DHANISHTA AMBWANI
Age 16 | Fredericton, New Brunswick
1st Place: Sanofi Biogenius Atlantic Competition 2018 | 1st Place Senior Award: River Valley Regional Science Fair | Finalist: Sanofi Biogenius National Competition | Canada Wide Science Fair Excellence Award: Intermediate Bronze Medal
Verticillium dahliae is a common soil-borne fungal plant pathogen that causes early senescence in tubers, affecting the quality and yield of potato crops. Currently, farmers with worries of Verticillium infection are fumigating their fields with chemicals that eradicate the fungus. Chemical fumigation is neither an economic solution, nor is it environmentally friendly. This project studies economic and environmentally friendly options for controlling V. dahliae. The objective of this project is to find a microbial organism that has an antagonistic relationship with fungi Verticillium dahliae in potatoes to be used as biocontrol. Soil bacteria found in potato fields and endophytic bacteria found in potato plants was tested for antagonism with V. dahliae. Three different strains of V. dahliae were plated on four different bacterial co-cultures; there were controls for each fungal strain. Growth of the colonies were analyzed after seven days. Endophytic bacteria 7D and soil bacteria BA37 were found to inhibit and decrease the amount of plated fungi for all three fungal strains. Endophytic bacteria 7D and soil borne bacterial strain BA37 are recommended as biocontrol microorganisms to control Verticillium dahliae. The bacteria have simply been isolated and not identified; the next step would be to identify bacteria 7D and BA37 through DNA sequencing analysis.
BACKGROUND
There are numerous microbial pathogens that cause plant disease, with fungi being the most problematic. Verticillium dahliae is a soil borne fungal plant pathogen that affects over 400 plant species including potatoes (Berlanger & Powelson, 2005). V. dahliae can live in soil for up to 10 years in as tiny, black hardened called microsclerotia. In potatoes, the fungi penetrate the outermost layer of the root, called the cortex and infest the water transporting vessels where fungus spores are formed. Colonization of the vascular system occurs when the spores are drawn into the plant along with water (Mol & Termorshuizen, 1995).
The tubers of a potato plant, which are formed from swollen underground projections of the stem, are widely harvested for human consumption. The green vines that make up the above-ground part of the potato plants die back each year in a process called senescence. The potato vines provide the nutrients needed for the plant to form tubers. V. dahliae causes early senescence in diseased plants that leads to decreased tuber formation (Nair, Weichel, Crump & Taylor, 2016). The fungi can then feed on the dead plant. Early death of these plants affects the yield and quality of tubers which is a point of distress for many Canadian farmers, and a concern for our agricultural systems (Tai et al., 2018). Currently, some farmers with worries of Verticillium wilt are fumigating their fields with chemicals to reduce the fungus. Chemical fumigation is neither an economic solution, nor is it environmentally friendly. Therefore, alternative strategies for controlling V. dahliae are needed.
Bio-control is a way of controlling pests, insects, mites, weeds and plant diseases such as V. dahliae through natural enemies otherwise known as antagonists. There are three generally accepted methods of bio-control: importation, augmentation, and conservation. Importation involves introducing a natural enemy of the pest to a place where it does not occur normally in attempt to gain control over the pest. Augmentation is the release of already locally occurring antagonists to the same environment as a preventative measure against the pest. Conservation is when actions are put into place to increase natural enemies such as planting nectarines at the ends of rice fields (Landis & Orr, 2015). This project studies options for biocontrol of V. dahliae. Soil bacteria found in potato fields and endophytic bacteria (non-invasive bacteria that live within a plant for part of its life cycle) found in potato plants were tested for antagonism with V. dahliae.
Research suggests that endophytic bacteria can strengthen plant defense mechanisms (Hardoim et al. 2015 and Guo et al., 2008); therefore, it was hypothesized that the endophytic bacterial strains would decrease fungal pathogen V. dahliae’s growth.
MATERIALS AND METHODS
Bacterial Cultures
Bacterial strains were obtained from Agriculture and Agri-Food Canada (AAFC). Bacterial strains 5A, 1B, and 7D were endophytes derived from potato stem tissue and BA37 was from the soil. To create a broth culture for the bacteria, bacteria were scraped from frozen glycerol stocks using a sterile loop and inoculated into 10 mL potato dextrose liquid with shaking overnight, with potato dextrose serving as a nutrient source for the growing bacteria and fungi. The next day, 0.1 mL was of each strain was spread on a 10 cm Petri dish with potato dextrose agar (PDA) in triplicate. There was an additional Petri dish with no bacteria as a control. The bacterial cultures and water were incubated at 25 °C for three days. Three 8 mm disks were then removed from the PDA of each Petri dish leaving four holes.
