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INVESTIGATING THE PO TENTIAL EXIST E NCE OF ENDOPHYTIC BACTERIA IN EARLY STAGE MAIZE KERNELS BY MATTHEW C. ANDERSON A Thesis Submitted to the Division of Natural Sciences New College of Florida in partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Dr. Amy Clore Sarasota, Florida May 2012
ii Acknowledgements First and fore most, I would like to thank Dr. Amy Clore and Mr. Joel Thurmond, M S for giving me the opportunity to work on this project. Joel, you were incredibly helpful every step of the way, and I truly appreciate all the time and effort you put into helping me o n my thesis research project. It was a pleasure to have worked with you, and I could not have done it without you Dr. Clore, thank you for all of the one on one support you have given me throughout my undergraduate career including i ndependen t s tudy p ro jects, classes labs, tutorials, and of course, this thesis. You have been an invaluable resource Thank you both for all that you have done for me. I would also like to thank Dr. Alfred Beulig and Dr. Christopher Hart for agreeing to be on my thesis committee and Dr. Joanne Dannenhoffer of Central Michigan University and Mr. Robert Buzzeo of University of South Florida Tampa for their help with confocal microscopy And, of course, thanks to the Committee of Academic Affairs for providing funding for this thesis research. Lastly, I would like to thank my friends and family for their continued support namely Gracelena Ignacio who was there every step of the way
iii Table of Contents Title Page Acknowledgements ii List of Tables and Figures v List of Acronyms and Abbreviations vi Abstract vii Chapter 1 Introduction 1.1 Overview of Endophytes and Their Benefits 1 1.1.1 Endophytic vs. Rhizobacteria 2 1.1.2 Systemic Acquired Resistance (SAR) and Induced Systemic Resistance (ISR) 3 1.1.3 Combinations of Plant Growth Promoting Bacteria (PGPB) 6 1.1.4 Phytoremediation 7 1.1.5 Indole 3 Aceti c Acid (IAA) Production 10 1.1.6 Siderophore Production 11 1.1.7 Modification of Genetic Expression 12 1.2 Specificity and Abundance of Endophytes 14 1.3 Maize and its Known Endophytes 17 1.4 Common Methods for Studying Endophytes 23 1.5 Specific Aims of Thesis Research 30 Chapter 2 Materials and Methods 2.1 Germination, Cultivation, and Pollination of Maize 32 2.2 Harvesting and Surface Sterilization 33 2.3 Culturing 3 3 2.4 Fluorescent In Situ Hybridization (FISH) 35 2.4.1 FISH with Homogenized Kernels 36 2.4.2 FISH with Sectioned Kernels 3 7 2.4.3 FISH Confocal Microscopy 3 8 2.5 DNA Isolation, Polymerase Chain Reaction (PCR), and Gel Electrophoresis 39 2.6 Terminal Restriction Length Polymorphism ( TRFLP ) 4 0 2.7 Statistical Analysis 42
iv Chapter 3 Results 3.1 Cultures 45 3.2 FISH 49 3.3 TRFLP 53 Chapter 4 Discussion 4.1 Cultures 59 4.2 FISH 62 4.3 TRFLP 65 4.4 Future Directions 6 9 Appendix A Recipes 7 4 Appendix B Supplementary Figures 7 5 Appendix C Chemical Structures 7 8 References 80
v LIST OF TABLES AND FIGURES Fig. 1: Pathways for SAR and ISR in Arabidopsis 5 Fig. 2: Common techniques for phylogenetic research on bacteria 26 Fig. 3: Hypervariable and conserved regions of 16S rDNA from a ligned bacterial 16S r D NA gene sequence s 26 Fig. 4: Primary and secondary structures of 16S rRNA of E. coli showing variable regions and target of EUB338 hybridization 31 Fig. 5: Orientation of maize kerne ls for Vibratome sectioning 38 Fig. 6: Target sequences and cut locations for HHaI and RsaI restriction enzymes 41 Table 1: Summarized microbial growth results from cultures 4 6 Fig. 7: Microbial growth results from 6 DAP kernels 47 Fig. 8: Microbial growth results from 0 DAP kernels 4 8 Fig. 9 : Representative confocal FISH images of E. coli control slides 51 Fig. 10 : Representative confocal FISH images of 2 DAP and 8 DAP kernels 52 Fig. 11 : Homogenized 12 DAP maize tissue spiked with E. coli 53 Fig. 12 : Representative electropherograms from 0 DAP and 8 DAP kernels 56 Fig. 13 : PCA and MDS ordinations from TRFLP results 57 Fig. 14 : Hierarchal dendrogram s constructed based on group average Bray Curtis similarity 58 Appendix B Supplementary Figures Fig. B 1: Vibratome sections of 3 DAP and 9 DAP maize kernels (CML 322 cultivar) showing nucellus and endosperm 7 5 Fig. B 2: Electropherograms from TRFLP 7 5 Fig. B 3: Illustrated longitudinal section through a developing maize kernel 77
vi List of Acronyms and Abbreviations ACC 1 aminocyclopropane 1 carboxylic acid BHI Brain Heart Infusion (agar) CFU Colony Forming Unit DAP Days After Pollination DAPI 4',6 diamidino 2 phenylindole ET Ethylene FISH Fluorescent I n S itu Hybridization FITC Fluorescein isothiocyanate ( a green fluorophore ) IAA Indole 3 acetic acid (a form of Auxin) ISR Induced Sy s temic Resistance JA Jasmonic Acid MDS (Non metric) Multi dimensional s caling NB Nutrient Broth PB (Sodium) Phosphate Buffer PCA Principal Component Analysis PCR P olymerase Chain Reaction PGPB Plant Growth Promoting Bacteria rDNA Gene encoding rRNA RFU Relative Fluorescence Units ROS Reactive Oxygen Species rRNA Ribosomal RNA (functional) SA Salicylic Acid SAR Systemic Acquired Resistance SS Surface Sterilized TRF Terminal Restriction Fragment TRFLP Terminal Restriction Fragment Length Polymorphism TSA Tryptic Soy Agar
vii INVESTIGATING THE POTENTIAL EXISTE NCE OF ENDOPHYTIC BACTERIA IN EARLY STAGE MAIZE KERNELS Matthew C. Anderson New College of Florida, 2012 ABSTRACT Endophytic bacteria reside inside of a host plant for part of or all of their life cycle without causing any apparent symptoms of disease. Most endophytes have a symbiotic relationship with their host, offering benefits such as increased nutrient uptake, stimulated phytohormone production, or improved resistance to pathogens and toxins. Endophytic bacteria have been well studied in various tissues of many different crops, but their potential presence has not been investigated in maize kernels at early developmental stages The aim s of this th esis research were to investigate the potential existence of endophytic bacterial communities in Zea mays L. (maize) kernels and then to address how any such bacterial communities change throughout the first 12 days of development following pollination. P otential endophytes were isolated from surface sterilized kernels at 0, 1, 2, 3, 4, 6, 8, and 12 days after pollination with culturing techniques. Maize kernels were also probed for potential e ndophytic bacteria with fluorescent in situ hybridization and confocal microscopy. Metagenomic DNA (i.e., total DNA content of a sample, potentially consisting of genomes of multiple organisms) was isolated from surface sterilized kernels at 0, 1, 2, 4, 6, and 8 days after pollination and the presence of endophytic bacteria was assayed using polymerase chain reaction (PCR)
viii with bacteria specific primers. Bacterial community profiles were obtained with terminal restriction fragment length polymorphism (TRFLP) techniques. The results were inconclusive but some aspects, particularly the T RFLP results, suggest the possibility of endophytic presence. The study of endophytic bacteria and how they influence development may lead to future studies that could yield promising agricultural applications to incre ase such aspects as crop yield, quality longevity, and sustainability, thereby potentially limiting the use of chemical agents. ______________________ Dr. Amy Clore Division of Natural Sciences
1 Chapter 1 Introduction 1.1. Overview of Endophytes and T heir B enefits Endophytes are defined as bacteria and fungi that reside inside of a host plant for part of or all of their life cycle without causing any visible harmful effects (Tan and Zou, 2001; Zinniel et al. 2002; Sarr et al. 2010; Da et al. 2012) First discovered in L olium temulentum L. ( the weed commonly known as darnel) in 190 4 (Freeman, 1904) endophytes and their ecological roles have been an increasingly important topic of research as more evidence is presented about their importance in plant development (T an and Zou, 2001) Endophytes have been found in xylem eleme nts, inter cellular regions, and sometimes intra cellular regions of many vascularized plants (Garbeva et al. 2001; Tan and Zou, 2001) Most e ndophytes have a symbiotic relationship with their host, offering such reported benefits as increas ed nutrient uptake, stimulat ed phytohormone production or improved resistance to pathogens toxins such as heavy metal s pests, drought, salinity, and frost and heat stresses (van Veen et al. 1997; Rajkumara et al. 2009; Sarr et al. 2010; Shin et al. 2012) In return, the endoph yte r eceive s residency and access to resources within the host (Zinniel et al. 2002) Because of the aforementioned benefits, e ndophyte research may lead to the development of agricultural applications to increase crop yield, sustainability, nutritional value, and longevity without the use of hazardous chemical agents (Misaghi and Donn delinger, 1990; Surette et al. 2003; Compant et al. 2005) Not only are e ndophyt ic bacteria nonpathogenic but they can also improve their resistance to pathogenic infections. Endophytes can do this by simply
2 outcompeting pathogenic bacteria for resources thereby preventing their growth and the risk of plant infection or they can prevent infection through more complex mechanisms (Choudhary et al. 2007) Endophyte s also compete with each other and may (i.e., their ability to influence the growth of other nearby plants ; Tan and Zou, 2001) Hence, when supplementing plants with endophytic bacteria, it is important to consider the compatibility between strains (Sundaramoorthy et al. 2012) 1.1.1. Endophytic vs. Rhizobacteria Endophytes can be organize d into two different groups: obligate endophytes which depend on the host for survival and spend their entire life cycle within the host, and facultative endophytes which spend part of their life cycle outside of the host (Rajkumara et al. 2009) Obligate endophytes can only transfer to other plants vertically (parent to seed) or via vectors and cannot thrive in the surrounding medium, while facultative endophytes originate outside of the host and enter via the r oot system, flowers, stems, stomates, cotyledons, radicles, or open wounds (Zinniel et al. 2002; Aravind et al. 2009; Rajkumara et al. 2009; Sarr et al. 2010) Some facultative endophytes such as Azoarcus communis and Klebsiella ornithinolytica c an even create their own openings in plants by secreting hydrolytic enzymes such as pectinase and cellulase to degrade cell walls (Sprent and Faria, 1988; Pariona Llanos et al. 2010) Rhizobacteria are bac teria found in the rhizosphere ( the surface of plant roots and their surrounding soil ) that form symbiotic relationsh ips with plants (Compant et al.
