Isolation, Identification and Characterization of

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2.3.2 Surface Sterilization of root and stem sample . ..... 14. 3.1. Isolate BSRI 1 under light microscope after gram staining. 32. 3.2. Growth of individual colonies ...

Isolation, Identification and Characterization of Sugarcane Associated Nitrogen Fixing Bacteria Obtained from Bangladesh Sugarcane Industry, Iswardi

M. S. Thesis

SUBMITTED BY DEPARTMENT OF MICROBIOLOGY UNIVERSITY OF DHAKA DHAKA-1000 JULY, 2016.

IMAM TASKIN ALAM EXAMINATION ROLL NO. 1504 REGISTRATION NO. HA- 1036 SESSION: 2014-2015

Isolation, Identification and Characterization of Sugarcane Associated Nitrogen Fixing Bacteria Obtained from Bangladesh Sugarcane Industry, Iswardi

A DISSERTATION SUBMITTED TO THE DEPARTMENT OF MICROBIOLOGY, UNIVERSITY OF DHAKA FOR THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN MICROBIOLOGY

DEPARTMENT OF MICROBIOLOGY UNIVERSITY OF DHAKA

SUBMITTED BY IMAM TASKIN ALAM

DHAKA-1000

EXAMINATION ROLL NO. 1504

JULY, 2016.

REGISTRATION NO. HA- 1036 SESSION: 2014-2015

Dedicated to...

My beloved parents and my brothers, who cherished my life with their blessings

Student’s Copy

Certification

It is hereby certified that student bearing Roll no. 1504, Registration No. Ha-1036 has carried out the research work entitled “Isolation, Identification and Characterization of Sugarcane Associated Nitrogen Fixing Bacteria Obtained from Bangladesh Sugarcane Industry, Iswardi” for the partial fulfillment of his Master of Science Degree in Microbiology from the University of Dhaka, Bangladesh, under my academic supervision in the Laboratory No. 206, Department of Microbiology, University of Dhaka.

..…………………………………………… Professor Humaira Akhter Department of Microbiology, University of Dhaka, Dhaka- 1000. Bangladesh.

Table of Contents Acknowledgement

I

Abstract

II

List of Tables

III

List of Figures

IV

Abbreviations

V

Chapter 1: Introduction and Literature Review .............................................................................. 1-15 1.1 General Introduction ..................................................................................................................... 2 1.2 Literature Review ......................................................................................................................... 3 1.2.1

Nitrogen fixation by Diazotrophs ........................................................................................ 3

1.2.2

Nitrogen Fixation by Endophytic Bacteria ........................................................................... 5

1.2.3

Overview of Biological Nitrogen Fixation (BNF) ................................................................... 7

1.2.3.1 The nitrogenase reaction ................................................................................................ 7 1.2.3.2 The energy requirement ................................................................................................. 7 1.2.4

Nitrogen fixation and sustainable agriculture ...................................................................... 9

1.2.5

Non-Symbiotic Nitrogen Fixers ........................................................................................... 9

1.2.5.1 Azospirillum .................................................................................................................. 10 1.2.5.2 Herbaspirillum .............................................................................................................. 10 1.2.5.3 Azoarcus ....................................................................................................................... 11 1.2.5.4 Rhizobia ........................................................................................................................ 11 1.2.6

Endophytic Nitrogen-Fixing Bacteria as Biofertilizer ........................................................... 12

1.2.7

Endophytic Nitrogen-Fixing Bacteria of Sugarcane ............................................................ 13

1.3 Bangladesh perspective .............................................................................................................. 14 1.4 Aims and objectives of the study ................................................................................................ 15 Chapter 2: Materials and Methods .................................................................................................... 16-29 2.1 Laboratory equipments and glassware ....................................................................................... 17 2.2 Culture Media ............................................................................................................................ 17 2.3 Isolation of Endophytes from Sugarcane (genus Saccharum) root and stem ................................ 17

2.3.1

Collection of plant material .............................................................................................. 17

2.3.2

Surface Sterilization of root and stem sample ................................................................... 18

2.3.3

Isolation of endophytes from sugarcane plant material .................................................... 18

2.3.4

Initial stocking of Isolates ................................................................................................. 19

2.3.5

Maintenance of working culture ....................................................................................... 19

2.4 Characterization of the Isolates .................................................................................................. 19 2.4.1

Morphological characteristics ........................................................................................... 20

2.4.2

Biochemical & metabolic characterization ........................................................................ 20

2.4.2.1 Cellulase activity ........................................................................................................... 20 2.4.2.2 Gelatin hydrolysis ......................................................................................................... 20 2.4.2.3 Starch hydrolysis .......................................................................................................... 21 2.4.2.4 Casein hydrolysis .......................................................................................................... 21 2.4.2.5 Catalase activity ............................................................................................................ 21 2.4.2.6 Nitrate reduction .......................................................................................................... 21 2.4.2.7 Production of H2S ......................................................................................................... 22 2.4.2.8 Indole production ......................................................................................................... 22 2.4.2.9 Citrate utilization .......................................................................................................... 22 2.4.3

Carbohydrate utilization test ............................................................................................ 22

2.4.4

Study of Plant growth promoting attributes ..................................................................... 23

2.4.4.1 Phosphate solubilization ............................................................................................... 23 2.4.4.2 Indole acetic acid (IAA) production ............................................................................... 23 2.4.5

Antibiotic sensitivity test .................................................................................................. 23

2.4.6

Molecular characterization of bacterial endophytes ......................................................... 24

2.4.6.1 DNA extraction ............................................................................................................. 24 2.4.6.2 Amplification of 16S rDNA from genomic DNA .............................................................. 25 2.4.6.3 nifH amplification ......................................................................................................... 26 2.4.6.4 Random Amplification of Polymorphic DNA assay ......................................................... 27 2.4.7

Identification of isolates using universal method .............................................................. 28

Chapter 3: Results .................................................................................................................................. 30-43 3.1 Morphological Characteristics .................................................................................................... 31 3.1.1

Cell Morphology ............................................................................................................... 31

3.1.2

Colony characteristics ...................................................................................................... 32

3.2 Metabolic characteristics ........................................................................................................... 33 3.3 Carbohydrate utilization pattern ................................................................................................ 34 3.4 Plant growth promontory attributes .......................................................................................... 36 3.4.1

Phosphate solubilization .................................................................................................. 36

3.4.2

Indole acetic acid (IAA) production ................................................................................... 37

3.5 Antibiogram profile ................................................................................................................... 37 3.6 Molecular characteristics ........................................................................................................... 39 3.6.1

16S rRNA gene profile ...................................................................................................... 39

3.6.2

nifH gene profile .............................................................................................................. 40

3.6.3

Random Amplification of Polymorphic DNA assay ............................................................ 41

3.7 Identification of isolates using universal method ....................................................................... 42 Chapter 4: Discussion and Conclusion ............................................................................................ 44- 49 4.1 Discussion ................................................................................................................................ 45 4.2 Future prospects and Conclusion ............................................................................................. 48 References .................................................................................................................................................... A-L Appendices ..................................................................................................................................................i-xii Appendix I ........................................................................................................................................ ii Appendix II ......................................................................................................................................vii Appendix III ...................................................................................................................................... x Appendix IV ..................................................................................................................................... xii

Acknowledgement First and foremost, I thank the Almighty Allah for allowing me to complete the research for this thesis work, although no words of gratitude can express His greatness enough. I must express my earnest respect and gratitude to my supervisor Prof. Humaira Akhter, Chairman, Department of Microbiology, University of Dhaka for her continuous support, valuable suggestion, supervision and cooperation to enable me to complete this research. My heartiest gratitude goes to Dr. Khandakar Mohiul Alam, Senior Scientific Officer, Soil and Nutrition Division, Bangladesh Sugarcane Research Institute, for giving me the opportunity to participate in this research project and providing me with preliminary materials and knowledge that helped me in completion of this research. I would like to convey my indebtedness to Dr. Anowara Begum, Professor, Department of Microbiology, University of Dhaka. Her presence, patience and suggestions were instrumental in the completion of this research. I am deeply grateful to Prof. Mahmuda Yasmin, Professor, Department of Microbiology, University of Dhaka, for coordinating the MS 2014-2015 course with dedication and skill. I would like to express my gratitude to Jannatul Ferdous and Zenat Zebin, PhD fellows, University of Copenhagen. This thesis would not come to light without their aid and suggestions that helped me successfully complete my dissertation without any serious flaw. My deepest gratitude goes to Ridwan Rashid, Lab senior, whose constant advice proved to be elemental in the completion of this study. I would also like to thank my lab members Sabera Saima, Rokaia Sultana, Sumaiya Zaman, Nusrat Nahar, Riajul Islam, Md. Masud Parvez and Md. Razib Hossain for cooperating with me during this research work. I would also like to express my gratefulness to my classmates and friends Sadikur Rahman, Md. Hassan-uz-Zaman, and Md. Abdullah-al-Mannan. Discussion with them was very helpful during my research work. Last but not least, I am thankful to my family. Their unparalleled love and support helped me get through turbulent times. July 2016

The Author

I

Abstract The present study was conducted with the intention to isolate and characterize endophytic bacterial diversity from stem and roots of sugarcane (Saccharum sp.) for the development of biofertilizer. Twelve endophytic bacteria were isolated from sugarcane samples obtained from the test field of Bangladesh Sugarcane Research Institute (BSRI), Iswardi, Bangladesh. The isolates were identified as members of Bacillus and Enterobacter group. The isolates were characterized by determining their metabolic characteristics, carbohydrate utilization pattern, antibiotic sensitivity, plant growth promontory attributes, and molecular characteristics. They were also tested for their ability to promote plant growth. A significant number of isolates were found to be able to produce IAA and solubilize phosphorus. Among molecular characteristics, the nifH gene, which is responsible for the production of nitrogenase reductase, a key enzyme for biological nitrogen fixation, was detected by using a universal degenerate primer. The genomic fingerprinting was conducted by RAPD method to construct a dendrogram that helped to better understand the relationship between the isolates. This study establishes that a significant percentage of the endophytic bacteria found in the sugarcane stem and root in Iswardi area are potential nitrogen fixer and plant growth promoter. Therefore, they have the feasibility to be used as bio-fertilizer.

II

List of Tables

Table No.

Title of the Table

Page No.

