BIODIVERSITY OF SOME ENDOPHYTIC AND … OF SOME ENDOPHYTIC AND ASSOCIATIVE BACTERIA ISOLATED FROM...

BIODIVERSITY OF SOME ENDOPHYTIC AND

ASSOCIATIVE BACTERIA ISOLATED FROM

SOME EGYPTIAN SOILS

By

Heba Belal Abd El-Samei Kandil B. Sc. Agric. Sci., (Soil Science), Fac. Agric., Cairo Univ., 2006

THESIS Submitted in Partial Fulfillment of the

Requirements for the Degree of

MASTER OF SCIENCE

In

Agricultural Sciences (Agricultural Microbiology)

Department of Agricultural Microbiology

Faculty of Agriculture

Cairo University

EGYPT

2012

APPROVAL SHEET



SOME EGYPTIAN SOILS

M. Sc. Thesis

In

Agric. Sci. (Agricultural Microbiology)

By


APPROVAL COMMITTEE

Dr. El-Shahat Mohamed Ramadan………………………………….. Professor of Agricultural Microbiology, Fac. Agric., Ain Shams University

Dr. Aziz Mohamed Aziz Higazy……………………………………..… Professor of Agricultural Microbiology, Fac. Agric., Cairo University

Dr. Mohamed Abdelalim Ali …………………………………….…….. Professor of Agricultural Microbiology, Fac. Agric., Cairo University

Dr. Mohamed Fayez Fouad Ibrahim……………………………...… Professor of Agricultural Microbiology, Fac. Agric., Cairo University

Date: / / 2012

SUPERVISION SHEET



SOME EGYPTIAN SOILS

M. Sc. Thesis In

Agric. Sci. (Agricultural Microbiology)

By


SUPERVISION COMNITTEE

Dr. Mohamed Fayez Fouad

Professor of Agricultural Microbiology, Fac. Agric., Cairo University.

Dr. Mohamed Abd El-Aleem Ali

Professor of Agricultural Microbiology, Fac. Agric., Cairo University.

Dr. Eman Ahmed Tantawy

Head Research of Agricultural Microbiology. SWERI, ARC, Giza.

Name of Candidate: Heba Belal Abd El-Samei Kandil Degree: M. Sc. Title of Thesis: Biodiversity of Some Endophytic and Associative Bacteria

isolated from Some Egyptian Soils. Supervisors : Dr. Mohamed Fayez Fouad

Dr. Mohamed Abdelalim Ali

Dr. Eman Ahmed Tantawy

Department: Agricultural Microbiology.

Approval: 25 / 12 /2012

ABSTRACT

In this work, endophytic bacteria were isolated from some wild plants;

Imperata cylindrical, Rumex vesicariu, Suaeda monoica, Triticum aestivum and

Zygophyllum spp. grown in highly salt-affected soil. According to cultural,

morphological and physiological characteristics as well as their BIOLOG GN2

Microplate analysis, the isolated endophytes were identified as Pantoea

agglomerans, Pseudomonas stutzeri, Bacillus mycoides, Bacillus thuringiensis,

Enterobacter cloacae, Brevundimonas diminuta, Pseudomonas aeruginosa and

Bacillus cereus. Production of indol acetic acid (IAA), exopolysaccharides (EPS)

and siderophores by the endophytic strains was in vitro examined. All strains

produced appreciable amounts of IAA and EPS approaching maxima values of

43.75 µg ml-1

and 26.77mg ml-1

with Bacillus cereus and B. mycoides, respectively.

Except with Bacillus strains¸ all endophytes produced siderophores. Plant growth

promoting ability of these bacteria was evaluated in a field experiment. Growth and

forage production of Sorghum biocolor L cv. (Sorghum hybrid local 102) were

improved under high saline soil stress at Gelbana, Sahl Eltina, North Sinai due to

inoculation with the endophytes. Plant fresh and dry weights, nitrogen, phosphorus

and potassium contents were increased in inoculated plants by >50% over controls.

Plant calcium content increased but sodium uptake decreased due to inoculation.

Results referred to a possible role of the isolated endophytes as inocula strains for

improving sorghum plant growth and forage production under high salinity stress.

Key words: Soil salinity, endophytes, plant growth promotion, Sorghum,

siderophore.

DEDICATION

I dedicate this work to whom my heart felt

thanks; to my parents, my brother (Ahmed), my sister (Aya) for their patience, help and for all the support they lovely offered along the period of my post graduation.

ACKNOWLEDGEMENT

Praise and thanks be to ALLAH, for assisting and directing

me to the right way.

