Colonisation of Perennial Ryegrass by Endophytic Bacteria

Colonisation of Perennial Ryegrass by Endophytic Bacteria 2. Materials and methods 2.1. Preparation of culture media Nutrient agar (NA) – NA (Oxoid, Basingstoke, Hants, UK) was prepared by weighing out 28g of nutrient agar powder and dissolving in 1L of deionised H2O, by warming on a hot plate. This was autoclaved at 121 °C and 15psi for 15 minutes. The medium was then aseptically dispensed in sterile petri dishes and allowed to solidify. Sucrose glutamate agar (SGA) – SGA was prepared by dissolving 20g of sucrose, 2g of glutamate, 15g of agar bacteriological (Agar No.1) and 1g of K2HPO4 in 1L deionised H2O. This was autoclaved as outline above. The medium was allowed to cool to approximately 60 °C at which 5ml of MgSO4 and 1ml of kanamycin was aseptically added to the medium using a Nalgene Syringe Filters. The medium was then poured into petri dishes and allowed to solidify. Nematode growth medium (NGM) – 1L of NGM was prepared by dissolving 3g of NaCl, 17g of agar, and 2.5g of peptone in 975ml of deionised H2O in a Duran bottle. This was autoclaved as outline above. The media was allowed to cool for 15 minutes at which 1ml of 1M CaCl2, 1ml of 5mg/ml cholesterol in ethanol, 1ml of 1M MgSO4 and 25ml of 1M KPO4 buffer were added aseptically in a Duran bottle. The bottle was swirled to ensure the medium was mixed properly and then aseptically dispensed in sterile petri dishes and allowed to solidify. 2.2. Culturing entomopathogenic nematodes (EPN) 9cm filter paper was placed in sterile petri dish and 1ml of stock nematode suspension was pipetted onto filter paper. Five live Galleria mellonella (wax moths) were placed onto petri dish and sealed with parafilm (Figure 1). The dish were kept in a dark and observed daily for the insect mortality. Once the G. mellonella were dead they were transferred to white traps. Figure 1 Galleria (wax moth) baiting technique. 2.3. Preparation of white traps White traps (White, 1927) were prepared which the lid of a small petri dish (35x10mm) was placed on top of the base and placed in a clear tub, this was then covered with filter paper. 30ml of water was added to the plastic container just to allow absorbing through the filter paper. The dead G. mellonella was placed on the moist filter paper and it was covered and placed in dark for 7 to 14 days (Figure 2). The white traps were observed daily for emergence of infective juveniles (IJs) by using stereoscope (Figure 3). Whenever the water around the stage became densely concentrated with nematodes, the water was decanted into a container and replaced with another 30ml of deionised H2O. The nematodes were stored at 4 °C in 25ml of distilled water until needed for use. Figure 2 – Galleria larvae on white trap. Figure 3 – Galleria larvae under stereoscope (20X). 2.4. Culturing Caenorhabditis elegans (C. elegans) on NGM (Couillault. C, 2002) A stock culture of C. elegans was cultures from a previous stock of C. elegans. In a laminar air flow, the plate was divided into equal sections. A sterilized scalpel was used to cut sections from the stock culture to the centre of a fresh NGM plate (Figure 4). The plates were sealed using a strip of parafilm and stored at room temperature or inside incubator at 21 °C for 3 days. The petri plates were observed regularly using stereoscope (Figure 5). Figure 4 – Culturing C. elegans on NGM (Chunking method). Figure 5 – Observation of C. elegans under stereoscope (20X). 2.5. Culturing of bacterial endophytes The endophytic bacterial strains used in this study were provided by IT Carlow stock collection and have been labelled with gfp (green fluorescent protein). The Pseudomonas strains used were F113, L321 and L228. 1L of Nutrient broth (NB) was prepared and 10ml was pipetted into Mc Cartney bottles and autoclaved. The Pseudomonas strains of L321gfp, L228gfp, and F113gfp were inoculated aseptically using a wire loop and incubated at 30 °C for 24hours in an orbital shaker incubator. The gfp strains in the nutrient broth were then streaked onto fresh non contaminated nutrient agar and SGA in duplicate for each of bacterial strains, using the quadrant streaking method. All plates were covered with parafilm, labelled and incubated at 30 °C for 24hours. A gram stain, catalase test, oxidase test, and observation of morphological characterisation were carried out for the Pseudomonas strains of L321gfp, L228gfp, and F113gfp. 2.6. Quantification of nematodes S. feltiae and C. elegans The stock suspension of nematodes was divided into 50ml samples. 