• No results found

To monitor the microbial biodiversity in soil within Uppsala

N/A
N/A
Protected

Academic year: 2022

Share "To monitor the microbial biodiversity in soil within Uppsala"

Copied!
51
0
0

Loading.... (view fulltext now)

Full text

(1)

21-X7

To

monitor the microbial biodiversity in soil within Uppsala

Tora Godow Bratt, Mathilda Stigenberg, Andreas Elenborg, Sarah Ågren, Andreas Medhage

Client: Biohacking

Client representative: Johan Ledin Supervisor: Lena Henriksson

1MB332, Independent Project in Molecular Biotechnology, 15 hp, spring semester 2021 Master Programme in Molecular Biotechnology Engineering

Biology Education Centre, Uppsala University

(2)

Abstract

This is an exploration of the potential for a citizen science project, with the goal to get the general public involved in microbial soil biodiversity around Uppsala, Sweden. Biodiversity serves an important role in how an ecosystem performs and functions. A large part of Earth's biodiversity exists below ground in soil, where microorganisms interact with plants. It would be beneficial to analyse the abundance and spread of some microorganisms in order to gain a better understanding of soil

biodiversity. We suggest that one species family to study could be Phytophthora. Phytophthora is a genus of oomycetes that often are pathogenic, causing disease in various trees and other plants. It is unknown exactly how widespread the genus is today, making it extra interesting for the proposed study. For the general public to be able to do this a device needs to be developed that is easy to use and preferably could be used directly in the field. An isothermal amplification method is suitable for identifying the microorganism under these conditions. Many isothermal amplification methods are expensive, perhaps too expensive for a citizen science study, but have great potential for easy field testing. We propose a device utilizing RPA and lateral flow strips. RPA - Recombinase Polymerase Amplification is a method for amplification that might be suitable since it is simple, sensitive, and has a short run time. It is however expensive, which is an issue, but isothermal amplifications are

expensive across the board. Lateral flow strips can be used to visualize the results. They utilize antibodies to detect the previously amplified amplicons, and give a positive or negative test answer that would be understandable to even untrained study participants. One of the biggest obstacles identified in this project concerns amplifying DNA from a soil sample, because an extraction step is necessary. The methods we have identified for extraction are not performable in the field, since they require centrifugation. In the proposition for a device a possible work-around for this is proposed, but since it has yet to be tested it is not yet known whether it will work or not.

(3)

Index

Abstract 1

Index 2

Abbreviations and word list 4

1. Background 7

1.1. Citizen science 7

1.2. Biodiversity 7

1.3. Isothermal amplification 8

2. Species of interest 10

2.1. Pathogens - harmful microbes 10

2.1.1. Sclerotinia sclerotiorum 10

2.1.2. Phytophthora 10

2.2. Beneficial microbes 11

2.2.1. Soil pathogen suppressiveness 12

2.2.1.1. Pseudomonas 13

3. Sampling of soil 14

4. Extraction of nucleic acids from soil 14

4.1. Dispersion 14

4.2. Lysis 15

5. Identifying microbial species 16

5.1. Loop-mediated isothermal amplification (LAMP) 17

5.2. Recombinase polymerase amplification (RPA) 18

5.3. Other methods 20

6. Design of primers 20

7. Detection and visualization 21

7.1. Lateral flow 21

7.2. Turbidimetry 22

7.3. Hybridization protection assay 22

7.4. Fluorescence 22

7.5. Bridge flocculation 23

7.6. Gel electrophoresis 23

8. Database and interface - tracking the species 23

9. Discussion 24

9.1. Selection of species of interest - Phytophthora 24

9.2. Selection of amplification method - RPA 25

9.3. How to extract without centrifugation 27

9.4. Selection of detection method - lateral flow 28

9.5. Other factors to consider 28

9.6. Citizen science and involving people 29

(4)

10. Design of the device 31

10.1. Components of the device 31

10.2. Alternative device procedures 32

11. Conclusion 33

12. Acknowledgements 33

13. Statement of Contribution 33

14. References 33

Appendix 41

Appendix 1: Project order 41

Appendix 2: Ethics 42

A2.1. Ethics of databases and citizen science studies 42

A2.2. Ethics concerning biodiversity 43

Appendix 3: Identifying microbial species 45

A3.1. In situ hybridization (ISH) 45

A3.2. Metagenomics 45

A3.3. Single primer isothermal amplification (SPIA) 46

A3.4. Transcription-mediated amplification (TMA) 46

A3.5. Nucleic acid sequence-based amplification (NASBA) 47

A3.6. Q-beta replicase reaction (QBRR) 47

A3.7. Helicase-dependent isothermal DNA amplification (HDA) 48

A3.8. Rolling circle amplification (RCA) 48

A3.9. Strand displacement amplification (SDA) 49

(5)

Abbreviations and word list

amplicon product of amplification

amplification copying of nucleic acids (DNA or RNA)

