SELECTION AND CHARACTERIZATION OF ENDOPHYTIC AND RHIZOSPHERIC

MICROORGANISMS OF CHRYSANTHEMUM

(Dendranthema grandiflora Tzvelve)

Thesis

by

KHUSHBOO SHARMA

Submitted in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

in

MICROBIOLOGY (Basic Sciences)

COLLEGE OF FORESTRY

Dr Yashwant Singh Parmar University of Horticulture and Forestry, Nauni

Solan – 173230 (HP), INDIA 2009

Dr Rajesh Kaushal Department of Basic Sciences Associate Professor College of Forestry Dr Y S Parmar University ofHorticulture and Forestry, Nauni-173 230, Solan (HP)

CERTIFICATE-I This is to certify that the thesis entitled, “Selection and

characterization of endophytic and rhizospheric microorganisms of

chrysanthemum (Dendranthema grandiflora Tzvelev)” 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 Khushboo Sharma

(F-2007-12-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: December, 2009 Major Advisor

CERTIFICATE-II

This is to certify that the thesis entitled, “Selection and

characterization of endophytic and rhizospheric microorganisms of

chrysanthemum (Dendranthema grandiflora Tzvelev)” submitted by

Ms Khushboo Sharma (F-2007-12-M) to Dr Yashwant Singh Parmar

University of Horticulture and Forestry, Nauni, Solan (H P) 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 Dr R Raina Dr R S Chauhan Major Advisor Dr Rajesh Kaushal Members of Advisory Committee Associate Professor Dr C K Shirkot Professor Dr S R Dhiman Associate Professor Dr (Mrs) Neerja Rana Associate Professor

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,

“Selection and characterization of endophytic and rhizospheric

microorganisms of chrysanthemum (Dendranthema grandiflora

Tzvelev)” submitted to Dr Y S Parmar University of Horticulture and

Forestry, Nauni-Solan (HP) by Ms Khushboo Sharma (F-2007-12-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

Dr A K Sharma Professor and Head

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

ACKNOWLEDGEMENTS

ride, praise and perfection belong to the irrevocable existence of divinity. I bow

my head and thank Thy for bestowing me with wits and courage to go through

this stupendous juncture.

On the spur of the moment, I owe my very existence to the towering peaks

of my life, my papa, mum, Golu and Suntu, whose blessings, selfless love,

constant encouragement, obstinate sacrifices have been the most vital source of

inspiration and motivation in my life.

With an overwhelming sense of legitimate pride and genuine obligation

which gives me exuberant pleasure and privilege to express my indebtedness to my

acuminous, prudent and dignified chairman of my Advisory Committee, Dr Rajesh

Kaushal, the noble, who taught me never to bend to accumulate false pride for his

impeccable guidance, immaculate suggestions, analytical rigors, swift execution

and finally scanning the manuscript in a scientific and meticulous manner.

I am deeply oblito Dr S R Dhiman for his willing assistance, critical and

valuable suggestions for preparing this manuscript. I am indeed beholden to the

other members of my advisory committee, Dr C K Shirkot and Dr (Mrs) Neerja

Singh Rana.

I seize this unique opportunity to earnestly thank Dr (Mrs) Mohinder

Kaur, Dr (Mrs) Nivedita Sharma, Mrs Anjali Chauhan, Seema mam for being

supportive and considerate. I express my loyal and sincere thanks to Dr A K

Sharma, Professor and Head, Department of Basic Sciences for providing all the

necessary facilities and means to carry out the experiments successfully.

I have been fortunate in getting the intelligent guidance by all my seniors

especially Varsha mam, Manoj sir and I also express my heartfelt gratitude for

enthusiastic co-operation by my guidemate Priyanka, Ruchi and all my classmates

are also acknowledged.

Heartiest considerations are also due to my ever dearest friends Nitin,

Happy, Sunil, Priyanka mam, Puja, Swati and all my juniors. Facilities and co-

operation provided by Dayaram ji, Satish bhaiya, Prem bhaiya, Ramu bhaiya

and field staff especially Chaman ji, Balak Ram and Yash Pal of Department

of Floriculture and Landscaping is thankfully acknowledged.

Last, but by no means the least, I am thankful to direct and indirect help

received from various other sources.

Needless to say, errors and omissions are solely mine.

Date: December, 2009 Place: Nauni, Solan (Khushboo Sharma)

P

Chapter Title Page(s)

1 INTRODUCTION 1-3

2 REVIEW OF LITERATURE 4-15

3 MATERIALS AND METHODS 16-35

4 EXPERIMENTAL RESULTS 36-68

5 DISCUSSION 69-76

6 SUMMARY AND CONCLUSIONS 77-80

7 REFERENCES 81-93

ABSTRACT 94

APPENDICES I-III

Table Title Page(s)

1 Enumeration of rhizosphere microbial population associated with chrysanthemum plants 37

2 Population of P- solubilizers associated with chrysanthemum plant

37

3 Enumeration of endophytic bacterial population associated with roots of chrysanthemum plants

38

4 Population of P- solubilizers associated with roots of chrysanthemum plants

38

5 Morphological characteristics of rhizospheric and endophytic bacterial isolates of chrysanthemum plants

39

6 Screening of bacterial isolates for multifarious plant growth promoting activities

40

7 Phosphorus solubilation efficiency of bacterial isolates on solid PVK medium

41

8 Dual culture compatibility assessment amongst ten bacterial isolates of cultivars (‗Ajay‘ and ‗Purnima‘) of chrysanthemum plants

42

9 Morphological, physiological and biochemical characteristics of Bacillus sp. (KS1, KS5, KS6, KS9)

44

10 Phosphorus solubilization efficiency of Bacillus spp. in liquid PVK medium

45

11 Effect of cell density on viable count of different bacterial isolates

46

12 Effect of liquid bacterial formulation on plant height (cm) of chrysanthemum

47

13 Effect of liquid bacterial formulation on root length (cm) 48

Table Title Page(s)

14 Effect of liquid bacterial formulation on plant biomass (g) 49

15 Effect of liquid bacterial formulation on number of cut stems 50

16 Effect of liquid bacterial formulation on length of cut stems (cm) 51

17 Effect of liquid bacterial formulation on number of leaves per cut stem 52

18 Effect of liquid bacterial formulation on days taken to

flowering

53

19 Effect of liquid bacterial formulation on number of flowers per plant 54

20 Effect of liquid bacterial formulation on flower diameter (cm)

55

21 Effect of liquid bacterial formulation on duration of flowering (days) 56

22 Effect of liquid bacterial formulation on vase life (days)

57

23 Physico-chemical properties, nutritional status and total bacterial count of soil mixture for net house experiment (initial status)

59

24 Effect of liquid bacterial formulation on pH & EC of soil

60

25 Effect of liquid bacterial formulation on OC %(Organic Carbon), PS% (Pore Space) & MWHC% (Maximum Water Holding Capacity of soil)

60

26 Effect of liquid bacterial formulation on available nitrogen in soil 61

27 Effect of liquid bacterial formulation on available phosphorus in soil 62

28 Effect of bacterial liquid formulation on available potassium in soil 63

29 Rhizospheric and endophytic bacterial population

associated with chrysanthemum plants (at the end of the experiment)

65

30 Correlation between NPK, rhizospheric and endophytic bacterial population, and root/shoot length and biomass of chrysathemum (cv. ‗Ajay‘ & cv. ‗Purnima‘)

68

Plates Title Between Page(s)

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

37-38

2 Purification of selected bacterial isolate on different medium (Streak Plate Method)

39-40

3 Multifarious plant growth promoting activities by different bacterial isolates a) P-solubilization b)

Siderophore production c) HCN production

41-42

4 Antifungal activity of bacterial isolates against fungal pathogens using dual culture technique

41-42

5 Synergistic & antagonistic activity of selected bacterial isolates on NA medium (Dual culture

compatibility assessment)

43-44

6 General view of experiment

45-46

7 Effect of bacterial inoculation on growth and flowering of chrysanthemum cultivars

47-48

8 Effect of liquid bacterial formulation on plant parameters of chrysanthemum cultivars

49-50

9 Rhizospheric and endophytic bacterial population associated with chrysanthemum at the end of

experiment

65-66

ABBREVIATIONS USED % : Per cent

0C : 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

CChhaapptteerr -- 11

IINNTTRROODDUUCCTTIIOONN

Chrysanthemum (Dendranthema grandiflora Tzvelev) a member of

the family Asteraceae (Anderson, 1987), is one among the top most cut

flowers and pot plants of the world. Chrysanthemum is believed to be a

native of the Arctic parts of the North and Central Russia, Japan and

China. In India, the first reference of Chrysanthemum is found in the

thirteenth century in Marathi literature ―Gyaneshwari‖ (1296 A.D.). It is

popularly known as ‗Guldaudi‘ (India) and ‗Glory of East‘ or ‗Mum‘

(U.S.A.).