1.1

Biological nitrogen fixation by diazotrophic endophytic bacteria

6

2.1

Sample code given to each isolate according to the site of isolation

18

2.2

Antibiotic disks a used to perform antibiotic sensitivity test

24

2.3

Primer sequences for the amplification of 16S rDNA of the isolates

25

2.4

PCR method for amplification of 16S DNA of the isolates

26

2.5

Primer sequences for the amplification of nifH gene region of the isolates

26

2.6

PCR method for the amplification of nifH gene region of the isolates

27

2.8

PCR condition for RAPD assay

28

3.1

Cell morphology and Gram’s characteristics of the isolates

31

3.2

Growth period and colony characteristics of the isolates

32

3.3

Metabolic characteristics of the isolates

33

3.4

Carbohydrate utilization pattern of the isolates

35

3.5

Result of phosphate solubilization and IAA production test

37

3.6

Antibiotic sensitivity profile of the isolates

38

3.7

Identification of the isolates

43

III

List of Figure

Figure No.

Title of the Figure

Page No.

1.1

Multiple applications of diazotrophic (N2 fixing) endophytic

4

bacteria in various fields, including agricultural practices, industries and environment (Modified from Hardoim et al., 2008) 1.2

Simplified reaction catalysed by nitrogenase in diazotrophs

7

1.3

A. diazotrophicus-like cells inhabiting cells of sugarcane

14

stems. The scanning electron micrograph shows sections of 5month-old plants grown with low N-fertilization 3.1

Isolate BSRI 1 under light microscope after gram staining

32

3.2

Growth of individual colonies of isolate Isd 8 on LB agar plate

33

3.3

Positive and negative results for different biochemichal tests

34

3.4

Different results of carbohydrate (fructose) fermentation test

35

3.5

A dendrogram constructed according to the carbohydrate

36

fermentation capability of the isolates 3.6

Positive and negative results of P-solubilization and IAA

37

production test 3.7

Inhibition zone observed as an effect of diffusion of different

38

antibiotic from antibiotic disks on Isd 6 and Isd 3 3.8

Agarose gel electrophoresis image of 16S rDNA amplification

39

products visualized in gel documentation system 3.9

Gel electrophoresis image of the amplified nifH gene products

40

3.10

Randomly amplified bands for RAPD assay visualized under

41

UV light in gel-doc 3.11

A dendrogram constructed using Jaccard similarity coefficient according to the band size and number recorded from RAPD assay

IV

42

Abbreviations

%

Percentage



Less than equal



Greater than equal

°C

Degree Centigrade

v/v

Volume per volume

µg

Microgram

µl

Microliter

g

Gram

M

Molar

ml

Milliliter

mm

Millimeter

mM

Millimolar

pmol

Picomole

FAO

Food and Agriculture Organization of the United Nations

US$

United States Dollar

N

Nitrogen

BNF

Biological Nitrogen Fixation

ATP

Adenosine triphosphate

DNA

Deoxyribonucleic Acid

BSRI

Bangladesh Sugarcane Research Institute

rRNA

Ribosomal Ribonucleic Acid

RAPD

Random Amplified Polymorphic DNA

IAA

Indole Acetic Acid

DEPC

Diethylpyrocarbonate

V

Chapter 1 Introduction and Literature Review

Introduction and Literature Review

1.1 General Introduction:

Nitrogen is an essential plant nutrient. It is the nutrient that is most commonly deficient, contributing to reduced agricultural yields throughout the world. Molecular nitrogen or dinitrogen (N2) makes up four-fifths of the atmosphere, but is metabolically unavailable directly to higher plants or animals. It is available to some microorganisms through biological nitrogen fixation (BNF) in which atmospheric nitrogen is converted to ammonia by the enzyme nitrogenase. According to statistics by FAO (2001), about 42 million tons of fertilizer N is being used annually on a global scale for the production of three major cereal crops. Crop plants are able to use about 50% of the applied fertilizer N, while 25% is lost from the soil–plant system through leaching, volatilization, denitrification and due to many other factors causing not only an annual economic loss of US$ 3 billion but also cause pollution to the environment. Some of the adverse environmental effects of excessive use of nitrogenous fertilizers are: (i)

metheamoglobinemia in infants due to NO3 and NO2 in waters and food,

(ii)

cancer due to secondary amines,

(iii)

respiratory illness due to NO3, aerosols, NO2 and HNO3,

(iv)

eutrophication due to N in surface water,

(v)

material and ecosystem damage due to HNO3 in rainwater,

(vi)

plant toxicity due to high levels of NO2 and NH4 in soils, and

(vii)

excessive plant growth due to more available N, depletion of stratospheric ozone due to NO and N2O.

If a BNF system could be assembled in the non-legume plants, it could increase the potential for nitrogen supply because fixed nitrogen would be available to the plants directly, with little or no loss. Such a system could also enhance resource conservation and environmental security, besides freeing farmers from the economic burden of purchasing fertilizer nitrogen for crop production. Thus, a significant reduction, in the relative use of fertilizer N can be achieved directly through an effective associative system with some of the characteristics of legume symbiosis. Recently, several approaches using techniques developed in the area of biotechnology have raised new hopes that success in this secondary objective may yet be realized. It is the author’s opinion that there are now sound reasons to anticipate that at least some non-leguminous 2|P a ge

Introduction and Literature Review

field crops may also become independent of soil nitrogen. This study approaches to isolate, characterize and identify the diazotrophic (N2 - fixing) bacteria that have the potential to act as a supplier of fixed nitrogen compound for increased plant growth.

Sugarcane is one of the major cash crops of Bangladesh. It is widely produced for both raw consumption and refined sugar production. There is also a great potential of using sugarcane as raw material for the production of bio-fuel, as done in many North American countries. In our country, nitrogen based chemical fertilizers are widely used in fields, which in terms can cause serious damage to our environment. Therefore, the use of naturally occurring diazotrophs of sugarcane as bio-fertilizer can prove to be a revolutionary idea for our agriculture and environment.

1.2 Literature review:

1.2.1 Nitrogen fixation by Diazotrophs

Biological nitrogen fixation is considered to be the most potential way to provide fixed form of nitrogen to the plants. However, nitrogen fixation is performed solely by prokaryotes (bacteria and cyanobacteria) and archeans. The diazotrophic bacteria are involved in the fixation process, in which these bacteria either in the free living form or in symbiosis can covert the atmospheric nitrogen into NH3 with the help of nitrogenase enzyme. Nodulated legumes with endosymbiosis with Rhizobia are among the most prominent nitrogen fixing system in agriculture. Although most of the biologically fixed nitrogen made available to the plants is contributed by Rhizobium sp. and cyanobacteria, their use is restricted only to certain plant species. Applications of plant growth promoting endophytic bacteria are being considered as a potential biofertilizer in recent years (Bhattacharjee et al., 2008; Akhtar and Siddiqui, 2010). This has driven intensive research towards in-depth characterization and better understanding of endophytic diazotrophic bacteria isolated from various plant species. Any bacterium could be considered as an endophytic diazotroph if: (i) it can be isolated from the surface of disinfected plant tissue or extracted inside the plants, (ii) it proves to be located inside the plant, either intra- or inter-cellularly by in-situ identification, and (iii) it fix nitrogen, as demonstrated by acetylene reduction and/or 15 N3|P a ge

Introduction and Literature Review

enrichment. This definition includes internal colonists with apparently neutral or saprophytic behavior as well as symbionts (Hartmann et al., 2000). Endophytic bacteria are better than their rhizospheric and rhizoplanic counterparts in terms of benefiting their host through nitrogen fixation as they can provide fixed nitrogen directly to their host (Cocking, 2003). As low partial oxygen pressure is necessary for the expression of the O2 sensitive enzyme, nitrogenase, endosphere of plant root is more amenable for N2 - fixation reaction. Moreover, endophytic bacteria are less vulnerable to competition with other soil microbes for scarce resources and remain protected to various abiotic and biotic stresses (Reinhold-Hurek and Hurek, 1998). In addition to diazotrophy, endophytic bacteria may enhance plant growth through one or more mechanisms which include phytohormone production, siderophore production, induced systemic tolerance and biocontrol potential. The applications of diazotrophic endophytes in various fields have been depicted in the Figure 1.1. The intimate relationship of endophytic bacteria with plant can be utilized in developing efficient biofertilizer and biocontrol agents for attaining sustainable agriculture (Sevilla and Kennedy, 2000).

Figure 1.1: Multiple applications of diazotrophic (N 2 fixing) endophytic bacteria in various fields, including agricultural practices, industries and environment (Modified from Hardoim et al., 2008). 4|P a ge

Introduction and Literature Review

1.2.2 Nitrogen Fixation by Endophytic Bacteria

In recent years, application of endophytic bacterial inoculants supplying N requirement have drawn attention for increasing plant yield in sustainable manner efficiently to the various crop plants. Percent contribution of plant nitrogen as a result of biological N2 - fixation by endophytic bacteria has been summarized in Table 1.1. Some of the promising endophytic biofertilizers include the members of Azoarcus, Achromobacter, Burkholderia, Gluconoacetobacter, Herbaspirillum, Klebsiella and Serratia (Rothballer et al., 2008; Franche et al., 2009). The efficient N supply by endophytic diazotrophic bacteria in sugarcane and kallar grass suggests the possible avenues of biological nitrogen fixation in interior niches of plants. In addition, bacteria isolated from non-leguminous plants like rice, wheat, maize, sorghum also fix the Endophytic nitrogen-Fixing Bacteria as Biofertilizer 187 N in endophytic manner. It is evident from the reports that the Gluconoacetobacter diazotrophicus (formally known as Acetobacter diazotrophicus) is the main contributor of endophytic biological nitrogen fixation in sugarcane, and it has the ability to fix the N approximately 150 Kg N ha-1 year

−1

(Dobereiner et al., 1993;

Muthukumarasamy et al., 2005). Azoarcus is recognized as another potential N2 - fixing obligate endophytic diazotroph. It dwells in the roots of kallar grass, and increased the hay yield upto 20– 40 t ha −1 year −1 (Hurek and Reinhold-Hurek, 2003). In addition, many energy plants (C4 plants) like Miscanthus sacchariflorus, Spartina pectinata and Penisettum purpureum have been found to harbor bacterial population, which have the potential to support the N nutrition of the plant (Kirchhof et al., 1997) . In a study, Herbaspirillum sp., inoculated into rice seedlings maintained in N-free Hoagland solution containing 15 N-labeled N, showed 15 N dilution amounting up to 40% increase in total N of plant (Baldani et al., 2000) . Growth stimulation of wheat, corn, radish, mustard and certain varieties of rice shoots following seed inoculation with a strain of Rhizobium leguminosarum bv trifolii in pot experiment has also been reported (Hoflich et al., 1995; Webster et al., 1997). These investigations suggest that endophytic diazotrophs have a considerable potential to increase the productivity of non-legumes including important cash crop plants.