3 2005) Rhizobacteria are well studied and have been shown to promote plant growth by processes such as nitrogen fixation (Zhang et al. 1996; O'Callaghan et al. 1997) The distinction between rhizobacteria and endophytic bacteria is that rhizobacteria reside on the exterior of a plant, while endophytes reside within the host. Rhizobacteria are known to play roles in phytoremediation or the use of plants to remove pol lutants from soil (Shin et al. 2012) In phytoremediation, water soluble pollutants are usually absorbed and volatilized by the plants themselves, but insoluble pollutants may require the help of rhizospheric microor ganisms (Ali et al. 2012) For example, mercury salts are water soluble and are taken in by certain plants to be reduced to Hg 0 which is then volatilized, but insoluble pollutants such as hydrocarbons are dependent on biodegradation by rhizospheric microorganisms (Ali et al. 2012) Rhizobacteria may also become facultative endophytic bacteria if at some point in their life cycle they penetrate the root cortex and colonize within t he roots, thus switching from rhizospheric residency to endophytic residency (Compant et al. 2005) Roles of endophytes in phytoremediation will be explored in section 1.1.4 but first, the roles of endophytes and rhizobacteria in eliciting plant defense responses against pathogens will be discussed. 1.1.2. Systemic Acquired Resistance (SAR) and Induced Systemic Resistance (ISR) The two most well defined plant defense responses are systemic acquired resistance (SAR) and induced systemic resistance (ISR) (Compant et al. 2005) Both of these responses result in an increased resistance to a broad range of pathogens and
4 parasites ( Fig. 1 ; Choudhary et al. 2007) SAR and ISR are differentiated by the eliciting bacterium and the regulatory pathways involved (Vallad and Goodman, 2004) SAR is typically induced by a pathogen and enhances r esistance in distal parts of the plant to further infection by the same or other pathogens (Rudrappa et al. 2010) ISR is phenotypically similar to SAR, but it is typically induced by rhizo or endophytic ba cteria (Choudhary et al. 2007; Rudrappa et al. 2010) Some bacteria can elicit both SAR and ISR, notably the endophytic Actinobacteria Streptomyces Micromonospora and Nocardioi des in Arabidopsis thaliana DC. (Conn et al. 2008) Eliciting both pathways can lead to a synergistic resistance against a broader spectrum of pathogens than either SAR or ISR alone (Choudhary et al. 2007) SAR results in the production of pathogenesis related (PR) proteins like PR 1, PR 2, PR 1,3 glucanase, and some peroxidases, while ISR results in the production of ph enylalanine ammoni a lyase polyphenol oxidase, chalcone synthase and/or low molecular weight substances called phytoalexins (Vallad and Goodman, 2004; Compant et al. 2005; Choudhary et al. 2007; Rudrappa et al. 2010; Sundaramoorthy et al. 2012) All of these products are implicated in plant defense ( Choudhary et al. 2007; Sundaramoorthy et al. 2012) ISR by rhizo or endophytic bacteria has been found to be regulated by the phytohormones jasmon ic acid and ethylene (see Appendix C for structures; Pieterse et al. 2007; Rudrappa et al. 2010) SAR elicited by pathogen infection is typically regulated by SA (salicylic acid ; see Appendix C for structure ) or SA analogs such as BTH (benzo(1,2,3) thiadiazole 7 carbothioic acid S met hyl ester) or INA (dichloroisonicotinic acid) (Rudrappa et al. 2010) ISR can be triggered by specific bacterial traits such as
5 flagellation or presence of certain lipopolysaccharides and leads to the activa tion of multiple defense mechanisms like the strengthening of cell walls and altered physiological and metabolic responses that increase the formation of PR plant defense chemicals (Compant et al. 2005) Figure 1 SAR and ISR transduction pathways in Arabidopsis SA = s alicylic a cid, JA = jasmonic acid ET = ethylene, PR = pat hogenesis related proteins NPR1 = the protein n onexpressor of p athogenesis r elated genes 1 Lower case italicized acronyms represent gene products that play roles in regulation Figure a dapted from Choudhary et al. 2007 The common intermediate between SAR and ISR is NPR1 ( n onexpressor of p athogenesis r elated genes 1 a kyrin repeat family protein ) (Choudhary et al. 2007) Induction of SAR upon pathogen infection involves SA regulating a redox reduction of oligomeric NPR1 in the cytoplasm to mon omers which are subsequently transported
6 into the nucleus. There, the monomers interact with TGA transcription factors (TGA being part of the DNA motif to which the transcription factors bind) to upregulate the production of PR proteins (Choudhary et al. 2007) The steps following the production of NPR1 in the ISR mechanism are not well understood, but acetoin (3 hydroxy 2 butanone ; see appendix C ) a volatile organic compound produced by endophytes such as Bacillus subtilis is believed to be involved (Rudrappa et al. 2010) Acetoin has been found to induce ISR through a SA/ET and NPR1 dependent mechanism (Rudrappa et al. 2010) 1.1.3. Combinations of Plant Growth Promoting Bacteria (PGPB) It has recently been found that applying specific combinations of endophytic and soil borne p lant g rowth p romoting b acteria (PGPB) increases the effectiveness of ISR in chili plants in response to a wilt inducing pathogen compared to both untreated controls and controls treated with only one PGPB (Sundaramoorthy et al. 2012) In Sundaramoorthy et al hili seeds were surfa ce st erilized with a 2% (v/v) sodium hypochlorite solution (bleach) and then rinsed with sterile water before being submerged for 24 hours in one of several PGPB suspension s After germination of the seeds, the nascent roots of seedlings were submerged in additional PGPB suspension for two hours, and the soil was supplemented with PGPB as well as the pathogenic fungus Fusarium solani The PGPB suspension that was found to be most effective in preventing wilting disease c au sed by F. solani cont ain ed a combination of the rhizobacterium Pseudomonas fluorescens strain Pf1 and the endophytic bacteri a B
7 subtilis strains EPCO16 and EPC5 (Sundaramoorthy et al. 2012) This effect appears to be due to the increased production of a wide array of antibiotics such as 2,4 diactylphloroglucionl, oligomycin, phenazine, pyolete roin, pyrrolnitrin, pyocyanin, l turin, bacilomycin, zwittermycin A, and surfactin (Sundaramoo rthy et al. 2012) A similar study involving the treatment of banana plants with combinations of various strains of B. subtilis and P. fluorescens also showed increased resistance to banana bunchy top disease caused by Banana bunchy top virus as dete cted by e nzyme l inked i mmunosorbent a ssay (ELISA), d ot i mmuno b inding a ssay (DIBA), and p olymerase c hain r eaction (PCR) (Harish et al. 2008) Expression of d efense enzymes (peroxidases, p olyphenol oxidase, phenylalanine ammonia lyase phenol, and PR proteins) were found to have been increased in banana plants treated with B. subtilis and P. fluorescens at significantly higher levels than in control plants. These results suggest that there is a synergistic effect between both rhizospheric and endophytic PGPB, namely between species from Bacillus and Pseudomonas genera 1.1.4. Phytoremediation Endophyti c bacteria can also increase the metals and the efficiency of heavy metal uptake (Sun et al. 2010) Heavy metals are associated with the form ation of reactive oxygen species (ROS) when in the presence of light (Sherameti and Varma, 2009) ROS can also form via the electron transport processes in photosynthesis and respiration. These ROS are usually short lived but are very reactive with organic substances and can be harmful to the plant. Plants prevent the formation
8 of ROS with antioxidant enzymes suc h as superoxide dismutases, catalases, ascorbate oxidases, glutathione peroxidases, and glutathione reductases, and many plants utilize tocopherol (Sheramet i and Varma, 2009) Some plants may rely on the help of endophytic bacteria to prevent the formation of harmful ROS (Sun et al. 2010) In a study by Sun et al. (2010), endophytic bacteria were isolated from the herbs Elsholtzia splendens Nakai and Commelina communis L. and they were tested for heavy metal resistance by culturing in media containing copper. Thirty two different copper resi stant species were fou nd : 15 from E. splendens and 17 from C. communis Of these 32 endophytes the E. splendens endophytes Burkholderia sp. GL12 and Bacillus megaterium JL35, and the C. communis endophytes Sphingomonas sp. YM22 and Herbaspirillum sp. YM23 had strong siderophore production ( siderophores bind and transport iron which will be further discussed in section 1.1.6) and 1 aminocyclopropane 1 carboxylic acid (ACC) deaminase activity ACC deaminase is an enzyme that helps regulate levels of the volatile plant hormone ethylene by cleaving ACC, the immediate precursor to ethylene (Compant et al. 2005; Glick, 2005; Rajkumara et a l. 2009; Li et al. 2010; Prischl et al. 2012) High levels of ethylene exacerbate stress symptoms, so by increasing the amount of ACC deaminase present via endophytic bacteria, the amount of ethylene is reduced and stress is relieved. Through this mechanism, the production of ethylene is modulated, ultimately allowing the host plant to live in an environment rich in otherwise toxic heavy metals (Sun et al. 2010) Other known mechanisms by which metal resistant bacteria can increase tolerance of metals
9 by plants are exclusion, active removal, biosorption, precipitation, and bioaccumulation (as reviewed in Rajkumara et al. 2009) All of these processes lower the toxicity of bioavailability to the plant (Rajkumara et al. 2009) Sun et al (2010) inocul ated rape plants ( Brassica napus L.) with aforementioned siderophore producing and ACC deaminase active bacteria by soaking germ inating rape seedlings in bacterial suspens ions or sterile water for control s Rape plants are fast growing and are capable of accumulating large biomasses, making them ideal for phytoremediation (Sun et al. 2010) All of these endophytes except for GL12 were found to significantly promote growth ( i.e., root and above ground tissue weights) and copper uptake of rape plants ( p < 0.05 ANOVA test ) For example, inoculation of plants with JL35 was associated with an 83% increase in above ground dry weight (and 155% increase in root weight) compared to the control group. Rape plants inoculated with GL12 had significantly less growth ( ~ 28% decrease) and copper upt ake ( ~ 80% decrease) compared to control groups ( p < 0.05, ANOVA) possibly due to compatibility issues with the rape plant or its other natural endophytes. ACC utilizing endophytes help increase the uptake of copper from soil and The copper tolerant plants act as copper sinks; once the plants absorb enough copper they can be removed from the environment. Once the copper is removed from the soil via these copper sink plants, other plants that are less copper resistant are then able to grow in the treated soil Phytoremediation can be employed as a cost
10 traditional metal cleanup techniques such as treating soil with acid and chelators (Rajkumara et al. 2009) 1.1.5. Indole 3 Acetic Acid (IAA) Production Another endophyte pro duced phytohormone is indole 3 acetic acid or IAA, a form of auxin which is also produced within the plant and promotes certain types of growth ( see Appendix C for structure ; Tan and Zou, 2001; Prischl et al. 2012) IAA is known to promote adventitious root formation through cell division and some elongation (Johnston Monje and Raizada, 2011) IAA promotes ethylene production by stimulating ACC synthase, the rate limiting enzyme in the ethylene biosynthetic pathway (Harish et al. 2008) Auxin producing endophytes ( namely Stenotrophomonas maltophilia Enterobact er asburiae and Enterobacter hormaechei ) have been found in Zea nicaraguensis a teosinte, which is a group thought to be the progenitor of cultivated maize (Johnston Monje and Raizada, 2011) Zea nicaraguensis grows in seasonally flooded coastal plains and estuaries in Nicaragua, and its roots are specialized with aerenchyma to supply oxygen to roots submerged in wate r. Auxin produced by endophytes help s promote the growth of these specialized roo ts (Johnston Monje and Raizada, 2011) Another teosinte influenced by IAA producing endophytes is Zea d iploperennis Zea diploperennis is a unique teosinte because it is perennial (lives longer than two years) and as such it requires a large, persistent root system. Auxin produced by Enterobacter hormaechei helps promote the growth of such a root system. Jala a giant Zea ma ys L. plant that can grow up to 25 feet tall was also found to harbor the
11 aux in producing endophytic bacterium Pantoea ananatis (Johnston Monje and Raizada, 2011) Such a giant plant requires an extensive root system, the growth of which is influenced by auxin produced by P. ananatis P. ananatis also has nitrogen fixing activity (Johnston Monje and Raizada, 2011) 1.1.6. Siderophore Production Root e ndophytic bacteria can also increase the uptake of essential nutrients such as iron. This is done by the production of siderophores which are strong iron ion binding agents that act to increase the uptake of iron (Neilands, 1995; Sun et al. 2010 ; see Appendix C for structural example) Iron is essential for metabolic activities such as DNA synthesis, but because iron has a very low solubility, it has a low bioavailability in soil. Therefore, the presence of siderophores is often required by plants to acquire enough iron (Sun et al. 2010) Some endophytic bacteria can also increase the bioavailability of micronutrients and phosphate from organic phosphate by secreting phosphatase or from inorganic phosphate by secr eting organic acids (Harish et al. 2008) Some endophytic bacteria, such as some Pseudomonas species, are also known to play roles in biological nitrogen fixation or the reduction of N 2 to ammonium, which can account for up to 30% of nitrogen content in cereals harboring such bacteria (Harish et al. 2008; Pariona Llanos et al. 2010) Bacteria that are involved in nit rogen fixation are called diazotrophs (Rajkumara et al. 2009)
12 1.1.7. Modification of Genetic Expression Endophytes can also increase plant growth by affecting the expression of genes. In a recent study by Da, Nowak, and Flinn, two potato plant varieties known as Red Pontiac and Superior were inoculated with the bacterial endophyte Burkholderia phtyofirmans strain PsJN suspended in phosphate buffer saline (PBS) (Da et al. 2012) This was done by subculturing potato plants from uninoculated stock plants by single node cutting T he nodal sections of subcultures were su bmerged in the PsJN suspension for one minute prior to planting After six weeks, Red Pontiac potato plants were observed to have a 68% increase in shoot length, 125% increase in shoot fresh weight, and 108% increase in root fresh weight over control plan ts which were submerged in PBS without any bacterial suspension I n contrast, Superior potato plants did not show any significant differences in growth six weeks after PsJN inoculation Genomic DNA CCGG inoculation in the two potato varieties. Demethylation of these sites in potatoes has been correlated with transcription of a variety of genes related to mer istem growth (Law and Suttle, 2003) The responsive Red Pontiac variety showed little to no difference in DNA methylation after being inoculated with PsJN, while the unresponsive Superior variety showed a significant i ncrease in DNA methylation after being inoculated with PsJN. These results suggest that the increased DNA methylation in Superior potato plants repressed transcription of PsJN induced genes related to plant growth, and overall plant response to endophytes (Da et al. 2012)
