Journal Journal

of Applied
Appl Journal of Applied Horticulture, 21(3): 195-200, 2019 Horticulture
DOI: https://doi.org/10.37855/jah.2019.v21i03.33 ISSN: 0972-1045

Seed treatment with liquid microbial consortia for germination

and vigour improvement in tomato (Solanum lycopersicum L.)

K. Raja1, K. Sivasubramaniam2 and R. Anandham3

1
Department of Seed Science and Technology, Tamil Nadu Agricultural University, Coimbatore - 641 003, India.
2
Agricultural College and Research Institute, Tamil Nadu Agricultural University, Kudumiyanmalai - 622 104, India.
3
Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore- 641 003, India.
*E-mail: kraja_sst@rediffmail.com

Abstract
Use of effective microorganisms as a pre-sowing seed treating agent is considered to be ecologically sound and beneficial to both seed
and environment. Therefore to ensure the benefits, studies were conducted in tomato seeds with different liquid microbial cultures. The
results revealed that the tomato seeds treated with liquid cultures viz., Azospirillum, phosphobacteria and Pink Pigmented Facultative
Methylotroph (PPFM) have showed significant increase in germination and vigour. The seeds soaked in equal volume of Azospirillum
@ 1:50 dilution for 24 h or phosphobacteria @ 1:50 dilution for 12 h or PPFM liquid culture @ 1:100 dilution for 18 h have registered
the higher germination and vigour. Among these microbial cultures, PPFM has performed well in enhancing the seed germination and
seedling vigour. Also, the viability and vigour of the inoculants infused seed were not much affected in three months storage. However,
consortia of these microbial cultures showed antagonistic effect in seed germination and seedling vigour. In addition, the seeds infused
with PPFM @1:100 dilution for 18 h followed by polymer coating @ 5 mL and carbendazim fungicide treatment @ 2 g kg-1 recorded
significant improvement in seed germination and vigour with minimal reduction in the microbial population. Therefore, it would be
possible to infuse the beneficial microbes into the seed through liquid cultures and also storing such seeds without much reduction in
the microbial population. Therefore, it is beneficial if the seeds treated with the effective microorganisms which favour the better seed
germination and seedling growth. Also, the microbes can easily be added into the soil along with the seed which may reflect on the
better coloization of the microbes in plant root zone.
Key words: Liquid biofertilizers, Azospirillum, phosphobacteria, PPFM, tomato seeds.

Introduction peat inoculant (Hynes et al., 1995). The bacterial cultures viz.,
Azospirillum, Pseudomonas, Azotobacter (Shaukat et al., 2006)
Seed is a prime input in agriculture and thus, the quality of the and Methylobacterium (Nkpwatt et al., 2006) promotes seed
seed used for sowing should be in high order. The quality can be germination and seedling growth. Qureshi et al. (2012) found that
achieved in many ways of which seed treatment is an important the co-inoculation of Rhizobium and Bacillus sp. increased the
method to get the higher productivity and plant population. root length, root mass, number of nodule and mass as compared
Among the seed treatments, pre-sowing management with to control in blackgram. Similarly, PPFM inoculated with a
organic products have proved very effective in controlling the diazotroph as individual and combined inoculant treatments
pathogens and increasing the seedling vigour. Nevertheless, has resulted in increased seedling vigor, dry matter production
use of effective microorganisms as a pre-sowing seed treating
and yield and this might be due to the increased rhizosphere
agent is considered to be ecologically sound and beneficial to
population of the inoculants (Raja and Sundaram, 2006). Based
both seed and environment. In general, about 5 to 30 per cent
on the background, it was decided to introduce the microbes into
yield increase has been recorded from various crops by such
the seed rather through surface coating which enables transfer of
bacterial inoculation (Datta et al., 1982). The application of
the microbial population to the soil for better seedling growth.
inoculum to the seeds is in vogue with carrier based bacterial
Therefore, the present study was contemplated to find out the
inoculants (Graham et al., 1987). Sometimes in order to improve
suitable liquid microbial culture for effective seed treatment
stickiness on the seed, adhesive is added (Jahuri, 2001). However
and the effect of seed treating chemicals on the survival of the
carrier-based inoculants have a short shelf life, poor quality and
inoculants in the tomato seed.
most of the carrier based inoculants production and application
procedure was found to be time consuming and difficult
Materials and methods
when used for large quantities of seed. Hence, alternate liquid
inoculants were developed for seed treatment as it is easy to use, Standardization of concentration and duration: Tomato
spreads well, mixes easily and needs no additional water supply seeds cultivar PKM 1 were collected from Vegetable Research
(Nethery, 1991). The liquid rhizobial inoculant for pea and lentil Station, Palur (India) and dried well for the purpose of
resulted in yield equal to or better than those obtained for the microbial treatments. The bacterial strains viz., Azospirillum,
Journal of Applied Horticulture (www.horticultureresearch.net)
196 Effect of liquid microbial consortia on germination and vigour improvement in tomato