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Introduction and Literature Review

Table 1.1 Biological nitrogen fixation by diazotrophic endophytic bacteria Endophytic bacteria

Associated plant

N derived from air

References

(%) Burkholderia

Rice

31

Baldani et al. (2000)

Herbaspirillum

Rice

19–47

Mirza et al. (2000)

Rhizobium

Rice

19–28

Biswas et al. (2000) and Yanni et al. (2001)

K. pneumoniae 324

Rice

42

Iniguez et al. (2004)

B. vietnamiensis

Rice

40–42

Govindarajan et al. (2008)

Beijerinckia, Bacillus, Klebsiella, Enterobacter, Erwinia Azospirillum, Herbaspirillum and Gluconaacetobacter Azospirillum

Sugarcane

18

Abeysingha and Weerarathne (2010)

Rice

9.2–27.7

de Salamone et al. (2010)

Pseudomonas, Stenotrophomonas, Xanthomonas, Acinetobacter, Rhanella, Enterobacter, Pantoea, Shinella, Agrobacterium and Achromobacter G. diazotrophicus, H.

Sugarcane

41.2–50.3

Taule et al. (2012)

Elephant grass

5.4–5.5

de Morais et al. (2012)

Microbacterium sp.

Sugarcane

5.4–6

Lin et al. (2012)

G. diazotrophicus, H.

Sugarcane

29–74

Urquiaga et al. (2012)

leguminosarum bv. trifolii

serpedicae and H. frisingense

seropedicae, H. rubrisubalbicans Burkholderia sp.

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Introduction and Literature Review

1.2.3 Overview of Biological Nitrogen Fixation (BNF):

Nitrogen fixation by various microorganisms involves two key features. These features are described below.

1.2.3.1 The nitrogenase reaction

Chemically, BNF is essentially the conversion of dinitrogen (N 2) to ammonia, catalysed by the enzyme nitrogenase. The reaction catalyzed can be represented as in Figure 1.2:

Figure 1.2: Simplified reaction catalyzed by nitrogenase in diazotrophs.

Of course, the process of N2 fixation is much more complex than this, but the biochemical reaction in Figure 1.2 contains the key ingredients and points to the major requirements. It indicates the dual needs for reducing potential and the substantial energy requirement in the form of ATP. It also suggests the need for a mechanism for ammonia utilization that will simultaneously neutralize the alkalinity generated.

1.2.3.2 The energy requirement

BNF requires energy. The free-living diazotrophs require a chemical energy source if nonphotosynthetic, whereas the photosynthetic diazotrophs utilize light energy. The free-living diazotrophs contribute little fixed nitrogen to agricultural crops. Associative nitrogen-fixing microorganisms are those diazotrophs that live in close proximity to plant roots (that is, in the rhizosphere or within plants) and can obtain energy materials from the plants. They may make a modest contribution of fixed nitrogen to agriculture and forestry. The symbiotic relationship 7|P a ge

Introduction and Literature Review

between diazotrophs called Rhizobia and legumes can provide large amounts of nitrogen to the plant and can have a significant impact on agriculture. The symbiosis between legumes and the nitrogen-fixing Rhizobia occurs within nodules mainly on the root and in a few cases on the stem. A similar symbiosis occurs between a number of woody plant species and the diazotrophic actinomycete Frankia. The plant supplies energy materials to the diazotrophs, which in turn reduce atmospheric nitrogen to ammonia. This ammonia is transferred from the bacteria to the plant to meet the plant’s nutritional nitrogen needs for the synthesis of proteins, enzymes, nucleic acids and chlorophyll. A number of studies have attempted to estimate energy requirements for BNF by comparing the rate of growth of legumes on nitrate with their growth on nitrogen. However, the energy requirement for N2 fixation is actually almost identical to that required for nitrate assimilation (Kennedy, 1988), the other main source of nitrogen for most field crops, except rice. Another important experimental approach has been the use of efflux of carbon dioxide from legumes growing with either nitrogen source (Silsbury, 1977). In general, greater carbon dioxide efflux has been observed on plants growing with dinitrogen than with nitrate (Schubert, 1982), suggesting an increased energy demand, as extra carbohydrate required. Unfortunately, none of these studies considered the fact that nitrate-grown plants produce bicarbonate rather than carbon dioxide in assimilating nitrate (Kennedy, 1988). When this difference in the metabolism of the two nitrogen sources is taken into account, the apparent difference in energy consumption could disappear. On the other hand, if the diazotrophic organism in an engineered system could have direct access to ample energy supply from the plant, such negative opinions about proposals to engineer non-legumes to fix dinitrogen based on energy requirement could be questioned. Substantial infection of the root cortex by organisms like Azospirillum would be a case in point. For such pessimism to be sustained, one would need to dismiss the positive role of leguminous nodules in fixing N2, which is absurd. To be effective, however, not only penetration but also adequate colonization with the diazotrophs and some kind of symbiosis would be required (Kennedy, 1992).

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Introduction and Literature Review

1.2.4 Nitrogen fixation and sustainable agriculture

By definition, biological N2 fixation is synonymous with sustainability. Advances in agricultural sustainability will require an increase in the utilization of BNF as a major source of nitrogen for plants. The process of BNF offers an economically attractive and ecologically sound means of reducing external nitrogen input and improving the quality and quantity of internal resources. Clearly, it is not realistic to consider sustainable agriculture on a broad scale in the absence of BNF; research is needed to optimize the contribution of BNF to sustainable agriculture. Nitrogen applied in fertilizers usually provides benefit to plants. However, if applied inefficiently, it can also have serious disadvantages in causing pollution. It is difficult to match nitrogen supply to actual requirements of a crop at a given ecosite and any excess may damage this or other ecosites. Excess reduced nitrogen (ammonium) in agricultural or forest ecosystems may lead to their acidification through the process of nitrification. In agricultural ecosystems, however, BNF fixation may usually be expected not to exceed the actual nitrogen requirements of an ecosystem, thus being less likely than fertilizers to cause pollution. Moreover, in the wake of deepening energy crisis and decontrol of fertilizer prices, the crucial role of organics and biofertilizers in sustaining soil productivity and ensuring food security must be realized.

As a society, we must find ways to reduce dependency on fossil fuels for economic, environmental and geopolitical reasons. Development of sources of renewable energy will be particularly important for agriculture. BNF will be a major component in the improvement of agricultural sustainability.

1.2.5 Non-Symbiotic Nitrogen Fixers The use of diazotrophic bacteria as ‘biofertilizer’ is particularly important in agricultural systems where fertilizer inputs are impractical, undesirable, or not possible. Experiments on inoculation of crops with often resulted in enhanced plant growth or nitrogen content under environmental conditions, improved nutrient assimilation, alter root size and function. Some of the known diazotrophs that have been subjected of various studies over the years are discussed below.

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Introduction and Literature Review

1.2.5.1 Azospirillum Bacteria of the genus Azospirillum are associative nitrogen (N2)-fixing rhizobacteria that are found in close association with plant roots. They are able to exert beneficial effects on plant growth and yield of many agronomic crops under a variety of environmental and soil conditions. In the case of rice, A. lipoferum and A. brasilense have been isolated from the roots and stems (Ladha et al., 1982) and A. amazonense has been isolated from the roots (Pereira et al., 1988). Microscopical evidence as to the endophytic nature of Azospirillum in rice has been presented (Baldan et al., 1993) and the colonization of 2,4-D-induced para-nodules by an ammoniumexcreting mutant of A. brasilense has been reported (Christiansen-Weniger, 1997). An ammonium-excreting mutant of A. brasilense (Wa3) promoted better growth of wheat plants compared to wild type (Christiansen-Weniger, 1991) and another strain (C3) was able to transfer the nitrogen fixed directly to the maize plant (Christiansen-Weniger, 1994) and the amount transferred was increased in para-nodulating plants obtained by treatment with 2,4-D. In maize plants treated with 2,4-D and Azospirillum, cob weight, 100-grain weight and grain cob–1 were found to be maximum and significantly higher (Saikia et al., 2004). Despite these different mechanisms exerted by facultative endophytic diazotrophs in association with graminaceous plants, increases in yield in the range of 5–30% have been observed in several inoculation experiments with Azospirillum (Okon and Gonzalez, 1994).

1.2.5.2 Herbaspirillum

Herbaspirillum is an endophytic nitrogen-fixing organism capable of colonizing intercellular spaces of maize, rice, sorghum and sugarcane. It has been found in the roots, stems and leaves of graminaceous plants. Colonization of rice plants by selected strains of H. seropedicae showed that the bacteria first colonize the epidermal cells of the root surface or secondary root emergence. H. rubrisubalbicans, another species of Herbaspirillum, was isolated from rice plants. Occurrence of H. rubrisubalbicans in rice plants may not be a surprise (Baldani et al., 1993) because inoculation of rice seedlings grown axenically with strain M4 increased nitrogen accumulation in the plant by about 30%.

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Introduction and Literature Review

1.2.5.3 Azoarcus

This obligate endophytic diazotroph has been isolated from kallar grass (Leptochloa fusca) grown in salinesodic soils in Pakistan. The strain BH 72 is capable of invading roots of the original hosts as well as rice plants, infecting the cortex region and is capable of expressing nitrogenase genes in the aerenchyma of rice roots (Egener, et al., 1999). Azoarcus is also able to colonize the interior of sorghum plants by means of its cellulotic enzymes (Stein et al., 1997).

1.2.5.4 Rhizobium

Soil bacteria of the genera Rhizobium, Azorhizobium and Bradyrhizobium (collectively referred to as Rhizobia) interact with leguminous plants to form N2-fixing nodules. Although not freeliving, the possibilities of extending the host range of Rhizobia to non-legumes were encouraged by the discoveries that Rhizobium forms nodules in Parasponia (Trinick, 1979) and that Rhizobium parasponium RP 501 and Bradyrhizobium CP 283 induce nodulation in oilseed rape (Cocking et al., 1992). In addition, recent evidence shows that Rhizobia are capable of colonizing the roots of non-legumes wheat, maize and barley, but the basis of the association between non-legumes and Rhizobia is so far not known. Rhizobia were found to have the ability to attach themselves to rice root hairs, elicite deformation of rice root hairs and to form nodulelike structures. Certain strains of R. leguminosarum bv. Trifolii could colonize intercellularly, multiply and migrate inside growing lateral roots (Prayitno, et al., 1999). The stimulation of lateral root development and colonization of lateral root cracks and xylem of rice roots by A. caulinodans (ORS 571) has been reported (Gopalaswamy and Kannaiyan, 2000). In addition to forming nodules after crack entry invasion of emerging lateral roots, it is able to fix nitrogen in the free-living state up to 3% (v/v) oxygen and without differentiation into bacteriods (Kitts and Ludwig, 1994). The maize plants inoculated with A. caulinodans alone had higher NPK content in grains and stover, and combination of A. caulinodans with auxin significantly enhanced the NPK content in the grains and stover (Saikia et al., 2003). Nodulated and Azorhizobium-treated plants also showed higher chlorophyll content in the leaf and enhanced nitrate reductase activity, leading to higher yield compared to the control plants (non-nodulated) (Saikia, et al., 2006).

