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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-2, Issue-6, Nov-Dec- 2017

http://dx.doi.org/10.22161/ijeab/2.6.58 ISSN: 2456-1878

Effect of bioformulations of Phosphate


Solubilizing Bacteria (PSB) on the Growth and
Biochemical Characters of the Gossypium
Hirsutum and Zea Mays
Tensingh Baliah N., Andal Priya S.

Post Graduate and Research Department of Botany, Ayya Nadar Janaki Ammal College, Sivakasi, Tamil Nadu, India

Abstract— Biofertilizers offer a new technology to Indian applied to the fields. They maintain soil health, minimizes
agriculture holding a promise to balance many of the short pollution of the environment by lowering the use of
comings of the conventional chemical based technology. chemicals (Tripti and Anshumali, 2012).
They are usually prepared as carrier based inoculants Phosphate solubilizing bacteria play an important role in
containing effective microorganisms. The present study was converting low grade insoluble inorganic phosphate sources
aimed at to study the nursery performance of different like rock phosphate, bone meal, basic slag and the
formulations of PSB in maize and cotton plants. The chemically fixed soil phosphorus into available form.
selected PSB was mass multiplied in the laboratory and Therefore, the use of phosphate solubilizing microbes in
incorporated into the nursery soil through different carrier agricultural practice would not only offset the high cost of
material such as coirpith, vermicompost, organic manure, manufacturing phosphatic fertilizers but would also
lignite and vermiculite. The effect of bioinoculants on the mobilize insoluble phosphorus in the fertilizers and soils to
growth and biochemical characters were studied from the which they are applied. The mechanism of solubilization of
control and treated seedling of Gossypium hirsutum and insoluble phosphate is ability to secrete organic acids and
Zea mays. The significant difference was observed in the phosphatase enzyme (Mahantesh et al., 2015).
growth and biochemical characters in both Gossypium The carrier based inoculants produced in India generally
hirsutum and Zea mays. The effect was differed with have a short shelf life, poor quality, high contamination and
reference to the nature of carrier materials used for the unpredictable field performance. High quality biofertilizers
preparation of bioformulations. The results indicated that would be expected to have higher population of desired
the bioformulation prepared by composted coirpith had microorganism, sufficient viability, and remain
superior in plant growth and development. uncontaminated for longer period of storage. Today,
Keywords─ PSB, carrier, bioinoculants, crop response, advances in inoculants technology were concerned with
cotton, maize. improving quality, extending useful shelf life and
developing new formulations for use under less favorable
I. INTRODUCTION conditions. Suggested that liquid inoculants and alginate
Biofertilizers are the bioinoculants of specific beneficial based granular formulations were two important new
microorganisms that promote the growth and development inoculants formulations which were an alternative to peat or
of plant crops by converting the unavailable form of lignite based ones (Brahmaprakash and Sahu, 2012). A cost
nutrients into available form. These biofertilizers also effective carrier materials which were nonpolluting,
improve the soil fertility (Sivasakthivelan and Saranraj, biodegradable, non-toxic, capable of maintaining high
2013). They can be used as supplements of chemical viable count and long shelf life amendable to nutrient
fertilizers; they were relatively inexpensive and renewable supplement and high water holding capacity (Gomare et al.,
sources of plant nutrient. Biofertilizers were selected strains 2013).
of microorganisms which were beneficial to the growth of Phosphate solubilizing bacteria of Pseudomonas and
the plants. These microorganisms were cultured in Bacillus species are producing phytohormones i.e. auxins,
laboratory, mixed with suitable carrier materials and then inhibition of deleterious pathogens or nutrient mobilization

