ThesisThesis - pdfs.semanticscholar.org€¦ · Mohinder Kaur and Mrs Anjali Chauhan for being supportive and considerate. I express my loyal and sincere thanks to Dr. C K Shirkot

EFFECT OF SELECTED PLANT GROWTH PROMOTING RHIZOBACTERIA ON GROWTH AND YIELD OF GINGER

(Zingiber officinale Rosc.)

ThesisThesisThesisThesis

by

KIRTI KAUNDAL

Submitted in partial fulfilment of the requirements

for the degree of

MASTER OF SCIENCE

in

MICROBIOLOGY

(BASIC SCIENCE)

COLLEGE OF FORESTRY Dr. Yashwant Singh Parmar University

of Horticulture and Forestry, Nauni, Solan-173 230 (H.P.) India

2012

Dr Rajesh Kaushal Department of Basic Sciences Associate Professor College of Forestry

Dr Y S Parmar University of Horticulture and Forestry, Nauni-173230, Solan (HP)

CERTIFICATE-I

This is to certify that the thesis entitled, “Effect of selected plant growth

promoting rhizobacteria on growth and yield of ginger (Zingiber officinale

Rosc.)” submitted in partial fulfillment of the requirements for the award of degree of

MASTER OF SCIENCE in MICROBIOLOGY (BASIC SCIENCES) to Dr Yashwant

Singh Parmar University of Horticulture and Forestry, Nauni, Solan (HP) is a record

of bonafide research work carried out by Ms Kirti Kaundal (F-2009-05-M) under my

guidance and supervision. No part of this thesis has been submitted for any other

degree or diploma.

The assistance and help received during the course of investigations has

been fully acknowledged.

Place: Nauni-Solan Dr Rajesh Kaushal Dated: , 2012 Major Advisor

CERTIFICATE-II

This is to certify that the thesis entitled, “Effect of selected plant growth


Rosc.)” submitted by Ms Kirti Kaundal (F-2009-05-M) to Dr Yashwant Singh

Parmar University of Horticulture and Forestry, Nauni, Solan (HP) in partial fulfillment

of the requirements for the award of degree of MASTER OF SCIENCE in

MICROBIOLOGY (BASIC SCIENCES) has been approved by the Student’s

Advisory Committee after an oral examination of the same in collaboration with the

external examiner.

Dean’s Nominee External Examiner Major Advisor Dr Rajesh Kaushal Members of Advisory Committee Associate Professor Dr C K Shirkot Professor Dr J . N Raina Professor Dr Ramesh Bhardwaj

Scientist

Professor and Head

Dept of Basic Sciences, COF, Nauni

Dean College of Forestry

YSPUHF, Nauni-Solan (HP)

CERTIFICATE-III

This is to certify that all the mistakes and errors pointed out by the external

examiner have been incorporated in the thesis entitled, “Effect of selected plant

growth promoting rhizobacteria on growth and yield of ginger (Zingiber

officinale Rosc.)” submitted to Dr Y S Parmar University of Horticulture and

Forestry, Nauni-Solan (HP) by Ms Kirti Kaundal (F-2009-05-M) in partial fulfillment

of the requirements for the award of degree of MASTER OF SCIENCE in

MICROBIOLOGY (BASIC SCIENCES).

Major Advisor

Dr Rajesh Kaushal

Associate Professor

Professor and Head

Department of Basic Sciences Dr Y S Parmar, UHF, Nauni-173230, Solan (HP)

ACKNOWLEDGEMENTS

With immense servility, I would like to pray and thank ‘GOD’ the almighty, who bestowed me patience, peace, understanding and courage enough for this stupendous academic adventure.

Every effort is motivated by an ambitions and inspirations behind; I owe this pride place to my parents, brother and sisters who always believed in giving a strong education wings to me. Words fail to express their sacrifices and heartful blessings which made this manuscript a remuneration to translate their dreams into reality.

Words betray to express my heartfelt sentiments towards the two towering peaks, my papa and mummy, whose blessings, selfless love, constant encouragement, obstinate sacrifices have been the most vital source of inspiration and motivation in my life.

I express my deep sense of gratitude to my esteemed teacher and chairman of my advisory committee, Dr. Rajesh Kaushal for his guidance, keen interest, critical analysis and valuable suggestions.

I emphatically express my venerable and profound thanks and heartfelt gratitude to the members of my advisory committee, Dr. C K Shirkot, Dr. J N Raina and Dr Ramesh Bhardwaj.

A word of special appreciation also goes to Dr.(Mrs) Nivedita Sharma, Dr.( Mrs) Mohinder Kaur and Mrs Anjali Chauhan for being supportive and considerate.

I express my loyal and sincere thanks to Dr. C K Shirkot Professor and Head, Department of Basic Science for providing all the necessary facilities and means to carry out the research successfully.

God was benevolent enough to bless me with friends like Charu, Shweta, Nisha, Sapna, Bharti, who stood by me through all my thick and thin and the memories of the time spent with them.

I have been fortunate in getting the intelligent guidance by my seniors and enthusiastic co-operation of friendly circle by my classmates and all my juniors are also acknowledged.

Facilities and co-operation provided by Dayaram Sir and Satish Bhaiya and other staff members of Department of Basic Sciences is thankfully acknowledged.

Needless to say, errors and omissions are solely mine.

Date: January, 2012 Date: January, 2012 Date: January, 2012 Date: January, 2012 Place: Nauni, SolanPlace: Nauni, SolanPlace: Nauni, SolanPlace: Nauni, Solan ( Kirti Kaundal)( Kirti Kaundal)( Kirti Kaundal)( Kirti Kaundal)

CCOONNTTEENNTTSS

CHAPTER TITLE PAGE(s)

1. INTRODUCTION 1-2

2. REVIEW OF LITERATURE 3-12

3. MATERIALS AND METHODS 13-27

4. EXPERIMENTAL RESULTS 28-42

5. DISCUSSION 43-50

6. SUMMARY AND CONCLUSION 51-53

7. REFERENCES 54-64

ABSTRACT 65

APPENDICES I-III

LLIISSTT OOFF TTAABBLLEESS

Table Title Page

1. Enumeration of rhizosphere bacterial population associated with ginger

29

2. Population of rhizospheric P – solubilizing bacteria associated with ginger

29

3. Enumeration of endophytic bacterial population associated with roots of ginger plants

30

4. Population of P - solubilizers associated with ginger roots

30

5. Morphological characterstics of rhizospheric and endophytic bacterial isolates of ginger roots

31

6. Phosphorus solubilization efficiency of bacterial isolates on solid PVK medium

32

7. Siderophore production efficiency of different bacterial isolates on CAS medium

32

8. Screening of selected bacterial isolates for multifarious plant growth promoting activities

33

9. Morphological, physiological and biochemical characteristics of selected bacterial (KK5) isolate

34-35

10. Effect of bacterial inoculums on plant parameter 36

11. Effect of bacterial inoculum on rhizomeparameters in net house

37

12. Physico-chemical properties, available nutrient content and total bacterial counts of soil mixture used for net house experiment (initial status)

38

13. Effect of bacterial inoculum on physico-chemical characteristic and available nutrient status of soil.

39

14. Rhizosphere and endophytic bacterial population associated with ginger (At termination of experiment)

40

15. Correlation between ginger plant microbial population and plant parameters at the end of the experiment

40

16. Correlation between ginger plant microbial population and soil parameters at the end of the experiment

41

17. Correlation coefficient (r-value) among the soil parameters and plant parameters from liquid based inoculants in net house at the end

42

LIST OF PLATES

Plates Title Between Page(s)

1. Isolation of microbes by modified replica plate method on different medium

31-32

2. Growth of bacterial (KK5) isolate on different medium by streaked plate method

31-32

3. P-solubilization by selected bacterial (KK5) isolate on PVK agar medium

33-34

4. Siderophore production by selected bacterial (KK5) isolate on CAS medium

33-34

5. HCN production by selected bacterial (KK5) isolate on King’s B medium.

33-34

6. Antifungal Activity of selected bacterial (KK5) isolate against Pythium spp.

33-34

7. Antifungal Activity of selected bacterial (KK5) isolate against Fusarium spp.

33-34

8. General view of net house experiment 35-36

9. Effect of bacterial inoculums on shoot length under net house conditions

37-38

10. Effect of bacterial inoculums on rhizome yield under net house conditions

37-38

LIST OF FIGURES

Plates Title Between Page(s)

1. IAA production (g/ml) by different bacterial isolate 33-34

2. Percent growth inhibition of fungus by different bacterial isolate

33-34

3. Effect of temperature on the growth of selected bacterial (KK5) isolate

35-36

4. Effect of pH on the growth of selected bacterial (KK5) isolate

35-36

5. Effect of bacterial inoculum on number of leaves of ginger under net house conditions

39-40

6. Effect of bacterial inoculum on shoot length of ginger under net house conditions

39-40

7. Effect of bacterial inoculum on number of tillers of ginger under net house conditions

39-40

8. Effect of bacterial inoculum on rhizome yield of ginger under net house conditions

39-40

9. Effect of bacterial inoculums on available nutrient contents in soil of ginger under net house conditions

39-40

10. Effect of bacterial inoculums on NPK uptake by ginger under net house conditions

39-40

ABBREVIATIONS USED

% : Per cent 0

C : Degree centigrade

CAS : Chrome-azurol-S

CF : Culture filtrate

Cfu : Colony forming units

cm : Centimeter

CRD : Completely randomized

design

CV : Coefficient of variation

FYM : Farm yard manure

g : Gram

h : hour

ha : Hectare

HCN : Hydrogen cyanide

IAA : Indole-3-acetic acid

K : Potassium

MEA : Malt extract agar

meq : milliequivalents

Min : minute

ml : millilitre

mm : millimeter

mM : millimolar

MTCC : Microbial type culture

collection

N : Nitrogen

NA : Nutrient agar

OC : Organic Carbon

OD : Optical density

P : Phosphorus

PGPR : Plant growth promoting

rhizobacteria

PGRs : Plant growth regulators

ppm : parts per million

psi : per square inch

PVK : Pikovskaya’s medium

rpm : Rotations per minute

spp. : Species

UV : Ultra voilet

cv. : Cultivar

v/v : volume/volume

w/v : Weight/volume

Chapter-1

INTRODUCTION

Ginger (Zingiber officinale Rosc.), member of family Zingiberaceae is a

selender perennial tuber crop consumed whole as a delicacy and spice. The

characteristic odour and flavour of ginger is due to a mixture of zingerone,

shogads, gingerols and volatile oil (Hara et al., 2007).

India is a leading producer of ginger in the world. It is grown in an area of

1.06 lakh hectares with a production of 3.70 lakh tonnes mainly in the states like

Kerala, Meghalya, Sikkim, Orissa, Arunachal Pradesh, Mizorum, Nagaland,

Orissa, Himachal Pradesh together contributes 70 per cent to the country’s total

production. Ginger can be grown in well drained, medium fertility loam soils

with pH value ranges from 6-7 (http://www.spices.res.in/package/ginger.pdf

2008). In Himachal Pradesh it is grown as rain fed crop at an altitude of 600-

1200m above mean sea level (Rai and Yadav, 2005) and cultivated in 1,850

hectares area with the annual production of 24,000 tonnes

(http://www.thaindian.com/newsportal/india 2009).

Ginger growing soils of the state are, in general, deficient in nitrogen and

having problems of availability of phosphorus nutrition. Further, low availability

of FYM / compost, other chemical fertilizers and problems of diseases like soft

rot and fusarium wilt of ginger have emerged as the major bottle necks for low

productivity and quality production of the crop.

In the present scenario of organic production of crops the application of

PGPR is the effective, ecofriendly, low volume, supplementing source of

nutrients not only for sustaining / increasing crop yields but also to sustain soil

health (Barakart and Gabr,1998 and Patra et al., 1989).

PGPR are free-living bacteria (Kloepper et al., 1989) and some of them

invade the tissues of living plants and cause unapparent and asymptomatic

infections (Sturz and Nowak, 2000). These rhizobacteria are referred to as

2

endophytes, and in order to invade roots they must first be rhizosphere

competent. PGPR’s are also known to produce plant growth promoting

compounds including phytohormones; auxins, cytokinins and gibberellins

(Garcia et al., 2001), as well as siderophores (Castignetti and Smarrelli ,1986).

PGPR may also increase plant growth by direct or indirect modes of

action (Beauchamp, 1993; Kloepper, 1993). Direct mechanisms include the

production of stimulatory bacterial volatiles and phytohormones, lowering of the

ethylene level in plant, improvement of the plant nutrient status (liberation of

phosphates and micronutrient cation from insoluble sources; non-symbiotic

nitrogen fixation). Indirect effects by stimulating ISR (Induced systemic

resistance) by acting as biocontrol agents or by degrading xenobiotics in

inhibitory contaminated or rhizoremediaters (Jacobsen, 1997).

Since there is no commercial biofertilizers PGPR formulations for the

ginger crop, therefore, there is an urgent need to develop an effective inoculum of

plant growth promoting rhizobacteria (PGPR) which can solubilize phosphorus,

fix atmospheric nitrogen and also act as anti-fungal agent to protect the crop from

soil borne pathogens (Cleyet Marcel et al., 2001).

Keeping this in view the present investigations were carried out with the

following objectives:

1. Isolation, enumeration and characterization of plant growth promoting

PGPR’s from rhizosphere and of ginger rhizome.

2. To study the efficacy of selected PGPR’s isolates on growth and yield of

ginger under controlled conditions.

Chapter-2

REVIEW OF LITERATURE

Soil bacteria are one of the major groups of microbes which are abundant

in rhizosphere soil ranging between 106 - 10

8 colony forming units (cfu) per gram

(Clark, 1967). The rhizosphere (a layer of soil around the root that is influenced

by the root) is the soil environment for the root growth extends up to a few

millimeters from the root surfaces into the surrounding soil (Rengel and

Marschner, 2005). A large microbial population is present in the rhizosphere and

only 7-15% of plant roots are colonized by soil microbes (Pinton et al., 2001).