11 | P a g e

Introduction and Literature Review

1.2.6 Endophytic Nitrogen-Fixing Bacteria as Biofertilizer

In this chapter, we review application, properties, ecology, and advances in biology of nitrogen fixing bacteria with reference to endophytic bacteria that colonize the interior of plant without exerting any substantive harm to their host plant. Nitrogen- fixing endophytic bacteria have edge over its rhizospheric counterparts because, being sheltered inside plant tissues, they face less competition and can make available the fixed nitrogen directly to plants. Moreover, the partial pressure of oxygen inside the plant tissue is more acquiescent for efficient nitrogen fixation. Nitrogen fixing endophytic bacteria have been isolated from several plant species and found to contribute up to 47% of nitrogen derived from air, which in turn enhance plant growth. Nitrogen fixing ability of bacteria can be evaluated by total nitrogen difference method, acetylene reduction assay, analysis of nitrogen solutes in xylem and other plant parts and N-Labeling Methods. Furthermore, molecular approaches such as amplification, analysis of nitrogen fixing genes (nif genes), and qualitative and quantitative estimation of their products can be used for evaluation of nitrogen fixing ability of the bacteria.

In addition to nitrogen- fixation ability, these bacteria can influence plant growth through one or more properties. These include production of phytohormones, siderophores, induced systemic tolerance through production of 1-aminocyclopropane- 1-carboxylase deaminase, induced systemic resistance and antagonistic activities. The make-up of endophytic bacterial communities depends on various factors such as soil type, soil composition, soil environment, plant genotype and physiological status, bacterial colonization traits, and agricultural management regimes. Colonization and abundance of different bacterial species varies widely with host plants. Endophytic bacterial community can be analyzed employing stable isotope probing as well as various modern molecular approaches which are based on analysis of 16S ribosomal deoxyribonucleic acid (DNA), gene encoding products for nitrogen fixation and repetitive DNAs.

Moreover, metagenomic approaches allow estimation and analysis of nonculturable bacteria at genomic as well as functional genomic level. Colonization process of an endophytic bacterium involves various steps which include migration towards root surface, attachment and 12 | P a g e

Introduction and Literature Review

microcolony formation on plant surface, distribution along root and growth and survival of the population inside plant tissue. Ongoing progress towards in-depth analysis of genomic and whole protein profile of some of the potential endophytic bacteria such as Azoarcus sp., Gluconoacetobacter diazotrophicus, Herbaspirillum seropedicae, Serratia marcesens can help understand mechanism involved in plant-endophyte interaction which in turn will be deterministic in use of suitable formulations of endophytic bacteria to be used as biofertilizer for sustainable agriculture.

1.2.7 Endophytic Nitrogen-Fixing Bacteria of Sugarcane

Sugarcane (Saccharum officinarum L.) is a major crop in the tropics, where it is grown for the production of biofuel as well as food. In Brazil, ethanol produced from sugarcane has been used extensively to replace petroleum fuel in cars. It was previously suggested that sugarcane could benefit from nitrogen fixing bacteria (Döbereiner et al., 1972; Boddey et al., 2003), and Acetobacter diazotrophicus (now renamed Gluconacetobacter diazotrophicus) was isolated from sugarcane (Boddey et al. 2003). Figure 1.3 shows how the bacteria inhibit in plant stem (Luis et al., 1998). Many species of endophytic nitrogen fixing bacteria have since been isolated from sugarcane (Baldani et al., 1992; Dong et al., 1994; Loiret et al., 2004) and other plants, e.g., rice, kallar grass and maize, and these bacteria supply fixed nitrogen (N) to their hosts (Hallmann et al., 1997; James 2000; Baldani et al., 2002). Other nitrogen fixing bacteria, including Azospirillum spp., Herbaspirillum spp., Burkholderia spp., Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae and Pantoea sp., were reported to have been isolated from the roots, stems and leaves of sugarcane (Govindarajan et al., 2007, 2008; Mendes et al., 2007). Symbiotic associations between sugarcane and its endophytic nitrogen fixing bacteria provide mutual benefits such as a combined N (NH3) supply to the plant and photosynthesis, refuge and transmission to the bacteria. Co-evolution of these partners to engage in symbiosis would be expected in advantageous environments.

13 | P a g e

Introduction and Literature Review

Figure 1.3: A. diazotrophicus-like cells inhabiting cells of sugarcane stems. The scanning electron micrograph shows sections of 5-month-old plants grown with low N-fertilization. (A) Section of sucrose storage parenchymatous cells located near to the stem cortex. (B) Section of a vascular bundle, a tracheary element (black asterisk) is surrounded by parenchyma cells (white asterisks). (Luis et al., 1998)

1.3 Bangladesh perspective:

Bangladesh, an agriculture based country, is in dire need of ways to increase food production as well as reduce environmental pollution. Traditionally used chemical fertilizers are not only costly but also harmful for the environment. Excessive chemical fertilizer from the farm field are washed out by rain and deposited in important water bodies, thus causing pollution and becoming a health hazard. The possibility of supplying the nitrogen demand of crops through BNF offers an advantageous way of protecting the environment and of reducing costs. Biofertilizers can be an attractive alternative of chemical fertilizer for agricultural use as it is echo friendly, cost limiting and easy to apply. The present study is a concern of Bangladesh Sugarcane Research Institute (BSRI) with the aim to mass produce nitrogen biofertilizer for sugarcane crop. In this era of life science development, this study could establish a ground work for further research in the agricultural industry of Bangladesh.

14 | P a g e

Introduction and Literature Review

1.4 Aims and objectives of the study:

The main goal of the present study was to isolate and identify the most abundant root/rizhosphere microbes and search for potential diazotrophs, and to better understand root and rhizosphere microbial assemblages and their functional association with sugarcane in relation to N assimilation and plant growth. The principle aims and objectives of this study are as follows – 1. Isolation of nitrogen-fixing bacteria from sugarcane root, stem and associated rhizosphere. 2. Determination of the metabolic characteristics of isolated microorganisms by various microbiological and biochemical method. 3. Presumptive identification of isolated microorganisms based on the morphological and metabolic characteristics. 4. Determination of growth promoting capabilities of the isolates e.g. IAA production, Phosphate solubilization etc. 5. Detection of Nitrogenase (nif gene) encoding gene by molecular methods. 6. Determination of the 16S rRNA genes of the isolates. 7. Genomic fingerprinting of the isolates by random amplified polymorphic DNA (RAPD) fingerprinting method. 8. Isolation of a suitable strain of microorganism for mass production of N-biofertilizer.

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Chapter 2 Materials and Methods

Materials and Methods

This chapter describes the general methods, apparatus used and special techniques adopted for the present study. The composition of media and other reagents are shown in the appendix. 2.1 Laboratory equipments and glassware: This study was performed in the Environmental Microbiology laboratory of the Department of Microbiology, University of Dhaka. All glassware was washed in concentrated sulfuric acid (1 M) and was washed under tap water afterwards. All glassware and micropipette tips were sterilized by autoclaving them under high pressure at 121° C for 15 minutes. Petridishes were further sterilized by heating them at 180°C for 1 hour in a hot air oven before each use. 2.2 Culture Media: The culture media used in this study includes different type of complex, selective and chemically defined media. These culture media are Luria Bertani agar & broth, Semi-solid LGIM medium (modified from Cavalcante and Dobereiner, 1988), Burk’s N-fee medium, Starch agar, Skim milk agar, Phenol red Carbohydrate broth, Sulphite Indole Motility (SIM) medium, Nitrate broth, Peptone water, Simmon’s Citrate agar, Nutrient Gelatin, Yeast extract tryptone broth, Carboxy methyl Cellulose (CMC) agar, Pikovaskya medium, and Yeast Peptone Mannitol broth.

2.3 Isolation of Endophytes from Sugarcane (genus Saccharum) root and stem: 2.3.1 Collection of plant material:

Sugarcane samples were collected from Bangladesh Sugarcane Research Institute (BSRI), Ishwardi. Endophytes were isolated from the root and stem portions of the plant samples (Dobereiner et al. 1988). Finally, 12 isolates were selected for further studies and were assigned the respective codes (Table 2.1). 17 | P a g e

Materials and Methods

Table 2.1 Sample code given to each isolate according to the site of isolation

Sample Code

In planta site

BSRI 1

Root

BSRI 2

Stem

BSRI 3

Stem

BSRI 4

Stem

Isd 1

Root

Isd 2

Root

Isd 3

Root

Isd 4

Root

Isd 5

Stem

Isd 6

Stem

Isd 7

Stem

Isd 8

Stem

2.3.2 Surface Sterilization of root and stem sample: Plant portions were washed under tapwater to get rid of the dirt. The plant portions were then soaked in 95% Ethanol for 5-10 seconds. The sample were then transferred into 3% solution of H2O2 and kept there for 3-5 minutes. The samples were then washed with sterile water for about 5 times to completely wash away any residual H2O2.

2.3.3 Isolation of endophytes from sugarcane plant material: The isolation of endophytes for this study was done by the procedure described by Dobereiner et al. (1988). The stem and root samples were individually cut into small pieces and crushed using mortar and pestle, then they were soaked in sterile water and mixed thoroughly. 1 ml of the 18 | P a g e

Materials and Methods

suspension was taken in a vial containing 9 ml sterile normal saline (0.85% NaCl) and a serial dilution (from a series of 10-2 to 10-7 dilutions) of the suspensions was made and formed a series of 10-2 to 10-7 dilutions. 0.1 ml of each dilution was spread into Burk’s N-free agar plates and was kept in 30°C incubator. After 3 days of incubation at 30°C, colonies were picked from the media and were subcultured into semi-solid LGIM media. The colonies that grow in both Burk’s media (neutral pH) and LGIM media (pH 5.5) were separately subcultured into Luria Bertani agar plates.

2.3.4 Initial stocking of Isolates: The 12 isolates were initially stocked by standard procedure of glycerol stocking. A total number of 12 sterile eppendorf tubes were taken and labeled. In each tube, 500 μl of 50% sterile glycerol solution was poured. Actively growing LB broth culture of each isolate (500 μl) was poured in each tube. The tubes were thoroughly mixed in a vortex mixer. All 12 tubes were kept in deep freezer at a temperature of -40°C.

2.3.5 Maintenance of working culture: The isolates were subcultured in LB agar plates at 15 days interval. Working cultures were kept in 4°C. It was observed that the isolates could be kept alive in 4°C for about month.

2.4 Characterization of the Isolates: All the isolates were characterized by determining their morphological, biochemical and molecular properties.

