Biocontrol of downy mildew disease of pearl

millet using Pseudomonas fluorescens

abstrak
Pseudomonas fluorescens was tested against pearl millet downy mildew disease by treating seeds with a pure
culture and formulated in talc powder. The bioagent was also tested as a foliar spray to pearl millet under
greenhouse and field conditions. Treated seeds increased seedling vigour and inhibited sporulation of downy
mildew pathogen. II fluorescens controlled downy mildew disease both by seed treatment and foliar
application, but efficacy was significantly higher when seed treatment was followed by a foliar application. Seed
treatment was better than foliar application alone. 0 1998 Elsevier Science Ltd. All rights reserved

Downy mildew of pearl millet [Pennisetum glaucum (L.) R. Br.], caused by

Sclerosporu gruminicolu (Sacc.) Schroter, is the most serious disease in major pearl millet
growing areas of the world. The economic significance of the crop has become more
accentuated than ever before, especially in relation to the high yielding cultivars (Shetty,
1990; Bangar and More, 1993; Singh et al., 1993a,b; Shetty et al., 199.5; Singh, 1995;
Sreedhara et al., 1995).
Pseudomonas puorescens (Trevisan) Migula is one of the most important antagonists
of Fusarium in cucumber (Liu et al., 1995), radish (Leeman et al., 1995) and in chick pea
(Vidhyasekaran and Muthamilan, 1995). Many greenhouse studies (Howell and Stipanovic,
1979; Thomshaw and Weller, 1988; Kaiser et al., 1989; Kraus and Loper, 1992), and a few
field experiments have been conducted (Trapero-Casas et al., 1990; Parke et al., 1991) to
show the efficiency of p JIuorescens in the management of many plant diseases.
Unsuccessful attempts to control downy mildew disease in pearl millet by using
chemicals have encouraged development of alternative means. The use of p jhorescens in
managing other plant diseases prompted us to test its efficiency in managing the downy
mildew disease in pearl millet, both under laboratory and field conditions.

Materials and methods

Seed material
Pearl millet seed material, HB3, which is highly susceptible to downy mildew ( > 90%
disease incidence when it is grown in plots infected with downy mildew) was obtained from the
International Crops Research Institute for the Semi-Arid Tropics, Hyderabad, India. The disease
reaction was confirmed by testing the seed sample following the procedure of Williams et al. (1981).
Antagonist culture
Pseudomonas jhorescens was isolated from the rhizosphere of pearl millet from a farmer’s
field at Mysore, India. After removing the loosely adhering soil from freshly excised roots, root
segments (1 g) were shaken in 100 ml of sterile distilled water for one hour (Vidhyasekaran and
Muthamilan, 1995). fluorescens was isolated and maintained using King’smedium B (KMB) (King
et al., 1954). The identity of the organism was further confirmed by conducting various tests specific
to fluorescens (Stanier et al., 1966). The 48 h-old-culture in KMB was centrifuged at 10,000 rpm
for 10 min. The pellets were resuspended in sterile distilled water and washing was repeated thrice.
The washed bacterial pellet was made into a turbid solution with sterile distilled water. The
optical density (OD) of the solution was adjusted to 0.45 (A610 nm) to obtain 1 x lo8 CFU/ml with
the help of a UV-Visible spectrophotometer (Hitachi U-2000, Japan). OD was adjusted to 0.225
(A610 nm) to get 1 x lo4 CFU/ml (Mortensen, 1992). The bacterial formulation was prepared by
mixing 400 ml of bacterial suspension containing 9 x lo8 CFU/ml to 1 kg of the purified talc powder
under sterile conditions, and -stored at polythene bags.

Pathogen and inoculation procedure

A suspension of S. Graminicola 4°C in sealed zoospores was prepared in distilled water
(Safeeulla, 1976). The zoospores (approximately 3O,OOO/ml) were inoculated to 5-day-old seedlings
maintained in clay pots by the whorl-inoculation technique of Singh and Gopinath (1985).

