Diversity of Frankia associated with Alnus nepalensis

and equisetifolia in West Bengal

A Thesis submitted to the U fNorth Bengal

For the Award of D octor of Philosophy in Botany By Debadin Bose

Under supervision of Dr. Arnab Sen

DRS Department of Botany, University of North Bengal Raja Rammohun Pur, Darjeeling West Bengal, India

July, 2012 1k 5 ~ ~ · ~ 0 ~ 5!./ll..( 1?1'13ci

2'1JOS5 0 7 JUN 1014

. ... DECLARATION

I declare that the thesis entitled "Diversity of Frankia associated with Alnus nepalensis and Casuarina equisetifolia in W es;-~engal" has been prepared by me \ under the guidance of Dr. Arnab Sen, Associate Professor of Botany, University of North Bengal. No part of this thesis has formed the basis for the award of any degree or fellowship previously.

Jt~~ose) DRS Department of Botany, University of North Bengal Raja Rammohun Pur, Darjeeling West Bengal, India

ii rr'liis wort is a tri6ute to my teacliers Late Sri Su6odli 1(umar .Jlc{fiikg_ri Late Sri .Jlrun Sartar SriSankg_rCDe6 qoswami et Sri 1(umarendra CJ3/iattacliarya

iii This present form of this thesis is the result of cwnulative effort of many individuals besides me. I cannot reach this destination without their inspirations and encouragements. I have no hesitation to say that a few words are not enough to explain the co-operation, inspiration, guidance and direction, which I got during this period.

First of all, I take this opportunity to express my deep sense of gratitude and sincere thanks to my supervisor Dr. Arnab Sen, Associate Professor, Department of Botany, North Bengal University for his mature, able and invaluable guidance and persistent encouragement.

I also express my sincere thanks to Dr. Asim Kr. Bothra, Associate Professor, Department of Chemistry, Raiganj University College, for the constant support and guidance throughout the work. I am extremely thankful to him for his invaluable suggestions and advice.

I am also thankful to my teachers, Prof. AP Das, Prof BN Chakraborty, Prof. PK.Sarkar Prof U Chakraborty, Dr. A Saha, Dr. SC Roy and Sri. P Monda! for their continuous support and encouragement.

I express my gratefulness to Prof LS Tisa, Department of Microbiology, University of Hampshire, USA, and Prof P Normand, University of Lyon, France for their kind help.

My sincere thanks to my lab mates Dr. BS Bajwa, Dr. Manprit Gill, Dr. BC Basistha, Sri Molay Bhattacharya, Dr. Saubashya Sur, Smt. Ritu Rai, Smt Subarna Thakur, Sri Arvind Kr. Goyal, Smt Tanmaye ~lishra, Sri Ayan Roy, Smt Sanghati Bhattacharya, Sri Manas Ranjan Saha and Krishanu Ghosh, whose presence was helpful and cooperative. I am also thankful to all the persons whose name is not in the figure out here for their help and support.

Lastly, I feel it very important & appropriate to acknowledge the contribution of my family, Smt Kakali Basu (elder sister), Er. Amitabha Basu (Brother-in-law), Ms Debleena Basu (niece), my wife Amrita Bose and especially my mother Smt Dali Bose. This septuagenarian noble lady did every bit of thing for me without caring her personal comfort. It was indeed difficult days in our family at that time & she did everything gracefully & silently for my carrier. Unfortunately I cannot bring back those days for her. I have no hesitation to say that I have taken away some valuable days from her life and returned nothing to her. I am sorry, Mom. Today, I also missed my father, Late Sri Baidyanath Bose, who wanted to see me at this position and I am sure he is enjoying this moment somewhere in heaven. I can visualize his smiling face when I look at the night skies, I can hear his soundless whisper "My son, my blessing is always with you" with every breezes passing by. My parents, my almighty, your blessing is my inspiration.

(])ate£ ~' Pface: ((])e'td;::(]3ose)

iv }l6stract

Dutch microbiologist and botanist Martinus Willem Beijerinck (1851-1931) first discovered the process of biological nitrogen fixation. It is the ability to convert the molecular nitrogen into cellular nitrogen. This process is a unique property of a group of free living, associative and symbiotic prokaryotic microorganisms. During this nitrogen fixation process, an enzyme complex called nitrogenase complex catalyzes an ATP dependent reduction of atmospheric nitrogen to ammonium. This fixed nitrogen is the primary source of available nitrogen in nature. It is very difficult to calculate any direct correlation among nitrogen fixing bacteria, however the process of biological nitrogen fixation and its enzyme system is very similar in all nitrogen fixers. They are distributed among 27 families and 80 genera of Eubateria (including cyanobacteria) and three thermophilic genera of archaebacteria.

The bacterium Frankia is an important member of biological nitrogen fixer. This bacterium is a microaerophilic, gram positive to gram variable; sporulating actinomycetes belong to the Darmatophilous group. They produce characteristic sporangia and vesicle. This morphology differentiates Frankia from other actinomycetes. Frankia make symbiotic association for biological nitrogen fixation with a number of woody dicotyledonous collectively known as actinorhizal .

The present work deals with the diversity of this bacterium isolated from coastal region to the Darjeeling hills. For diversity study the bacteria was isolated and characterized. Frankia were isolated from the root nodules of Casuarina equisetifolia and Alnus nepalensis using a newly . standardized technique. The isolated strains showed typical Frankia culture characteristics in

liquid Qmod as well as in nitrogen free defined propionate minimal medium (DPM). The vesicles were produced only in DPM medium. Visible nodule and typical root hair deformation was observed on Casuarina seedlings in reciprocal inoculation. However the germination of Alnus and its survival, in both in vitro and in vivo, is a serious challenge to scientists as the germination process is very slow. Mature of Alnus were collected from the healthy plants of Darjeeling hills. Some of the surface sterilized seeds were overnight soaked in aerated water, three different media WPM, MS and Hoagland in half and full strength was used. For the study of effects of hormones in seed germination, the media were separately supplemented with l-5mgll NAA and rnA singly or in combinations. Maximum germination was observed in Hoagland media for both treated and untreated seeds, whereas minimum in Y, MS for treated and \12 WPM for untreated seeds. rnA and

v NAA+ffiA were found to be more effective than NAA in terms of germination percentage and rooting. Good rooting and its growth were maximum in the media supplemented with ffiA(@ 3, 4 & 5mg/L) These experiments showed that germination of A. nepalensis seeds have specific hormonal requirements, which is fulfilled by mycorrhizal and associative fungal association.

Frankia strains isolated from both C. equisetifolia and A. nepalensis were subjected to various concentrations of heavy metal salts to study the heavy metal resistance pattern of Frankia. This experiment was a part of physiological characterization of Frankia of these regions. Frankia strains isolated from C. equisetifolia were studied in sensitivity to nickel, copper, lead, cadmium and cobalt salts. They were found to be resistant to high concentration of cadmium and cobalt salts. Heavy metal salt resistant patterns of Frankia isolated from the A. nepalensis were also prepared. The strains were highly resistant to cadmium chloride and lead nitrate, moderately resistant to cobalt chloride and less resistant to nickel chloride and copper sulphate. The heavy metal resistant Frankia colonies were re-isolated from various plates and preserved for further studies. A characteristic heavy metal induced either purple or red or both type of pigment production was noted in Frankia strains isolated from the A. nepalensis in all the cases except in lead at MIC or higher concentrations. This type of pigment production was totally different from the pigment production of the strains isolated from C. equisetifolia growing in North Bengal University campus where a faint red pigment was produced only in lead. It was found that Frankia isolated from C. equisetifolia could tolerate higher heavy metal salt concentration than Frankia strain isolated from A. nepalensis.

The availability of complete genome sequences of Frankia strains from different bio­ geographic locations gives us an opportunity to analyze the codon usage of the heavy metal resistance genes, predict their expression level in comparison to protein coding genes and ribosomal protein genes. CLUSTAL W was employed to the nucleotide sequences of studied heavy metal resistance genes to find out the diversity. All the protein coding genes, ribosomal protein genes, and genes for heavy metal resistances were analyzed by the software CodonW (Ver. 1.4.2) and CAl calculator 2. The heavy metal resistance genes clustered along with the ribosomal protein genes and exhibit strong codon bias. The Nc/GC3 plot was done, which demonstrated an effective technique for investigating codon usage variations among the genes. Genes coding ribosomal proteins that are known to be highly expressed were highlighted in the NC/GC3 plots. Compared to the ribosomal protein genes

vi the heavy metal resistance genes in the genomes showed some difference. Especially the heavy metal resistance genes of Ccl3 were less biased compared to the ACN14a and EANlpec strains. Most of the ribosomal protein genes and heavy metal resistance gen for all the Frankia genomes were found to be clustered at lower ends of the plot suggesting a strong codon bias for these genes. Genes with effective number of codons value <40 had much stronger codon bias than be simply explained in terms of mutational bias. Ribosomal protein genes and those associated with metal resistance had a lower mean N c value than that obtained for all of the protein coding genes suggesting a higher degree of bias in the former. These values indicated a strong codon bias over mutational bias. This codon bias was aroused due to natural selection for translational efficiency.

Codon Adaptation Index is a simple, effective measure of synonymous codon usage bias. The index uses a reference set of highly expressed genes from a to assess the relative merits of each codon, and a score for a gene is calculated from the frequency of use of all codons in that gene. In ACN and EAN heavy metal genes and ribosomal protein genes had higher mean CAl values compared to that of the protein coding genes. On an average the CAl values of protein coding genes are high. High CAl values indicate better expression levels. Especially the comparatively higher expression levels for heavy metal resistance indicated the ability of Frankia genomes to survive in stressed environments and subsequent adaptability. The result obtained in this study put an additional support to the hypothesis .that Frankia strain Ccl3 is more symbiotic than saprophytic.

The heavy metal resistance genes of Frankia evolved in a single major clade and two subclades. Clustering of copper resistance and tellurite resistance genes of the different Frankia strains suggests that they have co-evolved as a unit. The Zn-Co-Cd resistance gene has a completely different origin and does not lie in a particular clade. Clustering of copper resistance and tellurite resistance genes of the different Frankia strains suggested that they had co-evolved as a unit. The putative Zn-Co-Cd resistance gene which is basically a cation diffusion facilitator family transporter had a completely different origin and did not lie in a particular clade. The study of heavy metal resistance has great significance since the heavy metal resistance is a more suitable marker for slow growing bacteria like Frankia as it is more stable than antibiotics resistance.

Eleven C. equisetifolia and A. nepalensis specific Frankia strains were isolated and characterized on the basis of organic acid decarboxilation, protease and ~-glucosidase

vii activity as well as utilization of twelve different carbon sources. Three strains isolated from C. equisetifolia and two strains isolated from A. nepalensis showed decarboxylation and protease activity. All the strains utilize the Na-propionate, Na-acetate and Na-succinate as carbon source in nitrogen free medium in general. The strains isolated from NBU Campus could also utilize fructose and sucrose in addition. The result thus obtained was analyzed with the software POPGENE to calculate the diversity among the Frankia strains by Nei genetic diversity, Shannon index, single locus component and two locus component and W ahlund effect. Frankia population present in this area shows very low genetic diversity and the total genetic diversity was intrapopulation in origin and divided into two different physiological groups.

The entire Alnus specific strains along with CeSilO, CeSill and CeSil2 cluster in one group, and the rest of the Casuarina strains made other group. Among the strains all and CeSilO, CeSill and CeSi12 belong to physiological group A and CeSt2, CeSt5 CeSt9 of Casuarina strains belong to physiological group B. Field emission scanning electron microscopy was also performed and Frankia specific structures were found.

For the molecular biological work, DNA were isolated and purified both from culture and nodules. A new 16s r-DNA specific primers were developed and a 520 bp long portion of the said region was amplified. The amplicon was digested with Alul, Taq l, Hae III, Mbol and Mspl restriction enzymes. The PCR-RFLP profile was photographed. The results were analyzed with the software POPGENE to calculate the diversity among the Frankia strains by Nei genetic diversity and Shannon index. The results again showed that Frankia population present in this area had very low genetic diversity and the total genetic diversity was intrapopulation in origin. From the dendogram based on PCR-RFLP profile, a more or less distinguished pattern emerged in case of Alnus based Frankia. The Casuarina based Frankia were places in different clades. The probable reason of Casuarina based Frankia behaving differently from Alnus based Frankia could be that all the Casuarina plantations in West Bengal are exotic and were perhaps brought from different places.

viii 3.18 Purification of PCP Product 66 3.19 Digestion of PCR Products 66-67 3.19.1 Analysis of Restriction Fragments 67 3.19.2 Scoring of data 67 Cliapter 4 ~su{ts e1. CDiscussion 68-89 4.1. Isolation of the Bacterium 68-69 4.2 Gennination of Seeds of Actinorhizal Plants 69-72 4.3 Infectivity Test 72 4.4 Colony Morphology 72-73 4.5 Field Emission Scanning Electron Microscopy 73 4.6 Biochemical Characters of Frankia 73-77 4.7 Statistical Analysis of the Biochemical Data by Software POPGENE 77-79 4.8 Heavy Metal Salt Resistance Pattern 79-83 4.9 Analysis of Heavy Metal Resistance Genes of Frankia 83-86 4.10 Isolation of DNA 86 4.11 Purification of DNA 86 4.12 Development of a New 16s Primer Sequence for Frankia 86-87 4.13 PCR Amplification of Frankia DNA and Study of Genetic Diversity of Frankia 87 4.14 Analysis ofPCR-RFLP Data 87-89 Cliapter 5 Condusion 90-92 ~ference 93-108 .Jlcad"emic .Jlcliievements 109 .Jlppentfi:{.(I e1, II} 110-116

X List ofTables Page no

Table 2.1 List of Actinorhizal plants. 13

Table 2.2 Classification of nodulated plants of Rosid I linages. 13

Table 2.3 List of species of genus Frankia proposed by Becking (1970). 34

Table 2.4 Synopsis of strains tested for DNA homology and 16S sequence is 35 available for phylogeny approaches. Table 3.1 List of Restriction enzymes used in present RFLP study. 67

Table 4.1 Utilization of different carbon source by Frankia isolated from Alnus 76 nepalensis and Casuarina equisetifolia. Table 4.2 Physiological characteristics of Frankia isolated from Alnus 77 nepalensis and Casuarina equisetifolia.

Tab 4.3 MIC and MTC of various heavy metal salts. 84

Table 4.4 Values of different codon uses indices for the heavy metal resistance 86 genes of Frankia.

xi List of Figures Page no

Fig 2.1 Comparison of the physical organization of nif and nif-Jlssociated genes 12 from Eu!Kl strain with those from the 3 full-genome sequenced Frankia strains.

Fig 2.2 Present-day native distribution of actinorbizal plant hosts. 14

Fig 2.3 Model A for regulation of nodulation in actinorhizal symbiosis in root 15 hair infection in Frankia.

Fig 2.4 Model B for regulation of nodulation m actinorbizal symbiosis 16 intercellular penetration of Frankia.

Fig 2.5 Complexity of host-Frankia interaction and symbiotic. 21

Fig 2.6. Correspondence between the plant genera RbcL (left) and Frankia 16S 24 rRNA (right) phylogenies. Fig 2. 7. Some of the early photographs of Frankia. 26

Fig 2.8 Micrograph of Frankia spores and sporangia. 31

Fig 2.9 Schematic of strains grouping based on infection of host plants. 34

39 Fig 2.10 Phylogenetic of Frankia and closest neighbours comprising soil sequences. 40 Fig 2.11 Phylogenetic tree of the Frankinae by the Neighbor-Joining method including all validly described genera and species of suborder Frankinae. Fig 2.12 Genome maps of the three Frankia strains. 41

Fig 3.1 A sample Data Collection Sheet. 47

Fig 3.2 (A). Casuarina eqnisetefolia plant. (B) & (C). Male of C. 48 eqnisetefolia. (D). Female flower of C. equisetefolia. (E) & (G) Collection sites of C. equisetefolia. (F) Nodules at the base of the C. equisetefolia.

Fig 3.3 A!tms nepalensis branches bearing fruits. 49

Fig 4.1 Effect of various hormone concentrations on Alnus ttepalmsis seed 71 germination percentage.

Fig 4.2 Generation time (G time), Generation percentage and survival rate of 71 treated and untreated Alnus nepalmsis seeds in different medium.

xii Fig 4.3(a) A young Casuatina equisetifolia seedling with nodule; (b) A young 72 Alnus nepalmsis seedling with nodule; (c) A young root hairs show ignition of root hair deformation due to Frankia infection: (d) A young root hairs show typical hook shaped root hair deformation due to Frankia infection.

Fig 4.4 (a) FESEM picture of multilocular sporangia of Frankia; (b) FESEM 75 picture of vesicle of Frankia.

Fig 4.5 Dendrogram Based on Nei's (t987) Genetic distance showing the 80 diversity of Frankia based on physiological results: Method = UPGMA Modified from NEIGHBOR procedure ofPHYLIP Version 3.5.

Fig 4.6 Effect of different heavy metal salts on growth of Frankia strain (CeSiS) 81 isolated from CaS/latina equisetifolia.

Fig 4. 7 Heavy metal resistance pattern of Frankia strain (AnMrt) isolated from 82 Alnus nepalmsis.

Fig 4.8 Dendrogram of the relationship of heavy metal resistance genes of the 83 Frankia strains.

Fig 4.9 (A): The NcGC3 curve of Frankia Strain EANtpac: Protein coding 85 gene: Orange; Ribosome protein gene: Green; Metal resistant genes: Pink; (B) : The NcGC3 curve of Fra11kia Strain Cci3: Protein coding gene: Pink; Ribosome protein gene: Yellow; Metal resistant genes: Green; (C): The NcGC3 curve of Frankia Strain ACNt4a: Protein coding gene: Blue; Ribosome protein gene: Yellow; Metal resistant genes: Green.

Fig 4.10 Agarose gel photograph ofPCR amplification of Frankia DNA; Lanet, 87 Lambda DNA EcoRI/ Hind III double digest DNA marker; Lane2, -ve control; Lane3-7, At-AS; Lane8-t2, C6-Ct0; Lanet3, tkb DNA marker.

Fig 4.11 (a). PCR product digested with Alui; Lane t, Minisize SObp DNA 88 ladder; Lane2-6, At-AS; Lane 7-tt, Ct-CS; Lane t3, Minisize SObp DNA ladder. (b). PCR product digested with Haeiii; Lane t, Minisize SObp DNA ladder; Lane2-6, At-AS; Lane 7-tt, Ct-CS.

Fig 4.12 Dendrogram Based Nei's (t972) Genetic distance: Method diversity of 89 Frankia based on PCR-RFLP results= UPGMA, Modified from NEIGHBOR procedure ofPHYLIP Version 3.S.

xiii Chapter 1 INTRODUCTION

Dutch microbiologist and botanist a nitrogen fixer. At the same time Martinus Willem Beijerinck (1851- Sergei Nikolaievich Winogradsky 1931) first discovered the process of (1856 - 1953) in his short working biological nitrogen fixation. While period in St. Petersburg (1891-1905) as working in Wageningen University and chief of the division of general Polytechnische Hogeschool Delft microbiology of the Institute of (Delft University of Technology), he Experimental Medicine, first identified thoroughly studied the various nitrogen the ability of obligate anaerobe fixing organisms and found that certain Clostridium pastorianu m fixing bacteria can convert atmospheric atmospheric nitrogen. Basically, m nitrogen into ammonia. He had isolated biological nitrogen fixation, the Bacillus rudicicola and proved that it prokaryotic organ1sms convert the forms nodules on the roots of members atmospheric nitrogen into ammonium of the family Fabaceae. Later he ion, which is absorbed by the plants. isolated Rhizobium species, studied Frankia is one of those prokaryotic nitrogen fixation, and demonstrated organisms which have this unique nitrogen fixation by free-living property. microorganisms, particularly The bacterium Frankia 1s a Azotobacter chroococcum (http:// microaerophilic, nitrogen fixing, gram en. wikipedia.org/wiki/ positive to gram variable, sporulating Martinus_Beijerinck). Some of his actinomycetes belong to the students also did excellent work in this Darmatophilous group. This bacterium field. Prof. A.H. Van Delden, who was has a high G+C content of 68-72% (An a student of Prof. Beijerinck, worked in et al, 1983). It produces characteristic this field of microbiology. He worked sporangia in submerged liquid culture on Bacillus oligocarbophilus, which is and also form vesicles. This INTRODUCTION 2 morphology differentiates Frankia and significant inhibition above 25K from other actinomycetes (Myrold, Pa of oxygen is noted (Rosendahl and 1994). This bacterium produces three Huss.Danell, 1988; Silvester et al, characteristic cell types, namely 1990). vegetative hyphae, sporangium and An (1985) was the first to use the DNA vesicle in the culture medium as well homology technique in classification of as in planta and it is assumed that these Frankia (An et al., 1983; An et al., structures are also produced in the soil. 1985; An et al., 1987). With the advent In solid agar medium Frankia produce of Field emission scanning electron submerged colonies without aerobic microscopy, Polymerase Chin Reaction hyphae. In liquid culture Frankia (PCR) technology, Sequencing produce submerged growth with out technology, DNA hybridization and any floating or aerial growth (Benson various bioinformatics software like and Silvester, 1993). It is an obligate CodonW, CAl calculator etc gave a heterotrophic, microaerophilic, significant boost in the Frankia slow growing bacterium with a research. Various Frankia specific doubling time of about 15hr or PCR primers targeting to 16s rRNA, more in culture; however their 16s-23s rRNA spacer and nijD-nijK growth in symbiotic condition seems to intergenic region have been developed. be unrestricted, because timing of root Workers used these techniques to study infection, nodule development, and both pure cultures as well as uncultured host cell infection are similar to that of Frankia directly from nodules (Benson rhizobia-legume system (Wall, 2000). et al., 1996; Normand et al., 1996; Murry et al., 1997; Jarnman et al., Vesicle is the site of the nitrogenase 1993; Jeong et al., 1999; Sur et al., activity of Frankia. The lipid coating 2006, 2007, Bose et al.,2007; Ventura layers of the vesicle provide the et al.,2007; Sen et al., 2008, 2010). necessary oxygen protection towards the nitrogenase. This property of the Thus, it has become easy to study the evolution, systematic and diversity of vesicle is revealed by Freeze fracturing technique of electron microscopy Frankia with the help of these molecular approaches. Recently the (Parsons et al, 1987). The actinorhizal whole genome of six Frankia strains nodules show an optimal nitrogenase activity at around 20K Pa of oxygen have been sequenced (e.g. Frankia INTRODUCTION 3 EAN1pec NC 009921; Frankia Eullc plants under the influence of Frankia NC 014666, Frankia NC 015657; helps them becoming key Frankia ACN14a NC 008278; Frankia representative of different ecosystem Ccl3, NC 007777; and Frankia and various agro forestry programme. EUN1f, NZ ADGXOOOOOOOO) (http:// These plants fixed sustainable amount www.ncbi.nlm.nih.gov/genome ). of molecular nitrogen, which ranges 1 In addition to these molecular from 240-350 kg N2 ha·'y- and techniques, traditional culture based comparable with those of leguminous approaches are also on to elucidate the plants (Dawson, 1990; Hibbs and mechanism of nitrogen fixation by Cromack, 1990; Wheeler and Miller, Frankia-actinorhizal system. Heavy 1990, Wall, 2000). This nitrogen metal resistance pattern (Richard et improves the nitrogen condition of the a/.,2002 Bose and Sen.,2006, 2007) soil and this nitrogen fixing capability and antibiotic resistance pattern (Tisa is advantageous to the yield of et a!., 1999) of Frankia have been associated plant group and reduces the established in the determine the use of chemical nitrogenous fertilizers. effective genetic marker in Frankia. They increase the yields of associated Unlike Rhizobium, Frankia make crops by way of soil nitrogen with symbiotic association with a number of adding litters. They also increase the woody dicotyledonous plants concentration of nutrients to the collectively known as actinorhizal surface of the soil by extracting them plants (action from actinomycetes, from the deeper layer. Y. R. rhiza the plant root) (Tjepkema and Domrnergues (1987) postulated SIX Torrey, 1979). To be precise Frankia important concepts for optimal infects about 194 species of utilization of biological nitrogen dicotyledonous plants belonging to 25 fixation in agro forestry which are as genera, 8 families and 7 orders. follows: Surprisingly there are representatives a. Plant species with high nitrogen of actirhizal plants in almost every fixing ability with good adaptability orders cutting across the evolutionary should be used. lineage (Benson and Silvester, 1993, b. IdentifY and multiplication of best Normand and Fernandez, 2006). The host. nitrogen fixing capability of those c. The best should show high nitrogen INTRODUCTION 4 fixing potential in field condition. as shade (Thiagalingam, 1983; d. The host should provide with Bourke, 1985). In India, C. required nutrients for expression of full equisetifolia plantations are associated nitrogen fixing potential. with crops such as peanuts, sesame and e. Secondary effects should not be vanous pulses (Kondas, 1981 ). overlooked Casuarina is planted as windbreaks. C f. The host should be analyzed through glauca is used in this purpose in short term and long term method. Tunisia and C. equisetifolia is used in Among the actinorhizal species Alnus Senegal. Casuarina has profuse and Casuarina. (Akkermans and nodulation property and this property Houvers, 1983; Gauthier et al., 1984; of Casuarina explains its high nitrogen Bond, 1983) are important for agro -fixing potential. A selected clone of forestry. Casuarina equisetifolia was reported to