phosphobacteria, Methylobacterium (Pink pigmented Facultative in phosphobacteria @1:50 dilution + polymer coating @ 5 mL
Methylotroph, PPFM) were obtained from the Department of kg-1 + carbendazim seed treatment @ 2 g kg-1 of seed; T10 - seed
Agricultural Microbiology, Agricultural College and Research soaking in PPFM @1:100 dilution; T11 - seed soaking in PPFM
Institute, Madurai (India). The strains were cultured in NFb, @1:100 dilution + polymer coating @ 5 mL kg-1 of seed; T12 - seed
nutrient broth and ammonium mineral salts medium supplemented soaking in PPFM @1:100 dilution + carbendazim seed treatment
with 0.5 % methanol. The liquid based bio-inoculant formulations @ 2 g kg-1 of seed; T13 - seed soaking in PPFM @1:100 dilution
were prepared by diluting at various concentrations viz., 1:1, + polymer coating @ 5 mL kg-1 of seed + carbendazim seed
1:10, 1:50 and 1:100 ratios along with undiluted one. Then, the treatment @ 2 g kg-1 of seed. The treated seeds were stored for
tomato seeds were soaked in these concentrations at different a week and evaluated for the germination, vigour and microbial
time durations viz., 6, 12, 18 and 24 h with equal seed to culture population.
ratio i.e. 1:1 ratio (v/v). After that the seeds were shade dried to
Statistical analysis: The data collected were subjected to
the original moisture. The germination test was conducted by
statistical analysis (Panse and Sukhatme, 1967) and the critical
placing 400 seeds in four replications as per the ISTA (1999)
difference values were calculated at 5 % probability level.
procedure and evaluated.
Microbial consortia on seed quality: The consortia of microbial Results and discussion
cultures viz., Azospirillum, phosphobacteria and PPFM were Standardization of concentration and duration: Tomato
prepared by diluting the cultures as standardized in the earlier seeds treated with liquid Azospirillum culture showed significant
experiment like Azospirillum @ 1:50, phosphobacteria @ 1:50 differences in germination in which, the Azospirillum culture
and PPFM @ 1:100 concentrations. The consortia were prepared diluted at 1:50 ratio increased the seed germination (92 %) at
by mixing the different cultures at 1:1 or 1:1:1 ratio. Then, the 24 h soaking period when compared with untreated control (74
medium vigour seeds were soaked for 18 h in the microbial %) (Fig. 1a). The undiluted (100 %) and higher Azospirillum
consortia in the equal volume by following the treatment schedule culture concentration (1:1 dilution) showed deleterious effect on
viz., T1 - control; T2 - seed soaking in water; T3 - seed soaking in germination irrespective of the soaking durations. Similarly, the
Azospirillum @1:50 dilution; T4 - seed soaking in phosphobacteria phosphobacteria @ 1:50 dilution for 12 h soaking recorded higher
@1:50 dilution; T5 - seed soaking in PPFM @1:100 dilution; T6 - germination (96 %) than the control (89 %). The germination
seed soaking in Azospirillum @1:50 dilution + phosphobacteria was declined if the concentration of the culture was increased.
@1:50 dilution (1:1); T7 - seed soaking in Azospirillum @1:50 Undiluted and 1:1 diluted phosphobacteria cultures severely
dilution + PPFM @1:100 dilution (1:1); T8 - seed soaking in affected the germination irrespective of the soaking periods.
Azospirillum @1:50 dilution + phosphobacteria @1:50 dilution Lower germination of 36 per cent was recorded when the seeds
+ PPFM @1:100 dilution (1:1:1). The seeds were dried to the soaked in undiluted phosphobacteria culture for 24 h (Fig. 1b).