I wish to express my sincere thanks, deepest gratitude and

appreciation to Dr. Mohamed F.Fouad; Dr. Mohamed.A.Ali,

Professors of Agricultural Microbiology, Faculty of Agriculture,

Cairo University and Dr. Eman A. Tantawy, Head Research of

Agricultural Microbiology. ARC, Giza, for suggesting the

problems, supervision, continued assistance and their guidance

through the course of study and revising the manuscript.

Deep appreciation is given to the Dr. Fekry.M.Ghazal,

Head Research of Agric. Microbiology, Soils, Water and

Environment, Research Institute, ARC., Dr. Olfat.S.Barakat,

Professor of Agricultural Microbiology, Faculty of Agriculture,

Cairo University and Dr. Belal. A. Kandil , Senior Researcher of

Agric. Microbiology, Soils , Water and Environment, Research

Institute, ARC, Giza.

Grateful appreciation is also extended to all staff members

and colleagues in the Department of Agricultural Microbiology,

Faculty of Agriculture, Cairo University and the Biofertilizers

Unit, Soils, Water and Environment, Res. Inst., ARC. and plant

fertility lab - Agricultural Fund budget - AR C.

I

CONTENTS Page

INTRODUCTION………………………………...……………………………....… 1

REVIEW OF LITERATURE…………………………..…………………....… 3

1. Soil salinity and plant growth.…………………………..…….…..…. 3

2. Endophytic bacteria and their role in plant growth...…….… 5

3. Plant growth promoting substances excreted by

endoPhytic bacteria………………………….......................................

8

4. Endophytic bacteria in saline soil ………………………….…… 16

5. Tolerance of sorghum plant to high soil salinity……....……... 17

6. Families and genera of endophytic bacteria …...………….... 19

a. Family Enterobacteriaceae……………………………….…. 19

b. Family Pseudomonadaceae …………………….................... 21

c. Family Bacillaceae…………………………….......................... 22

MATERIALS AND METHODS………………………………………..….. 25

1. Experimental site and soil sampling………………………..….… 25

2. Irrigation water …………………………………………………...…. 25

3. Plant material……………………………………………………….……... 25

4. Plant sampling and isolation of endophytic bacteria…….… 27

5. Identification of the endophytic isolates…………………….… 28

6. Assessment of plant growth promoting ability of the

isolated endophytes…………………………………………….……… 32

1. In vitro indole acetic acid estimation …..……………….……. 32

2. Siderophores production assay …………….……….……….…... 34

Solution 1 (Fe-CAS indicator)………………............................. 34

Solution 2 (buffer solution)………………….……………...…... 34

Solution 3………………………………………………………….….... 34 Solution 4…………………………………………………….…….…... 35

3. Exopolysaccharide estimation…………………………………... 35

7. Field experiment…………………………………………....................... 36

a. Inocula preparation……………..………………………..……….. 36

b. Planting and plant analysis…………………………………….. 36

8. Statistical analysis……………………………………………………… 37

9. Culture media……………………………………………......................... 38

a. Media used for isolation…………………………………………. 38

1. Yeast Extract Mannitol (YEM) broth medium Vincent (1970)…………………………………….....................................

38

II

2. King's B medium Deshmukh (1997)……………………. 38

3. Yeast Extract peptone (YEP) medium Sambrook et al.

(1989)………………………………………………………………...

39

4. Luria-Bertani (L.B) agar medium Bertani (1951)….. 39

b. Media used for IAA production assay…………….. 40

RESULTS …………………………………………………………………………. 41

1. Isolation and identification of endophytic bacteria................. 41

2. Production in vitro of plant growth promoters substances

by the isolated endophytic…………..………………………………...

58

a. Indole acetic acid (IAA) production………...………………… 60

b. Exopolysachraides(EPS) …………………………………..……… 62

c. Sidrophore……………………………………………………………... 63

3. Endophytic bacteria as appropriate inocula for improving

sorghum growth under high saline soil condition…...........

63

DISCUSSION……………………………………………………………………... 77

SUMMARY....…………………………………………………………………….. 85

REFERENCES…………...……………………………………………………… 87

ARABIC SUMMARY…………………………………………………………

III

LIST OF TABLES

No. Title Page

1. Physical and Chemical properties of the experimental

soil in Sahl El-Tina Plain…………………..………………….…

26

2. Chemical analysis of El-Salam Canal water……….……… 27

3. The definition of plant samples……………………….………. 29

4. Layout of the field serial treatments……………………….… 37

5. The composition of YEM medium…………………………... 38

6. The composition of King's B medium………….…………… 39

7. The composition of YEP medium……………………………. 39

8. The composition of L.B medium…………………….……….. 39

9. The composition of L.B medium+L-trptophan……........... 40

10. Morphological and biochemical traits of endophytic bacteria isolated from some wild-grown plants……...…..

43

11. Characteristic reaction pattern of the Biolog GN2

Microplate for identification and characterization of

Pantoea agglomerans isolate……………………………..…….