13 It is also possible to genetically modify an endophytic bacterium to produce a desired result. For example, Fahey (1988) was the first to insert a gene in to an endophyte to control pests (Zhang et al. 2011) The bacterium Bacillus thuringiensis (Bt) is found in soil (Guidi et al. 2011) and is a natural endophyte of sugar cane (Goryluk et al. 2009) that expresses endotoxin CryIAc which kills lepidopteran s (Fahey, 1988) Fahey (1988) isolated the gene encoding CryIAc from Bt and inserted it into the natural maize endophytic bacterium Clavibacter xyli ssp cynodontis Tomasino et al (1995) tested th e ability of this recombinant C. xyli ssp cynodontis to resist damages from the European corn borer Ostrinia nubilalis C orn crops were inoculated with a suspension of recombinant C. xyli ssp. cynodontis (or PBS for controls) via injection into the stem of mature plants and/ or by soaking of seeds in suspension. I t was found that plants inoculated with the C. xyli (by either or both methods) reduced damage done by the European corn borer significantly more th an control PBS injections ( p < 0.05 ANOVA test ) (Tomasino et al. 1995) In a more recent study, the natural endophytic fungus Chaetomium globosum Kunze was isolated from rape plants and used as a living vector for expression of Pinellia ternate agglutinin (PTA), a n anti pest lectin (Zhang et al. 2011) This endophyti c fu ngus is able to colonize rape plants, but unable to c olonize rice plants. The gene coding for PTA was then isolated from the fungus C. globosum and inserted into the rice endophytic bacterium Enterobacter cloacae (strain SJ 10) The recombinant SJ 10 strain was inoculated into rice plants and PTA expression was observed. Rice plants were then exposed to ten 3rd instar nymphs of white backed planthoppers Sogatella
14 furcifera and then covered with insect proof netting to preven t their escape. Two sets of control plants were used, including rice seedlings inoculated with wild type SJ 10 and seedlings inoculated with sterile water. Numbers of WBPHs were counted for 26 days, after which all control plants died, but the plants ino culated with rSJ 10 survived. Furthermore, the maximum amount of WBPH observed in each group were significantly different ( p < 0.01), with the fecundity of WBPH in the rSJ 10 group decreasing by 86.1% and the SJ 10 (wild type) group by 25.6% as compared t o the sterile water control group (Zhang et al. 2011) 1.2. Specificity and A bundance of E ndophytes Like all natural environments, resources are limited within a host, and up to several hundred different bacterial and fungal endophytes may be present within a given host (Tan and Zou, 2001) This creates heavy competition between and among endophytic species and the abundance of each species is dependent on its ability to adapt t o its ecological niche (Compant et al. 2005) Since each species of host offers different resou rces, e ndophytic communit ies (or relative abundances of different endophytic species ) differ among hosts (Rajkumara et al. 2009) Furthermo re, different host tissues offer different resources for endophytes, so each tissue type may have a unique endophytic community (Pariona Llanos et al. 2010; Johnston Monje and Raizada, 2011) Thus, each endophytic community is specific to both tissue type and the genotype of the plant, and both endophytes and hosts have co evolved over time (Misaghi and Donndelinger, 1990; Johnston Monje and Raizada, 2011)
1 5 It has been suggested that endophyte s may have evolved from pathogens (Tan and Zou, 2 001) This may have occurred via a balanced pathogen host antagonism. For example, the pathogenic fungus Colletotrichum magna causes anthracnose (a disease which causes brown lesions of dead tissue) in cucurbit plants but a single single site prevents it from causing anthracnose while still allow ing the fungus to thrive inside of the plants (Redman et al. 1999) The fact that the switch from a pathogenesis lifestyle to a commensalism lifestyle is controlled by a single gene suggests that there is a genetic simplicity in the symbiosis between plants and fungi (Redman et al. 1999) This also suggests that this in teraction may be fragile and can be affected by environmental conditions Additionally, endophytic communities are dynamic and d ependent on factors external environment al conditions, time of year (season), age of plant, soil structure, and types of fertilizer used (Tan and Zou, 2001; Li et al. 2010; Pariona Llanos et al. 2010; Da et al. 2012) Howeve r, the general opinion of current research ers factor in determining endophytic community structure (Hardoim et al. 2011; Johnston Monje and Raizada, 2011; Prischl et al. 2012) Interestingly, a recent study showed that the endophytic community of maize stems was more dependent on the ecological location than was the endophytic community within maize s eeds (Johnston Monje and Raizada, 2011) The use of o rganic fertilizers have also been found to increase the number of diazotrophic bacteria in sugarcane compared to the use of conventional fertiliz ers or no fertilizer (Pariona Llanos et al. 2010)
16 Endophytes have been found in an array of vascularized plant species and are hypothesized by many to be present in nearly all plants (Tan and Zou, 2001; Johnston Monje and Raizada, 2011; Shin et al. 2012) Densities of endophytic bacteria in potato stem bases and roots have been found to be between 10 3 and 10 6 colo ny forming units (CFU) per gram of fresh weight, with the highest concentrations found near the roots (Garbeva et al. 2001) Densities of endophytic bacteria in cotton have been found to be between 0.4 X 10 3 and 11.6 X 10 3 CFU per gram fresh weight with higher densities in germinating radicles, roots, and flowers (Misaghi and Donndelinger, 1990) D ensities were found to be between 6.0 X 10 3 and 4.3 X 10 4 CFU per gram fresh weight in alfalfa root xylems (Gagn et al. 1987) Again, the highest densities were found nearest to the roots. In fact, this seem to be true of nearly all vascularized plants that have been s tudied, suggest ing that the soil is a major source of endophy tes (Prischl et al. 2012) Endophytic bacteria residing in the root portions are usually not only more abundant but have also been observed to be via ble for longer periods of time (Pariona Llanos et al. 2010) In general, the larger a bacterial population the larger its contribut ion to its interactions with its host (Pariona Llanos et al. 2010) However, an ideal inoculum size may exist for certain PGPB For example, in Arabidopsis Pseudomonas thivervalensis was found to have an ideal inoculum number of 10 5 CFU/mL (Persello Cartieaux et al. 2001; Pariona Llanos et al. 2010) Plants inoculated with 10 6 or higher CFU/mL resulted in irreversible damage (Pariona Llanos et al. 2010)
17 1.3. Maize and its Known Endophytes The d omesticated m aize plant is an important high yielding crop known for its nutritious fruit Domesticated m aize ( Zea mays ssp. mays ) is a giant grass domesticated from teosintes about 9,000 years ago in southwestern Mexico (Johnston Monje and Raizada, 2011) Maize is a cereal grain since its mature fruit consists primarily of a nutritious t issue called endosperm (Shewry, 2007) Immature (un fertilized ) maize kernels are typically filled with nucellus tissue (Randolph, 1936) This nucellus tissue is gradua lly repl aced by endosperm during the first eight or so days after pollination (DAP) (see appendix Figure B 1 ; Randolph, 1936; Sonchaiwanich, 2012) Endosperm contains large amounts of starch and protein and provides nutrition for the developing embryo (Coleman et al. 1997; Prioul et al. 2008) Endosperm results from a double fertilization event: two male gametes from a single pollen grain combine with two female gametes the haploid egg and diploid central cells, to form the diploid embryo and triploid endosperm respectively (Faure et al. 2003) Comparisons of characteristics of modern maize with extant teosintes suggest that the domestication of maize has led to modifications (Johnston Monje and Raizada, 2011) Since its domestication, seeds from the genus Zea have changed dramatically, and its associated microbial community has also probably gone through dramatic changes (Johnston Monje and Raizada, 2011) For example, the Zea seed has grown in size over time and it used to have a protective hard fruit case that has since been eliminated by artificial selection from ancient peoples (Wang et al. 2005; Johnston Monje and Raizada, 2 011) T he husk leaves protecting the maize cob have also been
18 enhanced and shoot branching has been decreased to allow for higher nutrient allocation to developing seeds (Johnston Monje and Raizada, 2011) Seed dispersal and germination have been altered to such an extent from human selection that domesticated corn ( those of ssp. mays ) can no longer survive in the wild (Johnston Monje and Raizada, 2011) The two most closely related wild growing extant species to domesticated maize are Zea mays ssp. parviglumis Iltis & Doebley and ssp. mexicana (Schrader) Also the more divergent ancestors of maize (teosintes) still exist in the wild today mostly in the mountains of Mexico and Central America (Johnston Monje and Raizada, 2011) Maize is cultivated in many different parts of the wor ld and is a staple to many in sub Saharan Africa Southeast Asia, and Latin America (Nuss and Tanumihardjo, 2010) Maize is also an important ingredient in livestock feed, espe cially in the United States. A bout 50% of total protein intake for humans (and up to 70% in developing countries) comes from cereal grains directly or indirectly through animal feed (Nuss and Tanumihardjo, 2010) Some cultivars of maize also contain sufficient amounts of essential micronutrients such as niacin (Burkholder et al. 1944) There a wide variety of industrial uses for maize including biodegradable plastics, b iofuels like ethanol, insulating materials, and adhesives, just to name a few ( http://texascorn.org/cornwebsite/education uses.html ). Naturally, it is beneficial to focus scientific research on such an important crop. Developing methods to grow maize in otherwise unsuitable areas, increase crop yield and efficiency and augment nutritional value can
19 ever increasing population as well as benefit many different agricultural and industrial fields There has been an abundance of past studies on maize involving genetic modification and the use of chemical agents to increase crop yield or decrease pest or environmental damage (McInroy and Kloepper, 1995; Laabs et al. 2000; Ngonyamo Majee et al. 2009; de Solla et al. 2011) However the use of genetic modifications and synthetic chemical agents is controversial. Chemical pesticides are expensive, can be non s us tainable ( may require frequent applications), and can be toxic to non target organisms (Compant et al. 2005; Aravind et al. 2009; Prischl et al. 2012; Sundaramoorthy et al. 2012) There has also been a public concern about the environmental safety of genet ically modified crops (see Wisniewski et al. 2002; Domingo and Gin Bordonaba, 2011; Prischl et al. 2012 for reviews) This has generated i nterest in developing methods of augmenting crop development through alternative means, such as the modification of biotic factors lik e s oil microbial communities or endophytic communities Endophytic bacteria have been previously discover ed in maize. One such endophyte is the diazotrophic p roteobacterium, Herbaspirillum seropedicae which is known to colonize maize, rice, sorghum, wheat, and sugar cane (Balsanelli et al. 2010) This root colonizing microorganism is known to stimulate growth in maize by producing phytohormones, preventing infection by outcompeting pathogenic microorganisms for resources, and supplying nitrogen via nitroge n fixation (Baldani et al. 1986; Gyaneshwar et al. 2002) H. ser o pedicae are facultative endophytic bacteria that associate with their host by first attaching to the surface of root s, then colonizing emergence points of
20 se condary roots and pen etrating through openings of the epidermis to colonize inter cellular spaces and aerenchyma, root xylem, and aerial portions (Balsanelli et al. 2010) This initial attachment of H. serpedicae to the surface of maize roots is dependent on components of the bacterial cell envelope such as lipopolysaccharides (Balsanelli et al. 2010) L ipopolysaccharides (LPS) contain antigens wh ose specif icity is modulated by flavonoids secreted in the root exudates and by the presence of the monosaccharide rhamnose in the O antigen of LPS (Duelli and Noel, 1997; Samuel and Reeves, 2003) It has been found that mutating the genes that encode rhamnose causes detrimental changes to the LPS of H. seropedicae preventing it from attaching to the surface of roots and thus preventing colonization ( Balsanelli et al. 2010) Other common bacteria that have been found in domesticated maize include those from the classes Actinobacteria, p roteobacteria including Enterobacteriaceae ( Enterobacter spp and Pantoea spp ), Pseudomonas spp p roteobacteria and Methylobacteria (Johnston Monje and Raizada, 2011; Prischl et al. 2012) In Johnston (2011) the most commonly observed endophytes in mature seed s were the p roteobacteria, while the most common root endophytes were the Actinobacteria and p roteobacteria. Prischl et al. (2012) also found that p roteobacteria were the most common root endophytes. Some but not all of these bacteria have been found in the wild growing ancestor s ( Z ea mays ssp. parviglumis and ssp. mexicana ) though the abundance of the se common endophytes may have been altered during evolution (Johnston Monje and Raizada, 2011) Using molecular based methods to identify endophytic bacterial communities, 76% of the phylotype s (a type