original moisture content. The germination test was conducted In addition, the seed infused with PPFM liquid culture at 1:100
by following the ISTA norms. The speed of germination was also dilution for 18 h soaking duration has recorded the highest
assessed during the germination test by following the formula, X1 germination (97 %) when compared with control (89 %) (Fig.
/ Y1 + X2-X1/ Y2 + …….+ Xn (Xn-1) / Yn, where, Xn - number of 1c). The germination was much affected if the PPFM culture
seeds germinated at nth count, Yn - number of days from sowing concentration is higher. Similarly, increase in soaking duration
on nth count (Maguire, 1962). The microbial populations in the has shown the negative effect on the seed germination. Decline
treated seeds were also assessed. In this regard, the treated seeds in mean germination in 24 h soaking (46.7 %) and undiluted
were first washed with sterile water for about four to five times to
higher concentration (60.5 %) has been recorded as the evidence
remove the chemicals adhering on the surface of the seeds. Then,
of the antagonistic effect. Similar findings of seed inoculation
the seeds were soaked in the sterile water and allowed in arbitrary
with plant growth promoting bacteria for increased germination
shaker for about one hour. The serial dilutions were prepared and
and seedling vigour were studied by many workers (Murty and
inoculated in the respective medium.
Ladha, 1988; Sharma et al., 2007; Meena et al., 2012; Bakonyi et
Compatibility with chemicals: Similarly, the effect of seed al., 2013). Adesemoye et al. (2009) opined that microorganisms
treating chemicals on the survival of microbes in tomato seeds or plant growth promoting rhizobacteria can help to reduce the
was assessed by infusing them with different liquid microbial application of inorganic fertilizers and contribute to improving
cultures for 18 h in equal volume. These bioinoculated seeds were soil fertility and reducing a negative environmental impact. The
shade dried to the original moisture content. Then, the seeds were positive effect of plant growth promoting bacteria on germination
treated with different chemicals as per the treatment details viz., and growth might be due to excreting phytohormones and
T1 - control; T2 - seed soaking in Azospirillum @1:50 dilution; T3 enhancing the nutrient mobilization from the seed (Murty and
- seed soaking in Azospirillum @1:50 dilution + polymer coating Ladha, 1988). Goes et al. (2012) found the growth-promoting
@ 5 mL kg-1 of seed; T4 - seed soaking in Azospirillum @1:50 activities particularly auxin synthesis in the plant growth-
dilution + carbendazim seed treatment @ 2 g kg-1 of seed; T5 - seed promoting bacteria. Among the cultures, PPFM performed better
soaking in Azospirillum @1:50 dilution + polymer coating @ 5 in increasing germination (87 %), speed of germination (2.6)
mL kg-1 + carbendazim seed treatment @ 2 g kg-1 of seed; T6 - seed and seedling length (17.2 cm) at 1:100 dilution (Table 1). Also,
soaking in phosphobacteria @1:50 dilution; T7 - seed soaking in no significant differences were observed in the germination and
phosphobacteria @1:50 dilution + polymer coating @ 5 mL kg-1 seedling vigour during the three months storage of this treated
of seed; T8 - seed soaking in phosphobacteria @1:50 dilution + seeds. But the reduction in microbial population was noticed
carbendazim seed treatment @ 2 g kg-1 of seed; T9 - seed soaking irrespective of the inoculants during seed storage. Pink pigmented
Journal of Applied Horticulture (www.horticultureresearch.net)
 Effect of liquid microbial consortia on germination and vigour improvement in tomato 197