The varied agro climatic conditions of our country are very much

suitable for its commercial cultivation throughout the year. It is being

grown almost in every state of the country and is being cultivated in

almost every district of Himachal Pradesh. The total area under

floriculture in Himachal Pradesh is 617.6 ha out of which about 82.75 ha

is under chrysanthemum cultivation (Anonymous, 2009).

In India, chrysanthemum is not only being grown for cut flower and

pot plant, its flowers are being used for making garlands, venis and

religious offerings and as important source of essential oil and

sesquiterpenoid. Some of its species are also cultivated as a source of

pyrethrum, an important botanical insecticide. In other parts of the world it

is eaten as delicacy after frying, the flowers are boiled to prepare a sweet

drink known as chrysanthemum tea which has many medicinal uses

including recovery from influenza.

The chrysanthemum growing soils of our state particularly in mid

hill regions are, in general, low to medium in organic matter, low in

nitrogen and also in phosphorus nutrition. Further the growers are also

facing problems regarding management of its diseases and pests.

http://en.wikipedia.org/wiki/Chrysanthemum_teahttp://en.wikipedia.org/wiki/Influenza

So, there is a need to develop renewable, low cost, ecofriendly

microbial inoculants which can supplement the nutritional requirements of

the crop and also manage the disease/pest problems. But in the absence

of any commercial formulation of biofertilizers/PGPRs particularly for

chrysanthemum, it has become imperative to isolate plant growth

promoting rhizobacteria.

The PGPR may induce growth promotion by direct or indirect

mechanisms. Direct influences include production of phytohormones,

liberation of phosphorus and micronutrients, nitrogen fixation and

stimulation of disease resistant mechanism. Indirect effects arise from

altered root environment and its ecology i.e. acting as biocontrol agents,

liberation of antibiotic substances that kill noxious microbes, competition

with deleterious agents, metabolism of toxic products etc.

The predominant PGPRs belong to Pseudomonas and Bacillus

genera as these bacteria can proliferate due to their rapid growth rate in

nutritionally diverse soil and have potential to be used in agriculture to

boost crop production and to sustain soil health.

The prospect of manipulating microbial population in crop

rhizosphere by inoculation of beneficial bacteria to increase plant growth

has shown considerable promise under controlled studies (Nelson, 2004).

An effective biocontrol agent often acts through the combination of

several different mechanisms (Bowen and Rovira, 1991). An attempt has

also been made by Liu et al. (2007) to control the Pythium root rot

diseases of chrysanthemum by the use of PGPRs.

Since there is no commercial PGPR inoculant formulation for

chrysanthemum, therefore, there is an urgent need to develop effective

location/cultivar specific inoculum for mid hill zones of H.P. So keeping

this in view the following objectives have been framed for the present

investigations.

Objectives:

i. Isolation, enumeration and characterization of beneficial

rhizospheric and endophytic microorganisms of

chrysanthemum.

ii. To study efficacy of selected PGPRs‘ isolates on growth

promotion of chrysanthemum rooted cuttings under controlled

conditions.

Chapter-2

REVIEW OF LITERATURE

An array of microbes is found in just a spoonful of soil.

Unfortunately, this diversity is not evenly distributed. Much of soil could be

viewed as a desert, but occasionally one comes to an oasis filled with life,

these oases are where plant roots are present (Banerjee, et al., 2006).

Soil adjacent to roots known as the rhizosphere (Hiltner, 1904) is

relatively nutrient rich and harbor diverse group of microbes depending

upon the plant species, age & environmental conditions.

The diverse groups of bacteria in close association with roots and

capable of stimulating plant growth by different mechanism(s) of action

are referred to as plant growth-promoting rhizobacteria (PGPR). They

effect plant growth and development directly or indirectly either by

releasing or altering endogenous levels of plant growth regulators (PGRs)

or other biologically active substances, enhancing availability and uptake

of nutrients, reducing harmful effects of pathogenic microorganisms on

plants and/or by employing multiple mechanisms of action (Khalid, 2009).

Plant growth in agricultural soils is influenced by a myriad of abiotic

and biotic factors. The growers routinely use physical and chemical

approaches to manage the soil environment and to improve crop yields.

The application of microbial products for this purpose is less common

(Nelson, 2004) even after such a long period of time i.e. about 60 years

ago that the application of microbes in agricultural practices was started.

The increasing evidences are there about enhancement of plant

resistance to adverse environmental stresses by these beneficial

microbes (Shen, 1997).

Rhizospheric soil is nutrient rich because as much as 40%

photosynthates moving into the roots are lost to the soil in the form of

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V67-4D3WR5M-2&_user=8240309&_coverDate=03%2F01%2F2005&_rdoc=1&_fmt=full&_orig=search&_cdi=5807&_sort=d&_docanchor=&view=c&_searchStrId=953500247&_rerunOrigin=google&_acct=C000052095&_version=1&_urlVersion=0&_userid=8240309&md5=3ed38e5635857f4391cd2de65551208e#bib31

soluble exudates, mucilage, cells or cell wall material (Lynch and Whipps,

1991). Therefore, populations of microbes in rhizosphere are enormous,

ranging from 1010 to 1012 cfu per gram of soil.

Rhizobacteria (PGPR) encompass all bacteria that inhabit plant

roots and exert a positive effect by various mechanisms i.e. direct

influence (e.g. increased solubilization and uptake of nutrients or

production of plant growth regulators) and indirect effect (e.g. suppression

of pathogen by producing siderophore or antibiosis). During the last two

decades the PGPR have received prominent attention because of their

multifarious activities to improve plant growth, primary production and

disease control (Glick et al., 1994; Kloepper et al., 1989, Panwar et al.,

2004, Senthil et al., 2003, Tomcyzak et al., 1999, Gutierrez Manero et al.,

1996).

Selection of efficient PGPR strains based on host plant specificity

or adaptation to a particular soil, climatic conditions or pathogens is vital

for achieving consistent and reproducible results under field conditions

(Bowen and Rovira 1999; Chanway et al., 1989).

The research work pertaining to PGPR/biofertilizers on

chrysanthemum and other closely related crops in India and abroad have

been reviewed in this chapter under the following headings:

2.1 PGPR as endophytic root colonizer

2.2 PGPR as plant growth promoter

2.3 PGPR as secondary metabolite, siderophore, HCN producer

2.4 PGPR as biocontrol agents

2.5 Effect of growing media, photoperiod, chemicals and

biofertilizers on growth of chrysanthemum

2.1 PGPR AS ENDOPHYTIC ROOT COLONIZER

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; Weller, 1984; Suslow and Schroth, 1982; Kloepper et al.,

1980).

A variety of bacterial traits and specific genes are known to

contribute to the process of root colonization, but only a few have been

identified. These include motility, chemotaxis to seed and root exudates,

production of pili or fimbriae, production of specific cell surface

components, ability to use specific components of root exudates, protein

secretion and quorum sensing (Benizri et al., 2001; Lugtenberg et al.,

2001).