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Materials and Methods

2.4.1 Morphological characteristics: Individual colonies of each isolates were obtained by streaking them on LB agar plates. The plates were incubated at 30°C for 3 days. After sufficient growth, colonies were observed to determine their morphological characteristics. Gram’s staining procedure was performed to observe the cell morphology. Isolates were heat fixed on glass slides, stained by crystal violet, followed by gram’s iodine solution. After washing under tap water, the isolates were destained with 70% ethanol solution and restained with safranin. The glass slides were then washed under running tap water and observed under light microscope. This microscopy determined their gram reaction and cell morphology. The endophytic isolates were also observed for exopolysaccharide production. If the bacterial growth on LB agar plate showed gummy, thick or milky creamy appearance, it was usually due to the production of exopolysaccharide which indicates the possibility of biofilm formation.

2.4.2 Biochemical & metabolic characterization: 2.4.2.1 Cellulase activity Freshly grown isolated cultures were streaked in a zigzag manner on nutrient agar plates supplemented with 0.2 % carboxy methyl cellulose (CMC). The plates were then incubated at 30 °C for 3 days. After sufficient growth, the plates were overlaid with Congo-red (1 μg/ml) solution for 15 min. The plate surface was washed with 1 M NaCl, and was observed for clear zone around the growth. The formation of clear zone indicated cellulose activity.

2.4.2.2 Gelatin hydrolysis Nutrient gelatin medium was inoculated with a loopful of actively growing bacterial culture and incubated for 3 days at 30 °C. Control tubes, with no bacteria in them, solidified when placed in ice. If any isolate can hydrolyze gelatin, the media will appear liquid after placing them in ice for about 20 minutes. The liquid state of the media indicates positive result, whereas solidification after chilling indicates negative result. 20 | P a g e

Materials and Methods

2.4.2.3 Starch hydrolysis Nutrient agar plates supplemented with 0.3 % soluble starch were inoculated with actively growing isolate cultures in a zigzag fashion. The plates were incubated for 3 days at 30 °C. When flooded with Gram’s iodine solution, a clear zone around the growth line indicates starch hydrolysis.

2.4.2.4 Casein hydrolysis Skim milk agar plates were streaked with actively growing cultures and incubated for 3 days at 30 °C. Clear zones around the inoculation spots indicate the ability of the isolate to hydrolyze milk casein.

2.4.2.5 Catalase activity Yeast extract tryptone broth tubes, inoculated with actively growing culture of the isolates, were incubated for 3 days at 30 °C. Catalase activity was observed by adding few drops of 3 % H2O2 solution to the broth cultures, kept on the glass slides. Formation of oxygen bubbles confirms catalase activity.

2.4.2.6 Nitrate reduction Nitrate broth with inverted Durham tube were inoculated with actively growing culture(s), and incubated for 2–3 days at 30 °C for the observation of gas production. After 3 days of incubation the inverted tubes were observed for bubble formation. If no bubble were to be observed, 5 drops of sulphalinic acid and α-napthylamine dissolved in acetic acid was added to the broth. Development of red color indicated partial nitrate reduction activity. Formation of bubbles in inverted tube indicated complete nitrate reduction activity.

21 | P a g e

Materials and Methods

2.4.2.7 Production of H2S SIM semi-solid agar media were inoculated by stab method in test tubes with actively growing culture. The tubes were incubated at 30°C for 3 days. Blackening along the growth of bacteria indicates the production of H2S. H2S negative isolates can grow but do not produce black color.

2.4.2.8 Indole production Yeast extract tryptophan broth was inoculated with actively growing bacterial culture. The tubes were incubated at 30°C for 3 days. Kovac’s reagent was added by 2-3 drops in the broth after sufficient growth. The development of cherry red color indicated the production of indole. Otherwise the reagent remains dark yellow.

2.4.2.9 Citrate utilization Simmon’s citrate agar slants were steaked with freshly prepared bacterial culture in a zigzag manner. The tubes were incubated for 3 days at 30°C. The development of deep blue color indicated the bacteria’s ability to utilize citrate.

2.4.3 Carbohydrate utilization test: The endophytes were tested for their fermentation ability with several carbohydrates. Pheol red Carbohydrate broths were prepared separately with sucrose, lactose, mannitol, dextrose, xylose and fructose. Inverted durhum’s tubes were place in each tube to observe gas production. The tubes were inoculate with actively growing bacterial culture and incubated at 30°C for 3 days. The development of yellow color of the broth indicates the low pH environment created by the bacteria’s fermentation activity. If the bacteria formed gas, bubbles would be observed in the Durhum’s tubes. The capability of fermenting different carbohydrates of a standard Gluconacetobacter diazotrophicus (MTCC-1224) was adapted from the study described by Singh et al., 2013. This data was used to construct a dendrogram that showed the carbohydrate utilization pattern and the relationship of the isolates with the standard culture. 22 | P a g e

Materials and Methods

2.4.4 Study of Plant growth promoting attributes: The following tests were performed with the test isolates to observe their plant growth promoting capabilities.

2.4.4.1 Phosphate solubilization Actively growing bacterial culture were spot inoculated on Pikovaskya medium agar plates, and incubated at 30 °C for 3 days. Positive isolates developed transparent zone against white opaque background.

2.4.4.2 Indole acetic acid (IAA) production Tubes of Yeast Peptone Mannitol broth (5 ml) with tryptophan (100 μg/ml) and its control were inoculated at 30 °C and 100 rpm shaking. The next day, cultures were centrifuged at 8,000 rpm for 10 min. Two millilitres of freshly prepared Salkowski reagent was added to 1 ml of culture supernatant. The reaction mixture was incubated at 30 °C for 25 min. Development of pink color indicated the production of IAA.

2.4.5 Antibiotic sensitivity test: Resistance and sensitivity of the isolates to different antibiotics was tested by the Kirby Bauer’s method (Barry and Throrns barry, 1985). Liquid cultures of each isolate were prepared and their turbidity was determined by photospectrometry. The isolates were then inoculated as a lawn on Muller-Hinton agar plates with sterile cotton swabs. After streaking, antibiotic disks were place on the surface of the agar. The antibiotics that were used in this test are listed in Table 2.2. The plates were incubated overnight at 30°C. After incubation, inhibition zones can be observed on the plates around the antibiotic disks. Diameter of these inhibition zones were measured and recorded. The sensitivity and resistance of the isolates were determined by interpreting these data with the reference table (Barry and Throrns barry, 1985).

23 | P a g e

Materials and Methods

Table 2.2 Antibiotic disks a used to perform antibiotic sensitivity test Name of Antibiotic

Concentration (μm)

Ceftriaxone

30

Tetracycline

30

Metronidazole

50

Amoxyclav

30

Gentamycin

10

Streptomycin

10

Penicillin G

10

Polymixin B

300

2.4.6 Molecular characterization of bacterial endophytes: 2.4.6.1 DNA extraction The genomic DNA template extraction for molecular characterization was performed by boil method (Maria et al., 2008). The procedure is described below: 

Enriched liquid culture of bacteria was prepared in LB broth, 1 ml of liquid culture was taken in 1.5 ml eppendorf tubes.



The tubes were centrifuged for 10 minute at 14000 rpm and supernatants were discarded.



Pellets were resuspended in 300 μl of DNase-RNase free distilled water (DEPC treated water) by a vortex mixer. 24 | P a g e

Materials and Methods



Tubes were again centrifuged at 14000 rpm for 10 minutes and supernatants were discarded.



Pellets were resuspended with 200 μl of DEPC treated water and the tubes were incubated at 100°C for 15 minutes on a heat block.



Tubes were immediately chilled on ice for 15 minutes. They were centrifuged for 5 minutes at 14000 rpm at 4°C.



In new sterile microfuge tubes, 200 μl of supernatant were transferred.



These tubes were incubated at 100°C for 5-10 minutes, immediately chilled on ice and stored at -20°C.

2.4.6.2 Amplification of 16S rDNA from genomic DNA Amplification of 16S rDNA from extracted DNA was performed using universal eubacterial primers. The master mix composition for this PCR is given in Table 5.27 in appendix section. The primer sequences and the PCR method are described in Table 2.3 and Table 2.5 respectively. The volume of genomic DNA template (extracted by the method described in section 2.4.6.1) in each PCR tube was 2.5 μl.

Table 2.3 Primer sequences for the amplification of 16S rDNA of the isolates Primer name

Primer sequence

Universal eubacterial F primer

5´-AGAGTTTGATCCTGGCTCAG-3´

Universal eubacterial R primer

5´-TACCTTGTTTTACGACTT-3´

25 | P a g e

Materials and Methods

Table 2.4 PCR method for amplification of 16S DNA of the isolates

40 cycles

Segments

Process

Temperature (°C)

Time (min)

Segment 1

Initial denaturation

94

7

Segment 2

Denaturation

94

1

Segment 3

Annealing

47

1

Segment 4

Extension

74

1

Final extension

72

10

Segment 5

After segment 1 was completed as described in the Table 2.4, segment 2, 3, and 4 were repeated for 40 cycles before the final segment, segment 5. After the program was completed, PCR amplicons were run on 1.5 % agarose gel and visualised under UV gel documentation system (VilberLourmat, France). 2.4.6.3 nifH amplification

nifH region with the size ranging from 360bp to 400bp (Saikia and Jain, 2007; Zehr and McReynolds, 1989) was amplified using degenerate primer sequences (Zehr and McReynolds, 1989), nifHf as forward nifHr as reverse primer. Master mix was prepared with the composition described in Table 5.27 in the appendix section, only the degenerate primer set was used instead of universal primer set. The primer sequences and PCR method are presented in Table 2.5 and Table 2.6 respectively. The volume of genomic DNA template (extracted by the method described in section 2.4.6.1) in each PCR tube was 2.5 μl. Table 2.5 Primer sequences for the amplification of nifH gene region of the isolates Primer name

Primer sequence

nifHf

5´-TGYGAYCCNAARGCNGA-3´

nifHr

5´-ADNGCCATCATYTCNCC-3´

(Where R = A or G, Y = C or T, D = A, G or T, N = A, C, G or T) 26 | P a g e

Materials and Methods

Table 2.6 PCR method for the amplification of nifH gene region of the isolates

35 cycles

Segments

Process

Temperature (°C)

Time (min)

Segment 1

Initial denaturation

95

10

Segment 2

Denaturation

94

1

Segment 3

Annealing

53

1

Segment 4

Extension

72

1.5

Final extension

72

10

Segment 5

After segment 1 was completed as described in Table 2.6, segment 2, 3, and 4 were repeated for 35 cycles before the final segment, segment 5. After the program was completed, PCR amplicons were run on 1.5 % agarose gel and visualised under UV gel documentation system (VilberLourmat, France).

