BIODIVERSITY OF SOME ENDOPHYTIC AND … OF SOME ENDOPHYTIC AND ASSOCIATIVE BACTERIA ISOLATED FROM...
Transcript of 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
BIODIVERSITY OF SOME ENDOPHYTIC AND
ASSOCIATIVE BACTERIA ISOLATED FROM
SOME EGYPTIAN SOILS
M. Sc. Thesis
In
Agric. Sci. (Agricultural Microbiology)
By
Heba Belal Abd El-Samei Kandil B. Sc. Agric. Sci., (Soil Science), Fac. Agric., Cairo Univ., 2006
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
BIODIVERSITY OF SOME ENDOPHYTIC AND
ASSOCIATIVE BACTERIA ISOLATED FROM
SOME EGYPTIAN SOILS
M. Sc. Thesis In
Agric. Sci. (Agricultural Microbiology)
By
Heba Belal Abd El-Samei Kandil B. Sc. Agric. Sci., (Soil Science), Fac. Agric., Cairo Univ., 2006
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
12. Characteristic reaction pattern of the Biolog GN2
Microplate for identification and characterization of
Pseudomonas stutzeri isolate…………………………….……..
46
13. Characteristic reaction pattern of the Biolog GN2
Microplate for identification and characterization of Bacillus mycoides isolate……………………………….………..
48
IV
14. Characteristic reaction pattern of the Biolog GN2
Microplate for identification and characterization of
Bacillus thuringiensis isolate…………………….……………..
50
15. Characteristic reaction pattern of the Biolog GN2
Microplate for identification and characterization of
Enterobacter cloacae isolate……………………….….………..
52
16. Characteristic reaction pattern of the Biolog GN2
Microplate for identification and characterization of
Brevundimonas diminuta isolate………………………………
54
17. Characteristic reaction pattern of the Biolog GN2
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.