100 µl of the infective juvenile suspension of each sample was pipetted using micropipette onto a counting tray and tally counter was used to count for nematodes under stereoscope (Figure 6). Once they were counted, the sample was discarded and washed with deionised H2O. This was repeated 10 times and the average number of nematodes was calculated. Figure 6 – Counting chamber containing suspension of nematodes. 2.7. Preparation of soil samples 2500g of soil was autoclaved as outlined in section 2.1. The soil was dried in an oven at 55 °C for 24 hours. The soil samples were prepared by weighing out 90g into plastic cups (Figure 7) and temporarily covered with tin foil to prevent any contamination. Figure 7 – Each plastic cup contains 90g of soil. 2.8. Preparation for Sodium alginate beads (Bashan, 2002) The microbeads stock suspension were prepared by dissolving 10g of Sodium alginate in 500ml of deionised H2O , 10g of Calcium Chloride in 500ml of deionised H2O, and 5g of powdered skimmed milk in 50ml of deionised H2O. All components were autoclaved separately and the skimmed milk was autoclaved only for 10 minutes. The working solution was prepared from the stock solutions as follows; 5ml of skimmed milk, 15ml of sodium alginate and 5ml inoculum. The components of the working solution were poured into a sterile petri dish and mixed using a sterile rod. Parafilm was used to plug the spout of 20ml syringe, the alginate mixture and grass seeds were subsequently added. The parafilm was then removed and a sterile rod was used to ensure the coated seeds dropped out individually, into a beaker containing Calcium Chloride on a magnetic stirring plate (Figure 8). The beads were washed at least three times with sterile distilled water and stored in a sealed petri dished until needed for use. Figure 8 – Beaker containing Calcium Chloride on a magnetic stirring plate. 2.9. Isolation of bacterial endophytes from alginate beads (Bashan, Y and Levanony, 1989) In order to isolate and enumerate bacterial endophytes from microbeads seed coating. Six alginate beads containing individual bacterium were dissolved in 10ml of 0.25M Potassium Phosphate buffer in a test tube and incubated at 30 °C for 24 hours. The bead was then shaken on a vortex for 5 minutes to break down the alginate. Using a serial dilution method, 1ml of bead samples containing bacteria was serially diluted in 9ml of sterile ringers from 10-1 to 10-10 (Figure 9); this was carried out onto SGA in triplicate and incubated at 30 °C for 24 hours. Figure 9 – Most probable number (MPN) method/Serial dilution method. 2.10. Isolation of bacterial endophytes from plants (Keogh, E, 2009) Each plant was removed from pots and excess soil was removed. Three samples were taken from each plant (stem, root, and rhizosphere). The stems and roots were surface sterilised with 1% of sodium hydrochloride and washed twice with sterile water. The stems and roots were cut with sterile scalpel and crushed with a pestle and mortar in 5ml of Ringers solution. 100 µl of suspension was added to 900 µl of sterile Ringers in 2.5ml microfuge tubes. The serial dilutions were carried out and the resulting dilutions of 30 µl were then pipetted onto SGA in triplicate and incubated at 30 °C for 24 hours. 3. Results 3.1. Characterisation of bacterial endophytes The classical approach to bacteria identification involves preliminary microscopic examination of the gram-stained preparation for its categorisation which would later form the basis for the selection of biochemical test to be performed to test their identity. Table (Table 1) and figures (Figure 10(a) to (f)) below shows the characterisation for each strain of endophyte. Figure 10 – Characterisation of bacterial endophytes. (a) Culture plate observation for F113. (b) Microscopy examination for F113. (c) Culture plate observation for L228. (d) Microscopy visualisation for L228. (e) Culture plate observation for L321. (f) Microscopy examination for L321. 3.2. Counting of nematodes S. feltiae and C. elegans The number of nematodes was counted per well in four weeks’ time (Table 2) and a chart (Figure 11) was produced comparing the S. feltiae and C. elegans. This was repeated 10 times and the average number of nematodes was calculated. Table 2 – Quantification of nematodes. Figure 11- Comparison between No. of IJ/100 µl with the time of S. feltiae and C. elegans. 