The nutrient status of rhizosphere and the nature of root exudates

have a direct effect on the composition of the rhizospheric microbial

community as well as the proliferation of introduced strain to that

environment (Klein et al., 1990; Lynch, 1990; Curl and Truelovac, 1986).

The most predominant root colonizing bacteria belong to the genus

Bacillus and Pseudomonas because of their rapid growth rate in

nutritionally diverse soil having varying amounts of organic matter

(Vijaypal et al., 1998).

Endophytic bacteria live in the plant tissues without causing harm

to the host or gaining any benefit other than a non-competitive

environment inside the host (Sharma et al., 2005). Singh et al. (2009)

explained the effect of plant genotype on the root endophytic colonization

ability of Pseudomonas striata, a plant growth promoting rhizobacteria

(PGPR).

James and Olivares (1998) have isolated bacterial endophytes

from surface–sterilized plant tissue or extracted from the internal plant

tissue of different plant species. A number of facultative endophytes have

been reported from rice (Biswas et al., 2000), maize (Rosenblueth, 2004;

Gutierrez-Zamora, 2001), wheat (Cooms and Franco, 2003; Larran et al.,

2002), sorghum (Baldani et al., 1997), cotton (Reva et al., 2002) and

Arabidopsis (Sessitsch et al., 2004; Hallmann et al., 2002).

Endophytic bacteria have been implicated in supplying biologically

fixed nitrogen in non-legumes and these associations can increase the

nitrogen economy of a crop by reducing the requirement for N fertilizers.

Bacterial endophytes have also been shown to prevent disease

development through endophyte-mediated de novo synthesis of structural

compounds and fungitoxic metabolites (Sturz, et al., 2000).

Bhatia et al. (2005) worked with ten isolates of fluorescent

pseudomonads and reported that only two namely Pseudomonas I and II

were most potential root colonizers. Positive influence on plant growth

and resistance to a broad range of plant pathogens have also been

observed for endophytes by Bacilio-Jimenez et al. (2001).

2.2 PGPR AS PLANT GROWTH PROMOTER

The beneficial effects of PGPR on germination, growth and yield of rice

(Ashrafuzzaman et al., 2009), tomato (Mena-Violantes et al., 2007) and

legumes (Remans et al., 2007) have been recorded under controlled and

field conditions.

Chatli et al. (2008) isolated P-solubilizing microorganisms (bacteria

and fungi) associated with Salix alba Linn and reported that morphological

characteristics of isolated bacteria were similar to Bacillus sp. and fungi to

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Penicillium sp. and Aspergillus sp. and compared their efficiency to

solubilize TCP under controlled conditions. They concluded that bacterial

isolates were more efficient in solubillizing TCP.

In recent years plant scientists have concentrated their efforts to

study potential of PGPR for improving crop growth and yield. There are

several ways by which PGPR help the plant growth and development.

They may fix atmospheric nitrogen; synthesize siderophores; synthesize

various phytohormones, including auxins and cytokinins; solubilize

insoluble form of phosphorus; synthesize enzymes etc. (Patten and Glick,

1996; Davison, 1988; Kloepper et al., 1986; Brown, 1974).

Several studies have demonstrated the potential of rhizosphere

microflora to synthesize plant growth regulators (PGRs) in vitro (Arshad

and Frankenberger, 1991). Tyler et al., (2008) reported primary (growth

and productivity) and secondary (disease reduction) effects of PGPR on

different crops.

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

promoting rhizobacterium (Acetobacter diazotrophicus) from 13 cultivars

of sugarcane had the ability to produce plant growth hormone indole

acetic acid (IAA). However the amount varied significantly with the strain,

which ranged from 0.14 to 2.42 µg IAA ml-1.

The application of mixtures of strains having synergistic nature is

known to significantly enhance the plant growth in terms of increased

seedling emergence (Dunne et al., 1998), plant height (Raupach and

Kloepper, 1998), and yield over single inoculation in different crop plants

(Duffy et al., 1996; Pierson and Weller, 1994)

Tilak et al. (2005) reported that plant-growth promoting

rhizobacteria (PGPR) in conjugation with efficient Rhizobium isolate,

increase the growth and nitrogen fixation in pigeonpea by increasing the

occupancy of introduced Rhizobium in the nodules of the legume.

The application of Bacillus subtilis strain BEB-13 and Glomus

fasciculatum to marigold flower increased inflorescence by 14-24 per cent

and had significant effect on flower fresh weight and flower diameter over

uninoculated controls (Flores et al., 2007).

Akhtar et al. (2009) conducted a study to assess the possible role

of the integrated use of seed inoculation with plant growth promoting

rhizobacteria (PGPR), compost and mineral fertilizers for improving

growth and yield of wheat sown at different plant spacing. The conjoint

application of PGPRs and chemical fertilizers increased significantly yield

and grain weight over uninoculated control.

2.3 PGPR AS SECONDARY METABOLITE, SIDEROPHORE, HCN PRODUCER

Microflora that are able to produce PGRs in vitro are present in

appraisable numbers in the rhizosphere of plants. Plant-growth-promoting

bacteria (PGPR) stimulate plant growth by producing and/or inducing the

plant to release secondary metabolites facilitating the uptake of nutrients

and/or inhibiting plant pathogenic organisms in the rhizosphere.

Among the factors involved in plant-microbe interactions as well as

in microbe-microbe interactions, in the rhizosphere, siderophores and

HCN production have received special attention. HCN is released as

product of secondary metabolism by several microorganisms and affects

sensitive organisms by inhibiting the synthesis of ATP-mediated

cytochrome oxidase (Knowles, 1976).

Under iron-limiting conditions, microorganisms produce a range of

iron chelating compounds or siderophores which have a very high affinity

for ferric ions. These bacterial iron chelators are thought to sequester the

limited supply of iron available in the rhizosphere making it unavailable to

pathogenic fungi, thereby restricting their growth (Loper and Henkel,

1999). Some PGPR strains go one step further and draw iron from

heterologous siderophores produced by cohabiting micro-organisms.

Sharma et al. (2003) suggested that siderophores produced by

Pseudomonas sp. may be used by the bacteria (homologously) or in

effecting plant nutrition (heterologously). The problem of iron non-

availability particularly in calcareous soils may be overcome by

incorporation of siderophore producing strains of fluorescent

psuedomonads (FLPs).

Bhatia et al. (2005) isolated ten isolates of fluorescent

Pseudomonas from the rhizosphere of sunflower, potato, maize & ground

nut and noted that all the isolates were phosphate solubilizers,

siderophores, HCN, IAA producers. Further they have studied the

enhancement of the plant growth and suppression of collar rot of

sunflower caused by Sclerotium rolfsii through fluorescent Pseudomonas.

Chakraborty et al. (2006) isolated Bacillus megaterium from tea

rhizosphere and tested its ability to promote growth and cause disease

reduction in tea plants. They have observed that siderophore production

was responsible for enhancing growth and disease suppression.

In view of Hossain et al. (2008) plant growth promoting

rhizobacteria (PGPR) effect plant growth by producing and releasing

secondary metabolites (plant growth regulators/ phytohormones/

biologically active substances) facilitating the availability and uptake of

certain nutrients from the root environment and inhibiting plant pathogenic

organisms in the rhizosphere.

Çakmakçi et al. (2007) studied the effect of plant growth-promoting

rhizobacteria on seedling growth in spinach and wheat and reported that

the enhanced plant growth could result from indole-3-acetic acid (IAA)

production by rhizobacteria. It was further reported that the application of

PGPR also improved the availability of N and P in rhizosphere which

might have improved the growth and activities of many enzymes.

http://www.essays.se/about/Md.+Shakhawat+Hossain/

Plant growth-promoting attributes like production of indole acetic

acid, HCN and siderophore, solubilization of inorganic phosphate and

strong antagonistic effect against Macrophomina phaseolina and

Fusarium oxysporum was also observed by Kumar et al., (2009) for

Pseudomonas aeruginosa isolate of tomato plant.