2.4.6.4 Random Amplification of Polymorphic DNA assay Random fragments of the extracted bacterial DNA were amplified by using the 1283 F primer (Akopyanz et al., 1992; Toni et al., 2001). DNA was extracted as mentioned in section 2.4.6.1 and PCR was performed using forward primer 1283 F (Table 2.7) according to the program described in Table 2.8. The composition of master mix for this PCR is described in Table 5.28 in the appendix section. The volume of genomic DNA template in each PCR tube was 3 μl.

Table 2.7 Primer sequence for RAPD assay Primer name

Primer sequence

1283 F

5´-GCGATCCCCA-3´

27 | P a g e

Materials and Methods

Table 2.8 PCR condition for RAPD assay

40 cycles

Segments

Process

Temperature (°C)

Time (min)

Segment 1

Initial denaturation

94

4

Segment 2

Denaturation

94

1

Segment 3

Annealing

32

1

Segment 4

Extension

72

2

Final extension

72

5

Segment 5

After segment 1 was completed as described in Table 2.8, segment 2, 3, and 4 were repeated for 40 cycles before the final segment, segment 5. After the program was completed, PCR amplicons were run on 1.5 % agarose gel and visualised under UV gel documentation system (VilberLourmat, France).

2.4.7 Identification of isolates using universal method: A universal method of identifying bacteria was applied to identify the endophytic isolates (Barghouthi, 2011). The 16S rRNA gene of each isolates was amplified with the universal eubacterial primer set. The amplicons (1.5 kb) were purified using Thermo-Scientific GeneJET PCR Purification Kit for DNA sequencing. Purification was performed according to the user manual. According to the manufacturer’s instruction the PCR products were purified by following procedure: 

Binding Buffer and PCR product was poured in spin columns at 4:1 ratio.



The columns were centrifuged at room temperature at 10,000 × g for 1 minute.



Flow through was discarded and the spin columns were placed into the collection tubes.



Wash Buffer with added ethanol was added with a volume of 700 μL to the each column.



The columns were centrifuged at room temperature at 10,000 × g for 1 minute. Flow 28 | P a g e

Materials and Methods

through was discarded from the collection tubes and the columns were placed in tubes. 

The columns were centrifuged at maximum speed at room temperature for 2–3 minutes to remove any residual Wash Buffer. The collection tubes were discarded.



Then the columns were transferred to a clean 1.5 ml microcentrifuge tube and 50 μL of Elution Buffer (10 mM Tris-HCl, pH 8.5) was added to the center of each column.



The columns were incubated at room temperature for 1 minute.



The columns were centrifuged at maximum speed for 2 minutes.



The elution tube contained the purified PCR product. The columns were removed. The recovered elution volume was approximately 50 μL and purified DNA stored at -20 ºC.

The concentration of each amplicons was adjusted according to the manufacturer’s instruction. The amplicons were then sealed in eppendorf tube with a volume of 30 μl. These sealed tubes were sent for sequencing procedure. After sequencing was complete, sequences for both forward and reverse primer was assembled using the software SeqMan 7.0 (DNASTAR Lasergene) to build a consensus sequence. Each of these consensus sequences were subjected to BLAST analysis with the NCBI database (URL http://www.ncbi.nlm.nih.gov/GenBank) to align them with existing 16S rRNA gene sequence data. A similarity of ≥ 98% with a species was accepted as result. If high scores were obtained (usually between 95 and 98%) with several species, only the genus name was accepted as result.

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Chapter 3 Results

Results

3.1 Morphological Characteristics: The morphological characteristics of cell and colony of the isolates were observed once the petridishes were incubated at 30°C for 2-3 days and individual colonies could be isolated as pure culture. 3.1.1 Cell Morphology: Observation under light microscope after gram staining revealed the cell morphology of the isolates. Table 3.1 shows the morphological characteristic of each isolates. Figure 3.1 shows the light microscopy image of isolate BSRI 1.

Table 3.1 Cell morphology and Gram’s characteristics of the isolates Sample

Gram’s Reaction

Shape

Arrangment

BSRI 1

Positive

Long rod (Bacilli)

Single

BSRI 2

Positive

Long rod (Bacilli)

Single

BSRI 3

Positive

Long rod (Bacilli)

Single

BSRI 4

Positive

Long rod (Bacilli)

Chain

Isd 1

Negative

Long rod (Bacilli)

Single

Isd 2

Negative

Sphere (Cocci)

Single

Isd 3

Negative

Sphere (Cocci)

Chain

Isd 4

Negative

Sphere (Cocci)

Single

Isd 5

Negative

Oval (Coccobacilli)

Cluster

Isd 6

Negative

Long rod (Bacilli)

Single

Isd 7

Positive

Long rod (Bacilli)

Cluster

Isd 8

Negative

Sphere (Cocci)

Single

31 | P a g e

Results

Figure 3.1: Isolate BSRI 1 under light microscope after gram staining.

3.1.2 Colony characteristics: Bacterial isolates appeared on LB agar media plates showing typical white, light or heavy orange-yellow colored colony. Table 3.2 lists the colony characteristics of the isolates. Figure 3.2 shows the growth of individual colonies of isolate BSRI 2 on LB agar plate.

Table 3.2 Growth period and colony characteristics of the isolates Isolate name BSRI 1 BSRI 2 BSRI 3 BSRI 4 Isd 1 Isd 2 Isd 3 Isd 4 Isd 5 Isd 6 Isd 7 Isd 8

Growth time on LB agar plates 24-36 hours 24-36 hours 24-36 hours 24-36 hours 24-36 hours 24-36 hours 24-36 hours 24-36 hours 24-36 hours 24-36 hours 24-36 hours 24-36 hours

Color White, opaque Orange, opaque Light-orange, opaque Orange, opaque White, translucent White, Translucent White, translucent White, translucent White, opaque Light-yellow, opaque Light yellow, opaque Light yellow, opaque

Form

Margin Elevation

Rhizoid Filiform convex Circular Entire convex Circular Entire convex Circular Entire umbonate Circular Entire convex Circular Entire convex Circular Entire umbonate Circular Entire convex Circular Entire convex Circular Entire convex Circular Entire convex Circular Entire convex

32 | P a g e

Results

Figure 3.2: Growth of individual colonies of isolate Isd 8 on LB agar plate.

3.2 Metabolic characteristics:

The isolates were characterized for their metabolic activity by standard biochemical tests. Results of these tests are summarized in Table 3.3. The results of different biochemical tests are shown in Figure 3.3.

Table 3.3 Metabolic characteristics of the isolates Tests performed H2S production Gelatin hydrolysis Starch hydrolysis Casein hydrolysis

BSRI BSRI BSRI BSRI 1 2 3 4 -

Isd 1 -

Isd 2 -

Isd 3 -

Isd 4 +

Isd 5 -

Isd 6 -

Isd 7 +

Isd 8 -

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

-

+

-

Nitrate reduction

-

+

+

-

+

+

+

+

-

+

-

+

Catalase activity Indole production Cellulase activity Citrate utilization

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

+

+

+

+

+

+

-

Where, (+) = positive result, (-) = negative result 33 | P a g e

Results

Figure 3.3: Positive and negative results for different biochemical tests. (A) Starch hydrolysis test, clear zone indicated positive result. (B) Casein hydrolysis test, clear zone indicated positive result. (C) Nitrate reduction test, development of red color indicated positive result. (D) Citrate utilization test, development of blue color indicated positive result.

3.3 Carbohydrate utilization pattern:

The ability of the isolates to ferment different carbohydrate was tested. The results are shown in Table 3.4. Figure 3.4 shows positive and negative result as well as gas formation in inverted Durhum’s tube in fructose broth.

34 | P a g e

Results

Table 3.4 Carbohydrate utilization pattern of the isolates BSRI

BSRI

BSRI

BSRI

Isd

Isd

Isd

Isd

Isd

Isd

Isd

Isd

1

2

3

4

1

2

3

4

5

6

7

8

Lactose

+

-

-

-

+

+

-

-

-

-

-

-

Xylose

-

-

-

-

+

+

+

+

+

+

-

+

Fructose

-

-

-

+

+

+

+

+

-

+

-

+

Dextrose

-

-

-

-

+

+

+

+

+

+

-

+

Sucrose

-

-

-

-

+

+

+

+

+

+

+

+

Mannitol

-

-

+

-

+

+

+

+

-

+

+

+

Carbohydrate

Where, (+) = positive result, (-) = negative result

Figure 3.4: Different results of carbohydrate (fructose) fermentation test

35 | P a g e

Results

A dendrogram was constructed using the UPGMA, a sub programme of online software found in the URL http://genomes.urv.cat/UPGMA/index.php?entrada=Example2 (Garcia-Vallve et al., 1999). The data input into the matrix include the data presented in Table 3.4 as well as carbohydrate utilization pattern of Gluconacetobacter diazotrophicus (MTCC-1224) (Singh et al., 2013). Jaccard similarity coefficient was used to compute the similarity matrix. The resulting tree is shown in Figure 3.5.

Figure 3.5: A dendrogram constructed according to the carbohydrate fermentation capability of the isolates. The tree also shows the relationship of the isolates with G. diazotrophicus on the basis of their carbohydrate fermentation pattern.

3.4 Plant growth promontory attributes:

The isolates were tested for their plan growth promontory attributes.

3.4.1 Phosphate solubilization Pikovaskya agar plates were observed for hollow zone to determine the phosphate solubilization capability of the isolates. Results of the test are listed in Table 3.5. Formation of hollow zone is shown in Figure 3.6.

