International Journal of Agricultural

Science and Research (IJASR)

ISSN(P): 2250-0057; ISSN(E): 2321-0087
Vol. 6, Issue 3, Jun 2016, 45-54
TJPRC Pvt. Ltd.

COMBINED EFFECT OF BIOPRIMING AND POLYMER

COATING AGAINST CHILLI DAMPING OFF
S. SATHYA1, S. LAKSHMI2 & S. NAKKEERAN3
1

Department of Seed Science and Technology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India
2,3

Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India

ABSTRACT
Chilli (Capsicum annuum) damping-off can be common under greenhouse conditions mainly caused by
Pythium ultimum Trow. this is estimated to be associated with 75% of the seed emergence problems. Seed treatments are
only effective against the soil borne pathogens in the seedling stages. Seeds were subjected to biopriming (B.
amyloliquefaciens and P. fluorescens) and polymer coating, dry seed treatment along with P. fluorescens, B.
amyloliquefaciens and metalaxyl. Biopriming with 6 per cent B. amyloliquefaciens and polymer coating @ 10 ml kg-1 of
seed was found higher for speed of germination, germination, shoot length, root length, vigour index, dry matter
production and population of biocontrol agents (6.9, 93 %, 4.7 cm, 11.2 cm, 1479, 28 mg seedlings-10, 25x108 cfu ml-1,
respectively) compared to other treatments. In greenhouse studies, seeds bioprimed with P. fluorescens and yellow
and dry seed treatment with metalaxyl @ 1 g kg-1 of seed showed 47 per cent reduction in the incidence of damping off
over control.
KEYWORDS: Bacillus Amyloliquefaciens VB7, Pseudomonas Fluorescens Pf1, Metalaxyl, Polymer Coating, Damping
Off

Original Article

polymer coating @ 10 ml kg-1 of seed, 6 per cent B amyloliquefaciens and yellow polymer coating @ 10 ml kg-1 of seed

Received: Mar 25, 2016; Accepted: Apr 07, 2016; Published: Apr 12, 2016; Paper Id.: IJASRJUN20167

INTRODUCTION
Chilli (Capsicum annuum) is one of the important spice crop cultivated around the world for its pungency
and colour. The pungency is due to the active principle capsicin content in the skin and septa of the fruit. It is also
used in beverages and preparation of medicines (Zagade et al., 2012).
Owing to the potentiality, intensive cultivation of chilli is attacked by several diseases leading to loss of
yield in terms of quality and quantity. Among these diseases, damping off incited by Pythium spp. is responsible
for 90 per cent of plant death either as pre or post-emergence in nurseries and fields (Sowmini, 1961). Pythium
species are soil borne plant pathogenic fungi, which causes pre and post emergence damping off (Shah Smith and
Burns, 1996). Damping off in chilli is caused by two species of Pythium namely, Pythium ultimum Trow. Fitz. and
Pythium aphanidermatum (Edson). Among them, P. aphanidermatum infects chillies grown under hydroponic
culture while, P. ultimum occurs relatively in higher frequency in dry areas (Zagade et al., 2012). Management of
Pythium damping off becomes as a key issue, because of the prolonged survival of propagules, rapid germination
of sporangia (Osburn et al., 1989) and quick infection (Whipps and Lumsden, 1991).
Though fungicides offer a greater degree of protection against pathogens, accumulation of residues in the

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S. Sathya, S. Lakshmi & S. Nakkeeran

fruits and their adverse effect on beneficial soil microorganisms and the environment cannot be ignored. Therefore,
biocontrol agents appear to hold promise in disease management. Since, biological control is a key component of
integrated disease management; it is active against specific pathogens for wider application (Nakkeeran et al., 2006).
Increase in the vigour and immunity of the crop plants via biopriming aids in curbing the pathogen at seed and
seedling stage itself. Hence, biopriming and coating the seeds with polymer based technique will be a viable strategy to
check the disease spread. Heydecker (1973) defined seed priming as a presowing seed invigouration treatment in which
seeds are soaked in osmotic solution that allows them to imbibe water and go through the first phase of germination, but
does not permit radicle or plumule protrusion through seed coat. Seed treatment with biocontrol agents along with priming
agents may serve as an important means of managing many of the soil and seed borne diseases, the process often known as
biopriming (Rao et al., 2007). Polymer coating is application of a thin, uniform layer of polymer over seeds without
significantly increasing seed size and weight. The film formed around the seed acts as a physical barrier, which has been
reported to reduce leaching of inhibitors from the seed coverings and may restrict oxygen diffusion to the embryo
(Vanangamudi et al., 2003).

