bacterial antagonism biocontrol lab report

Biocontrol of Bacteria For Antagonistic Potential Within Lettuce

Woody ArnoldTechnische Universität Graz

Department of Environmental Biotechnology

Table of Contents

Introduction pg. 1

Materials and Methods pg. 2 - 3

Results pg. 4

Discussion pg. 5 - 6

References pg. 7

Appendix pg. 8 - 14

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Introduction

Throughout the scientific and medical communities, people have been studying different types of bacterial species because of their effects on the environment and on the daily lives of our human civilization. Bacteria have benefited us by introducing us to processes like fermentation for the creation wine, beer, and bread; as well as the pasteurization and cures for different types of diseases. Because of this, bacteria play a large role and act as a type of symbiosis to the human body when it comes to our diet while even protecting us from other harmful bacteria and viruses. In addition to how bacteria effect us as a species directly, they also play a huge role in the environmental cycles that help to keep our environment in equilibrium and help in optimal growth for our crops. Like animals, who harbor complex microbiomes, plants are increasingly being recognized as having diverse bacterial communities. Bacterial communities associated with above ground portion plants can be found on the leafy surface of a plant or phyllosphere and within plant tissues. (Jackson, Randolph, Osborn, Tyler, et al. 2013)

To further explain why it is important to study bacteria within crops, in a study conducted at the University of Colorado in Boulder, CO, USA, showed that there is a broad diversity of bacteria within different types of fruits and vegetables. Even in handling between conventionally and organically farmed produce, there maybe also differing effects on human health. (Leff, Fierer, et al. 2013) The Gram negative bacterial family Enterobacteriaceae, which includes many of the human pathogens associated with plant foods, for example Escherichia, Salmonella, and Shigella, also contain a number of genera of plant pathogens such as Enterobacter, Erwinia, Pantoea, Pectobacterium, etc. that cause plant diseases such as blights, wilts, and soft rots. (Fletcher, Leach, Eversole, Tauxe, et al. 2013). In recent years, an increasing number of disease outbreaks in humans have been associated with consumption of contaminated vegetables, and most of the outbreaks during 1996 to 2008 were associated with leafy green vegetables, where Escherichia coli O157:H7 and Salmonella spp. accounted for 72% of the pathogens involved. Sprouts were the likely source of a large European outbreak of Escherichia coli O104:H4 originating in Germany, and a multi state (United States) disease outbreak of Escherichia coli O157:H7 in October 2011 was linked to romaine lettuce. (Jensen, Storm, Forslund, Baggesen, Dalsgaard, et al. 2013) For example, within leafy vegetables such as lettuce, there have been investigations in the US that show that there is a broad diversity of microorganisms in the phyllosphere of lettuce. In addition, the produce has been associated with various outbreaks of food-borne illnesses and now regarded as a significant vector of human pathogens that are traced back to Salinas Valley, CA, USA. (Williams, Moyine, Harris, Marco, et al. 2013).

Based on these findings, there have been recent efforts to address this problem related to public health and food safety. The aim of this study was to test various biological control agents against other pathogens to find inhibition in hopes of applying these findings for more biological solutions to inhibit bacteria that cause spoilage of our crops and cause outbreaks.

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Materials and Methods

Materials

• 30ml flasks• Petri-dishes• Eppenforf BioPhotometer• 1ml cuvettes• GFL Water Bath• Pilot-Shake• Incubation rooms set at 30° C and 37° C• Large flasks• Nutrient Broth II (NBII) (Sifin; Berlin, Germany)• Agarose (Agar) (Roth; Karlsruhe, Germany)

In addition to the materials used, the following biological control agents (BCAs) and pathogens were used:

BCAs: Pathogens:

Serratia plymuthica 3Re4-18 Enterobacter agglomerans 4Rx13

Serratia plymuthica HRO-C48 Pantoea ananatis BLBT1-08

Paenibacillus sp. GNDBL 71/2 Serratia marcescens

Pseudomonas trivialis 3Re-2-7 (Salavida) Klebsiella pneumoniae

Bacillus subtilis Co1-6 Salmonella thypimurium LT2

Escherichia coli K12

Raoultela ornithinolytica RE2-3-4

Methods

First, the BCAs were tested against Enterobacter agglomerans 4Rx13 and Escherichia coli K12. 30ml flasks with NBII and 300ml flasks filled a mixture of agar and NBII were prepared and then autoclaved to ensure sterility. The unused 300ml flasks filled with the NBII agar were stored in a 60° C heater. Then petri-dishes of pure NBII agar were prepared and the BCAs along with Enterobacter agglomerans 4Rx13 and Escherichia coli K12 were streaked out and then inoculated overnight at 30° C and 37° C respectively to ensure that pure cultures could be used during the experiments. On the next day, over a sterile environment, two 30ml flasks of NBII were used with one flask containing a colony of Enterobacter agglomerans 4Rx13 and the other with Escherichia coli K12. Then both were put onto a shaker at 37° C and inoculated overnight. From there, the two flasks were taken out of the incubation room and then the initial OD600 values were calculated

