Diazotroph bacteria are known to have a symbiotic relationship with legume plants whereby the bacteria thrive while providing energy to the plant3. Little research has been done about relationships between Diazotrophs and non-legume plants, especially during the germination stage. We investigated the use of diazotroph bacteria as a germination aid for cereal crops.
Using homemade equipment we carried out germination and growth experiments over 11 months. We analyzed the performance of more than 9,500 seed samples, recording over 120,000 manual measurements in 125 experimental sets. Based on our extensive experimental results we succeeded in showing statistically that two strains of Rhizobium bacteria can significantly accelerate the rate of crop germination (+40% for r.leguminosarum and 28% for r.japonicum; (p<0.0001). R.japonicum also increased the subsequent dry mass of barley by 70% (p<0.0001). We believe that the biochemical mechanism that produced the noted improvements is triggered when flavonoids from the crops prompt the release of lipochitooligosaccharides which catalyse accelerated seed growth.
These results have significant potential for increasing yields of food crops and reducing losses due to adverse weather conditions. They also offer opportunities for reducing the environmental footprint of agriculture by reducing fertilizer usage. As demand for cereals increases with population growth16, this discovery could act as a partial solution to the impending food poverty crisis. There is potential for future work in this area and we plan to investigate the biochemical mechanism involved and carry out more extensive field trials.
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We are 10th grade students in Kinsale Community School, Ireland. We have all been active scientists from a young age, taking a great interest in how the world works and how we can help those around us.
In 9th grade we entered our national science fair. We were awarded first place from 2000 entries, which allowed us to represent Ireland in the European Contest for Young Scientists in September 2013, where we also won first place.
As females we feel strongly about gender imbalance in STEM, and so one of our main role models is Anne-Marie Imafidon (founder; Stemettes). Another of our role models is Dr Tony Scott (founder; BT Young Scientist), as he has selflessly dedicated his life to youth science. Someday we hope to emulate their inspiring scientific contributions.
We each have other pursuits in music and sport. Emer and Ciara are accomplished musicians, and Sophie is a talented athlete. We are all currently considering careers in the biological sciences.
Winning the Google Science Fair would be brilliant, amazing, incredible!! :) Projecting our ideas into the public domain would give us the opportunity to work with experts in the area and to utilize resources which we wouldn’t otherwise have access to. This way our discovery may be used in agriculture, especially in developing countries, which really excites us. We have put no IP restrictions on our project so it may be used by everyone in need worldwide.
It’s OUR turn to Change the World!
Our STEM achievements:
Question / Proposal
If we innoculate crop seeds with Diazotroph bacteria, will their germination speed be increased?
We became aware of the food poverty crisis after the Horn of Africa famine in 2011. Food security is an ever growing issue in today’s world as due to rising populations food demand is rapidly increasing. The idea for our project was inspired by this problem and our desire to make a difference.
We found out about Diazotroph bacteria when Emers mom was gardening and found nodules on the roots of her pea plants. After conducting research we discovered that the nodules contained a natural bacteria called rhizobium.
Our research indicated that rhizobium forms a symbiotic relationship with legumes and thereby assists their growth, however we noticed a deficit of research on the interaction between rhizobium and non-legume plants. There was a particular lack of work during the germination stage of crop growth. We felt that more work was required in this area and decided to carry out this research. We chose cereal crops as our non-legume plants for our experiments as demand is increasing for crops high in starch such as cereals, particularly in the developing world.
After research outlined later, our hypothesis was:
If we innoculate cereal crop seeds with Diazotroph bacteria, their germination speed will be increased.
Should our above hypothesis be proved correct, our second hypothesis will be:
If we innoculate cereal crop seeds with Diazotroph bacteria at an early stage, the plant growth will be positively affected.
- Diazotroph bacteria form a symbiotic relationship with legume plants and fix nitrogen to enable them to grow faster. Nitrogen fixation is the process by which atmospheric nitrogen is converted into organic compounds such as ammonia for the plant. When the bacteria senses flavonoids in the roots of a legume plant it releases LCO’s or nod factors (lipochitooligosaccharides) which trigger the formation of nodules to house the bacteria2.