Casuarina, a member of fix 42.5 g ofN2 per tree during the first , consists of 82 species, nine months following plantation. mostly from Australia, South-East Asia Casuarina can tolerate high degree of and the Pacific islands. Johnson (1982) salt concentrations. C. obesa can recognizes four genera under this tolerate 15,000 ppm of chloride. So, family: Casuarina (e.g C. equisetifolia, they are considered as good candidates C. cunninghamiana, · C. obesa, C. for the reclamation of salt-affected junghuhniana, syn. C. montana, C. soils (Bajwa, 2004). Oligodon, C. glauca,); Allocasuarina On the other hand, genus Alnus (e.g., A. torulosa, A. littoralis, A. belongs to the family . fraseriana, A. stricta, A. decaisneana ); Important Alnus species are A Gymnostoma (e.g., G. rumphiana, G. nepalensis, A. viridis, A. glutinosa etc. deplancheana, G. papuana), and They are also good candidates for agro Ceuthostoma. Casuarina is suitable for forestry. Alnus prefer cool and moist agroforestry systems. One classical climate with mean annual temperature example is found in Papua New of 13-26° C and mature trees are frost Guinea, where C. oligodon and G. tolerant. They can grow up to 3000m papuana are used in the highlands and with annual rainfall of 500-2500mm. C. equisetifolia used in the lowlands, as They can also grow on non fertile hill intercrop with food crops, and are used soil. Alnus is introduced in several INTRODUCTION 5 countries for species trial m agro is the absence of a universal media for forestry which include Bolivia, isolation and maintenance of pure Uganda, Burundi and Java (Bajwa, culture. Frankia is a poor competitor 2004). for carbon sources in comparison to its The improvement of soil texture and contaminants. The cortical layers of environmental condition by actinorhizal root nodules are heavily actinorhizal nitrogen fixation depend contaminated with soil upon the improvement of host and microorganisms. The growth rate of symbiont, that is Frankia. A well which is generally faster than the characterized Frankia with good host Frankia. So, the prerequisite for can change the scenario of nitrogen successful isolation of Frankia was to deficient soil in the countries like eliminate these contaminants without India. damaging the endophyte in the cortical Therefore The Research Needs- tissue. Lechevalier and Lechevalier Difficulties in Frankia Research (1990) suggested simple tap water In comparison to other field, modem agar, define propionate minimal Frankia research has not grown up medium to complex Qmod (Lalonde satisfactorily. The slow growth rate of and Calvert, 1979) medium for Frankia and presence of isolation and maintenance. Bose and contaminations are responsible behind Sen (2006) pointed out the problems in this phenomenon (Benson and Schultz, using the two types of media The

1990). The growth rate IS the minimal medium suppressed the characteristic of an organism and a contaminations, which may grow upon very little is left in the hand of workers. transfer into richer medium and on the However researcher can reduce or other hand richer medium invite eliminate the contamination. Sodium contaminations from environment. hypochlorite and osmium tetra-oxide Frankia also need various nutritional are the potent candidates for this supplements for growth (Lechevalier et purpose (Benson and Silvester, 1993). a/., 1982). The isolation problem also Corrosive nature of osmium tetra-oxide inhibits the sequencing based diversity creates problem in the isolation of work. Limited work is done in recent Frankia. years and six strains of Frankia Another difficulty in Frankia research genome have been sequenced (e.g. INTRODUCTION 6

Frankia EAN1pec NC 009921; isolation of Frankia in pure culture is Frankia Eullc NC 014666, Frankia one of the prime necessities of Frankia NC 015657; Frankia ACN14a NC research. 008278; Frankia Ccl3, NC 007777; • Physiological and morphological and Frankia EUNlf, NZ characterization of Frankia strains ADGXOOOOOOOO) (http:// have not been done widely and in India www.ncbi.nlm.nih.gov/genome) this kind of characterization work is The criteria used in classifYing Frankia almost absent. Thus physiological and include the morphology, chemistry, morphological characterization of plant infectivity, serology and DNA Frankia is another important field of homology (Lechevalier, 1984). Frankia research which need Although m order to differentiate immediate attention. strains a few physiological tests have • The genetic diversity of Frankia is been developed (Lechevalier et a/., that kind of field where a considerable 1983; Horriere, 1984; Weber et a/., work has been done, but satisfactory 1988). But limited work had done in result needs more rigorous work in this this field of microbiology. The field with new primers and statistical molecular biological work is also very methods. limited in this field. Overall very limited work has been done in Indian Thus The Objective of The Present Study Frankia and particularly in Frankia associated with C. equisetifolia. A few • Survey and collection of work has been done in the Alnus germplasms from various parts of associated Frankia (Sen, 1996; Bajwa, Drujeeling hills for Alnus based 2004). So, there are mammoth work is Frankia and different regions of left in the field of Frankia research in coastal Bengal for Casuarina based India. In short the difficulties are Frankia. summarized under the following heads: • Isolation of Frankia in pure culture. • After almost thirty years of its • Morphological characterization of isolation in pure culture, the isolation pure culture of Frankia with field of Frankia in pure culture still remains emission scanrung electron a very difficult job to do. Development microscopy. of a suitable easy technique for • Biochemical characterization of INTRODUCTION 7 isolated Frankia found in hilly and with Elaeagnus strain. coastal Bengal. • Isolation of genomic DNA directly • Preparation of heavy metal stress from nodules and strains. resistance profile of Frankia found in • PCR amplification of parts of 16s hilly and coastal Bengal. rRNA gene of Frankia with newly • Study of GC composition, codon designed pruner and restriction usage profile, codon preferences and digestion of the amp !icon. codon adaptation index of heavy metal • Determination of diversity among resistance genes of Frankia of both cultured and uncultured Frankia strains Alnus and Casuarina based Frankia from hilly and coastal Bengal from the strains found in public domain along data generated. Chapter 2 REVIEW OF LITERATURE

2.1 Relevance of Biological Nitrogen molecular nitrogen into cellular Fixation Research nitrogen is called biological nitrogen All the living organisms required fixation. This is an unique property of a nitrogen for their daily needs like group of free living, associative and preparation of proteins, enzymes etc. It symbiotic prokaryotic microorganisms. is a triple bonded, stable, almost inert This fixed nitrogen is the primary colourless, odorless gas. In Earth's source of available nitrogen in the atmosphere there is about 77% of nature. nitrogen which is conserved through In the modern technological era, nitrogen cycle (Rosswall, 1981). The scientists invented another industrial cycle includes various steps which method, called Haber-Bosch process to includes nitrogen assimilation ' produce nitrogenous fertilizer as an mineralization, nitrification, artificial source of nitrogen. This denitrification and biological nitrogen process is very energy consuming and fixation. Most plants and microbes complicated. However it is very preferably assimilate the ammonium demanding as the demands for salts as a source of nitrogen through nitrogenous fertilizer increases day by either glutamate dehydrogenase (GDS) day as nitrogen (N) is the most or glutamine synthetase/ glutamate commonly used limiting nutrient used synthase (GS/GOGAT) cycle. This GS/ for plant growth. Ladha and Reddy GOGAT cycle is widely and preferably (1995) estimated the average energy used by organisms (Miflin and Lea, used in ammonia production in Haber­ 1977). The ability to convert the Hosch process. It is well known that REVIEW OF LITERATURE 9 the efficiency of the industrial process agriculture throughout the world. In varies considerably, but it is estimated India it is generally known as "Green that about 1.3 ton of oil (or equivalent revolution". This green revolution amount of energy) is required to fix 1 occurred due to introduction of

ton of NH3• The annual production of genetically improved cultivars of crop 6 about 77xl0 ton of NH3- N in the year plants as well as use of chemical 1996 requires about 0.1 x 109 ton oil or pesticides and of course use of huge equivalent. This is about 1.4% of the amount of nitrogen fertilizers. These total fossil fuel consumption in the year cultivars are very nitrogen-responsive 1995. (Patyk and Rheihardt, 1997; varieties and produce large Scholz et al., 1997). The combustion of reproductive structures which can such oil or coal add a huge amount of accommodate more grain. This is the pollutant to the environment. reason for increase of use of total Unfortunately our chemically produced nitrogenous fertilizer between 1950 nitrogenous fertilizer dependent green and 1990. The increase was about 10 revolution can not afford any fold. Recent studies show that the minimization of such figure which may yields of the major cereal crops and result in food shortages due to inability utilization of nitrogenous fertilizer did of the industry to produce nitrogenous not increase in the last decade. In some fertilizers. instances like Japan, United States of

After the Second World War, colonial America, Western Europe it decreased powers lost their control over major slightly. On the other hand, farmers in parts of the globe and either democratic Asian countries like India and or socialist governments came to the uses huge quantity of nitrogen power almost throughout the world. fertilizer. It is estimated that there is a The main challenge of these massive gap between the nutrient responsible governments was to fight inputs from external sources and the against hunger and poverty and feed constant drain of nutrients from the soil their population that is to give the due to crop removals and soil erosion. minimum food security to them. In the In India, the estimated nutrient deficit 20th century grain crop yields have was about 10 million tons in 1990, and constantly increased and reached a is likely to touch new summit with remarkable level in many areas of more intensive cropping systems and REVIEW OF LITERATURE 10 increasing soil degradation and finally So, it is established that nitrogen leading to loss of soil fertility (Bumb fertilizer reached its peak and left very and Baanante, 1996). limited potential for growth in the

If the fertilizer demands of next few present century and it is the time to years are considered, it is found that search an alternate source of fertilizer the food needs will increase and it will which would be environment friendly come from a nearly static land base. and have higher potential for growth. Thus, a more intensive agriculture with In a global scenario of reaching more and more use of fertilizer is nitrogen resource to a plateau, rising expected. Bumb and Baanante (1996) concerns over possible environmental based on an econometric model effects of chemical fertilizers, as well estimated 1.2% increase in the world as their cost for small-scale farmers in fertilizer use from 1990 to 2020. It is developing countries, it is essential to estimated that the expected fertilizer expand the use of the biological use will rise from 143.6 million tons to nitrogen fixation (BNF) technologies 208 million tons from 1990 to 2020. It that offer the greatest environmental is very interesting to note here that the and economic benefits for each specific rate of increase of nitrogen fertilizer is agro-ecosystem. much lower than that compared with Biological nitrogen fixation is the period of 1960-1990 (Buckley, generally found in prokaryotic system. 2000). Bumb and Baanante (1996) Eriphorum vaginatum, an eukaryotic explained this lower rate as 'a higher plant, is the only exception (Chapin et base, limited potential for further al., 1993). It is very difficult to growth and changing policy calculate any direct correlation among environment. It is because of the nitrogen fixing bacteria, however the already high application rates, process of biological nitrogen fixation environmental concerns, reduction in and its enzyme system is very similar farm support programme and trade in all nitrogen fixers. They are liberalization'. Buckley (2000) distributed among 27 families and 80 supported the calculation of Bumb and genera of eubateria (including Baanante (1996) and estimated an cyanobacteria) and 3 thermophilic annual 1.1% increase in nitrogenous genera of archaebacteria (Elkan, 1992). fertilizer use from 1990 to 2070. This process has four universal REVIEW OF LITERATURE 11 requirements, which are as follows: Ludden, 2008). Dinitrogenase

1. An enzyme system, called reductase is a 60KDa homodimer that nitrogenase enzyme complex. is encoded by the gene nifH. It specifically reduces dinitrogenase, 2. A high energy requirement and apparently transferring elements to the availability (ATP). P-cluster, which then channels them to 3. Anaerobic condition for nitrogenase FeMo-Co metalocluster. In addition to activity. this catalytic role, dinitrogenase 4. A strong reductant. reductase is also involved in the

During the nitrogen fixation process biosynthesis of FeMo-Co and the nitrogenase complex catalyzes the ATP maturation of dinirtogenase (Filler et dependent reduction of atmospheric al., 1986). nitrogen to ammonium and is The biological nitrogen fixing composed of two protein called organisms can be grouped into three dinitrogenase and dinitrogenase categories- freeliving, associative and reductase. Dinitrogenase is the most symbiotic. Free living diazotrophs was important enzyme of the system, which first described by Winogradrodsky. reduces the atmospheric dinitrogen into They can fix sustainable amount of ammonia at appropriate temperature nitrogen either by any specialized and pressure (Peters and Szilagyi, structure like heterospore of 2006). The dinitrogenase protein is a cyanobacteria or in anaerobic or partial a2~2 tetramer of 240 KDa that are anaerobic condition in nature. The encoded by the gene nijKD. The symbiotic organisms form an complex also contains two types of association with a particular host plant metal centres, the P clusters (8Fe-8S), and fix nitrogen with in a specialized which bridges the a and ~ subunits and structure called nodule. Associative an unique iron -molybdenum co-factor nitrogen fixing bacteria are newly (FeMo-Co), which is buried within the invented group of micro organisms. a subunit and is the site of substrate They are the predominant microbial reduction (Dean et al, 1993). The a and flora of root rhizosphere of particular

~ subunits of nitrogenase is linked with host plant, but do not enter into the each other by cysteine residues of host or host do not produce any molybdo-ferro protein (Rubio and specialized structure for them (Elkan, REVIEW OF UTERATURE 12

B D K E N XOitf'~W':(Z':c::::'B'=>CU:x""M:':nCS~=~=X~"::>''a === :::> FrD11UlJ sp. EuiKl ""' (J7.8kb)

H D K E N X Ill'- WZ B ~s~ ~-- 01ta on Fob =c=:>~~C>D Frt~~~kiasp.EANipec nP. V :;rr,._,....., {17.3 kb) ===

V H D K E N Xltr.WZ 8 H.P. ~ ... ~ Oib Oll.b H.l'.Fd• c::::>c::::>c:=:> ~ ~SCD:;C:>c:=:::>Dc::>£:==>~GG Fr(lll/dflalrriACNI4a u.r. u.. " (18.S kb)

FraniJa sp. Ufl'Ccll ... (11.7 kb} Fig 2.1 Comparison of the physical organization of nif and nif-associated genes from EuiKI strain with those from the 3 full-genome sequenced Frankia strains. Gene organization in the nif clusters are highly conserved among Frankia strains, except with regards to nifV location. In Alnus infecting strains, nijV was located just upstream of nifH, whereas the gene was found in a different genomic location in Casuarina and Elaeagnus infecting strains, including EuiKI strains. Bold letters repre­ sents the nif gebnes, H.P = Hypothetical protein, Fe-S assembly accessory proteins (in EAN I pee strains) (Adopted from: "Organization of nif gene cluster in Frankia sp. EuiKI strain, a symbiont of Elaeag­ nus umbellate' Chang Jae Oh • Ho Bang Kim • Jitae Kim •Won Jin Kim • Hyoungseok Lee • Chung Sun. An Arch Microbial (2012) 194:29-34DOI 10.1007/s00203-0ll-0732-7) 1992). pneumoniae is not found. They are first

2.1.1. Bacterial Genes Responsible For identified in the symbiotic organism Biological Nitrogen Fixation Rhizobium meliloti (Corbiun et al., Primarily three gene families are 1983; Earl et al., 1987; Ruvkun et al., associated with the process of 1982). Their presence was noted in biological nitrogen fixation. They are several other rhizobia latter on, which nif, nod and fix gene families. The nif includes Bradyrhizobium japonicum, genes are responsible for the (Fuhrmann et al., 1985; Gubler and production of nitrogenase (nijD and Hennecke, 1986) R. leguminosarum bv. nijK) and dinitrogenase reductase trifolii, (Iismaa et al., 1989) etc. Unlike (nifH) enzymes. Several nif genes are the nif genes the fix genes are situated identified and are sequenced from on a single operon and associated various groups of microorganisms, electron transport to nitrogenase. which include nijE, nijN, nifV, nijB, Important fix genes are fixA, fixB, fixH, nijS, nifA etc. They are structurally fixN, .fixO, fixX, fixR etc (Fischer, conserved (Fischer, 1994). The fix 1994). The nod genes are associated genes are those genes which have a with the process of nodulation in definite role in nitrogen fixation and legume- rhizobia system. Frankia found in rhizobia, but their genome contains some putative nod homologous counterpart in Klebsiella like genes, however, true nod gene is REVIEW OF LITERATURE 13 Table 2.1 List of Actinorhizal plants ((Benson DR and Silvester WB, 1993; Sen 1996). Plant Order Plant family Host plant genus Betulaceae Alnus Pro tales Elaeagnaceae Elaeagnus, Hippophae, Shepherdia Rbarnnales Rbarnnaceae Ceanothus, Colletia, Kentrothamnus, Retanilla, Telguenea, Trevoa, Discaria, Myricales Myricaceae Myrica, Comptonia* Casuarinales Casuarinaceae Casuarina, Allocasuarina, Gymnostoma, Ceuthostoma Ranunculales Coriariaceae Coriaria Rosales Rosaceae Drayas, Purshia, Cercocapus, Cowania, Chamaebatia

Violales Datiscaceae Datisca

* Frankia was first isolated from this plant Table 2.2: Classification of nodulated plants of Rosid I linages (YI all. 2000). Nitrogen fiXing Nitrogen fixing plant Families plant lineage Lineage I Myricaceae, Casuarinaceae and Betulaceae Lineage IT Elaeagnaceae, Rbarnnaceae and Rosaceae as well as Parasponia (Ulmaceae member, being infected by Bradyr­ hizobium) Lineage ill Coriariaceae and Datiscaceae Lineage IV Fabaceae (Includes non actinorhizallegume plants) absent in its genome (Franche et al., distributed among eight orders, eight 2009). The nif genes of Frankia are families, 24 genera and 194 species of highly conserved. The size varies from land plant (Table 2.1) (Benson and 17.3kb (Frankia sp. EulK1) to 18.5 kb Silvester, 1993, Normand and (Frankia strain ACN14a) (Chang et al., Fernandez, 2006). These plants are 2012). The physical organization of nif found in different hemisphere of the and nif-associated proteins of various earth ranging from arctic Tundra . Frankia genomes are shown in Fig (Dryas spp.) and alpine forest (Alnus 2.1. sp., Coriaria sp. etc) to costal and xeric

2.2 The Actinorhizal Plants condition (Casuarina sp.)(Fig 2.2). Frankia in symbiosis with actinorhizal A group of dicotyledonous plants that plants fixed sustainable amount of are nodulated by Frankia is called molecular nitrogen, which ranges from actinorhizal plants. They are REVIEW OF LITERATU RE 14

c B M+B M E E+M R

Fig 2.2 Present-day native distribution of actinorhizal plant hosts. (A) Distribution of plant ho t for Ccl3, including Casuarina and Allocasuarina of the Casuarinaceae (C). (B) Di tribution of plant ho t for ACN, inc luding Alnus p. in the Betulaceae (B) and Myricaceae (M) and their overlap (M+B). (C) Di tribution of plant ho ts for EAN including members of the Elaeagnaceae (E), Myricaceae (M), and the acti­ norhizal Tribe Colletieae of the Rhamnaceae in South America, Au tralia, and New Zealand (R). Elaeagnaceae and Myriceae (E+M) overlap in some areas. Maps were drawn with information from Silve ter (1977) and from the Missouri Botanical Gar­ den Web ite (www.mobot.org). (Adopted from 'Genome characteri tic of faculta­ tively symbiotic Frankia sp. strains reflect host range and ho t plant biogeography' Philippe Normand eta/, 2007 Genome Res. 2007 January; 17(1): 7-15.) REVIEW OF UTERATURE 15

Jlool rol>m~Jiool tin ~~~(~~------~ ~ Slort allll d!form.dJOO!Iair

hd'!dilnof~

~ (·) C.pllltll5j!lh!les

Sl; + Ji.oompound liilmt>n NH )NH ~) 1-->-7- Plant Nl 3 4 COilJIOund (Phlom) Biomass I t (+) SoilNitro;m

Plmphl!e Fig 2.3 Model A for regulation of nodulation in actinorhizal symbiosis in root hair infection in Frankia. Thin lines means transient pathway. *=Second symbiotic interaction and molecular recog­ nition step involving flavonoids. ** = First molecular signal involving the root hair deformation fac­ tor.(-)= Inhibition,(+)= Activation. S =Signal molecule, I= Inhibitor. (Wall, 2000) REVIEW OF UTERATURE 16

B· interce!Mar penetration

llrol ro~m~Root .&ion • ~1-(ii.'l ______,

rxtoollilirphn! as matrix; Frankia inten:!llJiaJ lllim!lllpmlifmtiJ~ 100! lll>lnil initiatiJn ~ ~; lll!lirtemaol mil primol'liagmwth

~ .· (-)

Frankialllim!llll~ in the infected cells; i!Sll! diff..miafun; ~!!lopiii!II! of oil!" pmtecliu!