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using a spectrometer. To ensure proper calibration of the spectrometer, a 1ml cuvette filled with pure NBII was used as a blank. To find the initial OD600 values, a 1:10 dilution was put into the cuvettes for each pathogen and then placed into the spectrometer to obtain the values. The reason for using a 1:10 dilution is because reasonable values will be easier to work with and if it isn't diluted prior to placing the cuvette in the spectrometer, a too large of an OD600 value is obtained. After obtaining the initial OD600 values, the values are then multiplied by 10 to get the true cell density. For example, if OD600 reading of 0.234 is obtained, then the true initial value would be 0.234(10) = 2.34. But if during the procedure, if, for example, the initial value was higher than 1 with a 1:10 dilution, then try the same procedure with a 1:100 dilution and then multiply that OD600 reading by 100 to get the true cell density. After that, to find the proper dilution amount, the following formula was used to calculate a theoretical value to obtain an OD600 reading of 0.2:

0.2(V )C

V = volume in NBII flaskC = true initial cell density

Sample

If V= 30ml and C = 3.4, then

0.2(30ml)3.4

=1.76

Based on the theoretical calculations, the proper dilution volumes were placed in two unused, 30ml flasks with NBII; one for each pathogen; and then placed onto the shaker at 37° C for 40 min. After the 40 min. incubation of the 30ml flasks with Enterobacter agglomerans 4Rx13 and Escherichia coli K12, OD600 readings were calculated again. If OD600 values weren't at or close to 0.4, the flasks were put back into incubation for 37° C for 20 min. to ensure there is optimal bacterial growth for mixture with NBII agar. Two 300ml flasks of NBII agar were then taken out of the 60° C heater and placed in a water bath calibrated to 55° C for at least 20 min. to ensure the bacteria in the 30ml NBII flasks won't get killed off by the temperature when placed in the warm agar. Petri-dishes with NBII agar containing the bacteria were poured into petri-dishes quickly under sterile conditions to avoid having the agar harden during pouring. Specifically, the pathogen with highest cell density after the 40 min. incubation was poured into the first 300ml flask of NBII agar, shaken to ensure a homogeneous mixture, and then swiftly poured into petri-dishes. After the petri-dises with the respective pathogens; Enterobacter agglomerans 4Rx13 and Escherichia coli K12; were prepared, the BCAs were then streaked onto the plates; where each BCA had two small linear streaks for each temperature at 30° C, 37° C, and room temperature; for each pathogen. All the streaked out plates were inoculated at 30° C overnight and prepared plates with the pathogens without any BCA streaked out on them were used as the control to show bacterial growth of the pathogens within the agar and be able to better analyze the plates for inhibition after the inoculation. Smaller streaks for each BCA were also used for that reason. Inhibition was also analyzed and recorded. The plates were then labeled for 30° C, 37° C, and room temperature and incubated at their respective temperatures overnight while just placing the room temperature labeled plates in a sterile area or parafilming around the plates to

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minimize the likelihood of contamination. Inhibition was again analyzed and recorded. The following method was also used for Pantoea ananatis BLBT1-08, Serratia marcescens, Klebsiella pneumoniae,and Salmonella thypimurium LT2. Once all the inhibition data was recorded for all BCAs and pathogens, graphs were created to better visualize the data for presentation.

Results

Based on the findings, Bacillus subtilis Co1-6 is shown to be the only BCA to inhibit Klebsiella pneumoniae and also shown antagonism to Pantoea ananatis BLBT1-08, but only after 48 hours. (Figures 1 and 2) This may be due to Bacillus subtilis Co1-6's nature to be very opportunistic to compete for nutrients. In addition both Serratia plymuthica HRO-C48 and Serratia plymuthica 3Re4-18 showed obvious antagonism with Serratia marcescans despite the fact that all of these bacteria are from the same family. This shows that even between the same genera and even between strains of the same genera, the Serratia strains will most likely compete with one another instead of working together to share access to nutrients. Serratia plymuthica 3Re4-18 also strongly inhibited Pantoea ananatis BLBT1-08 and Raoultela ornithinolytica RE2-3-4, but showed no inhibition with Klebsiella pneumoniae. Within Serratia marcescans plates, Serratia plymuthica 3Re4-18 showed antagonism after 48 hours. (Figures 3, 4 and 11) On the Escherichia coli K12 and Enterobacter agglomerans 4Rx13 plates, Bacillus subtilis Co1-6 and Serratia plymuthica 3Re4-18 were found to totally inhibit both enterobacteria. Paenibacillus sp. GNBL ½ showed no inhibition with any of the BCAs during the first 24 hours, but Enterobacter agglomerans 4Rx13 was the only BCA to show inhibition to Paenibacillus sp. GNBL ½ after 48 hours of incubation. (Figures 7, 8, and 12)