AMV avian myeloblastosis virus

BIP backward inner primer

BOP backward outer primer

buffer solution resistant to change in pH buoyant density density relative to fluid

chemotaxi ability to follow chemical gradients

DNA deoxyribonucleic acid

DNase enzyme breaking down DNA

dNTPs nucleoside triphosphates, building blocks for DNA dsDNA double-stranded DNA

eDNA environmental DNA

endosphere environment inside a plant root

enzyme protein with catalysing biological function

EPCM engineered phase change Material

exudate secreted substance

FIP forward inner primer

fluorescence emits light when shone on

fluorescence probe detection method utilizing fluorescence and probes food-web model of food chains

FOP forward outer primer

HDA helicase-dependent isothermal DNA Amplification helicase enzyme separating strands

hybridization two molecules binding together intellectual property intangible creation owned by company

ISH in situ hybridization

isothermal reaction under constant temperature ITS internal transcribed spacer regions LAMP loop-mediated isothermal amplification

(6)

lateral flow detection method utilizing antibodies lysozyme antimicrobial enzyme

macroscale larger scale, in contrast to micro microbiome the microbes living in an environment microfluidics how liquids act in a microscale microscale (µ) small scale, 10-6

NASBA nucleic acid sequence-based amplification NCBI the national center for biotechnology information NDGC nycodenz density gradient centrifuge

NINA non-Instrumental nucleic acid

NTPs nucleoside triphosphates, building blocks for RNA nuclease enzyme breaking down nucleic acid

nucleic acid RNA and DNA

Oomycetes "fungi like" eukaryotic organism group pathogen harmful organism

PCR polymerase chain reaction

phyllosphere environment on leaves

polymerase enzyme synthesising DNA/RNA

primer RNA fragment from where DNA synthesis starts, binds DNA probe used for finding specific sequence

protease enzyme breaking down protein

QBRR the Qβ replicase reaction

quorum regulation microorganism signalsystem

RCA rolling circle amplification

recombinase enzyme modifying genome

restriction enzyme enzyme cutting nucleic acid at specific restriction site rhizobium the microbes associated to a plant root

rhizosphere environment of microbes associated to a plant root ribosome organelle responsible for translation of mRNA to protein

RNA ribonucleic acid

RNase enzyme breaking down RNA

RPA recombinase polymerase amplification

(7)

SDA strand displacement amplification SDGC sucrose density gradient centrifuge SDS sodium dodecyl sulfate - lysis agent SPIA singel primer isothermal amplification ssDNA single-stranded DNA

throphic group position in food-web

TMA transcription-mediated amplification

turbidimetry detection method through cloudiness of solution viriome all viruses in environment

X spp. sub-species of X

(8)

1. Background

This project aims to investigate the possibility of studying biodiversity around Uppsala, using citizen science in order to generate interest in the area of biology. The exact project order is available in Appendix 1. Biohacking, the project’s client, is according to their website (biohackeri) a non-profit organisation with the goal to spread interest in biology and self-experimentation. They wish to create a workspace where people of all disciplines can work with “do it yourself” biology, and inspire the general public to participate in solving problems by making use of citizen science. An ethical analysis is included in Appendix 2. The goal of this report is to give advice in designing a device for detection of a specific microbe. Several methods are discussed with focus on isothermal amplification. Due to the time frame of the project, the project group will only be able to make a review of already made studies and will not be able to create a prototype of the device. Neither will designing primers for the project be possible. As the project mostly focuses on methods identifying specific species, deciding on possible species of interest is part of the project. To summarize, the report will contain an outline for a potential isothermal device to identify a species of interest.

1.1. Citizen science

Citizen science is a good tool for gathering large amounts of data (Bahls 2014, Kissling et al. 2018, Hermans et al. 2019). The term means to involve the general public in a study, often by having them gather and send in samples. However, there are also some challenges associated with studies

performed by private citizens, such as the computing costs and the data storage needed, as well as the need for an intuitive input system and interface (Bahls 2014, Kissling et al. 2018, Baker et al. 2019, Hermans et al. 2019). Baker et al. (2019) discuss at length the possible biases and misunderstandings that may occur when untrained study participants report their findings. The article concludes that the most common biases are the accessibility of sampling sites and the public awareness of a specific interest in a site. Sites already known to contain a species of interest are overrepresented in reports.

The article suggests utilizing statistical methods to validate the findings and weighing the submissions based on submission history. Many of the articles express a need for further research in the field and call for the construction of open databases where knowledge can be gathered and accessed by anyone (Kissling et al. 2018, Hermans et al. 2019, Edge et al. 2020).

1.2. Biodiversity

Biodiversity serves an important part in how an ecosystem performs and functions (Wagg et al. 2014).

A large part of Earth's biodiversity exists below ground in soil, where microorganisms interact with plants. Above ground, plant diversity influences many ecosystem functions. Thus, loss of microbial biodiversity in soil could constitute a major threat to the general biodiversity on Earth.

Our understanding of biodiversity has long been limited to above ground and under water (Thakur et al. 2020). Above ground, biodiversity can be the mechanism behind the performance of an ecosystem and its ecosystem services (Wagg et al. 2014). However, little is known about the biodiversity below ground, especially the microbial diversity. Soil interacts with multiple complex food-webs, and as such, any changes to biodiversity in a trophic group may vastly affect the abundance, diversity, and function of another. Soil is a nonrenewable resource (Griffiths & Philippot 2013), and the patterns of biodiversity vary on a spatial scale (Thakur et al. 2020).