Sl; I. !I,

P)Dsplille Fig 2.4 Model B for regulation of nodulation in actinorhizal symbiosis intercellular penetration of Frankia. Thin lines means transient pathway. * = Second symbiotic interaction and molecular recog­ nition step involving flavonoids. ** = First molecular signal involving the root hair deformation fac­ tor.(-)= Inhibition,(+)= Activation. S =Signal molecule, I= Inhibitor.. (Wall, 2000) REVIEW OF LITERATURE 17

240-350 kg N2 ha·'y-' , which is Casuarinaceae. The second subgroup, comparable to those of leguminous includes symbiotic taxa from the plants (Dawson, 1990; Hibbs and families Oatiscaceae and Coriariaceae. Cromack, 1990; Wheeler and Miller, The third subgroup, includes 1990; Wall, 2000). Since actinorhizal symbiotic taxa from the families plants are the early visitors of marginal Rhamnaceae, Rosaceae and soils, they are considered as ptoneer Elaeagnaceae (Tab 2.2) (Swensen, species tn the landslides and other 1996). The nodules formed by threatened areas. These plants are also members of different actinorhizal used as , fiber, food, chemicals, plants differ considerably regarding etc (Benoit and Berry, 1990; Diem and organization of infected cells in the Dommergues, 1988, 1990; Hibbs and cortex (i.e. symbiotic, nitrogen-fixing Cromack, 1990; Myrold, 1994). In cells), oxygen protection mechanisms India, research on actinorhi zal plants is for nitrogenase, patterns of Frankia limited. An excellent work has been differentiation, infection mechanisms, done by Basistha et al. (20 I 0) on and organization of carbon and

Hippophae salicifolia growing tn nitrogen metabolism (Pawlowski and Lachen and Lachung valleys of North Bisseling, 1996; Berry et al. , 2004, Sikk:im where snowfall, heavy rain and 201 1; Schubert et al., 20 I 0, 2011 ). landslides are very common. They The actinorhizal root nodules are found that this actinorhizal plant is established through a series of growmg as dominant species in inte ractions between the host different ecological and topological actinorhizal plant and the bacterium, condition in that region. Recently, Frankia. The actinorhizal root nodules Bargali (20 11) studied habit, habitat, are perennial with coralloid structure. It distribution and possible ecological consists of multiple lobes. The Myrica significance of eight actinorhi zal plants and Ceanothus lobes are discrete and in found in Kumaun Himalayan region. Alnus, they are densely packed. Each Three major phylogenetic subgroups of nodule is a modified lateral root. The actinorhizal plants have been identified actinorhizal roots contain vascular (Swensen, 1996). The first subgroup, cylinder, cortical tissue and periderm. includes symbiotic taxa from the The cortical cells get infected with the families Betulaceae, Myricaceae and Frankia. From the apex of the mature 27J065 0 7 JUN Z014 REVIEW OF LITERATURE 18 root nodule negatively geotrophic al., 1978, 1979; Berry and Torry, 1983) nodule root is developed. This nodule or intracellularly as in the case of root does not take part in the nitrogen Ceanothus, Elaeagnus, etc. Some fixation process (Bajwa, 2004). strains can follow both the pathways

The ideal condition for root infection for infection (Miller and Baker, 1986). and subsequent nodule development is The signal molecule of these plants not well understood in Frankia­ bear same basic chemical feature. Actinorhizal system. Frankia hyphae Actinorhizal plants undergo feedback were embedded in the mucilage layer regulation of nitrogen fixing symbiosis of the root hair. It is established that a with Frankia, which involves at least single infection is enough for nodule two different and consecutive signal development. However, in the natural molecules that leads to a mechanism condition more than one infection controlling root nodulation m occur in the nodule formation. In actinorhizal plants (Wall, 2000). laboratory conditions the frequency of The mechanism of infection in nodulation is directly proportional to actinorhizal plants follows two the amount of inocula (Newcomb and different ways - Wood, 1987). It is interesting to note i) root hair infection (Casuarina sp.) that root hairs of actinorhizal plants are and deformed by several non-symbiotic soil ii) intercellular infection (Shepherdia bacteria (Knowlton et al., 1980). These sp.).(Fig 2.3 and 2.4) soil bacteria play an important role in the Frankia-actinorhizal interaction. Frankia has very little or no host They are called 'Helper' organisms. specificity as found in case of other Pseudomonas sp. helps in the nodule nitrogen fixing bacterium like formation in Alnus and Casuarina in Rhizobium. In rhizobia-legume controlled condition (Knowlton et al., symbioses nod signal molecules helps 1980). in the nodulation and form the basis of host specificity (Oldroyd et al., 2009) The bacterium follows two different These kind of nod factors are absent in pathways for infection of the host Frankia. The bioassay of reporter gene tissue. It takes place either by root hair is also absent in Frankia (Ceremonie et deformation as found in Alnus, al., 1999). On the basis of the study of Casuarina, Myrica etc (Callallam et REVIEW OF LITERATURE 19 actinorhizal root hair deformation et al., 1995). However Casuarina (Gherbi et al., 2008; Markmann et at., infecting Frankia strains are capable 2008) and signal factors of arbuscular enough to produce vesicle. In fact mycorrhizal fungi (Maillet et al., Casuarina - Frankia does produce 2011), it is predicted that Frankia nod vesicle to protect their nitrogenase factor equivalents are a chitin-based from the toxic effect of oxygen, when molecule. Recently a LysM-type growing in pure culture (Sen, 1996). mycorhizal receptor was found in the Frankia strains have been shown to infection process of Rhizobium synthesize truncated hemoglobins for symbiosis of the nonlegume protection of nitrogenase from oxygen Parasponia andersonii (Op den Camp (Tjepkema et al., 2002; Beckwith et et al., 2011 ). This finding suggested al., 2002; Coats et al., 2009). that Frankia might have developed a Pawlowski et al. (2007) showed that in unique pathway of synthesis of a novel Datisca glomerata nodules, the plant chitin-based signal molecule unrelated also produces truncated hemoglobin in to Rhizobium which functions in a nod infected cells. It is involved in nitric factor-independent infection oxide (NO) detoxification, not in 0 2 mechanism (Pawlowski et al., 2011). transport as class I hemoglobin. The In nodule, Frankia followed a unique presence of truncated hemoglobin in pathway for nitrogen utilization. the D. glomerata cells infected with Unlike rhizobia! symbiosis, the Frankia, and the presence of large primary nitrogen assimilation might amounts of a class I hemoglobin in take place in Frankia and stored as alder (Sasakura et at., 2006) and arginine (Berry et al., 2011). Myrica gale nodules (Heckmann et al.,

Frankia is also able to produce a 2006) indicates that, as in legume special structure called vesicle to nodules (Horchani et al., 2011), large amounts of nitric oxide are produced in protect its nitrogenase from 0 2 • Exception is Casuarina nodules, which actinorhizal nodules, leading to high provide a partially anoxic environment levels of stress. Class II haemoglobins for Frankia to fix nitrogen by are generally believed to contribute to producing oxygen diffusion barrier by oxidative stress in nodules by class II hemoglobin (Berg and producing reactive oxygen species 2 McDowell, 1987, 1988; Jacobsen-Lyon (ROS) such as superoxide anions (0 -) REVIEW OF LITERATURE 20

and hydrogen peroxide (HzOz) (Becana found that it possess high antioxidant et al., 2000). This has been confirmed activities, which is helpful in by the fact that RNAi inhibition of preventing or slowing the progress of leghemoglobin gene transcription m oxidative stress.

nodules of the legume Lotus japonicus. From the above discussion it is It not only increase free 0 2 and the loss apparent, that there are two different of nitrogenase and nitrogen fixation in ways of mechanism with two different nodules, but also reduces HzOz sets of receptor and signal molecules. contents (Giinther et al., 2007). For C. The intercellular infection takes place glauca, it has been suggested that at a much faster rate than that of root CgMT 1, a class I type 1 hair infection. A flavonoid compound metallothionein protein, could be part is involved in this process (Hughes et of the antioxidant system to prevent al., 1999). However, the identity of the ROS accumulation in the nitrogen­ compound is not yet clear. There are fixing cells of the nodule (Obertello et several environmental and bacterial al., 2007). Additionally, Tavares et al. factors involved in nodulation. The (2007) have shown that Frankia environmental factors involve light, contributes to ROS production in water, availability of nitrogen and nodules. Thus, both legume and phosphate, soil pH, pCOz and pOz. The actinorhizal nodules have to cope up bacterial factors are concentration of with high levels of stress. Altogether, it the inocula, physiological state of the should be pointed out that actinorhizal strain and nitrogen fixing ability of the plants have a greater ability to tolerate bacterium. These factors play extreme environmental conditions. important role in the function, Progress on research on growth regulation and development of the condition of actinorhizal plants nodules in actinorhizal plants (Fig 2.5). indicates that they grow in relatively From the work of Valverde and Wall adverse conditions than legumes, and (1999), it is found that the tap root therefore, it is also possible that they system consists of a temporary window possess enhanced antioxidant based of susceptibility for nodulation. Wall defense systems (Pawlowski et al., (2000) proposed two different models 2011). Goyal et al. (2011) studied for the regulation of nodulation in various extracts of H. salicifolia and actinorhizal plants which involved two REVIEW OF LITERATURE 21

Actinorhizal host genotype Franl-".ia genotype

-E------7 - Climatic factors , ------;,.. ~ Edaphic factors ~ -~ ' Actinorhizal ~ Frankia , Phenotype -?I lnternction Phenotype , f- ' Actinomizal .... Franroa-host capability I Infection of Frank;a dependency

. ' . . . Functional compattblhty I

Symbiotic effectiveness Fig 2.5 Complexity ofhost-Frankia interaction and symbiotic. (Adopted from "Frankia-actinorhizal symbiosis with special reference to host-microsymbiont rela­ tionship." By Verghese and Misra, 2002) unknown signal molecule (S 1, S2) and Normand and his co-workers had an unknown inhibitor molecule (I). proved the existence of the bacterium These are two-step models and the Frankia in more than 100 Million years signal and inhibitor molecules regulate ago on the basis of nifH gene sequence the whole process by three independent (Normand and Bousquet, 1989). The pathways as follows:- actinorhizal symbiosis may evolve

• Opening and closing of almost in similar time (Sen, 1996). susceptibility window for There are two different hypotheses that interaction. have been projected regarding the evolution of the actinorhizal symbiosis. • Arresting nodule primordia at According the first hypothesis, widely stages before host cell invasion by related plant genera acquired the Frankia and vascular bundle capacity of making symbiotic differentiation. relationship with Frankia to achieve • Inhibiting nodule development m selective advantages in certain the growing root (Fig 2.3 & 2.4). REVIEW OF LITERATURE 22 ecological niches. So, acquisition of group may be subgrouped into four the symbiotic character had hardly linages, of which three are actinorhizal taken place before the divergence of and the fourth one is Fabaceae (Soltis families into their respective genera. et al., 1995). The fossil records, The second hypothesis stated that the geographical distribution, available soil nitrogen was limited in morphological and anatomical studies the early Cretaceous period (Bond, extended supports to this hypothesis 1983) and a group of plant was forced (Wall, 2000). However, on the basis of to make association with nitrogen 16s r-DNA sequence, Frankia can be fixing soil bacteria Frankia to survive divided into three clades and one in struggle for existence with other 'Frankia like clade'. The Frankia wind pollinated angiosperms largely strains have been successfully isolated belonging to Magnoliidae. Some of from Clade I and Clade II; however these plants later evolved into the there is no isolates from Clade III actinorhizal plants. During the last Frankia. The existence of this clade is lOOM years the availability of nitrogen proved by PCR amplification of the in the soil increased due to nitrogen 16s-rDNA. The analysis of data shows fixation and some of the ancient plants that the Frankia-Actinorhizal lost their symbiotic nitrogen fixing symbiosis follows a polyphyletic line habit. Although this ancient property of evolution and this occur at least remains as a selective advantage in three or four times in the history of some pioneer plants which are now evolution which clearly rejects the idea called actinorhizal plants. The DNA of Doyle (1998). This new idea was hybridization study by Bousquet et al. supported by Swenson (1996), Benson (1989) on host plants support the and Clawson (2000) and Jeong et al second hypothesis. (2000). There are a few Frankia rm

The rbcL gene sequence shows that the gene sequences available for the study. actinorhizal nitrogen fixing plants are From these sequences the evolutionary grouped in Rosid I linages of the seed distances can be measured. This type plants (Fig 2.6). The property of of study was made in Frankia by nitrogen fixation in nodules may Normand et al. (1996), Jeong et al. evolve in a single time in the earth's (1999) and Clawson et al. (2004) with history (Doyle, 1998). This Rosid I different conclusions. The Ochman's

., REVIEW OF LITERATURE 23 metric (Ochman and Wilson, 1987) is molecule that have same chemical recognized as the best estimate for this backbone as well as common type of work (Normand and Fernandez, characters. The primitive actinorhizal 2006) and according to this metric, plants, namely Myrica and ancestor of Frankia evolved at about Gymnostoma produce nodules when 350 MY ago from a group of soil infected by both Clade I and Clade II actinomycetes. During this period the Frankia. This is a notable exception first trace of land plant was found. This and this observation established ancestors of Frankia underwent a primitiveness of these plants and on second radiation about lOOMY ago this basis they can also be considered giving birth of Frankia. During this as the most primitive actinorhizae. A period the first dicotyledonous plant probable explanation of this infection families started to appear in the earth. lies in the habitat of Gymnostoma and This estimation that is very close to the Casuarina. Casuarina is native to drier fact that Frankia clusters emerged at Australian continent while 100-200MY ago, which corresponds Gymnostoma diversified in the wetter well to the appearance of the oldest Melanesia region from the tertiary era actinorhizal plant genera like Myrica beginning 65 MY ago. and Alnus (Normand and Fernandez, 2.3. The Frankia 2006). The root nodules of non-leguminous Compared to rhizobia, it is found that plants invite attentions of researchers Frankia has relaxed host specificity. from early nineteenth century. Meyen Frankia has very broad host range and (1829) first speculate on the nature of restricted in clade-to-clade interaction. the cause of nodule and stated that it is The plants belong to Hammamelidae a result of . parasitic infection in the clade is nodulated by the Frankia root. Woronin (1866) studied the strains belong to clade-I. Elaeagnaceae detailed anatomy of the nodules and and Rhamnaceae clade is nodulated by found some round vesicle and hyphae clade II Frankia and clade III Frankia like structure within it. He found that nodulate the Rosaceae plants. So in the hyphae were passing through the conclusion, it can be said that this intracellular region. He also noticed specificity largely resides on a group of that there are some hyphae with round signal molecules or a group of vesicular swelling tips .. Woronin REVIEW OF UTERATURE 24

Plant Orders Plant Gll'nera Franki:1 "'Clusters'"

Figure 2.6. Correspondence between the plant genera RbcL (left) and Frankia 16S rRNA (right) phylogenies. Heavy lines - infection via root hair. Lighter lines - root penetration. ((Clawson et al., 2004);web.uconn.edu/mcbstafflbenson!Frankia/ PhylogenyFrankia.htm). The oblique lines join the plant and bacterial taxa when they can establish symbiotic structures, dashed lines indicate laboratory only sym­ bioses not found in nature. (photo curtsey: Prof. Phillippe Normand, University of Lyon!, France). considered the endophyte as a fungus Frank changed his idea on the nature of and coined the name Schinzia alni as endoiphyte and along with Brunchorst this unknown organism showed considers Frankia as a fungus. On the resemblance with a fungal parasite other hand, the name Frankia alni was called Schinzia cellulicolia (Sen, also coined by Von Tubeuf (1895) as a 1996). Brunchorst, (1886) studied the tribute to A.B. Frank. Several cytological difference of leguminous synonyms of Frankia have been and non-leguminous roots and named proposed later on, which includes the endophyte as Frankia subtilis to Plasmodiophora alni (Woronin) honor his teacher eminent Swiss (Moller 1885), Frankiella alni microbiologist A.B. Frank (1885), (Woronin) (Maire and Tison 1909), who, ironically did not believe in the Aktinomyces alni (Peklo 191 0), presence of living microorganism in Actinomyces alni (von Plotho 1941), any kind of nodules and considered the Nocardia alni (Waksman 1941), structures as protein granules. Later Proactinomyces alni (Krassil'nikov, REVIEW OF LITERATURE

1949, 1959), Streptomyces alni nitrogen accumulator they are certainly (Fiuczek, 1959) (Normand & not the nitrogen fixer on the soil and Fernandez, 2006). another organism must be involved in

Hiltner (1898) for the first time the process of nitrogen fixation. identified the endophyte as member of In another land mark study, Beijerinck actinomycetes which is a close alley of isolated bacteria from legume root Streptomyces while studying the roots nodules which failed to infect non of Alnus and Elaeagnus. (http:// leguminous nitrogen accumulator and www.mcdb. ucla.edu/Research/Hirsch/ other plants proved that leguminous i m a g e s b I microsymbionts and non-leguminous HistoryDiscoveryN2fixing0rganisms.p microsymbionts are two different df; Quispe1, 1990). Hellriegel and organism. (Brewin, 2002; Pawlowski Wi1farth published two papers during and Bisseling,1996). Finally Krebber 1886-1888 on the fixation of identified non-leguminous atmospheric nitrogen by leguminous microsymbiont as actinomycetes m nodules with the help of the bacteria 1932 (Quispel, 1990). residing in the cortical layer. In their Pommer (1956) was probably the first paper they introduced two terminology worker who was successful to isolate a 'nitrogen user' and 'nitrogen slow-growing actinobacteria from accumulator' and showed differences nodules of Alnus glutinosa (Fig:2.7) between them. They identified which had unique morphological as nitrogen accumulator, and features like hyphae, multilocular undoubtedly established source of sporangia and vesicles in pure culture. nitrogen for growth of plants. Their About 0.6mm colonies of that work opened up a new avenue of bacterium were obtained after 2-3 research in plant microbial science weeks of growth in glucose-asparagin (Bottomley, 1912; Quispel, 1988; Sen, agar described by Waksman for 1996). actinomycetes (1950). Unfortunately Hiltner in his later work with Alders this strain was lost before independent demonstrated that young Alders can studies in different laboratories. In the not survive in nitrogen free soils year 1964, electron microscopic study without root nodules. This result of root nodules of Alnus glutinosa and pointed out that though Alders are Myrica cerifera reestablished that these REVIEW OF LITERATURE 26

Fig 2.7 Some of the early photo­ graphs of Frankia. (photos pro­ vided by Prof. Phillippe Nor­ mand, University ofLyon1, France and Prof Louis S Tisa, University of New Hampshire, USA

00 c Goo g <> c c c

(Diem and Dommergues, 1983; Diem Because of slow growth of Frankia and et al., 1983 and Sarma et al., 1998). frequent contamination by other fast Lalonde (1978) worked on Frankia growing organisms, fungi and other REVIEW OF LITERATURE 27 actinomycetes are often mistaken as 2.3.1 Parts of Frankia

Frankia. To overcome this problem A. Hyphae Lechevalier and Lechevalier, (1984) The actively growing cell type of proposed following definition of Frankia is hyphae (0.5-2!1 diameter) Frankia: which form a mycelial mat by "Actinomycetic, nitrogen fixing, repeated branching. The vesicles and nodule forming endophytes or sporangia are produced from hyphae endoparasite that have grown in pure by differentiation. Aerial hyphae are culture in vitro and that: absent on solid medium. The structure a. induce effective or ineffective of Frankia hyphae, both in vitro and nodules in a host plant and may be re­ in symbiosis, was extensively isolated from within the nodules of reviewed by Newcomb and Wood that plant, and (1987). Free-living organisms under b. produce sporangia containing non­ the light microscope show branched motile spores in submerged liquid hyphae. Under phase contrast and culture, and may also form vesicles. dark-field microscopes a large number of bright areas are seen within it c. free living actinomycetes having no (Benson and Silvester, 1993). They known . nodule forming or nitrogen have type III cell wall and type I fixing capacity, but that show the phospholipid content (Lechevalier et morphology described above." al., 1982). Cell wall in chemically Frankia produces three different kinds fixed materials appears to be of cellular structure during its growth composed of electron dense base layer in pure culture or in symbiotic (Lalonde, 1979; Baker et al., 1980; condition i.e. vegetative hyphae, Horriere et al., 1983; Lancelle et al., multilocular sporangia containing 1985). Cross walls originate from the spores and vesicle (Myrold, 1994, base layer. A membranous layer some Benson and Silvester, 1993; and time may be visualized outside the Akkermans and Hirsch, 1997). outer wall layer (Newcomb et al., However, Casuarina infecting 1979; Horriere et al., 1983; Newcomb Frankia does not produce vesicle in and Wood, 1987) and the cell wall, planta (Sen, 1996). that may also contain spherical or REVIEW OF LITERATURE 28 ovoid inclusions which can be B. Vesicles visualized in both chemical! y fixed and Vesicles are the most definitive and freeze-substituted materials (Lancelle characteristic structure of Frankia et al., 1985). Internilly hyphae cells which is a unique developmental contain numerous rosette-shaped structure designed for physiological glycogen granules (Benson and compartmentation and is totally absent Eveleigh, 1979; Lancelle et al., 1985) in any other prokaryotic group (Benson and lipid droplets. Individual and Silvester, 1993). In pure culture, ribosomes and polyribosomes are seen vesicles are spherical, thick-walled and in freeze-substituted hypha! cells short stalked structure (2-6!-l diameter) cytoplasm as relatively large (300nm) and in planta, they have variable globular bodies. A large number of shapes and their shapes are determined cytoplasmic tubules are present at the by their host. The multilarninate thick periphery of the freeze-substituted wall of the vesicle acts as a barrier to hypha! cell cytoplasm. They are oxygen and protects the nitrogenase circular in cross section (45 nm in enzyme from toxic effect of oxygen diameter). These structures underlie the (Parson et al., 1987; Silvester et al., cell membrane both at cell septum and 1990). These vesicles are the site of at the outside wall (Benson and nitrogen fixation in free-living and Silvester, 1993; Lancelle et al., 1985). symbiotic Frankia (Tisa and Ensign, An extracellular multilayered envelope 1987; Huss-Danell and Bergmann, was first identified in hyphae of free­ 1990). The vesicles show a living Frankia alni HFPCpll characteristic metabolism (Tisa and (Newcomb et al., 1979). This structure Ensign, 1988; Tisa, 1998). Although is also present in vesicle of Frankia spores form the main mode of and this multilayered envelope has propagation, the vesicles can also give been identified in symbiotic hyphae of rise to vegetative hyphae (Schultz and Frankia (Lalonde et al., 1976; Berg Benson, 1989). They are lipid and McDowell, 1987; Abeysekera et encapsulated, born either terminally or al., 1990). This indicates that lipid laterally to hyphae by a short enveloped hyphae may be common encapsulated stalk and generally among the Frankia (Benson and produced in nitrogen deficient medium Silvester, 1993). REVIEW OF LITERATURE 29 (Benson and Silvester, 1993). growing in culture, but neither of them

Vesicles developed in nitrogen free appears essential in symbiosis (Bens~m medium first as a terminal swelling of and Silvester, 1993). hyphae or on short side branches. C. Sporangia and spores

These early structures are separated by Frankia strains are readily recognized a septum near the base and are termed by production of multilocular provesicle, which rapidly develop in to sporangia located either terminally or mature vesicles (2-4 Jl in diameter) in an intercalary position (Newcomb et (Fontaine et al., 1984). Provesicles are al., 1979) on the hyphae in liquid spherical cells, with dense cytoplasm culture. This morphology differentiates (1.5-2J.! in diameter), and they may Frankia from other actinomycetes show the initiation of internal (Myrold, 1994). Sporangia (10 x 30-40 compartmentation. The provesicles are !l) contain multiple ovoid spores and unable to convert molecular nitrogen are generally produced in stationary into ammonia nitrogen (Fontaine et al., phase of growth (Myrold, 1994). They 1984). However some workers have are also formed inside nodules of some reported the formation of vesicle in actinorhizal plants (Schwintzer, 1990) presence of available nitrogen in the The Frankia sporangia were first medium (Gauthier et al., 1981; identified in the nodules of Alnus Meesters et al., 1987). Vesicles are glutinosa (Van Dijk and Merkus, observed as a bright structure under 1976). They remain infective in dry phase contrast microscope (Fontaine et soil for a long period (Tortosa and at., 1984) and also shows birefringence Cusato, 1991 ). On the basis of presence under polarized light microscope or absence of sporangia within a root (Torrey and Callaham,I982), and can nodule, Frankia strains have been be seen as a bright halo under dark classified as either spore+ or spore· field microscope (Parsons et al., 1987). (Schwintzer, 1990) (Fig 2.8). The vesicle envelope and internal Spore+ strains appear to be much septations are the necessary more infective than spore· strains prereqnisite for nitrogenase activity (Normand and Lalonde, 1982), both and identified as two important and spore+ or spore· strains have been characteristic structural elements of characterized at the molecular level mature vesicle in Frankia cells (Simonet et al., 1994). Filaments, REVIEW OF LITERATURE 30 vesicles and sporangia have the -51! in diameter (Benson and Silvester, infection potential; however the 1993) (Fig:2.8). spores are the best material to 2.3.2 Growth and physiology of Frankia infect. They are the major means Frankia grow as filamentous colony for Frankia propagation in nature on agar plates and they are generally (Wall, 2000). grown in liquid culture In static batch Chemically fixed developing spores culture the bacterium grow as show an electron translucent nucleoid submerged colonies with out any region with dispersed fibrils, like the floating or aerial growth. They are hyphae residue of a laminate envelope obligate heterotrophic and numerous lipid droplets (Newcomb microaerophilic bacterium. Frankia et al., 1979). However the nucloid is a slow growing bacterium with a region is not evident in freeze­ doubling time of about l5hr or substituted preparation. The cytoplasm more m culture (Benson and of the developing sporangia dispersed Silvester, 1993); however their growth like that of the hyphae. The tubules are in symbiotic condition is seems to be absent in the developed spores unrestricted, since timing of root (Newcomb et al., 1979). The sporangia infection, nodule development, and developed by hypha! thickening host cell infection are similar to that of followed by segmentation by septa rhizobia-legume system. Thus originating from the inner layer of a difficulties to grow the bacterium in double layered sporangia! cell wall culture or isolating the bacterium are (Horriere et al., 1983). This type of due to lack of know ledge in their sporogenesis has been termed as growth conditions and growth enterothalic sporogenesis (Locci and requirements (Wall, 2000). Sharples, 1984). The immature spores Some Frankia strains may produce red, are densely packed and are arranged in yellow, orange, pink brown, greenish the middle portion of the sporangium, and black pigments in the medium. where as the mature spores are However, their pigment production arranged in the peripheral region of the depends on the strain, medium used sporangia (Benson and Silvester, and the age of the culture (Lechevalier 1993). Mature spores are nonmotile, and Lechevalier, 1984). A common red spherical or ovoid and measure about I pigment has been identified as 2-methy REVIEW OF LITERATURE 31