21 that that classifies an organism by its phylogenetic relationship to other organisms) observed in mature seeds and 79% in stems of ssp. par viglumis were observed in mature domesticated maize (ssp. mays ) in these respective tissues (Szkely et al. 2009; Johnston Monje and Raizada, 2011) Similarly, 78% of mature seed phylotypes and 92% of stem phylotypes of ssp. mexicana were found in mature domesticated maize in their respective tissues S pecies specific endophytic bacteria have also been found in both maize and teosintes. T hree distinct phylotypes of bacteria in maize (predicted in silico to be Chloroflexi Bradyrhizobium and Paenibacillus caespitis ) and eight in teosinte were recently discovered none of which were culturable or found in clone sequence libraries (Johnston Monje and Raizada, 2011) Johnston Monje and Raizada (2011) also investigated whether there was a distinct microbiota in mature seeds compared to stems of maize, and they found that there was not a significant difference ( p > 0.05 ) in the number of phylotypes represented in each. However, they did find one phylotype ( predicted in silico to be Burkholderia phytofirmans or Pantoea ) in maize seeds but not in stems, and reciprocally, they found one phylotype (believed to be from genus Burkholderia Clostridium or Sphingobacterium ) in maize stems but not in seeds. The most common benefits offered by culturable mature Zea seed endophytes (i.e. traits that were the most conserved between endophytic species) are increased solubility of phosphate, secretion of acetoin (involved in the induction of ISR) and nitrogen fixation (Johnston Monje and Raizada, 2011) The secretion of cellulase and pectinase w as also a common trait found among these bacteria. These enzymes are involved in the breaking down of cellulose and pectin (component of cell walls in
22 plants), respectively, for the successful colonization of endophytic bacteria (Reinhold Hurek and Hurek, 1998; Pariona Llanos et al. 2010) Other, moderately common benefits of these Zea endophytes include ACC deaminase activity which decreases ethylene levels to reduce s tress symptoms, antibiosis against bacteria or yeast, and secretion of RNase which may play a role in viral defense (Li et al. 2010; Johnston Monje and Raizada, 2011) Other, less c o mmon traits of these bacteria include auxin production and siderophore secretion (Johnston Monje and Raizada, 2011) Interestingly, about 50% of the Enterobacter spp isolated in Johnston Monje and Pantoea spp were found to do so, even though they are closely related to Enterobacter spp (Johnston Monje and Raizada, 2011) Some P antoea spp are pathogenic (and thus not endophytic) but other strains are endophytic and have anti fungal properties Pseudomonas spp. are largely responsible for nitrogen fixation ( Vermeiren et al. 1999; Pariona Llanos et al. 2010) and also have ACC deaminase activity (Glick, 2005) iron chelation abilities by siderophore production (Kloepper et al. 1980) and secrete RNase, cellulase, and pectinase (Johnston Monje and Raizada, 2011) Pseudomonas have low acetoin and no auxin production, so they probably do not modulate phytohormone production in maize (Johnston Monje and Raizada, 2011) Methylobacteria are conserved across many maize genotypes and are believed to modulate airborne signals emitted by stomata by metabolizing volatile methanol (Romanovskaia et al. 2001; Abanda Nkpwatt et al. 2006) Met h ylobacteria also fix nitrogen, produce phytohormones, and have ACC deaminase activity (Johnston Monje and Raizada, 2011)
23 1.4. Common Methods for S tudy ing Endophytes When conducting research on endophytic microbes contaminat ion is always a major concern. It is very important to be able to determine the source of an isolated bacterium i.e. ensuring that an isolate is of endophytic origin and not the result of a n outside or surface contamination. The most common way of dealing with this problem is to first sterilize the exterior of a plant tissue sample. Even the earliest research on endophytes (Freeman, 1904) i nvolved surface sterilization techniques. To surface sterilize a plant tissue sample, the sample is first immersed in a sterilizing agent for several minutes and then washed with sterile water Freeman (1904) used mercuric chloride as a sterilizing agent bu t current research usually involves the use of a 0.5 % to 6 % (v/v) s olution of sodium hypochlorite (Garbe va et al. Romero, 2001; Aravind et al. 2009; Balsanelli et al. 2010) The sodium hypochlorite (bleach) acts to kill off any microbes residing on the exterior of the tissue sample without affecting microbes inside of the sample (endophytes) All subsequent methods of study are then conducted under aseptic conditions with sterilized equipment and reagents to ensure that any isolated microbes originated from within the tissue sample and not from the surface or so me external source Another issue with studying endophytes is determining whether or not an isolated bacterium is pathogenic By definition, an endophyte does not cause any apparent symptoms of disease, so in order for the bacterium to be classified as endophytic, it must not be pathogenic. Differentiating between an endophyte and a pathogen can sometimes be difficult. For exam ple, some Pantoea spp. are known
24 pathogens that cause soft rot and can also cause human disease, but other spp. are not pathogenic and even have anti fungal properties (Johnston Monje and Raizada, 2011) Wh en conducting endophytic research, it is important to only collect sample s from healthy plants that do not exhibit any apparent symptoms of disease. Earl ier research on endophytes centered on culturing techniques to isolate and identify microbes (Misaghi and Donndelinger, 1990; McInroy and Kloepper, 1995; Coombs et al. 2004; Pontes et al. 2007) Surface sterilizing tissue samples and then partially embedding in a growth medi um is a common low budget way to isolate and identify some endophytes, but this technique i s severely limited Diffe rent microbes require different nutrients such as carbon sources elements like nitrogen, phos phorus, and sulphur, various minerals, and amino acids (Tortora et al. 2004) Different microbes may also require different growing conditions ( e.g. temperature and pH ) Since no one growth medi um can account for all nutrition and growt h conditions, all growth media are inherently selective and dependent on experimental conditions (Prischl et al. 2012) Furthermore, not all of the bacteria may be cultur able for example it is estimated that only a very small percentage of all microbes within an environmental sample are able to be cultured (0.001% to 15% depending on type of sample) (Amann et al. 1995) In order to obtain a more complete understanding of natural bacterial communities research has recently shifted towards the utilization of molecular based methods of study ( Fig. 2 ; Woese et al. 1990; Pontes et al. 2007) Molecular techniques can be isolation and culture independent and are able to detect a much broader range
25 of bacteria through the use of DNA sequencing, particularly of the ribosomal subunit gene s (Mocali et al. 2003) Ribosomal RNAs are present in all Eubacteria and they contain highly conserved regions as well as variable regions (Amann et al. 1990) Likewise, the genes that encode the ribosomal subunits (referred to as rDNA) have conserved and variable regions ( Fig. 3 ). rDNA is not appreciably affected by horizontal gene transference or environmental changes so its mutation rate due to environmental effects is very low (Pontes et al. 2007) The ribosomes of Eubacteria are 70S, each consisting of a small 30S subunit and a large 50S subunit (Cox et al. 2012) The small subunit ( 3 0S) consists of a 16S R NA (size = 1540 nt ) subunit bound to 21 proteins, while the large subunit ( 50 S) consists of a 5S subunit (size = 120 nt) a 23S subunit (size = 2900 nt) and 3 6 proteins (Cox et al. 2012) The rDNA encoding the 5S, 16S, and 23S subunits are usually organized in an operon that has internal transcribed spacers (ITS) between the genes that can vary in length and sequence (Pontes et al. 2007) Up to 15 copies of this rDNA ope ron may be present in a (Pontes et al. 2007)
26 Figure 2. Common techniques used for phylogenetic research on bacteria RFLP = restriction fragment length polymorphism, ARDRA = amplified rDNA restriction analysis, DGGE = denaturing gradient gel electrophoresis, TGGE = temperature gradient gel electrophoresis, SSCP = single stranded conformation polymorphism, FISH = fluor escent in situ hybridization M ethods used for this thesis research are highlighted in yellow (see Chapter 2) Figure from Pontes et al. 2007 Figure 3. A representation of the highly conserved and hypervariable regions within the 16S rDNA gene based on aligned 16S sequences The plotted blue line represents differences in aligned 16S rDNA gene sequences of many different prokaryotes, with peaks representing hypervariable regions (V1 V9) and tro ughs representing conserved regions Black line is an approximate extension to include the complete 16S rDNA gene ( ~ 1540 bp). Arrows at positions 9 and 1509 show the targets of the PCR primers used in this thesis. See Fig. 4 for where the product hyperv ariable regions are located on the 16S r RNA secondary structure. Figure m odified from Siqueira et al. 2012
27 The gene encoding the 16S subunit in particular has several characteristics that make it ideal for phylogenetic study (Pontes et al. 2007) It is of a suitable length ( approximately 1540 bp ), it contains highly conserved regions as well as variable regions, it is present in all Eubacteria and it is easily manipulate d but not affected by horizontal gene transference. Bacterial diversity can be assessed from an environmental sample via DNA isolation and PCR a mplification of the 16S r D NA gene (Pontes et al. 2007; Rudrappa et al. 2010; Shin et al. 2012) A method known as terminal restriction fragment length polymorphism (TRFLP) involves isolation and amplification of the 16S rDNA with primers specific to the 16S rDNA gene (Kitts, 2001) One or both of the primers ha ve s labeled with a fluorescent molecule (if both they must be labeled with different fluorophores ) The present study used 6 FAM ( see Appendix C for structure) as a label for forward primer. The forward and reverse primers are designed to anneal to highly conserved regions of the 16S rDNA and encompass nearly the entire gene (size = approximately 154 0 bp) (Fuchs et al. 1998; Kitts, 2001) The space between the primers should contain hypervariable regions ( Fig. 3 ) which are specific to each phylotype of bacteria, usually the genus level (Kitts, 2001) Due to these hypervariable regions, the resulting mixture will contain amplicons of various sequences each of which is specific to a different phylotype of bacteria A tetranucleotide restriction enzyme digest ( which cut DNA, on average, once every 256 bp; Lynn et al. 1980) is performed on the amplicons to yield fluorescently labeled terminal restriction fragments ( TRF s) that vary in size due to the variance in
28 sequence of the PCR amplicons (Kitts, 2001) Once the amplicons are digested, the TRF s are separated by size via electrophoresis and each TRF fluorescence intensity is determined by a fluorescence detector. only the terminal fragments are read by the fluorescence detector. TRF s of different sizes will represent different phylotypes, and t he abundance of each TRF (as me asured by its relative fluorescence level) will be directly related to the abundance of the bacteria from which it originated (Osborn et al. 2000) P rofiles of microbial communities can be constructed as electropherograms which show the fluorescence of each TRF observed in a sample (in relative fluorescence units or RFU) vs. the size of each TRF (in bp) (Szkely et al. 2009) These data give the relative abundanc es of each prokaryotic phylotype (each TRF) represented within a sample. M ultivariate statistics procedure s such as principal component analysis (PCA) and non metric m ulti d imensional s caling (MDS) can then be conducted on the electropherogram data (using the area under each peak of fluorescence as the abundance of its repr esentative TR F) to create plots visualizing the relatedness between the microb ial communities of each sample (Dollhopf et al. 2001) PCA and MDS will be further discussed in section 2. 7 T RFLP is a rapid and sensitive method of detecting bacterial diversity without the need to isolate or culture. However, amplification of the 16S rDNA is not able to differentiate between very close ly related bacteria (i.e. individual species within a genus) though resolution can be improved by using multiple restriction enzymes. Other limitations to 16S rDNA PCR amplification include the dependency on efficiently
29 extracting metagenomic DNA ( i.e., all of the DNA present within a sample potentially consisting of genomes of many organisms) possible PCR biases such as some template DNA with weaker homology not competing well for primers and thus being underrepresented and the difficulty in est imating numbers of bacteria within a sample. Finally, 16S rDNA amplification can give relative abundances of different phylotypes above the species level, but it cannot be used to assess raw numbers of bacteria (Pontes et al. 2007) In order to localize bacteria in a sample bacteria can be labeled with a fluorophore and visualized with fluorescent or confocal microscopy with a technique called fluorescent in situ hybridization (FISH) (Banerjee et al. 2002) This technique also exploits highly conserved regions of rRNA which are stable under most conditions and very abundant in all Eubacteria (Coleman et al. 2007) A DNA probe tagged with a fluorophore and having a sequence complementary to a highly conserved region of the prokaryotic 16S rRNA is allowed to hybridize for several hours under controlled conditions (Coleman et al. 2007) A commonly used probe is EUB338 which targets a highly conserved region of the 16S rRNA from positions 338 to 355 ( Fig 4 ; Amann et al. 1990) This selectively labels 16S rRNA molecules of Eubacteria within a sa mple. The resulting 16S rRNA DNA fluorophore hybrid is then excited with a laser and visualized with either fluorescent or confocal microscopy. This technique can be used to visualize and sometimes quantify bacteria present within a n environmental or eukaryotic tissue sample since the probe specifically labels prokaryotic rRNA but not eukaryotic rRNA (Babot et al. 2011; Lopez et al. 2011) However, nonspecific binding of the DNA probe
30 can sometimes occur, so it is important to process negative controls in parallel with the experimental samples, including usi ng a labeled probe that has identical sequence as the conserved region of the 16S rRNA (instead of complementary sequence) and samples without probe (Osborn et al. 2000) 1.5. Specific Aims of Thesis Research Previous studies of endophytes in maize have largely focused on endophytes residing in the roots or mature kernels (Johnston Monje and Raizada, 2011; Prischl et al. 2012) The aim of this thesis was to detect the presence of endophytes within early developmental stage B73 maize kernels and assess how the endophytic community changes throughout the early phases of development. The inbred maize line B73 was selected becaus e it is commonly used to breed present day commercial varieties (Candela and Hake, 2008) and its genome has been fully sequenced (Schnable et al. 2009) Specifical ly, the potential presence of endophytes residing in developing B73 maize kernels (at 0, 1, 2, 3, 4, 6, 8, and 12 DAP ) was studied using culturing techniques, TRFLP, and FISH.