MATERIAL AND METHODS

Chilli cv. K 2 seeds were subjected to the following dry seed treatments, biopriming, combination of biopriming
and polymer coating treatments.
Treatment Details
T1: Control
T2: Dry seed treatment with metalaxyl @ 1 g kg-1 of seed.
T3: Dry seed treatment with P. fluorescens Pf1@ 10 g kg-1 of seed.
T4: Dry seed treatment with B. amyloliquefaciens VB7 @ 10 g kg-1 of seed.
T5: Biopriming with 8 % P. fluorescens Pf1 + yellow polymer coating @ 10 ml kg-1 of seed.
T6: Biopriming with 6 % B. amyloliquefaciens VB7 + yellow polymer coating @ 10 ml kg-1 of seed.
T7: Biopriming with 8 % P. fluorescens Pf1.
T8: Biopriming with 6 % B. amyloliquefaciens VB7.
The experiment was carried out with four replications in Completely Randomized Design (CRD).
Seeds were bioprimed with liquid formulation of B. amyloliquefaciens VB7 and P. fluorescens Pf1 at
concentrations of 6 and 8 %, respectively. After the soaking duration of 12 h, the seeds were removed from the solutions
and shade dried at room temperature. Similarly after biopriming, seeds were removed and immediately coated with
polymer (10 ml kg-1 of seed) and then shade dried at room temperature (28 2C). The control as non primed seeds. The
control and treated seeds were evaluated for seed and seedling quality parameters.
Effect of Biopriming and Polymer Coating for Enhancing Seed Quality
Population of Biological Agents
The number of colony forming unit (cfu) ml-1 solution of seed wash was enumerated using KingsB medium
Impact Factor (JCC): 4.7987

NAAS Rating: 3.53

Combined Effect of Biopriming and Polymer Coating Against Chilli Damping Off

47

(King et al., 1954) (Peptone - 20.0 g; K2HPO4 - 1.5 g; MgSO4 - 1.5 g; Glycerol - 10.0 ml; Agar - 20.0 g; Distilled water - 1
lit) and nutrient agar medium (Peptone - 5.0 g; Beef extract - 3.0 g; NaCl -5.0 g; Agar - 20 g; Distilled water - 1 lit) for both
P. fluorescens Pf1 and B. amyloliquefaciens VB7, respectively. One gram of treated seeds was mixed with 10 ml of sterile
water and shaked well at 150 rpm for two minutes. Ten fold serial dilutions were prepared for each sample and 1.0 ml
aliquots from 10-8 dilutions were transferred to sterile Petri plates. The slimy (P. fluorescens Pf1), irregular and wrinkled
colonies with serrated margins (B. amyloliquefaciens VB7) appeared after 48 h of incubation were counted and the
population was expressed as cfu ml-1 of seed wash.
Speed of Germination
Four replicates of twenty five seeds each were used to test the germination speed of seeds from different
treatments. The seeds showing radicle protrusion were counted everyday from the second day after sowing until fourteenth
day. From the number of seeds germinated on each day, the speed of germination was calculated using the following
formula and the results were expressed in number (Maguire, 1962).

Speed of germination =

X1 X 2 X1
X X n 1
+
+ ........... + n
Y1
Y2
Yn

X1: Number of seeds germinated at first count; X2- Number of seeds germinated at second count
Xn: Number of seeds germinated on nth count; Y1- Number of days from sowing to first count
Y2: Number of days from sowing to second count; Yn- Number of days from sowing to nth count
Germination
Germination test was conducted by following the procedure outlined in ISTA Seed Testing Rules (2011) using
paper (between paper) medium. Four replicates of 100 seeds each were germinated in a germination room maintained at 25
2C temperature and 90 3 % relative humidity. At the end of fourteenth day after sowing, the number of normal
seedlings in each replication, pertaining to various treatments was counted and the germination was calculated and
expressed in percentage.
Root Length
At the time of germination count, ten normal seedlings were selected at random from each replication and used for
measuring the root length of seedlings. Root length was measured from the collar region to the tip of primary root. The
mean values were calculated and expressed in centimetre.
Shoot Length
The seedlings used for measuring root length were also used for measuring shoot length. The shoot length was
measured from the collar region to tip of the primary leaves and the mean values were expressed in centimetre.
Dry Matter Production
Ten normal seedlings selected at random were placed in paper cover and dried in shade for 24 h and then, they
were kept in an oven maintained at 103 2C for 16 1 h. The dried seedlings were weighed and the mean values were
expressed in mg seedlings-10.