2.4 PGPR AS BIOCONTROL AGENT

In vitro screening of organisms for antibiosis production towards

targeted pathogen is the most frequent method to select organism

biocontrol agent.

Many Bacillus strains are known to suppress fungal growth in vitro

by the production of one or more antifungal antibiotics (Hashidoko et al.,

1999; Kim et al., 1999; Milner et al., 1995).

Most naturally occurring biological controls are likely to result from

mixtures of antagonists rather than from populations of a single

antagonist. Similarly, application of mixture of introduced biocontrol

agents would more closely mimic the natural situation and might broaden

the spectrum of biocontrol activity and enhance the efficacy and reliability

of control (Duffy and Weller, 1995).

A field study was undertaken to study the possibility of controlling

the disease using three biocontrol agents viz., Glomus mosseae,

Pseudomonas fluorescens, Trichoderma viride, singly and in combination.

Inoculation with Trichoderma viride + Glomus mosseae gave the best

result in controlling the disease which was even better than the

application of Emisan (0.2%) fungicide (Boby and Bagyaraj, 2003).

The application of Pseudomonas fluorescens to black pepper is

known to enhance root proliferation and fibre root production in addition to

act as biocontrol agent against foot rot caused by Phytophthora capsici

(Paul and Sarma, 2006).

The capacity of several strains of root-colonizing bacteria

belonging to genera Bacillus and Pseudomonas were authenticated to

suppress root rot diseases caused by Pythium spp in chrysanthemums. It

is noted that the application of Bacillus and Pseudomonas isolates

reduced the infection (root rot pathogens) by 83 per cent and 72 per cent

respectively (Liu et al., 2007).

Harish et al. (2009) performed an experiment in which plant

growth-promoting rhizospheric and endophytic bacterial strains were used

to induce systemic resistance against BBTV in tissue-cultured banana

plantlets. Application of mixtures of Pseudomonas fluorescens strain Pf1

and strain CHA0 (rhizobacteria) and Bacillus subtilis strain EPB22

(endophyte) showed reduction of infection by 80 per cent over control

plants.

Wang et al. (2009) reported that the application of the cultural

filtrate of Bacillus sp. strain CHM1, isolated from a paddy field inhibited

the mycelia growth of different fungi (Fusarium oxysporum, Rhizoctonia

solani, Botrytis cinereapers, Gibberella zeae, Dothiorella gregaria and

Colletotrichum gossypii) under laboratory conditions. They further

reported that the application of culture filtrate under the field conditions

protected maize (Zea mays) and horsebean (Vicia faba) against infection

by Bipolaris maydis and R. solani, respectively.

Recep et al. (2009) isolated 17 PGPR strains belonging to Bacillus

spp. and tested them against antifungal activity (in vitro and in vivo) of dry

rot disease caused by Fusarium spp. They have further reported that the

isolate Burkholderia cepacia has greatest potential to be used as effective

biocontrol agent against F. oxysporum and F. culmorum under storage

conditions.

2.5 EFFECT OF GROWING MEDIA, PHOTOPERIOD, CHEMICALS

AND BIOFERTILIZERS ON GROWTH OF CHRYSANTHEMUM

Growing media is the key input for attaining optimum crop

productivity on sustainable basis. Marcussen (1979) recommended

peat/sawdust supplemented with fertilizers for commercial growing of

chrysanthemum. A mixture of peat and sand in a ratio of 3:1 (v/v) was

found best for growth of chrysanthemum when different combinations of

peat, sand and coal ash were compared (Huang et al.,1989).

De Sauza et al. (1995) evaluated the substrate for ‗White Polaris‘

chrysanthemum cultivation in pots and found that the plant and flower

development were best in soil:sand:carbonized rice husk in ratio of 2:1:4

or 4:1:4.

Sita Ram and Sehgal (1993) reported that no flowering bud

formation was observed under natural conditions in December, February

& April planted chrysanthemum cultivars ‗Aparajita‘ and ‗Rajini‘. They

further observed good flowering when artificial short days (photoperiod)

was provided by thick dark tarpaulin.

Hanke (1996) observed much earlier flowering in 20 cultivars of

chrysanthemum when subjected to short days (13 h dark period )

compared to plants grown under natural daylength.

The search for PGPR and investigation of their modes of action are

increasing at a rapid pace as efforts are made to exploit them

commercially as biofertilizers. Biofertilizers are products containing

living/latent cells of different types of microorganisms, which have an

ability to convert nutritionally important elements from unavailable to

available form through biological processes (Hegde et al., 1999 and

Vessey, 2003).

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V67-4D3WR5M-2&_user=8240309&_coverDate=03%2F01%2F2005&_rdoc=1&_fmt=full&_orig=search&_cdi=5807&_sort=d&_docanchor=&view=c&_searchStrId=953500247&_rerunOrigin=google&_acct=C000052095&_version=1&_urlVersion=0&_userid=8240309&md5=3ed38e5635857f4391cd2de65551208e#bib14http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V67-4D3WR5M-2&_user=8240309&_coverDate=03%2F01%2F2005&_rdoc=1&_fmt=full&_orig=search&_cdi=5807&_sort=d&_docanchor=&view=c&_searchStrId=953500247&_rerunOrigin=google&_acct=C000052095&_version=1&_urlVersion=0&_userid=8240309&md5=3ed38e5635857f4391cd2de65551208e#bib37

These biofertilizers have emerged as an important component of

the integrated nutrient supply system and hold a great promise to improve

crop yields through environmentally sound nutrient supplies. Wu et al.

(2005) recommended biofertilizers as an alternative to chemical fertilizer

for crop production and sustainable soil fertility. The mechanisms of

growth promotion by PGPR-biofertilizers include production of plant

growth activators such as IAA, release of volatile growth-stimulating

compounds, and inhibition of deleterious rhizobacteria via competition for

iron. Mechanisms of biological control by PGPR-biofungicides include

production of antifungal compounds, including many types of antibiotics,

and induction of host defences.

Woltz (1960) reported that organic materials (Caster pomace,

Peruvian guano, sewage sludge and slaughterhouse tankage) are better

sources for N to chrysanthemum over water soluble fertilizer. Further,

Ying and Joiner (1961) emphasized the use of both organic and inorganic

sources of fertilizers for N supply in chrysanthemum.

Srivastava and Govil (2005) investigated the effect of Azotobacter,

Phosphate Solubilizing Bacteria (PSB), VA-mycorrhiza (VAM) and Farm

Yard Manure on growth and flowering in gladiolus cv. ‗American Beauty‘

and observed that the conjoint application of these biofertilizers

significantly improved different vegetative and floral characters as

compared to control.

The application of VAM fungi and PSM have become important

biotechnological tools and are being employed to reduce the input of

chemical fertilizers and irrigation water. Chandra et al. (2009) evaluating a

number of species of VAM and PSM recommended the conjoint

application of Glomus fasciculatum (VAM ) and Aspergillus niger (PSM)

to chrysanthemum crop.

Lee et al. (2005) studied the effect N:K ratio (14.3:3.9 to 17.9:3.9

meq/L) on flowering and vase life of Tulip. They have reported that higher

doses of N not only increased the flowering of Tulip but also increased

vase life significantly over control.

Roychowdhury and Roychowdhury (1995) studied the effect of

different sources and levels of K on quality and vase life of gladiolus and

reported that the application of K2SO4 is best for both flower production

and vase life.

Under green house conditions, the conjoint application of

biofertilizers, chemical/mineral fertilizers significantly increased growth

parameters of Matthiola incana and it also proved to be the best to get

these plants established under nutrient deficient soil (Eid et al., 2009).

CChhaapptteerr -- 33

MMaatteerriiaall aanndd mmeetthhooddss

The present investigations entitled “Selection and

characterization of endophytic and rhizospheric micro-organisms of

chrysanthemum (Dendranthema grandiflora Tzvelev)” 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 2007-09. A brief account of the materials used

and methodologies adopted are discussed in this chapter.