31 Figure 4. The primary and se condary structures of the 16S ribosomal RNA of Escherichia coli V1 V9 show the approximate locations of hypervariable regions not conserved between different bacteria (Neefs et al. 1993 ; see Fig. 3) The highlighted area ( green ) shows the rRNA sequence to which the DNA probe EUB338 hybridizes ( inset bottom hybridizes to position 33 8 This region in highly conserved among Eubacteria (Amann et al. 1990) Figure modifed from Case et al. 2007
32 Chapter 2 Materials and Methods 1 2.1. Germin ation Cultivation and Pollination of Maize The maize ( Zea mays ssp. mays L.) inbred line studied was Iowa based B73 Stock s eeds which we re ordered from the Maize Stock Ce nter (North Central R egional Plant Introduction S tation Ames, Iowa) were subjected to a 15 minute treatment in 0.53 % (v/v) sodium hypochlorite in dH 2 O prior to germination Seeds were germinated in a growth chamber at 25C until the radicles were about 3 cm in length Seedlings were planted in 3 5 gallon pots (one plant per pot) containing soil media consisting of 29% peat moss, 29% vermiculite, 7% top soil (Scotts Premium Potting Soil), 20% sand (Sakrete Multi Purpose Sand), and 15% perlite. Maize plants were grown in greenhouse conditions on campus o f New College of Florida in Sarasota with supplemental lighting to give a 16 hour light cycle and 8 hour dark cycle Plants were fertilized three times per week with 300 mL of Southern Ag 20 20 20 S oluble F ertilizer w ith M i nor E l ements, and twice per wee k with 300 mL of 2 mM magnesium sulfate. Surface p ests and fungal growth were controlled with topical applications of n eem oil and Thuricide (Farm and Garden Sarasota, FL ) which contains active toxins isolated from Bacillus thuringiensis ssp. kurstaki (B t ) that target larval lepidopteran s (Federici, 2005) In order to control pollination, n ascent cobs and mature anthers were covered until the time of pollination Mature plants were self pollinated or sib pollinated (which gives rise to similar progeny given that the line is nearly isogenic) covered, and harvested after a set number of days after pollination (DAP). 1 Chemicals were obtained from S igma Aldrich unless otherwise noted.
33 2.2. Harvesting and Surface Sterilization Maize cobs were harvested at 0, 1, 2, 3, 4, 6, 8, and 12 DAP and brought into the lab to harvest kernels under aseptic conditions. This entailed sterilizing work surfaces, gloves, and tools with 70% ethanol. All work was conducted near an open flame, and tools were flame sterilized before and after each use. The husk of the maize cob was also gently wiped down with 70% ethanol to avoid contamination All pipette tips reagents, and glassware were autoclaved prior to use K ernels were harvested from t hree different maiz e plants for each DAP T hose that were surface sterilized were first wa s hed with five quick submersions in 50 mM sodium p hosphate b uffer pH = 7 ( PB; see Appendix A) Kernels were t hen surface sterilized with a solution containing 1 % (v/v) sodium hypochlorite amended with 0.1% Tween 20 ( polyoxyethylenesorbitan monolaurate ) in 50 mM PB for 1 5 minutes under gentle agitation (Ayorinde et al. 2000) Finally, kernels were washed with t hree 5 minute changes of sterile 50 mM PB under gentle agitation Control k ernels that were not surface sterilized were harvested directly from the cob Portions of harvested kernels (for each DAP) were either cultured in growth media fixed in 4% (v/v) formaldehyde (Ted Pella) in 50 mM PB and s tored at 4C or frozen at 80C for DNA isolation. 2.3. Cultur ing Tryptic soy agar (TSA ), brain heart infusion agar (BHI ), and nutrient broth (NB Fisher EMD ) were used as growth
34 media for micro organisms (see appendix A ) These media are used for cultivation of a wide variety of aerobic and anaerobic micr o bes including fastidious form s of bacteria, yeasts, and mold s (Leavitt et al. 1955; MacFaddin, 1985; Vanderzant and Splittstoesser, 1992) and have been used in previous studie s involving culturing endophytes (Mocali et a l. 2003; Seghers et al. 2004; Rijavec et al. 2007) In order t o verify surface sterilization, control kernels that had been surface sterilize d were placed directly on TSA and/or BHI and incubated at room temperature for 5 days. If no growth was obs erved after five days, the kernels were said to be effectively surface sterilized For comparison some kernels were harvested and placed directly in growth media without surface sterilization The internal tissue of surface sterilized kernels ( mainly nucellus in unfertilized or very early DAP kernels and endosperm in older kernels see appendix F igure B 1 for micrographs showing developmental stages ; Randolph, 1936; Sonchaiwanich, 2012) w as analyzed for endophytes by extraction with a sterile blade under aseptic conditions (as described previously) followed by direct placement of this tissue on TSA or BHI However, if kernels were very immature (0 to about 2 DAP kernels), very little tissue could be extracted making it difficult to place this tissue directly on TSA or BHI. In these cases internal tissue was extracted and homogeniz ed with a sterile pestle in 5 mL of NB Broth suspensions were incubated at 25 C on a New Brunswick Scientific Controlled Environment Incubator Shaker with moderate agitation until bacterial growth was observed (typically 3 5 days). A sterile loop was then used to streak broth suspensions onto TSA or BHI media. TSA and BHI pl ates were incubated in a growth chamber at 25 C until growth was observed (typically 5 8 days)
35 Pure bacterial colonies were isolated from m ixed colony TSA and BHI plates by touching single colonies with a sterile loop and streaking onto fresh TSA or BHI plates. After 2 3 days of incubation, t hese pure colonies were then inoculated into fresh NB and incubate d for another 1 2 days Once growth was observed, b acteria were f rozen at 80C in a 1:1 mixture of NB bacterial suspension and sterile 50% (v/v) glycerol in sterile water for long term storage 2.4. Fluoresce nt I n S itu Hybridization (FISH) Potential e ndophytic bacteria were probed for using 16S r RN A targeting DNA oligonucleotide EUB 338 2 ( GCTGCCTCCCGTAGGAG ) tagged with fluorescein isothiocyanate (FITC see Appendix C for structure ) ordered from Invitrogen The E UB 338 probe targets most Eubacteria by binding highly conserved regions of the 16S ribosomal RNA at positions 338 2 to 35 5 ( Fig. 4 ; Amann et al. 1990) T he complementary control probe NON 338 ( A CTCCTACGGGAGGCAGC ) also tagged with FITC was used as a negative control to detect non specific binding since it should not hybridize to the 16S rRNA target (Wallner et al. 1993) Kernels were surface sterilized a s described previously and fixed overnight in 4 % formaldehyde in 50 mM PB under rotation and stored at 4C. They were then washed with three 15 minute changes of 50 mM PB prior to processing for hybridization After the PB washes, two different methods were attempted to probe for endophytic bacter ia 2 to 355. A basic local alignment search tool (BLAST) search of the probe sequence against the Zea mays ssp. mays genome revealed no significant sequence similarity (all E Altschul et al. 1997)
36 (see sections 2.4.1 and 2.4.2). Most kernels were probed as described in 2.4.2. 2.4.1. FISH with Homogenized Kernels The first attempt to fluorescently label potentially endophytic bacteria using FISH involved cutting the kernels in half with a sterile blade under aseptic conditions, and the internal tissue was extracted and homogenized with a sterile pestle in 40 L of nan opure water. A volume of 5 L of the homogenized internal tissue was added t o each sample well of an 8 w ell glass slide Positive control wells were then spiked with fixed whole cell Escherichia coli which were prepared as follows. E. coli were cultured overnight in NB tubes at 37 C, collected by centrifugation at 6000 X g and resuspended in 50 mM PB, then fixed overnight with three volumes of 3.7% (v/v) formaldehyde in 50 mM PB (pH = 7.2) to one volume of bacterial suspension. The E. coli were then wa shed with two 15 minute changes of 50 mM PB and stored in a solution of 50 mM PB and ethanol (1:1 by volume) at 4C. Once the samples and E. coli controls were pipetted into the wells, the slides were allowed to air dry at room temperature and then were s ubjected to a dehydration series in 50%, 80%, and 95% ethanol (3 minutes each). To each sample to be probed, 8 L of hybridization buffer ( 20 mM Tris, 0.9 M NaCl, 0. 0 1% SDS, and 3 5% w/v formamide 3 ; see appendix A for recipe ) and 1 L of either EUB338 or NON338 probe (50 ng/L in nanopure water) was applied. T o each sample without probe (negative controls), 9 L of hybridization buffer was added. 3 Formamid e concentrations of 15%, 20%, and 35% (v/v) were tested. A concentration of 35% (v/v) gave rise to the best stringency level for labeling E. coli control slides. None of these concentrations significantly affected fluorescence levels in kernels.
37 Humidity chambers equilibrated with 1 M NaCl were prepared by soaking filter pa per in 1 M NaCl and placing it as a liner around the inside of 50 mL conical tubes. The chambers were preheated to the hybridization temperature of 48C (Banerjee et al. 2002; Coleman et al. 2007) and sample slides were then enclosed in the humidity chambers and permitted to hybridize in a hybridization oven for 3 4 hours at 48C. Once hybridization was complete, excess probe in hybridization buffer was removed with 5 mL of wash buffer preheated to 48C (20 mM Tris, 1 0 0 mM NaCl, 5 mM EDTA trace SDS, and 0. 4 nM DAPI or 4',6 diamidino 2 phenylindole which can penetrate cell walls and stain s DNA see Appendix A for recipe ; Banerjee et al. 2002) Slides were then immersed in additional wash buffer for 20 minutes to further remove excess probe and hybridization buffer. A final wash with nanopure water was followed by a step in which the slides were permitted to air dry in the dark. SlowFade Antifade reagent ( Molecular Probes/Invitrogen ) in 50% (v/v) glycerol/PB S containing 1.5 g/mL DAPI as a nuclear counterstain was used as the mounting med ium (Component A, kit S 24635) Coverslips were sealed with colorless nail polish and slides were stored in the dark at 4C. 2.4.2. FISH with Sectioned Kernels The second attempt to fluorescently label bacteria using FISH involved sectioning kernels with a V ibratome Series 1000 rather than homogenizing the kernel (as described in sections 2.4 and 2.4.1) The majority of kernels probed with FISH were sectioned F ollowing fixation and the subsequent washing with PB, kernels were sectioned with a n
38 ethanol sterilized V ibratome filled with sterile 50 mM PB chilled to 4C. The kernels were cut into approximately 200 m thick sagittal longit udinal sections by first adhering them to the cutting block with Instant Krazy Glue such that a lateral surface and the embryo faced downwards ( Fig. 5 ) Figure 5 Sagittal longitudinal orientation of kernel for V ibratome sectioning. Figure prepared by author. Each s ection w as then placed in a separate well of an 8 well glass slide and allowed to air dry, then was subjected to probe hybridization This was performed as described i n section 2.4.1, however, sections were not dehydrated with ethanol ; rather, probe/hybridization buffer was added directly on top of the sections After hybridization was complete secti ons were transferred to the wells of a sterile ceramic plate for the was hing and counter staining steps After washing/counter staining was complete sections were then transferred back to the 8 well glass slides and mounted with antifade, sealed with nail polish and stored in the dark at 4C as described in 2.4.1. 2.4.3. FISH Confoc al Microscopy Homogenized and sectioned FISH samples were visualized with a PerkinElmer UltraView ERS Spinning Disk Confocal Microscope (located at University of South
39 Florida Tampa ) an Olympus Fluoview 300 Confocal Laser Scanning Microscope (located at Central Michigan University), or a Nikon Eclipse Ti Inverted Confocal Microscope (located at Central Michigan University) All sectioned samples (section 2.4.2) were visualized wit h the spinning disk confocal scope, and homogenized samples (section 2.4.1) were visualized with the Olympus laser scanning scope and the Nikon inverted scope. 2.5. DNA Isolation, Polymerase Chain Reaction (PCR) and Gel Electrophoresis Metagenomic DN A was isolated from homogenized internal tissue extracted from surface sterilized kernels with the Ul t ( MO BIO Carlsbad, CA ) optimized for the isolation of bacteria l DNA in the presence of plant residues and other potential interfering compounds DNA samples were stored at 80C until PCR amplification. The 16S r D NA gene was amplified in triplicate for each sample (i.e., three separate PCR runs per sample which included metagenomic DNA from 0, 1, 2, 4, 6, and 8 DAP kernels ) via PCR in 50 L reactions utilizing the 16S rD NA primers U9f 6 FAM 6FAM GAGTTTGATYMTGGCTC GYTACCTTGTTACGACTT both ordered from Invitrogen S ee Fig ure 3 in section 1.4 for the target positions on the 16S rDNA of this DNA primer pair (Cunning et al. 2008) The PCR reagent s used were from the Clontech Advantage 2 PCR kit includ ing 5 L 10x Advantage 2 buffer, 1 L 50x dNTP mix, and 1 L 50x Adv antage 2 polymerase (Taq) per reaction. In addition, each PCR reaction contained 40 L nanopure water, 1 L forward primer (U9f ; final
40 conc entration = 2 M ) 1 L reverse primer (U1509 r ; final concentration = 2 M ) and 1 L of DNA extract Sigma E. coli strain B type VIII genomic DNA (1 g/ L) was used as a template for positive controls and sterile PCR grade water was used for negative controls (no template DNA) Thermocyclin g was conducted with a MJ Research PTC Cycler with an initial denaturation step at 94.0C for 4 min followed by 30 cycles of denaturation at 94.0C for 30s, annealing at 60.0C for 30 minutes, and extension at 72.0C for 90s, with a final extension step of 72.0C for 10 minute s. PCR amplicons were stored at 20 C Amplification of the 16S r D NA gene was confirmed for each PCR run with electrophoresis on a 0.8% (w/v) agarose gel containing et h idium bromide ( EtBr ) A volume of 5 L PCR product w as loaded with 2 L 6X loading dye and 5 L nanopure water Gels were ru n with 1 X TAE buffer at ~ 90 V for 30 minutes and imaged with a Genomic Solutions gel imager Once PCR amplification was confirmed by the presence of a ~ 1500 bp band the three products were pooled for terminal restriction fragment length polymorphism ( TRFLP ) analysis. 2.6. Terminal Restriction Length Polymorphism ( TRFLP ) Following metagenomic DNA extraction and triplicate PCR amplification and confirmation (see section 2.5), the three PCR products of each sample were combined and then purified with the Qiagen QIAquick PCR Purification Kit (a kit optimized for
41 removing ex traneous primers, nucleotides, polymerases, and salts without affecting PCR amplicons) After purification, the combined PCR amplicons were digested with the tetranucleotide restriction enzymes RsaI and HhaI ( Fig. 6 ; both ordered from New England BioL abs ) Figure 6 Target sequences recognized by RsaI and HhaI restriction enzymes. Black arrows are where hydrolysis occurs. Figure prepared by author. For the RsaI digestions, 0.5 L RsaI (5 units where one unit is defined as the amount of restriction enzyme necessary to fully digest 1 g of substrate DNA in a 50 L reaction in 60 minutes ), 2 L 10X NE buffer (New England Biolabs proprietary buffer) and 9.5 L nanopure water were added to 8 L purified PCR product T he reaction was allowed to digest for 1.5 hours at 37C, then an additional 0.5 L of RsaI ( 5 units) was added and the digest ion continued for another 1.5 hours. For the HhaI digestions, 0.25 L HhaI (5 units), 2 L 10X NE buffer, 0.2 L 100X B ovine Serum Albumin (BSA) and 9.55 L nanopure water were added to 8 L PCR product The reaction was allowed to digest for 1.5 hours at 37C, then an additional 0.25 L of HhaI (5 units) was added and digest ion continued for another 1.5 hours. After the 3 hour digestion by RsaI or HhaI, samples were stored at 20C until further
42 processing. Digested s amples were then shipped to the Core DNA Facility of the University of Illinois where fragments were separated by size via electrophoresis and the rela tive fluorescence of each terminal 37 30xl Genetic Analyzer GeneMapper v 4 software. 2.7. Statistical Analysis Terminal restriction fragment ( TRF ) peaks identified by Gene Mapper software from individual TRFLP electropherogram s were compiled and manually aligned to create large data matrices for each restriction enzyme digest Only TRFs between 50 and 1050 bp in size with peaks matching the followi ng criteria were used for statistical analysis : height of over 50 relative fluorescence units ( RFU ) minimum peak half width of 2 pts polynomial degree of 3, and peak window size of 15 pt s (all default settings) TRFs smaller than 50 bp and larger than 1 050 bp were removed due to uncertainties with fragment sizes and to avoid the detection of primers (as done by Klaus et al. 2007) TRFs that did not match the peak criteria listed above were considered background noise and culled from statistical analysis The area under each peak was transformed to a logarithmic scale 4 For prin c ipal component analysis (PCA), all areas were also normalized 5 such that the sum of all peak areas o f identified TRF s equaled zero 4 Log(X + 1), where X is the peak area. 5 The mean area was subtracted from each variable, and then each term was div id ed by the standard deviation.