Bacteria that effectively colonize root surface and promote plant growth,

as a result of some signal communications between the host plant and the bacteria

are termed rhizobacteria (Bianciotto et al., 2000; Schroth and Hancock, 1982).

Root colonization is the process where bacteria survive on seeds, multiply in

spermosphere in response to seed exudates rich in carbohydrates and amino acids

attach on to the root surfaces and colonize the developing root system. (Suslow,

1982).

Kloepper et al. (1989) coined the term plant growth promoting

rhizobacteria (PGPR) to include bacteria inhabiting the root and rhizosphere soil

which have the ability to increase plant growth. Inoculation of crop plants with

certain strains of PGPR at early stage of development improves biomass

production, through direct effects on root and shoots growth.

PGPR include a wide range of soil microbes including the microbes,

which are in symbiosis with their host plant like rhizobiums, fixing atmospheric

N2 and which are not in symbiosis association with their host plant such as

Pseudomonas spp., Bacillus spp., Azospirilum spp. and Burkholderia spp. (Glick

et al., 1998).

Somers et al. (2004) classified plant growth promoting rhizobacteria

(PGPR) as biofertilizers (increasing the availability of nutrients to plant),

4

phytostimulators (plant growth promoting, usually by the production of

phytohormones), rhizoremediators (degrading organic pollutants) and

biopesticides (controlling diseases, mainly by the production of antibiotics and

antifungal metabolites). Bashan and Holguin (1998) proposed the division of

PGPR into two classes: biocontrol-PGPR and PGPR. A large number of bacterial

strains have been isolated, screened and were evaluated for plant growth

promotion of different plant species (Chanway and Holl 1993; Lifshtiz et al.,

1987; Chanway et al., 1989; Glick et al., 1997; Bashan, 1998).

The research work pertaining to the effect of PGPR on ginger rhizome

and other related crops all over the world have been reviewed in this chapter

under the following heading.

2.1 PGPR as endophytic root colonizer

2.2 PGPR as secondary metabolite producers

2.3 PGPR as biofertilizer for enhanced growth and yield of crop

2.1 EFFECT OF PGPR ON ROOT COLONIZATION

Plant growth promoting rhizobacteria (PGPR) were first defined by Kloepper

and Schroth (1978) to describe soil bacteria that colonize the roots of plant following

inoculation onto seed or soil that enhance plant growth. In the process of root

colonization bacteria multiply in the spermosphere (region surrounding the seed) in

response to seed exudates rich in carbohydrates and amino acids, then these get attached

to root surface and colonize the developing root system (Weller, 1983; Suslow, 1982 ;

Suslow and Schroth, 1982; Kloepper et al., 1980).

Hallmann et al. (1997) reported that endophytic bacteria originate from

the epiphytic bacterial communities of the rhizosphere and phylloplane, as well

as from endophyte-infested seeds or planting materials. They gained entry to

plants through natural openings or wounds and also actively penetrate plant

tissues using hydrolytic enzymes like cellulase and pectinase.

Gray and Smith (2005) studied larger number of PGPR strains and

reported that a few of these PGPR can enter root interior and establish

5

endophytic populations. Many of them are able to transcend the endodermis

barrier, crossing from the root cortex to the vascular system, and subsequently

thrive as endophytes in stem, tubers, and other organs.

Microorganisms enters into plant roots by three putative pathways i.e root

tips, point of emergence of developing lateral root and axils of emerging or

developing lateral roots and also reported that endophytes either become

localized at the point of entry or are able to spread throughout the plant and such

isolates can live within cells in the intercellular spaces or in the vascular system

(James, 2000).

Yuming et al. (2002) isolated fourteen strains of putative endophytic

bacteria, excluding endosymbiotic Bradyrhizobium strains, inducing root

nodulation in soybean (Glycine max. (L.) Merr.) root nodules. Three isolates

(NEB4, NEB5, and NEB17) were able to increase soybean yields when plants

were co-inoculated with one of the isolates and Bradyrhizobium japonicum under

nitrogen-free conditions, as compared with plants inoculated with B.

japonicum alone.

Seed treatment of tomato with endophytic bacterium Bacillus pumilus

strain SE 34 prevented the entry of vascular fungus Fusarium oxysporum into the

vascular stele and observed that mycelia growth was restricted to the epidermis

and outer root cortex of the plant root (Benhamou et al., 1996).

Vetrivelkalai et al. (2010) conducted a detailed survey to isolate

endophytic bacterial isolates in different agroecosystems comprising Tamil Nadu.

Nineteen endophytic bacterial isolates were obtained from surface-sterilized roots

of different crops. Study on the morphological, phenotypic and biochemical

characterization of endophytic bacteria revealed that eight isolates viz., EB1 to

EB8 belong to the group of Pseudomonas sp., ten isolates viz., EB9 to EB18

belong to the group of Bacillus sp. and isolate EB19 belongs to

Methylobacterium sp. On seed bacterization with nineteen endophytic bacterial

isolates, four isolates viz., EB3, EB16, EB18 and EB19 significantly enhanced

6

the germination percentage, shoot and root length and vigour index of bhendi

seedlings by roll towel technique and pot culture studies.

2.2 PGPR AS A SECONDARY METABOLITE

Effect of PGPR on P- solubilizing

Phosphorus (P) is major essential macronutrient for biological growth and

development. The improvement of soil fertility is one of the most common

strategies to increase agricultural production. The ability of some microorganisms

to convert insoluble phosphorus (P) to an accessible form, like orthophosphate, is

an important trait in a PGPB for increasing plant yields (Rodriguez et al.,

2006).The use of phosphate solubilizing bacteria as inoculants increases the P

uptake by plants ( Igual et al., 2001).

Kandasamy et al. (1985) inoculated the seeds of chilli and brinjal and

recorded increase in seedling length in chilli (19.7 cm) and brinjal (14.9 cm)

when soil was inoculated with mycorrhiza and phosphobacteria in nursery.

Sattar and Gaur (1987) reported that P-solubilizers improved the plant

growth and development by the production of plant growth hormones like indole

acetic acid (IAA), gibberellic acid (GA) and cytokinins in addition to phosphate

solubilization.

Glick et al. (2007) reported that PGPR influence plant growth by

synthesizing plant hormones or facilitates the uptake of nutrients from the soil

through different mechanisms such as atmospheric nitrogen fixation, solubilizatin

of phosphorus (P) and synthesis of siderophore for iron sequestration making

available to plants from root environment .

Cattelan et al. (1999) conducted in vitro studies on rhizosphere of

Soyabean and found to solubilise Phosphorous along with other plant growth

promoting traits and increases the soyabean growth. The use of PGPR isolates as

inoculants is beneficial for rice cultivation as they enhance the growth of rice and

phosphorus solubilisation (Ashrafuzzaman et al., 2009).

7

Effect of PGPR on siderophore / IAA/HCN Production

Iron is the most important micronutrient used by microorganisms and is

essential for their metabolism, being required as a cofactor for a large number of

enzymes and iron-containing proteins (Dave et al., 2006). Siderophores are low

molecular weight, extracellular compounds with a high affinity for ferric iron,

that are secreted by microorganisms to take up iron from the environment (Hofte,

1993) and their mode of action in suppression of disease were thought to be

solely based on competition for iron with the pathogen (Bakker et al., 1993)

Bakker et al. (1986) reported that the siderophores of fluorescent

pseudomonads suppressed the plant pathogens by competition for iron between

pathogens and siderophores of fluorescent pseudomonads has been implicated in

the biocontrol of wilt diseases caused by Fusarium oxysporum (Kloepper et

al.,1980) and Pythium root rot of wheat (Becker and Cook, 1988) Pyoverdines

chelate iron in the rhizosphere and deprive pathogens of iron which is required

for their growth and pathogenesis (Leong, 1986).

Rhizobacteria produce various types of siderophores (Pseudobactin and

ferrooxamine B) that chelate the scarcely available iron and thereby prevent

pathogens from acquiring iron (Loper et al., 1991). Leeman et al. (1996) found

that pyoverdin type pseudobactin siderophore, produced by pseudomonads was

responsible for induced systemic resistance against fungal diseases.

Siderophore production by a plant growth promoting fluorescent

Pseudomonas sp. RBT 13 effective against several fungal and bacterial pathogens

has been demonstrated (Dileep and Dubey, 1993). Five strains of fluorescent

pseudomonads exhibited growth promotion of lentil and biocontrol of wilt caused

by F. oxsporum f.sp. lini with siderophore production as the mechanism (Rao et

al., 1999).

HCN is produced by many rhizobacteria and play a role in biological

control of pathogens (Defago and Haas, 1990). Production of HCN by certain

strains of fluorescent pseudomonads has been involved in the suppression of soil

8

borne pathogens. The cyanide producing strain CHAO of Pseudomonas

stimulated root hair formation and altered plant physiological activities (Voisard

et al., 1989). Under in vitro conditions Pseudomonas fluorescens releases HCN

which inhibited the mycelial growth of Pythium (Westsleijn, 1990). Four of the

six PGPR strains produced HCN and resulted induced systemic resistance in

cucumber against Colletotrichum orbiculare (Wei et al., 1991).

The HCN production is found to be a common trait of Pseudomonas

(88.89%) and Bacillus (50%) in the rhizospheric soil and plant root nodules and

is a serious environmental pollutant and a biocontrol metabolite in Pseudomonas

species (Ahmad et al., 2008).

Wani et al. (2007) conducted studies on rhizosphere isolates for HCN

producing ability under in vitro condition and found that most of the isolates

produced HCN and helped in the plant growth. The isolates from the rhizospheric

soil of chickpea also exhibits more than two or three PGPR traits including HCN

production, which promotes plant growth directly or indirectly or synergistically.

The rhizosphere competent Mesorhizobiumloti MP6 produces

hydrocyanic acid (HCN) under normal growth conditions and enhances the

growth of Indian mustard (Chandra et al., 2007). The Pseudomonas fragi

CS11RH1 (MTCC 8984), a psychrotolerant bacterium produces hydrogen

cyanide (HCN) and the seed bacterization with the isolate significantly increases

the per cent germination, rate of germination, plant biomass and nutrient uptake

of wheat seedlings (Selvakumar et al., 2009).

IAA (indole-3-acetic acid) is the member of the group of phytohormones

and is generally considered the most important native Auxin. There are numerous

soil microflora involved in the synthesis of auxins in pure culture and soil

(Barazani and Friedman, 1999). The potential for auxin biosynthesis by

rhizobacteria can be used as a tool for the screening of effective PGPR strains.

The strains which produce the highest amount of auxins i.e. indole acetic acid

(IAA) and indole acetamide (IAM) in non-sterilized soil, causes maximum

increase in growth and yield of the wheat crop (Khalid et al., 2004). IAA is

9

phytohormone which is known to be involved in root initiation, cell division and

cell enlargement (Salisbury, 1994).

Vessey (2003) has reviewed the production of this hormone and

implicated it in the growth promotion by PGPR. However, the effect of IAA on

plants depends on the plant sensitivity to IAA and the amount of IAA produced

from plant associated bacteria and induction of other phytohormones (Peck and

Kende, 1995). Besides its hormonal function, indole-3-acetic acid (IAA) is

involved in the stimulation of the ethylene synthesis (Glick, 1995).

Bastian et al. (1998) reported that all 18 strains of plant growth promoting

rhizobacterium, Acetobacter diaztrophicus from 13 cultivars of sugarcane had the

ability to produce indole-3-acetic acid (IAA) ranging from 0.14 to 2.42 µg IAA

ml-1

.

2.3 PGPR AS BIOFERTILIZERS FOR ENHANCED GROWTH AND

YIELD OF CROP

Biofertilizers are living microorganisms, which supplement nutrients in

the rhizosphere and promote plant growth. These are supplied as carrier based or

liquid inoculants. Quality of biofertilizers is judged on the basis of population of

viable bacteria. They enhance the productivity of soil by converting nutritionally

important elements from unavailable to available form through atmospheric

nitrogen fixation or by solubilizing soil phosphorous or by stimulating plant

growth through synthesis of growth promoting substances which help in

expansion of root system and better seed germination.

Kabesh et al. (1987) observed that mineral Phosphorus + biofertilizers or

biofertilizers alone significantly increased the plant height, leaf area index and

dry weight of soybean in pot experiment.

Patten and Glick (2002) demonstrated that bacterial IAA from P. putida

played a major role in the development of host plant root system and stimulatory

effect on the plant growth. It was observed that when sweet potato is inoculated

with the isolates capable of IAA production and there was a significant increase

10

in the plant growth by the N, P, K, Ca and Mg uptake (Farzana and Radizah,

2005).

When cucumber, tomato and pepper are inoculated with different strains

of PGPR which produce IAA, there is a significant increase in the growth of the

vegetables (Kidoglu et al., 2007).

Nath and Korla (2000) reported that two biofertilizers i.e. Azofert slurry

at 250g/10kg seeds and natrin slurry at 250g/10kg seeds were used and gave

better results in respect of plant stand, plant height, number of tillers and leaves

per plant in ginger cv. Himgiri over the normal rate of NPK and the control. The

Azofert biofertilizer found to be most effective for growth and development of

rhizomes and yield. The net returns and benefit cost ratio was also highest with

Azofert treatment.

Significant increase in the yield of many crops such as wheat, maize and

potato have been observed in response to inoculation with PGPR (Javed et al.,

1998; Zahir et al.,1998; Khalid et al.,1997; Iswandi et al., 1987).

Shukla et al. (2009) studied effect of inorganic and biofertilizers on yield

of tomato and revealed that application of NPK+ phosphate solubilizing bacteria

results in early initiation of flowering, more number of fruits per cluster and

good quality of tomatoes.