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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-2, Issue-6, Nov-Dec- 2017
http://dx.doi.org/10.22161/ijeab/2.6.58 ISSN: 2456-1878
and ammonification. The plant growth benefits due to the 2.2. NURSERY EXPERIMENT
addition of PSB include increases in germination rate, root A nursery experiment was conducted to study the nursery
growth, yield, leaf area, chlorophyll content, tolerance to performance of different formulations of PSB in Gossypium
drought, shoot and root weight. Such groups of bacteria are hirsutum and Zea mays. The seeds with uniform size, colour
termed as phosphate solubilizing bacteria and inoculation and weight were chosen for the experimental purpose and
with PSB as biofertilizers enhances P accumulation and surface sterilized with 0.1% HgCl2 for 1 minute and
biomass production of plants (Abbasi et al., 2015). Single thoroughly washed with distilled water 3-5 times. Seeds
inoculation of Pseudomonas moraviensis and Bacillus were pre-soaked for 12 hours in distilled water and were
cereus with maize straw and sugarcane husk increased plant sown in sterilized soil mixture. The soil mixture was
height and fresh weight and protein, proline, sugar contents prepared by mixing black soil, red soil and sand in the ratio
and antioxidant activities. Inoculation of PSB further of 1:1:1. The PSB formulations were applied 10g each at
increased plant growth, physiology and yield characters top soil of the pots. The treatment details were: 1.Control
over single inoculation with different carriers. It is inferred 2.Coir pith 3.Vermicompost 4.Organic manure 5.Lignite
that carrier based biofertilizer effectively increased growth, 6.Vermiculite. The growth characters such as seed
maintained osmotic balance and enhanced the activities of germination percentage, germination index, seedling vigour
antioxidant enzymes and yield parameters (Hassan and index I and II, shoot length, root length, number of leaves,
Bano, 2016). fresh weight and dry weight were analyzed in the treated
The cultivation of cotton and maize requires higher amount and untreated plants. Biochemical characters such as total
of phosphatic fertilizer for their growth as well as Chlorophyll (Wellburn and Lichtenthaler, 1984), Protein
sustainable yield. But the added phosphatic fertilizers are (Lowry et al., 1951), glucose (Jayaraman 1981), amino acid
rapidly transformed to fixed form or unavailable forms. (Jayaraman, 1981) and NR activity (Jaworski, 1971) were
This is mainly due the nature of soil. These fixed forms of estimated.
phosphates are unavailable to plants. It is directly affected 2.3. STATISTICAL ANALYSIS
the plant growth, development and yield of cotton and The data were reported as mean ± SE and in the figure
maize. These problems can be easily solved by application parentheses represent the per cent activity. Values are
Phosphate Solubilizing Bacteria (PSB). These beneficial expressed as means ± standard deviation of three
microorganism can able to soluble the fixed forms of independent data.
phosphate into soluble/available form and can make it
available to the plants. The application/amendment of PSB III. RESULTS
with cotton and maize will increase the soil fertility and 3.1 ISOLATION AND MASS MULTIPLICATION OF
crop yield. Further, the sustainable yield can be achieved by BIOINOCULANTS
application biofertilizers like PSB. Phosphate solubilizing bacteria (PSB) were isolated based
on the solubilization zone production in the hydroxy apatite
II. MATERIAL AND METHODS medium from the rhizosphere soils of Brinjal. Totally 10
2.1. ISOLATION AND MASS MULTIPLICATION OF PSB strains were isolated. Based on preliminary screening,
BIOINOCULANTS PSB strain BP1 was selected as elite strain and used for
Isolation and enumeration of PSB was carried out by further studies. The selected BP1 strain was mass multiplied
dilution plate technique using hydroxy patite medium. The in the laboratory with different carrier materials and used
selected PSB isolate was multiplied in the culture flask with for nursery experiments. The carrier materials such as
nutrient broth. The broth culture was mixed with sterilized composted coirpith, vermicompost, organic manure, lignite
carrier materials. The viable count in the inoculum was and vermiculite were used for mass multiplication. A viable
checked before mixing the inoculums with carrier materials. count ranged from 109 to 1010 ml-1 preferred for the
Various organic materials such as composted coir pith, preparations of bioformulation. Three day old culture was
lignite, organic manure, vermicompost and vermiculite mixed with the sterilized carrier materials and used for the
were used as carrier for the mass multiplication of PSB. The nursery experiment.
carrier materials were sterilized, sieved and maintained 3.2 EFFECT OF PSB FORMULATIONS ON THE
proper water content in the carrier materials. The mass GROWTH CHARACTERS
multiplied liquid culture was mixed with the carrier Effect of bioformulations on the growth characters were
materials and used for nursery experiments. studied from the control and treated seedlings of