- LCOs follow a common pattern of sugar pieces based on an acylated chitin oligomeric backbone. LCOs are chemical messengers to the plant and are present in minute concentrations, having an effect at a trillionth of a mole. Hence measurement of them is extremely difficult.
- The Diazotroph family of bacteria3 includes a number of groups. We decided to use Rhizobacteria as this was the group specifically mentioned by our science teacher. We used one acidic strain (r.leguminosarum) and one basic strain (r.japonicum).
- Seed dormancy ends when the embryo absorbs water and hydrolysis starts. Gibberellins diffuse from the embryo to thealeurone layer and initiate the creation of α-Amylase. The α-Amylase then hydrolyses starch, providing sugars such as maltose for energy for the radical to grow causing the testa to break. Once this happens germination is considered to have occurred.
- Dr D Smith in McGill University Canada confirmed that direct application of LCOs enhance barley germination11. Studies of grass species in Mexico and Iran showed that they contained almost 20 flavonoids12,13. They are poaceae and bouteloua species similar to the crop types we are studying. We hypothesise from this information that when the rhizobium senses the flavonoids in the crop seeds it will excrete LCOs which will enhance the germination based on Dr D Smith's research.
- A review of the use of plant growth promoting hormones15 stated that benefits might be related to a combination of effects attributed to secondary compounds produced by the rhizobial strains, including exopolysaccharides (EPSs), plant hormones in addition to lipo-chitooligosaccharides (LCOs). The results emphasize the potential of using secondary metabolites of rhizobia to improve the growth and yield of grain crops.
- The EU IMPACT Project8 proposes that if bacteria could be used as an alternative to fertilizers it would benefit the environment. It considers the use of beneficial bacteria like rhizobia as a way of improving the rhizosphere.
- The Irish agricultural research institute (Teagasc) have expressed great interest in our investigation given the potential to give flexibility to farmers to sow crops quicker when weather conditions are changeable. Once the germination stage passes seedlings are more robust and not impacted as significantly by disease and ground conditions. Improvements that increase yield (such as more ears per stalk), or allow early season sowing in cooler climates would be of notable benefit.
- This can also be used to address the growing issue of food security. According to researchers, we will need 50% more food by 205017. Growth promoting natural bacteria such as rhizobium can combat this challenge or at least provide a partial solution.
Method / Testing and Redesign
Our experiments took place in our home (spare bedroom) lab. We built temperature controlled incubators and an aseptic transfer chamber.
- A natural strain of Rhizobacteria was purified by centrifugation and re-suspended in sterile water.
- The bacterial concentrations were measured by nephelometer based turbidity measurements.
- The crop seeds were sterilised in a 3% hypochlorite solution.
- The samples were arranged on plastic lab containers, inoculated with 0.05ml of culture and blinded.
- Control samples were prepared using sterile water instead of inoculant.
- The containers were placed in a constant temperature incubator and manually inspected for germination at fixed time intervals.
Small Scale Field Trials
- 12 growing trays were filled with soil.
- A hole of standard size was made and a germinated seed was inserted (either treated or untreated).
- It was then filled with soil and 5ml of water added.
- The trays were held for 3 weeks in a greenhouse.
- The plants were harvested and their length was recorded before being weighed after drying at 70oc.
Large Scale Field Trials
- Natural rhizobia bacteria was separated by centrifugation and added to sterile water.
- 7x340 seeds were mixed with diluted syrup (1:3).
- 37.5mls of suspension was added to 37.5g of three carriers (sodium polyacrylate, peat and cellulose) in 6 containers (two concentrations/carrier).
- The syrup-coated seeds were added and coated in inoculated carrier.
- 2 other groups of seeds were planted and sprayed with 37.5 ml aqueous bacteria suspension.
- A control group was sprayed with 37.5ml water when planted. Carrier control groups without bacteria were also run.
- The seeds were planted in 10 soil-filled crates positioned equidistant from an irrigation supply.
- The dry weight of the mature plants was recorded.
Over 11 months more than 125 experimental runs were carried out involving 9,500 seed samples and over 120,000 manual measurements.
Sources of Variation
- The irrigation of the seeds:
- We used sterile pipettes and watering systems to standardise water and innoculant added to the seeds.
- The moisture levels of samples during our lab experiments:
- We used plastic lab containers (ISO 9001 std) with tight fitting lids.