3.3. Soil samples inoculation In order to make sure the soil samples free from contamination, the serial dilutions were carried out and the resulting dilutions of 30 µl were then pipetted onto SGA in triplicate and incubated at 30 °C for 24 hours (Figure 12). The results indicated no growth in the soil samples. Figure 12 – No growth in the soil samples. 3.4. Isolation of bacterial endophytes from alginate beads In order to isolate bacterial endophytes from alginate seed coating, the beads were plated onto SGA and incubated at 30 °C for 24 hours. The results indicated that fluorescent which present of green colour pigment (Figure 13). Figure 13 – SGA changed to green colour. 3.5. Colonisation and enumeration of endophytic bacterial within plant tissues Inoculated perennial ryegrass (Lolium perenne) was allowed to grow for 4 weeks (Figure 14) before sampling took place. Total bacterial population of gfp expressing were determined for each of the tissues examined. Endophytic bacteria are considered to be those isolated from the internal tissues of surface sterilised plants. However, it is difficult to determine whether an organism is truly endophytic or merely a survivor of the surface sterilisation process. To ensure that the sterilisation processes were adequate, the sterilised tissues were pressed against the surface of a sterile SGA plate and samples of the third water rinsing were also plated onto SGA plates (Figure 15). Bacterial counts (Figure 16 and 17) on these plates were always between 10-1 to 10-4 CFUs per ml (Table 3 and 4), which was considered to be a good indication that the surface was successfully sterilised. However, under epifluorescent microscopy, the gfp expressing from inoculated plants. Pseudomonas species str ain L321 was detected only in the rhizosphere and the interior root tissues of inoculated plant (Figure 18(a) and (b)). Figure 14 Lolium perenne was allowed to grow. Figure 15 – Bacterial count on SGA plates Table 3 –Plate counts on Pseudomonas strain of L321. Figure 16 – Bacterial counts between S. feltiae and C. elegans. Table 4 – Plate counts on Pseudomonas strain of F113. Figure 17 – Bacterial counts between S. feltiae and C. elegans. Figure 18 – Visualisation under epifluorescent microscope. (a) L321gfp bacteria (400X). (b) L321gfp bacteria (100X). 4. Discussion Bacterial colonisation of the internal tissues of plants has been described in almost all plant species examined so far. Although many of these bacteria are phytopathogenic, a considerable number have also been found that colonise the plant without causing disease. Such bacteria are referred to as bacterial endophytes. Colonisation may take place at the local tissue level or throughout the plant, with bacterial colonies and biofilms residing latently in the intercellular spaces and inside the vascular tissues. This project describes the isolation, identification and colonisation efficiency of perennial ryegrass by gfp labelled bacterial endophytes. Furthermore, this study has shown the successful colonisation of perennial ryegrass by three endophytic bacterial strains under controlled conditions. The Pseudomonas strains, L321 demonstrated efficient colonisation resulting in high population numbers within the plant tissues. This experiment shows that the L321 bacteria endophyte worked successfully with the C. elegans to increase the plant colonisation. In this project, L228 was discarded due to the lawns were very poor and did not fluorescence very well so the experiment carried out only with L321 and F113. During the characterisation of bacteria endophytes, the results were obtained which the genus Pseudomonas appeared in Gram negative bacilli motile by polar flagella. In addition, in catalase test shows positive formed the bubbles when comes into contact with Hydrogen Peroxide. On the other hand, the results show that they are oxidase producing which will be oxidised to deep purple colour. Also, when nematodes had been put on plates and timescale had begun it was noticed that some plates start to dry out which may be due to the media drying out so to overcome this this, the plates have to seal with parafilm to prevent from dry out. Furthermore, other notice when the plates rinsed with water, I noticed crystals formed in the media and this may be due to the tem perature problem. Generally in the experiment there is no physical quantity can be measured with perfect certainty; there are always errors in any measurement. For example, the systematic errors are due to poorly calibrated instrument; observational for example, errors in judgment of an observer when reading the scale of a measuring device to the smallest division. 