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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-2, Issue-6, Nov-Dec- 2017
http://dx.doi.org/10.22161/ijeab/2.6.58 ISSN: 2456-1878
Gossypiyum hirsutum and Zea mays. The germination rate aminoacid content in the leaves of G. hirsutum and Z. mays
was higher in the plants treatment with coirpith formulation in all treated plants. Among them, the effect was higher in
over the control plants. Among the bioformulations, the low coirpith formulation over other formulations and control.
germination rate was organic manure in G. hirsutum and The NR activity was maximum in plants treated with PSB
vermiculite formulation in Z. mays. The germination index as coirpith formulation over other treated plants in both G.
was varied according to the bioformulation treatment. Little hirsutum and Z. mays (Table 3 and table 4).
difference was shown in germination index among different
bioinoculant treatments but the effect was higher to the IV. DISCUSSION
control. The values of the seedling vigour index of the 4.1. MASS MULTIPLICATION OF PSB STRAIN
Gossypium hirsutum and Zea mays varied according to the Biofertilizers are usually prepared as carrier based
bioformulation treatment. The highest value of seedling inoculants containing effective microorganisms.
vigour index was observed in the plants treated with Incorporation of microorganisms in carrier material enables
coirpith formulation. In both G. hirsutum and Z. mays, easy-handling, long-term storage and high effectiveness of
seedling vigour index II was significantly increased in the biofertilizers. Basically, the carrier based inoculant of these
treatment of coirpith formulation followed by bacteria can be prepared by a common procedure. In the
vermicompost formulation. The result revealed that the bioinoculants preparation, various carrier materials were
shoot length was higher in the plants of G. hirsutum and Z. used such as lignite, vermiculite, charcoal, agro industrial
mays treated with PSB with coirpith as carrier followed by waste, compost etc. The efficiency of different types of
vermicompost formulation. The effect was least with bioinoculants was varied based on the nature of carrier
vermiculite bioformulation and control. Results also material used for preparation of bioinoculants. Therefore,
indicated that the root length was higher in all treated plants selection of carrier material was important one in the
over control. It was observed that the plants grown with biofertilizer production as well as in the crop response
coirpith formulation produced taller roots than other (Uma Maheswari and Kalaiyarasi, 2015).
treatments in both G. hirsutum and Z. mays. The number of The nature of carrier material, shelf life and inoculums
leaves per plant was significantly influenced by potential were important in the quality of bioinoculants.
bioformulation treated plants than untreated control plants. Quality of bioinoculants was one of the most important
The highest number of leaves was found in plants treated factors deciding their performance. A good carrier material
with coirpith formulation in both G. hirsutum and Z. mays. was one which can keep up the viability of microbes for a
Among different formulation tested, coirpith formulation longer period by providing organic food base to the
significantly increased the plant fresh weight followed by organisms and retaining the moisture content (Yadav and
vermicompost and least in vermiculite formulation. Like Chandra, 2012). Presently lignite powder was being used as
fresh weight, same trend was observed in plant dry weight carrier material by most of the bioinoculant producing units
also (Table 1 and table 2). in India. Availability of quality lignite powder was also in
3.3. EFFECT OF PSB FORMULATIONS ON THE doubt because of adulteration by agents and improper mesh
BIOCHEMICAL CHARACTERS size in the pulverizing unit. Several scientists have
In the nursery experiment, the inoculation of PSB with suggested compost as carrier material for biofertilizers. But
Gossypium hirsutum and Zea mays increased the the role of good compost is maintaining microbial
biochemical characters such total chlorophyll, protein population. The role of vermicompost as carrier in
content, aminoacids content, glucose content and NR maintaining shelf life of inoculated bacterial culture has
activity. The total chlorophyll content was higher in plants been studied for selecting as alternative carrier material to
treated with coirpith formulation followed by vermicompost lignite (Gandhi and Sivakumar, 2010; Murray et al., 2003).
formulation and least in organic manure formulation. In The quantity of culture required for mass multiplication was
both G. hirsutum and Z. mays, the protein content was differed based on the physical properties of carrier
significantly higher in plants treated with PSB as coirpith materials. It was also determine the quality of bioinoculants.
formulation and least in organic manure. The result revealed Based on the water holding capacity and particle size of the
that there was marked difference in the glucose content carrier materials, the quantity of requirement of culture was
among the treatments. In both G. hirsutum and Z. mays varied. The quantity of culture filtrate varied with the
glucose content was higher in plant treated with coirpith carrier material with their water holding capacity. Coirpith
formulation. Application of biofertilizer increased the required higher concentration of culture than others;

