpetuating stage of the coffee rust and more eco-friendly biopesticide and plant based
fungus. Wet weather, wind and intermittent rain and products. Many fungi and bacteria have the potential to
sunshine favours the disease development. Urediospores act as biological control agents. The indigenous strains

© JBiopest. 63
S. Daivasikamani1 and Rajanaika2 95
of Bacillus subtilis and Pseudomonas fluorescens appear stereoscopic microscope. The total number of urediospore
to function as better antagonists in disease control as present in each microscopic field and the number of
they are well adapted to local conditions. Rangeshwaran germinated urediospore were counted. Based on the
and Prasad (2000) reported the effectiveness of P. count, the percentage inhibition over control was
fluorescens against chickpea wilt pathogen. Not much estimated.
work on biocontrol of coffee leaf rust pathogen has been
undertaken. The present investigation was carried out to Field evaluation of bacterial antagonists
assess the antagonistic effect of B. subtilis and P. Field experiments were conducted for two years during
fluorescens, isolated from rhizosphere soils of coffee. The 2006 and 2007 at Gundikhan estate near CCRI. The bacteria
bacterial antagonists were tested in vitro and in vivo for tested under in vitro conditions were further evaluated in
their effectiveness either in suppressing or controlling the field for their antagonistic effect against coffee leaf
the coffee leaf rust pathogen. rust (CLR) pathogen. The experiment was laid out in a
randomized block design (RBD). Water sprayed plots were
MATERIALS AND METHODS considered as control and fungicide sprayed plots were
Evaluation of bacterial antagonists under in vitro used for comparison. There were ten treatments with a
conditions plot size each of 9.0 x 5.4 m2 with three replications. The
The bacterial antagonists B. subtilis and P. fluorescens plant material used was 15 years old arabica coffee variety
isolated using standard techniques from coffee Cauvery. The agronomic practices were carried out as per
rhizosphere soils of Chikmagalur coffee zone were used standard recommendations (Anonymous, 2003).
for the present study. Laboratory studies were carried out The biocontrol agents, B. subtilis and P. fluorescens were
at the Central Coffee Research Institute (CCRI), Bale tested individually at 1 x 10 7, 1 x 10 8 and 1 x 109 cfu ml-1
honnur, Chikmagalur, Karnataka during the year 2006. The concentrations. In combination spray the dose of the
experiment was laid out in a completely randomized design agents was kept at 1 x 108 cfu ml-1 each. Bordeaux mixture
(CRD). There were ten treatments with five replications. was used at the recommended dose of 0.5% while Bayleton
The bacterial antagonists were tested at three different 25 WP was used at 0.02% a.i. Check plants were treated
doses (1 x 107, 1 x 108 and 1 x 109 cfu ml-1) individually and with sterile water. The foliar spray was imposed with high
in combination (both the bacterium at 1 x 108 cfu ml-1). The volume ASPEE – Gator sprayer during the last week of
May (season I = Pre monsoon) and first week of September
recommended fungicides like Bordeaux mixture at 0.5%
(season II = Post monsoon) in the year 2006 and 2007.
and Bayleton 25 WP (triadimefon) at 0.02% a.i. were
The adjuvant, Indtron-AE (Indofil Chemicals Company,
included as treatments for comparison along with a control.
Mumbai) a non-ionic spreader, sticker and activator was
Rust infected leaves were collected from the most used at 0.3 ml per litre of spray solution. For each treatment,
susceptible arabica coffee genotype (Cauvery) grown at 1500 litres of spray mixture (containing bioagents or
the CCRI farm. The urediospores were collected from fungicides in water) per hectare was used. Observations
urediosori of the infected leaves and ten milligram of on percentage of rust incidence in the experimental plot
urediospores was weighed and added to each of the test was recorded prior to treatment and thereafter at 15 days
dose containing bacterial suspensions and fungicide interval up to 90 days. In each treatment, fifteen plants
solutions taken in a 10 ml glass test tube (Corning make). were selected for scoring of leaf rust incidence. The per