43 PCA and non metric m ulti d imensional s caling (MDS) of TRFs were conducted with PRIMER v 6 (PRIMER E) mu ltivariate statistic software. PCA is a parametric ordination technique that reduces the number of variables of a set of data down to the fe w th at best account for variability between samples (Pett Ridge and Firestone, 2005) and has been commonly utilized for TRFLP analysis of microbial profiles (Wang et al. 2004; Park et al. 2006; Zhang et al. 2007; Szkely et al. 2009; Johnston Monje and Raizada, 2011) It achieves this by generating called Principal variables (data) such that PC1, PC2, etc. are uncorrelated PC1 accounts for the most variability observed between all samples, and PC2 accounts for the second most variability while being uncorrelated to PC1. By doing this PCA reduces high dimensional data down to two or three dimensions (for visualization) with minimal information loss. F rom PCA, ordination plots were generated showing PC1 vs. PC2 (the two PCs that account for the most variability observed between samples) Ordination by MDS was also conducted in order to more easily visualize differences between samples PCA ordinations show a plot of PC1 vs. PC2, but since PC1 and PC2 account fo r different percentages of total variability (i.e., they carry different weights), it is harder to visualize similarities between microbial profiles of different samples. MDS ordinations are based on cluster analysis using Bray Curtis similarity 6 S ample s that appear closer together on MDS ordinations ( i.e., they cluster together ) 6 The similarity between two samples is defined by where C ij is the sum of the lesser v alue for only those TRFs detected in both samples, and S i and S j are the total number of TRFs detected in both samples.
44 have more similar microbial communities Dendrograms were also created with the PRIMER v 6 software to show hierarchical clustering (also Bray Curtis) of the bacterial communities. MDS has also been a commonly method of analyzing TRFLP results (Harder et al. 2004; Klaus et al. 2007; Thiyagarajan et al. 2010)
45 Chapter 3 Resu l ts 3.1. Culture s The e fficacy of surface sterilization of maize kernels was tested using culturing techniques The results of the culturing techniques are summarized in Table 1 and examples of various growths are shown in F ig ures 7 and 8 In general, the surface sterilizatio ns were reasonably successful. T he surface sterilized kernels tended to display less growth than both the non surface sterilized kernels and the internal tissue extract ed from surface sterilized kernels for both TSA and BHI media. Intact s urface sterilized kernels tended to show no or very little growth, while the non surface sterilized kernels showed the most growth. Plates streaked with internal tissue extracted from surface sterilized kernels displayed various amounts of growth There did not appear to be an obvious difference in the abundance of microbial g rowth between TSA and BHI media for any of the experimental groups when both were used In some cases, only TSA was used due to limiting quantities of kernels. Although both bacteria and fungi were found to grow from kernel tissues, I chose to focus on potential bacterial endophytes for the remainder of the analyses.
46 Table 1. Microbial growth after 5 8 days of incubation. Commas separate individual kernels, and forward slashes (/) separate bacterial and fungal growths from same kernel (if both present). Each row represents kernels harvested from a different cob. Blank table cells are lack of data typically due to a limited numb er of kernels from a particular harvest. TSA = tryptic soy agar; BHI = brain heart Infusion agar; DAP = days after pollination; SS = surface sterilized kernels partially embedded in growth medium; Non SS = non surface sterilized kernels partially embedded in growth medium; Endo = potentially endophytic growth from homogenized internal tissue extracted from surface sterilized kernels and streaked onto growth medium b = very little bacterial growth, bb = moderate bacterial growth, bbb = abundant bacterial growth (f, ff, fff represent fungal growths, minus means no growth see figures 7 and 8 for examples ). TSA BHI DAP SS Non SS Endo SS Non SS Endo 0 bbb/ff, bbb/ff, bbb/f, bbb/f bb, fff, bbb/fff f, 0 b, b bb, bbb/f bb, bb b, ff, b 1 b, bb, bb 2 bb, bbb ff bb, bb 4 bb, bb bbb, bb 6 ** fff, ff 6 bbb bbb bbb, bb, bb/f, bb/f bb 8 b, b bb, bbb 12 b bbb, fff b/ff Shown in Fig. 8 ** Shown in Fig. 7
47 Figure 7 Examples of microbial growth arising from various treatments of B73 6 DAP kernels (all from same cob) partially embedded in growth media after 13 days of incubation. Black arrows (1 4) indicate kernels or remnants of smeared internal tissue in the case of streaks (5). Translucent white ovals indicate approximate streak zones. A: Surface sterilized kernels in TSA; no bacterial or fungal growth ( ) on 1 or 2 B: Non surface sterilized (surface microbes) kernels in TSA; fungal growth around 3 ( fff) contaminant bacterial growth ( white arrow) on side of culture No growth around 4 ( ) C + D: Internal tissue (mostly endosperm) extracted and homogenized from surface sterilized kernels streaked on BHI ; one possible endophytic fungal growth (ff) plus one contaminant fungal growth ( centered outside of streak zone ; ff) See Table I for rest of data f = very little fungal growth, ff = moderate fungal grow th, fff = abundant fungal growth minus means no growth.
49 Figure 8 Examples of microbial growth arising from various treatments of B73 0 DAP kernels (all from same cob) partially embedded in growth med ia after 5 days of incubation. Black arrows (1 8) indicate kernels or remnants of smeared internal tissue ( mostly nucellus in this case ) in the case of streaks (9). Translucent white ovals indicate approximate streak zones. A: Surface sterilized kernels in TSA; low level of bacterial growth around 1 and 2 (both b) B: Surface sterilized kernels in BHI; low level of bacterial growth around 3 (b) No growth around 4 ( ) C: Non surface sterilized (su rface microbes) kernels in TSA; bacterial growth around 5 (bb) multiple bacterial growths (bbb) and one fungal growth around 6 (f ) D : Non surface sterilized ( surface microbes) kernels in BHI; fungal and bacterial growth around 7 (fff) little bacterial growth around 8 (b) E: Internal tissue (mostly nucellus) extracted from surface sterilized kernels streaked on TSA; bacterial growth on both streaks (both bb) F: Internal tissue (mostly nucellus) extracted and homogenized from surface sterilized kernels streaked on BHI; no growth. b = very little bacterial gr owth, bb = moderate bacterial growth, bbb = abundant bacterial growth (f, ff, fff represent fungal growths), minus means no growth. investigating sections of kernels from the same cob under a microscope, it was determined that they were not fertilized (pollen was not viable), and thus these samples were counted as 0 DAP. 3.2. FISH C ontrol slides containing only E. coli were first analyzed to verify probe efficacy. These slides showed positive results ( Fig 9 ) DAPI (blue) counterstaining was visible o n all E. coli samples and was not affected by the presence or absence of the probe. Positive control E coli slides with EUB338 showed visible FITC (green) fluorescence, negative control E. coli slides with N ON 338 displayed very little FITC fluorescence (nonspecific binding of probe) and the negative control E. coli slides with no probe showed no FITC fluorescence. Therefore, the probe appeared to function as expected. However, d espite the attempt of two methods for visualizing endophytic bacteria using fluorescently labeled probes, no such bacteria were able to be observed with confocal microscopy in sectioned or homogenized kernels. All homogenized ( data n ot shown ) and sectioned kernels ( Fig. 10 ) exhibited significant amounts of autofluorescence in the FITC channel Levels of FITC
50 autofluorescence were similar in all homogenized and sectioned samples including those probed with EUB338 those probed with NON 338 and those without any probe Since EUB338 is supposed to selectively label Eubacteria with FITC, the large amount of autofluorescence in the FITC channel made it difficult to assess the potential presence of endophytic bacteria Figure 1 1 shows a homogenized 12 DAP kernel that was intent ion ally spiked with E. coli and probed with EUB338 to show relative levels of fluorescence from maize tissue and bacteria
51 Bright F ield DAPI FITC Figure 9 Representative FISH images of E. coli control slides visualized with the UltraView ERS Spinning Disk Confocal Microscope The left column shows bright field images, the middle column shows DAPI images, and the right column shows FITC images. The top row was probed wi th EUB338, the middle row was probed with NON338, and the bottom row had no probe. Note that the DAPI counterstain was present in the wash buffer and mounting medium for all preparations. EUB338 NON 338 No Probe
52 DAPI FITC DAPI + FITC (overlay) Figure 1 0 Representative FISH images of sectioned kernels visualized with the UltraView ERS Spinning Disk Confocal Microscope The left column shows DAPI images, the middle column shows FITC images, and the right column shows overlaid FITC + DAPI images. The top row was a 2 DAP kernel probed with EUB338, the middle row was an 8 DAP probed with NON338, and the bottom row was a 2 DAP with no probe. Note that the DAPI counterstain was present in the wash buffer and mounting medium for all preparations. The tissue shown the 2 DAP images is nucellus (maternal) tissue which initially fills the kernel and is replaced by endosperm by about 8 DAP (middle row ). 2 DAP kernel probed with EUB338 8 DAP kernel probed with NON338 2 DAP kernel without any p robe
53 Figure 1 1 Structures consistent in morphology with r od shaped E. coli in homogenized 12 DAP maize tissue that had been intentionally spiked (indicated by red circle) labeled by EUB338 as visualized with a fluorescent microsco pe. Mag. = 630 X. 3.3. TRFLP Amplification of 16S rDNA from metagenomic DNA isolated from internal tissue of surface sterilized kernels ( or E. coli genomic DNA for positive controls ) via PCR was confirmed by the presence of a ~1540 bp band visualized on agarose gel s while n o amplification was observed in negative controls (data not shown) TRFs were separated by size with electrophoresis and the relative fluorescence of each TRF was measured. From these data, electropherograms were generated ( representations are shown in Fig. 1 2 a nd the remaining electropherograms are in appendix Fig. B 2 ). In total, 23 different
54 TRFs were detected between the two restriction enzyme digests of DNA obtained from 7 different kernels (0, 1, 2, 4, 4, 6, and 8 DAPs) PCA was conducted to generate ordinations which display levels of similarit ies between profiles of the represented TRFs in each kernel ( Fig. 1 3 ). Note that the two axes carry different weights, so cluster ing of samples by PC2 i s less significant than clustering of samples by PC1. Bray Curtis similarity between samples is also shown by the colored outlines (each indicating a percentage of similarity of TRF profiles between samples). Only the first two principal components are shown (PC1 and PC2) accounting for 73.1 % of total variability for HhaI digests (PC1 = 45.6 % and PC2 = 27.5 %) and 90.6% of total variability for RsaI digests (PC1 = 46.2 % and PC2 = 25.1 %) Except for the 4 DAP samples, b oth restriction enzymes produc ed similar results. The unfertilized kernel (0 DAP) was separated far from the rest of the kernels mostly by PC1 for both RsaI and HhaI digests For the Hh aI digest s one 4 DAP kernel ( labeled 4A ) was separated from the 1, 2, 4 B 6, and 8 DAP kernels mainly by PC2 For the Rs aI digests, the two 4 DAP kernels seemed to swap places compared to the Hha I digests: t he 4 DAP kernel labeled 4B was sep arated from the 1, 2, 4A 6, and 8 DAP kernels mainly by PC2 The MDS ordinations showed similar results to the PCA ( Fig. 1 3 ) The MDS ordinations are nonmetric and do not have axes. Kernels are clustered in two dimensional space based on their Bray Curtis similarity; kernels that appear closer together have more similar TRF profiles. Dendrograms show a hiera rchal representation of the same Bray Curtis similarity data ( Fig. 1 4 ). Again, the unfertilized kernel (0 DAP) diffe red the most for both digests having between 60 % and 70% similarity with all other
55 kernels for HhaI digests and between 50 % and 60% for R saI digests All other samples clustered between 70 % and 80% similarity for HhaI digests and between 60 % and 70% similarity for RsaI digests. The greatest similarity observed was about 92 93 % (group average from Fig. 1 5 ) between the 1 6, and 8 DAP kernels for HhaI digests.