V. dahliae Cultures
V. dahliae fungal strains 6882, 6883 and 2934 were obtained from the AAFC. These strains were all shown to cause early dying in potatoes. Each V. dahliae strain was spread on 10 cm PDA culture dishes and grown at 25 °C until fungi formed a tight even layer covering the entire dish. An 8 mm diameter disk of PDA with each fungal culture was removed with biopsy coring device. An additional dish of PDA with water was also prepared as a control. Three disks of each V. dahliae strains 6882, 6883, 2934 were placed in each of three holes in the PDA with and without bacterial culture Petri dishes and incubated at 25 °C. The water control disks were placed on the water control PDA. All V. dahliae cultures were also grown on PDA media without bacteria as well to serve as controls. The growth of V. dahliae co-cultured with and without bacteria was monitored. Photographs of each dish were taken at zero and seven days after the V. dahliae disk was added. The growth of fungus beyond the 8mm disk was measured using Image J, an open source media analysis software, to determine the area in pixels covered by V. dahliae. The 8mm disk was measured as 465 pixels, thus percentage growth was calculated with the formula: (number of pixels covered by V. dahliae / 465) * 100.
RESULTS
All V. dahliae strains showed growth without bacterial co-culture (PDA, Fig. 1) with 6882 having the best growth increase at 163%.
The bacterial strains had different growth patterns when spread on PDA. Some showed clumping growth patterns (1D, 5A and 7D) and BA37 produced an evenly distributed lawn (data not shown).
All of the bacteria affected the growth of V. dahliae, however, these effects varied between bacterial strain. Two of the V. dahliae strains showed growth when co-cultured with bacterial stain 1B; however, there was a slight decrease in growth compared to the fungal growth on PDA controls (Fig. 1). Average growth of V. dahliae strains 6882 and 6883, but not 2934 were decreased when co-cultured with bacterial strain 5A relative to the PDA control.
Figure 1. Combined bar graph of the percentage growth of V. dahliae colonies seven days post inoculation.
The average area of the colonies of the strains with and without bacterial co-culture after seven days is shown in Figure 2. The average area of all V. dahliae strains was decreased when co-cultured with bacterial strains 1B, 7D and BA37 (Fig. 2).
Figure 2. Combined bar graph of the average area of V. dahliae colonies seven days post-inoculation.
DISCUSSION
The bacterial strains that were most effective in inhibiting growth of V. dahliae were BA37 and 7D as growth of all three V. dahliae strains were considerably inhibited by the bacteria. BA37 was a bacterial strain found in the soil. Since V. dahliae inhabits the soil, a soil bacterium can be an effective biocontrol agent. The potential application of BA37 as a biological control can involve importation to new areas and augmentation to areas with existing colonization.
7D was an endophytic bacterium that was found within the potato plant. Endophytic microorganisms have been documented to enhance plant defense and produce beneficial bioactives, important chemicals found in plants (Hardoim et al. 2015 and Guo et al., 2008). The discovery of an endophytic bacteria in potato with antagonism against V. dahliae is highly novel and suggests that strategies for biocontrol can include inoculation of biocontrol bacteria into plants.
Endophytic bacteria 7D and soil borne bacterial strain BA37 are recommended as biocontrol microorganisms to control V. dahliae. Consideration can also be given to using both strains together with BA37 applied to soil and 7D applied to the plant. Testing a bacteria cocktail of BA37 and 7D for antagonism against V. dahliae may be subject of future experimentation, as using a varied biocontrol strategy can provide greater protection against the pathogen. Furthermore, the bacteria tested have been isolated but not yet identified; thus, an additional next step would be to identify bacteria 7D and BA37 through DNA sequencing analysis.
CONCLUSION
Bacteria with potential application in biological control against a major crop pathogen, V. dahliae, were found. Of four bacterial strains tested, endophytic bacterial strain 7D and soil bacteria BA37 show promise as biological control agents. It was hypothesized that the three endophytic bacteria would have stronger antagonistic relationships with V. dahliae. This proved to be partially true since 7D was endophytic; however, BA37 was a soil bacterium and it displayed greater antagonism than the other two endophytic bacteria tested. The discovery opens the door to alternative practices for controlling fungal pathogens that reduce utilization of chemicals, which are good for both environmental and economic sustainability of crop production.