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S. Sathya, S. Lakshmi & S. Nakkeeran

Vigour Index
Vigour index values were computed using the following formula and the mean values were expressed in whole
number (Abdul-Baki and Anderson, 1973).
Vigour index = Germination per cent x Total seedling length (cm).
Effect of Biopriming and Polymer Coating for Healthy Seedling Production under Greenhouse Condition
Pathogenicity of Damping off Pathogen- Pythium Ultimum
The pathogenicity of the pathogen P. ultimum was proved by Kochs postulates using the chilli cv. K 2. Sandmaize medium containing sand and maize @ 9:1 ratio respectively, was sterilized in autoclave at 121C, 15 psi for 2 hours.
The isolate of P. ultimum was inoculated into sand-maize medium and incubated for 15 days at room temperature (28
2C) for multiplication (Riker and Riker, 1936). Potting soil mixture (red soil: sand: vermicompost: coirpith @ 1:1:1:1
w/w/w/w) was sterilized in autoclave at 121C, 15 psi for two hours for two consecutive days. Pathogenicity of the isolated
pathogen was tested by artificially inoculating the sand-maize culture in the potting soil mixture at 10 per cent (w/w). The
potting soil mixture incorporated with the fungus was filled in pots. Seeds of chilli subjected for dry seed treatment,
biopriming with biocontrol agents and combination of biopriming and polymer coating were sown @ 25 seeds per pot
separately. Three replications were maintained.
Assessment of Damping off Incidence
The observation on damping off incidence was monitored everyday under greenhouse conditions up to 25 days
after sowing.

Number of infected plants

100
Percentage of disease incidence = Total number of plants

RESULT AND DISCUSSIONS

Effect of Biopriming and Polymer Coating for Enhancing Seed Quality
The result of present study was given by enumerated population of biocontrol agents and seed quality parameters
(Table 1). In the present study, significant differences were observed in population of biocontrol agents between dry seed
treatments, biopriming and polymer coating. Seeds bioprimed with 6 per cent B. amyloliquefaciens VB7 along with
polymer coating recorded higher population of 25x108 cfu ml-1 followed by 8 per cent P. fluorescens + polymer coating (22
x108) compared to other treatments.
Table 1: Influence of Dry Seed Treatment, Biopriming and Combination of
Biopriming and Polymer Coating in Chilli Seeds

Treatments

T1
T2
T3
T4
T5

Population of
Biocontrol
Agents (108 cfu
ml-1 Solution of
Seed Wash)*
0 (0.71)
0 (0.71)
10 (3.24)
12 (3.53)
22 (4.74)

Impact Factor (JCC): 4.7987

Speed of
Germination

Germination
(%)**

Root
Length
(cm)

Shoot
Length
(cm)

Vigour
index

Dry Matter
Production
(mg Seedlings-10)

4.8
5.0
5.2
5.6
6.8

81 (64.16)
87 (68.87)
85 (67.22)
87 (68.87)
91 (72.54)

8.6
9.8
10.0
10.4
11.0

4.0
4.2
4.2
4.4
4.5

1021
1218
1207
1288
1411

22.1
23.4
24.7
25.6
27.5
NAAS Rating: 3.53

Combined Effect of Biopriming and Polymer Coating Against Chilli Damping Off

25 (5.05)
6.9
93 (74.66)
17 (4.18)
6.6
88 (69.73)
20 (4.53)
6.6
89 (70.63)
T
T
T
0.05
0.17
2.67
SEd
0.11
0.35
5.51
CD (P=0.05)
* - Values in parentheses are square root transformed values
T6
T7
T8