100 a cd
90 6h 12 h 18 h 24 h
80
b bc bc bcd bc bcd bcd bcd a def bcbcd bcd
bcd bcd
a g bc cde
70
Germination (%)

ghi gh fgh
60 hi ijk

i
50
40
30
20
10
0
Control Water Undiluted 1:1 ratio 1:10 ratio 1:50 ratio 1:100 ratio
Azospirillum Concentration
110 b
100 bc abc 6h 12 h 18 h 24 h h a
ab ab
90
def ab a jk
80 def
cde fg ij
70
Germination (%)

ab
60 ghi
hij ijk
50 ijk efg ijkl
l abc ijk
40 k ij
30
20
10
0
Control Water Undiluted 1:1 ratio 1:10 ratio 1:50 ratio 1:100 ratio
Phosphobacteria Concentration

110 c
100 ab a
e 6h 12 h 18 h 24 h
ab a abc abcd
90 abc ab abc
c a a abc
d bc ab
80 ab
e
Germination (%)

70 cd
60
50 f f
f f
40 f f
30
20
10
0
Control Water Undiluted 1:1 ratio 1:10 ratio 1:50 ratio 1:100 ratio
PPFM Concentration

Fig. 1. Effect of seed infusion with a) Azospirillum, b) phosphobacteria and c) PPFM cultures on germination in tomato
facultative methylotroph recorded the highest population during and seedling vigour. However, the combination of two or more
initial (40 x 105 cfu g-1 of seed) as well as three months storage microbial cultures showed the decrease in the germination and
(22 x 105 cfu g-1 of seed). The available seed moisture might have seedling vigour. In contrast, Qureshi et al. (2012) found that the
supported the viability of the microorganisms in the seed. co-inoculation of phosphate solubilizing bacteria and rhizobia
increased the root length, root mass, number of nodule and mass
Microbial consortia on seed quality: Based on the results in mash bean. Combined inoculation of phosphate-solubilising
obtained, the microbial consortia were prepared with 1:50 bacteria and Azotobacter exhibited beneficial effect on yield, as
(Azospirillum and Phosphobacteria) or 1:100 (PPFM) dilutions well as on nitrogen and phosphorous storage in different crops
and seeds were soaked for 18 h in the consortia. The consortia (Kundu and Gaur 1984; Monib et al., 1984). Being small in size,
seed treatment resulted negative effect in germination and tomato seeds was not able to tolerate the higher concentrations
seedling vigour. In which, the consortia Azospirillum and during consortia formulation, therefore, the soaking of tomato
phosphobacteria or Azospirillum and PPFM or Azospirillum, seeds in microbial consortia had negative impact on the seed
phosphobacteria and PPFM recorded 68, 68 and 59 per cent quality. Also, the seed infusion treatment with different bacterial
germination, respectively compated to untreated control (78 %). cultures showed positive response in penetration of the colonies
Nevertheless, monoculture treatments improved the germination into the seed. In this regard, the microbial population was higher
of medium vigour seeds to certain extent like 85, 84 and 89 in PPFM seed treatment in which a load of 41 x 105 cfu g-1 of
% in Azospirillum, phosphobacteria and PPFM, respectively seed was recorded. Similar findings of enhanced seed germination
(Table 2). Among the cultures, PPFM performed better in by seed coating or seed inoculum of methylotrophs were
terms of enhancement in germination, speed of germination recorded earlier (Anitha, 2010 and Meena et al., 2012). Corpe
Journal of Applied Horticulture (www.horticultureresearch.net)
 198 Effect of liquid microbial consortia on germination and vigour improvement in tomato

and Rheem (1989) reported that the

Microbial population in

PPFMs are present in the rhizosphere

41 x 104
22 x 105
seed (cfu g-1 of seed)

9 x 105

41 x 105

30 x 105
3MAS

PPFM
and phyllosphere regions of plants and
-

-
-
-

23 x 105
Microbial population in seed (cfu g-1 of seed)
even on the surface of the seeds of
various plants. Nkpwatt et al. (2006)
found that the cell-free supernatant of
40 x 105
22 x106
44 x104
Initial

the Methylobacterium bacterial culture

Phosphobacteria
-

stimulated germination, suggesting

20 x 104

17 x 104
30 x 10
the production of a growth-promoting

-
-
-

-
agent by the methylotroph. Pink
Mean
12.8
16.2
14.9
17.1

pigmented facultative methylotroph

Seedling length

mediate the cytokinin or other plant

growth promoting substances on
3MAS
(cm)