Fig 2.8 Micrograph of Frankia spores and sporangia. (Photographs are kindly pro­ vided by Prof Louis S Tisa, University of New Hampshire, USA) 1-4, 7, 9, 12, tetrahydroxy-5, 6- protease and amylase activity and dihydrobenzo [a] naphthacene-8, 13- utilize various carbon sources which dione, a carboxylated derivative and a include arabinose, glucose, maltose, characteristic sugar 2-0-methyl-D­ sucrose, trehalose, xylose, acetate, mannose (Mort et al., 1983) have been propionate, pyruvate and succinate. identified in Frankia (Gerber and The Gr-B Frankia are more symbiotic Lechevalier, 1984). Since pigmentation and physiologically less active. is highly variable, it is a poor criterion 2.3.3 Heavy Metal Resistance of Frankia for placement in to genus but may also Heavy metals are essential trace prove useful as a phenotypic trait for elements as they act as a cofactor of species recognition (Benson and many enzymes and essential for growth Silvester, 1993). in plants and microbes, but generally

Lechevalier divided the Frankia in two they are toxic at higher concentrations physiological groups (Lechevalier et to all forms of life (Silver and Phung, al., 1983; Lechevalier and Ruan, 1996). The heavy metal resistance 1984); namely Gr-A and Gr-B. The Gr­ pattern arose much earlier than A Frankia is more saprophytic, more evolution of human beings on earth. aerobic and can be maintained in the This system arose soon after the life slants. They show comparative rapid began in the world already polluted by growth and diversity on serological and volcanic activities and other genetical basis. They also show geographical sources (Silver and REVIEW OF LITERATURE 32 Phung, 1996). As this system has toxicity. The second subgroup contains evolved over a long time so it attained Zn, Ni, Cu, V, Co, Wand Cr, which a good diversity. (Bose et al., 2007). are generally toxic but acts also as These heavy metal resistance systems trace elements. Third subgroup contain are important survival strategy for the well known toxic Ag, Sb, Cd, Hg, Pb bacteria. Bacterial chromosomes and and U ions which have little or no plasmids contain genes for resistance trace elemental activity (Nies, 1999). to metal ions. They are frequently The importance of study of heavy generating strong reactive oxygen metal resistance pattern is three fold, species (ROS) and directly or firstly they acts as a genetic marker, indirectly causing gene mutations or secondly in bio-mining of expensive protein damage. (Spain and Alm, metals and thirdly heavy-metal­ 2003). More than fifty elements are resistant bacteria may be used for available in the periodic table, which bioremeditation of heavy metal are designated as heavy metals. But the contaminated environments (Nice, microbiologists are interested to only 1999). 17 of them. This is because rests are The mechanisms of heavy metal either not available or available in trace resistance are generally resides in to the biological system. Weast (1984) efflux pumping and enzymatic estimated that elements at an average detoxification of metal ions. concentration lower than 1 nM are very Occasionally, bio-accumulation or un-likely to have any useful or toxic sequestration is a mechanism of effect in biological system, and a resistance. Efflux pumps are the major resistance gene will not evolved for currently known group of resistance these metals. Tri- or tetravalent cations systems with both plasmid and have very low solubility in water and chromosomal systems. They can be because of the low solubility they have either ATPase (e.g., the Cadmium and no biological significance. The Copper ATPase of gram positive and remaining 17 heavy metals are Arsenite ATPase of plasmids of gram important for study and grouped into negative bacteria) or chemiosmotic three subgroups. The first subgroup (e.g., the divalent cation efflux systems contains Fe, Mo and Mn, which are of soil Alcaligenes and the Arsenite important trace elements with low efflux system of the chromosome of REVIEW OF LITERATURE 33 the gram negative bacteria and of on in planta morphology; vesicles plasmid of gram positive bacteria) morphology and he also considered (Silver and Phung, 1996). this bacterium as an obligate symbiont.

In Frankia research the use of heavy However many subsequent workers metal resistance is very limited and challenged Becking's view. After the focused on into genetic marker study successful isolation of Frankia, a good only as there is a paucity of genetic number of isolates were available for marker in Frankia. In order to classical taxonomical approaches for determine a genetic marker in Frankia classifying this bacterium. Lechevalier system beside heavy metal resistance (Lechevalier and Lechevalier, 1984; pattern (Richards et al., 2002, Bose and Lechevalier and Ruan, 1984) proposed Sen , 2006, 2007) and antibiotic several taxonomic criteria for resistance pattern (Tisa et al., 1999) of classification of Frankia which were Frankia have also been determined. based on ecology, infectivity, 0 morphology, cell chemistry, Frankia strain ACN1A , Cc1.17, Cci3, CN3, Cpll-P, Cpll-S, DC12, physiology, serology, DNA homology EANlpec, ElSe, Eullc, EUNlf, and and 16S rRNA profile. He proposed QA3 were studied for heavy metal two Frankia types, "A" and "B". The resistance pattern. It is found that most type "A" correspond to Elaeagnaceae­ of the Frankia strains are resistant to infective (i.e. cluster 3) Frankia strains elevated levels of several heavy metal and the type "B" corresponds to Alnus­ ions. The heavy metal resistance infective (cluster l) strains. However pattern is proved to be more effective this classification scheme did not meet than antibiotic resistance pattern due to satisfaction. Baker (1987) proposed a the non degradable nature of heavy classification scheme (Fig 2.9) based metal ions for long time of incubation on host infectivity which is as follows:-

(Richards et al., 2002). 0 Alnus and Myrica infective group,

2.3.4 and Diversity of Frankia 0 Casuarina and Myrica, infective Becking (1970) was for the first time group tried to classify the family Frankiaceae 0 Elaeagnaceae and Myrica infective with 10 species of unisolated group and symbionts in genus Frankia (Tab 2.3). 0 Those infective only on Elaeagnus His tentative classification was based REVIEW OF LITERATURE 34

Table 2.3. List of species of genus Frankia proposed by Becking (1970). (Normand and Fernandez, 2006) (Prof. Phillippe Normand, University of Lyon, France kindly provides this figure). Plant Order Plant family Host plant Frankia Vesicles (size,shape) genus s~ecies Fagales Betulaceae Alnus alni (type) 3-8).1m, spherical Rhamnales Elaeagnaceae Elaeagnus elaeagni 2-4J.lm, spherical " Rhamnaceae Ceanothus ceanothi 1.5-3J.lm, spherical " Discariaceae Discaria discariae 4J.lm, sphaerical Myricales Myricaceae Myrica brunchorstii 7.5-12.5xl.6-2.4J.lm, club Casuarinales Casuarinaceae Casuarina casuarinae 3-4x0.6-l.SJ.lm, club Coriariales Coriariaceae Coriaria coriariae 9-12xl.2J.lm, club Rosales Rosaceae Dryas dryadis 1.5-5xl.5-2J.lm, club " " Purshia purshiae ND " " Cercocarpus cercocarpi ND (Fig 2.9). taxonomical scheme based on the

This classification was based on the system proposed by Lechevalier, but he finding that Myrica is a promiscuous used the names alni and elaeagni to host, which was infected by most of the refer to host plants. He further Frankia, with exception with Myrica subdivided F. alni into two species gale, this species can be nodulated by namely pommerii and vandickyi, which only A/nus-infective strains (Huguet et was based on the spore positive (sp+) al., 2005). and spore negative (sp·) characters. But this idea was later discarded due to Lalonde et al. (1988) proposed another uncertainties on spore characters and

4-E/aeagnus

3-Eiaeagnus 1-A/nus +Myrica +Myrica

2-Casuarina +Myrica

Fig 2.9 Schematic of strains grouping based on infection of host plants (Baker, 1987). (Prof. Phillippe Normand, University of Lyon, France kindly provides this picture). (Normand and Fernandez, 2006) REVIEW OF LITERATURE 35

Table 2.4. Synopsis of strains tested for DNA homology (Fernandez et al., 1989; Lu­ mini et al., 1996), and 168 sequence is available for phylogeny approaches. (Prof. Phillippe Normand, University of Lyon, France kindly provides this scheme). (Normand and Fernandez, 2006)

Species by Fer­ Ref. strains Other strains 168 avai­ Infectivity nandez et al., 1989 tested lable

Genomic species 1 AcoN24d Cpll, Arl3, ACN14a Alnus, Morella ACN1AG Genomic species 2 AV22C AVN17o AVN17s " Genomic species 3 ARgP5Ag ARgP5Ag " Genomic species 4 Ea1-12 HR2714· Ea1z, Ealz, Elaeagnaceae, Ea26, Ea33 EalzEa33 Morella Genomic species 5 TX31eHR EAN1pec, EAN1pec Morella HRX401a Genomic species 6 EUNlf Morella+Alnus Genomic species 7 HRN18a Morella+Alnus Genomic species 8 Ea50-1 Morella Genomic species Morella+Alnus 10 Genomic species Morella+Alnus 11 Genomic species SCNlOa Morella 12 Genomic species 9 CeD ORS020607, CeD, Casuarina (ORS02060 Cj 1-82, Allll Ccl3 6) absence of molecular evidence in favor infective isolates, 1 species with a of spore characters. single Elaeagnus-infective isolate and

An et al published a series of papers on several unc1ustered isolates. The Alnus­ the use of DNA homology technique in infective isolates belong to the cluster 1 classification of Frankia (An et al., and the Elaeagnus-infective isolate 1983; An et al., 1985; An et al., belongs to the cluster 3 of this 1987; ). This bacterium has a high G+C bacterium. Later Fernandez et a[. content of 68-72% (An et al., 1983). (1989) worked in the same way with Prof. An examined 19 isolates and 43 isolates and proposed 9 Frankia proposed 1 species with 9 Alnus- species among them, in which 3 REVIEW OF LITERATURE belongs to the group of Alnus-infective homologous as evident by DNA (i.e. belongs to cluster I), 5 belongs to homology values (above 70%; the group of Elaeagnus-infective (i.e. Fernandez et al., 1989) and have a very belongs to cluster 3) and the last one in conserved PCR-RFLP patterns in the the group of Casuarina-infective ( also rm and nif operons (Rouvier et al., in cluster I) strains (Tab 2.4). Dobritsa J992, 1996). When a Frankia strain is and her co-workers ( Akimov et al., isolated from the C. equisetifolia out 1991; Akimov and Dobritsa, 1992;) side Australia, the strains showed partially extended the study by variations. Thus it can be concluded Fernandez et al. and found 5 from the above discussion that there genospecies m the Alnus infective are about 3 to 7 or more strains and four Frankia genospecies genomospecies infective on Alnus and on Elaeagnus infective strains. Myrica and from 8 to 12 or more However, in both the group tested few genomospecies infective on strains and therefore it is impossible to Elaeagnaceae and Gymnostoma. So, a conclude if these numbers add to those minimum of 12 species are available in previously described or not. The isolates under the genus Frankia. So, it exception of this case is the Frankia is possible to that there exists over alni strains Cpll and Ar13. Lumini et hundred species of Frankia which are al (1996) worked on DNA homology distributed in the four clusters. further with other Elaeagnus-infective Isolation of Frankia from Rosaceous strains, particularly with those strains members or from Datisca or from that can infect both of Elaeagnus and Coriaria is very difficult. According to Alnus, and they described three further Becking (1970) there were at least species. These species have no pure three Frankia species present in culture existence and their Rosaceous plants. Mirza et al. (1994) differentiations are based only on DNA first showed that the Frankia isolated hybridization. They are called from Datisca could nodulate Coriaria, genomospecies or genospecies and both have the similar 16S rRNA (Normand and Fernandez, 2006). Host sequence. He presumed that these infectivity or host species of origin are bacterium infecting both Datisca and studied in this species. The Casuarina Coriaria may be the same bacterium. infective strains are closely Bosco et al. (1994) on the basis of 16S REVIEW OF liTERATURE 37 rRNA sequence concluded that the compared with corresponding gene of symbiont isolated from Dryas (a Streptomyces for taxonomic purpose. member of the family Rosaceae) are The length of 16s rDNA is 1513nt, 23s close to those of Datisca and Coriaria rDNA is 3099nt and 5s rDNA is 200nt symbionts. These plants can also be in Frankia. The 16s rDNA region of nodulated by cluster 3 genomospecies Frankia is highly conserved (Misra and under laboratory conditions. These Verghese, 2004) cluster 3 strains are cosmopolitan and Hahn et al (1989) were the first to use can cross infect with other actinorhizal 16S rRNA sequences of Frankia to plants while other strains are selective. investigate the phylogenetic The prime obstacle of Frankia research relationship of Frankia with its lies in its difficulties in the isolation. neighbors. On the basis of their study it With invention of polymerase chain was revealed that Frankia was close to reaction (PCR) technology (Mullis et Geodermatophilus, a dry soil al., 1986) identification of Frankia actinomycetes, in possessing similar become easier. First PCR amplification multilocular sporangia and to of Frankia was done with universal Blastococcus, a sea microbe. But there primer FGPS849 and FGPS1176 and is little description about the genus the size of amplicon was 325bp Blastococcus (Normand and (Nazaret et al., 1991). The first 16s Fernandez, 2006). Marechal et al rDNA specific primers (FGPS989ac (2000), based on their studies on, recA and FGPS989e) targeting the helix 31 marker on this regard found an of domain ill of 16s rRNA gene was interesting finding that a thermal spring developed by Bosco et al. (1992). microbe, Acidothermus cellulolyticus is According to Misra and Verghese the closest phyletic neighbor of (2004) the ITS region of rm genes Frankia rather than shows good diversity among Frankia Geodermatophilus . Recent studies also strains. Simonet et al (1991) and Mirza confirm that Frankia can be subdivided et al (1994) constructed primers for into four clusters (Fig .2.1 0 and 2.11) that region and successfully amplified Frankia is world wide distributed and the ITS region of rm gene of Frankia. faces various physiochemical soil Normand et al (1994) studied the properties like soil pH, aeration, structure of rm operon in Frankia and moisture etc. Even this bacterium may REVIEW OF LITERATURE persist in soil in absence of the host 5,433,6284499bp. The Frankia strain plants (Huss-Danell and Frej, 1986; ACN 14a has intermediate genome size Smolander and Sundman, 1987). It has of about 7,497,9346786bp (Normand et been found that actinorhizal plants gets al., 2007). Normand et al. (2007) nodulated far away from their original found 2,810 genes common in the habitat. This indicates the presence of genome of Ccl3, ACN14a and that particular Frankia in that area EANlpec strain of Frankia. So it can (Dawson et al., 1989; Smolander, be stated that the diversity of Frankia 1990; Jamann et al., 1992, Huguet et strain lies in their variable genome al., 2004, McCray Batzl et al., 2004; size. So the conclusion on the diversity Normand and Fernandez, 2006). of Frankia can be drawn from the word

J amann et al. ( 1992, 1993) working on of Normand ''The full genetic diversity nifD-nifK intergenic region of Frankia of Frankia may not yet have been from Elaeagnus shows that the revealed due to limits brought about by diversity which is measured by the the approaches used. Indeed, the time­ Shannon-Weaver diversity index differ consuming isolation step not only in accordance with soil pH. Soil strongly limits sampling, it also pollutant also has a role in Frankia induces biases, under representing non­ diversity, which was assessed by or poorly nodulating strains and Ridgway et al. (2004) in Frankia selecting those isolates that are easily strains associated with Alnus incana. It culturable. A previous facilitating but was found that different metal bias-inducing step often seen is the use concentration play a ml\ior role in of an intermediate host inoculated in distribution of Frankia associated with the laboratory by crushed nodules from Gynmostoma (Navarro et al., 1999). the field. Indeed, it was reported that the genetic diversity observed in The recent whole genome sequencing natural populations can be totally data of the three strains of these different from that observed following bacterium shows that there are huge greenhouse inoculations with soil or variation in the genome size (Fig 2.12). field nodules (Huguet et al., 2005). The Frankia strain EANlpec has a Direct detection has been used in genome size of 9,035,218,7976bp several recent attempts to describe while the Frankia strain Ccl3 has Frankia diversity in nodules or soil of genome size of about REVIEW OF LITERATURE 39

Franlda alni

Frankia Casuarina­

infective N ~-, E:ipsier"l

Frankia genosp #3

Frankia genosp #2

P':'~''~:;"""'~::;:-·----~'1:' ,"_IJrankiaceae ... , ;:·":t_,,•- ·~:-·· Frankia genosp ·Cluster-~ #4

Franlda genosp #5

.;~i~ster 2· -".~-:.': ">-··- ". "~..! Franlda Rosaceous­ " .~, "",.; - '•·"-- -- infective •< ·r_.; •

Frankia AgBI-9

Acidothemms cellu­ /olyticus

soil isolate

soil isolate

Fig 2.10: Phylogenetic tree (Saitou and Nei, 1987) of Frankia and closest neighbors comprising soil sequences. I: Alnus and Casuarina infective strains; 2: Rosaceaous-, Datiscaceae- and Coriariaceae-unisolated symbionts; 3: Elaeagnus-infective strains; 4: a divergent group of ineffective or non-infective strains. (Normand and Fernan­ dez, 2006) ). ((Prof. Phillippe Normand, University of Lyon, France, kindly provides this dendogram) REVIEW OF LITERATURE 40

I'"

P;imkiilceae - -

Acidotllennus cellulolyticus 4cidothef~'

M :maceo!!

Geodermotophilus ' obsmrus

B/astococcus aggregatus

Modestobacter - multiseptatus

Sporichtliya polymorpha i '-:SpoTichthya-_ ' c'eae;- Sporichthya brevicatena '1'<· ·< Kineosporia - aurantiaca Kineosporia - succinea - Kineosporia rhizophila

Kineosporia rhamnosa

Kineococcus ~ · .'{Ci~eO~pori::_ -~ -" aurantiacus ,~;;;:f;: .· ' < Kineococcus - radiotolerans

Cryptosporangium arvum

Cryptosporangium c. japonicum Cryptosporangium - minutisporangium Cryptosporangium aurantia­ cum

Nakamurella multipar­ tita

Fig 2.11: Phylogenetic tree of the Frankinae. by the Neighbor-Joining (Saitou and Nei, 1987) method including all validly described genera and species of suborder Frankinae (Prof. Phillippe Normand, University of Lyon, France kindly provides this dendogram). (Normand and Fernandez, 2006) REVIEW OF LITERATURE 41

0

•.#' •-' ·I l~tt.t, .,._ IL;ran kia sp _ \ _ _, Ccl3 _§ !>433&28 ...... ' ~, , ·t~ \..

Fig 2.12 Genome maps of the three Frankia strains. Circles, from the outside in, show (1) gene regions related to symbiosis including shcl, hup2, hup 1, and nif; (2) the coordinates in Mb beginning at 0 = oriC; (3) regions of synteny (syntons) calcu­ lated as a minimum of five contiguous genes present in all strains with an identity >30% over 80% of the length of the shortest gene (syntons are tagged with a spec­ trum-based [red-yellow-green] color code standardized on ACN to indicate regions where syntons have moved in the other strains); (4) IS elements and transposases. Circles were drawn using Gen Vision Software from DNAStar.(Adopted from 'Genome characteristics of facultatively symbiotic Frankia sp. strains reflect host range and host plant biogeography' Philippe Normand et al, 2007 Genome Res. 2007 January; 17(1): 7-15.

PCR-amplified DNA fragments, but Actinomyce tales (Normand and these methods are often based on one Fernandez, 2006). or two conserved genes (generally rrn 2.3.5 Bioinformatics of Frankia and nif genes) and cannot but fail to Bioinformatics is essentially a marriage describe the full extent of biological between biol ogy, informatio n diversity." (Normand and Fernandez, technology and computer science. In 2006). Thus according to the present the present era, bioinformatic analysis taxonomic status of this bacterium is virtually required in every aspect of belongs to the monogeneric family bioscience and Frankia research is no Frankiaceae (Normand et al. , 1996), exception. Perhaps the first Frankia which is a member of suborder partial sequence submitted to the Frankinae together with 5 other public domain was a 243bp long families namely Acidothermaceae, sequence o f rDNA (GenBank Geodermatophi l aceae , accession no S70679.1) done by Lyon Sporichthyaceae, Kineosporiaceae and group (Nick et al., 1992). Since then Microsphaeraceae, and the suborder on, several sequences have been Frankineae is a member of the order submitted in various DNA depositories REVIEW OF LITERATURE 42 like GenBank, EMBI database, DDBJ, index) to investigate codon usage etc. The first DNA sequence which pattern of whole genome of Frankia appear from India was done by AK with special reference to nif genes. Sur Misra group (GenBank: S70135.1). et a[. (2006, 2007) studied the codon

Two groups came forward for Frankia usage profiling and analyze the whole genome sequencing project. intergenic association of Frankia These are LS Tisa and D. Benson from EuiKl nif genes and compared the USA, who have done the sequencing of result with Bradyrhizobium. On the Frankia strains Cci3 and EANlpec in basis of codon usage data they collaboration with DOE, JGI, USA and concluded that Frankia strain show P Normand, who did the sequencing of high level of codon preference. In Frankia strain ACN14a with another study (Sur et a[., 2007), this Genoscope, France. With the advent of group found that the expression level Frankia genome project various other of nif genes of Frankia are moderately workers started working on high and show mutational bias. The life bioinformatics of Frankia. Prominent style pattern of various Frankia strains among them are Sen and his group in was studied using codon usage pattern University of North Bengal, Ventura et analysis (Sen et al., 2008). It was found al. (2007) and McEwan and Gatherer that in Frankia, the highly expressed (1999). McEwan and Gatherer (1999) genes are more biased in comparison to first used codon indices to predict the the other protein coding genes. The expression of nif operon of Frankia. analysis of COG profiles of the Sen and his group published a series of predicted highly expressed genes paper on bioinformatics of Frankia further highlighted the differences (Sur et al., 2006, 2007; Bose et al., amongst the Frankia strains. The 2007; and Sen et al., 2008, 2010). They results obtained in their study further used certain parameters like GC supported the hypothesis that Frankia content, GC3 content (G or C Cci3 is slowly becoming a symbiotic nucleotide in the third codon position), specialist. Besides such sequence effective number of codon (Nc), analysis studies, structurally analysis of relative synonymous codon usage proteins of Frankia have also been (RSCU), Fop (frequency of optimal carried out. The three-dimensional codon) and CAl (codon adaptation structure of NifH protein of Frankia REVIEW OF LITERATURE 43 was resolved using homology locate any polysaccharide degrading modeling technique (Sen et al., 2010). enzyme within it. They attributed The metal binding sites and important property of secretome to a group of functional regions of this protein have esterase, lipases and hydrolases which also been determined. The structure help in the process of nodulation. provided valuable insight into the 3D Niemann and Tisa (2008) found two framework and structure-function truncated hemoglobin genes (hboN and relation of this protein. Beside nif hboO) in Frankia strain Ccl3 genome. genes other genes of Frankia also got These genes help in the oxygen serious attention from the workers. protection in Frankia (Misra and Sen, Kosawang (2009) studied the 2011). Mastronunzio and Benson regulation of hydrogenase using (2010) used mass spectroscopy and comparative genomics of Frankia two dimensional liquid strain Ccl3, ACN14a and R43. Frankia chromatography in proteome research strain R43 show an interesting feature in Frankia and identified 1300 proteins in its hydrogenase protein. It works in of Frankia isolated from Alnus incana, bidirectional mode and evolved 1,100 proteins from Elaeagnus hydrogen which is absent in Ccl3, angustifolia and 100 proteins from ACN14a. In this decades the Ceanothus americanus. They found knowledge and technologies of that the iron proteins are the most structural bioinformatics, protrome and abundant proteins in Frankia secretome research was generated (Sur Recently Sen et al. (2011) explored the et al., 2010) which gave a boost in significance of rare TTA codon understanding the biological nitrogen containing genes in Frankia. Amongst fixation by Frankia. The proteome of the high GC content genomes of Frankia was first studied by Alloisio et Actinomycetes, Frankia genomes had al. (2007) in both under nitrogen fixing the highest percentages of TTA and nitrogen depleted condition. This containing genes. They have found that study reveals that total of 126 proteins Frankia genomes retain large number is associated with nitrogen assimilation of such genes in several important and oxidative defense system. functional groups such as metabolism Mastronunzio et al. (2008) studied the and cellular process. Many of these secretome of Frankia and unable to genes had ortholog in other Frankia REVIEW OF UTERATURE 44 genomes. Their study on evolutionary Frankia started and gathered a good significance and codon-anticodon chunk of information about this interaction further point to the fact that organism, which reached its peak with so many of the Frankia genes hold on whole genome sequencing of the to this rare codon without organism. In the recent past post compromising their translational sequencing work using bioinformatics efficiency. tools started and Frankia research

The study of morphology and grew up. However, many opportunities physiology of Frankia was started of study have been left in this field, from the time of first isolation. In the which invites serious attention of last decade of the last century genetics workers. and molecular biology based work of Chapter 3 MATERIALS & METHODS

3.1 Isolation and Identification of Howrah, Hooghly and North 24 Frankia pargana district. 3.1.1 Survey of Actinorhizal Plants 3.1.2 Collection of Gerrnplasm The present study included two Germplasm were collected from actinorhizal plants growing in West different places of West Bengal, which Bengal. These are Alnus nepalensis includes the coastal as well as hilly D.Don and Casuarina equisetifolia L. regions of the state. There are two A. nepalensis is common in the hills of actinorhizal plants present m this Darjeeling district (Eastern region- A. nepalensis and C. ) at an altitude between 1670 equisetifolia. A. nepalensis (up to and 2040 m (up to 3000 m) with a 3000m) is growing in the hilly regions temperate climate (Sharma and of the Darjeeling district and C. Ambasht, 1986). It prefers moist, cool equisetifolia is mainly growing in the climate with mean annual temperature coastal regions. Although C. of 13-26°C. On the other hand, mature equisetifolia can be seen in the other C. equisetifolia is common plant in sea parts of Bengal in planted condition shore. It is cultivated all through the and they are growing luxuriantly. sea shore of South India, Odisha and Nodules (germplasm for Frankia) of West Bengal. In West Bengal this tree the A. nepalensis samples were is very common in Midnapur and collected in the way of Siliguri - South 24-Pargana district. However, Darjeeling - Siliguri route via Mirik­ scattered plantation can be found in all Pasupati- Ghoom- Sonada. This plant over the state, particularly in Kolkata, is found in abundance as road side tree, MATERIALS AND METHODS while C. equisetifolia germplasm were 3.1.3 Isolation of the Bacterium: collected from several places like Procedure 1: Direct Isolation North Bengal University, Siliguri In the first procedure nodules were Teesta barrage campus, Kolkata and collected from different places of West surrounding areas, Diamond harbor, Bengal. For surface sterilization, the Namkhana, Digha and Bok-kbali. The collected nodules were subjected to the fresh, light whitish brown colored, treatment of 1%HgCb and 30%H20 2 young nodules were collected from for different time span. The HgCb mainly young healthy trees. The light treatment lasted 1 minute to 5 minute , colored nodules indicate the active and with 30% H20 2 it was for 10 growth and youngness (Myrold, 1994). minute to 30 minutes. The effective Essential field data of the tree and the HgCb treatment was 1 minute and it locality were collected in data was 20 minute for 30%H20 2• After the collection sheet (Fig 3.1) which treatment the nodule lobes were includes habit, habitat, vegetation in washed with sterile distilled water for surrounding areas, soil type and several times. After final wash nodule nodules. Photographs of the plants and lobes were peeled off with two sterile surrounding vegetations were taken needles. Each lobe was then washed (Fig 3.2 & 3.3). The vegetation were twice with sterile distilled water and studied and recorded in a tabular form. was crushed in a 100 mL bottle with 25 The percentage of nodulation was mL of Defme propionate minimal calculated by the following formula of media (DPM-nitrogen free) (Baker and Raman and Elumalai, 1991: O'keefe, 1984)) without nitrogen.