Although Raoultela ornithinolytica RE2-3-4 was the only test pathogen to not go through with the48 hour incubation period like the other test pathogens. (Figure 13) Serratia plymuthica 3Re4-18, Serratia plymuthica HRO-C48, and Pseudomonas trivialis 3Re-2-7 (Salavida) showed antagonism withBacillus subtilis Co1-6 showing the most antagonism toward Raoultela ornithinolytica RE2-3-4.But, besides Bacillus subtilis Co1-6 slow inhibition growth toward Pantoea ananatis BLBT1-08 after 48 hours. There were no significant results with the procedure of initially inoculating every plate at 30° C after 24 hours and then incubating the plates with some plates at room temperature, 30° C, and 37° C for another 24 hours. (Figures 14-16) Based on the pictures shown in the appendix, different colonies on each of the plates had different cell densities where some were small, some obviously big, some close to no growth visually. Although this result maybe based on how the plates were streaked, it may also be due to the competitive nature between the bacteria in the plates to inhibit one another.

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Discussion

Based on the results, by conducting biocontrols of various bacteria, biological solutions could be proposed to prevent wilting of lettuce based on the bacteria. Not only just lettuce, but the same method can be used for various other crops and fruits and can be applied for testing for fungal inhibition. For example, in a study at the Kasmir University of Agricultural Sciences and Technology using commercial products that include, Biocon, Biogaurd, Ecofit, F-Stop, Soilguard etc with Tricoderma sp. as active ingredient, and Mycostop, Rhizoplus Subilex etc utilizing various Bacillus species as active ingredient. Biological control can achieve the objective of disease suppression through a number of ways such as antibiosis, competition, mycoparasitism, cell wall degradation and induced resistance, plant growth promotion and rhizosphere colonization capability. As the bioagent represents a living system, it needs to be mass produced and formulated into various commercial products in a way it remains viable for at least two years. (Junaid, Dar, T.A. Bhat, A.H. Bhat, M.A. Bhat; et al 2013) In addition, in another study related to soybeans, 31 endophytic bacteria belonging to Pseudomonas, Bacillus, Enterobacter, Klebsiella, Acetobacter Burkholderia, Rhizobium andXanthomonas were isolated from soybean (Glycine max (L) Merril) and were screened in vitro for the antagonistic activity against soil-borne fungal pathogens of soybean viz., Rhizoctonia solani, Fusarium oxysporum, Sclerotium rolfsii, Collectotruchum truncatum, Macrophomina phseolina and Alternaria alternata. The effective antagonists were further screened for their plant growth promoting (PGP) activity viz., production of plant growth regulators (auxins, gibberellins and cytokinins), siderophores and HCN. Five isolates JDB 3 (Pseudomonas sp.), JDB 5 (Pseudomonas sp.), JDB 9 (Bacillus sp.), JDB 11 (Bacillus sp.) and JDB 14 (Bacillus sp.) were found to exhibit maximum number of plant growth promoting traits. Hence, they are considered as efficient endophytic isolates as they possess dual abilities i.e. antagonistic and plant growth promotion with the view of plant health and productivity. Thus, these promising endophytic bacterial isolates obtained from the present study may be commercially formulated as effective biocontrol agents for the management soil-borne fungal pathogens of soybean. (Dalal, Kulkarni; et al 2013)