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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-2, Issue-6, Nov-Dec- 2017
http://dx.doi.org/10.22161/ijeab/2.6.58 ISSN: 2456-1878
because the water holding capacity was more with coirpith secondary fruit and total number of fruit by the application
than other carrier materials. High level of organic matter of PSB in strawberry (Zargar et al., 2008). Biochemical
content increased the water holding capacity and neutral pH parameters of chlorophyll, total carbohydrate, total protein
for better survival of the microorganisms (Roychowdhury et and total fat contents found higher in biofertilizer enriched
al., 2015). vermicompost treatments. Increased amount of chlorophyll
4.2 CROP RESPONSE OF PSB contents seems to correlate the increased photosynthetic
Ahemad and Kibret (2014) stated that mobilization of properties (Khomami and Moharam, 2013). Application of
mineral nutrients like P in soil by bacteria could be the main PSB increased the plant growth and also the dry matter
mechanism for increased growth and development of plants content. It also increased the physiological parameters like
which makes these nutrients in more readily plant available total chlorophyll, protein, amino acids, glucose and NR
forms. Improvement in shoot length, root length, shoot activity. The response was varied based on the nature of
fresh weight, shoot dry weight, root length, root fresh carrier material used for preparation of bioinoculants. From
weight, root dry weight, yield per plant and P contents of these, it was clear that PSB not only solubilize the P but
root and shoot by inoculation of PSB strains in combination also increased plant growth and development. PSB was not
with organic amendment was more prominent as compared only due to the release of plant available phosphorus but
to sole inoculation of PSB strains PSB containing also produce the biologically active substances like indole
phosphatase enzymes liberate P through mineralization of acetic acid, gibberellins, cytokinin. The favourable effect of
organic matter which promote the growth and yield of the inoculants on plant growth and nutrient uptake was due
plants (Walpola and Yoon, 2013). to the improved phosphate nutrition and production of
A significant difference in the root, shoot length and shoot growth promoting substances by PSB (Yadav and Chandra,
dry weight of tomato plants was observed at 60 days of 2014).
plant growth due to various inoculation treatments. The PSB application significantly increased the biochemical
treatment receiving inoculation of PSB5 recorded maximum parameters like the Chlorophyll a, and b, carotenoid, protein
root length and was significantly superior over all other and abscorbic acid (Singh et al., 2014). Ferreira et al.
inoculation treatments followed by PSB7. All treatments (1987) reported that wheat plants inoculated with PSB
showed significant increase in root growth over absolute showed greater activity of the nitrate reductase enzyme.
control (Assefa and Fenta, 2017). Phosphate solubilizing Inoculation with nitrogen fixing bacteria always increased
bacteria improved the plant growth, yield and phosphorus leaf NRA suggesting a greater supply of NO3 to the plants
content of several crops and used as bioinoculant to enhance over uninoculated control. The increased NO3 uptake may
the sustainable production. All strains showed a positive relate to increased root development in response to
effect on plant growth. A significant increment in plant production of hormones. The biochemical characters of
height, shoot dry weight was determined in plants treated protein contents of leaves of pot grown plants was 33-35%
with Pseudomonas tolaasii, while Pseudomonas koreensis higher over control when plants were inoculated with
has remarkably increased P content compared to the Bacillus cereus and Pseudomonas moraviensis. Co-
uninoculated control. Pseudomonas strain was selected and inoculation of both Bacillus cereus and Pseudomonas
evaluated under field conditions in combination with triple moraviensis significantly increased protein content. Further,
superphosphate as P fertilizer. The presence of sugar contents of leaves were also significantly increased
Pseudomonas strain stimulated seedling emergence, shoot (Galal, 2003).
length, grain yield, grain weight, total dry biomass and P
content of maize plants. In general, P. tolaasii, inoculation V. CONCLUSION
was more efficient as bioinoculant without P fertilizer The isolated PSB strains were screened in vitro for the
(Viruel et al., 2014). selection of elite strain. The selected strain of PSB was
The application of PSB either individually or combined, mass multiplied and used for nursery experiment. PSB
effected on the growth and biomass production of several strain was incorporated into the nursery soil through
crops (Hariprasad and Niranjana, 2009). Biofertilizer different carriers. The bioinoculation of PSB was increased
produced the plant growth regulating substances, which the growth and biochemical characters of Gossypium
promotes root growth found the significant effect on hirsutum and Zea mays. The response was varied with
number of primary flowers number of secondary flowers, carrier materials used in bioinoculation of PSB.
total number of flowers, number of primary fruit, number of