, with 0,oof no du lat. ron :;;; No. of plants nodules XIOO No. of plants observed Procedure II: Isolation of Frankia In The collected nodules were stored in Alginate Beads plastic bags containing moist tissue In this procedure nodules were papers for the maintenance of active sterilized as per protocol standardized water potential and kept in ice box to by Sarma et al (1998). About 0.5 minimize the tissue degradation. The grams of nodule lobes were taken in to soil samples were also collected in the the tissue homogenizer with 1OmL of same manner and both the samples 3% PVP in phosphate buffer saline were kept in -2o•c for further use. (PBS). The nodule lobes were crushed MATERIALS AND METHODS 47

Data Collection Sheet. Sample no- J>1..R 5

A. General Information- !. Collection Site: .Y\J?l..i~ · 2. Date: 23· 3. ::WO\) 3. Time: 12.tt\l p?"n · •• n~~ 4. Scientific name of the host: 1\\'lWYl v"'F=--- 5. Local name: Oo \fs. . 6. Nodule collected: ~o. 7. Rootcollected: •Ye~ 8. Seeds collected: \.lfes/No. 9. Soil collected: ~o. I 0. Twig collected: 1..-Vlr's/No.

B. Habits: I. I,.Jrl0Shrub/Herb 2. Flowering time: ...... 3. Seeding time: ...... 4. Planting time: ......

C. Habitat and area of the vegetation:

I. Rain fall season: From ...M.mt c1L- to 6cl- 2. Altitude (mt): 1011'0 3. Temperature: COC) (a) Soil: 13' C (b) Air: 15' 'C 4. Topography: Swamp/ Plaini..!:Jill5'1 Others 5. Vegetation type: Natural forest/ Road side/ Soc~st/ others 6. Management: Cutting! Burning! Natural/ Habitat~ed.

D. Specific collection site: I.· Site cover: Bearup to 20%/21-40o/o/41-60o/o/~o/81-IOO%. 2. Soil pH: 5 •Cf 3. Color of the nodule: Red/ Yellow/ Re-own/Gray. 4. Drainage: Flooded! poorly drained/ we~d.

E. Nodules: I. Location: Crown areal Tap root/ La~oot. 2. Growth form: ~edt Scattered.

by (!) titf'm (/Jose ~eardi Scnofar, !Mofecu{arfienetics LaO. !Department ofiJJotany, !NIP/V.

Fig 3.1. A sample Data Collection Sheet. MATERIALS AND METHODS

Fig 3.2 A. Casuarina equisetifo/ia plant. Fig 3.2 8 & C. Male fl ower of C. equisetifolia. Fig 3.2 D. Female flower of C. equisetifolia. Fig 3.2 E & G Collection sites of C. equi set ifol ia. Fig 3.2 F Nodules at the base of the C. equisetifolia. MATERIALS AND METHODS 49

Fig 3.3 Almts nepalensis branches bearing fr uits properl y and the debris were al lowed to dropped asepticall y through a terile settle down for 10 mm. The upper yringe into the terile CaCI~ olution aqueou phase was then taken in a and kept for half an hour in the terile conical tl a k and kept over night refrigerator for hardening. The bead in a refri gerator to get rid of the were then harvested and washed with phenolic present in the nodule di stilled water and finally with fresh (Leechvalier and Leechva li er, 1990). med iu m. The beads were then ext day, l g of a-alginate wa distributed into aliquots with fresh dis olved in 40mL of liquid med ium by medium. keeping it in boiling water bath. Along Procedure Ill with th i , 250mL of I% CaC I ~ solution In thi s procedure, fresh, young and wa al o prepared. Both the soluti ons light brown colored root nodules from were teri li zed by autoclaving for 20 mature actinorhizal plant were 2 min at 1. 1k g/cm . Then the aqueou collected. The woody nodule lobe pha e of the crushed nodul es wa were urface sterilized with a erie of mi xed with a-alginate when the surface steril izing agents like 4°/o temperature of the soluti on came odium hypochlorite solution for 7 down below 30°C. They were then min, 70% ethanol for 2 min, 0.1% MATERIALS AND METHODS 50 mercuric chloride solution for 7 min complex Qmod (Lalonde and Calvert, and finally in 30% hydrogen peroxide 1979) medium. Define propionate for 30 min in dark. Each step was minimal medium is also a very good followed by repeated washing with medium for maintenance of the sterile distilled water. The nodule lobes endophytes in long term. were then incubated with Qmod 3.3. Maintenance of Pure Culture (Lalonde and Calvert, 1979) medium The pure cultures were maintained in supplemented with 3% activated both minimal and complex medium charcoal for 48h ·· at 3 0°C. After and named accordingly, i.e. first two incubation, the nodule lobes were letters from the genus and the species washed with sterile distilled water to of the host plant with the . first one remove charcoal. Sterilization process capitalized and the subsequent two was repeated once again. Epidermal letters from the collection site. For layers were removed and nodule lobes example the strains isolated from were chopped into small pieces. Each Siliguri and adjoining area from the nodule piece was incubated with 1OmL nodules of C. equisetifolia were named of Qmod (Lalonde and Calvert, 1979) as CeSi I, CeSi5 etc. Similarly the medium supplemented with Vit B1 and strains isolated Mirik and adjoining

B12 in 30 mL screw cap test tube at areas from the nodules of A. nepalensis 28°C for one month or more in dark were named as AnMr1, AnMr2, etc. without shaking (Bose and Sen, 2006). The pure cultures were maintained in. 3.2 Media for Isolation and Maintenance DPM (Baker and O'keefe, 1984) and Even a very complex isolation Qmod (Lalonde and Calvert, 1979) medium for Frankia is available, a medium in 100mL screw cap bottles in variety of medium were tested. These 10mL of requisite medium and sub were Defined propionate minimal culturing were done in every 2-6 media (DPM) (Baker and 0 Keefe, months. The Frankia cultures grow 1984), F-medium (Simonet et al., best in liquid culture between 25° to 1985), OS-1 medium (Dobritsa and 35°C. New stains often slow grower Stupar, 1989) and Qmod medium than once that have been in culture for (Lalonde and Calvert, 1979), but the some times and strains can be adopted best medium for isolation of Frankia to grow more rapidly by frequent beside Frankia isolation medium is the transfers. The hyphae were fragmented MATERIALS AND METHODS 51 by passing throw a sterile needle. The and non-soaked were used for the fragmentation helps in the faster experiment. Three different media growth of this bacterinm. The culture woody plant medinm (WPM, Hi­ characteristics of the bacterium were Media, Cat#PT1 05) (pH-5 .6), also observed. Murashige and Skoog medium (MS) 3.4 Plant Infectivity Test (Hi-Media, Cat#PT0018) (pH-5.6) and 3.4.1 Germination Of Seeds Hoagland solution (pH-7) (Hoagland The seeds of the two hosts were and Arnon, 1950) in half and full collected from healthy host plants from strength were used in the study to see the field conditions. The seeds of A. the effect of media composition. 1 mL nepalensis were collected in the month of media was poured in each culture of March from hills of Darjeeling tube carrying a strip of filter paper and districts and the seeds of C. was autoclaved at 121 oc for 20 min at equisetifolia were collected in the 1.08 Kg/cm2 pressure. A few surface month of May from Namkhana, sterilized Alnus seeds were placed on Kanning and Diamond harbor. Both the the filter paper in the culture tube and seeds need two different conditions for were incubated at 25±1 °C under white germination. fluorescent light. For the study of A. nepalensis seeds were pretreated effect of hormones in seed with aerated water for over night and germination, the media were separately C. equisetifolia seeds needed no such supplemented with 1-5mg/L NAA and pretreatment and germinate directly IBA. Sterilized seeds were also placed (Myrold, 1994). The seeds were taken on sterile vermicompost for separately in two different sterile germination and growth. Double layer conical flasks and surface sterilized culture media were prepared by

with 30% H20 2 for 10 minutes and pouring Hoagland medinm (0.4 % washed with sterile distilled water agar) on MS medinm (1.2 % agar), several times. WPM (1.2 % agar) and Hoagland Mature seeds of Alnus were used in the medinm (1.2 % agar) supplemented study of the effect of different with hormones as described earlier. hormones and media in germination The C. equisetifolia seeds were kept in condition. Two treatments of seeds­ a sterile plastic box in free floating overnight soaked with aerated water condition in BOD incubator at 26 ± MATERIALS AND METHODS 52 3°C for germination. solution was decanted and the samples Fifteen day to one month old seedlings were washed with the same buffer for were then placed in sterile blotting three times. The samples were treated paper supported by stainless steel with I% Osmium tetra oxide solution supporter in magenta box (Planton, in phosphate buffer (pH 7 .2) for I hour Tarsons make) containing sterile and washed with the phosphate buffer Hoagland Solution without nitrogen of (pH 7.2) for three times. The Frankia various concentrations (1/2, 1/4, 1/8, samples were passed through with l/I6). graduated ethanol from 50% to For the plant infectivity tests seedlings absolute for I 0 minute each for were inoculated with fresh crushed dehydration. The 95% ethanol wash nodule suspension and treated it as +ve was performed twice. After control. Seedlings were inoculated with dehydration the samples were kept in lOOJ.tL of 30 days Frankia culture absolute alcohol for microscopy (Sen, under examination. A set of 1996; Bajwa, 2004) uninoculated seedlings were used as - The samples were critical point dried ve control. The seedlings were kept in for 2h. Each sample was mounted on plant growth chamber at 26°C with copper tape and was platinum coated approximately 90% humidity and IIOO by JFC 1600 platinum caster, (JEOL, lux illumination. Japan) and viewed on JEOL JSM- 3.5 Field Emission Scanning Electron 6700F field emission scanning electron Microscopy microscope at an accelerating voltage For field emission scanning electron 2kV. microscopy, one month old Frankia 3.6 Physiology of Frankia cultures were used. The bacterial cells Eleven Frankia strains were selected were taken in a micro centrifuge tube and used in the study of physiology of and centrifuge at I 000 rpm for 5 the bacterium. The reference strain minute. The media was then discarded used in this study was ACNI AG which and cells were washed with distilled was kindly provided by Prof. Louis S water. The bacterial mycelium were Tisa, Department of Microbiology, treated with 4% glutaraldehyde University of New Hampshire, USA. solution in phosphate buffer (pH 7 .2) Frankia strains were characterized on for 4h at 30°C. The glutaraldehyde the basis of organic acid MATERIALS AND METHODS 53 decarboxilation, protease and /3- (Baker and O'keefe, 1984)) was glucosidase (Horriere, 1984; Weber et supplemented with 12% of gelatin and al., 1988) activities as well as 5mL of such sterile medium was utilization of twelve different carbon inoculated with 1mL of thick hyphal sources. suspension of Frankia and incubated at 3.6.1 Decarboxylation of Organic Acids 28°C in dark for 42 days. The tubes Decarboxylation of organic acids were were checked every alternate day. determined as described by Leechvalier 3.6.3 Esculin Hydrolysis Test et al. (1983). Organic acid A five week old Frankia suspension decarboxylations were determined in a was inoculated in 5mL of DPM (Baker basal medium containing in gL -I: and O'Keefe, 1984) medium agar tube peptone, 5; NaCl, 1; MgS04.7H20, 0.2; supplemented with 0.1% ferric citrate

KH2P04, 0.2; CaCl2.2H20, 0.01; Fe Na and 0.1% esculin. The esculin was EDTA,0.01 and phenol red indicator hydrolyzed to a /3-glucosidase activity 1 (0.04% sol) 20mL L- • The pH was and gave rise to coumarine. This maintained at 6.8. The sodium salts of coumarine react with ferric citrate to organic acids were added at 0.2% prior form a brownish black complex. The to the autoclaving. The organic acid tubes were incubated at 28°C in dark salts were Na-citrate, Na-propionate, for 42 days and checked every alternate Na-acetate and Na-pyruvate. day. Decarboxylation leads to a rise in pH 3.6.4 Utilization of Different Carbon and assessed as change in color of the Source indicator from yellow - orange to red. For utilization of single carbon source 5mL of the sterile decarboxylation as source of energy the basal salt medium was inoculated with lmL of solutions were as same as the DPM thick hyphal suspension of Frankia and (Baker and O'Keefe, 1984) medium incubated at 28°C in dark for 42 days. (without nitrogen and propionate). The The tubes were checked every alternate carbon source was added to this basal 1 day. medium separately at 20 gL- • The 3.6.2 Gelatin Hydrolysis Test carbon sources were glucose, sucrose, The protease activity of the Frankia fructose, lactose, mannitol, Na-acetate, strains were tested with gelatin as Na-propionate, Na-succinate, Na­ enzyme substrate. DPM (N2 free) pyruvate, Na-citrate and tween 80. MATERIALS AND METHODS 54 1 Tween 80 was added at lmL 1· • 5mL chloride (NiCI2), cadmium chloride

of basal medium was supplemented (CdClz), lead nitrate (Pb(N03) 2), cobalt

with the carbon source as stated above. chloride (CoCI2) and copper sulphate

A five week old Frankia culture was (CuS04). The metal salts were used at taken and the colonies were collected the concentration of 0.005, 0.01, 0.05, by centrifugation (at 12000rpm, for 2 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4 and 5 min) and the colonies were crushed in mM. Two week old culture was used as 1.5 mL of sterile distilled water into a inoculum. The hyphae were collected hyphal suspension. Each tube of basal by centrifugation at 10,000 rpm for 2 medium supplemented with the carbon min and fragmented by passing source was inoculated with 50J.1L of through an aseptic syringe. 2% DPM Frankia hyphal suspension. The tubes (Baker and O'keefe, 1984) agar plates were incubated at 28°C in dark for 60 containing various heavy metal salts at days to see the visible growth. The different concentrations were prepared media pH was maintained at 7. The separately. Half (1/2) mL of hypha! results thus obtained were analyzed suspension was mixed with 5 mL of with the free software POPGENE to 0.8% sterile water agar at 45°C. The calculate the diversity among the hypha! suspension mixed with water Frankia strains by Nei genetic agar poured uniformly on 2% DPM diversity (Nei, 1987), Shanon index (Baker and O'keefe, 1984) agar plates (Shannon, 1948; Weaver and Shannon, containing various heavy metal salts at 1949), single locus component and two different concentrations separately. locus component (Brown and Feldman, Before pouring the content of the tube 1981) and Wahlund effect (Brown and were mixed by agitation using a vortex Feldman, 1981). All the above mixer, but vortexing should be done experiments were done in triplicate and carefully to avoid foaming in the agar. same results were found. (Bose et al., Plates were incubated at 28°C in dark 2011) for 14 days.

3.7 Heavy Metal Salt Resistance Pattern Colonies of Frankia appearing on the plates were taken as resistant and Frankia cultures were subjected to counted. The colony count was plotted heavy metal stress to determine its against the increasing concentration of sensitivity by growth inhibition assay. various heavy metals. The heavy metal salts included nickel MATERIALS AND METHODS 55 The mam advantages of the double expanding genome which helps the layer technique over the liquid culture bacterium to adopt in new niches and media are as follows:- with new substrates with its host. On !. Contaminants do not necessarily the other hand the strain Ccl3 with its over grow the Frankia colonies. reducing genome became more 2. Microaerophilic Frankia colonies specialized to a particular environment are not directly exposed to the in terms of host and substrates. atmosphere. Codon usage and codon preferences 3. Frankia colonies can be detected are the interesting tools for studying under microscope or even under naked the variations among these kinds of eye. organisms (Grantham et a!., 1981; 4. Colony morphology of Frankia can Karlin et a!., 2001, Sur et at., 2008). be done. (Bose and Sen, 2005, 2006) The G + C composition variations are 3.8 Analysis of Heavy Metal Resistance the results of either translational Genes of Frankia selection or mutational bias or both and The genome sequences of the three it fix the path of codon variation strains of Frankia, namely ACN14a, (Knight et a!., 2001 ). The highly Ccl3 and EAN1pec, were available expressed genes are greatly affected by (Normand eta!., 2007). They showed a translation selection and show higher degree of homogenecity in the 16s degree of biasness in the codon usage rRNA sequence (98%-99% identity), pattern (Ikemura, 1981; Sharp and Li, however the size of these genome 1987; Dos Reis eta!., 2003; Baneijee varies significantly. These variations et a!., 2004). The codon usage is mainly arise due to the result of gene nonrandom and non specific and in duplication and gene deletion microorganisms, it is produced by (Normand et a!., 2007). These gene directional mutational pressure and duplication and gene deletion are natural selection acting at the level of associated with host diversity, translation (Gouy and Gautier, 1982). biogeographic location of the strains The strength and direction of these and its their life cycle pattern (Sur et forces fluctuate within and across the a!., 2008). From these whole genome organisms; depending on their life style sequence data of Frankia strains it was and genomic G+C content. The evident that the strain EANI pee had an effective number of codon (Nc) MATERIALS AND METHODS s6 (Wright, 1990) and codon adaptation dendrogram was constructed to find index are alternate measures of such out the diversity amongst them. All the biasness in organisms. The codon protein coding genes, ribosomal adaptation index (CAl) is a relative protein genes, and heavy metal measurement of a gene with a resistances were analyzed by the particular set of reference gene, that software CodonW (Ver. 1.4.2) (http:// also denotes the biasness at rnRN A www.molbiol.ox.ac.uk/ level (lkemura, 1981; Sharp and Li, win95.codonW) and CAl calculator 2 1987; Dos Reis eta/., 2003; Wu eta/., (http://www.evolvingcode.net/codonl 2005). CalculatorCAis.php). The effective The heavy metal resistance system is number of codons (Nc) was calculated an important survival strategy for the as per Wright (1990). This index is a bacteria. This system arose soon after simple measure of overall codon bias. the life began (Silver and Phung, 1996) Its value represents the number of and attained a good diversity. The aim equal codons that would generate the of this study was to explore the same codon usage bias that was diversity of the heavy metal resistant observed. Under random codon usage gene, their codon use pattern and the expected value of Nc was predict the expression level of that calculated by the following formula of gene in comparison with protein Wright, (1990):

2 coding and ribosomal protein genes. Nc=2 +S+ {29/[1 {s'+(+ (1-S/) }} ••••••••••• (1) The complete genome sequence of Where, S represents GC3 values. three Frankia strain were obtained A commonly used measure of codon from IMG website (http:// bias in prokaryotes and eukaryotes is www.img.jgi.doe.gov) (Markowitz et the codon adaptation index (CAl). It is a/., 2006). The GenBank accession a measurement of relative adaptedness number of the strain EAN1pec is NC of a gene's codon usage towards the 009921, ACN14a is NC 008278, and codon usage of highly expressed genes. Ccl3 is NC 007777. At first a multiple The relative adapted-ness (ro) of each sequence alignment was performed codon is the ratio of the usage of each using CLUSTAL W taking the codon, to that of the most abundant nucleotide sequences of the studied codon within the same synonymous heavy metal resistance genes and a family. MATERIALS AND METHODS 57 CAl (Sharp and Li, 1987) is calculated protein gene. as follows: 3.9 Isolation of Genomic DNA of Frankia 3.9.1 Isolation of Genomic DNA From CAl= exp(_!_ ±In mk) ... (2) Pure Culture L k=I Following protocol (Bajwa, 2004) was

Where, rok is the relative adapted-ness followed for isolation of genomic DNA of the kth codon and L designate the from pure culture of Frankia : number of synonymous co dons in the • One mL of I month old Frankia gene. culture was taken in a micro The software CodonW was developed centrifuge tube. by Peden (1999) which is used to • The tube was centrifuged at calculate the GC3 and effective I OOOOrpm for 10 minutes and the number of codon. The GC3 denotes the supernatant was discarded. number of guanine and cytosine in the • The pellet was resuspended in lmL third position of the codon and the of TE buffer (pH 8) (see appendix effective number of codon produce a for composition) and transferred to overall picture of codon biasness. In a 1.5mL microcentrifuge tube highest conserved organism the value (Tarsons, India). of effective number of codon is 20. • It was centrifuged at I OOOOrpm for That indicates the use of a single codon 15 minutes and the pellet was for a particular amino acid, when other dissolved in lmL of TE buffer (pH alternative options are available. In 8.0) highest biased case the value is 61. It • The solution was forced through signifies that the particular organism is 5m1.. sterile syringe needle of 0.56 utilizing all the codon equality. The X 25mm to break the colonies. codon adaptation index or CAl is a • To this solution !Omg/mL of measure of codon uses within a gene molecular biology grade lysozyme relative to a reference set of genes ( Sigma, USA. Cat#L-6876) and a (Sharp and Li, 1987). Its value ranges pinch of acromopeptidase (Sigma, from 0 - 1. The high value indicates USA. Cat#A3422) were added and that the expression of the gene of was incubated at 20°C for an hour. interest is similar to the reference set of • 250J.LL of 20% SDS was added to gene, which is a generally ribosomal this solution and it was incubated at MATERIALS AND METHODS 58