The spores were soaked for 5 minutes in each of the cent disease index was calculated based on the total
bacterial suspension and fungicide solutions. In sufficient number of healthy leaves and total number of infected
numbers of sterilized Petri plates (9 cm dia.), 15 ml each of leaves from the tagged branches and the data were
2% water agar medium was poured and allowed to solidify. tabulated.
To each Petri plate, one ml of the fungicide or bacterial
suspension containing urediospores were added and Statistical analyses
uniformly spread over the agar medium. The Petri plates Data collected from laboratory and field experiments were
analysed statistically using standard statistical
containing only 2% agar inoculated with urediospores of
procedures. The percentage values were subjected to arc-
CLR pathogen served as the control. The inoculated Petri
sine transformation. The treatment means were compared
plates were incubated over night in darkness at room
using least significant difference (LSD) and Duncan’s
temperature (24 ± 2oC). After 18 hrs of incubation, the multiple range tests (DMRT) at 5% level of significance
inoculated plates were observed directly under a (Gomez and Gomez, 1984).
Biological control of coffee leaf 96
RESULTS AND DISCUSSION Field efficacy of biological control agents
Bioefficacy under laboratory conditions Based on the mean values of individual season, the
The results on in vitro bacterial antagonisms of B. subtilis percentage of disease reduction in a season by the
and P. fluorescens are presented in Table 1. Per cent bacterial antagonist at different doses and also the
inhibition of urediospore germination over control in treatment effect of fungicides were compared with water
different treatments is presented in the table. The sprayed control. The data are presented in Table 2.
percentage germination of urediospores of H. vastatrix The cumulative data on mean per cent disease reduction
in different test doses of the bioagents was compared of two seasons over a period of two years indicated that
with the fungicides as well as untreated control. Lowest during season-I (pre-monsoon period), maximum reduction
spore germination (6.82%) was observed in Bayleton in disease index was recorded in fungicide Bayleton
fungicide treatment. Among the two bacterial antagonists, treatment (71.84%) followed by Bordeaux mixture (53.37%).
B. subtilis recorded less spore germination (19.78%) Among the bioagents, B. subtilis recorded maximum
compared to P. fluorescens (22.08%) at the higher disease reduction (42.98%) at a test dosage of 1 x 109 cfu
concentrations (1 x 10 9 cfu ml -1) tested. A maximum ml-1 followed by P. fluorescens (33.65 %) at the same test
percentage of (62.21%) germination of uredospore was dose. P. fluorescens and B. subtilis in combination at 1 x
observed in control treatment. 108 cfu ml-1 have reduced the disease incidence to an extent
Among the fungicides, the maximum per cent inhibition of 26.45% in the field. The cumulative disease reduction
over control was observed in Bayleton (89.03%) followed during the post-monsoon period (season-II) in fungicide
by Bordeaux mixture (80.64%). The rhizobacterium, B. Bayleton treatment was 55.29% followed by Bordeaux
subtilis inhibited the growth of urediospores (68.20%) at mixture 35.42%. Among the bioagents, B. subtilis recorded
a test dosage of 1 x 109 cfu ml-1 followed by P. fluorescens maximum disease reduction (28.40%) at a test dosage of 1
(64.50%) at the same test dose. Combination of P. x 109 cfu ml-1 followed by P. fluorescens (22.54 %) at the
fluorescens and B. subtilis at 1 x 108 cfu ml-1 inhibited the same test dose. P. fluorescens and B. subtilis combination
growth of urediospores of the CLR pathogen to an extent at 1 x 108 cfu ml-1 have reduced the disease incidence to an
of 61.46%. The laboratory studies indicated that the
extent of 17.68% under field conditions. Between seasons
antagonists either P. fluorescens or B. subtilis could be
used alone or in combination for the management of coffee within a year, the season I (pre-monsoon period) in both
leaf rust disease as a possible alternative to fungicides the years of experiment recorded considerably higher
after appropriate field evaluation.