44



Pseudomonas stutzeri isolate…………………………….……..

46


Microplate for identification and characterization of Bacillus mycoides isolate……………………………….………..

48

IV



Bacillus thuringiensis isolate…………………….……………..

50



Enterobacter cloacae isolate……………………….….………..

52



Brevundimonas diminuta isolate………………………………

54


Microplate for identification and characterization of Bacillus cereus isolate………………………….…………………

56

18. Botanical species, the isolated endophytic strains,

cultural media used for isolation and EC of rhizospher

soil…………………………………………………………………...…..

59

19. Indole acetic acid (IAA), exopolysaccharide (EPS) and

sidrophore production by endophtic bacterial

isolates…………………………………………………………………

64

20. Effect of endophytic bacterial inoculation on forage

sorghum plants height, fresh and dry weights…….……… 67

21. Effect of endophytic bacterial inoculation on N, P&K contents……………………………………………………...………… 73

22. Effect of endophytic bacterial inoculation on plant Na

and Ca contents……………………………………………..………. 74

23. The ratio of plant elements……………………………………… 75

V

LIST OF FIGURES

No. Title Page

1. Plant sterilization test after 48 h………………………….………… 31

2. Standard curve for IAA……………………………………………..… 33

3. (1-13) Color changes in plate cultures endophytic strains

treated with Salkowski due to IAA production on L-B

tryptophan agar …………………………………………………....…….

61

4. Indole acetic acid (IAA) production by endophytic

bacterial strains……………………………..…………….………………

65

5. Effect of endophytic bacterial inoculation on forage

sorghum plants height (cut 1& 2) ………………………....………. 68

1

INTRODUCTION

Salinity and sodicity are among the permanent problems in

Egypt as well as in all arid and semiarid regions due to harsh climatic

conditions of high temperature and low rainfall (Sharma et al., 2004).

According to a report by the USAID (1980) approximately 28% of the

currently cultivated land in Egypt is affected by salinity while Kishk

(1986) classified at least 50% of Egyptian cultivated land as saline soil.

Soil salinity is caused by accumulation of salts in soil leading to

a sharp decrease in soil fertility. Salt concentration left in plant

capillaries, with insufficient amount of nourishing substances leads to

plant dying. Salinity becomes a problem when salts accumulate in the

root zone in an amount high enough to negatively affect plant growth.

Excess salts in the root zone hinder plant roots from withdrawing water

from surrounding soil (Nikos et al., 2003).

The impact of soil salinity on the physicochemical and

biological properties renders the salt-affected soils unsuitable for

microbial processes to support plant growth (Munns, 2002; Rengasamy

et al., 2003). Under these conditions,the average crop yield in saline

areas particularly in Egypt is much lower than in normal soils.

Some bacteria canlivein plant tissues or inner plant parts without

any visible harm. Those are called endophytes and are known

tostimulate plant growth via indole-3-acetic acid (IAA), siderophores

and exopolysaccharides (EPS) production in addition to increasing

2

disease resistance, improving plant’s ability to withstand environmental

stresses and enhancing N2-fixation (Sturz and Nowak, 2000).

Endophytic bacteria were identified as bacteria that can be

isolated from surface-disinfected plant tissues or extracted from inner

plant parts and do not cause visible harm to the host or external visible

structures. The endophytes are either localized at their point of entry or

spread throughout the plant tissues wherein they are physically

protected from biotic and abiotic stresses (Hallman et al., 1997).

Endophytic bacteria isolated from rice grown in highly saline soils

showed an ability to tolerate high soil salinity, nitrogen fixation and

plant growth promoting substances (PGPS) production. Among the

latter, indol acetic acid (IAA) plays a key role in the regulation of plant

growth and development of root elongation and increase plant mineral

absorption as a result and thus help alleviating salt stress in plants

growing in saline environments (Tantawy, 2009).