Also, the results show that the Serratia strains show antagonism between one another despite being part of the same genera. A study of Serratia plymuthica from a raw vegetable processing line shows that Serratia strains have the potential to inhibit one another. None of the Serratia spp. type strains produced a halo except for Serratia proteamaculans, which caused a weak inhibition ofSerratia grimesii. However strain RVH1 caused complete inhibition of Serratia entomophila, Serratia ficaria, Serratia grimesii, Serratia odorifera, Serratia plymuthica, Serratia proteamaculans, and Escherichia coli ESS, but not Serratia fonticola, Serratia liquefacians, and Serratia quinivorans.Preliminary tests suggested that the antibacterial activity can be ascribed to a protein and possibly due to differences in growth temperature and other experimental factors. (Van Houdt, Moons, Jansen, Vanoirbeek, Michiels; et al 2005) Therefore, based on findings in this study, a Serratia strain could possibly be used to treat infections caused by another Serratia strain; even for applications at neonatal intensive care units in hospitals. To support this opinion, a hospital study done from 2006 – 2011 where 11 patients with Serratia marcescens sepsis, a blood infection, and 47 patients colonized due to the spread of various clones were observed. The strength of this study was the 6 year long period of observation and the stepwise introduction of interventions that made it possible to identify a breakpoint along with continuous monitoring of one of the interventions and staff compliance. (Samuelsson,

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Isaksson, Hanberger, Olhager; et al 2014) To address the weakpoints of this study, the introduction of the application of other Serratia strains, could therefore be possible biological solutions to patients with sepsis caused by Serratia marcescans. This proves that conducting biocontrol tests to check for antagonism of enterobacteria related to the food we eat such as lettuce, can also show us solutions for other applications in public health.

Besides the significant results shown by the Serratia strains inhibiting one another during the courseof the experiment, the ability of bacteria to show antagonism is dependent on the cell density, growth rate, and the temperature. Growth rate could be a possible factor that shows gradual inhibition because different bacteria based on the results shown with Bacillus subtilis Co1-6 and Kleibsiella pneumoniae, show that bacteria may need more time to grow and be able to show antagonism against other bacteria.Competition between these bacteria also play a huge role because although bacteria are able to co-exist with other bacteria based on resource usage, antimicrobial compounds, and resource acquisition, a study on Escherichia coli O157:H7 and epiphytic bacteria, which are bacteria that live on plants that get moisture and nutrients from the air and rain and live on other plants; show that competition for nutrients and end products of metabolism are important for reducing the growth of Escherichia coli O157:H7 in vitro and on epiphytic surfaces. A pH reduction was also observed for the majority of antagonistic bacteria. In addition it was also reported in a 2009 study by Johnston titled,“Microbial antagonists of Escherichia coli O157:H7 on fresh-cut lettuce and spinach” , that acid production by lettuce and spinach bacterial isolates act as a possible mechanism for growth inhibition of Escherichia coli O157:H7. However in a 2010 study titled, “ Reduction of Escherichia coli O157:H7 in fresh spinach using lactic acid bacteria and chlorine as a multihurdle intervention” byGragg and Brashears, didn't detect production of organic acids by lactic acid bacteria that was inoculated onto spinach stored at refrigeration temperatures suggesting that other compounds such as bacterocins, hydrogen peroxide and carbon dioxide would provide more effective growth deterrents on leaf surfaces. (Lopez-Velasco, Tydings, Boyer, Falkinham, Ponder; et al 2012). Therefore, competition played a huge role that determines antagonistic potential between the BCAs and pathogens used in this biocontrol experiment.

Although significant results were found, especially the finding of the antagonistic nature between the Serratia strains, from just only culturing the bacteria and streaking them on to NBII agar, if this study were to be conducted again from the beginning, inoculation should be done overnight with samples stored at room temperature, 30° C, and 37° C for the first 24 hours and then do the same for 48 hours. The reason being is because it would give a more clear observation of inhibition growth at different temperatures consistently at the time periods of 24 hours and 48 hours. In addition, even though visually inspecting the petri-dishes after inoculation of 24 hours and incubation at 48 hours, using additional methods would have given more quantifiable values for the results to show different levels of inhibition and cell densities. All and all, it would be great to see more solutions using biological means for the health problems plaguing animals and our crops. The more we understand the bacteria that affect our lettuce and other produce, the more we can understand how we can keep our produce safe for the public naturally.

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References

1) Fletcher, Jacqueline, et al. "Human pathogens on plants: Designing a multidisciplinary strategy for research." Phytopathology 103.4 (2013): 306-315.

2) Williams TR, Moyne A , Harris LJ, Marco ML (2013) Season, Irrigation, Leaf Age, and Escherichia coli Inoculation Influence the Bacterial Diversity n the Lettuce Phyllosphere. PLoS ONE 8(7): e68642. doi:10.1371/journal.pone.0068642

3) Jackson, Colin R., et al. "Culture dependent and independent analysis of bacterial communities associated with commercial salad leaf vegetables." BMC microbiology 13.1 (2013): 274.

4) Jensen, Annette Nygaard, et al. "Escherichia coli Contamination of Lettuce Grown in Soils Amended with Animal Slurry." Journal of Food Protection® 76.7 (2013): 1137-1144.