Table 1. Effect of bacterial antagonists on germination of urediospores of H. vastatrix

Treatments Germination percentage `% inhibition over control

T1 - P. fluorescens @ 1 x 109 cfu ml -1 22.08 (27.92)bc 64.50 (53.10)cd

T2 - P. fluorescens @ 1 x 108 cfu ml-1 25.03 (30.01)bc 59.76 (50.25)de
T3 - P. fluorescens @ 1 x 107 cfu ml -1 32.58 (34.37)d 47.62 (43.20)f
T4 - B. subtilis @ 1 x 10 9 cfu ml -1 19.78 (25.81)b 68.20 (55.40)c
T5 - B. subtilis @ 1 x 10 8 cfu ml -1 24.33 (29.07)bc 60.89 (50.81)de
T6 - B. subtilis @ 1 x 10 7 cfu ml -1 27.12 (31.22)cd 56.40 (48.50)e
T7 - P. fluorescens @ 1 x 108 cfu ml-1 23.97 (28.76)bc 61.46 (51.00)de
+ B. subtilis @ 1 x 108 cfu ml-1
T8 - Bordeaux mixture @ 0.5% 12.04 (20.17)a 80.64 (63.11)b
T9 - Bayleton 25 WP @ 0.02% a.i. 6.82 (14.18)a 89.03 (70.63)a
T10 - Un-treated control (Sterile distilled water) 62.21 (51.84)e -
SEM ± 3.12 2.98
CD (P=0.05) 5.85 6.41
Figures in parentheses are arc-sine transformed values; In a column, means followed by same letter(s) are not significantly
different at P=0.05 as per LSD.
S. Daivasikamani1 and Rajanaika2 97
Table 2. Effect of bacterial antagonists on coffee leaf rust pathogen (Cumulative mean of two years and four seasons)
Per cent disease reduction over control
Treatments Pooled Mean
Year 2006 Year 2007
Season I Season II Season I Season II Season I Season II

T1 - P. fluorescens 36.67 (36.85)d 21.06 (27.25)cd 30.63 (33.20)c 24.01 (29.32)cd 33.65 (34.15)d 22.54 (28.19)d
@ 1 x109 cfu ml-1
T2 - P. fluorescens 20.53 (26.66)ef 9.74 (17.75)ef 11.35 (19.47)ef 11.41 (19.46)ef 15.94 (22.72)f 10.58 (18.27)fg
@ 1 x 108 cfu ml-1
T3 - P. fluorescens 10.49 (18.71)g 3.72 (9.87)f 5.31 (12.85)f 6.05 (14.13)f 7.90 (15.12)g 4.89 (10.15)h
@ 1 x 107 cfu ml-1
T4 - B. subtilis 46.75 (42.67)c 26.85 (30.63)bc 39.21 (38.66)c 29.94 (32.49)bc 42.98 (40.21)c 28.40 (31.72)c
@ 1 x 109 cfu ml-1
T5 - B. subtilis 22.03 (27.92)e 10.74 (18.39)ef 14.37 (21.96)de 13.91 (21.78)ef 18.20 (25.09)f 12.33 (20.19)f
@ 1 x 108 cfu ml-1
T6 - B. subtilis 12.26 (20.26)fg 4.82 (11.66)f 7.85 (15.31)ef 8.53 (16.38)f 10.06 (18.17)g 6.68 (14.22)gh
@ 1 x 107 cfu ml-1
T7 - P. fluorescens 31.46 (33.86)d 16.42 (23.55)de 21.44 (28.27)d 18.93 (25.12)de 26.45 (29.53)e 17.68 (24.12)e
+ B. subtilis @ 1 x 108 +
1 x 108 cfu ml-1
T8 - Bayleton 25 WP 76.81 (60.63)a 55.79 (47.86)a 66.87 (54.33)a 54.78 (47.25)a 71.84 (58.32)a 55.29 (46.39)a
@ 0.02% a.i.
T9 - Bordeaux mixture 56.51 (48.43)b 34.34 (35.63)b 50.22 (45.03)b 36.49 (36.83)b 53.37 (46.20)b 35.42 (36.09)b
@ 0.5%
Figures in parentheses are arc-sine transformed values; In a column, means followed by same letter(s) are not
significantly different at P=0.05 as per DMRT.

percentage of disease reduction over season II (post- ACKNOWLEDGEMENT

monsoon period). This could be due to the reason of higher The authors are grateful to Dr. Jayarama, Director of
levels of rust incidence generally recorded in the Research, Central Coffee Research Institute, Balehonnur
plantation during the post-monsoon period (season II). for providing the facilities to carry out the experiments.
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