3.1 COLLECTION OF SAMPLES Isolation of the microorganisms was carried out from the

rhizosphere, bulk soil and roots of chrysanthemum (Dendranthema

grandiflora Tzvelev)” collected from two locations at Solan (Himachal

Pradesh) i.e. Nauni and Kandaghat by selecting three sites at each

location and two cultivars (cv. ‗Ajay‘ & cv. ‗Purnima‘) at each site.

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

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 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 (PDA) 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

3.3 CHEMICALS AND REAGENTS

Analytical grade chemicals and reagents obtained from standard

company were used for present investigations.

3.4 MICROBIOLOGICAL METHODS

3.4.1 Sterilization

Glassware used were thoroughly washed in detergent water,

running tap water followed by rinsing in distilled water. Glassware 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 work.

3.4.2 Isolation and enumeration of rhizospheric and endophytic

bacteria

Modified replica plating technique was used to isolate PGPRs from

rhizosphere and roots.

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 medium. After incubation of

24 to 48 h, the isolated colonies that developed on enriched medium

(master plate) were replica plated onto the selective media: Nitrogen free

medium (Jensen, 1992) 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 (Plate.1). At the end of the

incubation period, the locations of the colonies appeared on the replica

plates were compared to the master plate. Populations were expressed

as colony forming unit (cfu) per gram of soil dry weight.

Isolation of endophytic bacteria

The root sample was surface sterilized by 0.2 per cent mercuric

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

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

surface sterilized roots in sterilized nutrient agar medium for 24 h at

35±10C. One gram of surface sterilized root sample was placed 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 to 48 h, the isolated colonies that developed on enriched medium

(master plate) were replica plated onto the selective media: Nitrogen free

medium (Jensen, 1992) 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 locations of the colonies appeared on the replica plates were

compared to the master plate. Populations were expressed as colony

forming unit (cfu) per gram of root weight.

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

fungal pathogens Rhizoctonia solani, Pythium ultimum and Fusarium

oxysporum were procured from Department of Mycology and Plant

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

cultures were maintained on malt extract agar and bacterial cultures on

nutrient agar at 40C. Sub-culturing of fungal cultures was done

periodically (ones in fortnight) on the respective medium at incubation

temperature of 28±10C.

3.4.3 Measurement of growth

Preparation of inoculum

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

grown

on nutrient agar broth at the rate of 10 per cent was used as inoculum in

all experiments, unless mentioned otherwise.

Turbidity

Growth was monitored by measuring the change in absorbance of

cells in the broth at 540 nm using uninoculated broth as blank.

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.

Dual culture compatibility assessment

A culture of bacterial isolate was spot inoculated on nutrient agar

medium. The plates were incubated for 48 h at 35±20C. The incubated

plates were over-layered with 2 ml of half strength molten nutrient agar

(sterilized) containing culture of another bacterial isolate.

Similarly, culture of another bacterial isolate was spot inoculated on

nutrient agar medium, followed by 48 h incubation at 35±20C and over-

layering with 2 ml of half strength molten nutrient agar (sterilized)

containing culture of bacterial isolate.

The plates were observed for inhibition zone after 48 h of

incubation at 35±20C and experiment was replicated thrice.

3.4.4 Screening of bacterial isolates

The screening of the bacterial isolates for various plant growth

promoting activities like P-solubilization, siderophore, HCN, auxin

production, growth on N-free medium were performed by adopting the

standard methods. The brief descriptions of these methods are as follows

Phosphate solubilizing activity

Each of the purified isolates were spot inoculated on PVK medium

as described by Pikovskaya (1948) and was incubated for 72 h at 35 ±

20C. Colonies showing solubilization halos (>0.1 mm diameter) were

selected for further experiments.

Nitrogen fixing activity Each of purified isolates were seeded in a straight line on Jensen‘s

medium and was incubated for 72 to 120 h at 35 ± 20C and the plates

showing growth in the form of bacterial colony were selected.

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 bromide (HDTMA solution prepared by

dissolving 72.9 mg 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 and

was autoclaved separately. It was mixed with Chrome azurol- S (100 ml)

and then the plates were prepared for further experiments.

Bit of 72 h old culture test bacterium was placed on prepoured blue

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

350C for 24 h and observed for production of yellowish-orange halo

around the bit.

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 solution of 0.5 per cent picric acid and 2 per cent sodium

carbonate and then placed on the lid of each petriplate. Petriplates were

sealed with parafilm and were incubated at 35+2oC for 1-4 days.

Uninoculated control was kept for comparison of results. Plates observed

for change of color of filter paper from yellow to orange brown.

Quantitative estimation of indole-3-acetic acid (Auxins)

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 72 h at 35+2oC on orbit shaker at 100 rpm.

The cultures were centrifuged at 15000 rpm for 20 minutes and

supernatant were collected and stored at 4oC.

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

determine the quantity of IAA equivalents in selected bacterial cultures. 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 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 was established by measuring the

intensity of this colour (Appendix 2.1).

Antagonistic activity of bacterial isolates against test fungus

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

bacterial isolates against test fungus. A loop full of 48 h old culture of

each isolate was streaked a little below the centre of the prepared MEA

petriplate and incubated at 350C for 24 h to check contamination. Mycelial

disc of 5 days old culture of the test fungal pathogen (Pythium ultimum,

Fusarium oxysporum and Rhizoctonia solani) was placed separately on

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

pathogen only was kept for comparison. The plates were incubated at

28±10C for 7 days and per cent growth inhibition was calculated as

described by Vincent (1947).

C-T I = x 100 C Where,

I = Per cent growth inhibition

C = Growth of fungus in control

T = Growth of fungus in treatment

3.4.5 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 isolates were

identified.

3.4.6 Characterization of bacterial isolates

Separate experiments were performed for optimization conditions

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

isolates.

Effect of pH on growth

3ml 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. Each tube was inoculated with 0.1 ml of 48 h old

bacterial cell suspension (OD 1.0 at 540 nm) of selected bacterial

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 the test tube.

Effect of temperature on growth

Growth curves were drawn by growing the culture at various

temperatures (25, 30, 35, 40oC). 3 ml of nutrient broth was taken in 5 ml

test tubes and inoculated with 0.1 ml of 48 h old selected bacterial cell

suspension (OD 1.0 at 540 nm). The optimum temperature selected in

present experiment for growth was maintained for further experiments.

The temperature suited for maximum growth was selected on the basis of

turbidity caused by the bacterial growth in the test tube.

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 PVK broth was dispensed in 250 ml of Erlenmeyer flask containing

0.5 per cent tri-calcium phosphate (TCP) and autoclaved it at 15 psi for 20

min. 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 (5ml) of the bacterial suspension (OD 1.0 at 540 nm) and incubated

at 35±2oC on rotary shaker at 100 rpm for 120 h. Flasks were withdrawn

at 0, 24, 48, 72, 96 and 120 h of inoculation and contents were

centrifuged at 15000 rpm for 20 min at 4oC. The culture supernatant was

used for determination of the soluble phosphorus as described by Bray

and Kartz (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 spectrophotometer and the results were extrapolated by standard

curve (Appendix I) drawn using potassium 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

ammonium molybdate was added and mixture was thoroughly shaken

and the evolved CO2 was released. The contents of the flasks were

diluted to 20 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 the colour and colour intensity was measured after 10 minutes at

660 nm using red filter on spectrophotometer.

3.4.7 Net house studies

Preparation of liquid formulation

The population density (1.5 O.D at 540 nm) that resulted in

formation of 108 CFU/ml of bacterial isolates was used for preparation of

liquid formulation.

Liquid bacterial formulation of rooted cuttings of chrysanthemum The rooted cuttings of chrysanthemum (cv. ‗Ajay‘ and ‗Purnima‘)

were dipped in the above prepared liquid bacterial culture (single as well

as consortium of synergistic isolates) as per treatments given below for

three hours before potting them in the pot size of 4 inches. The liquid

formulation was applied to the potting mixture after every thirty days till

flowering was attained.