In this workit was planned to isolate and identify the endophytic

bacteria from wild plants grown in highly-salt-affected soil. In addition,

to examinein vitro the plant-growth-promoting capacity of these

isolates. Moreover, to challenge the plant growth promoting isolates as

sorghum biofertilizers in highly salt-affected soil in Sahl El-Tina, North

Sinai.

3

REVIEW OF LITERATURE

1. Soil salinity and plant growth

Salinity is an important land degradation problem. United States

Salinity Laboratory USSL (1954) divided salt-affected soils into three

main categories depending upon the electrical conductivity (EC),

slightly saline soils with EC from 4 to <8 dSm-1

, moderately saline

from 8 to <16 dSm-1

and strongly saline that more than 16 dSm-1

compared with salt free soil which less than 4 dSm-1

. According to the

USDA salinity laboratory, saline soil can be defined as soil having an

EC of the saturated paste extract of 4 dSm-1

(4 dSm-1

≈ 40 mM NaCl)

or more. Most grain crops and vegetables are glycophytes and are

highly susceptible to soil salinity even when the soil ECe is 4 dSm-1

.

Salinity affects plant growth by the osmotic effect of salts in the

outside solution and ion toxicity due to salt build-up in transpiring

leaves in a second phase in addition to induction of nutrient

deficiencies (Wyn Jones, 1981).

Under conditions of soil salinity, rapid reduction in net

photosynthesis, inhibition of growth, disturbance of anatomical

structure, alterations in cell membrane and K+ deficiency have been

reported. Both soil salinity and water logging alter root and shoot

hormone relations e.g. decreases cytokinins and gibberellins as well as

increases abscisic acid contents, Gadallah (1999). Soil salinity reduces

yield production of most crops, and hence soil salinity is an important

4

environmental stress posing threat to agriculture and food supply

(Flowers, 2004).

Salinity becomes a problem when enough salts accumulate in

the root zone to negatively affect plant growth. Excess salts in the root

zone hinder plant roots from withdrawing water from surrounding soil

(Nikos et al., 2003).

Sodium toxicity under saline conditions is particularly common

in graminaceous crops and results in a range of disorders in protein

synthesis and enzyme activation (Tester and Davenport, 2003).

Ahloowalia et al. (2004) showed that soil salinity is one of the

main problems for agriculture, especially in countries where irrigation

is an essential aid to agriculture.

As reported by Mathur et al. (2007) the impacts of soil salinity

on agricultural yield are enormous as it affects the establishment,

growth and development of plants leading to huge losses in

productivity.

At present, out of 1.5 billion hectares of cultivated land around

the world are saline soils and about 77 million hectares is affected by

excess salt content as reported by Evelin et al. (2009). Also, Jadhav et

al. (2010) documented that nearly 40% of world’s surface has salinity

problems.

Butale et al. (2010) stated that salinity in soil is developed due

to accumulation of excessive salts. Predominant nitrogen fixing

5

bacteria in the salt deposited soils were found difficult to fix nitrogen

due to high pH and salinity.

Problems associated with salinity not only affect agriculture but

also the biodiversity of the environment. This situation is more

alarming in arid and semi arid environments (Fernandez-Aunión et al.,

2010).

2. Endophytic bacteria and their role in plant growth

Endophytic bacteria are bacteria, live in plant tissues or the inner

plant parts without any visibly harm, and can be isolated from surface-

disinfected plant tissues, or extracted from inner plant parts. These

bacteria help plant to withstanding adverse conditionsvia production

plant growth promoting substances (PGPS). Also, the mass of soil

bacteria that associated with plant roots have a lot of endophytic

bacteria that could crack and entre the plant tissue.

Hallmann et al. (1997) identified endophytic bacteria for the

first time as bacteria that can be isolated from surfaces disinfected plant

tissues or extracted from inner plant parts, and do not cause visible

harm to the host or external visible structures with either become

localized at the point of entry or spread throughout the plant.

Endophytic bacteria can not only promote plant growth and act

as biocontrol agents, but also produce natural products to control plant

diseases (Guan et al., 2005).

6

Endophytic potential to actas a biocontrol agent against

phytopathogens has been reported by Sturz et al. (1998) and against

insects by Azevedo et al. (2000).

Tan and Zou (2001) reported that every plant has some specific

endophytic bacteria which can produce organic compounds or

secondary metabolites.

According to Morris et al. (2001) and Gofar (2004) plant tissue

is an optimal habitat for both pathogen and non-pathogen microbes.

Beneficial effects from interaction between non pathogen microbes and

host plant is growth promotion of host plant because the microbe can

produce phytohormones.

Bacteria that live in the interior of plants without causing

diseases to their hosts are called endophytic (Azevedo et al., 2000).