11.2
10.5
10.8
T
0.32
0.65

49

4.7
4.5
4.5
T
0.13
0.27

1479
1320
1362
T
23.22
49.22

28.0
25.9
26.4
T
0.60
1.26

** - Values in parentheses are arcsine transformed values

In seed quality parameters, speed of germination and germination observed in all biopriming treatments were on
par with each other. Higher speed of germination (6.9) and germination (93 %) was observed in seeds bioprimed with 6 per
cent B. amyloliquefaciens + polymer coating which was on par with 8 per cent P. fluorescens + polymer coating (6.8 & 91
%), 6 per cent B. amyloliquefaciens (6.6 & 89 %), 8 per cent P. fluorescens (6.6, 88 %) compared to dry seed treatment and
control.
The root length and shoot length was significantly influenced by biopriming treatments and dry seed treatments.
The longest root length (11.2 cm) was observed in biopriming with 6 per cent B. amyloliquefaciens + polymer coating
which was on par with 8 per cent P. fluorescens + polymer coating (11.0 cm), as well as biopriming with 6 per cent B.
amyloliquefaciens (10.8 cm). Whereas, the control recorded the shortest (8.6 cm) root length. Significantly longest shoot
length (4.7 cm) was recorded in seeds bioprimed with 6 per cent B. amyloliquefaciens and polymer coating and was on par
with 8 per cent P. fluorescens and polymer coating (4.5 cm) and also biopriming with 6 per cent B. amyloliquefaciens (4.5
cm), 8 per cent P. fluorescens (4.5 cm). Control produced lower root length (4.0 cm).
Statistically significant variations were observed for dry matter production and vigour index due to seed
biopriming treatments and dry seed treatments. Highest dry matter production (28 mg seedlings-10) was observed in 6 per
cent B. amyloliquefaciens + polymer coating which was on par with 8 per cent P. fluorescens + polymer coating (27.5 mg
seedlings-10). While, control recorded significantly lowest (22.1 mg seedlings-10) dry matter production. Highest vigour
index (1479) was registered in 6 per cent B. amyloliquefaciens + polymer coating followed by 8 per cent P. fluorescens +
polymer coating (1411) and biopriming with 6 per cent B. amyloliquefaciens (1362). Significantly lowest vigour index
value of 1021 was recorded in control.

Figure 1: Influence of Dry Seed Treatment, Biopriming and Polymer Coating on

Germination (%), Vigour Index and Population of Biocontrol Agents

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S. Sathya, S. Lakshmi & S. Nakkeeran

It is evident from the present study that biopriming with 6 per cent B. amyloliquefaciens and polymer coating is
the best treatment in the case of rhizosphere colonization and seed quality parameters. The improvement in seed
germination by B. amyloliquefaciens was also found in other works and it was shown that some PGPR induced increase in
seed emergence and in some cases achieving increase up to 100 per cent greater than control was observed. These findings
may be due to the increased synthesis of hormones like IAA and the high lipid band could assume that it is related to the
increase of the carbonyl bond around 1743 cm-1, which would have triggered the activity of specific enzymes such as
amylase, that promoted early germination and brought an increase in availability of starch assimilation. Besides, significant
increase in seedling vigour would have occurred by better synthesis of auxins (Buensanteai et al., 2008b). These results are
also similar to the findings of Buensanteai et al. (2008a) who assessed the inoculation effect of B. amyloliquefaciens strain
KPS46 on growth of vegetable soybean. They observed that inoculated plants resulted in better germination, early
development and flowering and also increased dry weight of both the root system and the upper plant parts. Similarly,
promotion in growth parameters and yields of various crop in response to inoculation with PGPR were reported by other
workers.
This result is in confirmation with the results of (Junges et al., 2013), in which biopriming with B. subtilis in
combination with polymer coating showed increase in germination by reduction of dead seeds, weak and abnormal
seedlings in maize.
Effect of Biopriming and Polymer Coating for Healthy Seedling Production under Green House Condition
Chemical fungicides are generally used for control of this pathogen. However, frequent and indiscriminate use of
chemicals leads to ill effects such as environmental pollution and development of resistance in the pathogens. Hence,
biocontrol has been advocated as the most promising alternate strategy to overcome these problems.
Incidence of damping off revealed significant variations between biopriming and dry seed treatments over the
control (Table 2). Seeds bioprimed with 6 per cent B. amyloliquefaciens + polymer coating, 8 per cent P. fluorescens +
polymer coating and dry seed treatment with metalaxyl @ 1 g kg-1 of seed showed very low disease incidence of damping
off (20 %). A very high range of damping off incidence was observed in control (38 %) when compared to other
treatments.
Disease incidence was also reduced by biopriming with 6 per cent B. amyloliquefaciens (22 %) and 8 per cent P.
fluorescens (24 %), followed by dry seed treatment with B. amyloliquefaciens @ 10 g kg-1 seed (24 %), dry seed treatment
with P. fluorescens @ 10 g kg-1 seed (28 %) when compared to control (38 %).
Table 2: Effect of dry Seed Treatment, Biopriming and Combination of
Biopriming and Polymer Coating on Disease Incidence of Chilli
Treatments
T1
T2
T3
T4
T5
T6
T7
T8
Impact Factor (JCC): 4.7987

Damping Off
Disease
Incidence (%)
38 (38.06) a
20 (26.57) e
28 (31.95) b
24 (29.33) c
20 (26.57) e
20 (26.57) e
24 (29.33) c
22 (27.97) d

Reduction Over
Control (%)
47 (41.14) a
26 (33.62) d
37 (37.74) c
47 (41.14) a
47 (41.14) a
37 (37.74) c
42 (39.47) b
NAAS Rating: 3.53