- The temperature the seeds were germinated at:
- We built polystyrene temperature controlled incubators. We used laboratory thermometers to monitor our incubators.
- The amount of bacteria in the suspensions used:
- We used a nephelometer to ensure that all samples had similar bacterial concentrations.
- Subjectivity and bias in the manual measurements:
- For consistency we set ‘germination criteria’. After our initial experiments we decided to blind our experiments.
- We were dealing with living organisms with natural variation:
- Each seed was handled identically. We avoided contamination by sterilising samples and using aseptic technique. We also ran multiple replicates.
- Amount of bacteria/control.
- Strain of bacteria.
- Type of seeds.
- Germination time.
- Plant length.
- Plant dry mass.
Rhizobacteria are naturally occurrring in the environment and are GRAS (Generally Regarded As Safe). We took other precautions and wore appropriate protective equipment when using hot and sharp materials.
The experimental results were analysed statistically using the SAS-JMP software package. The statistical tests we used were the Student-t test and Dunnett test, and Analysis of Variance (ANOVA). We also identified some non-parametric data sets by using the Kolmogorow-Smirnoff and Shapiro-Wilkes Tests. For these we needed different statistical tests (Dunn and Wilcoxon). Full graphical summaries of our results are appended
- Initial investigations were analyzed using the Chi Square statistical test and found that seeds germinated better in dark conditions vs light to a statistically significant level (95%). (ChiSq, p<.0001)
- Salts present in bacterial cultures suppressed germination as the cereal crops are glycophytes (10%>germination time; p<0.03 ).
- Wheat seeds were also used however their small size meant that assessing if our germination criteria was met was difficult and these results were considered unreliable and excluded from our work.
- Both r.japonicum and r.leguminosarum reduced germination time of the crops, both at 16oc and at 25 oc when shorter germination times were observed.
- The optimum concentration of r.japonicum for the germination of barley seeds was found to be 2x107CFU/ml (13% reduction; ANOVA p<0.0264). R.leguminosarum had a positive effect on the germination of Barley and reduced germination time by approximately 40% at 25oc (ANOVA p<0.0001).
- For Oats, an optimum concentration of 4x106 CFU/ml of r.japonicum was observed to be most efficient and resulted in a reduction in germination by 22 hours (28% Reduction; ANOVA p<0.0001).
- Lower concentrations of r.leguminosarium were most effective on oat germination. A concentration of 16x104 CFU/ml reduced germination times from 86 to 66 hours (23% reduction; Dunnett test p<0.0001).
Amylase and Maltose Tests
- Testing for variations in the levels of Alpha-Amylase and Maltose during germination ( treated vs controls) showed trends that were indicative of an accelerated generation of Alpha-Amylase and reduced quantity of Maltose, implying a changed rate of seed metabolism. The sample size was small and inadequate to determine if such trends were statistically significant. The high cost of the materials for such testing prevented us doing additional biochemical analytical work.
Small Scale Agricultural Tests
- R.japonicum was seen to have a positive effect on the length and dry mass of barley crops. (+10.4% length increase:+13% dry mass; p<0.0328), the effect was more notable at higher concentrations.
- It was observed that Oats treated with a higher concentration of r.japonicum (4x106 CFU/ml) produced a greater dry mass (p=0.0248) and longer length (p=0.0043) than water treated seeds.
- The small scale growth experiments unanimously proved that treating seeds with either r.leguminosarum or r.japonicum in an aqueous suspension did not impact negatively on post-germination growth. These strains of bacteria could be used as a germination aid with no later impact on plant growth.
Large Scale Agricultural Tests
- Lower concentrations of r.japonicum (3x109 CFU/ml) with peat as a carrier were the most successful treatments (ANOVA p<.0001) and resulted in an average increase in plant dry mass of 0.284g/5 seedlings (74%).
- Spraying the seeds with aqueous culture post planting increased dry mass by a mean of 44% (Dunn p<0.0001)
Conclusion / Report
- In all test groups seeds treated with r.japonicum and r.leguminosarum germinated faster by approx 50% (p<0.001).
- Both bacterial strands increased crop yield by an average of 30% with some results exceeding 70% (p<0.001).
- This will be of benefit to agriculture and the developing world as it has the potential to increase crop yields and assist food production in challenging climates.