ACKNOWLEDGEMENTS
I would like to extend my sincerest thanks to Dr. Helen Tai from Agriculture and Agri-Food Canada for being my mentor and for her support throughout my project. I would also like to thank Alexa Creelman from Agriculture and Agri-Food Canada for assisting me in executing the experiment.
REFERENCES
Berlanger, I., & Powelson, M. (2005). Verticillium Wilt. Verticillium Wilt. doi:10.3897/bdj.4.e7720.figure2f
Guo, B., Wang, Y., Sun, X., & Tang, K. (2008). Bioactive natural products from endophytes: A review. Applied Biochemistry and Microbiology, 44(2), 136-142.
Hardoim, P. R., van Overbeek,L. S., Berg, G., Pirttilä, A. M., Compant, S., Campisano, A., Döring,M., and Sessitsch, A. (2015) The Hidden World within Plants: Ecological and Evolutionary Considerations for Defining Functioning of Microbial Endophytes. Microbiol Mol Biol Rev. 79(3): 293–320 doi: 10.1128/MMBR.00050-14
Landis, D. A., & Orr, D. B. (2015, July 01). Biological Control: Approaches and Applications. Retrieved from https://ipmworld.umn.edu/landis
Mol L., Termorshuizen A.J. (1995) Life cycle and ecology of Verticillium dahliae in potato. In: Haverkort A.J., MacKerron D.K.L. (eds) Potato Ecology And modelling of crops under conditions limiting growth. Current Issues in Production Ecology, vol 3. Springer, Dordrecht. doi: 10.1007/978-94-011-0051-9_16
Nair, P. V., Wiechel, T. J., Crump, N. S., & Taylor, P. W. (2016). Role of Verticillium dahliae and V. tricorpus naturally infected tubers in causing Verticillium wilt disease, contribution of soil pathogen inoculum and subsequent progeny tuber infection. Australasian Plant Pathology,45(5), 517-525. doi:10.1007/s13313-016-0442-3
Tai, H. H., D. De Koeyer, M. Sønderkær, S. Hedegaard, M. Lagüe, C. Goyer, L. Nolan, C. Davidson, K. Gardner, J. Neilson, J. R. Paudel, A. Murphy, B. Bizimungu, H. Y. Wang, X. Xiong, D. Halterman, and K. L. Nielsen. 2018. Verticillium dahliae Disease Resistance and the Regulatory Pathway for Maturity and Tuberization in Potato. Plant Genome 11. doi:10.3835/plantgenome2017.05.004
BIBLIOGRAPHY
Bowers, J. H., Nometh, S. T., Riedel, R. M., & Rowe, R. C. (1996). Infection and Colonization of Potato Roots byVerticillium dahliaeas Affected byPratylenchus penetransandP. crenatus. Phytopathology,86(6), 614. doi:10.1094/phyto-86-614
Eljounaidi, K., Lee, S. K., & Bae, H. (2016). Bacterial endophytes as potential biocontrol agents of vascular wilt diseases – Review and future prospects. Biological Control,103, 62-68. doi:10.1016/j.biocontrol.2016.07.013
Government of Canada. (2014, June 10). Verticillium wilt. Retrieved April 05, 2018, from http://www.agr.gc.ca/eng/science-and-innovation/agricultural-practices/agroforestry/diseases-and-pests/verticillium-wilt/?id=1367500568532
Government of Canada. (2017, November 23). Guide for the Importation and Release of Arthropod Biological Control Agents. Retrieved from http://agr.gc.ca/eng/about-us/offices-and-locations/agricultural-pest-management/agricultural-pest-management-resources/guide-for-the-importation-and-release-of-arthropod-biological-control-agents/?id=1511380291353
DHANISHTA AMBWANI
Dhanishta is a grade 11 student with a passion for helping others; she does this in as many ways she can by volunteering for multiple local and international organizations. Furthermore, Dhanishta has always fostered a fascination for science and the curious nature of what people’s minds are capable of questioning, molding, and connecting in the field. Her love for science and helping others is why she has always aspired to pursue a career in Medicine. In her spare time, Dhanishta enjoys playing piano, painting, and playing rugby and basketball; due to her participation in these activities she was awarded the Duke of Edinburgh’s Bronze and Silver awards, and she is currently working towards her Gold Award.