Treatment details

Treatment name Treatments

Control Uninoculated control

KS1 Rhizosphere isolate of cv. ‘Ajay’

KS5 Rhizosphere isolate of cv. ‘Ajay’

KS6 Rhizosphere isolate of cv. ‘Purnima’

KS9 Rhizosphere isolate of cv. ‘Purnima’

KS1+KS

6 Consortium of isolate from cv. ‘Ajay and ‘Purnima’

KS1+KS

9 Consortium of isolate from cv. ‘Ajay and ‘Purnima’

KS5+KS

6 Consortium of isolate from cv. ‘Ajay and ‘Purnima’

KS5+KS

9 Consortium of isolate from cv. ‘Ajay and ‘Purnima’

Procurement of rooted cuttings

The rooted cuttings of chrysanthemum (cv. ‗Ajay‘ & cv. ‗Purnima‘)

were procured from the experimental farm of Department of Floriculture

and Landscaping, University of Horticulture and Forestry, Nauni, Solan

(H.P.).

Preparation of potting mixture

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

of the 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.

Physico-Chemical properties of potting mixture

Freshly prepared potting mixture was analyzed for important

physico-chemical & available nutrient status by adopting the following:

pH

pH was determined in soil, water ratio of 1:2.5 by a pH meter as

described by Jackson (1973).

Electrical conductivity

Electrical conductivity in 1:2.5 soil suspensions was measured by

systronic‘s conductivity meter and was expressed in dSm-1.

Organic carbon

Organic carbon was determined by Chromic acid titration method of

Walkley and Black (1934).

Bulk density and Particle density

The bulk density and particle density was determined by the

method described by Singh (1980).

Pore space

The pore space was calculated by:

Maximum water holding capacity (MWHC)

Water holding capacity was determined by Keen Raczkowski Box

method (Piper, 1966).

The MWHC was calculated by:

MWHC (%) = Maximum water absorbed by the soil

x 100 Oven dry weight of the soil

Available Nitrogen (Subbiah and Asija, 1956)

Five gram of soil was weighed and moistened with 2ml of distilled

water and was added to Kjeldahl distillation flask. 25 ml of 0.32 % KMnO4

and 25ml of 2.5% NaOH solution were added to the assembly and the

cork was fitted immediately. Take 20 ml of 0.02N H2S04 in a conical flask

and add 3 drops of methyl red indicator into the conical flask. Hot plate

was switched on to distill ammonia gas and 30ml of distillate in 0.02N H2

SO4 was collected. The excess of H2SO4 in the conical flask was titrated

against 0.02N NaOH and the change in colour was noted (pink to yellow).

Where,

Available Nitrogen percentage = (10-A) x 0.00028

x 100 Weight of soil

A = Volume of 0.02N NaOH used ppm of available Nitrogen in soil =Available Nitrogen percentage x 10,000 Available Nitrogen kg/ha = ppm x 2.24

Pore space (%) = 1 – ) ×100

Available Phosphorous ( Olsen‟s et al., 1954)

One gram of soil was transferred to a 100 ml conical flask, followed

by the addition of a pinch of Darco - G 60 and 20 ml 0f 0.5N Sodium

bicarbonate. The contents were shaken for 30 minutes and thereafter

filtered to obtain clear filtrate To 5 ml of the filtrate , 5 ml of ammonium

molybdate was added. The mixture was thoroughly shaken to remove the

CO2 evolved The contents of the flask were diluted to about 20 ml. Added

one ml of working solution of SnCl2 and its volume was made to 25 ml in

the volumetric flask. The contents were mixed thoroughly and the blue

colour intensity was measured after 5 minutes at 660 nm and appropriate

blank was kept.

ppm of available P in soil = A x Total dilution

Where,

A = Concentration of P read from the standard curve.

Available Phosphorous kg/ha = ppm x 2.24

Available Potassium (Merwin and Peech, 1951)

5 gm of soil was transferred to a 150 ml of conical flask. 25 ml of

neutral normal ammonium acetate solution was added and the contents

were vigorously shaken on electric shaker for 5 minutes. The contents of

the conical flask were filtered and the filtrate was fed to the automizer of

the flame photometer. The flame photometer was standardized by feeding

standard solution of known concentration prepared by KCl. The standard

curve was prepared by the standard fed to the equipment and reading of

the test sample was extrapolated.

ppm of available K in soil = Y x Total dilution

Where,

Y = ppm as read from the standard curve.

Available Potassium kg/ha = ppm x 2.24

Some of the chemical characteristics of the potting mixture

Parameters Values

1. PHYSICO-CHEMICAL PROPERTIES

a. pH

b. Electrical conductivity (dSm-1

) c. Organic carbon (%) d. Pore space (%) e. Maximum water holding capacity (%)

6.77 0.39 1.27 53.7

36.79

2. NUTRITIONAL STATUS

Available Nutrients (Kg/ha) N P K

315.3 29.6

226.9

3. Total Bacterial Count

NA (× 104

cfu g-1

soil)

SEM (× 105

cfu g-1

soil)

76.33 87.66

Control of photoperiod

The potted chrysanthemum plants were provided photoperiod after

45 days of planting. To provide artificial short days, semi-circular tunnel

shaped metallic frame (3×1.5×1.65 m) completely covered with thick dark

coloured tarpaulin, was placed over the pots for 16 h daily (5p.m. to 9

a.m.). The cover was continued upto the stage till 60-70% flower buds on

a plant showed colour, which was found satisfactory on the basis of

previous studies (Sita Ram, 1991; Sita Ram and Sehgal,1999).

Artificial short days were provided by reducing the total day length

hours below 12 per day. Under Solan-Nauni conditions, this was required

to be provided from second week of July to second week of September,

2009 because during this period natural day length was more than 12 h.

Plant parameters‟ studies Chrysanthemum plants were analyzed for different traits such as:

leaf, shoot and root characteristics at the end of the experiment.

Shoot characteristics

Plant height

Shoot length (cm) was recorded in centimeters from the soil line to

the base of apical bud of stem.

Plant weight The plants were cut at collar with a secateur and plant fresh weight

(g) was taken. Shoot biomass was noted after drying to constant weight in

an oven at 65±5oC for 72 h.

Number of cut stems per plant

Plants of both cultivars were pinched after planting and cut stems

formed thereafter were counted at the time of peak flowering.

Length of cut stems

Length (cm) of cut stems was recorded at the time of peak

flowering from the point where it was attached to the main stem upto the

base of flower.

Number of leaves per side shoot

The number of leaves per side shoot were counted at the time of

peak flowering.

Days taken to flowering

Days were counted from planting to the stage till first flower bud on

the plant shows colour.

Diameter of flower

Flower diameter (cm) was recorded at the time of peak flowering

as ―average of the distance between the apices of petal in East to West

direction and the distance between apices of petal in North to South

direction.

Duration of flowering

Duration of flowering (days) was recorded from colour showing

stage till flower remains presentable on the plant.

Number of cut flowers per plant

In cultivar Purnima (standard variety) disbudding was done at the

early stage and only one flower bud was left on each cut stem, whereas

no disbudding was done in cultivar Ajay. Cut flowers per plant were

counted at full opening stage of all the buds on the plant.

Vase life

Vase life (days) was recorded in ordinary tap water. The days were

counted from the date of placing flowers in the vase to the stage till they

remain presentable.

Root characteristics

Root length

The length (cm) of the root was recorded in centimeters using

measuring scale by placing it horizontally on the ground.

Root weight

The plants roots were washed with excess of water and wiped out

by placing it between the folds of filter paper. Then the plants were cut at

collar with a secateur and root fresh weight (g) was taken. Root biomass

was noted after drying to constant weight in an oven at 65±5oC for 72 h.

Plant analysis

The oven dried samples of shoot and root were ground and sieved

(40 mesh) for estimation of total NPK content.