We can conclude from these results that our hypotheses: ‘If we inoculate cereal crop seeds with Diazotroph bacteria, their germination speed will be increased,’ and ‘If we inoculate cereal crop seeds with Diazotroph bacteria at an early stage, the plant growth will be positively affected,’ have been successfully statistically proven at a 95% confidence level. We are confident in the statistical validity of our results due to our large sample sizes and experimental controls.
The presence of flavonoids within the poaceae species raises the possibility that the rhizobia are triggered to produce lipochitooligsaccharides (LCOs) in crops in a similar way to nodulation in legumes. We believe that the LCOs may then be assisting seed germination based on the work of D. Smith11. Testing of bacteria for the release of LCOs or other growth promoters in the presence of seeds would be the appropriate next step in the project.
Agriculturalists have stressed that any technique for speeding up germination would be beneficial as seeds are more at risk the time prior to radical emergence. Being able to lengthen a growing season by promoting germination and growth at lower temperatures would be a significant innovation if this were successfully achieved with rhizobacteria as it may assist the growing of cereal crops in more adverse climates.
Dr. John Spink, Head of Crop Science, Teagasc, Irish National Agri-research Center indicated that barley is considered to have too few stalks per unit area. Rapid early season growth is desirable to increase ear number per plant and the effects seen would have the potential to significantly increase crop yields. Dr Hans-Joerg Lutzeyer, Research Program Officer with the European Union Directorate for Research and Innovation plans to discuss our work at a forthcoming meeting in Brussels with representatives of Teagasc.
The potential use of our work in the malting step of the industrial brewing process has also been identified and a patent application for this particular commercial innovation has been submitted. All other uses are unprotected by IP restrictions.
We aim to investigate our observations at a trace biochemical level to fully understand why the use of rhizobium bacteria is speeding up the germination rate. Testing of bacteria in the presence of seeds for the release of LCOs or other growth promoters would be the appropriate next step in the project. For this we will need advanced analytical equipment and facilities. We also plan to carry out extensive field trials to fully investigate the viability of rhizobium bacteria as a growth promoter. We hope to experiment with different diazotroph bacteria and different crop species to expand the use of this discovery.
Bibliography, References and Acknowledgements
Bibliography and References
Youtube Video Links
For any extra information about the project please check out our Google+ or Youtube accounts!
Ref 1: Baset Mia M.A., Shansuddin Z.H. and Mahmood M. (2010). ‘Effects of rhizobia and plant growth promoting bacteria inoculation on germination and seedling vigor of lowland rice’. African Journal of Biotechnology.
Ref 2: Brewin N.J. (2010). ‘Root Nodules (Legume-Rhizobium Symbiosis)’. Norwich, UK (John Innes Center).
Ref 3: Somasegaran and Hoben H.J. (1985). ‘Methods in Legume-Rhizobium Technology’. University of Hawaii.
Ref 4: Graham P.H. and Vance C.P. (2003). ‘Legumes: Importance and Constraints to Greater Use’. American Society of Plant Biologists.
Ref 5: Bhattacharjee R.B., Singh A. and Mukhopadhyay S.N. (2008). ‘Use of nitrogen-fixing bacteria as biofertiliser for non-legume: prospects and challenges’. The journal of applied microbiological biotechnology.
Ref 6: Craighead M.D. ‘Effects of Fertilisers on Seed Germination in New Zealand’. Christchurch, New Zealand.
Ref 7: Diaz-Miguel M., Serrano F. and Rosúa J.L. (2011). ‘Improvement of Germination of Three Endemic Species of the Sierra Nevada (S. Spain)’. Environmental Earth Sciences.
Ref 8: O’Gara F., Economidis I., Moenne-Loccoz Y. and Dowling D.N. ‘Biotechnology & Ecology of Microbial Inoculants’. Commission of the European Communities Directorate-General XII Science, Research and Development. (IMPACT: Interactions between Microbial inoculants and resident Populations in the rhizosphere of Agronomically important Crops in Typical soil).
Ref 9: Ni B. R. and Bradford K. J. (1992). ‘Quantitative Models Characterizing Seed Germination Response to Abscisic Acid and Osmoticum’. University of California, USA.