Plant kingdom is colonized by diverse endophytic bacteria which

benefit plants via stimulating plant growth, increasing disease

resistance, improving the ability of plants to which stand stresses and

enhance N2-fixation (Sturz and Nowak, 2000).

Endophytes have an excellent potential to be used as plant

growth promoters with legumes and non-legumes (Bai et al., 2002).

Under salinity conditions, the problem facing agricultural

production is how to optimize a suitable root zone. Bio-fertilizers play

an important role in promoting plants to tolerate salt stress and toxicity,

(Ghoulam et al., 2002).

7

The endophytic bacteria can infect host plant not only via root

but through flowers, stems, and cotyledon (Zinniel et al., 2002).

Bacteria are common inhabitants on the surface as well as inside

tissues of the plants, exerting various effects on the development of

their hosts (Selosse et al., 2004).

Bacterial endophytes colonizing an ecological niche similar to

that of phytopathogens make them suitable as biocontrol agents (Berg

et al., 2005).

Endophytic bacteria supply essential vitamins to plants, along

with osmotic adjustment, stomatal regulation, modification of root

morphology and enhanced uptake of minerals and alteration of nitrogen

accumulation (Compant et al., 2005).

Endophytic bacteria, co-evolved with plants, have been found in

virtually every plant studied, where they colonize the internal tissues of

their host plant and can form a range of different relationships

including symbiotic, mutualistic and trophobiotic (Robert et al., 2008).

Endophytic bacteria have attracted more and more attention as

novel resources of biocontrol of plant diseases and plant growth

promoters (Lin et al., 2009).

Tadych and White (2009) reported that endophytic organisms

associated with plants are diverse and complex. Endophytic microbes

occupy a relatively privileged niche within plant and usually contribute

8

to plant health. Some groups of endophytic microorganisms are

believed to protect plants against biotic stresses.

Jalgaonwala et al. (2011) reported that exploitation of

endophyte-plant interactions can results in the promotion of plant

health and play significant role in low input sustainable agriculture

applications for both food and nonfood crops. An understanding of the

mechanisms of endophytic bacteria to interact with plants will be

essential to achieve the biotechnological potential of these microbes.

The challenge is to be able to manage microbial communities to favor

plant colonization by beneficial endophytic bacteria.

3. Plant growth promoting substances excreted by endophytic

bacteria

Endophytic bacteria supply essential vitamins to plants. The

production of auxin-like compounds increases seed production and

germination (Rodelas et al., 1993).

A siderophore is an iron chelating compound secreted by

microorganisms (bacteria and fungi) and many plants; it works as iron

binding that bind iron and transports it into the plant cell (Neilands,

1995).

As monitored by Patten and Glick (1996), production of

phytohrmone indole acetic acid (IAA) is widespread among endophytic

bacteria and its role in stimulating plant growth and phytopathogensis

is well-considered.

9

Iron is an essential nutrient element for all living organisms. The

scarcity of bioavailable iron in soil habitats and on plant surfaces

foments a furious competition (Loper and Henkels, 1997). A myriad of

environmental factors modulate siderophores synthesis. These include

pH, level of iron and the form of iron ions, the presence of other trace

elements, and an adequate supply of carbon, nitrogen, and phosphorus

(Duffy and Défago, 1999).

Hedge et al. (1999) mentioned that in general, bio-fertilizers are

environment friends, low cost agricultural input with maximum output.

These bio-fertilizers play an important role in enhancing crop

productivity through nitrogen fixation, phosphate solubilization, plant

hormone productivity, ammonia excretion, siderophore formation and

control various plant diseases.

Under iron-limiting conditions, plant growth promoting bacteria

(PGPB) produce low-molecular-weight compounds called siderophores

to competitively acquire ferric ion (Whipps, 2001).

Direct stimulation of PGPB may include providing plants with

fixed nitrogen, iron that has been sequestered by bacterial siderophores,

soluble phosphate and other nutrients, and the ability to produce the

correct amounts of plant hormones such as IAA, gibberellic acid and

cytokinins (Bloemberg and Lugtenberg, 2001). Hubbell and Kidder,

2001) argued that endophytic bacteria could increase nitrogen content

of plant host.

Date post:	03-May-2018
Category:	Documents
Upload:	hahuong
View:	217 times
Download:	1 times

BIODIVERSITY OF SOME ENDOPHYTIC AND … OF SOME ENDOPHYTIC AND ASSOCIATIVE BACTERIA ISOLATED FROM...

Documents