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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-2, Issue-6, Nov-Dec- 2017
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ACKNOLDGEMENT [10] Hassan, T. U., & Bano, A. (2016). Biofertilizer: A
The authors are thankful to Management and the Principal novel formulation for improving wheat growth,
of Ayya Nadar Janaki Ammal College, Sivakasi, Tamil physiology and yield. Pak. J. Bot. 48(6): 2233-2241.
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Table.1: Effect of PSB formulations on the growth characters of Gossypium hirsutum


Treatment Seed Germinatio Seedlin Seedling Shoot Root Number Fresh Dry
Germinatio n Index g Vigour Length Length of leaves Weight Weight
n Vigour Index II (cm) (cm) (per (g) (g)
(%) Index I plant)
Control 76 0.92 18.2 1.12 24 9.5 15 4.25 1.48
±0.49 ±0.02 ±0.30 ±0.32 ±0.15 ±0.02 ±0.13
(100) (100) (100) (100) (100) (100) (100)
Coirpith 96 2.04 36.5 5.21 38 17.3 33 12.83 5.43
formulation ±0.37 ±0.06 ±0.32 ±0.41 ±0.30 ±0.75 ±0.47
(200) (465) (158) (182) (220) (302) (367)
Vermicomp 94 1.85 34.8 3.49 36 16.8 32 11.40 3.80
ost ±0.35 ±0.60 ±0.15 ±0.72 ±0.40 ±0.47 ±0.65
formulation (191) (312) (150) (177) (213) (268) (257)
Organic 90 1.53 30.2 1.98 28 13.2 22 6.67 2.65
manure ±0.49 ±0.54 ±0.35 ±0.40 ±0.47 ±0.12 ±0.18
formulation (166) (172) (117) (139) (147) (157) (179)
Lignite 96 2.04 32.6 3.42 34 15.6 25 9.98 3.57
formulation ±0.15 ±0.01 ±0.25 ±0.35 ±0.37 ±0.10 ±0.13
(179) (305) (142) (164) (166) (235) (241)
Vermiculite 91 1.59 30.4 2.22 33 12.8 27 8.14 2.42
formulation ±0.47 ±0.02 ±0.49 ±0.30 ±0.58 ±0.05 ±0.27
(167) (198) (137) (135) (180) (192) (164)

Table.2: Effect of PSB formulations on the growth characters of Zea mays


Treatment Seed Germinati Seedlin Seedling Shoot Root Number Fresh Dry
Germinati on Index g Vigour Lengt Length of leaves Weight Weight
on Vigour Index II h (cm) (per (g) (g)
(%) Index I (cm) plant)
Control 75 0.78 13.4 0.70 20 9.0 13 3.55 1.05
±0.15 ±0.03 ±0.25 ±0.09 ±0.28 ±0.11 ±0.01
(100) (100) (100) (100) (100) (100) (100)
Coirpith 93 1.97 33.5 2.14 36 15.7 25 8.41 2.31
formulation ±0.40 ±0.01 ±0.15 ±0.20 ±0.35 ±0.34 ±0.07
(250) (305) (180) (174) (192) (237) (220)
Vermicompo 90 1.82 26.1 1.68 30 14.0 22 6.92 1.93
st formulation ±0.25 ±0.07 ±0.40 ±0.15 ±0.40 ±0.71 ±0.03