Digestion of samples

The digestion of 0.20 g samples for estimating nitrogen was carried

out in concentrated H2SO4 by adding digestion mixture of following

chemicals:

Potassium sulphate (K2SO4) = 400 parts

Mercuric oxide (HgO) = 3 parts

Copper sulphate (CuSO4.5H2O) = 20 parts

Selenium powder (Se powder) = 1 part

For the estimation of other elements i.e. P and K, the digestion was

carried out in diacid mixture prepared by mixing nitric acid and perchloric

acid

( 4:1) taking all relevant precautions as suggested by Piper (1966).

Estimation of nutrient elements

The nitrogen was estimated in Kjeltec Auto 1030 Analyzer (Tecator

AB, Sweden). Phosphorus was determined by Vanado molybdo-

phosphoric yellow colour complex method by using spectrophotometer

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

Nutrient uptake

The total nutrient uptake by plant on biomass basis was worked out

by using the formula:

% Nutrient x Biomass (g)

Nutrient uptake (mg/plant) = _________________________________________ x 1000 100

3.4.8 Reisolation and enumeration of rhizospheric and endophytic

bacteria

The bacterial population was determined in chrysanthemum plants

at the end of the experiment.

Reisolation of rhizospheric bacteria

Rhizosphere bacterial population was determined in 1.0 g of

rhizosphere soil. Chrysanthemum plants collected from net house were

shaken vigorously to remove the soil tightly adhered to the roots. One

gram of soil was placed in 9 ml sterilized distilled water under aseptic

conditions. The soil suspension was diluted in 10 fold series and the

bacterial count was determined by using standard spread plate technique.

Populations were expressed as colony forming units (CFU) per gram of

dried soil.

Reisolation of endophytic bacteria

The endophytic bacterial count was determined by taking whole

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 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. One gram of root sample was placed in 9 ml of

sterilized distilled water and was ground to produce slurry using mortar

and pestle under aseptic conditions. The root suspension was diluted in

10 fold series and bacterial count was determined by standard spread

plate technique. Populations were expressed as colony forming units

(CFU) per gram of wet root weight. Isolates were maintained on specific

medium for further studies.

3.5 STATISTICAL ANALYSIS

The data recorded under the laboratory and net house conditions

for 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.

CChhaapptteerr -- 44

EXPERIMENTAL RESULTS

The results obtained during the course of investigations are

presented in this chapter under the following sections:

4.1 Isolation and enumeration of microbial population 4.2 Screening of bacterial isolates for multifarious plant growth

promoting activities 4.3 Identification and characterization of selected bacterial isolates 4.4 Effect of liquid bacterial formulation on growth and flowering

of chrysanthemum 4.4.1 Standardization of inoculum density of different

bacterial isolates 4.4.2 Effect of liquid bacterial formulation on plant

parameters of chrysanthemum plants (cv. „Ajay‟ & cv. „Purnima‟)

4.4.3 Effect of liquid bacterial formulation on nutrient uptake of chrysanthemum plants (cv. „Ajay‟ & cv. „Purnima‟) 4.5 Effect of liquid bacterial formulation on physico-chemical properties, available nutrients and microbial population in soil 4.6 Correlation (r) studies: (i) Microbial population and plant parameters

(ii) Microbial population and available nutrients

(iii) Available nutrients and plant parameters

4.1 ISOLATION AND ENUMERATION OF MICROBIAL POPULATION

Isolation (Plate 1) of the microorganisms was carried out from the

rhizosphere and roots of the two cultivars (‗Ajay‘ and ‗Purnima‘) of

chrysanthemum (Dendranthema grandiflora Tzvelev) collected from

different locations (Nauni and Kandaghat) of Solan district of Himachal

Pradesh.

4.1.1 Microbial population in rhizosphere of chrysanthemum plants.

A summary of microbial population colonizing chrysanthemum

rhizosphere at different locations is presented in Table 1. The results

revealed that the rhizospheric microbial population differed with locations

and cultivars. The highest microbial count (180.66 cfug-1 soil and 152.33

cfug-1 soil) for cultivars ‗Ajay‘ and ‗Purnima‘ respectively was recorded at

Nauni (Solan) location. The soil of Nauni location also harbor the highest

N-fixers (33.33 cfug-1 soil and 31.00 cfug-1 soil) and P-solubilizers (86.99

and 82.88) for both cultivars ‗Ajay‘ and ‗Purnima‘ respectively (Table 1).

The highest per cent P-solubilizers to total PVK count for ‗Ajay‘

(67.05 cfug-1 soil) was recorded at Nauni whereas highest for ‗Purnima‘

(65.80 cfug-1 soil) was recorded at Kandaghat (Table 2).

Table 1. Enumeration of rhizosphere microbial population

associated with chrysanthemum plant

Location Cultivar Microbial count ( × 103 cfug-1 soil )

Nutrient agar (NA)

Jensen‟ Medium (JM)

Pikovskaya‟s Medium (PVK)

Nauni Ajay 180.66 33.33 86.99

Purnima 152.33 31.00 82.88

Kandaghat Ajay 153.00 31.00 81.77

Purnima 146.99 27.66 81.55

Table 2. Population of P- solubilizers associated with

chrysanthemum plant

Location Cultivar Pikovskaya’s medium

(PVK)

PSB + ve colonies

% P solubilizer to PVK count

Nauni Ajay 86.99 58.33 67.05

Purnima 82.88 52.00 62.74

Kandaghat Ajay 81.77 55.33 67.66

Purnima 81.55 53.66 65.80

Microbial growth on NA medium

(Master Plate)

Jensen medium NA medium PVK medium

Plate 1. Isolation of microbes by modified replica plate method

on different medium

Wooden block

Velvetin cloth

4.1.2 Microbial population in the roots of chrysanthemum plants

The results represented in Table 3 revealed that roots of plants

collected from different sites harbored different bacteria capable of growth

on different medium. The highest total endophytic bacterial count (102.66

cfug-1 root) was recorded for cv. ‗Ajay‘ at Nauni and cv. ‗Purnima‘ (99.99

cfug-1 root) at Kandaghat . The endophytic P-solubilizers‘ count was more

as compared to counts of N-fixers. The highest endophytic bacterial

population on the PVK medium was recorded at Nauni (76.99 cfug-1 root

for cv. ‗Ajay‘ and 72.88 cfug-1 root for ‗Purnima‘) and minimum was at

Kandaghat (71.55 cfug-1 root for cv. ‗Ajay‘ and 71.77 cfug-1 root for cv.

‗Purnima‘).

The highest per cent P-solubilizers to total PVK count for cultivars

‗Ajay‘ and ‗Purnima‘ (32.90 and 38.87) were recorded at Nauni (Table 4).

Table 3. Enumeration of endophytic bacterial population associated

with roots of chrysanthemum plants

Location Cultivar Microbial count ( × 101 cfug-1 root)

Nutrient agar (NA)

Jensen‟ medium

(JM)

Pikovskaya‟s medium

(PVK)

Nauni Ajay 102.66 27.33 76.99

Purnima 96.33 21.66 72.88

Kandaghat Ajay 94.00 25.66 71.55

Purnima 99.99 22.00 71.77

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

chrysanthemum plants

Location Cultivar Pikovskaya’s medium

(PVK)

PSB + ve colonies

% P solubilizer to PVK count

Nauni Ajay 76.99 25.33 32.90

Purnima 72.88 28.33 38.87

Kandaghat Ajay 71.55 22.66 31.67

Purnima 71.77 21.00 29.26

4.1.3 Selection of morphologically similar colonies

The isolates capable of growth on PVK and nitrogen free medium

having similar morphological features were grouped together to represent

one isolate. A total of ten isolates from both cultivars (five each from cv.

‗Ajay‘ and cv. ‗Purnima‘) were selected from existing twenty three purified

isolates (Plate 2). Among the five isolates of ‗Ajay‘, four isolates (namely

KS1, KS2, KS3, KS5) from the rhizosphere and one (KS4) from the roots

were selected, whereas for ‗Purnima‘, four isolates (KS6, KS7, KS9, KS10)

were rhizospheric and one (KS8) was endophytic. The data on colony

morphological characteristics of these isolates are summarized in Table 5.