12.7
15.9
14.7
16.9
15.1

NS
0.2
S

germinating seeds (Holland and

Polacco, 1994) and that might be the
Azospirillum

22 x106

12 x105

20 x106

7 x106
reason for enhanced germination in
Initial
12.9
16.4
15.0
17.2
15.4

0.3
0.6

-
-

-
-
tomato seeds. The increased seedling
T

vigour by the production of IAA by

Methylobacterium was confirmed
Mean
2.0
2.0
2.1
2.6

earlier in tomato (Subhaswaraj et al.,

Seedling length

2017). Agafonova et al. (2013) found

germination

the phosphate-solubilizing activity of

Speed of

(cm)
13.5
14.7
17.0

13.7
17.1
15.9

12.4

15.8
3MAS

1.0
2.2
0.08
NS
2.0
2.1
2.2
2.6
2.2

methylobacteria in phytosymbiosis.
S

Seed germination and seedling vigour

In addition, methylotrophs play a

major role in phosphorus acquisition,
Table 2. Seed infusion with liquid microbial consortia on germination, vigour and microbial population in tomato seed
Initial
2.0
1.9
1.9
2.6
2.1

0.1
0.2

nitrogen fixation, phytohormone

T

production, iron chelation and plant

germination
Speed of

growth promotion and therefore,

1.6
1.7
2.1

1.9
2.4
2.1

1.6

1.5

0.1
0.2
Table 1. Seed infusion with liquid bioinoculants on seed viability and microbial population in tomato seed

Mean
82.7
85.0
83.0
86.5

co-inoculation of these bacteria as

biofertilizers can result in viable
Seed germination

agriculture practices (Manish Kumar

3MAS*

et al., 2016).
84.0
(%)

Germination (%)
NS
0.7
83
84
83
86

Compatibility with chemicals: In

15.3
7.0

another experiment, tomato seeds

78
82
85

84
89
68

68

59
Initial

infused with PPFM liquid culture

84.8

1.1
2.2
83
86
83
87

@1:100 dilution for 18 h followed by

polymer coating @ 5 mL kg-1 and seed
T3 - Seed soaking in Phosphobacteria liquid culture @1:50 dilution for 18 h

T6-Seed soaking in Azospirillum @1:50 dilution + Phosphobacteria @1:50

T7-Seed soaking in Azospirillum @1:50 dilution + PPFM @1:100 dilution

T8-Seed soaking in Azospirillum @1:50 dilution + Phosphobacteria @1:50

T4-Seed soaking in Phosphobacteria liquid culture @1:50 dilution for 18 h

treatment with carbendazim @ 2 g kg-1

T2 - Seed soaking in Azospirillum liquid culture @1:50 dilution for 18 h

T3-Seed soaking in Azospirillum liquid culture @1:50 dilution for 18 h

dilution + PPFM @1:100 dilution liquid cultures (1:1:1) for 18 h

of seed recorded higher germination

(96 %), speed of germination (3.0) and
T4 - Seed soaking in PPFM liquid culture @1:100 dilution for 18 h

T5-Seed soaking in PPFM liquid culture @1:100 dilution for 18 h

seedling length (17.8 cm) compared to

control (Table 3). Other seed treatments
with PPFM culture combination like
polymer @ 5 mL kg-1 or carbendazim
@ 2 g kg-1 of seed have also recorded
the better germination and seedling
vigour. Similarly, Azospirillum
Treatments

dilution liquid cultures (1:1) for 18 h

and phosphobacteria infused seeds

treated with chemicals like polymer
T2-Seed soaking in water for 18 h

or carbendazim showed advantages

liquid cultures (1:1) for 18 h

in seed quality enhancement than

untreated control. However, the
advantage was higher in PPFM infused
T1 - Untreated Control

* months after storage

seeds than the others as discussed

earlier. While, analyzing the microbial
density in the seed, carbendazim
CD (P=0.05)
CD (P=0.05)
Treatments

T1-Control

fungicide treatment was found to

have negative impact. Seeds treated
Mean

SEd
SEd

with carbendazim recorded lowest

Journal of Applied Horticulture (www.horticultureresearch.net)
 Effect of liquid microbial consortia on germination and vigour improvement in tomato 199