56 Figure 1 2 Representative electropherograms from 0 DAP and 8 DAP kernels. Each peak used in statistical analysis is labeled by an arrow and its TRF size. The blackened areas from 0 to 50 bp and from 1050 to 1200 bp were culled from analysis due to uncertainties with fragment sizes and to avoid detection of primers. Unlabeled peak s were deemed by the GeneMapper software to be background no ise and/or artifacts. Full scale electropherograms are included in a ppendix Fig. B 2
57 Figure 1 3 PCA and MDS ordinations for kernels at 0, 1, 2, 4 6 and 8 DAP. Two different kernels from different cobs were tested for 4 DAP (4A and 4B). Each kernel is labeled by its DAP. Note that the PCA ordinations in A and B carry different weights between their x and y axes. The MDS ordinations in C and D are nonmetric and d o not have axes K ernels that are closer together (two dimensionally) have more similar TRF profiles Simila rities are shown as percentages based on Bray Curtis similarity (cluster analysis). A: PCA ordination for HhaI samples. B: PCA ordination for RsaI samples. C: MDS ordination for HhaI samples. Note that t hree kernels (1, 6, and 8) were very similar (> 90%) and largely o verlap ped (indicated by *) D: MDS ordination for RsaI samples
58 Figure 1 4 Dend r ogra m s from Hh aI (top) and RsaI (bottom) digest data computed based on Bray Curtis similarity Hierarchal similarities are based on group averages (e.g., for the Rs aI digests, sample 6 had approximately 88 % similarity with the Bray Curtis average linkage between samples 2 and 8 ) Theoretically, these rela tionships should correspond to the levels of similarities between the bacterial phylotypes in these different samples.
59 Chapter 4 Discussion 4.1. Cultures Bacterial growth was observed in both TSA and BHI media streaked with internal tissue extracted from surface sterilized kernels. Control plates containing partially embedded, surface sterilized kernels tended to have little or no growth. Together, these results suggest that the bacteria observed in the surface sterilized, internal tissue streaked plates may have originated from within the kernels However, since there was growth observed around some surface sterilized (negative control) kernels, a firm conclusion cannot be made as to the origin of these bacteria Ideally, surface sterilization should completely sterilize the surface of all samples without affecting potential endophytic microorganisms (Aravind et al. 2009) However, o ne possible explanation for the observance of growth around surface sterilized kernels is that f reshly harvested kernels tended to be very delicate especially those earlier in development (low DAP) and they were sometimes damaged during processing Although care was taken to minimize damage, s mall ruptures in the surface of kernels sometimes occurred while tr ansferring kernels from surface sterilization solution or sterile wash water to growth media. These ruptures may have served as a means for endophytic bacteria to exit the inner tissue and appear as gro wths around some surface sterilized kernels Conversely, such minute openings could allow for entry of s urface bacteria into the inner tissue of kernels but it should be noted that v ery f ew kernels had obvious ruptures and damage occurred in the st e ps f ollowing surface sterilization
60 Another possible explanation for growt hs occurring around the surface sterili zed kernels is that the surface sterilization solution used was not completely effective. S amples in this study were treated with a solution containing 1% (v/v) sodium hypochlorite prior to culturing or DNA extraction which has been effectively used in previous studies involving the isolation of endophytes (Misaghi and Donndelinger, 1990; Garbeva et al. 2001; Rijavec et al. 2007; Harish et al. 2008) However, some previous studies have used more concentrated sol utions containing up to 6% (v/v) sodium hypochlorite (Balsanelli et al. 2010) The length of time that samples are imme rsed in surface sterilization solution also var ies greatly in the literature from as low as 10 seconds (Zinniel et al. 2002) to about 2 0 minutes (Balsanelli et al. 2010) In addition, s ome researchers precede and/or follow sterilization with sodium hypochlorite with immersion of samples in ethanol (Aravind et al. 2009; Balsanelli et al. 2010) No previous studie s h ave involved the isolation of endophytes from early developmental stage maize kernels, so a sterilization procedure had to be developed for thi s study. Since tissues of early stage kernels are delicate, a lower concentration of sodium hypochlorite was used in order to minimize the risk of solution penetrating surface and killing potential endophytes. An ethanol treatment step was n ot used in this study since ethanol can severely dehydrate such delicate tissue thereby affecting the morphology of the kernel s A relatively long sterilization time (15 minutes) was used to compensate for th e lower concentration of sodium hypochlorite Kernels that were not surface sterilized (embedded in media immediately after harvesting) a lways displayed the most growth, suggesting that the highest
61 concentration of microorganisms was on the surface of kernels. These bacteria and fungi may be argued t o still be since many researchers use to simply refer to bacteria and fungi that live inside of a plant without causing observable harmful effects (Zinniel et al. 2002 ; Da et al. 2012) Maize kernels are firmly encased by several overlapping layers of husk, so bacteria residing on the surface of kernels are still technically inside of a developing structure It should also be noted that s ome endophytes are able to transfer between tissues (Sprent and Faria, 1988; White et al. 1995) O nly kernels from apparently healthy plants were harves ted for analysis No plants that displayed obvious signs of disease were used in this study Therefore, if any bacteria isolated in this study did in fact originate from within the plant they fit the definition of endophytic (not causing apparent symptoms of disease ), but that is not to say that these bacteria a re completely innocuous Any bacterium or fungus isolated from within the maize may have some s well being without the plant displaying obvious symptoms of disease. This may be because said bacterium/fungus is in very low abu ndance or has only very recently infected the plant Any bacterium or fungus isolated from within a surface sterilized plant requires further investigation before it can be deemed non pathogenic even if it fits the definition of
62 4.2. FISH The results of the FISH samples were inconclusive due to the difficulty of detecting the possible presence of FITC labeled bacteria with in tissue that itself brightly emits in the FITC (green) channel DAPI staining on all control and maize samples showed expected results that is staining of cell walls and nuclei of all cells. DAPI staining was not affected by presence or absence of either probe. Control slides that contained only E. coli showed good FITC labeling S amples probed with EUB338 showed a r easonable level of fluorescence (indicating that the probe was able to hybridize with the bacterial rRNA target) while samples probed with NON338 showed very little fluorescence (due to nonspecific binding of the pr obe), and samples without any probe show ed no fluorescence ( Fig. 9 ) However, all samples that contained maize tissue showed very bright fluorescence in the FI TC channe l There was not a n observable difference in the amount of fluorescence between samples probe d with EUB338, NON338, and those without any probe. Since even samples without any probe showed bright emission in the FITC channel, the emission may be attributed to autofluoresce nce of the tissue and not nonspecific binding of the probe. Hybridization c onditions were adjusted to optimize results for the E. coli control slides. A hybridization time of about 4 hours at 48 C produced the best results for these slides, but the various temperatures (46 C to 48 C ) and lengths (4 hours to 24 hours) attempted d id not significantly change the levels of fluorescence of the samples that contained maize tissue. A few of the homogenized tissue samples were intentionally spiked with E. coli prior to hybridization with EUB338 in order to verify that the probe
63 wa s able to bind to bacteria within the maize tissue. Some rod shaped E coli were visualized within these samples ( Fig. 1 1 ) but finding these bacteria was a difficult task even when samples were spiked with large amounts. Some structures from the maize tissue were of somewhat similar size, shape, and fluorescent intensity as rod shaped bacteria (resembling the E. coli ) but no structures were able to be definitively deemed endophytic bacteria in the non spiked samples The str ingency of the hybridization buffer was also adjusted from 15% (v/v) formamide to 35% (v/v) formamide to try to reduce the background fluorescence but these various stringency conditions did not significantly affect fluorescence levels. In addition, a ba sic local alignment search tool (BLAST) search against the Zea mays ssp. mays genome of the target sequence to which EUB338 binds showe d no significant sequence similarity to endogenous maize sequences (all E .3 ; Altschul et al. 1997) These results also suggest that the observed fluorescence was not due to nonspecific binding of the probe but rather autofluorescence of the tissue. Previous work by Dr. Clore suggested that maize kernels do not autofluor esce significantly in the FITC channel (Clore et al. 1996) therefore initially we thought that this probe would not pose a problem and were surprised by the high level of autofluorescence in these young B73 kernels. I did a survey of previous literature to investigate maize kernel autofluorescence further : a f ew p revious studies have used fluorescent and/or confocal microscopy to visualize maize kernels (Felker and Muhitch, 1990; Sen et al. 1994; Clore et al. 1996; Duncan and Howard, 2010) but none have investigated early stage kernels from the B73 cultivar. An older study by Felker and
64 Muhitch (1989) u sed phycoerythrin conjugated antibodies (which fluoresce orange when excited) to label glutamine synthetase in developing maize kernels ( 8 to 32 DAP; cultivar not specified) They noted significant b road spectrum autofluorescence ( mainly ye llow to green c o lors ) that varied in intensity between tissues and DAPs. The authors stated that the least amount of autofluorescence was found in the lower endosperm, and that autofluoresence increased in the upper endosperm from 24 to 32 DAPs (Felker and Muhitch, 1990) A more recent study by Duncan and Howard (2010) characterized infection of mature maize kernels by the pathogenic fungus Fusarium verticillioides by forcing expression of the fluorophore ZsGreen by the fungus and visualizing its infection with confocal microscopy. T he authors n oted green autofluorescence within the stylar canal of mature maize k ernels from the AD38 cultivar as well as red autofluorescence in the pericarp of mature kernels. However, t he authors did not observe significant red or green autofluorescence in the internal endosperm tissue (Duncan and Howard, 2010) In a study by Sen et al. (1994), b lue autofluorescence was observed in pericarp aleurone, and embryo of cryostat sections of mature kernels from the hybrid maize cultivar known as Ritchie This blue autofluorescence was attributed to the presence of phenolic acids The authors were able to quench th is blue autofluorescence by stai in aleurone cells and the embryo (believed to be due to enhanced aromatic amines ). Green autofluorescence in the endosperm tissue was however not visible in samples stain (Sen et al. 1994)
65 On the other hand, a n image 7 found on the Nikon M icroscopy U website shows an example of significant autofluoresc ence emission from a thin section of a maize kernel (cultivar and stage of development not specified ). This unpublished image was observed with a Nikon DAPI FITC Texas Red florescence filter combination (without any staining). The caption on the website sugg ests that the autofluorescence may be due to the endogenous lignins, carotene, and/or xanthophyll. In summary, except for the Nikon image and the Felker and Muhitch reference (1990), these studies reported green autofluorescence mainly in the aleurone lay er and pericarp, but little to none in the endosperm. However, these studies investigated more mature kernels from different cultivars than were investigated by the present study. The abundant autofluorescence observed in endosperm in this study may be specific to the B73 maize line and/or to early developmental stage kernels. Also, in order to visualize very small bacteria, a greater magnification was required than has been used in any of the previous studies. It is possible that autoflu orescence intensity in the endosperm or nucellus effectively becomes a larger problem with increased magnification 4.3. TRFLP Theoretically, each TRF in a single sample (or set of samples from the same restriction enzyme digest) represents a different phylot ype of Eubacteria (Dunbar et al. 7 Nikon image URL: http://www.microscopyu.com/articles/fluorescence/filtercubes/triple/dapifitctexasred/dapifitctexasredz ealarge.html