Combined Effect of Biopriming and Polymer Coating Against Chilli Damping Off

51

Means in a column followed by same superscript letters are not significantly different according to DMRT.
(Values in parentheses are arcsine transformed values)
Seeds bioprimed with 6 per cent B. amyloliquefaciens + polymer coating, 8 per cent P. fluorescens + polymer
coating and dry seed treatment with metalaxyl @ 1 g kg-1 of seed showed higher percentage of reduction of damping off
over control (47 %). Percentage reduction of damping off over control was influenced by biopriming with 6 per cent B.
amyloliquefaciens (42 %), followed by biopriming with 8 per cent P. fluorescens (37 %), dry seed treatment with B.
amyloliquefaciens @ 10 g kg-1 seed (37 %), dry seed treatments with P. fluorescens @ 10 g kg-1 seed (26%).
The results of the study are also in agreement with the findings of many workers having tested metalaxyl against
pre-emergence damping off in various crops (Deshpande, 1986; McDonald et al., 1987; Rama, 1980; Dahiphale, 2006).
Bucherau et al. (1981) obtained good control of pre-emergence damping off with metalaxyl caused by Pythium ultimum in
alfafa. Efficacy of metalaxyl has been confirmed as seed dresser against Pythium ultimum in case of carrot, chick pea,
cotton, cucumber, pea and sugarbeet (Deshpande, 1986; McDonald et al., 1987; Rama, 1980; Kaiser and Hannan, 1983;
Lal et al., 1979; Paulus and Nelson, 1977). Palakshappa et al. (2010) reported that metalaxyl-m + mancozeb (4+64) at all
concentrations were found effective and recorded low seedling death due to chilli damping off caused by Pythium
aphanidermatum.
Various isolates of P. fluorescens have been found to be effective seed bioprotectants against Pythium spp. in a
number of crop species (Howell and Stipanovic, 1980; Loper, 1988; Weller and Cook, 1996). Muthukumar (2008)
observed that the endophytic bacteria significantly reduced the colony growth of Pythium aphanidermatum. Muthukumar
and Bhaskaran (2007) revealed that P. fluorescens was highly effective in inhibiting the mycelial growth of Pythium spp.
Similarly, Harris et al. (1994) reported that chilli seeds treated with P. fluorescens reduced the damping off of Capsicum.
Muthukumar et al. (2010) recorded the lowest incidence of pre and post-emergence damping off in chilli (9.10 and 12.33
per cent, respectively). B. subtilis as seed treatment reduced the percentage of damping off incidence of sugar beet under
greenhouse conditions (Abo-Elnaga, 2006).
Nakkeeran et al. (2006) reported that P. chlororaphis and B. subtilis showed the maximum inhibitory effect on the
mycelial growth of Pythium aphanidermatum causing chilli damping off and Pythium ultimum causing cucumber and sugar
beet damping off (Georgakopoulos et al., 2002). B. amyloliquefaciens and P. aeruginosa were used to protect cucumber
seedling against Pythium aphanidermatum (Elazzazy, 2012).
Bacillus subtilis reduced the damping off and increased dry biomass in both brinjal and chilli crops. These bacteria were
equal or superior in effectiveness as compared to metalaxyl (Bohra et al., 2006). Similarly biopriming with P. fluorescens
protected sh2 and se sweet corn seeds at a level equivalent to that obtained by treatment with the fungicide metalaxyl
(Callan et al., 1990).
Pseudomonas spp. has been reported to produce 2, 4-diacetylphloroglucinol (Shanahan et al., 1992), phenazines
(Thomashow et al., 1990), pyoluteorin, pyrrolnitrin (Howell and Stipanovic, 1980), Bacillus spp. to produce iturin A
(Paulitz and Belanger, 2001; Kloepper et al., 2004), mycosubtilin (Leclere et al., 2005) and zwittermicin A (Smith et al.,
1993) by which biocontrol agents suppressing damping off (Pythium spp.) and metalaxyl would prevent growth of the
germ tube, thus preventing seedling infection by Pythium spp. (Babadoost and Islam, 2002).

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S. Sathya, S. Lakshmi & S. Nakkeeran

CONCLUSIONS
In view of above findings, seeds bioprimed with 6 per cent B. amyloliquefaciens VB7 and yellow polymer coating
@ 10 ml kg-1 of seed was found safe and effective and may be used as an efficient & eco-safe alternative of synthetic
fungicides for the management of damping off disease of chilli and for obtaining higher yields.
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Combined Effect of Biopriming and Polymer Coating Against Chilli Damping Off
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