5. Conclusion In conclusion, this study has shown the successful colonisation of perennial ryegrass by three endophytic bacterial strains under controlled conditions. The Pseudomonas strains, L321 demonstrated efficient colonisation resulting in high population numbers within the plant tissues. Hence, none of the introduced strains showed any signs of pathogenicity towards their host plant and others tested. Many studies have shown that the colonisation levels in field trials are less successful than those in laboratory trials. This is probably due to increased microbial competition and less favourable environmental conditions. Therefore, additional long-term field trials need to be carried out in order to gain a better understanding of the colonisation pattern and population dynamics of endophytic bacteria in the perennial ryegrass. If time permitted future work would include, the carrying out of plant biomass which is a time consuming method that involves drying of cells and to perform by weighi ng the dry and fresh weight of each plant. 6. References Brown, R.H. and Kerry, B.R. (1987). Principles and Practice of Nematode Control in  Crops. Academic Press, Sydney. 447 pp. Evans, D., Trudgill, D.L. and Webster, J.M. (1993). Plant Parasitic Nematodes in  Temperate Agriculture. CAB International, Wallingford. 648 pp.   Luc, M., Sikora, R.A. and Bridge, J. (2005). Plant Parasitic Nematodes in Subtropical  and Tropical Agriculture, 2nd edn. CAB International, Wallingford. 871 pp. Mai, W.F. and Mullin, P.G. (1996). Plant Parasitic Nematodes. A Pictorial Key to  Genera, 5th edn. Comstock, London and Cornell University, Ithaca. 276 pp. Ahmad, F., Ahmad, I., Khan, M.S. 2008. Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiol. Res. 163, 173-181. Barraquio W L, Ladha J K, Watanabe I. Isolation and identification of a N2 fixing Pseudomonas associated with wetland rice. Can J Microbiol. 1983;29:867–873.

Eye movements are a reflection of cognitive processes Essay -- Psychol

Introduction The mind is an intriguing element of the human life not only because of its complexity and capability but mostly because of its opacity. How does psychology begin to understand something so obscure and complex? Evidently, by observing and measuring the product of the cognitive processes that occurs through an interaction between the external and internal world. The term ‘cognitive processes’ is a rather collective term referring to a range of mental processes such as perceiving, thinking, speaking, acting, planning and imagining (Ward, 2006). The processes themselves are immensely difficult to measure directly, if possible at all. However, one may gain insight into these mental processes through observing the covert products, such as haemodynamic and electrophysiological changes and the overt products, such as behaviour, accuracy, response times and eye movements. The scope of this essay is to review the evidence that demonstrates exactly how eye movements reflect cognitive processes. Due to the vastness of the topic, this essay will focus on only one of the aforementioned cognitive processes. There has been much attention and extensive literature and reviews regarding eye movements as a tool for understanding the processes of reading, visual perception, visual search and attention (e.g., Rayner, 1998; Liversedge and Findlay, 2 000; Schutz, Braun and Gegenfurtner, 2011). All the mentioned research fields involve measuring eye movements while view some form of visual stimulus, this dissertation will explore a more aberrant field, evidence will be based on literature relating to eye movements when sensory visual input is absent, during visual mental imagery. While reviewing the literature basic themes relating eye m... ...nformation processing: 20 years of research. Psychological Bulletin, 124, 372-422. Schutz, A.C., Braun, D.I., & Gegenfurtner, K.R. (2011). Eye movements and perception: A selective review. Journal of Vision, 5, 1-30. Shallice,T. (1988). From neuropsychology to mental structure. New York: Cambridge University Press. Sima, J.F., Lindner, M., Schultheis, H., & Barkowsky, T. (2010). Eye movements reflect reasoning with mental images but not with mental models in orientation knowledge tasks. Spatial Cognition, 10, 248-261. Spivey, M.J., & Geng, J.J. (2001). Oculomotor mechanisms activated by imagery and memory: eye movements to absent objects. Psychological Research, 65, 235-241. Ward, J. (2006). The students guide to cognitive neuroscience. New York: Psychology Press. Watson, J.B. (1913). Psychology as the behaviourist views it. Psychological Review, 20, 158-177.