Zehra et al. (2009) studied the yield response of potato to inoculation

with Bacillus sp. OSU-142 at three levels of N fertilization (0, 120, and 240 kg

ha-1) under field conditions .Tuber inoculation with Bacillus sp. OSU-142

significantly affected yield and yield components. Tuber yields and yield

components were higher at all levels of nitrogen fertilizer in the inoculated plots

as compared to the control. However, beneficial effect of Bacillus sp. OSU-142

on tuber yield was noted at 120 kg N ha-1, possibly indicating either more

effective of inoculation in the low-N input agriculture or an interaction of

Bacillus sp.OSU-142 with higher yielding seasonal conditions. In general, more

response to inoculation was observed in the absence of major crop growth

11

limitations, suggesting the complementary contribution of the Bacillus sp.OSU-

142 treatment to more efficiently developing higher yielding potato.

Adesemoye et al. (2008) observed that application of microbial

consortium (Bacillus amyloliquefaciens IN937a and Bacillus pumilus T4)

inoculation performed better than individual inoculation. Further it was revealed

that consortia (Rhizobium, PSB and PGPR) registered the maximum (4532 kg

ha-1

) grain yield with reduced level of N and P (75%) which was found to be

higher than control with full doses of N and P (100%).

Mena and Portugal (2007) studied the effect of inoculation of tomato

roots with plant growth-promoting rhizobacteria (Bacillus subtilis) on yield and

fruit quality and demonstrated that PGPR had positive effects on tomato fruit

quality attributes, particularly on size and texture.

Minorsky (2008) isolated Pseudomonas flouresence B16 as a PGPR from

the roots of graminaceous plants which when inoculated to the plant resulted to

increase plant height, flower number, fruit number and fruit weight of tomato

family. Peudomonas putida and Pseudomonas flouresence increased root and

shoot elongation in canola, lettuce and tomato (Glick et al., 1997; Hall et al.,

1996) as well as crop yield in potato, radishes, rice, sugarbeet, tomato, apple,

citrus, beans and wheat.

Farzana et al. (2007) conducted field experiment to determine the effects

of different local strains of PGPR and nitrogen fertilizer on growth and yield of

sweetpotato. Four PGPR strains (Klebsiella sp. UPM SP9, Erwinia sp. UPM parts

were determined. Plants inoculated with the PGPR together with 1/3 of the

normal rate (33 kg N ha-1

) gave the highest storage root dry weight and increased

concentrations of N, P and K in shoots and storage root compared to non-

inoculated control plants.

Singh (2001) was conducted an experiment to evaluate the N efficiency

with bio-fertilizers inoculation. Combined inoculation of Azotobacter and

phospho-inoculant culture together significantly increased the potato tuber yield.

12

When these bio-fertilizers inoculated seperately Azotobacter gave 7.9 % and

phospho-inoculant gave 8.6% higher tuber yield significantly up to maximum

level (150 kg N/ha). Combined inoculation of both fertilizers along with 150 kg

N/ha was the highest yielder and gave the tuber production in the ratio 22: 62: 16

of large, medium and small size tubers, respectively, corresponding to that

control had a ratio of 9: 66: 25. The highest net return was also obtained from this

treatment.

Gracia et al. (2004) investigated the effect of inoculation under natural

and green house conditions on different crops. The application of bacterium

significantly increased the leaf area, fruit yield and biomass. The effect was more

pronounced on pepper as compared to tomato. They further reported that

colonization and competitive ability of the bacterium enable it to use as

biofertilizers or biocontrol agent and increase in yield of bean (Lee, 2005) and

brinjal (Bhakare et al., 2008).

Mena and Portugal (2007) also demonstrated that PGPR have positive

effects on tomato fruit quality attributes, particularly on size and texture. They

also reported that PGPR (Bacillus subtilis BEB-ISbs) treatment increased the

yield per plant as well as fruit weight and length as compared to uninoculated

control.

SOLAN

Nauni Kandaghat

A1 A2 B1 B2

Rhizosphere Soil Roots and Rhizome of Ginger

Chapter-3

MATERIALS AND METHOS

The present investigations entitled “Effect of selected plant growth


Rosc.)” were conducted in the section of Microbiology (Basic Sciences) and Soil

Microbiology Laboratory (Soil Science and Water Management) at Dr. Y. S.

Parmar University of Horticulture and Forestry, Nauni-Solan, Himachal Pradesh

during the years 2010-2011. A brief account of the materials used and

methodologies adopted are discussed in this chapter.

3.1 COLLECTION OF SAMPLES

The rhizospheric soil and roots of ginger (Zingiber officinale Rosc.) plants

were collected from Solan and Sirmour districts of Himachal Pradesh. In each

district two sites and two sub-sites were selected for sampling purposes. The

samples were placed in plastic bags and stored in Soil Microbiology Laboratory

of Department of Soil science and Water Management for further isolation and

analysis work.

3.2 MEDIA

Composition of the media (Atlas, 1995) used for the study are as follows:

3.2.1 Nutrient Agar (NA)

Constituents : quantity / litre

Beef extract : 3g

Peptone : 5g

SIRMOUR

14

NaCl : 5g

Agar : 20g

pH : 6.5 ± 0.01

3.2.2 Malt Extract Agar (MEA)

Malt extract : 20g

Agar : 20g

pH : 5.6 ± 0.01

3.2.3 Pikovskaya’s (PVK) Broth

Glucose : 10g

Ca3 (PO4)2 : 5g

(NH4)2 SO4 : 0.5g

KCl : 0.2g

MgSO4.7H2O : 0.1g

MnSO4 : Trace

FeSO4 : Trace

Yeast extract : 0.5g

Bromocresol purple : 0.01%

3.2.4 Pikovskaya’s (PVK) Agar

Pikovskaya’s broth + 20 g agar

3.2.5 Jensen’s Medium (N-free medium)

K2HPO4 anhydrous : 1g

MgSO4.7H2O : 1g

NaCl : 0.5g

FeSO4 : 0.1g

Sucrose : 20g

Ca (CO3)2 : 2g

Agar : 20g

15

3.2.6 Chrome-azurol-S agar

CAS : 0.06g

HDTMA : 0.07g

HCl : 0.02ml

FeCl3 : 0.2g

Agar : 20g

3.2.7 King’s Medium B

Proteose peptone : 20g

K2HPO4(anhydrous) : 1.5g

MgSO4.7H2O : 1.5g

Glycerol : 15g

Agar : 20g

pH : 7.2 + 0.01

3.2.8 Potato Dextrose Agar Medium

Dextrose : 20g

Potatoes : 200g

Agar : 20g

3.2.9 Luria Bertani (LB) Agar

Tryptophan : 10g

Yeast extract : 5g

NaCl : 5g

Agar : 20g

pH : 7.5 + 0.01

3.2.10 Soil Extract Medium

Glucose : 1g

K2HPO4 : 0.5g

Agar : 20g

Soil extract : 100ml

pH : 6.8 + 0.01

16

3.3 CHEMICALS AND REAGENTS

Analytical grade (AR) chemicals and reagents obtained from standard

company were used for present investigations.

3.4 MICROBIOLOGICAL METHODS

3.4.1 Sterilization

Glasswares used were thoroughly washed in detergent water, running tap

water followed by rinsing in distilled water. Glasswares were sterilized in hot air

oven at 180oC temperature for 30 minutes. All the media, water blanks etc. were

sterilized in autoclave at 15 lbs per square inch pressure of pure steam for 20

minutes, unless mentioned otherwise. Laminar airflow chamber was sterilized by

disinfectant followed by ultra violet (UV) irradiation for 30 minutes before start

of the work.

3.4.2 Isolation and enumeration of rhizospheric and endophytic

rhizobacteria

Modified replica plating technique was used to isolate PGPR isolates

from rhizosphere and roots of ginger.

3.4.2.1 Isolation of rhizobacteria

One gram of the rhizosphere soil was placed in 9 ml of sterilized distilled

water under aseptic conditions. The serially diluted suspension of soil was spread

on pre-poured nutrient agar (NA) medium. After incubation of 24–48 h, the

isolated colonies that developed on enriched medium (master plate) were replica

plated onto the selective media: Nitrogen free medium (Jensen, 1987) for

nitrogen fixing activity, Pikovskaya medium (Pikovskaya, 1948) for phosphate

solubilizing ability. All colonies were transferred to same position as the master

plate with the help of wooden block covered with sterilized velvetin cloth

(Plate1). At the end of the incubation period, the location of the colonies

appeared on the replica plates were compared to the master plate. The microbial

count was expressed as colony forming units (cfu) per gram of soil.

17

3.4.2.2 Isolation of endophytic rhizobacteria

The root sample was surface sterilized by 0.2 per cent mercuric chloride

(HgCl2) for two minutes followed by repeated washing in sterilized distilled

water. The surface sterility of rhizome was cross checked by incubating the

surface sterilized rhizome in sterilized nutrient agar medium for 24 h at 35± 20C.

One gram of surface sterilized rhizome sample was crushed in 9 ml of sterilized

distilled water and was crushed to produce slurry using pestle and mortar under

aseptic conditions. The serially diluted suspension of soil was spread on pre-

poured nutrient agar medium. After incubation of 24-48 h, the isolated colonies

that developed on enriched medium (master plate) were replica plated onto the

selective media: Nitrogen free medium (Jensen, 1987) for nitrogen fixing

activity, Pikovskaya medium (Pikovskaya, 1948), for phosphate solubilizing

ability. All colonies were transferred to same position as the master plate with the

help of wooden block covered with sterilized velvetin cloth. At the end of the

incubation period, the location of the colonies appeared on the replica plates were

compared to the master plate. The microbial growth was expressed as colony

forming units (cfu) per gram of root.

3.4.3 Maintenance of the cultures

The isolated cultures were purified by streak plate method and maintained

on the slants of respective medium at 40C in refrigerator. The culture of Pythium

spp. and Fusarium spp. were procured from Department of Mycology and Plant

Pathology, University of Horticulture and Forestry, Nauni-Solan. Fungal cultures

were maintained on malt extract agar at 40C. Sub-culturing of fungal cultures was

done once in fortnight on respective medium at incubation temperature of

28+10C.

3.4.4 Measurement of growth

3.4.4.1 Preparation of inoculum

A bacterial cell suspension (OD 1 at 540 nm) of 48 h old culture grown on

nutrient broth at the rate of 10 per cent was used as inoculum in all experiments,

unless mentioned otherwise.

18

3.4.4.2 Turbidity

Growth was monitored by measuring the change in absorbance of cells in

the broth at 540 nm using un-inoculated broth as blank.

3.4.4.3 Viable count

Appropriate dilutions of bacterial cell suspension were used to seed the

NA plates. The number of viable cells in the initial population was obtained by

counting the number of colonies that developed after incubating the plates and

multiplying this figure by dilution factor.

3.4.5 Screening of bacterial isolates

The screening of the bacterial isolates for various plant growth promoting

activities like P-solubilization, siderophore, HCN, growth on N-free medium,

auxin production and antagonism against Pythium spp. and Fusarium spp. were

performed by adopting the standard methods. The brief description of these

methods are as follows:

3.4.5.1 Phosphate solubilizing activity

Each of purified isolate were seeded in a straight line on PVK medium as

described by Pikovskaya (1948) and was incubated for 72 h at 35 ± 20C. Colonies

showing solubilization halos (>0.1mm diameter) were selected.

3.4.5.2 Nitrogen fixing activity

Each of the purified isolate were seeded in a straight line on Jensen’s

medium and was incubated for 72 to 120 h and the plates showing growth of

bacteria in the form of bacterial colony were selected.

3.4.5.3 Siderophore production

Siderophore production was detected by CAS plate assay method

(Schwyn and Neilands, 1987). Sterilized blue agar was prepared by mixing CAS

(60.5 mg/50ml distilled water) with 10 ml iron solution (1mM FeCl3.6H2O in 10

mM HCl). This solution was slowly added to hexadecyltrimethyl ammonium

19

bromide (HDTMA) solution was prepared by dissolving 72.9 mg HDTMA in

40ml distilled water). Thus, 100 ml CAS dye was prepared. 750 ml nutrient agar

was mixed with 1, 4 piperazine diethane sulphonic acid (30.24 g) and pH 6.8 was

adjusted with 0.1N NaOH. It was autoclaved separately and then mixed with

Chrome azurol- S (100 ml) under aseptic conditions and then the plates were

prepared for further experiments.

A bit of 72 h old culture of each test bacterium was placed on prepoured

blue coloured chrome-azurol-S agar (CAS) plates. Plates were incubated at 30+

20

C for 24 h and observed for production of orange halo around the bit.

Per cent siderophore efficiency =

3.4.5.4 HCN Production

Bacterial isolates were screened out for the production of hydrogen

cyanide (HCN) as per method described by Bakker and Schippers (1987).

Bacterial cultures were streaked on prepoured plates of King’s medium B

amended with 1.4 g/l glycine. Whatman No.1 filter paper strips were soaked in

0.5 per cent picric acid in 2 per cent sodium carbonate and were placed in the lid

of each petriplates and plates were sealed with parafilm and were incubated at

35+2oC for 1-4 days. Uninoculated control was kept for comparison of results.

Plates were observed for change in color of filter paper from yellow or orange

brown.

3.4.5.5 Quantitative estimation of indole-3-acetic acid

Bacterial cultures were grown in modified Luria Bertani broth amended

with 5 mM L-tryptophan, 0.065 sodium dodecyl sulphate and 1% glycerol for 24,

48 and 72h at 35oC under shaking conditions. The cultures were centrifuged at

15,000 rpm for 20 minutes and supernatant were collected and stored at 40C.

The method described by Gorden and Palleg (1957) was used to

determine the IAA equivalents, 3 ml of supernatant was pipetted into test tube

and 2 ml Salkowski reagent (2 ml 0.5 M FeCl3 + 98 ml 35% HClO4) was added

20

to it. The tubes containing the mixture were left for 30 minutes (in dark) for

colour development. Intensity of colour was measured spectrophotometrically at

535 nm. Similarly, colour was also developed in standard solution of IAA (10 -

100 µg/ml) and a standard curve (Appendix 2.1) was established by measuring

the intensity of this colour.