36 | P a g e

Results

3.4.2 Indole acetic acid (IAA) production Supernatant of YMP broth from the broth cultures were taken in separate labeled vials. In 1 ml supernatant, 2 ml freshly prepared Salkowski reagent was added. The vials were kept in dark for 30 minutes. The development of pink color indicated IAA production. The results are listed in Table 3.5 and Figure 3.6 shows positive and negative result. Table 3.5 Result of phosphate solubilization and IAA production test Tests performed Phosphate

BSRI

BSRI

BSRI

BSRI

Isd

Isd

Isd

Isd

Isd

Isd

Isd

Isd

1

2

3

4

1

2

3

4

5

6

7

8

-

-

-

-

-

+

+

-

-

+

-

+

-

-

-

-

+

+

+

+

-

+

+

+

solubilization IAA production

Where, (+) = positive result, (-) = negative result

Figure 3.6: Positive and negative results of P-solubilization (left) and IAA production (right) test

3.5 Antibiogram profile:

The antibiogram profile of the isolates was determined by Kirby-Bauer disk diffusion method as described in Materials and Methods chapter (section 2.4.5). The antibiotic sensitivity and resistant patter, designated R = Resistant, I = Intermediate and S = Sensitive obtained from test with different antibiotics are shown in Table 3.6. The inhibition zone created as the result of diffusion of antibiotic is shown in Figure 3.7. 37 | P a g e

Results

Table 3.6 Antibiotic sensitivity profile of the isolates Sample code

AMC

CTR

MTZ

TE

P

PB

S

GN

BSRI 1

I

I

R

I

S

I

I

I

BSRI 2

S

S

R

S

S

I

I

I

BSRI 3

I

I

R

I

S

I

I

I

BSRI 4

S

S

R

S

S

I

R

S

Isd 1

I

S

R

S

R

I

I

I

Isd 2

I

S

R

S

R

I

R

I

Isd 3

S

I

R

R

R

R

I

I

Isd 4

I

S

R

I

R

R

R

I

Isd 5

I

I

R

I

R

R

R

R

Isd 6

I

S

R

I

R

R

R

I

Isd 7

S

S

R

S

R

I

I

I

Isd 8

I

S

I

I

R

I

I

S

Where, AMC = Amoxiclav, CTR = Ceftriaxone, MTZ = Metronidazole, TE = Tetracycline, P = Penicillin G, PB = Polymyxin B, S = Streptomycin, and GN = Gentamycin; S = Sensitive, I = Intermediate, and R = Resistant

Figure 3.7: Inhibition zone observed as an effect of diffusion of different antibiotic from antibiotic disks on Isd 6(left) and Isd 3 (right) 38 | P a g e

Results

3.6 Molecular characteristics:

3.6.1 16S rRNA gene profile

16S rRNA gene of each isolates was amplified by a universal primer. Gel electrophoresis of the resulting PCR products show that all of the genes were 1.5 kb long. The gel documentation image of the PCR is given in Figure 3.8. Lambda DNA (extended) marker was used to identify the size of the amplified products.

Figure 3.8: Agarose gel electrophoresis image of 16S rDNA amplification products visualized in gel documentation system.

39 | P a g e

Results

3.6.2 nifH gene profile

All isolates were observed to grow in nitrogen free media. Therefore, detection of nifH gene by molecular method was needed. A universal primer for nifH gene was used to amplify and detect the nitrogenase gene in all isolates. The presence of non-specific band was the result of using degenerate primer. Lambda DNA marker was used to identify the size of the amplified products. Approximately 360 bp to 400 bp long band was found in all isolates. The gel documentation image of the PCR is shown in Figure 3.9.

Figure 3.9: Gel electrophoresis image of the amplified nifH gene products. (N = negative control)

40 | P a g e

Results

3.6.3 Random Amplification of Polymorphic DNA assay

Random amplification of polymorphic DNA (RAPD in short) was performed by amplifying random DNA fragments of the isolates. The resulting multiple bands were recorded carefully by their band size. Lambda DNA (extended) marker was used to identify the size of the amplified products. A dendrogram was constructed using the band size and number data collected from the gel documentation image using the UPGMA, a sub programme of online software found in the URL http://genomes.urv.cat/UPGMA/index.php?entrada=Example2 (Garcia-Vallve et al., 1999). Cluster analysis of the isolates was done using Jaccard similarity coefficient. The gel documentation image is shown in Figure 3.10 and the dendrogram is presented in Figure 3.11.

Figure 3.10: Randomly amplified bands for RAPD assay visualized under UV light in gel-doc.

41 | P a g e

Results

Figure 3.11: A dendrogram constructed using Jaccard similarity coefficient according to the band size and number recorded from RAPD assay.

3.7 Identification of isolates using universal method:

The results of the BLAST analysis of amplified 16S rRNA gene product of the isolates are shown in Table 3.7. The table shows the name of the bacteria, 16S ribosomal RNA sequence of which microorganism shows the highest score in identity with the test isolates’ 16S rRNA gene sequences.

42 | P a g e

Results

Table 3.7 Identification of the isolates Isolate Code

Bacteria with the highest score in identity

BSRI 1

Bacillus mycoides (99%)

BSRI 2

Planomicrobium sp.*

BSRI 3

Planomicrobium sp. (95%)

BSRI 4

Bacillus sp.*

Isd 1

Kosakonia oryzae (100%)

Isd 2

Enterobacter sp. (90%)

Isd 3

Pantoea sp. (96%)

Isd 4

Leclercia sp. (99%)

Isd 5

Enterobacter asburiae (99%)

Isd 6

Pantoea dispersa (99%)

Isd 7

Enterobacter sp.*

Isd 8

Pantoea anthophila (99%)

The bacteria marked * were presumptively identified according to their metabolic relationship with the other isolates. This was done due to the low quality results yielded from the sequencing procedure.

43 | P a g e

Chapter 4 Discussion and Conclusion

Discussion and Conclusion

4.1 Discussion:

This study was aimed to isolate and characterize different diazotrophic and endophytic organism from sugarcane stem and root. The screening of diazotrophs was done in N- free chemically defined media. To distinguish the acid tolerant diazotrophs group from other groups, media with both low pH (pH 5.5) and neutral pH (pH 7.0) was used. Isolates that were not acid tolerant couldn’t grow in low pH, but could grow in Burk’s media with neutral pH. Only the acid tolerant diazotriphs could grow in low pH. Based on the reported growth characteristics of Gluconacetobacter spp. 12 suspected bacterial endophytes from different sugarcane samples were recovered, purified and assigned the respective codes (Table 2.1). These were observed for their shape and size along with the colony and cell morphology of individual isolates (Table 3.1 and 3.2). Yellow/orange pigmented pellicle appeared after 7-10 days of incubation in LGIM-agar plates; however, it took only 2–3 days in air tight vials indicating their higher nitrogenase activity under microaerophilic environment (Cavalcante and Dobereiner, 1988). Eight of the isolates were able to grow in semisolid LGIM medium (pH 5.5) indicating the necessity of acidic environment for these cultures (Baldani and Baldani, 2005). The exceptions were the isolates provided by BSRI (isolate BSRI 1, BSRI 2, BSRI 3 and BSRI 4). However, they did grow in N-free Burk’s media with neutral pH, indicating their nitrogenase activity at neutral pH. Heavy or moderate mucous secretion was observed in all the isolates, which might have help to maintain optimum O2 concentration without inhibiting nitrogenase activity and cell metabolism (Dong et al., 2002). Biochemical activities of the isolates were found to be diverse. Majority of the isolates could reduce nitrate, but could not produce gaseous N2. Except the BSRI isolates, all the other isolates could utilize citrate compound. The isolates showed mostly negative results for all the other biochemical tests. However, Isd 4 and Isd 7 showed H2S production, Isd 5 and Isd 7 showed casein hydrolysis capability and BSRI 4 showed starch hydrolysis capability (Table 3.3). These

data helped to understand the metabolism of the isolates. They were also helpful in presumptive identification of the genus of the isolates.

45 | P a g e

Discussion and Conclusion

The carbohydrate utilization assay produced response patterns that distinguished among widely disparate samples and among dissimilar sample types within larger categories. Carbon source utilization profiling showed a significant relatedness among the isolates (Figure 3.5). The isolates were compared with the standard G. diazotrophicus. UPGMA hierarchical clustering showed close similarity between all the isolates coded Isd (except Isd 7) and standard G. diazotrophicus. However, Isd 7 was more similar to BSRI 3. The BSRI isolates were placed in separate groups in the phylogenetic tree.

The plant growth promontory activity is accomplished by different mechanisms, such as, solubilization of essential minerals, increased nutrient uptake, production of certain phytohormones like IAA, vitamins, enzymes and suppression of pathogens through siderophores or by bio-control agents.

Some of the Isd isolates including Isd 2, Isd 3, Isd 6 and Isd 8 showed clear zones on Pikovaskya medium (Pedraza, 2008), indicating positive test for phosphate solubilization (Table 3.5). In spite of the fact that soil usually contains high amounts of total phosphate, its availability to plants is always scarce and thus a limiting factor. Therefore, an organism which is intended to be used as bio-fertilizer, must have the ability to solubilize total phosphate from soil to make it nutritionally available to the plant. About 50% of the Isd isolates (able to survive at low pH) was shown to have the ability. But the BSRI isolates that did not grow at low pH cannot solubilize phosphate at all, indicating the potential of the Isd isolates as plant growth promoter.

Out of 12 isolates used in this study, all the Isd isolates except Isd 5 showed IAA production (Table 3.5). When all the IAA production capability of the isolates was compared by measuring O.D., it was found that Isd 8 has the highest production. The YPM medium supplemented with tryptophan acts as precursor of IAA.

The antibiotic sensitivity test showed that all isolates, except Isd 8, were resistant to Metronidazole. The isolates showed significant sensitivity to Ceftriaxone and Amoxyclav. Penicillin G inhibited the growth of all BSRI isolates, but showed no effect on Isd isolates. The other antibiotics showed diverse effect on the growth of the isolates (Table 3.6). 46 | P a g e

Discussion and Conclusion

The amplification of nifH gene was observed in all the recovered isolates, with a common band of nearly 400 bp (Figure 3.9). Multiple bands were observed in all isolates. This may have happened due to the use of degenerate primers which amplify some of the non specific regions along with nifH gene (Ando et al., 2005). nifH, being the oldest existing functional genes in evolutionary history, can be used for evaluating the relatedness among the isolates. nifH PCR amplicons reflect genetic potential and thus also reflect genetic potential for nitrogen fixation in a particular environment. PCR using degenerate primers enables diverse group of microorganisms to be detected. Moreover, it also signifies the agricultural relevance of the isolates as potential nitrogen fixers. If the isolates could be identified with absolute confidence, species specific primers could be designed to amplify only the nifH region and can be subjected to various molecular assays to better understand their characteristics.

