Plant Microbiome Project

Plant Microbiome ProjectBy Thomas Brinkman

Hello, today I am going to be talking about my version of the plant microbiome project. I know a few of the members of the lab are doing this, and jenny spoke about this last week. 1

Introduction

Monsanto video on microbes and how they help the world2

BackgroundWhat is unknown:What makes the microbe beneficialWhat determines if a microbe is a symbiont or a phytopathogen Trends between these beneficial microbes from different plants

BackgroundBacteria found in Arabadopsis, corn, duckweed and soybean

DefinitionsRhizosphere microbiome beneficial for plant growth and health. Organisms found in the rhizosphere include bacteria, fungi, oomycetes, nematodes, protozoa, algae, viruses, archaea and arthropods. Most of the organisms in the rhizosphere biome belong to a complex food web that utilizes the large amount of nutrients released by the plant.

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BackgroundOnly 33% of bacterial species detected in the molecular analysis were present for the culture dependent analysis (Matsuzawa, H., et al., 2010).Bacterial species may live interchangeably as a symbiotic endophyte or phytopathogenFungal antagonist Fusarium oxysporum in lettuce (Moretti et al., 2012).

Genetic sequencing showed that there are common genetic systems that allow these bacteria to colonize in a plant.The problem is that many of these bacteria require the plant host to survive, so they are not detected in culture dependent studies. This is why the 16s rRNA analysis is good. It is very sensitive and will amplify these microbes. This is a culture dependent analysis though since they were grown on LB.

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BackgroundAfter inoculating a beneficial microbe it was shown to increase pathogen resistance in Pinus sylvestris, potato and tomato varieties (Ardanov et al., 2012).Microbes reduce the need for fertilizers and pesticides (Horrigan et al., 2002).

Goal of the StudyFind common genetic precursors Determine how these microbes slip past the plants innate immune system See if microbes from different plants can be used to increase growth of other plants

We would like to see if there is something about their DNA that shows they will be beneficial or harmful to the plant

(is it that theyre undetectible or can the plant discriminate the beneficial microbes from pathogenic)

We will take these microbes and grow them on arabadopsis and on duckweed to see if they have benefits to these land plants even though seaweed is from the sea.

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My partSequencing endophytes from seaweed Inoculate microbes on Arabadopsis thaliana and duckweedSequence entire genome of bacteria that display beneficial growth

Seaweed name= ulva lactuca8

Isolated endophytes from seaweed and seawater in BrazilGrown on LB with glycerol and stored in -80C16S Colony PCR Sanger sequencing

Methods

He isolated the endophytes the same way as duckweed except he had to cut the seaweed up first since its large. Then bleached in about 15% bleach and rinsed with rinse solution and then distilled water 3 times. Then the plants were plated on LB and grown up and transferred to glycerol stocks9

9 bacteria colonies as endophytes and 6 from the water of grumari beach10

Trindade Beach

4ednophyte bacterial colonies from trindade beach11

4 endophyte colonies and 12 bacterial colonies from the water here.12

16S rDNA

Conserved in almost all bact-variable enough to distinguish to certain levelShows phlyum and genus of bacteria but loses power to more specific you get.The black parts are the conserved regions and the squiggles are the variable regions that allow us to distinguish between them. We are using the DNA that makes the RNA to study it.

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Results

1000 800 1000800 control control control control control control control control 100 BP ladder100 BP ladder

These are supposed to be around 925 bp. The controls in both sets have contamination and in the second picture there are no bands in the bacteria but there are in the controls.14

Results

control control control NTC

Kennys Samples1000 800 100 BP ladder

Sorry kenny15

Actual Results

16S Colony PCR

ExoSAP-IT clean up7501000 750

Actual Results

1 kb ladder1000750PDJ-1PDJ-2PDJ-3PDJ-4

PDJ-1PDJ-2PDJ-3PDJ-4100 BP ladder1000 800 16S Colony PCR ExoSAP-IT clean upcontrol control control

ResultsSAM IDFROM:PHYLUMGENUSG2-4Grumari BeachFirmicutesBacillusG2-5AGrumari BeachFirmicutesBacillusG2-5BGrumari BeachFirmicutesBacillusG2-6Grumari BeachFirmicutesBacillusTrin-1Trindade BeachFirmicutesBacillus; GeobacillusTrin-2Trindade BeachFirmicutesBacillus; GeobacillusTrin-3Trindade BeachFirmicutesBacillus; GeobacillusTrin-4Trindade BeachFirmicutesBacillus; Geobacillus

THE DIFFERENCE BETWEEN BACILUS AND GEOBACILLUS18

Differences in Locations: Grumari Beach

Trindade Beach

Out-group

Duckweed vs. Seaweed: Duckweed

Seaweed

Out- Group

DiscussionMistakes happenDuckweed vs. Seaweed endophytesMore results needed

Future WorkInoculate cultures on Arabadopsis and duckweedSequence ones that have an effect on the plantExpand study to other locations and plantsQuestions?