Nature is such a beautiful place Essay

Life is getting hard and expensive as the years are passing by. The prices on food, and other utility prices are going up. People have school, work and a family to look out for and do not have the time to enjoy the simple things in life that earth has to offer which is nature. In the essay of â€Å"An entrance to the Woods† Wendell Berry. He admits to living such a fast paste of life that is hard to come back down to the ground and enjoy what is in front of him. People pass by things so quickly and don’t pay attention that they fail to appreciate them. Once a person slows down, they could see what surrounds them. Nature is a good way to find yourself because it is easy to escape from the influence society has. Nature has away of taking away the things that are stressing us and giving away a chance to become refreshed. â€Å" In the middle of the afternoon I left off being busy at work, and drove sixty or seventy miles an hour, hardly aware of the country I was passing through, because on the freeway one does not have to be. The landscape has been subdued so that one may drive over it as seventy miles per hour without any concession whatsoever to one’s whereabouts. One might as well be flying. Though one is Kentucky one is not experien cing Kentucky. One is experiencing the highway, which might be in nearly any hill country east of the Mississippi.† (Berry, 88) During berry’s time in the woods, he writes â€Å"A man enters and leaves the world naked. And it is only naked–or nearly so that he can enter and leave the wilderness. If he walks, that is; and if he doesn’t walk it can hardly be said that he has entered. He can bring only what he can carry– the little that it takes to replace for a few hours or a few days an animal’s fur and teeth and claws and functioning instincts. And comparison to the usual traveler with his dependence on machines and highways and restaurant and motels–on the economy and the government, in short–the man who walks into the wilderness is naked indeed. He leaves behind his work, his household, his duties, his comforts–even, if he comes alone, his words. He  immerses himself in what he is not. It a kind of death.† (Berry, 90) We came to this world naked, and that is how we should enter and leave the wilderness. If we walk using our feet and explore the wilderness that’s how we can say that we entered the wilderness. The little that we bring to survive, it is gone in a few hours or days. An animal uses its fur, teeth and animal instincts to survive versus a traveler that needs machines, highways and restaurants and motels. To be in the wilderness one has to leave behind the work, the household, duties and comforts and even his words. One has get out of its comfort zone, and use your own abilities to survive the wilderness. It is like taking a risk of death. Wendell Berry own experience where he is up and left civilization. The work setting to enter a nature environment devoted to being self efficient on nature. Berry loved and respected nature, he wanted to develop his appreciation further, as he wanted it to evolve and understand for it. He wanted to know how nature worked, and learn it’s reason for being. Berry’s goal was met through this act and it was gratifying experience to escape from moderation and become organic. Berry realizes the purpose of the trip, but the connection to nature cannot replace to connection to man. In the essay of â€Å" Why I went to the woods† by Henry David Thoreau. Thoreau tried to produce his own crops, to live from the labor of his own hands, and to get rid of all the complicating things that distracted him from life’s true meaning by living in the woods. †Thoreau wanted time to read, write, and think. He wanted to make time for nature. And he wanted to test himself, to see just how much he could simplify his life, to determine how much time he could save to do what he really wanted to do with every minute of everyday†( Thoreau, 700) Thoreau mentions that he wants to live a deliberately life. To live deliberately means to take care and think of everything that you do in life, and not to do anything just for the heck of it. Everything that we do has to have a purpose and a meaning, and that is not a waste of time. â€Å"slow down rather than to speed up, to saver a few things fully rather than sample many things fleeting, and have time to decide what, in the long run of his short  life, matters most and why.