60°C for 30 minutes and at room 3.9.2 Isolation of Genomic DNA of temperature for 15 minutes. Frankia From Host Nodules • The solution was then divided into Five A. nepalensis and five C. two equal parts of 625!-lL each. equisetifolia trees were chosen from • To each tube equal volume of different collection sites. The A. molecular biology grade nepalensis trees were selected from equilibrated phenol (SRL Cat# different locations of Darjeeling hills. 1624262) was added and mixed Plants were selected from Mirik (A1), gently. Fatak (A2), Sukbiapukhuri (A3), • The tubes were then centrifuged at Ghoom (A4) and Sonada (AS). C. 1OOOOrpm for 10 minutes and the equisetifolia were selected from upper aqueous phase so obtained Jalpaiguri (Cl), North Bengal was taken to a fresh tube. University Campus(C2), • An equal volume of chloroform Diamondharbor(C3), Bokkbali (C4) (600!-lL) (E Merck Ind Ltd. and Digha (CS) region of south 24- Cat#822265) was added gently and Pargana district. centrifuged at I OOOOrpm for 5 Procedure minutes. • About 0.5 grams of tap water • The upper aqueous phase was taken washed nodule tissue were taken in in a fresh tube and to it 360 IlL of a I OOmL beaker. isopropyl alcohol (E Merck Ind. • The nodule tissues were incubated Ltd. Cat# 17813) was added and with I OmL of TE buffer the tube was kept in room supplemented with 2% activated temperature overnight. charcoal for 24h. • The tubes were then centrifuged at • After incubation the tissues were 4°C for 20 minute at 12000rpm The taken out from the TE buffer and supernatant was discarded. washed with distilled water and • The pellet was washed with 70% dried properly with a paper towel. ethanol. • The nodule tissues were grinded • Finally the pellet was dried in into a fine powder using liquid vacuum desiccators and the DNA nitrogen and a mortar and pestle. was resuspended in I 0 IlL of Liquid nitrogen facilitates the pyrogen free water. grinding at extremely low MATERIALS AND METHODS 59 temperature (- 196°C). rpm for 10 minutes and upper • Transfer the powder in to IS mL of phase was taken in a fresh tube. prewarmed (at 65°C) DNA • In this tube, double volume of ice extraction buffer (see appendix II cold ethanol and 100 J.!L of for composition). Ammonium acetate (1OM) (Merck, • Incubate it for 1hr in 65°C. Cat no#61750105001730) was • Add equal volume of chloroform: added and the mixture was isoamyl alcohol (24: 1) to it and incubated for 2hr in -2o•c and mixed it properly. The mixture was centrifuge for 12,000 rpm for 30 centrifuged for I 0 minutes at 6000 minutes at 4•c. rpm. • The supernatant was discarded and • Take the upper phase to a fresh the pellets were washed with 70% tube. cold ethanol. • To this tube, 0.6 volume of ice cold • Finally the pellets were dried and isopropanol was added and stored resuspended in 20J.!L of TE buffer at -20°C over night. and stored at -20° C for further use. • In the next day DNA was The DNA extracted by the above precipitated at 10,000 rpm for 10 procedure was contaminated with minutes. RNA, so the extracted DNA were • The pellet was dried and dissolved treated with RNase to digest the RNA in SOOJ.!L of TE buffer and transfer present in the preparations. in to 1.5 mL centrifuge tube. 3.10 RNase Treatment of DNA Sample • Equal volume of phenol: In the process of RN ase treatment, chloroform: isoamyal alcohol IOmg /mL RNaseA (Sigma Cat# R- (25:24:1) was added to the tube and 4875) were added in distilled water and mixed properly. The tubes were boiled for 20 minutes to dissolve. The centrifuged at 1O,OOOrpm for I 0 solution was then cooled and stored in minutes and upper phase was taken at -20°C for further use. in a fresh tube. The following steps are taken to purify • Equal volume of chloroform: the DNA with RNase A solution: isoamyl alcohol (24:1) was added • In the DNA samples isolated so far, to the tube and mixed properly. The RNase A solution were added @ tubes were centrifuged at 10,000 SOJ.!g/mL and incubated for one MATERIALS AND METHODS 6o hour at 3 7°C in dry bath( Genei, dissolved in 2.5M aqueous solution of India). NaCI. • The RNaseA treated DNA samples The following steps are taken to purify were extracted with equal volume the DNA with PEG 8000 of phenol: chloroform: isoamyl (Polyethylinglycol 8000, Hi-Media, alcohol (25:24:1). The tubes were Cat no #RM7402): centrifuged at IO,OOOrpm for IO • DNA samples were taken and make minutes and upper phase was taken the volume up to 50 J.1L by adding in a fresh tube. fresh TE buffer. • Equal volume of chloroform: • In this tube 50 J.1L of 20% PEG isoamyl alcohol (24:1) was added solution was added and mixed to the tube and mixed properly. The properly. tubes were centrifuged at • The mixture was incubated at 37°C lO,OOOrpm for IO minutes and for I5 minutes. upper phase was again taken in a • The DNA-PEG mixture was fresh tube. centrifuged at I7,OOOrpm for I5 • In this tube double volume of ice minutes at room temperature. cold ethanol and 100J.1L of • The supernatant was discarded Ammonium acetate was added and carefully. centrifuged for 12,000 rpm for 30 • In this tube I25 J.1L of ice cold 80% minutes at 4 °C. ethanol was carefully added at the • The supernatant was discarded and wall of the tube and washed twice. the pellets were washed with 70% • Ethanol was dried off completely. cold ethanol. • The DNA was resuspended in 25 • Finally the pellets were dried and J.1L of fresh IX TE buffer and resuspended in 20J.1L of IX TE stored at -20° C for further use. buffer and stored at -20° C for 3.12 Purification of DNA By Glass Milk further use. • In I L glass beaker, 400g of The RNase treated DNA was further borosilicated glass powder was purified with PEG, glass milk and resuspended in 800mL double Sigma kit to eliminate plant phenolics. distilled water. 3.11 PEG Purification of DNA • The preparation was stirred for I Requisite amount of PEG8000 was hour. MATERIALS AND METHODS 61 • Stir plate was then turn off and • Continue washing and spinning of allowed the slurry to settle for 90 glass particles was continued and minutes. spinning as above until the pH of • The supernatant was removed, the slurry was neutral. which contained the fine glass • After slurry was neutralized, the particles, which can be used for glass particles were finally pelleted glass milk, and put into a 250mL by spinning at 6000rpm for 10 centrifuge bottle. minutes. • The glass particles were pelleted by • The glass pellet was resuspended in spinning at 6000rpm for 10 double distilled water to make 50% minutes. slurry based on volume. • The pellet was resuspended m • The glass milk solution was stored 250mL double distilled water. at room temperature. • Concentrated nitric acid was added • DNA solution was taken in a in a to the glass particles suspension 1.5mL microcentrifuge tube. and make it 50%. • Glass milk solution was vortexed to • The solution was stirred and heated resuspend. gent! y and turns on the heat to • In a tube l11L glass milk solution "high". was added per l11L DNA and • The temperature was brought near mixed well and 3 volume of the boiling point of the slurry and saturated Sodium iodite solution then turn off the heat. was added. • The slurry was brought back to • The mixture was incubate for 5 room temperature with continuous minutes at room temperature and stirring. mixed occasionally to keep the • The glass particles were pelleted as glass milk in suspension. in step 5. • The tubes containing the solution • The glass particles were were centrifuged at full speed for 5 resuspended in 250mL of double seconds. distilled water. • Pellets were washed for 3 times • The glass particles were pelleted by with S0011L wash solution and spinning at 6000rpm for 10 centrifuged at full speed for 5 minutes. seconds. MATERIALS AND METHODS 62 • The pellets were dried at room minute. The elute was discarded temperature for 5-l 0 minutes. and the collection tube was • To elute the DNA, glass pellet was retained. resuspended the in an equal volume • Binding colunm was replaced into of I X TE buffer. the collection tube and 0.5mL of • Spin tube for 30 seconds at top wash solution was applied in to the speed in a microfuge to pellet glass column and the column was milk. centrifuged at 12,000rpm for I • Supernatant was carefully removed minute. The elute was discarded with eluted DNA into a fresh tube and the collection tube was and stored at -20° C for further use. retained. 3.13 Purification of DNA with Kit • Binding colunm was replaced into The DNA was finally purified by the collection tube. The colunm GenElute™ PCR Clean-up Kit was centrifuged at 12,000rpm for 2 (GenElute™ PCR Clean-up Kit, PLN- minute, without any additional 70) provided by SIGMA, which are as wash solution, to remove excess follows (as per the guide line provided ethanol. The residual elute as well by SIGMA): as collection tube was discarded. • A GenElute™ m1mprep binding • The colunm was transferred to a

colunm with a blue o-ring was fresh 2mL collection tube and 50~1 inserted info a collection tube. In of elution solution or purified each miniprep colunm, 0.5mL of sterile water was added to the the colunm preparation solution centre of each colunm. The tube was added to each miniprep was incubated at room temperature column and centrifuged at for I minute. 12,000rpm for 30 seconds to I • DNA was centrifuged at 12,000rpm minute. Elute was discarded. for !minute. The eluate is ready for • In this tube 5 volume of binding immediate use or storage at -20°C. solution was mixed with 1 volume 3.14. Quantification of DNA of DNA solution and the solution Quantification of DNA was done with mixture was transferred to the UV vis-spectrophotometer (Thermo binding colunm. The colunm was Electro Corporation, England). centrifuged at 12,000rpm for 1 Spectrometer for DNA measurement MATERIALS AND METHODS was set to the wave length 260 run and C12H2oBrN3 MW 394.33, Sigma 280nm. Cat #E 8751) was added to the gel Procedure for the staining of DNA. 600J.1L of IX TE buffer was taken in a • The gel was cast on a gel platform clean cuvette as blank (-ve control), set (100x70mm) (Tarsons, Cat no # zero. Six (6) JlL of buffer was replaced 7030) keeping the well end towards with same amount of DNA and mixer the cathode side. thoroughly. Optical density (OD) was • The gel was submerged in tank by taken at both 260nm (OD260) and 280 adding IX TBE buffer (See nm(OD2so). The ratio of OD26o/OD2so appendix II for composition). was calculated to assess the purity of • Carefully 2J.1L of DNA was mixed DNA. with 3J.1L of loading buffer 3.15 Agarose Gel Electrophoresis of (Typeiii, Sambrook and Russell, Genomic DNA 2001) (For composition and The size of the isolated DNA samples preparation see appendix II) and were analyzed by agarose gel was loaded into wells. electrophoresis as described below: • Lambda DNA /EcoRII Hind III • Agarose gel was prepared using double digest was used as a molecular biology grade, DNase molecular marker (Genei Cat no free agarose (gelling temperature #MBD 31). 36°C) (Sigma Cat no # A 9539). • The DNA was made to run a For agarose gel electrophoresis of constant volt of 60V applied with genomic DNA as well as PCR Electrophoresis Power Supply Unit product 0.8% gel is used. (Tarsons, Cat no# 7090). • For 30mL gel, 0.24 gm of agarose • After one hour of run, the gel was was dissolved in 30mL of IX TBE viewed on a UV Transilluminator buffer (See appendix II for (Genei, Cat no# SF850). composition) by heating m 3.16 Development of A New 16s Primer microwave oven (LG make). Sequence for Frankia • The gel was cooled up to 40°C and DNA obtained from the root nodules of 0.5Jlg/mL Ethidium Bromide (3,8- A. nepalensis and C. equisetifolia were diamino-1 0-ethyl-9- composed of mixture of DNA from phenylphenaanthridinium bromide, the host plant, the Frankia and possibly MATERIALS AND METHODS contaminating bacteria, which might and Frankia sp. EANlpec (NC not have been eliminated during the 009921) available in GenBank (http:// surface sterilization and peeling of www.ncbi.nlm.nih.gov/sites/entrez? nodules. Hence Frankia specific db=genome and cmd=search primers were required for obtaining andterm=Frankia). The efficiency of exclusive Frankia genome band. A the primers is were checked with the new I 6s specific PCR primer sequence software 'In Silico PCR was developed with the help of in Amplification' (http://insilico.ehu.es/ sillico PCR amplification of Frankia. PCRI). (Bikandi et al., 2003). A 520 Initially primers FGPS989ac (Bosco et bp long amplicon was expected. al 1992) Annealing temperature depends upon 5'GGGGTCCGTAAGGGTC3' and the size and nature of the primers and primer FGPSI490 (Normand et al., was calculated with the following 1996) were chosen. However, the formula. primer pair failed to amplify any of the Annealing temperature ("C) = complete Frankia genome sequence in {2(A+T) +4(G+C)} -5 silico. Therefore the primers have been Though the two primers (Forward and modified suitably to make them Reverse) had different annealing compatible to Alnus and Casuarina temperature, the lowest one (58°C) was isolates and modified accordingly that used. can amplify the reference strains 3.17 PCRAmplification of DNA ACN14a and Ccl3 only. These The Polymerase Chain Reaction was modified primers were originally carried out in a 50 !!L volume. The designed, synthesized commercially. reaction mixture contains the following The primers were as follows: forward ingredients:- -5'-GGGTCCGTAAGGGTC-3' The dNTP mixture = 0.2 11M of each and reverse dNTP. 5'-AAGGAGGTGATCCAGCCGCA- Taq DNA polymerase = 2U per 3'. The software Priroer3 (Rozen and reaction. Skaletsky, 2000) developed those Primers = 1 11M each. primers from the 16s sequence of PCR Buffer (lOX)= 5 !!L per reaction. Frankia sp. Ccl3 (NC 007777), Template DNA= l!!L per reaction(< 1 Frankia alni ACN14a (NC 008278) !!g/1 00 !!L). MATERIALS AND METHODS 65

The reaction mixture was adjusted to a • For 30mL gel, 0.24 gm of agarose final volume of 50!11 by adding was dissolved in 30mL of IX TBE pyrogen free water. The polymerase buffer (See appendix II for chain reaction was performed on a composition) by heating m Applied Biosystem thermal cycler microwave oven (LG make). (model 2700). The amplification • The gel was cooled up to 40°C and reaction was performed with a negative 0.5!lg/mL Ethidium Bromide (2, 7- control. This negative control did not diamino-1 0-ethyi-9- contain any template DNA. The phenylphenaanthridinium bromide, amplification cycles consisted of CnHzoBrNJ, MW 394.33, Sigma following steps: Cat #E 8751) was added to the gel 1. First Cycle: Denaturation at 94°C for for staining of DNA. 5min, primer annealing at 58°C for 2 • The gel was cast on a gel platform minute and primer extension at 72°C (100x70mm) (Tarsons, Cat no # for 2 minute. 7030) keeping the well ends 2;Cycle 2-34: Denaturation at 94°C for towards the cathode side. The gel lmin, primer annealing at 58°C for 2 was submerged in tank by adding minute and primer extension at 72°C IX TBE buffer (See appendix II for for 2 minute. composition). 3. Cycle 35: Denaturation at 94°C for • 101-11 of DNA was mixed with lmin, primer annealing at 58°C for 2 2.5!11 of loading buffer (Typeiii, minute and primer extension at 72°C Sambrook and Russell, 2001) (For for 7 minute. composition ·and preparation see 3.17.1 Electrophoresis of Amplified DNA appendix II) and was loaded into The PCR products were analyzed by wells. agarose gel electrophoresis as • Lambda DNA EcoRI/ Hind III described below: double digest and 1kb DNA marker • Agarose gel was prepared usmg were used as a molecular marker molecular biology grade, DNase (Genei Cat no #MBD 31). free agarose (Sigma Cat no # A • The DNA was made to run at 9539). constant volt of 60V applied with • For agarose gel electrophoresis of Electrophoresis Power Supply Unit PCR product 0.8% gel is used. (Tarsons, Cat no# 7090) MATERIALS AND METHODS 66 • After two hour of run the gel was was centrifuged at 12,000rpm for 1 viewed on a UV Transilluminator minute. The elute was discarded (Genei, Cat no# SF850). and the collection tube was • The size of the PCR product was retained. determined with UltraLum Gel • The column was again centrifuged documentation software. at 12,000rpm for 2 minute, without 3.18 Purification of PCP Product any additional wash solution, to The PCR products were further remove excess ethanol. The purified by GellElute™ PCR Clean-up residual elute as well as collection Kit (GenElute™ PCR Clean-up Kit, tube was discarded. PLN-70p) rovided by SIGMA as per • The column was transferred to a the guide line provided by SIGMA, fresh 2mL collection tube. 50f.lL of which are as follows: elution solution or water to the • A GenElute m1mprep Binding centre of each column. Incubate at column with a blue o-ring was room temperature for 1 minute. inserted into a collection tube. . • DNA was centrifuged at 12,000rpm 0.5mL of the column preparation for lminute. The elute was ready solution was added to each for use or stored at -20°C. miniprep column and centrifuge at 3.19 Digestion of PCR Products 12,000rpm for 30 seconds to 1 • In a sterile microcentrifuge tube minute. Elute was discarded. 1Of.lL of PCR amplified product • Five volumes of Binding solution was taken. was mixed with l volume of PCR • In this tube 2f.lL of 1OX restriction reaction mix and the solution was enzyme digestion buffer (supplied transferred to the binding column. with Bangalore Genei Pvt Ltd • The column was centrifuged at restriction enzyme) was added and l2,000rpm for 1 minute. mixed thoroughly. • Elute was discarded and the • The PCR products were subjected collection tube was retained. to 5U of restriction digestion with • Binding column was replaced into various restriction enzymes, which the collection tube. 0.5mL of includes Alui, Taq I, Hae III, Mbol diluted wash solution was applied and Mspi (Bangalore Genei Pvt in to the column and the column Ltd) (Table 3.1). MATERIALS AND METHODS 67 Table 3.1: List of Restriction enzymes used in RFLP study Restriction Enzymes Sequence Reaction Volume Reaction Tern- Amount of with Cat# (Bangalore perature ('C) REused Genei, India) Alu I (MBE -17-S) AGLCT 20Jll 37 5U Taql ( MBE -7S) TtCGA 20Jll 65 5U Haeiii ( MBE -I OS) GGLCC 20Jll 37 5U Mho! ( MBE -27S) LGATC 20Jll 37 5U Msf!.l ( MBE-31S) qcoo 20[!1 37 5U • The tubes were incubated in a dry Minisize 50bp DNA ladder (Norgen bath at indicated reaction Biotech, Canada, Cat # 11200) was temperature for 1 hour. used as molecular weight standards. • The reaction was stopped by The gel was viewed on a UV adding 0.5 M EDTA (pH 8.0) to a Transilluminator (Bangalore Genei, final concentration of 1OmM. The Cat no# SF850) and photographed digested DNA was stored at -20'c using Kodak digital science DC 120 for further use. digital camera. 3.19.1 Analysis of Restriction Fragments 3.19.2 Scoring of data RFLP analysis was done performing The PCR RFLP data was scored as the gel electrophoresis of the digested present (1) or absent (0) using the product. A 2 %(w/v) molecular biology SIMQUAL program and DICE grade high resolution agarose (Sigma coefficients. A matrix was computed Cat no # A 9539) gel was prepared in and dendograme was developed by a IX TBE buffer for a 15x10 em casting UPGMA clustering of the POPGENE tray. Electrophoresis was done in mini to calculate the diversity among the submarine electrophoresis unit (Tarson Frankia strains by Nei genetic Cat # 7050). 20 J.lL of digested DNA diversity (N ei, 1987), Shannon index was electrophoresed in 3V/cm electric (Shannon, 1948; Weaver and Shannon, field. 1949). Chapter 4 RESULTS & DISCUSSION

4.1. Isolation of The Bacterium contaminant and the vegetative The cortical layers of actinorhizal root bacterial and fungal cells could be nodules are heavily contaminated with killed at high rate but the chemical soil microorganisms. The growth rate resistant bacterial spores might of which is generally faster than the withstand this treatment and they might Frankia. So, the prerequisite for germinate later on in culture medium. successful isolation of Frankia was to When the nodule lobes were incubated eliminate these contaminants without with rich Qmod (Lalonde and Calvert, damaging the endophyte in the cortical 1979) medium for 48 hr at 30°C tissue. Of the three procedure tried to temperature the spores might germinate isolate the endophyte, procedure III and the nascent vegetative cells might (Bose and Sen, 2006) was found as the be killed by subsequent chemical most suitable for easy isolation of treatment (Bose and Sen, 2006). Frankia. In this procedure a series of Another problem in Frankia isolation mild and easily available surface was the presence of plant phenolics sterilizing agents were used to which leached out in the medium from eliminate these contaminants, which the piece of nodule during isolation. was followed by incubation in rich The plant phenolics were potential Qmod (Lalonde and Calvert, 1979) inhibitor of the growth of Frankia. The medium for 48 hr at 30°C temperature activated charcoal minimized the effect with 3% activated charcoal. The of phenolics by absorbing it and thus surface sterilizing agents used were increased the efficiency of Frankia useful against the bacterial and fungal isolation. RESULTS AND DISCUSSION

The advantage of using Qmod but Vit B12 at 0.2% concentration in

(Lalonde and Calvert, 1979) in the post aqueous solution (lmLIL) and Vit B1 at isolation maintenance of the bacterium 0.5% concentration in aqueous solution was its nutritional richness, in which (I mLIL) gave the best result. any contamination can not remain in 4.2 Germination of Seeds of Actinorhizal dormant condition. Thus, Qmod Plants (Lalonde and Calvert, 1979) medium The seeds of Alnus nepa/ensis and visibly ensure an absolute Casuarina equisetifo/ia need different contamination free condition for pure conditions for germination. No special culture. Defme propionate minimal treatment was needed for germination medium (DPM) (Baker and of C. equisetifolia seeds and they were O'keefe,1984) is also a very good germinated on sterile distilled water in medium for maintenance of Frankia floating condition. cultures but this medium have some Generally, the seeds of A. nepalensis disadvantages in isolation of the need a special kind of treatment in the endophytes. The absence of nitrogen in form of overnight soaking in aerated the DPM (Baker and O'keefe,l984) water for better germination. The helped selectively in the growth of treated and non-treated conditions, Frankia while the spore forming media nutrients and contamination of contaminants remain in the dormant fungus do not have any effect on the condition for a long period. These duration of seed germination. In all the contaminants might germinate latter cases the seeds germinated within 4 while transferring in nutritionally rich weeks. Contaminations were reported medium. in mineral rich medium under treated Frankia colonies came as minute and non-treated conditions. The cottony white dots at the bottom of the percentage of germination varied with tube after one month incubation in the media used (Fig: 4.1) in A. dark. Each colony was collected and nepalensis seeds. Maximum subcultured m separate tubes germination was observed in Hoagland containing Qmod (Lalonde and media for both treated and non treated Calvert, 1979) medium. Although seeds while minimum in Y:. Murashige several concentrations of VitB 12 and and Skooge medium (MS) for treated

VitB 1 were played in isolation medium and Y:. woody plant medium (WPM) RESULTS AND DISCUSSION 70 for non treated seeds. The germination were observed in the media was probably inhibited by high nutrient supplemented with IBA 3, 4 and 5mg/ concentrations. For treated seeds, L and NAA + IBA (I+ 1 mg/L). The maximum (80%) survival rate was best result was recorded in the medium reported in Hoagland media while supplemented with 4mg/L IBA. In the minimum (0%) survival rate was medium supplemented with NAA + reported in WPM. For non-treated IBA (2+2 mg/L) rooting was poor but seeds, maximum (100%) survival rate the roots showed good growth. was reported in Hoagland while In the study of seed germination in m1mmum (20%) survival rate was sterilized vermicompost, good reported in half strength WPM. germination rate was reported within Moderate germination and survival rate 10 days but the seedlings did not with low contamination were reported survive. This death event was due to in water used as germination medium poor root development. In double layer (Fig 4.2). culture experiment both the The hormones had profound but germination percentage and survival variable effect on the seed germination rate were very low although the media and rooting of A. nepalensis. IBA and contained required hormones as NAA+IBA were found to be more described in earlier experiments. effective than NAA in terms of These experiments reveals that germination percent and rooting. germination of A. nepalensis seedlings Moderate germination percentages have specific hormonal requirements. (above 50%) were observed in the These hormonal requirements are media supplemented with IBA I and 2 fulfilled by mycorrhizal or associative mg/L and NAA+IBA 1+1 and 2+2 mg/ fungi during germination and growth in L. Maximum (90 %) germination was nature. In vermicompost the Alnus observed with the application of IBA seedlings did not survive though there (1 mg/L) and NAA + IBA (1+1 mg/L). were plenty of nutrients. The nutrients The germination percent increased with were not absorbed due to poor root the increase of NAA concentration development which require external while declined with the increase of hormonal supply. This plant A. IBA and NAA + IBA concentrations. nepalensis, thus begins its life with an Good rooting and good growth of roots association with fungi and at its RESULTS AND DISCUSSION 71

Germination percentage 100 90 -"" T \ r\ m 80 Cl \ \ sc 70 m u \ ~ 60 m c. \ I. c 50 ..0 \ \ ~ 40 c E 30 \ m~ \ \ CJ 20 ' ...... j v \ ~ 10 / "'-"' 0 ' ' ' ' ' I I I I I 1 4 5 1 2 3 1+1 2+2 3+3 4+4 NAA+IBA Honnones in mg/1

Fig 4.1: Effect of various hormone concentrations on A. nepalensis seed germination percentage.