3.4.5.6 Antagonistic activity of bacterial isolates against test fungus

Agar streak plate method was used to test the efficacy of bacterial isolates

against the test fungus. A loop full of 48 h old culture of each isolate were

streaked a little below the centre of the prepared MEA Petri plate and incubated

at 350C for 24 h to check contamination. Mycelial disc of 5 days old culture of

the test fungal pathogen (Pythium spp. and Fusarium spp.) was placed separately

on one side of the streak in each plate. A check inoculated with the test pathogen

only was kept for comparison. Then plates were incubated at 28±10C for 7 days

and per cent growth inhibition was calculated as described by Vincent (1947).

C-T I =

C X 100

Where;

I = Per cent growth inhibition

C = Growth of fungus in control

T = Growth of fungus in treatment

3.4.6 Identification of bacterial antagonist

On the basis of morphological, cultural and biochemical characteristics

and as per the criteria of Bergey’s Manual of Systematic Bacteriology (Claus and

Berkeley, 1986), the selected isolate was identified.

3.4.7 Characterization of bacterial isolates

Separate experiments were performed for optimization conditions

(physical, chemical and nutritional) for growth of selected bacterial isolates.

3.4.7.1 Effect of pH on growth

3 ml nutrient broth was taken in 5 ml test tubes. The medium was adjusted

to various pH (5, 6, 7and 8) using 0.1 N NaOH or 0.1 N HCl as the case may be.

21

Each tube was inoculated with 0.1 ml of 48 h old bacterial cell suspension (OD

1.0 at 540 nm) of selected isolates. The experiment was carried out in triplicates.

The pH suited for maximum growth was selected on the basis of turbidity caused

by the bacterial growth in test tube.

3.4.7.2 Effect of temperature on growth

Growth curves were drawn by growing the culture at various

temperatures. 3 ml of nutrient broth was taken in 5 ml test tubes and inoculated

with 0.1 ml of 48 h old bacterial cell suspension (OD 1.0 at 540 nm). The

optimum temperature for growth was selected on the basis of turbidity caused by

the bacterial growth in test tube.

3.4.7.3 Estimation of P- solubilization in liquid PVK medium containing

TCP (tri-calcium phosphate)

PVK broth was used to study the solubilization of phosphorus. 50 ml of

medium was dispensed in 250 ml of Erlenmeyer flask containing 0.5 per cent tri-

calcium phosphate (TCP) and autoclaved at 15 lbs per square inch of pure steam

for 20 minutes. The bacterial inoculum was prepared by streaking 48 h old

growth of organism on nutrient agar slants. The flasks were inoculated with 10

per cent (5 ml) of the bacterial suspension (OD 1.0 at 540 nm) and incubated at

35±2oC on rotating shaker at 100 rpm for 72 h. Flasks were withdrawn at 72 h

and contents were centrifuged at 15,000 rpm for 20 minutes at 4oC. The culture

supernatant was used for determination of the soluble phosphorous as described

by Bray and Kurtz (1945).

The procedure essentially consists of estimating soluble phosphorus

formed by the action of phosphate solubilizing bacteria on tri-calcium phosphate.

The soluble phosphorus formed was estimated by using spectrophotometrically

and the results were extrapolated by standard curve (Appendix 1) drawn using di-

hydrogen phosphate. An aliquot (0.1-1.0 ml) from the culture supernatant was

taken in 25 ml volumetric flask and diluted to about 5 ml with distilled water.

Then 5 ml of ammonium molybdate was added and the mixture was thoroughly

shaken and evolved CO2 is released. The contents of the flasks were diluted to 20

22

ml. Added 1.0 ml of working solution of SnCl2 and immediately made up the

volume to 25 ml. Kept it for 5-10 minutes to develop colour and coloured

intensity was measured after 10 minutes at 660 nm using red filter on

spectrophotometer.

3.5 PREPARATION OF LIQUID FORMULATION AND RHIZOME

TREATMENT

3.5.1 Procurement of seeds

Rhizome of ginger (Zingiber officinale Rosc.) was procured from Seed

Technology and Production Centre, Dr Y S Parmar University of Horticulture

and Forestry Nauni, Solan (H.P.)

3.5.2 Rhizome surface sterilization

Rhizomes were surface sterilized in 0.2 per cent mercuric chloride

(HgCl2) solution for 5 minutes and rinsed several times with sterilized distilled

water.

3.5.3 Preparation of liquid formulation

The population density (1.5 OD at 540 nm) that resulted in formation of

108 cfu/ml of bacterial isolates were used for preparation of liquid formulation.

3.6 NET HOUSE STUDIES

3.6.1 Preparation of potting mixture

Soil obtained from a furrow slice (0-15 cm depth) from a forest block of

Department of Silviculture and Agroforestry, UHF, Solan was sieved through 2

mm sieve and used for pot culture experiment. The potting mixture was prepared

by mixing sand, soil and farm yard manure (FYM) in a ratio of 1:1:2. The

mixture was then filled in the pots and moistened to one third of its maximum

water holding capacity.

3.6.2 Experiment details

The following treatments were applied in triplicate under net house

conditions

23

Treatments

T1 Absolute control (uninoculated)

T2 Uuninoculated control (with recommended doses of fungicides)

T3 Inoculation with KK1







Total treatments - 9

Replication - 5

Experimental Design = Completely Randomized Design (CRD)

3.6.3 Sowing of rhizome

Ginger rhizomes were sown in pots containing potting mixture: sand, soil

and FYM (1:1:2). Healthy rhizome having at least two well differentiated buds

dipped in the bacterial cell suspension of 1.5 O.D. were sown at a uniform depth

of 2 cm in each pot. After 2 months rhizome was sprouted.

3.6.4 Physico-Chemical properties available nutrient and microbiological

status of potting mixture

Potting mixture was analyzed before and after the treatments for

important physic-chemical and available nutrient status by adopting the

following:

3.6.4.1 pH and electrical conductivity

The soil pH was determined in 1:2.5 soil: water suspension and the

electrical conductivity of the supernatant liquid was recorded and expressed in

dSm-1

(Jackson 1973).

24

3.6.4.2 Organic carbon

Organic carbon was determined by Chromic acid titration method of

Walkley and Black (1934).

3.6.4.3 Available Nitrogen

Available nitrogen was determined by alkaline permagnate method of

Subbiah and Asija (1956).

3.6.4.4 Available Phosphorous

0.5 N NaHCO3 at 8.5 pH was used to extract available phosphorus

(Olsen’s et al., 1954) and determined by spectrophotometrically

3.6.4.5 Available Potassium

Available potassium was extracted by normal neutral ammonium acetate

(Merwin and Peech, 1951) and determined on flame photometer.

3.6.4.6 Total Microbial Count

The soil was analyzed for total microbial count. 1g of soil mixture was

taken in 9 ml of sterilized water blank and the soil suspension was diluted in 10

folds series, then microbial count was determined by standard pour plate

technique on different media as described by (Subba Rao, 1999).The population

was expressed as colony forming units (cfu/g soil)

3.6.5 Morphological characteristics

3.6.5.1 Number of leaves

Number of leaves was counted at fortnightly intervals from the tallest

shoot of the selected plants and average number of leaves per shoot was worked

out.

3.6.5.2 Number of tillers

Number of tillers per plant was counted at fortnightly intervals from the

selected plants and average number of tillers per plant was worked out.

25

3.6.5.3 Shoot length

The shoot of the plant was measured in centimeters (cm) from soil line to

the highest tip of the plant, at peak growth stage.

3.6.5.4 Rhizome yield per plant

The fresh weight of all the plants per plot after removing the aerial part,

roots and soil were recorded in g/plant.

3.6.5.4.1 Nitrogen, Phosphorus and Potassium (NPK) analysis

3.6.5.4.1.1 Sample collection

The samples collected were immediately weighed and brought to the

laboratory in paper bags. All the samples were washed in series first with tap

water then 0.1 N HCl followed by bags. All the samples were washed in series

first with tap water then 0.1 N HCl followed by distilled water. The washed

samples were allowed to dry in air subsequently in oven at 600 C till constant

weight. Oven dried samples of shoot, root and rhizome were ground and sieved

(40 mesh) and stored in butter paper bags for chemical estimation.

3.6.5.4.1.2 Digestion of samples

Total Nitrogen

The digestion of 0.50 g samples for estimating nitrogen was carried out in

the concentrated H2SO

4 in presence of by adding digestion mixture of following

composition:

Potassium sulphate (K2SO4) = 400 parts

Mercuric oxide (HgO) = 3 parts

Copper sulphate (CuSO4.5H2O) = 20 parts

Selenium powder (Se powder) = 1 part

Total P and K

For the estimation of P and K 0.5g plant sample was digested in diacid

mixture prepared by mixing nitric acid and perchloric acid (4:1) taking all

relevant precautions as suggested by Piper (1966).

26

3.6.5.4.1.3 Estimation of nutrient elements

The nitrogen was estimated in Kel Plus Classic Dx. Auto Analyzer.

Phosphorus was determined by Vanado molybdo-phosphoric yellow colour

method and potassium was determined by flame-photometer (Jackson, 1973).

3.6.5.4.1.4 Nutrient uptake

The total nutrient uptake by plant on dry weight basis was worked out by

using the formula:

% NC x Biomass (g) (root) + % NC x Biomass (g) (shoot) + % NC x Biomass (g) (rhizome)

NU (mg/plant)= x 1000

100

3.6.5.5 Reisolation and enumeration of endorhizobacteria and

rhizosphere soil bacteria

The bacterial population was determined in rhizosphere and roots of

ginger plants grown in net house.

3.6.5.5.1 Reisolation of rhizosphere soil bacteria

Ginger plant collected from net house was uprooted and shaken

vigorously to remove the soil tightly adhered to the roots. Resolution was done

by the serial dilution and pour plate technique was described by Subba Rao

(1999). Populations were expressed as colony forming units (cfu) per gram of

soil.

3.6.5.5.2 Resolution of endophytic rhizobacteria

The endophytic bacterial count was determined by taking whole rhizome

root system. The roots were washed with tap water to free the rhizosphere soil.

Washed roots were surface sterilized by 0.2 per cent mercuric chloride (HgCl2)

solution for 1-2 minutes and rinsed several times with sterilized distilled water.

Surface sterilization of roots was cross checked by incubating the surface

sterilized roots in sterilized nutrient broth. The bacterial growth, if any, around

the roots were recorded after 24 h of incubation. The serial dilution and pour

plate technique was described by Subba Rao (1999). Populations were expressed

27

as colony forming units (cfu) per gram of roots. Isolates were maintained on

specific medium for further studies.

3.7 STATISTICAL ANALYSIS

The results under laboratory and glass house conditions on various

parameters were subjected to statistical analysis as per method outlined by

Gomez and Gomez (1976). The CD at 5 % and 1 % level was used for testing the

significant differences among the treated means.

Chapter-4

EXPERIMENTAL RESULTS

The results obtained during the course of investigations are presented in

this chapter under the following headings:

4.1 Isolation and enumeration of microbial population

4.2 Screening of bacterial isolates for multifarious plant growth

promoting activities

4.3 Biochemical characterization and Identification of selected bacterial

isolate

4.4 Effect of bacterial inoculum on number of tillers, shoot length and

rhizome yields of ginger

4.5 Effect of bacterial inoculum on total nutrient uptake

4.6 Effect of bacterial inoculum on physico-chemical properties, available

nutrients status and microbial population

4.7 Correlation (r-value) studies:

4.1 ISOLATION AND ENUMERATION OF MICROBIAL

POPULATION

Isolation of microorganisms was carried out from the rhizosphere, rhizome

and roots of the ginger (Zingiber officinale Rosc.) collected from different

locations/sites of Solan (Kandaghat and Nauni) and Sirmour (Narag and Rajgarh)

districts of Himachal Pradesh.

4.1.1 Microbial population in rhizosphere of ginger

A summary of population of microorganisms in rhizosphere of ginger at

different locations/sites is presented in Table 1 The data revealed that the

rhizosphere soil had great variation in total microbial counts at different

sites/locations. Among various sites, Narag (Sirmour) had highest (222.40 x105

cfu/g soil) bacterial counts on NA medium and Kandaghat (Solan) had the lowest

(150.62 x105 cfu/g soil) counts. The population on soil extract medium was

29

highest (279.69 x105

cfu/g soil) at Narag (Sirmour) and the lowest (252.77 x105

cfu/g soil) at Nauni( Solan). The population on the Jensen’s medium which is

specific for asymbiotic N-fixers was highest (75.34 x105

cfu/g soil) at Narag

(Sirmour) and lowest (56.65 x105 cfu/g soil) at Kandaghat (Solan).

The population on the Pikovskaya’s medium specific for P-solubilisation

was highest (82.35 × 105cfu/g soil) at Nauni (Solan) and lowest (65.28 x10

5 cfu/g

soil) in Rajgarh (Sirmour).

Table 1. Enumeration of rhizosphere bacterial population associated with

ginger

Microbial count (×105 cfu / g soil ) Locations Sites

Nutrient

agar (NA)

Jensen’s

medium

(JM)

Pikovskaya’s

medium

(PVK)

Soil extract

medium

(SEM)

Nauni 163.55 66.94 82.35 252.77 SOLAN

Kandaghat 150.62 56.65 72.09 271.72

Rajgarh 214.33 64.35 65.28 266.33 SIRMOUR

Narag 222.40 75.34 73.95 279.69

The data pertaining to P-solubilizing microbes are presented in Table 2

The percentage of phosphate solubilizing microorganisms to total PVK count

ranged from 63.88 per cent (Nauni, Solan) to 78.36 per cent (Rajgarh, Sirmour).

Table 2. Population of P - solubilizers associated with ginger

Location Sites Pikovskaya’s

medium (PVK)

PSB + ve

colonies

% P solubilizer to

PVK count

Nauni 82.35 48.67 63.88 SOLAN

Kandaghat 72.09 54.08 73.84

Rajgarh 65.28 62.00 78.36 SIRMOUR

Narag 73.95 56.99 70.78

4.1.2 Microbial population in roots of ginger

The data presented in Table 3 revealed that the ginger roots collected from

different location/sites harboured variable microbial population. The endophytic

bacterial population was highest on NA medium followed by PVK and lowest on

Jensen’s medium.