Table 3. Chemical treatment on germination, seedling vigour and microbial population in bioinoculants infused tomato seed
Treatments Seed Speed Seedling Microbial
germination of length population
(%) germination (cm) (cfu g-1 of seed)
T1-Control 80 1.4 11.9 -
T2-Seed soaking in Azospirillum liquid culture @1:50 dilution for 18 h 91 1.9 15.3 17 x 105
T3-Seed soaking in Azospirillum liquid culture @1:50 dilution for 18 h + 92 2.2 15.7 12 x 105
Polymer coating @ 5 mL kg-1 of seed
T4-Seed soaking in Azospirillum liquid culture @1:50 dilution for 18 h + 85 2.3 16.7 6 x 105
Carbendazim seed treatment @ 2 g kg-1 of seed
T5-Seed soaking in Azospirillum liquid culture @1:50 dilution for 18 h + 81 2.6 16.3 8 x 105
Polymer coating @ 5 mL kg-1 + Carbendazim seed treatment @ 2 g
kg-1 of seed
T6-Seed soaking in Phosphobacteria liquid culture @1:50 dilution for 18 h 90 1.9 12.8 52 x 104
T7-Seed soaking in Phosphobacteria liquid culture @1:50 dilution for 18 91 1.8 14.9 56 x 104
h + Polymer coating @ 5 mL kg-1 of seed
T8-Seed soaking in Phosphobacteria liquid culture @1:50 dilution for 18 92 2.0 16.3 48 x 104
h + Carbendazim seed treatment @ 2 g kg-1 of seed
T9-Seed soaking in Phosphobacteria liquid culture @1:50 dilution for 18 93 2.0 16.1 50 x 104
h + Polymer coating @ 5 mL kg-1 + Carbendazim seed treatment @
2 g kg-1 of seed
T10-Seed soaking in PPFM liquid culture @1:100 dilution for 18 h 93 2.9 16.5 40 x 105
T11-Seed soaking in PPFM liquid culture @1:100 dilution for 18 h + 95 2.9 17.1 31 x 105
Polymer coating @ 5 mL kg-1 of seed
T12-Seed soaking in PPFM liquid culture @1:100 dilution for 18 h + 95 2.9 17.4 7 x 104
Carbendazim seed treatment @ 2 g kg-1 of seed
T13-Seed soaking in PPFM liquid culture @1:100 dilution for 18 h + 96 3.0 17.8 32 x 104
Polymer coating @ 5 mL kg-1 of seed + Carbendazim seed treatment
@ 2 g kg-1 of seed
SEd 2.3 0.2 0.9
CD (P=0.05) 4.8 0.4 1.8
population (7 x 10 4 cfu g -1 seed). Likewise, population of seeds infused with more than one culture. Nevertheless, seed
Azospirillum and phosphobacteria was reduced drastically due soaking in PPFM liquid culture @1:100 dilution for 18 h followed
to this fungicide (Table 2). Fortunately, the polymer coating by polymer coating @ 5 mL and carbendazim treatment @ 2 g
followed by carbendazim treatment recorded the minimum kg-1 of seed has recorded the higher germination and seedling
reduction in the microbial population. This might be due to the vigour with minimum reduction in the microbial population.
barrier effect of polymer between the microbes and carbendazim. The study revealed that the beneficial microbial inoculants can
Similar findings on the survival of the bioinoculants in the be introduced into the seed and transferred to the field without
chemical treated seeds were observed in many crops (Dunfield coating with carrier based inoculants.
et al., 2000; Bikrol et al., 2005; Mehta et al., 2011; Tariq et al.,
2016). Khalequzzaman (2008) opined that the inoculation of Acknowledgement
lentil and chickpea seeds with Rhizobium followed by bavistin
treatment significantly decreased foot and root rot incidence and The authors thank the University Grants Commission (UGC),
increased plant stand and grain yield. However, seed inoculation New Delhi for financial assistance to carry out this work under
techniques used for research purposes are often not feasible at a the major research project.
commercial scale and there are significant technical challenges
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Received: May, 2019; Revised: May, 2019; Accepted: June, 2019

Journal of Applied Horticulture (www.horticultureresearch.net)