120

100

80

60

40

20

0 1 2 3 4 5 6

Fig 4.2: Generation time (G time), Generation percentage and survival rate of treated and untreated A. nepalensis seeds in different medium. RESULTS AND DISCUSSION 72

Fig 4.3: a) A young C. equisetifolia seedling with nodule; b) A young A. nepalensis seedling with nodule: c) A young root hairs show ignition of root hair deformation due to Frankia infection: d) A young root hairs show typical hook shaped root hair deformation due to Frankia infection. maturity make another association for in A. nepalensis and C. equiset~folia surviva l with an actinomycetes bacteria seedlings grow in aseptic condition. Frankia. C. equisetifolia, did not have This indicates the presence of Frankia thi s problem. in the medium. 4.3 Plant Infectivity Test 4.4 Colony Morphology T he c ha racterist ic root hai r Medium used: Defined propionate deformation and visible nodule minimal medium (Baker and O'keefe, fonnation (Fig 4.3) were observed both 1984) RESULTS AND DISCUSSION 73

Reaction pH: 7 4.5 Field Emission Scanning Electron A. Size: Filamentous. Microscopy Temperature: 28°C. Age: 30 days The hyphae, sporangium, and vesicles B. Motility : Not observed are the most important structures of C. Sporangia and spores: Present, Frankia. The multilocular sporangium, sporangia multilocular, spore round round shaped vesicle structures were shaped smooth walled in older culture. found. The structure of sporangium 2mM Pb increase sporulation. was ±1.5 x 0.7 !!· The mean diameter D. Agar colonies : of the hyphae was ± 0.5!!. The mean Temperature : 28°C diameter of vesicle was ± 0.7!!. These Age: 14 days. results were nicely fitted with a Location : Deep colony classical frankial structure (Fig 4.4a & Form : Spindle shaped 4.4b). Edge : Filamentous 4.6 Biochemical Characters of Frankia Optical character: Opaque Eleven Frankia strains were selected Pigmentation : Red in 2mM Pb and used in the study of physiology of concentration. the bacterium. Out of these eleven E. Broth: strains six were isolated from C. Temperature : 28°C equisetifolia and the rest from A. Age: 14 days. nepalens is.

Odor :None The three Frankia strains (CeSil 0, Clouding : Absent CeSi11, CeSi 12) isolated from C. Surface growth: Absent equisetifolia showed rapid Sediments : Flanky decarboxylation activity with Amount of sedimentation: Scanty propionate and pyruvate and very slow F. Forms and arrangement: decarboxylation activity with acetate Filamentous and citrate. Decarboxylation leads to a • G. Gram staining : Positive rise in pH assessed as change in colour H. Biological relationship: Symbiotic of the indicator from yellow - orange to to actinorhizal plants. red. These strains showed high I. Isolated From : DPM (Baker and protease activity which were O'keefe, 1984) lead plates containing demonstrated by the liquefaction of 2mM Lead. (Bose and Sen, 2005) gelatin. None of these strains showed~ RESULTS AND DISCUSSION 74 -glucosidase activity. They could not recognize tween compounds which 0 utilize a variety of organic acids and a include ACN1A , EANlpec, and Eu!Ic few carbohydrates. They had utilized etc. A possible explanation of the propionate, acetate, succinate, sucrose difference between the two groups of and fructose as sole carbon source as Frankia is that they lack, an active they produce visible colonies within 60 carbohydrate transport system (Prof. days of incubation, but they can not Normand, personal communication). utilize lactose, glucose, pyruvate, From the above result it could be mannitol, tween80 and citrate. concluded that the three strains namely The other three Frankia strains (CeSt2, CeSilO, CeSill and CeSi12 belongs to CeSt5, CeSt9) isolated from C. the physiological group 'A' of the equisetifolia showed no Frankia which is more saprophytic decarboxylation activity with than symbiotic while the other strains propionate, pyruvate, acetate and namely CeSt2, CeSt5, CeSt9 belongs citrate. None of these strains showed P­ to the physiological group 'B' of the glucosidase activity, though they Frankia which are physiologically less showed very slow protease activity. active and more symbiotic. Overall the They could utilize propionate, acetate strains utilize the propionate and and succinate as sole source of carbon acetate as best carbon source. So in this but they could not utilize glucose, area results showed diversity among pyruvate, mannitol, tween 80, citrate, the Frankia on the basis of standard sucrose, fructose and lactose. All the physiological parameters. These tests above experiments were done in can be a useful addition to the methods triplicate and same results were found . used in differentiating Frankia strains The results are summarized in table 4.1 isolated from C. equisetifolia. and table 4.2. Similar physiological experiments It was very interesting to note that were also performed with the Frankia these Frankia strains could not utilize strains isolated from A. nepalensis. the tween compounds which is a very Two Frankia strains (AnMrl, AnMr 5) common carbon source for Frankia. isolated from the root nodules of A. But non utilization of tween compound nepalensis growing in Datjeeling Hills is not uncommon in Frankia strains. showed rapid decarboxylation activity Several well recognized strains could with propionate and pyruvate and RESULTS AND DISCUSSION 75

Fig 4.4: a) FESEM picture of multilocular sporangia of Frankia; b) FESEM pic­ ture of vesicle of Frankia another three Frankia strains (AnMr2, decarboxylation activity with AnMr2a AnMr2b) isolated from the propionate and pyruvate and the strains root nodules of the same plants also AnMr2b showed slow decarboxylation growing in the same area showed no activity with acetate and citrate as RESUlTS AND DISCUSSION

Table 4.1: Utilization of different carbon source by Frankia isolated from A. nepalensis and C. equisetifolia. Alnus specific strains AnMrl AnMr5 AnMr2 AnMr2b AnMr2a Propionate + + + + + Pyruvate + + + + + Acetate + + + + + Succinate + + + + + Glucose (-) (-) (-) (-) (-) Sucrose (-) (-) (-) (-) (-) Lactose (-) (-) (-) (-) (-) Fructose (-) (-) (-) (-) (-) Tween 80 + + + + + Glucose+ Tween 80 (-) (-) (-) (-) (-) Mannitol + + + + + Citrate + + + + + Casuarina specific Frankia strains CeSi 10 CeSi II CeSi 12 CeSt2 CeSt5 CeSt9 Propionate + + + + + + Pyruvate (-) (-) (-) (-) (-) (-) Acetate + + + + + + Succinate + + + + + + Glucose (-) (-) (-) (-) (-) (-) Sucrose + + + (-) (-) (-) Lactose (-) (-) (-) (-) (-) (-) Fructose + + + (-) (-) (-) Tween 80 (-) (-) (-) (-) (-) (-) Glucose+ Tween 80 (-) (-) (-) (-) (-) (-) Mannitol (-) (-) (-) (-) (-) (-) Citrate (-) (-) (-) (-) (-) (-) +-Visible colony occur within 30 days of incubation; (-) -No visible colony occur within 30 days of incubation; shown earlier by Frankia isolated from ~-glucosidase activity. These strains C. equisetifolia. The AnMrl and could utilize a variety of organic acids AnMr5 show high protease activity and a few carbohydrates. They utilized which IS demonstrated by the propionate, acetate, succinate, liquefaction of gelatin while rest of the pyruvate, mannitol, tween80 and citrate strains shows very slow protease as sole carbon source as they produce activity. None of these strains showed visible colonies within 60 days of Table 4.2. Physiological characteristics of Frankia isolated from A. nepalensis and C. equisetifo/ia ACNli\G' AnMrl AnMrS AnMr2b AnMr2 AnMr2a CeSi!O CeSill CeSil2 CeSt2 CeStS CeSt9

Dec. of acetate (-) + + + + + + + + (-) (-) (-) Dec. of propionate (-) ++ ++ (-) (-) (-) ++ ++ ++ (-) (-) (-) Dec. of citrate (-) + + + + + + + + (-) (-) (-) Dec. of pyruvate (-) ++ ++ (-) (-) (-) ++ ++ ++ (-) (-) (-) Protease activity ++ ++ ++ (-) (-) (-) ++ ++ ++ (+) (+) (+) p-glucosidase activity (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

Dec.= Decarboxylation;+= Positive result (witb in two to three weeks);++= High activity (positive result with in two weeks);(+)= Vety slow (positive result with in four "m weeks); (-)=Negative result with in 42 days of incubation; *=Reference strain. c !:j"' "')>z c c (') C/J. cr' 0.. , (') >o (') >o (') 'T1 ~· ~· Cii lXI i:l (') 0 0 :::;: ~· s 0 ~ ::r So 0 ... 8' > (') ::r '< ~ 0 ""'...... "' ~ ~ - "' -~ - c - 0 < ... 0 >::: 1:> ~ ~ ~ i:l § n s ~ , .., ""1:;• ::1 " § a. " s (') " (') - 0 " i:l ... G) ::r - 0 cr' >o ~ "' z ... "s: "'~· " >o ::r "' s - p.. - ~ "' 0 " i:l " " ::r en -0 0 0 i:l ~· 6' t;l a- " .g. So "' m .... 0 "' "'B i:l ... 0 ~· q'"' § CD ... "' 1:> 0.. ~ 0 0.. . "'~=t. z (') cr' "'" " Dl 0 - ~· " " z ~ ""-· .g ..§. >-3 (;' " 0 i:l at> "' 3 c at> 0.. "'0 (') at>- ::r - .," at> 0 e. m 0.. ::r i:l ...... ~· ~· cr' i:l "' ~· - - ~ - 0 ~ (') § " § ...., " .., " (') 0 e; 0 ... " ~ 0 . ... t:;· (') " - (') s 0.. ~· " ~ 0 i:l i:l " So 0.. - 5 -...., Dl ... "' " " cr' >o ,.....,- i:l ... "' ~" ~ 0 e. -s· ~ " ;i "0 0 c:t. 0.. .., § " "' " ~ Cl 0.. (') -~· 0 C/J. ~ " - Dl "' "' >o- " ;i Cj!j at> a to i:l 1:> cr' So 0.. c "0.. C/J. < -~ § "' ... ::r X 0 " 0 So 0.. 0 i:l " 0.. ::1 '< c - ~ ""' l'l If If . eL ~ ~ ·" So " ~· " Dl "' @ .§ :r: " - ~ ~· " 0.. ~· " " "' § ,....., < t:;· "t:: "' ... )> ::1 ~· c ~ cr' :::: ... 2' " Dl :;;· s· 8. - ~ iv ::J, (') ~ ~ - 0 C/J. "" 0 "'" l'l "' " ~ 0 = "' ~ So (') § .? ::r " "t:: Dl ~ ~. g: ~ "' "'" 8 s 0 ~ 1:> 0.. 0.. ..." (') "' 5· ::;" " - 0 ~· -... ~ cr' 0.. C" '< a "' - - ~· "' " 0 " 0 "'c "'c ~· 0.. "' "' " "' "~ "' "'0 '< ... ~· q' "' ...., "'c;;· " 0.. \0 i:l ~ 0.. :::! "' 0 " ~ >oa 0 ~· ... " 0.. - 0 iii' !;l " ~· ...., @ ~ " 0 " "' -" z 0.. -0 "" ... 0 - ~. ::r -c § c \0 c at> ~· - (') " i:l i:l at> - 'T1 - "'" ~ en " " §' - "' 8. 0 y . ~· ~ .P· (') 0 0 ~ c ~ 8 " a 0 " 0 " "' 0 "' 1Jl "' (;' §. -" " ~· "' i:l ~ ~ 0.. N "' "' e. ... 0 - 0 " E. - ~ (') - - \0 s - " at> a 0.. 0 \0 ~ 8. 0.. c:t. "' :e ~· § - " ~· - 0.. - cr' S" (') s· - co -1 "'~· 0 -Dl 8. - - <>" So " a at> c ::r i:l No 8' ~ " co -~ - So l'l So So cr' ~· ... c " .. (JQ ~ - § "' - ? .."'" ;::! 0.. If CD CD n "'!Z 0.. " ~ s i:l !" ~·I :::l ""' -" " "' " " " -"' " " " " " RESULTS AND DISCUSSION

1981) and Wahlund effect (Brown and total number of individuals. The Feldman, 1981). Among the Frankia Shannon index has moderate sensitivity population the total genetic diversity towards the sample size. In the present had been found to be 0.281 (SD ± study the value of Shannon index had 0.0547), which showed low level of been found as 0.3982 (SD= ± 0.3293), genetic diversity. The estimated gene which indicated a moderate level of flow within the population was zero. diversity. The coefficient of gene differentiation Another important diversity among population indicated what measurement index used in this study percentage of total genetic diversity was the single locus component and came from interpopulation and in this and two locus component. The single case, there was no lateral gene transfer locus components are the average and in Frankia strains. So, the entire the variation among the population in genetic diversity came from gene diversity and variance among the intrapopulation source and hence the population in aile! frequency. The two value of coefficient of gene locus component include the mean and differentiation was 1. variance of disequilibria, the Shannon index is used to study the covariance of aile! frequencies, over diversity of the population. It is population and various interactions. assumed that all species are Wahlund effect is used to explain the represented in the sample and they are single locus component and and two randomly sampled. The advantage of locus component. It refers to the this index is that it takes into the reduction in the heterozygocity in the account the number of species and the population caused by subpopulation evenness of the species. The index is structure. The Frankia population increased either by having additional showed Wahlund effect 2.7071 for unique species or by having greater single locus effect and 8.901 for the species evenness. The value of two locus effect. If the subpopulations Shannon index will be maximum if the have the same gene frequency the evenness present in the population is value of Wahlund effect will be zero. maximum. It has specific importance in In this case the value represented very the expression of equal proportion low level of genetic diversity among character in the sample rather than the Frankia population. RESULTS AND DISCUSSION 79 The dendrogram Based on Nei's were plotted against the increasing (1987) Genetic distance (Method = concentration of various heavy metal UPGMA Modified from NEIGHBOR salts (Fig 4.6; 4.7) and the minimum procedure of PHYLIP Version 3.5) inhibition concentrations (MIC) (Fig 4.5) showed that the Frankia calculated (Table 4.3). Figure 4.6 population was divided in to two major shows the growth pattern of Frankia clades. The first clade included the strains isolated from C. equisetifo/ia in bacterium isolated from C. presence of different metal salts. The equisetifolia and the second clade results show that the strain was highly included bacterium isolated from A. resistant to cadmium chloride (5mM), nepa/ensis. Each clade was again sub moderately resistant to cobalt chloride divided into two minor clades. This and lead nitrate (3mM) and less pattern of the dendrogram supported resistant to nickel chloride and copper the presence of two physiological sulphate (0.5 mM). This results were groups among the bacteria isolated compared with the American strains from each host. and found that the present strain was Thus from the result of Nei genetic much more tolerant to cadmium diversity (Nei, 1987), Shannon index chloride and cobalt chloride. While the (Shannon, 1948; Weaver and Shannon, MIC value of USA strains Ccl3 and 1949), single locus component and two Cc117 for CdClz was 0.1 mM and for locus component (Brown and Feldman, CoClz 0.1 and 0.05 mM respectively. 1981) and Wahlund effect (Brown and This strain showed a MIC value of 5

Feldman, 1981), it can be concluded and 3 mM for CdClz and CoC12 that that the Frankia population present respectively. However, USA strains in this area shows very low genetic grew at an elevated concentration of diversity and the total genetic diversity CuS04, NiCiz and Pb (N03) 2 than the was intrapopulation in origin and studied strains (Richard et a/., 2002). divided into two different physiological The heavy metal resistant Frankia group (Fig4.5). (Bose eta/., 2011) colonies were re-isolated from various

4.8 Heavy Metal Salt Resistance Pattern plates and preserved for further studies. Each experiments were repeated twice Heavy metal salt resistant colonies of and were found to produce similar Frankia appeared after around 14 days results. The strain used in the study of incubation. The numbers of colonies RESULTS AND DISCUSSION So

+CeSi10 +--1 +------2 + CeSl11 ! 2462 ! + CeSi12 +-----9 284 + CeSt2 +--3 +------4 + CeSt5 24.62 ! -10 + CeSt9

+ AnMr1

+------59 11 ! · +AnMr5 +------9 18.34 ! + AnMr2 +--6 +------7 +AnMr2a 9.11 ! + AnMr2b

Fig 4.5: Dendrogram Based on Nei's (1987) Genetic distance showing the diversity of Frankia based on physiological results: Method = UPGMA Modified from NEIGHBOR procedure ofPHYLIP Version 3.5. Numbers indicated in the bottom of the lines are root distance. were found compatible with elevated (2mM) and lead nitrate (5mM) concentration of cadmium, cobalt and moderately resistant to cobalt chloride lead salts. (0.75mM) and less resistant to nickel Similarly, the same protocol was used chloride(0.05mM) and copper sulphate for the heavy metal resistance pattern (0.5 mM). This results were also study in Frankia isolated from the A. compared with the American strains nepalensis. Heavy metal salt resistant (Richards et al., 2002) and found that colonies of Frankia isolated from the the present strains were much more A. nepalensis appeared similarly after tolerant to cadmium chloride and around 14 days of incubation. The copper sulphate. While the MIC value numbers of colonies were counted of USA strains for CdC12 was 0.5 mM against the increasing concentration of and for CoCh 0.25, the strain, studied various heavy metal salts and plotted in strains showed a MIC value of 2 and the graph. Fig 4.7 shows the growth 0.5 mM for CdC1 2 and CoC1 2 pattern of Frankia strain AnMrl in respectively. However, USA strains presence of different metal salts. The grew at an elevated concentration of results show that the strains were NiCh and Pb(N03) 2 than the studied highly resistant to cadmium chloride strain. The heavy metal resistant RESULTS AND DISCUSSION 81

9000

~ .2 8000 ~

~c 7000 ::I g> 6000 E ~ 5000 c>- 0 4000 0 (.) ... 3000 ....0 Ql .c 2000 E :::J z 1000

0 0 0.005 0.01 0.05 0.1 0.25 0.5 0. 75 1 2 3 5 Cone. of metal salts (mM) Fig 4.6. Effect of different heavy metal salts on growth of Frankia strain (CeSiS) isolated from C. equisetifolia; ; ~ +- nickel chloride, •-cadmium chloride, .& - cobalt chloride, •-lead nitrate and X-copper sulphate (Bose and Sen, 2006).

Frankia colonies were re-isolated from When these two results were compared various plates and preserved for further it was found that Frankia strain studies. Each experiment was repeated isolated from A. nepalensis were less twice and was found to produce similar resistant to the heavy metal salts. It was results. hypothesized that the Frankia isolated A characteristic heavy metal induced from A. nepalensis facing less heavy either purple or red or both type of metal stress than Frankia isolated from pigment production was noted in all the C. equisetifolia. As the latter was the cases except in lead at MIC or higher inhabitant of coastal region which was concentrations. This type of pigment facing the more metal concentrations production is totally different from the due to metal pollution and become pigment production of the strains more resistant to heavy metal ions. On isolated from C. equisetifolia growing the other hand hilly forest region of the in North Bengal University campus Darjeeling district is relatively free (Bose and Sen, 2005, 2006) where a from metal pollution and hence native faint red pigment was produced only in Frankia were less exposed to heavy lead plates. metal ions. One of the strain AnMrl RESULTS AND DISCUSSION 82

'""'

--+-Cu _._Ni -+-Co __,._Cd

~Pb

""'

·~------~--~~~~~~------4~------~~OrrM 0.00~ 0.01rrM 0.05rrM 0.1nt.1 0.251TM O.Srrt.'l 0.75rrM 1rrM 2rrM 3rrM 4rrM Snt.'l Concentrations of differet heavy metal salts (mllll)

Fig 4.7: Heavy metal resistance pattern of Frankia strain (AnMrl) isolated from A. nepalensis. (Bose and Sen, 2007). was found compatible with elevated The tellurite and tellurium resistant concentration of cadmium and lead genes clustered together and the copper salts. genes clustered together with the This had great significance since the exception of one copper resistance heavy metal resistance is a more gene from Frankia Cci3. Clustering of suitable marker for slow growing copper resistance and tellurite bacteria like Frankia as it is more resistance genes of the different stable than antibiotics resistance Frankia strains suggests that they have (Richards eta/., 2002). co-evolved as a unit. The Zn-Co-Cad 4.9 Analysis of Heavy Metal Resistance resistance gene had a completely Genes of Frankia different origin and did not lie in a The heavy metal resistant genes of particular clade. (Bose eta/., 2007). Frankia are listed in the Tab 4.4. The heterogenecity of the codon usage Figure 4.8 showes the dendrogram was significantly related to the obtained by the multiple sequence translation of that particular gene. The aligrunent of the studied genes of the high! y expressed genes, like ribosomal three strains of Frankia. It was seen protein genes some have high degree of from the dendrogram that there was a codon bias, depending on their single major clade and two subclades. effective number of codon (Nc) RESULTS AND DISCUSSION

C3

E3

C2

A1

E2 Fig 4.8: Dendrogram of the A7 relationship of heavy metal resistance genes of the .------AS Frankia strains.