30

The highest total bacterial counts (116.25× 101cfu/g roots) and lowest

(103.33 ×101cfu/g roots) was noted for Rajgarh (Sirmour). The maximum (52.32

×101cfu/g roots) endophytic bacterial population capable of N-fixation as shown

by their growth on jensen’s medium was recorded for Rajgarh (Sirmour) and

lowest (40.82 ×101cfu/g roots) counts was recorded for Nauni (Solan). The

maximum (65.98 ×101cfu/g roots) endophytic bacterial counts of P-solubilization

was recorded at Narag (Sirmour) and minimum (56.34 ×101cfu/g roots) was

noted for Nauni (solan).

Table 3. Enumeration of microbial population associated with ginger roots

Microbial count (×10

1 cfu / g roots ) Locations Sites

Nutrient agar

(NA)

Jensen’s

medium (JM)

Pikovskaya’s

medium (PVK)

Nauni 116.25 40.82 56.34 SOLAN

Kandaghat 110.66 49.65 59.27


Narag 111.70 45.68 65.98

The data embedded in Table 4 clearly revealed that per cent phosphate

solubilizers to the total population on PVK medium at different location/sites

varied from 62.42 per cent at Narag (Sirmour) to 79.00 per cent at Kandaghat

(Solan).

Table 4. Population of P - solubilizers associated with ginger roots

Location Sites Pikovskaya’s

Medium (PVK)

PSB + ve

colonies

% P solubilizer to

PVK count

Nauni 56.34 61.98 77.11 SOLAN

Kandaghat 59.27 87.72 79.00


Narag 65.98 61.98 62.42

4.1.3 Selection of morphologically similar colonies

The isolates capable of growth on different media with similar

morphological features were grouped together to represent one isolate. A total of

seven isolates (three from rhizosphere and four from roots) were selected out of

32 purified isolates. The data pertaining to morphological characteristics of these

isolates are presented in Table 5.

31

Table 5. Morphological characterstics of rhizospheric and endophytic

bacterial isolates of ginger roots Isolates Form Elevation Margin Surface Gram’s

reaction

Shape

KK1 Circular Raised Undulate Smooth + Rods

KK2 Irregular Flat Entire Smooth + Rods

KK3*

Circular Raised Entire/curled Smooth + Rods

KK4*

Circular Convex Erosed Rough + Rods

KK5*

Irregular Circular Curled Smooth + Rods

KK6*

Rhizoidal Umbonate Lobate Smooth + Rods

KK7*

Circular Convex Entire Rough + Rods

* Endorhizobacteria

4.2 SCREENING OF BACTERIAL ISOLATES FOR MULTIFARIOUS

PLANT GROWTH PROMOTING ACTIVITIES

The selected seven isolates were screened for their ability to perform

multifarious plant growth promoting activities i.e. P-solubilization, N-fixation,

siderophore, auxin, HCN production and antagonism against major fungal

diseases i.e soft rot (Pythium spp.) and Fusarium wilt (Fusarium spp.).

4.2.1 Tricalcium phosphate solubilization by selected bacterial isolates

The phosphate solubilizing activity of the selected bacterial isolates were

compared on the basis of their per cent solubilizing efficiency (%SE) on PVK

agar medium and in PVK broth (Table 6). The results revealed that the P-

solubilization efficiency of different isolates had great variations, it’s values

ranged from 56.0 to 86.6 per cent on agar medium and from 63.0 to 202.2 µg/ml

in liquid medium.

The maximum (86.6 %) P-solubilization efficiency was noted for KK5

isolate, however, it was statistically at par with KK3 (76.1 %), KK6 (76.8 %),

KK7 (73.7 %) isolates and the minimum (56.0 %) efficiency was recorded for

KK2 isolate. In liquid PVK medium, maximum (202.2 µg/ml) P-solubilization

was recorded for isolate KK6, which is statistically at par with KK2 (200.3

µg/ml), KK4 (195.2 µg/ml), KK3 (198.6 µg/ml) isolates.

32

Table 6. Phosphorus solubilization efficiency of bacterial isolates on solid

PVK medium

Isolates Colony size (C)

(mm)

Zone size (Z)

(mm)

% P-Solubilization

Efficiency (% SE)

P-solubilization in

liquid medium

(µg/ml)

KK1 2.33 3.83 64.37 (53.33) * 63.00

KK2 3.25 5.07 56.00 (45.24) 200.33

KK3 2.47 4.35 76.11 (60.62) 198.66

KK4 2.83 4.32 52.65 (48.48) 125.25

KK5 3.67 6.85 86.65 (68.62) 202.22

KK6 3.20 5.66 76.87 (60.29) 195.67

KK7 3.74 6.50 73.79 (60.61) 79.62

CD0.05 0.38 0.04 8.37 12.43

Z-C

% SE = x 100, where C C = colony size , Z = Halozone size

C

*Figures in the parentheses are arc sine transformed values

4.2.2 Siderophore production efficiency of different bacterial isolates on

CAS medium

The siderophore production efficiency of the selected bacterial isolates

was checked by Chromeazurol plate assay method. The results revealed that all

the selected isolates were positive for siderophore production (Table 7). The

maximum (86.79%) siderophore production efficiency was noted for isolate KK5,

however, which was at par with isolates KK3 (75.89%) and KK6 (73.98 %)

isolates.

Table 7. Siderophore production efficiency of different bacterial isolates

on CAS medium

Isolates Colony size (C)

(mm)

Zone size (Z)

(mm)

% Siderophore production

efficiency (% SE)

KK1 4.24 5.84 37.74 (37.90)*

KK2 3.25 4.85 49.23 (44.54)

KK3 2.24 3.94 75.89 (60.27)

KK4 4.46 6.85 53.58 (47.10)

KK5 3.56 6.65 86.79 (68.32)

KK6 2.16 3.76 73.98 (60.97)

KK7 2.15 3.55 65.11(54.00)

CD0.05 0.09 0.05 8.98

Z-C

%SE = x 100, where C = colony size, Z = Halozone size

C

*Figures in the parentheses are arc sine transformed values

33

4.2.3 IAA production

The results depicted in Fig.1 and presented in Appendix 2.1 revealed the

IAA equivalents produced by different isolates varied from 18.67 to 29.62 µg/ml.

Table 8. Screening of selected bacterial isolates for multifarious plant

growth promoting activities

Antagonistic

activities**

Isolates P-

Solubilization*

N-free

medium

Auxin

Production**

Siderophore

Production*

HCN

Production

Pythium

spp.

Fusarium

spp.

KK1 +++ + +++ + - ++ ++

KK2 ++ ++ ++ + + - ++

KK3 +++ +++ ++ ++ +++ ++ +++

KK4 ++ + + ++ + ++ -

KK5 +++ +++ +++ +++ ++ +++ +++

KK6 +++ +++ ++ +++ ++ +++ +++

KK7 ++ ++ ++ ++ + - +++

*values ranging from 40-70 % (++), ≤ 40% (+), ≥ 70% (+++), no activity (-)

** values ranging from 20-40 (++), ≤ 20 (+), ≥ 40 (+++), no activity (-)

The IAA production by KK5 isolate was maximum (29.62 µg/ml) after 72

h of incubation.

From the data presented in Table 8, it is clear that only KK5 bacterial

isolate exhibited the maximum plant growth promoting traits i.e production of

siderophores, HCN production, auxin production, solubilization of phosphorus

and growth on nitrogen free medium. The isolate was also capable of inhibition

of mycelial growth of Pythium spp. and Fusarium spp. (Fig.2) Hence, the isolate

was characterized and identified upto genus level.

4.3 BIOCHEMICAL CHARACTERIZATION AND IDENTIFICATION

OF SELECTED BACTERIAL (KK5) ISOLATE

The morphological, physiological and biochemical characteristics of KK5

isolate are presented in Table 9. The isolate on NA medium has circular colony

Fig 1. IAA production (µg/ml) by different bacterial isolate

Fig 2. Percent growth inhibition of fungus by different bacterial isolate

IAA

pro

du

ctio

n (

µg/m

l)21.22

18.97

28.27

23.96

29.6728.27

19.83

0

5

10

15

20

25

30

KK1 KK2 KK3 KK4 KK5 KK6 KK7

Isolates

34

configuration having rough surface, umbonate elevation, erose margin. The cells

are gram positive, spore forming and motile rods occurring singly.

The organism grew at a wider temperature range i.e 100C - 40

0C but

showed no growth at 450C. (Fig 3). The colony growth was observed from pH 4-

8 , however, optimum pH for its growth was recorded as 7.0 (Fig 4).

The isolate was found positive for Voges Proskauer test, catalase test,

fermentation of carbohydrates and gelatin hydrolysis; however, it showed

negative reaction for indole test, methyl red test, starch hydrolysis, casein

hydrolysis and citrate utilization test, hydrogen sulphide production and urease

test.

On the basis of morphological, physiological, and biochemical

characteristics and by criteria of Bergey’s Manual of Systematic Bacteriology

(Claus and Berkeley, 1986) the isolate KK5 is tentatively identified as Bacillus

spp.

Table 9. Morphological, physiological and biochemical characteristics of

selected bacterial (KK5) isolate

A. Morphological characteristics

Tests Results

Colony morphology

Form Circular

Margin Erose

Elevation Umbonate

Surface Rough

Gram’s reaction +ve

Cell shape Rods

Arrangement Occurring singly

Spore(s)

Endospore formation -

Position Subterminal

Mobility +ve

35

B. Physiological characteristics

Temperature (°C) Growth pH Growth

10 + 5 .0 +

25 + 6.0 +

30 + 7.0 +

35 + 8.0 +

40 + 9.0 -

45 -

C. Biochemical tests

Tests Results

Indole test -

Methyl red test -

Voges Proskauer test +

Starch hydrolysis -

Casein hydrolysis -

Gelatin hydrolysis +

Hydrogen sulphide production -

Catalase test +

Citrate utilization test -

Urea hydrolysis -

Fermentation of Glucose +

4.3.1 Characterization of selected bacterial (KK5) isolate

4.3.1.1 Effect of temperature and pH on growth of KK5 isolate

The experiment was conducted by taking different temperatures (10-

450C) and pH (5.0-9.0) to determine the optimum temperature and pH for the

growth of selected bacterial (KK5) isolate. From the data depicted in Fig. 3 and

Fig 4 it is cleared that after 72h of incubation the optimum growth (on the basis

of maximum O.D at 540 nm) for KK5 isolate was found to be best at a

temperature of 350C and pH 7.0.

4.3.1.2 Effect of bacterial inoculum on selected plant parameters

The data pertaining to plant parameters are presented in the Table 10. The

application of different bacterial isolates significantly increased number of leaves,

shoot length, number of tillers, over C1 (uninoculated absolute control) and C2

(fungicide treated control).

Fig 3. Effect of temperature on the growth of selected bacterial (KK5) isolate

Fig 4. Effect of pH on the growth of selected bacterial (KK5) isolate

36

Among the isolates, KK5 isolate registered maximum (80.63) shoot

length, however which was at par with KK3 (78.40) and KK6 (79.57) isolates.

The maximum values for number of tillers (5.66) were recorded for KK5 isolate

treated plants which was at par with KK3 (4.66) but significantly higher than

other tried isolates and both the controls i.e C1 (uninoculated absolute control)

and C2 (fungicide control).

The effects of bacterial inoculation on number of leaves was significant

over uninoculated control. The maximum (24.33) number of leaves was recorded

with KK5 isolate and the minimum with C1 (15.67) isolate.

Table 10. Effect of bacterial inoculums on plant parameter

Isolates Plant Parameters

Shoot length (cm) Number of tillers Number of leaves/ plant

C1 54.60 1.00 15.67

C2 58.40 2.00 16.67

KK3 78.40 4.66 21.00

KK4 70.63 3.00 19.00

KK5 80.63 5.66 24.33

KK6 79.57 4.33 22.00

CD0.05 0.37 0.72 2.05

4.3.1.3 Effect of bacterial inoculum on rhizome yield under net house

conditions

An inquisition of data recorded for weight of rhizome per plant presented

in Table 11 clearly revealed a significant increase in weight of rhizome by the

application of bacterial inoculums. The maximum per cent increase (48.39) was

recorded with the inoculation of KK5 isolate over C1 (uninoculated control) and

minimum 29.56 % was noted for KK4. The maximum yield/plant (0.24 kg/plant)

was recorded for KK5 isolate, however which was statistically at par with KK6

(0.23 kg/plant) isolate and KK3 (0.23 kg/plant) and the minimum (0.16 kg/plant)

was noted for C1 (uninoculated absolute control). The KK5 bacterial isolate

registered an increase of 48.39 % and 43.48 % over C1 (uninoculated absolute

control) and C2 (uninoculated fungicide control), respectively (Fig 8).

37

Table 11. Effect of bacterial inoculum on fruit parameters in net house

Isolates Weight of

rhizome/ plant (g)

Yield/Plant

(kg)

% increase in

yield/plant over C1

% increase in

yield/plant

over C2

C1 58.62 0.16 0.00 -4.25

C2 60.63 0.17 4.44 0.00

KK3 80.67 0.23 45.55 38.33

KK4 72.50 0.21 29.56 25.33

KK5 81.37 0.24 48.39 43.48

KK6 78.37 0.23 42.93 38.40

CD0.05 0.38 0.01 0.54 0.49

4.4 EFFECT OF BACTERIAL INOCULUM ON PLANT NUTRIENT

UPTAKE

4.4.1 N

A perusal of data given in the Appendix 2.5 and depicted in Fig 10

revealed that total N content was significantly influenced by different bacterial

isolates over C1 treatment (uninoculated control). The maximum (105.5 mg/plant)

N uptake was recorded with KK5 isolate whereas, the minimum (86.53 mg/plant)

was recorded for C1 treatment (uninoculated absolute control) .