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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-2, Issue-6, Nov-Dec- 2017
http://dx.doi.org/10.22161/ijeab/2.6.58 ISSN: 2456-1878
Treatment Seed Germinati Seedlin Seedling Shoot Root Number Fresh Dry
Germinati on Index g Vigour Lengt Length of leaves Weight Weight
on Vigour Index II h (cm) (per (g) (g)
(%) Index I (cm) plant)
(195) (240) (150) (156) (169) (195) (182)
Organic 82 1.30 28.0 1.12 35 14.3 23 5.18 1.41
manure ±0.30 ±0.02 ±0.20 ±0.45 ±0.41 ±0.60 ±0.13
formulation (209) (160) (175) (159) (177) (146) (134)
Lignite 87 1.55 27.8 1.42 32 13.7 20 5.77 1.64
formulation ±0.73 ±0.03 ±0.55 ±0.40 ±0.81 ±0.49 ±0.05
(207) (203) (160) (152) (154) (163) (156)
Vermiculite 81 1.28 18.2 0.92 25 12.2 19 4.39 1.26
formulation ±0.20 ±0.02 ±0.65 ±0.55 ±0.40 ±0.11 ±0.24
(136) (131) (125) (136) (146) (123) (120)

Table.3: Effect of PSB formulations on the biochemical characters of Gossypium hirsutum


Treatment Total Protein Glucose Aminoacid NRA
Chlorophyll (mg/g LFW) (mg/g LFW) Content (µmol NO2
(mg/g LFW) (mg/g LFW) formed/g LFW/h)
Control 1.07 2.91 11.13 1.64 1.68
±0.01 ±0.03 ±0.16 ±0.10 ±0.04
(100) (100) (100) (100) (100)
Coirpith 2.83 6.80 22.35 3.90 3.13
formulation ±0.02 ±0.19 ±0.42 ±0.35 ±0.17
(264) (234) (201) (238) (186)
Vermicompost 2.32 5.66 19.92 2.94 2.95
formulation ±0.07 ±0.23 ±0.21 ±0.32 ±0.09
(217) (195) (179) (179) (175)
Organic manure 1.35 3.85 14.70 2.35 1.95
formulation ±0.04 ±0.04 ±0.32 ±0.11 ±0.14
(126) (132) (132) (143) (116)
Lignite 1.94 4.36 17.43 2.21 2.32
formulation ±0.05 ±0.07 ±0.17 ±0.17 ±0.05
(181) (150) (157) (134) (138)
Vermiculite 1.45 4.14 19.86 3.12 2.72
formulation ±0.06 ±0.09 ±0.25 ±0.11 ±0.16
(135) (142) (178) (190) (162)

Table.4: Effect of PSB formulations on the biochemical characters of Zea mays


Treatment Total Protein Glucose Aminoacid NRA
Chlorophyll (mg/g LFW) (mg/g LFW) Content (µmol NO2
(mg/g LFW) (mg/g LFW) formed/g LFW/h)
Control 0.92 2.65 9.81 1.12 1.18
±0.03 ±0.05 ±0.03 ±0.02 ±0.02
(100) (100) (100) (100) (100)
Coirpith 2.69 5.43 21.40 3.14 2.55
formulation ±0.05 ±0.26 ±0.72 ±0.03 ±0.17
(292) (204) (218) (280) (216)

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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-2, Issue-6, Nov-Dec- 2017
http://dx.doi.org/10.22161/ijeab/2.6.58 ISSN: 2456-1878
Treatment Total Protein Glucose Aminoacid NRA
Chlorophyll (mg/g LFW) (mg/g LFW) Content (µmol NO2
(mg/g LFW) (mg/g LFW) formed/g LFW/h)
Vermicompost 2.24 4.71 17.09 2.21 2.04
formulation ±0.11 ±0.07 ±0.53 ±0.11 ±0.07
(243) (178) (174) (197) (173)
Organic manure 1.12 3.15 13.62 1.47 1.48
formulation ±0.24 ±0.09 ±0.75 ±0.04 ±0.06
(121) (119) (139) (131) (125)
Lignite 1.63 4.32 15.18 1.85 2.18
formulation ±0.05 ±0.57 ±0.15 ±0.05 ±0.09
(177) (163) (155) (165) (184)
Vermiculite 1.38 3.22 16.33 2.51 1.96
formulation ±0.06 ±0.13 ±0.42 ±0.14 ±0.05
(150) (122) (166) (224) (166)

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