† (Thoreau, 700) â€Å" I went to the woods because I wished to live deliberately, to front only the essentials fact of life, and see if I could not learn what it had to teach, and not, when I came to die, discover that I had not live I did not wish to live what was not life, living is so dear; nor did I wish to proactive resignation, unless it was quite necessary. I wanted to live deep and suck out all the marrow of life, to live so sturdily and Spartan-like as to put to rout all that was not life, to cut a broad swath and shave close, to drive life into a corner and reduce it to its lowest terms, and if it proved to be mean, why then to get the whole and genuine meanness of it and publish its meanness to the world, or if it were sublime, to know it by experience, and be able to give a true account of it in my next excursion.† ( Thoreau, 701) By living in the woods he will be living a simple life away from civilization and to see his strength and weakness. By not being accustomed to that type of lifestyle he might die. When all hope is gone he realizes there are many things he have not tried. The simplest things in life could be and mean much more. Life is precious.

Summer Solstice in the Northern Hemisphere

June 20-21 is a very important day for our planet and its relationship with the sun. June 20-21 is one of two solstices, days when the rays of the sun directly strike one of the two tropical latitude lines. June 21 marks the beginning of summer in the northern hemisphere and simultaneously heralds the beginning of winter in the southern hemisphere. In 2014, the summer solstice occurs and summer begins in the Northern Hemisphere on Friday, June 21 at 6:51 a.m. EDT, which is 10:51Â  UTC. The earth spins around its axis, an imaginary line going right through the planet between the north and south poles. The axis is tilted somewhat off the plane of the earths revolution around the sun. The tilt of the axis is 23.5 degrees; thanks to this tilt, we enjoy the four seasons. For several months of the year, one half of the earth receives more direct rays of the sun than the other half. When the axis tilts towards the sun, as it does between June and September, it is summer in the northern hemisphere but winter in the southern hemisphere. Alternatively, when the axis points away from the sun from December to March, the southern hemisphere enjoys the direct rays of the sun during their summer months. June 21 is called the summer solstice in the Northern Hemisphere and simultaneously the winter solstice in the Southern Hemisphere. Around December 21 the solstices are reversed and winter begins in the northern hemisphere. On June 21, there are 24 hours of daylight north of the Arctic Circle (66.5Â ° north of the equator) and 24 hours of darkness south of the Antarctic Circle (66.5Â ° south of the equator). The suns rays are directly overhead along the Tropic of Cancer (the latitude line at 23.5Â ° north, passing through Mexico, Saharan Africa, and India) on June 21. The Reason for Seasons Without the tilt of the earths axis, we would have no seasons. The suns rays would be directly overhead of the equator all year long. Only a slight change would occur as the earth makes its slightly elliptical orbit around the sun. The earth is furthest from the sun about July 3; this point is known as the aphelion and the earth is 94,555,000 miles away from the sun. The perihelion takes place about January 4 when the earth is a mere 91,445,000 miles from the sun. When summer occurs in a hemisphere, it is due to that hemisphere receiving more direct rays of the sun than the opposite hemisphere where it is winter. In winter, the suns energy hits the earth at oblique angles and is thus less concentrated. During spring and fall, the earths axis is pointing sideways so both hemispheres have moderate weather and the rays of the sun are directly overhead the equator. Between the Tropic of Cancer and the Tropic of Capricorn (23.5Â ° latitude south) there really are no seasons as the sun is never very low in the sky so it stays warm and humid (tropical) year-round. Only those people in the upper latitudes north and south of the tropics experience seasons.