The RAPD assay requires no prior knowledge of the DNA sequence of the targeted genome, as the primers will bind randomly somewhere in the sequence, but it is not certain exactly where. This makes the method a very useful preliminary step of molecular characterization of an unknown microorganism. In recent years, RAPD has been used to characterize, and trace the phylogeny of diverse organisms. In this study, the 1283 F primer was used for random amplification. The amplicons do not give us any positional or functional information about the genome, but it can help us compare the genome of different microorganisms and construct a dendrogram according to the number and size of the amplicons (Figure 3.11). The tree divided the 12 isolates into two major groups. The large group consisted most of the isolates, except BSRI 2, Isd 2 and Isd 8. These isolates that did not belong to the larger group had their own relatedness to each other. Isd 2 was shown to be more related to BSRI 2 than Isd 8. Isd 8 was also the most distant from all the other isolates, which is surprising considering all the Isd isolates were thought to be belonging in the same group. It is also worth mentioning that the two dendrogram shown in this study (Figure 3.5 and Figure 3.11) did not resemble each other, as they were constructed by interpreting very different data sets. Also, neither of them showed the actual evolutionary relationship between the isolates. The purpose of these trees was to compare the isolates with each other and, in case of carbohydrate utilization assay, with a standard strain, to help us characterize them. 47 | P a g e

Discussion and Conclusion

The universal method of identification yielded expected results from the isolates, as most of the species identified by this method corresponded with previous investigations (Abeysingha and Weerarathne, 2010; Taule et al., 2012). In these investigations, endophytic bacteria belonging to the class Gammaproteobacteria (which consists most of the Entarobacter species found in this study) was found to be potential nitrogen fixer and plant growth promoting factor producer. Although quantitative assay for measuring the amount of nitrogen fixation by the isolates was not performed in this study, it is likely that they would prove to play an important role in providing the host plant sufficient amount of fixed nitrogen, as seen in previous studies. There was some difficulty in applying the universal method to some isolates as they were poorly sequenced. These isolates were presumptively identified by their relationship with the other isolates in terms of metabolic relatedness and their genomic fingerprinting data obtained from the RAPD assay. The method revealed that most of the isolates belonged to Enterobacteriaceae class with the exception of the BSRI isolates, which were found to be belonging to the Bacilli class. According to the sequencing results, the anticipated G. diazotrophicus, or any bacteria related to it was not found among the isolates. The bacteria might have been isolated if the sample size was larger than 12 isolates.

4.2 Future prospects and Conclusion:

Over the past quarter of a century, since the original discovery of the bacterium, a lot of research has been conducted on diazotrophs as the potential key to attaining BNF within various crops. The discovery of endophytes living within sugarcane stem and root opened up a new frontier in agricultural science. There is evidence of similar BNF systems within various other plants such as rice, maize, coconut etc. While the majority of studies in the past have primarily focused on understanding the bacterium, its traits, and characteristics, recent studies have moved toward a more molecular focus. The development of modern molecular techniques in recent years will only further the efforts in the molecular field, potentially unlocking the door to successful BNF in crops. Some aspects of these endophytes which can be investigated are listed in the next page:

48 | P a g e

Discussion and Conclusion

1. Effect of different temperature, pH, salt concentration etc. on the growth of the isolates 2. Effect of plant pesticide on the isolates 3. Isolation and sequencing of species specific nif gene 4. Plasmid profiling 5. Plant infection study 6. Quantitative study of phytohormones (such as IAA) production 7. Quantitative study of nitrogen fixation ability 8. Qualitative study of siderophore production 9. Study of plan disease controlling attributes 10. Genetic manipulation of the isolates to increase nitrogen fixation ability, mineral sollubilization, phytohormone and other growth promoting substance production.

Bangladesh is a developing country with a number of social and economic hardships. We have vast resources, but not enough knowledge to utilize those resources. Only with proper research and study we can overcome these problems and rise as a resourceful and progressive country in the world. In the light of increasing concern about environmental pollution caused by excessive use of chemical fertilizer, it is the author’s hope that this study will prove to be an important preliminary step toward the establishment of the use of bio-fertilizer to prevent pollution in our agricultural industry.

49 | P a g e

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L

Appendices

Appendices

Appendix I

5.1 Culture Media: The composition of all the media used for this study is listed below. Distilled water was used as solvent in all media. Table 5.1 Luria Bertani agar & broth Ingredient

Quantity (gL-1)

NaCl

10

Tryptone

10

Yeast extract

5

Agar*

15

*No agar was added in Luria Bertani broth Table 5.2 Semi-solid LGIM medium (modified from Cavalcante and Dobereiner, 1988) Ingredient

Quantity

Sucrose

100 gL-1

K2HPO4

0.2 gL-1

KH2PO4

0.6 gL-1

MgSO4.7H2O

0.2 gL-1

CaCl2

0.2 gL-1

Na2MoO4

0.002 gL-1

FeCl3

0.01 gL-1

Bromothymol blue

5 mlL-1 (0.5% in 0.2 M KOH)

Agar

2 gL-1

pH = 5.5

ii

Appendices

Table 5.3 Burk’s N-fee medium Ingredient

Quantity (gL-1)

MgSO4

0.2

K2HPO4

0.8

KH2PO4

0.2

CaSO4

0.13

FeCl3

0.00145

Na2MoO4

0.00253

Sucrose

20

Agar

15

pH = 7.0 Table 5.4 Starch agar Ingredient

Quantity (gL-1)

Peptone

5

Beef extract

3

Starch (soluble)

2

Agar

15

Table 5.5 Skim milk agar Ingredient

Quantity (gL-1)

Peptone

5

Skim milk powder

100

Agar

15

iii

Appendices

Table 5.6 Phenol red Carbohydrate broth Ingredient

Quantity (gL-1)

Trypticase

10

NaCl

5

Carbohydrate

10

Phenol red

0.02

pH = 7.0 Table 5.7 Sulphite Indole Motility (SIM) medium Ingredient

Quantity (gL-1)

Peptone

30

Beef extract

3

(NH4)2Fe(SO4)2.6H2O

0.2

Na2S2O3.5H2O

0.025

Agar

3

Table 5.8 Nitrate broth Ingredient

Quantity (gL-1)

Peptone

5

Beef extract

3

NaCl

5

KNO3

1

Table 5.9 Peptone water Ingredient

Quantity (gL-1)

Peptone

10

NaCl

5

iv

Appendices

Table 5.10 Simmon’s Citrate agar Ingredient

Quantity (gL-1)

NH4H2PO4

1

K2HPO4

1

Sodium Citrate

2

MgSO4

0.2

Bromothymol blue

0.08

Agar

15

pH = 6.9 Table 5.11 Nutrient Gelatin Ingredient

Quantity (gL-1)

Peptone

30

Beef extract

3

Gelatin

120

Table 5.12 Yeast extract tryptone broth Ingredient

Quantity (gL-1)

Tryptone

16

Yeast extract

10

NaCl

5

Table 5.13 Carboxy methyl Cellulose (CMC) agar Ingredient

Quantity (gL-1)

Peptone

5

Beef extract

3

Carboxy methyl Cellulose

2

Agar

12 v

Appendices

Table 5.14 Pikovaskya medium Ingredient

Quantity (gL-1)

Dextrose

10

Yeast extract

0.5

(NH4)2SO4

0.5

Ca3(PO4)

5

KCl

0.2

MgSO4

0.1

MnSO4

0.0001

FeSO4

0.001

Agar

15

pH = 7.0 Table 5.15 Yeast Peptone Mannitol broth Ingredient

Quantity

Mannitol

25 gL-1

Peptone

3 gL-1

Yeast extract

5 gL-1

Tryptophan

100 μg/ml

vi

Appendices

Appendix II

5.2 Reagents: All the reagents used in this study is listed below with their composition.

Table 5.16 50% Glycerol solution Ingredient

Quantity (ml)

Glycerol

50

Distilled water

50

The solution was autoclaved under high pressure at 121° C for 15 minutes. Table 5.17 Congo red solution Ingredient

Quantity

Congo red

100 μg

Distilled water

100 ml

Table 5.18 Gram’s Iodine solution Ingredient

Quantity (ml)

Iodine

1

Potassium iodide

2

Distilled water

300

vii

Appendices

Table 5.19 3% H2O2 solution Ingredient

Quantity (ml)

35% H2O2 solution

1

Distilled water

11

Table 5.20 Sulphalinic acid solution Ingredient

Quantity

Sulphalinic acid

0.16 g

5N Acetic acid

20 ml

Table 5.21 α-napthylamine solution Ingredient

Quantity

α-napthylamine

0.12 g

5N Acetic acid

20 ml

Table 5.22 Kovac’s reagent Ingredient

Quantity

Concentrated HCl

25 ml

Amyl alcohol

75 ml

Paradimethylamino-benzaldehyde

5g

Table 5.23 Salkowski reagent Ingredient

Quantity (ml)

70% perchloric acid

49

0.5M FeCl3

2

Distilled water

49 viii

Appendices

Table 5.24 TAE buffer (50X) Ingredient

Quantity

Tris base

242 g

Glacial acetic acid

57.1 ml

0.5 M EDTA

100 ml

Deionized water was added to the mixture upto 1 liter. The pH of the solution was adjusted to 8.3. Table 5.25 TE buffer Ingredient

Quantity (ml)

1 M Tris-HCl

1

0.5 M EDTA

0.2

Double distilled water was added to the mixture upto 100 ml. pH of the solution was adjusted to 8.0. The solution was autoclaved under high pressure at 121° C for 15 minutes.

Table 5.26 DEPC treated DNase-RNase free water Ingredient

Quantity (ml)

0.1% Diethylpyrocarbonate (DEPC)

1

Distilled water

1000

The solution was autoclaved under high pressure at 121° C for 15 minutes. The autoclave process was done 3 times to ensure the denaturation of all DNase and RNase.

ix

Appendices

Appendix III

5.3 PCR master mix composition: The PCR master mix compositions for the PCR reactions done in this study are presented below.

Table 5.27 PCR master mix composition for 16S rRNA gene and nifH gene Component

Volume for each

Number of reaction

Total volume in

tube (μl)

tube

master mix (μl)

2.5

12

30

1

12

12

dNTP mix

0.2

12

2.4

Forward primer

0.625

12

7.5

Reverses primer

0.625

12

7.5

Taq polymerase

0.05

12

0.6

DEPC treated PCR grade water 10x Buffer with added MgCl2

x

Appendices

Table 5.28 PCR master mix composition for RAPD assay Component

Volume for each

Number of reaction

Total volume in

tube (μl)

tube

master mix (μl)

8

12

96

1.25

12

15

dNTP mix

0.25

12

3

1283 F primer

1.25

12

15

Taq polymerase

0.25

12

3

DEPC treated PCR grade water Buffer with added MgCl2

xi

Appendices

Appendix IV

5.3 Apparatus The apparatus used for this study are listed below. Table 5.29 Apparatus Used Apparatus Name

Manufacturer

Autoclave (Model HL-42E)

Tokyo, Japan

Centrifuge machine

Sigma, USA

Class-11 Al biological safety cabinet

Thermo Forma, USA

Electric balance model no. 2 1 OS

Sartorius, Germany

Freezer (-30°C)

Thermo Forma, USA

Fridge (4°C)

West frost

Fridge 8°C model no. M1R-253

Japan

Gel Documentation

VilberLourmat, France

Incubator

Memmert, Germany

Incubator, WTB binder, model no. D-78502

Germany

Microcentrifuge, Eppendrof centrifuge

Germany

Microscope, Leicazom 2000

China

Microwave oven, model no. CE2933N

Samsung, Korea

PCR machine

MJ Research.

Power supply

BIO-RAD, USA

pH meter, model no. MP220.

Toledo, Germany

Shaker incubator

Thermo Forma, USA

Sterilizer

Memmert, Germany

Vortex Mixer

Fisher Brand, England

xii

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