4.4.2 P

It is evident from the data presented in Appendix 2.5 and Fig 10 that

different bacterial isolates influenced total P content significantly over C1

treatment (uninoculated absolute control). The maximum (84.4 mg/plant) total P

uptake was recorded by KK5 isolate. The minimum (20.47 mg/plant) P uptake

was recorded for C1 treatment (uninoculated absolute control).

4.4.3 K

K content was also influenced significantly by all the tried bacterial

isolates over absolute control (Appendix 2.5 and Fig. 10). The maximum (149.3

mg/plant) K uptake was recorded for KK5 treated plants and the minimum (91.14

mg/plant) K content was noted for C1 (uninoculated absolute control).

Plate 9. Effect of bacterial inoculums on shoot length under net house conditions

Plate 10. Effect of bacterial inoculums on rhizome yield under net house conditions

C1C2 KK3 KK4

KK5 KK6

C1C2 KK3 KK4 KK5 KK6

38

4.5 EFFECT OF BACTERIAL INOCULUM ON

PHYSICOCHEMICAL PROPERTIES, AVAILABLE NUTRIENT

STATUS AND MICROBIAL POPULATION BEFORE AND

AFTER THE EXPERIMENT

The important physico-chemical properties were recorded at the start and

termination of the experiment under net house conditions. The data pertaining to

initial status of the soil presented in the Table 12 revealed that the soil was nearly

neutral in reaction with pH value 7.56, EC in normal range (0.49 dSm-1

), high in

organic carbon (1.25%). The available N and K were medium range whereas,

available P was in high range. The total microbial counts on NA were 96.8 × 105

cfu/g soil and for soil extract medium the count were 110.7 × 105

cfu/g soil

respectively.

Table 12. Physico-chemical properties, available nutrient content and total

bacterial counts of soil mixture used for net house experiment

(initial status)

PARAMETERS VALUES

1. PHYSICO-CHEMICAL PROPERTIES

a. pH (1:2-5)

b. Electrical conductivity (dSm-1

)

c. Organic carbon (%)

7.56

0.49

1. 25

2. NUTRITIONAL STATUS

Available Nutrients (kg/ha )

N

P

K

315.6

28.3

235.2

3. TOTAL BACTERIAL COUNT

NA (× 105

cfu / soil)

SEM (× 105

cfu / soil)

96.8

110.7

The data pertaining to effect of different bacterial isolates on physico-

chemical properties of soil at the end of experiment are depicted in Table 13. The

data indicated that none of the bacterial isolates influence pH, EC and OC of soil

significantly over uninoculated control. However, a little variation in soil pH and

EC values were recorded by the application of different isolates over their

respective initial values.

39

Table 13. Effect of bacterial inoculum on physico-chemical

characteristic and available nutrient status of soil

Pysico-chemical properties of soil Isolates

pH

(1:2.5)

EC

(dSm-1

)

OC

(%)

N

(kg/ha)

P

(kg/ha)

K

(kg/ha)

C1 7.68 0.42 0.89 290.3 33.15 251.4

C2 7.85 0.39 1.13 305.3 34.12 262.4

KK3 7.85 0.46 1.24 323.2 37.20 277.5

KK4 7.75 0.37 1.14 317.5 36.66 267.6

KK5 7.91 0.44 1.38 330.3 39.73 279.6

KK6 7.71 0.41 1.02 327.0 38.34 275.6

CD0.05 NS NS NS 0.28 0.34 0.39

The amount of available NPK increased significantly by inoculation of

different bacterial isolates. The highest amounts of available N (330.3 kg/ha), P

(39.73 kg/ha) and K (279.6 kg/ha) were observed by the inoculation of KK5

isolate whereas, the lowest amount was recorded in C1 treatment uninoculated

control. The available NPK content of the soil, in general increased by 25 per

cent, 23 per cent and 18 per cent respectively, over uninoculated control and over

fungicide control (Fig 9 ; Appendix 2.6)

The results presented in Table 14 revealed that microbial counts was more

on SE medium as compared to NA medium. The maximum microbial counts was

recorded on NA (176.6× 105 cfu/g soil ) and SE (197.6× 10

5 cfu/g soil) medium

in rhizosphere of plants whose rhizome were treated with KK5 isolate. However,

the minimum (114.4× 105 cfu/g soil and 155.2 × 10

5 cfu/g soil) counts on NA and

SE medium was noted for C2 treatment (uninoculated fungicide control).

The maximum endophytic microbial counts on NA and SE medium were

(127.2 × 101 cfu/g roots) and (129.4× 10

1 cfu/g roots), respectively. However,

their respective minimum (77.60× 101 cfu/g roots and 73.20 × 10

1 cfu/g roots)

counts was recorded in C2 treatment (uninoculated fungicide control).

Fig 5. Effect of bacterial inoculum on number of leaves of ginger under

net house conditions.

Fig 6. Effect of bacterial inoculum on shoot length of ginger under net

house conditions.

Fig 7. Effect of bacterial inoculum on number of tillers of ginger under net

house condition

Fig 8. Effect of bacterial inoculum on number rhizome yield of ginger

under net house condition

Fig 9. Effect of bacterial inoculum on available nutrient contents in soil of ginger

under net house conditions.

Fig 10.Effect of bacterial inoculum on NPK uptake by ginger under net house

conditions.

40

Table 14. Rhizosphere and endophytic bacterial population associated

with ginger (At termination of experiment)

Rhizosphere population

(×105 cfu/g

soil)

Endophytic bacterial

population (× 101 cfu/g

root)

Isolates

NA SE NA SE

C1 125.2 160.6 81.20 97.60

C2 114.4 155.2 77.60 73.20

KK3 178.4 196.6 120.6 122.8

KK4 125.4 190.2 98.20 94.20

KK5 176.6 197.6 127.2 129.4

KK6 178.4 192.8 121.8 123.4

4.6 CORRELATION (r-value) STUDIES:

A significant positive correlation (r) was noted between rhizospheric

bacterial population and plant parameters (r=0.83 for weight of rhizome), (r=0.86

for number of tillers), (r=0.87 for shoot length and r=0.89 for number of leaves),

endophytic bacterial population and plant parameters (r= 0.70 for weight of

rhizome/ plant and r=0.83 for yield/plant) at 1 per cent level of significance

(Table 15).

Table 15. Correlation between ginger plant microbial population and

plant parameters at the end of the experiment

Parameters weight of

rhizome/plant yield

/plant number of

tillers /plant shoot

length number of

leaves

Rhizosphere

bacterial

population

0.83*

0.92*

0.86*

0.87*

0.89*

Endophytic

bacterial

population

0.70*

0.83*

0.79*

0.73*

0.77*

*significant at 1 % level

A significant positive correlation (r) between rhizospheric bacterial

population and pH (r=0.52) and endophytic bacterial population and pH (r=0.43)

at 5 % level of significance. Similarly a significant positive correlation between

41

endophytic bacterial population and available nitrogen(r=0.76), endophytic

bacterial population and available phosphorus (r= 0.79), endophytic bacterial

population and available potassium (r=70) at 1 per cent level of significance

(Table 16)

Table 16. Correlation between ginger plant microbial population and soil

parameters at the end of the experiment

Parameters pH EC OC Available

N Available

P Available

K

Rhizosphere

bacterial

population

0.52**

0.68*

0.89*

0.90*

0.97*

0.86*

Endophytic

bacterial

population

0.43**

0.61*

0.70*

0.76*

0.79*

0.70*

**significant at 5% level

* significant at 1% level

A significant positive correlation (r) between plant parametrers and

available nutrients shoot length and available N (r=0.97), available P (r=0.96),

available K (r= 0.96), number of leaves and available N (r=0.90), available P (r=

0.95 ), available K (r=0.89) while number of tillers and available N(r=0.92),

available P(r=0.95), available K (0.96) at 1 per cent level of significance (Table

17).

A significant positive correlation (r) between available nutrients and plant

parameters weight of rhizome per plant and available N(r=0.95), available

P(r=0.95), available K(r=0.79), yield per plant and available N(r=0.85), available

P(r=0.88), available K(r=0.85) at 1 per cent level of significance (Table 17).

A significant positive correlation (r) between soil parameters and plant

parameters pH and weight of rhizome per plant (r=0.82), yield per plant (r=0.92),

shoot length (r=0.81). Similarly a significant positive correlation (r) between EC

and number of tillers per plant, OC and number of leaves at 1 per cent level of

significance (Table 17).

42

Table 17. Correlation coefficient (r-value) among the soil parameters and

plant parameters from liquid based inoculants in net house at

the end

Soil

Parameters

weight of

rhizome/

plant

yield

/plant

number of

tillers

/plant

shoot

length

number

of leaves

N uptake

(mg/

plant)

P uptake

(mg/

plant)

K uptake

(mg/

plant )

pH 0.82* 0.86* 0.85* 0.81* 0.79* 0.71* 0.67* 0.67*

EC 0.97* 0.92* 0.93* 0.97* 0.89* 0.94* 0.84* 0.85*

OC 0.94* 0.99* 0.89* 0.96* 0.89* 0.74* 0.93* 0.85*

N 0.95* 0.85* 0.92* 0.97* 0.90* 0.95* 0.90* 0.93*

P 0.95* 0.88* 0.95* 0.96* 0.95* 0.96* 0.92* 0.95*

K 0.79* 0.85* 0.96* 0.96* 0.89* 0.93* 0.87* 0.88*

*significant at 1% level of significance

Chapter-5

DISCUSSION

With the rapid growth of world population the use of chemical inputs

have tremendously increased to produce more and more food grains, this

indiscriminate use has led to environmental pollutions and degradation of soil

health. Hence, there is a need to use chemical fertilizers inputs in conjugation

with other sources to fulfill crop nutrient requirements, to sustain soil

productivity and to minimize the risk of soil and water pollution. The alternatives

of soil fertilization are organic sources, including the addition of bulky manure,

enriched composts and plant crop residue, use of legumes as green manures and

use of soil beneficial microbial inoculants (Adesemoye and Kloepper, 2009a,b).

The thin layer of soil 1 - 2 mm thick surrounding crop roots and the

volume of soil occupied by roots is known as the rhizosphere which supports

large and active microbial population capable of exerting beneficial, neutral or

detrimental effects on plant growth (Bowen , 1999). The rhizosphere is known to

be preferred ecological niche for soil microorganisms due to rich nutrient

availability where PGPR colonize plant roots and exert beneficial effects on plant

growth and development (Ashrafuzzaman et al., 2009) by increasing the

availability and uptake of nutrients (Gaskin et al., 1985) and by suppressing soil

borne plant pathogen and other deleterious rhizosphere microorganisms (Schroth

and Hancock, 1982).

Ginger (Zingiber officinale Rosc.) occupies considerable value because of

its importance in the agricultural economy of the country. In Himachal Pradesh, it

ranks as one of the most important cash crops particularly in midhills. Ginger

being heavy feeder and exhaustive crop requires large quantities of inorganic and

organic fertilizers. Inorganic or chemical fertilizers besides being costly are also

injurious to plants, ground water and environment (Nath and Korla, 2000).

Biofertilizers have emerged as promising components of nutrient supply system.

44

It is not only low-cost input but also gives high returns under favourable

conditions (Pradhan, 1994).

The experimental results obtained from the present investigations entitled

“Effect of selected plant growth promoting rhizobacteria on growth and yield of

ginger (Zingiber officinale Rosc.)” conducted during year 2010-11 have been

discussed in foregoing section in the light of pertinent literature under the

following subheadings:

5.1 Isolation and enumeration of microorganisms

5.2 Screening, characterization and identification of bacterial isolates

5.3 Effect of bacterial inoculum on plant growth promotion

5.4 Effect of bacterial inoculum on plant nutrient content and their

uptake

5.5 Effect of bacterial inoculum on physico-chemical properties and

nutrient status of the soil

5.6 Effect of bacterial inoculum on rhizosphere and endophytic bacterial

populations

5.1 ISOLATION AND ENUMERATION OF MICROORGANISMS

The rhizosphere and phyllosphere of the plants under natural conditions

harbors a large and varied population of the microorganisms. It has been

demonstrated that bacteria belonging to genera Bacillus and Pseudomonas, not

only proliferate rhizosphere, but also reside inside the root tissue of agriculturally

important crop species (Hallmann, 1997).

The rhizosphere serves as a very dynamic habitat for microorganisms.

The ginger rhizospheric and roots/ rhizome samples were collected from its

natural habitat for isolation of PGPR’s isolates.

The data presented in Tables 1-4 reveals a great variations in the

microbial counts both in rhizosphere and roots of ginger collected from different

45

locations in mid hills of Himachal Pradesh. The total microbial counts, in general,

was more in rhizosphere (279.69 to 252.77 × 105 cfu/g soil) as compared to

endophytic (111.70 to 103.33× 101 cfu/g root) counts. Our results corroborate

with the similar studies performed by Hallmann et al. (1997) who, reported that

under natural conditions, the rhizosphere and rhizoplane of the plants harbour a

large and varied population of the microorganisms.

The variation in microbial population both in rhizosphere and roots may

be attributed to climatic conditions of the location, age of plant, variety/cultivar

type, time of sampling and physico-chemical properties of soil. The results are

further in line with those of Shishido et al. (1999) who has also reported variation

in microbial population with respect to location/plant parts. Zhang et al. (2006)

showed that different environment parameters, content of soil organic carbon,

total nitrogen and altitude could affect the diversity of soil flora.

Endophytic bacteria reside within the plant tissues and thus have a natural

and intimate association with plants. The internal tissues of plants provide

relatively uniform and protected environment when compared with rhizosphere

and rhizoplane (Sharma et al., 2005).

The data on abundance and diversity of the population of P-solubilizers in

rhizosphere and roots for different locations/sites are presented in Table 2 and

Table 4. The population of P-solubilizers varied from 62.42 to 78.36 per cent of

the total bacterial counts. Kapoor et al. (1989) also reported that the population of

phosphate solubilizing microorganisms, in general, varied from 20-24 per cent of

the total population and in some soils it may be upto 85 per cent of the total

population. In other studies conducted by Kundu et al. (2002) reported about 16

per cent of the total bacterial population in rhizosphere was P-solubilizers. The

solubilization of phosphorous in the rhizosphere is the most common mode of

action implicated in PGPR that increase nutrient availability to host plants

(Richardson, 2001).