C4

AS

A2

A4

(Ikemura 1981; Lafay et al., 2000). genome showed low value of effective Thus Nc was plotted against the GC3 number of codon ( <40). to investigate the diversity of codon The values of the codon usage indices usages in the genomes (Peden, 1999). calculated for the studied strains are It is a common fact that high GC shown in Table 4.4. From the results it containing organism show considerable was seen that the G+C content of the codon usages diversity (Wu et al., Frankia strains were high. Due to this 2005). The ribosomal protein genes property, it was hoped that the GC3 were considered as a reference in this content and the Nc values for the genes work. These proteins were clustered at from Frankia genomes might have the lower end of the graph which was some degree of heterogeneity. It was an indication of strong codon bias. The seen that the Nc values decrease with heavy metal resistance genes also increase in GC3 content indicating that clustered along the ribosomal protein codon bias increases with the increase genes and a strong codon bias was in GC3 content. The Nc/GC3 plot indicated in these cases. The Frankia demonstrated an effective technique for RESULTS AND DISCUSSION

investigating codon usage variations among the genes. The Nc value of the genes showed a range for all the genomes suggesting that these highly GC rich genomes exhibited considerable amount of heterogeneity m codon usage. Genes coding ribosomal proteins that are known to be highly expressed were highlighted in the NC/GC3 plots. Compared to the ribosomal protein genes the heavy metal resistance genes in the genomes showed some difference. Heavy metal resistance genes of Ccl3 were less biased compared to the ACN14a and EANlpec strains. Most of the ribosomal protein genes for all the Frankia genomes were found to be clustered at lower ends of the plot suggesting a strong codon bias for these genes. However, heavy metal resistance genes of Ccl3, ACN14a and EANI pee strains were clustered at lower ends of the plot (Fig 4.9A,B,C). Genes with low Nc (value <40) have much stronger codon bias than be simply explained in terms of ~ mutational bias. Ribosomal protein u0 genes and those associated with metal resistance had a lower mean Nc value than that obtained for all of the protein coding genes suggesting a higher degree of bias in the former. These

RESULTS AND DISCUSSION 86 bands .. downstream reactions the samples were 4.11 Purification of DNA further purified with glass milk, The genomic DNA 1s generally PEG8000 and sigma kit. These contaminated by various proteins, purified DNA was used for RNA and polysaccharides. During the polymerase chain reaction. The DNA extraction process the proteins purified DNA was subjected to were eliminated by phenol and spectrophotometric quality testing. All chloroform. The RNA were removed the samples were found to be by RNaseA treatment. The qualitatively pure with a ratio of A2r,o/ polysaccharides were removed by the Azso of~ 1.8. standard DNA extraction protocol 4.12 Development of A New 16s Primer (Bajwa, 2004). To get rid of extra Sequence for Frankia phenolics which in turn hinders the A 20 bp long primer was developed by

Table 4.4- Values of Different Codon usages Indices for the Heavy Metal Resistance Genes of Frankia Genes Gene ID Abbn. CAl CBI Fop Nc GC3 GC Copper resistance D [Ccl3] 637880116 C1 0.228 0.241 0.54 35.2 0.89 0.72 Copper resistance protein Cope [Ccl3] 637880115 C2 0.251 0.203 0.53 38.8 0.82 0.74 Tellurite resistance pro- tein TerB [Cc13] 637880329 C3 0.334 0.226 0.56 33.3 0.91 0.64 Putative tellurium resis- tance protein TerA [Cc13] 637881001 C4 0.342 0.357 0.63 33.4 0.91 0.69 Tellurium resistance protein TerA [Ean1pec] 641239986 E1 0.332 0.369 0.63 29.7 0.94 0.71 Copper resistance protein Cope [Ean1pec] 641246078 E2 0.26 0.327 0.6 32.8 0.92 0.74 Tellurite resistance TerB [Eanlpec] 641240861 E3 0.207 0.197 0.54 36.6 0.86 0.7 Tellurite resistance TerB [Eanlpec] 641241347 E4 0.195 0.192 0.52 34.4 0.91 0.77 Putative copper resis- tance protein [ACN14a] 638100993 AI 0.229 0.219 0.53 38.9 0.81 0.69 Tellurium resistance protein [ACNI4a] 638101739 A2 0.327 0.331 0.62 28.3 0.96 0.68 Tellurium resistance protein terE [ACNI4a] 638098466 A3 0.13 0.168 0.49 49.9 0.55 0.77 Tellurium resistance protein terE [ACNI4a] 638102547 A4 0.282 0.168 0.5 42.4 0.68 0.67 Tellurium resistance protein terA [ACN14a] 638102544 A5 0.246 0.165 0.49 38 0.7 0.71 Putative Cobalt-zinc- cadmium resistance pro- tein [ACN14a] 638100325 A6 0.264 0.288 0.57 30.7 0.95 0.75 Putative Copper resis- tance domain [ACNI4a] 638096856 A7 0.204 0.124 0.49 35.3 0.91 0.77 RESULTS AND DISCUSSION

Fig 4.10 Agarose gel photograph ofPCR amplification of Frankia DNA; Lane!, Lambda DNA EcoRII Hind III double digest DNA marker; Lane2, -ve control; Lane3-7, Al-AS; Lane8-12, C6-CIO; Lanel3, lkb DNA marker (for details refer section No. 3.9.2 ofMM).

Primer3. The newly designed primer 4.14 Analysis of PCR-RFLP Data set could amplify a product of 520 bp The above results were analyzed with various from the distal part of 16s rDNA statistical methods of POPGENE package as mentioned earlier. Among the Frankia portion of Frankia infecting both A. population the total genetic diversity had been nepalensis and C. equisetifolia . The found to be 0.2689 (SO± 0.1858), which sequence was as follows: showed low level of genetic diversity. This low forward-5'-GGGTCCGT AAGGGTC-3' and level of diversity indicates that total genetic reverse-5'-AAGGAGGTGATCCAGCCGCA- diversity comes from inter population source 3' and in this case, there were no lateral gene 4.13 PCR Amplification of Frankia DNA transfer occur in Frankia strains. The distal part of the 16s rDNA of Shannon index is used to study the Frankia was the target sequence of the diversity of the population. It is newly developed primer. The primer assumed that all species are pair then amplified a single DNA band represented in the sample and they are of expected length (520bp). The PCR randomly sampled. The advantage of products were subjected to restriction this index is that it takes into account digestion with various restriction the number of species and the evenness enzymes, which includes Alui, Taq I, of the species. The index is increased Hae III, Mba! and Mspi. Relatively either by having additional umque low level of polymorphism was found. species or by having greater species RESULTS AND DISCUSSION 88

showed that the Frankia population of Alnus based and Casuarina based were divided into three major clades. In the first clade Alnus based germplasm · collected from Mirik (AI), Sonada (AS) and loosely Fatak (A2) were there. These places are within the geographic regionof I 0-20 km. In the Fig4.lla. PCRproduct digested withA/ul; Lane land 13- SObp DNA ladder; Lane 2-6, AI second clade, we have Alnus based -AS; Lane 7-11, Cl-CS. (for details refer sec­ tion No. 3.9.2 ofMM) Sukhiapukhuri (A3) and Ghoom (A4) evenness. The value of Shannon index which are also geographically close. will be maximum if the evenness Among the Casuarina based present in the population is maximum. population we found them scattered in It has specific importance in the different clades. Casuarina nodules expression of equal proportion collected from Diamondharbor (C3) character in the sample rather than the went with Alnus nodules of Mirik (A 1) total number of individuals. The and Sonada (AS) where as Casuarina Shannon index has moderate sensitivity based Frankia of Bakkhali (C4) were towards the sample size. In the present clubbing with Alnus based Frankia of study the value of Shannon index has Sukhiapukhuri (A3) and Ghoom (A4). been found as 0.4062 (SD= ± 0.2619). On the other hand Casuarina based This value also indicated the low level Frankia collected from Jalpaiguri (CI), of genetic diversity. Digha (CS) and North Bengal

Thus from the result of statistical analysis, it could be concluded that the Frankia population present in this area shows very low genetic diversity and the total genetic diversity was intra population in origin.

The dendrogram based on Nei's (1972)

Genetic distance (Method = UPGMA Fig 4.11 b. PCR product digested with Haelll; Modified from NEIGHBOR procedure Lane I, SObp DNA ladder; Lane2-6, Al-AS; Lane 7-11, CI-CS. (for details refer section No. of PHYLIP Version 3.S) (Fig 4.I2) 3.9.2 ofMM) RESULTS AND DISCUSSION Sg +------Al +------2 +------4 +------C3 +--7 +------AS +------A2 +------8 +------A3 +------1 +------6 +------A4 --9 +------C4 +------Cl +------3 +------5 +------cs +------C2 Fig 4.12 Dendrogram Based Nei's (1972) Genetic distance: Method diversity of Frankia based on PCR-RFLP results= UPGMA, Modified from NEIGHBOR pro­ cedure ofPHYLIP Version 3.5 University Campus (C2) comprised of of Casuarina based Frankia behaving third clade (Fig 4.12). differently from Alnus based Frankia While from the above result a more or could be that all the Casuarina less distinguished pattern emerged in plantations in West Bengal are exotic case of Alnus based Frankia. The and were perhaps brought from Casuarina based Frankia were places different places. in different clades. The probable reason Chapter 5 CONCLUSION

Before I conclude my work, I must heavy metal induced pigment make a summary of it. So, I summarize production of Frankia strains was my whole work in a point wise fashion, reported. which is as followes : •Codon usage and codon preferences, •Frankia strains from different parts G+C composition, effective number of of West Bengal were isolated from the codon (Nc) and codon adaptation index nodules of C. equisetifolia and A. of heavy metal resistance genes of nepalensis by a newly standardized Frankia strain EANlpec, ACN14a and isolation technique. Ccl3 were calculated. From these study • The culture characteristics and the life cycle pattern of Frankia strains Field Emission Scanning Electron were predicted. microscopy of the bacterium was • A new pair of PCR pnmers performed. specific for distal part of 16s rDNA • Effect of different media and region of Frankia were developed and hormone concentrations on the subsequently, a 520 bp long portion of germination of seed of A. nepalensis Frankia genome were amplified with were studied. those primers followed by PCR-RFLP • The characteristic root hair profiling revealed the diversity of deformation and. visible nodule Frankia in West Bengal. formation in A. nepalensis and C. At the time of starting of my work, equisetifolia seedlings authenticate the a simple but basic question was in my presence of Frankia in the medium. mind, that was the problem of studying • Physiological characterization of the diversity of Frankia and I set my Frankia strains were performed. mind to work on it. After starting the • Heavy metal resistance profiling work, I found that isolation of Frankia of Frankia strains were performed and was a big problem and there was no CONCLUSION 91 easy technique for isolation of the facing less heavy metal stress than the endophyte from the nodule. Ironically Frankia isolated from C. equisetifolia. the solution of this question opens up In the year 2007, three Frankia many avenues. I choose a bidirectional whole genome sequences were approach to find the answer of the available in public domain. I took this diversity related questions. One was opportunity to start studying the metal strain based work and other was resistance genes of Frankia with metagenomic work. At this point of bioinformatics tools like Codon W, study, I decided that I should stick to ClustalW, CAl calculator, etc. The common parameters only for studying metal resistance genes of Frankia had the diversity of both Alnus based high CAl values which was indicating Frankia and Casuarina based Frankia. better expression levels. These genes I selected standard biochemical and also showed strong codon bias, the metal resistance parameters for them. result obtained in this study put an On the basis of these results it was additional support to the hypothesis found that Frankia population of this that Frankia strain Ccl3 is more region was divided in to two major symbiotic than saprophytic, since they clades. The first clade included the remain largely in the nodules (a bacterium isolated from C. protective place for them) and hardly equisetifolia and the second clade grow in the soil. This kind of work was included bacterium isolated from A. first of its kind in India. nepalensis. Each clade was again sub For metagenomic work, it was divided into two minor clades. This found that there was no PCR primer pattern supported the presence of two that had the ability to amplify the physiological groups among the portion of 16s r-DNA of both Alnus bacteria isolated from each host. The based and Casuarina based Frankia. results of the metal resistance work So, we had developed a new PCR were also interesting. Frankia strains primer for that and found positive isolated from A. nepalensis were less results. To study the diversity, the resistant to the heavy metal salts than amplicons were digested with the strains isolated from C. restriction enzymes and PCR-RFLP equisetifolia . It was hypothesized that profiles were obtained. Based on those Frankia isolated from A. nepalensis PCR-RFLP profiles, it was found that a CONCLUSION 92 more or less distinguished pattern Now, it is the time to answer the emerged in case of Alnus based question which I had asked at the time Frankia. The Casuarina based Frankia of staring of my work. I shall answer it were places in different clades. The in a positive mood. I have successfully probable reason of Casuarina based detected the diversity of both Alnus Frankia behaving differently from based and Casuarina based Frankia Alnus based Frankia could be that all sticking on the common parameters. the Casuarina plantations in West Although their diversity was totally Bengal are exotic and were perhaps intrapopulation ·in origin and do not brought from different places. cross the line of speciation. REFERENCES

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Waksman SA (1950) The Actinomycetes, potential uses of actinorhizal plants in their nature, occurrence, activities, and Europe. In: The biology of Frankia and importance. Percaline Rouge, Waltham, Actinorhizal plants (Schwintzer CR & Mass., USA. Tjepkema JD eds). Academic press, San Wall LG (2000) The actinorhizal symbiosis. J. Diego. pp 365-389. Plant growth Regul, 19: 167-182. Wright F (1990) The effective number of Wayne LG, Brenner DJ & Colwell RR codons used in a gene, Gene, 87,(1), 23- (1987) Report of the ad hoc committee on 29. reconciliation of approaches to bacterial Woese CR (1987). Bacterial evolution. systematics. Int. J. Syst. Bacteriol. 37: 463 Microbiol Rev 51:221-271. -464. Woronin MS (1866) Uber die bei der Weber A, Smolander A, Nurmiaho-Lassila Schwarzerle (Alnus glutinosa) und bei der E & Sundman V (1988) Isolation and gewohnlichen Garten-Lupine (Lupinus characterization of Frankia strains from mutabi/is) auftretenden Alnus incana and Alnus glutinosa in Wurzelanschwellungen. Memoires de Finland. Symbiosis. 6:97-116. I'Academie Imperiale des Sciences de St. Weast RC (1984) CRC handbook of chemistry Petersbourg, VII Ser. 10: 1-13. and physics, 64 edn. CRC, Boca Raton, Wu G, Culley DE & Zhang W (2005) Fla. Predicted highly expressed genes in the Weaver W & Shannon CE (1949) The genomes of Streptomyces coelicolor and mathematical theory of communication. Streptomyces avermitilis and the Urbana Illinois; University of lllinois. implications of their metabolism. Wheeler CT & Miller 1M (1990) Current and Microbiology 151:2175-2187. ACADEMIC ACHIEVEMENTS

Paper published from the thesis 1. Debadiu Bose and Arnab Sen (2005) 'Heavy metal resistance pattern of Frankia isolated from sub Himalayan West Bengal'. In: Stress Biology: Current Research Scenario (Chakraborty U and Chak­ raborty BN eds), Narosa Publication House, New Delhi, pp 154-157. 2. Debadin Bose and Arnab Sen (2006). 'Isolation and heavy metal resistance pattern of Frankia from Casuarina equisitifolia nodules'. Indian Journal of Microbiology, New Delhi. voi46(I),pp- 9-12 3. Debadin Bose , Saubashya Sur, Asim Kr. Bothra and Arnab Sen (2007) 'Study of the diversity of heavy metal resistance genes and their codon usage profiling' The Icfai Journal of Biotechnology, Icfai University Press, Hyderabad, Vol!, No 3, pp 49-59. 4. Debadin Bose and Arnab Sen (2007). 'Heavy metal induced pigment production by Frankia iso­ lates from Mirik Hills '. Journal of Hill Research , Gangtok. vol 20, no- I, pp 36-38. 5. Debadin Bose, B. Bajwa, M. Bajwa and A Sen (20 II). "Physiological characterization of Frankia strains isolated from sub-Himalayan West Bengal, India reveals two distinct groups". Biotechnology, Vol3, No I, pp 6-15.

Technology Generated 1. A nobel technique for isolation of Frankia in pure culture.

Paper accented for presentation in International Seminar in overseas for oresentation 1. Debadin Bose, B. Bajwa, M. Bajwa and A Sen. "Physiological characterization of Frankia strains isolated from sub-Himalayan West Bengal, India reveals two distinct groups" In 16th International Meeting on Frankia and Actinorhizal plants & International Symposium on Franineae in city of Porto, Portugal, on 5'" to 8,. September, 2010.

Paper oresented in International Seminar in overseas: 1. Debadin Bose, S Sur, AK Bothra and A Sen (20 10), "Evolution of genes for heavy metal resis­ tance in Frankia -A bioinforrnatics approach", in Annual Botanical Conference 2010, Rajshahi, Bangladesh; on I Ilh December, 2010, organized by the Department of Botany, University of Ra­ jshahi, Bangladesh. Appendix -I

Composition of Buffer

Composition of the Buffers used 1. DNA extraction buffer (C-TAB Extraction buffer) Ingredients 2% (WN) C-TAB (Hi-media Cat#RM164) 1.4M NaCI (Hi-media Cat#I150) 20mM EDTA (pH-8.0) (Hi-media Cat#RMI 197) IOOmM Trizma Base (pH-8.0) (Sigma Cat#T1503) I% Polyvinylpyrrolidone (PVP)(WN) (Sigma Cat#P5288) 0.2% P-Mercaptoethanol (Hi-media Cat#RM2895), added just before use. Procedure 12.11 grams of molecular biology grade Trizma Base (Sigma Cat#T1503, Tris

(hydroxyrnethyl) aminomethen, C4H11N03.MW- 121.1) was dissolved in 600 ml of sterile double distilled water and the pH is adjusted to 8.0. The solution was made upto 800 m1 with sterile double distilled water and divided into two parts; to one part

7.44 gm of EDTA (Ethylenediaminetetra acetic acid, C10H14N2Na20s.H20) was added. In other part 81.82gm of Sodium chloride (NaCI), 20gm of C-TAB

(Hexadecyl trimethyl ammonium bromide,C 1 9~2NBr) was added. Then both the solutions were mixed and to it 1% Polyvinylpyrrolidone was added. The volume was made upto llit with sterilized double distilled water. P -Mercaptoethanol (0.2%) was added just before the use of the buffer.

2. DNA Loading buffer Type III{6X concentration) {Fermentas Cat#R0611) Ingredients 0.25% Bromophenol Blue (Hi-media Cat#RM914) 0.25% Xylene cyanol FF (Hi-media Cat#RM1877) 30% Glycerol (Hi-media Cat#MB060) in Double distilled water. APPENDIX 111 Procedure Two and half gram of Bromophenol Blue and Xylene cyanol was dissolved in 1000 ml of30% Glycerol in Double distilled water. 3. TE· buffer (Tris EDTA Buffer, pH-8) Ingredients lOmM Tris Trizma Base (pH-8.0) (Sigma Cat#T1503). 1OmM EDTA (pH-8) (Hi-media Cat#RM1197). Sterile double distilled water. Procedure 1.21 gm of molecular biology grade Trizma Base (Sigma Cat#T1503, Tris

(hydroxymethyl) arninomethen, C4HuN03.MW- 121.1) was dissolved in 400 m1 of sterile double distilled water and pH was adjusted with concentrated Hydrochloric acid (Hi-media, Cat#RM5955) to 8.0 and sterilized by autoclaving. Similarly 0.372 gm of Di-sodium salt of EDTA (Ethylenediarninetetra acetic acid,

CwH,4NzNa20s.H20) was dissolved in 400 m1 of double distilled water. The solution was stirred properly and the pH was adjusted with sodium hydroxide pellets (NaOH, Hi-media, Cat#RM1183) sterilized by autoclaving. Both the solutions were then mixed and the volume was made upto llit sterilized double distilled water. 4. TBE· buffer (Tris Borate EDTA Buffer, pH-8) (5X Concentration) Ingredients Tris (Trizma Base, Sigma Cat#T1503) 54 gm/lit. Boric acid (Hi-media Cat#MB007) 27.5 gm/lit. 20 mL of 0.5M EDTA (Hi-media Cat#RM1197). Sterile double distilled water. Procedure 54 gm of molecular biology grade Trizma Base (Sigma Cat#T1503, Tris

(hydroxymethyl) arninomethen, C4HuN03, MW- 121.1) and 27.5 gm of Boric acid (Hi-media Cat#MB007) was dissolved in 800mL of sterile double distilled water. 20 mL of 0.5 M EDTA (pH 8.0) was added. The volume was adjusted up to llit with sterile double distilled water. The working concentration ofTBE buffer is IX, so the 5X stock solution is diluated with sterile distilled water to 1X. APPENDIX 112 5. Taq buffer (10X Concentration, supplied with Taq polymerase, Finnzymes Cat#F501L) Ingredients 10 mm Tris-HC1 (pH- 8.8). 1.5mM MgCb(Hi-media Cat# MB040). 50 mM KCI. (Hi-media Cat# RM697). 0.1% Triton X-100. (Hi-media Cat# MB031). 6. Washing solution: Ingredients 70% ethanol (E Merck Cat# 101076HBD). !OmM Ammonium acetate (Hi-media, Cat#MB033) 7. PBS- PhosphateBuffered saline (pH-7.4) 137 mM NaCl (Hi-media Cat#1150). 2.7mM KCl (Hi-media Cat# RM697).

!OmM Na2HP04(Hi-media Cat#RM 1416). 1.8mM KH2P04(Hi-media Cat#RM 2951). Procedure 8 gm of NaCl, 0.2g of KCl, 1.44gm of Na2HP04 and 0.24gm of KH2P04 were dissolved in 800mL of sterile double distilled water. The pH was adjusted to 7.4 with HCI. Volume was made up to 1 lit with sterile double distilled water. 8. Peeling Buffer (pH-7.4): NaCl (Hi-media Cat#1150)- 8gm. KCl (Hi-media Cat# RM697)- 0.2gm. Na2HP04(Hi-media Cat#RM 1416) - 1.44gm. KH2P04(Hi-media Cat#RM 2951)- 0.24gm. Polyvinyl pyrollidone (PVPP) (Sigma Cat#P5288) - 30 gm. Procedure The compounds, except PVPP were dissolved in 800mL of sterile double distilled water and the pH was adjusted to 7.4. 30 gm of Polyvinyl pyrollidone (PVPP) was added to this solution. The final volume was made up to llit with sterile double distilled water and autoclaved. Appendix -II

Composition of Medium Qmod (Lalonde and Cal- DPM (Baker and O'keefe, vert, 1979) 1984) Macronutrients (gL-•)

K2HPO. 0.3 KH2P04 1.0 NaH2P04 2H20 0.23 MgS04,?H20 0.2 0.1 KCI 0.2 CaCh 0.01 Yeast extract 0.5 Peptone 5.05 Lecithin 0.04 Sodium propiona- - 1.2 te Glucose 10.0

1 Micronutrients and iron (mgL- )

H3B03 1.5 2.86 MnS04,H20 0.8 MnCh 1.8 ZnS04,7H20 0.6 0.22 CuS04,5H20 0.1 0.08 Na2Mo04 0.025

(N~)s(Mo702) 0.2 4,4H20

CoS04,7H20 0.025 CoCh.?H20 0.025 C6Hs01,H20 10 CsHs07Fe,3H20 10 FeNaEDTA 13.20 APPENDIX 114

F-medium (Simonet et al., 1985) pH= 7.00. Constituents Stock solution. (gmllit) Working solution (11M/lit)

Macronutrients lOx

KH2P04 5.0 2871

MgSO, 2.0 1148

CaC!,,2H20 !.0 2127

D-glucose 100.0 5550

Casein hydrolysate 40.0

Yeast extract 0.50

Oligo quispel lOOOX

H,BO, !.50 24.3

MnSO,,H20 0.80 4.70

ZnSO,,?H20 0.60 2.10

CuS04,5H20 0.10 0.40

(NH,),Mo,02,4H20 0.20 0.16

CoS04,?H20 0.025 0.09

Iron lOOOX

C8H,0,,H20 10.00 47.6

C,H80 7Fe,3H20 10.00 33.5

Vitamines lOOOX

Thiamine HC! 0.01 0.03

Nicotinic acid 0.05 0.04

Pyridoxine HC! 0.05 0.24

Tween 80 lml APPENDIX 115

OS-1 medium (Dobritsa and Stupar, 1989)

Constituents Stock solution. (gmllit) Working solution (ltM/Iit) Macronutrients lOX

KH2P04 1.36 999.00

MgS04 3.38 3039.00 Micronutrients lOOOX H,BO, 2.86 46.30

MnCI2,4H20 1.81 9.10

ZnS04,7H20 0.22 0.80

CuS04,5H20 0.08 0.30

Na2Mo04 0.025 0.01

CoCI,,7H20 0.025 O.ot Iron

FeS04,7H20 10.008 370.00

Na2EDTA 13.41 36.00

Hoagland solution (pH=7, Nitrogen free) (Hoagland and Arnon, 1950)

Constituents Stock solution. (gmllit) Working solution (J&MIIit) Macronutrients lOX

KH2P04 1.36 999.00 MgSO, 3.38 3039.00 Micronutrients lOOOX H,BO, 2.86 46.30

MnCI2,4H20 1.81 9.10

ZnS04,7H20 0.22 0.80

CuS04,5H20 0.08 0.30

Na2Mo04 0.025 0.01

CoCI2,7H20 0.025 0.01 Iron

FeS04,7H20 10.008 370.00

Na2EDTA 13.41 36.00 A PPENDIX 116 ------Frankia Media MPN= MOPS + phosphate + nitroe:en 4.2g MOPS buffer (mw 209.3)

1.7g K2HP04 I OmL NH4Cl (0.5M stock) bring up to I OOOmL pH to 7

Growing Frankia In a SOOmL flask Add I OOmL MPN Add carbon source Autoclave Add 1.7mL Trace Elements Mix Add homogenized Frankia

Trace Elements Mix

20mL ofO.I M stock Na2Mo04 8mL of 0.5M stock MgS04 4mL of20mM stock FeClr NTA 2mL of I OX stock MOD-MBA trace salts

MOD-MBA trace salts 2.5g F ez(S04)3, ?H20

5g MnC)z,2H20

0.25g CuC12,2H20

lOg CaC12,2I-{z0 0.5g H3B03 lg ZnS04,?H20 0.2g CoCI2,6H20 bring up to I OOOmL in O.lN HCl