Table 5. Morphological characteristics of rhizospheric and

endophytic bacterial isolates of chrysanthemum plants Isolates Form Elevation Margin Surface Gram‟s

reaction Shape Pigment

KS1 Punctiform Flat Entire Smooth + Rods White

KS2 Circular Flat Erose Smooth + Rods White

KS3 Rhizoidal Raised Filamentous Rough + Rods White

KS4 Circular Raised Entire Smooth + Rods White

KS5 Irregular Flat Undulate Smooth + Rods White

KS6 Irregular Flat Undulate Smooth + Rods Cream

KS7 Irregular Flat Erose Smooth + Rods White

KS8 Circular Raised Undulate Rough + Rods Cream

KS9 Circular Flat Undulate Smooth + Rods White

KS10 Irregular Flat Lobate Rough + Rods White

4.2 SCREENING OF ISOLATES FOR MULTIFARIOUS PLANT

GROWTH PROMOTING ACTIVITIES The bacterial isolates were screened for multifarious plant growth

promoting activities i.e. growth on Pikovskaya‘s (PVK) medium, N-free

medium, production of siderophore (CAS medium), auxin (LB broth), HCN

(King‘s B medium) and antagonism against Pythium ultimum, Fusarium

oxysporum and Rhizoctonia solani of chrysanthemum plant.

Growth on Nutrient agar Growth on PVK medium

Growth on Jensen medium

Plate 2. Purification of selected bacterial isolate on different

medium (Streak Plate Method)

Table 6. Screening of bacterial isolates for multifarious plant growth promoting activities

Isolates

Phosphorus solubilization

Growth on

N- free medium

Auxin productio

n

Siderophore production

HCN production

Antagonism against

Fusarium oxysporum

Pythium ultimum

Rhizoctonia

solani

KS1 ++ ++ ++ + - - - -

KS2 + + + - - - - -

KS3 + + + - - - - -

KS4 + ++ + - - - - -

KS5 +++ +++ ++ + + - + -

KS6 +++ +++ ++ ++ ++ ++ + ++

KS7 + ++ + - - - - -

KS8 ++ + + - - - - -

KS9 ++ ++ + + - - - -

KS10 ++ ++ + - - - - -

+++ very good activity, ++ good activity, + fair activity, - no activity

All the ten isolates were P-solubilizers, auxin producers and

capable of growth on N-free medium. However, only four isolates

produced siderophore and two of them (KS5 and KS6) produced HCN

(Plate 3.b,c) out of which KS5 showed antagonism against Pythium

ultimum whereas KS6 isolate inhibited the mycelial growth of all the three

fungal pathogens Pythium ultimum, Fusarium oxysporum and Rhizoctonia

solani (Table 6) (Plate 4).

The results further revealed that only one bacterial isolate (K6)

exhibited the concurrent production of siderophores, solubilization of

phosphorus, growth on nitrogen free medium, HCN production and

effective inhibition of the mycelial growth of Fusarium oxysporum,

Rhizoctonia solani and Pythium ultimum.

4.2.1 Phosphorus solubilization efficiency on solid PVK medium

All isolates were found to solubilize TCP in PVK agar medium

(Table7). The P- solubilization efficiency had great variation with the value

ranging from 36.82 (KS2) to 88.97 (KS6) per cent (Plate 3.a).

Table 7. Phosphorus solubilization efficiency of bacterial

isolates on solid PVK medium

Isolates Colony size (c) (mm)

Zone size (z) (mm)

%P-Solubilization efficiency (% SE)

KS1 3.3 5.8 76.02 (60.70)* KS2 2.2 3.1 36.82 (37.35)

KS3 2.5 3.8 50.67 (45.38)

KS4 2.1 3.3 58.73 (50.03) KS5 2.2 3.9 80.47 (63.94) KS6 3.4 6.4 88.97 (70.79) KS7 2.3 3.6 57.95 (49.58)

KS8 2.5 4.1 61.18 (51.48) KS9 3.0 5.0 67.03 (54.97) KS10 3.1 4.4 41.04 (39.84)

CD0.05 0.12 0.09 3.98 *Figures in the parentheses are arc sine transformed values

Z-C (% SE) = X100 C

Where, C = Colony size Z = Halozone size

Control

a) P- solubilization by bacterial isolates

Control Yellowish orange zone

b) Siderophore production by bacterial isolates

Control Colour change of filter paper

(Yellow to orange brown)

c) HCN production by bacterial isolates

Plate 3. Multifarious plant growth promoting activities by

different bacterial isolates a) P-solubilization b)

Siderophore production c) HCN production

Control Treated Treated

Pythium ultimum Pythium ultimum

Pythium ultimum + +

KS6 isolate KS5 isolate

Control Treated

Fusarium oxysporum

Fusarium oxysporum +

KS6 isolate

Control Treated

Rhizoctonia solani

Rhizoctonia solani +

KS6 isolate

Plate 4. Antifungal activity of bacterial isolates against fungal

pathogens using dual culture technique

Zone of contact inhibition;

% growth inhibition=23%

KS5 KS6

Zone of no growth; % growth inhibition=33%

KS6

Zone of contact inhibition; % growth inhibition=25%

KS6

4.2.2 IAA production

The data on production of IAA by different bacterial isolates is

embodyed in Fig 1 (Appendix 2.1) revealed that highest (26.25 µg/ml) IAA

equivalents were produced by KS6 isolate followed by KS5 (25.50 µg/ml),

KS1 (22.34 µg/ml) and KS9 (23.17 µg/ml) bacterial isolates respectively at

72 h of incubation.

4.2.3 Dual culture compatibility assessment All the ten isolates were screened for their antagonistic and

synergistic activities amongst themselves. Table 8 (Plate 5) revealed that

isolate KS6 showed synergism with rest of isolates except KS4. Isolate KS7

showed antagonism to five isolates (KS1, KS2, KS3, KS4 and KS5).

Table 8. Dual culture compatibility assessment amongst ten

bacterial isolates of cultivars („Ajay‟ and „Purnima‟) of chrysanthemum plants

Isolates used for

overlayering

Synergism with bacterial isolates

Antagonism with bacterial isolates

KS1 KS2, KS3, KS5, KS6, KS8, KS9, KS10 KS4, KS7

KS2 KS1, KS3, KS5, KS6, KS8, KS10 KS4, KS7, KS9

KS3 KS1, KS2, KS4, KS5, KS6, KS9, KS10 KS7, KS8

KS4 KS1, KS2, KS5, KS7, KS9, KS10 KS3, KS6, KS8

KS5 KS1, KS2, KS3, KS4, KS6, KS9, KS10 KS7, KS8

KS6 KS1, KS2, KS3, KS5, KS7, KS8, KS9, KS10 KS4

KS7 KS6, KS8, KS9, KS10 KS1, KS2, KS3, KS4, KS5

KS8 KS4, KS5, KS6, KS7, KS9, KS10 KS1, KS2, KS3

KS9 KS1, KS3, KS5, KS6, KS7, KS8, KS10 KS2, KS4

KS10 KS1, KS4, KS6, KS7, KS8, KS9 KS2, KS3, KS5

On the basis of results of screening (Table 6) of various isolates to

multifarious plant growth traits, only four isolates (KS1, KS5, KS6 and KS9)

were selected for identification and characterization.

4.3 IDENTIFICATION AND CHARACTERIZATION OF SELECTED BACTERIAL ISOLATES (KS1, KS5, KS6, KS9)

4.3.1 Morphological, physiological and biochemical characteristics of the isolates

The morphological, physiological and biochemical characteristics of

the isolates Bacillus spp. after 48 h of incubation are presented in Table 9.

The isolated colonies of the KS1 isolate on NA medium were punctiform in

colony configuration having smooth surface, flat elevation, undulate

margin and cream colour. Isolate KS5 had an irregular colony

configuration having smooth surface, fl