5) Flores, Gilberto E., et al. "Diversity, distribution and sources of bacteria in residential kitchens." Environmental microbiology 15.2 (2013): 588-596.

6) Samuelsson, Annika, et al. "Late-onset neonatal sepsis, risk factors and interventions: an analysis of recurrent outbreaks of Serratia marcescens, 2006–2011." Journal of Hospital Infection 86.1 (2014): 57-63.

7) Junaid, Jan Mohd, et al. "Commercial biocontrol agents and their mechanism of action in the management of plant pathogens." Int. J. Modern Plant & Anim. Sci1.2 (2013): 39-57.

8) Dalal, Jitendra, and Nikhilesh Kulkarni. "Antagonistic and Plant Growth Promoting Potentials of Indigenous Endophytic Bacteria of Soybean (Glycine max (L) Merril)." Current Research in Microbiology and Biotechnology 1.2 (2013): 62-69.

9) Houdt, Rob, et al. "Genotypic and phenotypic characterization of a biofilm‐forming Serratia plymuthica isolate from a raw vegetable processing line."FEMS microbiology letters 246.2 (2005): 265-272.

10) Lopez-Velasco, Gabriela, et al. "Characterization of interactions between Escherichia coli O157: H7 with epiphytic bacteria in vitro and on spinach leaf surfaces." International Journal of Food Microbiology 153.3 (2012): 351-357.

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Appendix

Figure 1. Bacillus subtilis Co1-6 after 24 hours.

Figure 2. Bacillus subtilis Co1-6 after 48 hours.

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Enterobacter agglomerans 4Rx13

Pantoea ananatis BLBT1-08

Serratia marcescens

Klebsiella pneumoniae

Salmonella thypimurium LT2


0 10 20 30 40 50 60 70 80 90 100

no inhibtionantagonism

N=6 / antagonistic [%]

Enterobacter agglomerans 4Rx13Pantoea ananatis BLBT1-08

Raoultella ornithinolytica RE2-3-4Serratia marcescens

Klebsiella pneumoniaeSalmonella thypimurium LT2


0 10 20 30 40 50 60 70 80 90 100



Figure 3. Serratia plymuthica 3Re4-18 after 24 hours.

Figure 4. Serratia plymuthica 3Re4-18 after 48 hours.

Figure 5. Serratia plymuthica HRO-C48 after 24 hours

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Enterobacter agglomerans...


Raoultella ornithinolytica R...

Serratia marcescens




0 10 20 30 40 50 60 70 80 90 100

no inhibtion

antagonism


Enterobacter agglomerans ...


Serratia marcescens




0 10 20 30 40 50 60 70 80 90 100





Raoultella ornithinolytica RE...

Serratia marcescens




0 10 20 30 40 50 60 70 80 90 100



Figure 6. Serratia plymuthica HRO-C48 after 48 hours.

Figure 7: Paenibacillus sp. GNDBL 71/2 after 24 hours

Figure 8: Paenibacillu sp. GNDBL 71/2 after 48 hours

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Serratia marcescens




0 10 20 30 40 50 60 70 80 90 100



Enterobacter agglomeran...


Raoultella ornithinolytica ...

Serratia marcescens




0 10 20 30 40 50 60 70 80 90 100

no inhibtion

antagonism




Serratia marcescens




0 10 20 30 40 50 60 70 80 90 100



Figure 9: Pseudomonas trivialis 3Re-2-7 (Salavida) after 24 hours

Figure 10: Pseudomonas trivialis 3Re-2-7 (Salavida) after 48 hours

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Raoultella ornithinolytica RE...

Serratia marcescens




0 10 20 30 40 50 60 70 80 90 100





Serratia marcescens




0 10 20 30 40 50 60 70 80 90 100

no inhibtion

antagonism


Figure 11: Serratia marcescans, Kleibsiella pneumoniae, and Salmonella thypimurium LT2 plates after inoculation after 48 hours at 37° C.

Figure 12: Enterobacter agglomerans 4Rx13 and Escherichia coli K12 plates after incubation after 48 hours at 37° C

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Figure 13: Raoultela ornithinolytica RE2-3-4 plates inoculated after 24 hours at 30° C

Figure 14: Pantoea ananatis BLBT1-08 incubated after 48 hours at 30° C

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Figure 15: Pantoea ananatis BLBT1-08 incubated after 48 hours at 37° C

Figure 16: Pantoea ananatis BLBT1-08 incubated after 48 hours at room temperature.

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Figure 17: Pantoea ananatis BLBT1-08 control incubated after 48 hours at 30° C.

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bacterial antagonism biocontrol lab report

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