Effect of P. fluorescens and its formulation on seed germination and seedling vigour under
laboratory conditions
Seeds were treated with the pure culture of II fluorescens at the rate of 1 x lo4 and 1 x lo8
CFU/ml. Wet seeds of pearl millet were gently shaken in fluorescens suspension prepared in sterile
distilled water for 24 h. Seeds soaked in sterile distilled water served as control. After 24 h, seeds
were blot-dried, plated on wet blotters and the germination test was carried out by the paper towel
method (ISTA, 1993). Vigour index was calculated by using the formula (mean shoot length+mean
root length) x per cent germination (Abdul Baki and Anderson, 1973). The experiment was carried out
with four replicates of 100 seeds each.
Seeds were treated with freshly prepared R jluorescens formulation at the rate of 6 and 10 g of the
formulation per kg seeds. Seeds wet with 5 ml of water were shaken overnight in the formulation.
Seeds shaken only with 5 ml of water served as control. The treated and control seeds were subjected
to germination test replicated four times with 100 seeds each and vigour index was calculated as
explained earlier.
Effect of P. fluorescens on the incidence of downy mildew disease under greenhouse
conditions
Seed treatment. Seedlings from the seeds treated either with the pure culture of R
fluorescens (at the concentration of 1 x lo4 and 1 x 10s CFU/ml) or its formulation (at the
concentration of 6 and 10 g/kg seed) were raised in clay pots. Seedlings raised from the untreated
seeds served as control. Five-day-old seedlings were whorl-inoculated with S. Graminicola zoospore
suspension. Pots were maintained in greenhouse conditions and the disease incidence (the number of
plants showing the typical symptoms of downy mildew disease such as stunted growth, sporulation,
malformation etc.) was recorded after 15 days and also at dough stage.

Foliar application
Pearl millet seedlings were raised in clay pots from untreated seeds. Whorl-inoculation of the
5-day-old seedlings with the pathogen was carried out. The pure culture of fluorescens (1 x lo4 and
1 x lo* CFU/ml) was sprayed to run off to 7-day-old and 16day-old plants. Disease incidence was
recorded as explained previously.

Seed treatment followed by foliar application

Pearl millet seedlings were raised in clay pots from the seeds treated either with the pure
culture of fluuorescens (1 x lo4 and 1 x 10’ CFU/ml) or with the formulation (6 and 10 g/kg).
Seedlings raised from the untreated seeds served as control. Five-day-old seedlings were whorl-
inoculated with S. Graminicola and 7-day-old and 14-day-old plants sprayed with p. fluorescens at
a concentration of 1 x lo8 CFU/ml. Disease incidence was recorded as explained previously.

Effect of P. fluorescens on S. graminicola

Effect on sporulation. Leaves infected with downy mildew were collected as already
explained. Pieces of washed leaf were attached with cellophane tape on the upper lid of a Petri plate
so that the sporulating side of the leaf was facing the lower lid. Under aseptic conditions, the upper lid
of a 2-day-old fluorescens culture plate was removed and was replaced with an upper lid containing
a leaf. The plates were kept for incubation under darkness at 22+ 1°C. The pieces of leaf were
observed for the effect of p.fluorescens on sporulation after 12 h incubated either with distilled water
or with KMB in the lower lid served as controls.

Effect of P. fluorescens on the incidence of downy mildew disease under field conditions
The field trials were carried out with all the treatments namely seed treatment, foliar
applications and seed treatment followed by foliar application as explained in the greenhouse studies.
All the trials were carried out in a plot infected with downy mildew, which has been maintained over
the last two decades, by a frequent showering of inoculum from dried, diseased pearl millet plants.
Thus downy mildew inoculum as oospores get into the soil every season. During screening, the highly
susceptible cultivar HB3, was sown as infector rows, 3 weeks before that of the test material.
Seedlings also received the inoculum from neighbouring infector rows.
In all the field experiments there were 16 replicates in a randomised block design with about
50-60 plants in each replicate and each experiment was repeated in two consecutive seasons. Normal
agronomical practices such as plants at every 15 cm in rows spaced 50 cm apart were followed with
the plot row length of 5 m.
Disease screening was done after 30 days and also at dough stage (Williams et al., 1981).
Table 1. Effect of P. fhorescens pure culture on seed germination and seedling vigour

Values are the means 2S.E. of four replicates of 100 seeds each.
MRL - mean root length; MSL - mean shoot length; VI - vigour index;
CFU - colony forming units.
The values in the column followed by same letter(s) are not significantly
different according to DMRT @ = 0.05).

Statistical procedures
Data on percentages were transformed to arcsin and analysis of variance was carried out with
transformed values. The means were compared for significance using Duncan’s multiple new range
test (DMRT, p = 0.05).

Results
Effect of P. fluorescens pure culture and its formulation on seed germination and seedling
vigour
There was no significant change in the germination of seeds treated with bacterial pure
culture over untreated ones (Table I), but the mean root length and mean shoot length increased
significantly due to the treatment and consequently vigour index increased. The increase was not
dependent on the bacterial cell count.
When the seeds were treated with the bacterial formulation, germination did not show a
significant change over untreated seeds (Ebke 2). However, the increase in mean root length and the
mean shoot length due to the formulation treatment was dependent on the dosage, with no significant
difference in the vigour at 6 g/kg, but, a significant increase at the dosage of 10 g/kg.
 More vigorous shoot and root growth can be seen in the bacteria treated plants (Figure Ib)
when compared to untreated ones (Figure la).