46

5.2 SCREENING, CHARACTERIZATION AND IDENTIFICATION

OF BACTERIAL ISOLATES

The bacterial isolates capable of growth on Pikovskaya’s (PVK) medium,

N-free medium were screened for production of siderophore, auxin, HCN and

antagonism against Pythium spp. and Fusarium spp. Data presented in Table 6

shows that all the seven isolates were P-solubilizers siderophore producers, auxin

producers, and nitrogen fixers.

All the seven isolates were capable of hydrolyzing tri-calcium phosphate

(TCP) in liquid as well as in solid PVK medium. The per cent P-solubilization

efficiency shown by different isolates had great variation with the value ranging

from 52.65 to 86.65 per cent in agar medium and from 63.00 to 202.22 µg/ml in

liquid medium after 72 hrs of incubation. The maximum (86.65 %) P-

solubilization efficiency was noted for KK5 isolate and the minimum (52.65 %)

for KK4 isolate in solid PVK medium, while in liquid PVK medium isolate KK5

solubilized maximum TCP with release of 202.22 µg/ml phosphorus after 72 h

of incubation (Table 6, Plate 3). Kundu et al. (2009) reported P-solubilization by

native isolates of chickpea, mustard and wheat in range of 5.9 to 123.8% and 2.2

to 227.2 µg/ml in solid and liquid Pikovskaya’s medium, respectively.

All the bacterial isolates were able to grow on CAS medium, showing

their ability to produce siderophore ( Table 7, Plate 4). The maximum (86.79 %)

siderophore production efficiency was observed for KK5 isolate, however, which

was statistically at par with KK3 (75.89 %) and KK6 (73.98 %) isolates. The

findings are in line with those of Neilands (1981) and Joseph et al. (2007) who

also reported that bacteria produces siderophore.

All the seven bacterial isolates produces indole-3-acetic acid ranging from

18.67 to 29.62 µg/ml in Luria Bertani broth after 72 h of incubation. IAA is

phytohormone which functions as important signal molecule in regulation of

plant development and considered as important native auxin. Our results are in

agreement with those of Beneduzi et al. (2008) who also reported that many

Bacillus spp and Paenibacillus spp produces IAA on Luria Bertani Broth.

47

Further, Sarwar and Kremer (1992) also reported that rhizospheric bacterial

isolates were more efficient auxin producers than endophytic bacterial isolates.

The selected bacterial isolates showed marked antagonism against

Pythium spp. (Plate 6) and Fusarium spp. (Plate 7) as it is evident from inhibition

zone formed on MEA medium plate. The formation of zone may be due to

secretion of antifungal substance that might have diffused in the medium and

inhibited the fungal growth. Our results are in agreement with those of

Giacomodonato et al. (2001) and Sadfi et al. (2002) who also reported that many

Bacillus strains have inhibitory effect against soil borne fungal pathogen.

5.3 EFFECT OF BACTERIAL INOCULUM ON PLANT GROWTH

PROMOTION

Since the discovery of the PGPR many studies have been made all over

the world to evaluate the mechanisms by which these bacteria renders benefits to

the plant (Kloepper et al.,1980; Van loon et al., 1998 and Kravechenco et al.,

2002).

The rhizome treatment with different bacterial cultures under net house

conditions increased shoot length (47.68%), numbers of leaves/plant (55.12%)

and numbers of tillers (183.3%) over C1 (uninoculated absolute control), C2

(uninoculated fungicide control). The possible reason for enhanced growth and

development may be because of better plant stand which is due to direct

contribution of PGPR in improving the nutrient status of soil particularly for N, P

and Fe. These findings are in conformity with those of Badawy and Amer (1974)

and Chaudhary et al. (1982) for vegetable crops particularly tomato and pea. The

present results are further supported by significant positive correlation between

rhizospheric bacterial population and shoot length (r=0.87), rhizospheric bacterial

population and number of leaves (r= 0.89) and rhizospheric bacterial population

and number of tillers (r= 0.86).

A significant increase was recorded in rhizome yield 48.39 % by the

application of KK5 isolate over C1 (uninoculated absolute control) and C2

(uninoculated fungicide control) (Table 11). The present results are supported by

48

significant correlation between rhizospheric bacterial population and weight of

rhizome per plant (r=0.83) and rhizospheric bacterial population and rhizome

yield per plant (r=0.92). These results are further, in agreement with the findings

of Thamizh and Nanjan (1998) in potato, Mahendran et al. (1996) and

Shashidhara (2000) in chilli crops.

The increased growth by Bacillus spp. (KK5) inoculation may be

attributed to the cumulative effects of phosphate solubilization, nitrogen fixation

and production of plant growth regulators (auxins) and the inhibition of soil

borne pathogens particularly Pythium spp. and Fusarium spp. Further it was

interesting to note that no disease symptoms appeared throughout the

experimental period bacterial in treated plant as compared to control plants. A

significant reduction have also been reported in indigenous bacterial and fungal

populations by the application of effective Bacillus inoculum in the rhizosphere

of potato (Kloepper and Schroth, 1981) and sugar beet (Suslow, 1982). The

increase in growth and biomass yield by the use of Bacillus spp. have been

reported for various crops by different workers (Kundu et al., 2002; Brown,

1993; Kloepper et al., 1989).

The indirect plant growth promoting effects of PGPR (Bacillus and

Pseudomonas) to inhibit deleterious microorganisms have been reported by

various researchers (Luna et al., 2002; Whipps, 2001; Compant et al., 2005).

5.4 EFFECT OF BACTERIAL INOCULUM ON NUTRIENT

CONTENT AND THEIR UPTAKE

Availability of nutrients in soils is greatly enhanced through microbial

activities which may lead to lowering of pH and release of organic acids to

solubilize inorganic complexes present in the soil matrix (Sridevi and Mallaiah,

2009).

The rhizome inoculation with different bacterial isolates not only

improved the nutritional content particularly NPK of plants but also increased

their uptake significantly over uninoculated controls (C1 and C2). The maximum

N (105.5 mg/plant), P (84.4 mg/plant), K (149.3 mg/plant) uptake was recorded

49

by application of isolate (KK5). This increase may be attributed to atmospheric

nitrogen fixation, phosphate solubilization in the rhizosphere and increase in root

length which might have explored the virgin horizons of soil for absorption of

nutrient elements. Further due to enhanced uptake by increase in specific ion

fluxes at the root surface in the presence of plant growth promoting rhizobacteria

has also been reported by Bertrand et al. (2000) and Bashan and Levanony

(1991).

Phosphate solubilizing bacteria are common in the rhizosphere and the

solubilization of P in the rhizosphere by PGPR through organic acid secretion

and phosphate production facilitated the conversion of insoluble forms of P to

plant available forms and ultimately increased availability to host plants (Kim et

al., 1998; Richardson, 2001).

5.5 EFFECT OF BACTERIAL INOCULUM ON PHYSICO-

CHEMICAL CHARACTERISTICS OF SOIL

The data on physico-chemical characteristic of soil mixture (initial and at

the termination of the experiment) are presented in (Table 12, Table 13). The soils

are slightly alkaline in reactions pH (7.68-7.91) and EC was in normal range (0.38-

0.46 dSm-1

). The OC content (0.89-1.38 %) was in high range.

The available N (290-330.3 kg/ha) content was medium whereas, available

P (33.15-39.73 kg/ha) and K (251.4-279.6 kg/ha) was in high range. The significant

correlation (r) between rhizosphere bacterial population and available N (r=0.90),

endophytic bacterial population and available P(r=0.79), rhizosphere bacterial

population and available K (r=0.86) was observed under net house conditions

(Table 16). Fierer and Jackson (2006) considered pH as the best predictor of soil

bacterial diversity and richness. Further results are in conformity with that of Chatli

et al. (2008).

5.6 EFFECT OF BACTERIAL INOCULUM ON RHIZOSPHERE AND

ENDOPHYTIC BACTERIAL POPULATIONS

The results in the present investigations revealed that KK5 isolate

tentatively classified as Bacillus spp. could become endophytically established in

50

ginger roots. The significantly higher populations of rhizospheric and endophytic

rhizobateria in treated plants over the untreated controls is an indication of the

aggressive nature of PGPR isolates (KK5 isolate) in colonizing the root system.

The inoculation of KK5 isolate significantly increased the total microbial

population in rhizosphere (Table 14). At the termination of the experiment

maximum rhizosphere population (197.6 × 105

cfu/g soil) and endophytic

bacterial population (123.8 × 101

cfu/g soil) was also observed in KK5 treated

plants. Alfonso et al. (2009) also recorded high microbial population level in the

rhizosphere of the inoculated plants over uninoculated control. These results are

in conformation with those of Hong et al. (2002) who also reported that soil

treatment with bacterial isolates resulted in significant increase in bacterial

population in rhizosphere and in the roots of plants.

Chapter-6

SUMMARY AND CONCLUSION

The present investigations entitled “Effect of selected plant growth


Rosc.)” was carried out during 2009-11. The studies were aimed to isolate and

characterize plant growth promoting and disease suppressing bacterial isolates

native to rhizosphere soil and/or endophytic tissues of ginger plants and their

effect to enhance plant growth and confer protection against soil borne fungal

diseases. Secondly, to study the effect of the inoculants’s on plant growth,

nutrient uptake, rhizosphere/endophytic bacterial population, soil physico-

chemical characteristics and available major nutrient contents. The salient

features of the investigations are as follows:

� There were significant variations in rhizospheric and endophytic total

bacterial counts of ginger collected from different locations/sites. The

bacterial counts in rhizosphere ranged from 252.77 to 279.69 ×105 cfu/g

soil and the endophytic counts varied from 56.34 to 65.98 ×101 cfu/g

roots.

� On the basis of morphological characteristics (form, elevation, margin,

surface and shape), Gram’s reaction, cell shape and arrangement,

phosphate solubilization, IAA production, siderophore production and

growth on N-free medium, only seven isolates were selected from total of

32 purified isolates isolated from rhizosphere and roots of ginger.

� Seven isolates namely (KK1, KK2, KK3, KK4, KK5, KK6 and KK7)

exhibited the maximum plant growth promoting attributes viz: phosphate

solubilization, siderophore production, growth on N-free medium, auxin

production and HCN production.

� Out of seven isolates, only six isolates were HCN-producers. Six isolates

showed antagonism against Pythium spp. and only five isolates inhibited

the mycelial growth of Fusarium spp. under in vitro conditions.

52

� On the basis of morphological (Gram +ve rods), physiological (optimum

growth at 35°C and 7 pH) and biochemical characteristics (Glucose

fermentation, production of gelatinase and catalase enzymes) possessed

by KK5 isolate and as per criteria of Bergey’s Manual of Systematic

Bacteriology the KK5 isolate was tentatively identified as Bacillus spp.

� The isolate was gram positive, rod shaped, spore forming and grew best at

35°C and neutral pH.

� All the selected isolates were capable of hydrolyzing tri-calcium

phosphate (TCP) in liquid as well as in solid PVK medium. However, the

per cent P-solubilization efficiency had great variation with the value

ranged from 64.37 per cent to 86.65 per cent. The maximum (86.65 per

cent) P-solubilization was noted with KK5 and followed by and KK6

(76.87 per cent) and KK3 (76.11 per cent) in solid PVK medium.

However, in the liquid medium, the P-solubilization ranged from

63.00µg/ml to 202.22µg/ml. The maximum (202.22 µg/ml) P -

solubilization in liquid medium was observed with KK5 isolate followed

by isolates KK3 (198.66 µg/ml) and KK5 (195.67 µg/ml).

� All the selected isolates were siderophore and auxin producers. The

values of siderophore and auxin ranged from 37.74 per cent to 86.79 per

cent and 18.67 µg/ml to 29.62 µg/ml, respectively. The maximum (29.62

µg/ml) IAA production on Luria Bertani broth after 72 h of incubation

was recorded with KK5 isolate. The isolate KK5 produced maximum

(86.79 per cent) siderophore on CAS medium.

� The rhizome inoculation with KK5 isolate, significantly increased the

shoot length (47.68 and 38.24 per cent), number of leaves (55.12 and

32.21 per cent), number of tillers (46.7 and 50.00 per cent) over C1

(absolute uninoculated control), C2 (uninoculated fungicide control)

treatments, respectively under net house conditions.

� The application of KK5 isolate registered maximum increase in weight of

rhizome/plant (38.85 and 34.75 per cent) and rhizome yield/plant (48.39

and 43.38 per cent) over C1 (absolute uninoculated control), C2

(uninoculated fungicide control) treatments, respectively.

53

� The soil inoculation with KK5 isolate also registered a significant increase

in total NPK uptake by ginger.

� The initial pH of soil mixture was slightly alkaline (pH 7.56), EC in

normal range (0.49 dSm-1

) and high in organic carbon (1.25%). The

available N and K contents were medium, whereas, available P was in

high range.

� The application of KK5 isolate inoculum significantly increased the

available NPK content of the soil at the termination of experiment i.e after

6 months. The increase was by 27, 19 and 23 per cent, respectively over

initial NPK content of the soil. However, there was no significant effect

of the inoculation on physic chemical properties.

The KK5 isolate had showed maximum plant growth promoting attributes

and excel all other tried isolates to enhance shoot length, number of leaves,

number of tillers and rhizome yield and also increased available nutrients (NPK)

content, microbial population (endophytic and rhizosoheric). The KK5 isolate

tentatively identified under Bacillus spp. is most predominant colonizer of roots

and rhizosphere of ginger plant and has good prospects to be used as biofertilizers

and biocontrol agents not only for enhanced growth and rhizome production but

also to sustain soil health under mid hill conditions of Himachal Pradesh.