Ref 10: Hall E., Watson S. and Paulsen E. (2005). ‘The Effects of NaCl on Zea mays – A study of Seed Germination, Dry Weight/Water Weight Ratio, Height, Chlorophyll Content, Fluorescence and Root Shoot Ratio’.
Ref 11: M. Miransari and D. Smith. ‘Rhizobial Lipo-Chitooligosaccharides and Gibberellins Enhance Barley (Hordeum vulgare L.) Seed Germination’, Biotechnology, Year: 2009 | Volume: 8 | Issue: 2.
Ref 12: Arrieta, Hernández, COFAA- Instituto Politécnico Nacional. ‘Flavonoids of the genus Bouteloua (Poaceae) from Mexico’, Núm.20, pp.17-29, ISSN 1405-2768; México, 2005.
Ref 13: Khanaziani amd Rahimine. ‘Study of phenolic constituents of triticum (poaceae) species in Iran’. Department of Botany, Faculty of Sciences, University of Shahrekord.
Ref 14: ‘Novozymes-Optimise’ (As of May 2014) http://www.bioag.novozymes.com/en/products/unitedstates/biofertility/optimize/
Ref 15: Marks, Noqueira and Hungria, ‘Biotechnological potential of rhizobial metabolites to enhance the performance of Bradyrhizobium spp. and Azospirillum brasilense inoculants with soybean and maize’. AMB Express 2013 April 17.
Ref 16: Khoury, Bjorkman et. al., ‘Increasing homogeneity in global food supplies and the implications for food security’ International Center for Tropical Agriculture; March 3rd 2014
Ref 17: ‘United Nations- Water for Life’ (As of May 2014) http://www.un.org/waterforlifedecade/food_security.shtml
- Mr. David Woods, University College Cork.
- Mr. John Dunne, Gold Crop Limited.
- Dr. Ewen Mullins, Teagasc.
We would like to thank University College Cork (UCC) for training us in aseptic technique and for giving us the pure natural rhizobia bacteria we needed for our experiments. We are extremely grateful to our school, Kinsale Community School and our science teacher Mr Shaun Holly who gave us guidance whenever we needed it. We would also like to acknowledge the support given to us by the principal Mr Fergal McCarthy. A big thank you to; Goldcrop Limited who supplied us with seeds (barley, wheat and oats) and Sarstedt and Sartorius who gave us sterile supplies, loaned us precision scales and a microcentrifuge. We would also like to thank Eli Lilly and Company for the industrial scale and laboratory centrifuge. Finally thank you to our parents, family and friends who supported us throughout the course of this project.
Equipment and Materials
- Sterile disposable pipettes. (Sartorius)
- Sterile disposable gloves. (Eli Lilly and Company)
- Eppendorf + eppendorf tips. (Sartorius)
- Sterile test tubes. (UCC, Eli Lilly)
- Sterile innoculation loops + hockey sticks. (Sartorius)
- Sterile plastic containers. (Sarstedt)
- Seeds: Barley, Wheat and Oats. (Goldcrop)
- Pure Natural Bacterial Strains: R. Leguminosarum & R. Japonicum. (UCC)
- Transfer Chamber. (Home-built)
- Incubators. (Home-built)
- Shaker table. (Home-built)
- Nephelometer. (Hach-Lange)
- Centrifuge. (Sartorius)
- Industrial scale centrifuge. (Eli Lilly)
- General Lab Equipment. (Kinsale Community School)
We made a McFarland standard in order to calibrate our nephelometer per literature based techniques using a known concentration of a Barium Sulphate suspension under supervision by a qualified professional.
Ingredients of YMB
Mannitol 10g 4g
K2HPO4 0.5g 0.2g
MgSO4.7H2O 0.2g 0.08g
NaCl 0.1g 0.04g
Yeast Extract 0.5g 0.2g
HzO 1.0L 400ml
Rhizobium Safety Statement
Equipment and Material certification
Wheat: Sparrow Variety/KWS UK/36TGW
Barley: Chance Variety/RAGT Fr./49TGW
Oats: Binary Variety/ Wiersum Plantbreeding Holland/35TGW
r.japonicum (Strain: CJI/T46B9)
r.leguminosarum (Strain: CL100/918AC)
Model 2100P kindly loaned to us by Hach –Lange