66 2001; Kitts, 2001) This premise is derived from the observation that the 16S ribosomal RNA present in all Eubacteria has highly conserved regions (found in all E ubacteria) as well as variable regions (Woese et al. 1990) These variable regions are conserved among species in a single phylotype of Eubacteria but differ between phylotypes (Du nbar et al. 2001) Therefore, PCR amplification of the near full length sequence of the gene encoding the 16S ribosomal RNA by using primers that anneal to conserved regions and encompass variable regions will result in a mixture of amplicons of the s ame size but with variable sequences. Subsequent digestion of amplicons by a restriction enzyme will yield patterns of cuts that are specific to a single phylotype. By labeling termina l restriction fragments (TRFs) will fluoresce during TRFLP analysis. To give some measure of the level of resolution (i.e., level of phylotype) TRFLP provides, Dunbar et al (2001) sequenced the TRFs resulting from digestion of amplicons generated with pr imers very similar to the ones used in the present study by HhaI and found that 68% were specific to less than four species of the same genus. In the present study, TRFs were detected in all samples suggesting that microbial communities exist in 0, 1, 2, 4, 6, and 8 DAP kernels. The TRF profiles of all fertilized kernels were more closely related to each other than they were to the unfertilized kernel. This suggests that a distinct microbial commun ity exists prior to fertilization. However, it should be noted that the unfertilized kernel (0 DAP) used in TRFLP analysis was originally thought to be 12 DAP, but after investigating sections of several kernels from the same cob, it was determined that f ertilization did not occur and
67 thus it was counted as a 0 DAP. Perhaps bacterial and/or fungal endophytes are able to colonize B73 maize kernels by somehow using ungerminated pollen as a vector or perhaps the pollen tubes were able to grow part of the way down the style. In order to test this hypothesis, TRFLP analysis should be conducted on an unfertilized sample that was not exposed to pollen. More replicates shou ld also be included for each DAP Previous researchers have observed that some fungal pat hogens can enter maize kernels via the stylar canal ( Fig. B 3 ) which is a remnant structure from a third carpel that does not fully develop (Duncan and Howard, 2010) Duncan and Howard characterized the infection of 8 to 11 DAP kernels of the AD38 cultivar by the fungal pathogen Fusarium verticillioides when inoculated via injection into the silk channel of the cob. Growth of F verticillioides on the surface of kernels was observed after 72 hours postinoculation, and infection of the kernels via the stylar canal was observed 7 days postinoculation (Duncan and Howard, 2010) Duncan and Howard also showed that such infection does not occur in mature kernels of the HT1 cultivar since these kernels exhibit a closed stylar canal (and are thus not susceptible). I was unable to find any references addressing whether or not B73 has an open or closed stylar canal, and Duncan and Howard required scanning electron microscopy to determi ne open versus closed stylar canals in the kernels of the AD38 and HT1 cultivars. If B73 kernels exhibit an open stylar canal, this may serve as a means for fungal and/or bacterial endophytes to colonize within kernels. In addition, an open stylar canal may have allowed the surface sterilization solution used in this study to enter the inside of kernels, potentially affecting endophytic bacteria. Future studies should investigate whether B73 has an
68 open stylar canal and if it does, they should investiga te its potential use as a route of entry for microorganisms. T hree PCR amplifications of 16S rDNA from each metagenomic DNA sample were confirmed to occur and amplicons were then combined and purified prior to restriction enzyme digestion. PCR was conducted in triplicate in order to minimize PCR biases such as preferential amplification of templates with good primer homology However, some previous studies have used up to six amplifications for every sample (Wang et al. 2004) If any PCR biases persisted throughout these amplifications, this would cause some TRFs to be over or under represented, and they would therefore not reflect the true abundance of their representative phylotypes. Multiple T RFs were detec ted in the electropherograms of all processed s ample s, indicating that the restriction enzyme s HhaI and RsaI successfully cut the PCR amplicons. However, d espite the purification step there is still a possibility that some fluorescently labeled primer st ill remained in the samples To reduce the risk of mistaking any potentially remaining primer or any uncut amplicons (full length = 1540 bp) only TRFs of sizes between 50 and 1050 bp were used for PCA and MDS (as was done by Klaus et al. 2007) In addition, t he amplitudes and shapes of peaks were used for selection of viable TRFs for PCA or M DS. This was done in order to detect only peaks that were above the background fluorescent level (noise) while avoid ing detection of any peaks that were more likely due to erro r (artifacts) Non viable peaks included those that were short in amplitude (baseline noise level) very thin in width, and/or having an odd shape ( defined by its polynomial degree)
69 These restrictions were necessary in order to differentiate TRFs from background noise, but some peaks that did not meet these arbitrary criteria may have represented endophytic bacteria For example, i f a particular phylotype of bacteria was in very low abundance within a kernel then its resulting TRF peak would have been very small and it could have blend ed in with the background noise Yet a nother limitation of TRFLP is that it is dependent on efficient extraction of metagenomic DNA from the kernels. If DNA was not efficiently extracted, then there may have been phylotypes that were present within the kernel but did not show up in the TRFLP data. 4.4. Future Dire ctions Much study remains to be done in order to get a more complete understanding of the source of the detected microbial communities and how they change throughout the development of maize kernels. Furthermore, t he individual isolates of bacteria currently sto red as subculture s need to be identified and the roles they may play remain to be determined Even if the bacteria cannot be definitively determined to be kernel versus cob endophytes (i.e., if they reside within the internal tissue of kernels or on their surface) their identification and characterization may still be informative. Gram stains and further characterizations need to be conducted on the isolates. These include determining the carbon and nitrogen sources utilized by each isolate, the su bstrates to which each binds, and the potential benefits each may provide to maize. In addition, fungal endophytes of maize need to be investigated. Fungal growths
70 observed in the present study were not saved, but future work may benefit from focus ing on the roles of fungal endophytes. Identifying bacterial isolates could be done by sequencing the genome s of the frozen bacteria, but this method would only lead to the identification of culturable bacteria. In order to identif y both culturable and nonculturable bacteria 16S rDNA clone libraries can be constructed from the metagenomic DNA (Leigh et al. 2010) This is done by first amplifying the 16S r DNA sequences from metagenomic DNA, then cloning thes e into plasmid vectors of E. coli to form competent cells containing the recombinant vector (Leigh et al. 2010) The E. coli is then streaked out on selective medium for identification of transformants (non transfo rmants die ), and each resulting colony is sequenced. This is a labor intensive and expensive procedure, but it could lead to a complete identification of all represented endophytic bacteria (Leigh et al. 2010) Me tagenomic shotgun sequencing might also be employed to sequence the near complete genomes of all of the represented bacteria, but this would require many samples in order to gather enough DNA to detect under represented bacteria (Tyson et al. 2004) A low budget way of characterizing culturable bacteria would be to grow isolates in various selective media to test for activities such as nitrogen fixation (growth on a nitrogen free medium; Elbeltagy et al. 2001) or ACC deaminase activity (growth on medium with ACC its sole source of nitrogen; Johnston Monje and Raizada, 2011) Other biochemical assays can be conducted to test for RNase activity, acetoin
71 production, auxin production, siderophore production, and pectinase and cellulase activities (Johnston Monje and Raizada, 2011) Since the FITC labeled EUB338 probe was s ufficient for labeling E. coli in the absence of maize tissue, but insufficient for visualizing potential endophytes within developing B73 maize kernels due to autofluorescence from maize tissue in the FITC channel, future studies may want to employ the us e of an EUB338 probe with a different fluorophore (one with an excitation wavelength outside of the FITC channel). Alternatively, perhaps an enzyme labeled EUB338 probe could be used to selectively label bacteria in order to avoid other possible autofluor escence issues. Previous research has shown that EUB338 probe linked to horseradish peroxidase is sufficient for labeling bacteria via the formation of a visible intracellular precipitate after the addition of the substrate diaminobenzidine (Amann et al. 1992) Other molecular based methods of pattern analysis (microbial fingerprinting similar to TRFLP ) that can be applied to amplified 16S rDNA include d enaturing g radient g el e lectrophoresis ( DGGE ) t emperature g radient g el e lectrophoresis (TGGE) and s ingle s tranded c onformation p olymorphism (SSCP) (Pontes et al. 2007) Both DGGE and TGGE separate DNA fragments of the same size but of different sequences based on the G+C content of each fragment by using a denaturant or temperature gradient, respectively (Anderson and Cairney, 2004) SSCP involves denaturing 16S rDNA amplicons into single stranded DNA fragments, flash freezing the m to preserve their resulting unique structures (which depend on the sequence of each), and then
72 separating them by their structures on a chilled polyacrylamide gel (Lowell and Klein, 2001) Of course, potential endophytes within other regions of maize, such as stems l eaves, and cob husks, also need to be characterized for a more complete understanding of the endophytic communities within maize. Other cultivars of maize might also be investi gated for their endophytic communities The effects of traditional pest control agents on the endophytic communities of maize are also currently unknown. Growth of plant material for t he present study necessitated topical applications of neem oil and Thu ricide to control rot inducing fungi and herbivory from lepidopteran s respectively. Either of these applications could have potentially affected the endophytic communities within the maize although the husks are still tightly wrapped at these young sta ges Finally, potential bacteria within and on the surface of mature pollen grains should be investigated. The present study provides evidence that pollen may serve as a vector during fertilization and introduce bacteria to developing maize kernels. Sim ilarly, the potential endophytic communities within maize anthers should be investigated and compared to the potential endophytes within the pollen. In conclusion, t he present study resulted in isolation of potentia l l y endophytic bacteria from early developmental stage of B73 maize kernels, showed that the FITC labeled EUB338 probe is insufficient for observing selectively labe led bacteria within developing B73 maize tissue because of significant autofluorescence of the plant tissue and profiled microbial communities corresponding to the early stages of kernel development. More work will need to be conducted to definitively determine if the
73 data obtained correspond to intra kernel endophytes and if so, if fertilization allows for the entry of some of these organisms.
74 Appendix A Recipes Brain Heart Infusion Agar (BHI) : 52 g/L of B BHI, autoclaved and poured into sterile growth plates under aseptic conditions, stored in original sleeves at 4C FISH Hybridization Buffer 2 mL : Fill to 2 mL with dH 2 O > add last to avoid precipitation FISH Wash Buffer 50 mL : 1 mL 1M Tris (pH 8. 0) 1.25 mL 4M NaCl 0.5 mL 0.5 M EDTA Fill to 50 mL with dH 2 O > add last to avoid precipitation Nutrient Broth (NB) : 8 g/L of Fisher EMD Nutrient Broth, autoclaved and poured into sterile test tubes under aseptic condition s, stored at 4C Sodium Phosphate Buffer (PB) 200 mL : 39.0 mL 2M monobasic sodium phosphate monohydrate 61.0 mL 2M dibasic sodium phosphate Fill to 200 mL with dH 2 O to make 1M sodium phosphate buffer at pH = 7. Surface Sterilization Solution (1% sodium hy pochlorite) 50 mL : 9.52 mL stock laundry bleach (5.25% sodium hypochlorite) Fill to 50 mL with dH 2 O Amend with 50 Tween 20 Tryptic Soy Agar (TSA) : 30 g/L of MP Biomedicals LLC Bacto Agar, autoclaved and poured into sterile growth plates under as eptic conditions, stored in original sleeves at 4C.
75 Appen dix B Supplementary Figures Figure B 1 Vibratome sections of 3 DAP ( A ) and 9 DAP ( B ) maize kernels of the CML 322 cultivar stained with toluidine blue Endosperm tissue (star) fills mu ch of the kernel thereby replac ing the surrounding nucellus tissue (Nu) by about 8 DAP (Randolph, 1936; Sonchaiwanich, 2012) Bars = 1 mm. Figure modified from Sonchaiwanich, 2012
76 Figure B 2 Electropherograms for all H haI and RsaI digested samples. TRFs from 0 to 1050 bp are shown. Note that the two 4 DAP kernels (4A and 4B) were from different cobs.
77 Figure B 3 Illustrated longitudinal section through a developing maize kernel showing an intact silk containing a pollen tube The carpels converg e with the rudimentary carpel to form the stylar canal which can be open or closed depending on the maize cultivar ES = embryo sac. Figure from Duncan and Howard, 2010.
78 Appendix C Chemical Structures 6 FAM ( isomer derived from 6 carboxy fluorescein ) Acetoin (3 hydroxy 2 butanone) DAPI ( 4',6 diamidino 2 phenylindole) Enterobactin (an example of a siderophore )
79 ET (Ethylene) FITC (Fluorescein isothiocyanate) IAA (a form of auxin) JA (Jasmonic acid) SA (Salicylic acid)
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