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65

DR Y S PARMAR UNIVERSITY OF HORTICULTURE AND FORESTRY

NAUNI-173 230, SOLAN (H P)

DEPARTMENT OF BASIC SCIENCE

Title of thesis : “Effect of selected plant growth promoting rhizobacteria

on growth and yield of ginger (Zingiber officinale Rosc.)” Name of student : Kirti Kaundal Admission Number : F-2009-05-M Name of Major Advisor : Dr. Rajesh Kaushal Major field : Microbiology Minor field(s) : 1) Vegetable Science

2) Soil Science and Water Management Degree awarded : M.Sc. Year of award of Degree : 2011 Number of pages in Thesis : 65 + III Number of words in Abstract : 373

ABSTRACT

Ginger being heavy feeder and exhaustive crop requires large quantities of nutrient elements.

Chemical fertilizers besides being costly and also causes degradations both to soil and environment. The

nutrient requirements and changing climatic conditions coupled with higher incidence of diseases such as soft

rot (Pythium spp.) and Fusarium wilt (Fusarium spp.) and pests infestation resulted in sizeable yields loss in mid

hills of Himachal Pradesh. The use of PGPR to supplement chemical fertilizers and pesticides is a potential

alternative but no commercial formulation is available for the crop. So, the present investigations entitled

“Effect of selected plant growth promoting rhizobacteria on growth and yield of ginger (Zingiber officinale Rosc.)” were carried out during 2009-2011. Rhizosphere and root samples of ginger were collected

from major ginger growing area by selecting four locations i.e. Nauni, Kandaghat and Narag, Rajgarh in Solan

and Sirmour districts of Himachal Pradesh. A total of 32 isolates were isolated by using modified replica plate

method. On the basis of colony morphological similarities seven isolates were selected for screening of

possession of multifarious plant growth promoting traits. Among which KK5 isolate possesses maximum plant

growth promoting attributes like P-solubilization (86.6%) on PVK solid medium, (202.2 µg/ml) in broth, growth

on Jensen’s medium, siderophore production (86.7%), auxin production (29.6 µg/ml), HCN production and

antagonism against Pythium spp. (43.2%) and Fusarium spp. (42.5%). On the basis of morphological (Gram +ve

rods), physiological (optimum growth at 35°C and 7 pH) and biochemical characteristics (Glucose fermentation,

production of gelatinase and catalase enzymes) KK5 isolate tentaviley identified as Bacillus spp. Rhizome

bacterization with KK5 isolate not only showed significant increase in plant parameters such as shoot length

(47.6 % and 38.0%), number of tillers (46.9% and 48.9%), number of leaves (43.1% and 39.7%) and yields

(48.3 % and 43.5 %) over C1 (uninoculated absolute control) and C2 (uninoculated fungicide control)

treatments, respectively but also increased available N, P and K by 26.5 per cent, 22.9 per cent and 18.8 per

cent, respectively over initial contents. On the basis of efficacy i.e. rhizocompetence and plant growth

promoting efficiency under net house studies the bacterial isolate KK5 has good potential to be used as

biofertilizer or biocontrol agent for ginger production in mid hills conditions of Himachal Pradesh.

Signature of Major Advisor Signature of the Student

Countsersigned

Professor and Head

Department of Basic Science Dr YSP, UHF, Nauni, Solan (H P)

i

APPENDIX -1

1.1 Preparation of standard curve (10-100 µg/ml) for IAA stock solution

(100 ppm):

10 mg of IAA (99.00 %) pure was dissolved in 50 ml distilled water and

the final volume was made to 100 ml in a volumetric flask.

IAA

(ml)

Distilled

water

(ml)

Final

volume

(ml)

Salkowski

reagent

(ml)

ppm

(ml)

Optical density

(OD) at 535 nm

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

2.7

3.0

2.7

2.4

2.1

1.8

1.5

1.2

0.9

0.6

0.3

0.0

3

3

3

3

3

3

3

3

3

3

2

2

2

2

2

2

2

2

2

2

10

20

30

40

50

60

70

80

90

100

0.12

0.27

0.32

0.42

0.51

0.68

0.72

0.74

0.78

0.79

Appendix I: Standard curve of indole-3-acetic acid

ii

1.2 Preparation of the standard curve for P- estimation

i) Stock solution (10ppm): KH2PO4 = 21.93/100 ml

ii) Chloromolybdic acid: 15 g of ammonium molybdate dissolved in 400 ml

of warm distilled water. Add 342 ml of conc. HCl and cool. Make up the

volume to one liter with distilled water.

iii) Chlorostannous acid (40%): Dissolve 10 gm of SnCl2 crystal in 25 ml of

conc. HCl and store in brown bottle.

iv) Working solution: Take 0.5 ml of solution and make up the volume to 66

ml with distilled water and mix before use.

Volume of

standard solution

(ml)

Concentration

(ppm)

0.5 1

1.0 2

1.5 3

2.0 4

2.5 5

3.0 6

3.5 7

+ 5 ml

Ammonium

molybdate

+1 ml.

working

solution

SnCl2

Final

volume

25 ml

ii) Estimation of phosphorous in sample

a) 50 ml PVK broth + 10 per cent inoculum (1 OD at 540 nm)

b) Incubation under shaking condition

c) Centrifugation

d) 0.1 ml culture filtrate + distilled water = 1 ml (dilution 10 times)

e) 1 ml of above CF (step d) + 5 ml Ammonium molybdate + 1 ml working

solution of SnCl2 + distilled water = final volume to 25 ml (dilution 10

times) OD at 660 nm.

Total dilution = 10 times

iii) Calculations:

Concentration (ppm) from standard curve × 10 (dilution factor) = P-

solubilized (ppm)

iii

Final concentration calculated after deducting from P-solubilized in control

Appendix I: Standard curve for phosphorus

iv

APPENDIX –II

Appendix 2.1 Production of indole-3-acetic acid by different bacterial

isolates

Isolates Indole-3-acetic acid production (µg/ml)

KK1 23.22

KK2 18.67

KK3 27.81

KK4 22.25

KK5 29.62

KK6 27.34

KK7 20.33

CD0.05 1.59

Appendix 2.2 Fungus per cent growth inhibition by different Bacterial

isolate

Isolates Per cent growth inhibition against

Pythium spp. Fusarium spp.

% %

KK1 26.84(30.84)* 21.95(23.29)*

KK2 0.00(0.00) 27.51(29.25)

KK3 42.47(40.67) 35.59(39.21)

KK4 24.62(26.73) 0.00(0.00)

KK5 43.27(45.98) 42.59(40.99)

KK6 40.16(39.16) 30.51(34.26)

KK7 0.00(0.00) 40.44(40.26)

CD0.05 0.40 0.39

*Figures in parenthesis are arc sine transformed values

v

Appendix 2.3 Effect of bacterial inoculum on per cent increase in shoot

length, number of tillers and number of leaves (net house

conditions)

Per cent increase in shoot

length

Per cent increase in

number of tillers

Per cent increase in

number of leaves

Treatments

Over

uninoculated

absolute

control

Over

uninoculated

fungicide

control

Over

uninoculated

absolute

control

Over

uninoculated

fungicide

control

Over

uninoculated

absolute

control

Over

uninoculated

fungicide

control

C1 0.00 -6.50 0.00 -10.00 0.00 -6.00

C2 6.95 0.00 10.0 0.00 6.36 0.00

KK3 43.59 34.24 36.67 31.65 34.02 25.97

KK4 29.36 20.95 16.26 14.49 21.25 14.95

KK5 47.68 38.07 46.94 48.96 43.12 39.75

KK6 45.72 36.24 33.34 27.58 40.58 36.62

Appendix 2.4 Effect of bacterial inoculation on per cent increase in weight of

rhizome and rhizome yield ( net house conditions)

Per cent increase in weight of

rhizome/plant

Per cent increase in rhizome

yield/plant

Isolates

Over

uninoculated

absolute

control

Over

uninoculated

fungicide

control

Over

uninoculated

absolute

control

Over

uninoculated

fungicide

control

C1 0.00 -3.43 0.00 -4.25

C2 3.46 0.00 4.44 0.00

KK3 37.65 33.04 45.55 38.33

KK4 23.71 19.57 29.56 25.33

KK5 38.85 34.74 48.39 43.48

KK6 33.73 29.24 42.93 38.40

vi

Appendix 2.5 Effect of bacterial inoculation on plant nutrient uptake

Nutrient uptake (mg/plant) Isolates

N P K

C1 64.14 42.76 81.12

C2 68.22 48.41 90.52

KK3 102.93 78.35 120.24

KK4 99.63 82.48 129.06

KK5 112.54 89.62 138.2

KK6 105.87 72.69 125.79

Appendix 2.6 Effect of bacterial inoculation on available nutrients (At

termination of experiment)

Isolates Per cent increase in available nutrients (kg/ha) over

initial

N P K

C1 12.36 2.64 6.76

C2 15.14 5.63 11.60

KK3 19.06 15.17 17.99

KK4 22.85 13.48 13.77

KK5 26.57 22.98 18.84

KK6 20.84 18.66 17.17

vii

APPENDIX-III

3.1 Anova of Table - 7 (Phosphorus solubilization efficiency)

MSS Source of

variation

DF

% P- solubilization

efficiency

P-solubilization in liquid

medium (µg/ml)

Treatment (T) 9 225.67 14682.28

Replication (R) 20

T*R 39.486

15.60

Total 29

3.2 Annova of Table - 8 (Siderophore production efficiency of different

bacterial isolates on CAS medium)

MSS Source of variation DF

Growth on LB-broth

Treatment (T) 9 712.25

Replication (R)

T*R 20 25.638

Total 29

3.3 Anova of appendix-2.1 (Production of indole-3-acetic acid by

different bacterial isolates)


Growth on LB-broth

Treatment (T) 9 157.63

Replication (R)

T*R 20 4.9810 × 10

-2

Total 29

3.4 Anova of appendix -2.2 (Antifungal activity of selected bacterial

isolates against fungal pathogens )


Pythium spp. (%) Fusarium spp.(%)

Treatment (T) 9 652.99 985.60

Replication (R)

T*R 20 4.2463 × 10

-2 4.1592 × 10

-2

Total 29

viii

3.5 Anova of Table-14 (Effect of bacterial inoculums shoot length,

number of tillers on number of leaves )

MSS Source of

variation

DF

shoot

length(cm)

number of

tillers

number of

leaves

Treatment(T) 8 5.1035 6.3984

2.8599

Replication(R)

T*R 36 4.5121 × 10-2

5.2219× 10-2

1.0478 × 10-2

Total 44

3.6 Anova of appendix 2.5 (Effect of bacterial inoculum on percent

increase in shoot length, number of tillers on number of leaves )

MSS

Shoot length(%) Number of tillers(%) Number of tillers(%)

Source of

variation

DF

Over

uninoculated

absolute

control

Over

uninoculated

fungicide

control

Over

uninoculated

absolute

control

Over

uninoculated

fungicide

control

Over

uninoculated

absolute

control

Over

uninoculated

fungicide

control

Treatment(T) 6 1769.5 1574.4 1772.2 1356.0 1388.3 1229.4

Replication(R)

T*R 28 1.5631 × 10-1 5.112 ×10-1 1.1818 ×10-1 2.6547×10-2 5.1015 1.9185 ×10-1

Total 34

3.7 Anova of Table-16 (Effect of bacterial inoculum on weight and yield

of rhizome in net house)


weight of

rhizome/plant (kg)

yield per plant (kg)

Treatment(T) 6 16.522 1.2881

Replication(R)

Error 28 7.1286 x 10-3 1.5333 x 10-1

Total 34

3.8 Anova of appendix 2.5 (Effect of bacterial inoculum on percent

increase in weight and yield of rhizome in net house)

MSS

weight of rhizome (%) yield per plant (%)

Source of variation

DF

Over

uninoculated

absolute

control

Over

uninoculated

fungicide

control

Over

uninoculated

absolute

control

Over

uninoculated

fungicide

control

Treatment(T) 6 1864.5 1928.2 2852.4 2548.5

Replication(R)

T*R 28 42.189 29.216 32.680 22.965

Total 34

ix

3.9 Anova of Appendix 2.6 (Effect of bacterial inoculum on plant

nutrient uptake)

MSS Source of

variation

DF

N (mg/plant) P (mg/plant) K (mg/plant)

Treatment(T) 6 1956.5 2179.1 3865.6

Replication(R)

T*R 28 4.2889 5.3504 9.981

Total 34

3.10 Anova of Appendix 2.7 (Effect of bacterial inoculum on available NPK

of the soil)

MSS Source of

variation

DF

N P K

Treatment(T) 6 330.95 21.201 684.50

Replication(R)

T*R 28 6.2287 × 10-2

4.8511

×10-2

44.283 ×10-1

Total 34

3.11 Anova of Appendix 2.8 (Effect of bacterial inoculums on available

nutrients over initial value)

MSS Source of

variation

DF

N (%) P (%) K (%)

Treatment(T) 6 466.82 253.69 212.15

Replication(R)

T*R 28 5.8991 × 10-2

9.1820 ×

10-2

1.6685 × 10-3

Total 34

3.12 Anova of Table 14 (Rhizospheric and endophytic bacterial population

associated with ginger in net house)

MSS

Rhizospheric

population

Endophytic

population

Source of

variation

DF

NA SEM NA SEM

Treatment(T) 6 322.67 515.55 99.675 68.185

Replication(R)

T*R 28 2.9962 ×

10-1

7.2411 ×

10-1

5.2237 ×

10-1

3.242 ×

10-1

Total 34

CURRICULUM VITAE

Name : Kirti Kaundal

Father’s Name : Late Sh Puran Dutt Kaundal

Date of Birth : Mar. 06, 1985

Sex : Female

Marital Status : Unmarried

Nationality : Indian

Educational Qualifications :

Certificate/Degree Class/Grade Board/University Year

Matriculation Second HP Board 2001

10+2 Second HP Board 2005

B.Sc.(Microbiology) First UCTBS 2008

Whether sponsored by some state/ : NA

Central Govt./Univ./SAARC

Scholarship/ Stipend/ Fellowship, any : NA

other financial assistance received

during the study period

( Kirti Kaundal )

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