Effect of P. fhorescens on the incidence of downy mildew disease under greenhouse

conditions
With seed treatment disease incidence in the untreated seeds was 87.6%, whereas it was
significantly (p = 0.05) reduced in seeds treated with the bacterial pure culture, by about 70% at the
lower dosage compared with 85% at the higher dosage (Figure 2a).
The disease incidence was reduced from 86% in the control to 30 and 14% when seeds were
treated with 6 g and 10 g/kg of the formulation, respectively (Figure 2d). Foliar applications reduced
disease incidence from 87% in untreated seeds to 40 and 32%, respectively, in the plants sprayed with
the bacterial suspension at 1 x lo4 and 1 x 10’ CFU/ml (F&re 2b). Disease incidence of 84% in the
untreated seeds significantly @ = 0.05) reduced to 25 and 9%, respectively, when seeds treated with
bacterial pure culture at the concentrations of 1 x 104and 1 x lo8 CFUlml, were subsequently sprayed
(Figure 2c). The reduction in the disease incidence increased with the increase in the bacterial load in
the treatments.
The bacterial formulation followed by foliar application also had a similar effect on disease
incidence (Figure 2e), the higher dosage being more effective.

Table 2. Effect of P. fluorescens talc powder based formulation on seed germination and
seedling vigour

Values are the mean &SE. of four replicates of 100 seeds each. MRL - mean root
length; MSL - mean shoot length; VI - vigour index; CFU - colony forming units. The
values in the column followed by same letter(s) are not Ggnificantly different
according DMRT 0, = 0.05).
Effect of P. fluorescens on S. graminicola
Effect of P. fluorescens on sporulation. Infected leaf tissue with the bacterial culture in a
Petri dish did not sporulate, whereas leaf pieces incubated either with the KEI medium or distilled
water sporulated well.

Effect of P. fluorescens on the incidence of downy mildew disease under field conditions
The disease incidence in the untreated seeds of 86.2% was reduced to 16 and 13%,
respectively, in the seeds treated with the bacterial pure culture at the concentrations of 1 x lo4 and 1
x lo8 CFU/ml (Figure 3a). Similarly when seeds were treated with bacterial formulation at the rate of
6 and 10 g/kg, disease incidence was significantly @ = 0.05) reduced to 24 and 18%, respectively
(Figure 3c). A foliar treatment also significantly (p = 0.05) reduced disease incidence from 81.6% in
the untreated seeds to 29 and 24%, respectively, when plants were sprayed with 1 x lo4 and 1 x lo8
CFU/ml bacterial suspension, (Figure 3b). The combination of seed treatment (10 g/kg seed) followed
by a foliar application reduced disease incidence from 90% in the untreated seeds to 8% (Figure 3d).
Figure 2. Effect of Pseudomonas fluorescens seed treatment and foliar spray on the
incidence of downy mildew disease under greenhouse conditions. The bar(s) represented by
the same letter(s) do not significantly differ at 5% level when subjected to DMRT. The lines
on each bar represent standard error

Discussion
Despite several studies showing the antagonistic effect of ,fluorescens on various pathogens
(Liu et al., 1995; Raaijmakers et al., 1995; Expert and Digat, 199.5) no study on biocontrol of S.
gruminicolu using this bacterium had been carried out. Our studies show that p.fluorecens can be
used effectively to manage the downy mildew disease of pearl millet both under greenhouse and field
conditions, whether applied as a pure culture or formulated with talc.
Laboratory studies indicated both root and shoot growth was prompted by treating seeds with
P. fluorecens. This growth promotion confirms reports on several other crops (Kaiser et al., 1989;
Parke et al., 1991; Traperocasas et al., 1990; Whipps and Lumsden, 1991).
While the bacterium could be used as a seed treatment or foliar application, the synergistic
effect of both seed treatment and foliar application was better than the individual methods of
treatment. The talc formulation of p fluorescens developed during the present study was equally as
efficient as pure culture in controlling the disease, both under greenhouse and field conditions. Thus
seeds can be treated with a pure culture or the formulation of p. fluorescens before supplying them
to farmers for sowing, as recommended by Vidhyasekaran and Muthamilan (1995).
The laboratory study also showed that exposure of the pathogen to p.fluorescens can inhibit
sporulation on the diseased leaves. p fluorescens may control the disease either by inhibition of the
pathogen, or by producing volatile compounds that affect sporulation.
According to Liu et al. (1995) the controlling mechanism might be due to the production of
antibiotics or related substances by p. fluorescens. Further studies are in progress to understand the
mechanism of the interaction between p fluorecens and S. gruminicola in pearl millet.
Figure 3. Effect of Pseudomonas fluorescens seed treatment and foliar spray on the
incidence of downy mildew disease under field conditions. The bar(s) represented by the
same letter(s) do not significantly differ at 5% level when subjected to DMRT. The lines on
each bar represent standard error