(9)

The microbiome contains bacteria, fungi, protozoa, nematodes, and viruses (Fierer 2017, Edge et al.

2020). The bacteria and fungi tend to dominate in biomass (Fierer 2017), but the exact distribution varies depending on the environment. Bacteria tend to be dominant in neutral to mildly acidic areas with relatively high nutrient availability and low organic content. Fungi dominance occurs in acidic areas with high organic mass and low resource quality.

Soil provides a number of different ecosystem services, many of which are dependent on various microbes. Microbes serve an important part in biogeochemical cycling, where they are needed to cycle nutrients - such as ammonium, phosphorus, and carbon (Fierer 2017, Edge et al. 2020), as well as serving a role in detoxifying metals and pollutants, and suppressing pathogens (Berendsen et al. 2012, Fierer 2017). However, some species of microbes in soil can be pathogenic and cause disease in plants. Plants can be directly affected by associated microbes or indirectly by free-living microbes in the bulk soil that regulates soil properties (Heijden et al. 2008). Other articles define direct effect as the microbes promoting/suppressing growth and indirect as when microbes affect soil properties or other microbes (Lugtenberg & Kamilova 2009, Glick 2014, Lobmann et al. 2016).

Soil is generally starved of resources. Many microbes therefore rely heavily on plant root exudates to survive (Lugtenberg & Kamilova 2009, Berendsen et al. 2012). Microbes find the root through

chemotaxis, following a gradient of exudates, and then proceed to colonize it (Lugtenberg & Kamilova 2009, Bulgarelli et al. 2013). The microbes then compete for a niche in the area near the roots (the rhizosphere), inside the root (the endosphere), or on leaves (the phyllosphere). Quorum regulation, secretion, and detection of signal molecules in the surroundings allows the microbes to communicate amongst each other (Lugtenberg & Kamilova 2009, Berendsen et al. 2012, Bulgarelli et al. 2013).

This allows for organization of advanced cooperative processes such as colonization, biofilm formation, spore formation, metabolic processes, and pathogenic infection.

1.3. Isothermal amplification

Traditionally, identification of bacteria was done through culture based approaches (Craw &

Balachandran 2012, Abbasian et al. 2016, Ghurye et al. 2016, Goordial et al. 2017, Ellingham et al.

2019, Brumfield et al. 2020). The process is however time consuming, often taking several days. The method is also limited by many microbes not being culturable. Today amplification is also available as a means for identification. One of the most common methods used for this is Polymerase Chain Reaction (PCR) (see Figure 1) (Akduman et al. 2002, Gill & Ghaemi 2008, Craw & Balachandran 2012, Barreda-García et al. 2016, Yang Q et al. 2018, Zatti et al. 2019, Brumfield et al. 2020, Xing et al. 2020). In PCR the DNA is first denatured by heating. Then primers can hybridize to sequences around the target by lowering the temperature. The temperature is then raised to an optimal

temperature for the DNA polymerase to extend the primers, thereby replicating the DNA. The system is then heated again and the cycle is repeated, creating multiple copies of the target. PCR can be used both for amplification of DNA and for identification. The latter through amplification of

microbe-specific sequences such as the highly conserved 16S rRNA. The identification can then be accomplished by detecting reaction products, revealing that amplification has occurred and therefore that the target is present. This is often done using gel-electrophoresis but can also be done using fluorescence. PCR however, requires advanced equipment, such as a thermocycler, and trained personnel.

(10)

Therefore the project will primarily focus on isothermal amplification techniques. They can identify microbes in the same way PCR does (Craw & Balachandran 2012, Yang Y et al. 2017, Yang Q et al.

2018, Zatti et al. 2019, Yang Q et al. 2020). However, due to their isothermal nature they eliminate the need for the thermal cycler, which is both expensive and too cumbersome to bring on field studies.

They also often have simpler protocols so even untrained personnel could use the methods. This can make some of the methods more applicable in a field study.

Most of the methods are owned by companies in the form of intellectual properties (Craw &

Balachandran 2012). Therefore many of the methodologies are not commercially available and require direct partnership with the provider. Some of the methods however, have commercially available kits, but they may in many cases be expensive. Craw and Balachandran (2012) speculate this may be the reason many of the methods are not

applied in more studies.

Many methods for isothermal

amplification utilize polymerases with strong strand displacement activity. The strong displacement polymerases synthesise a new complementary strand to the parent strand and displace the existing downstream complementary DNA (Walker et al. 1992, Walker et al.

1996, Boldinova et al. 2020) (see Figure 2). The already bound complementary strand is then replaced with the newly synthesised

(11)

2. Species of interest

As the proposed method in this project focuses on a specific microbe to study, the choice of what species should be studied is important. Therefore what microbes might be of interest needs to be discussed. Note however, that the method can be adapted to other species as needed. The chosen microbe to study is only a suggestion for a good starting point.

2.1. Pathogens - harmful microbes

Soil supports a large number of organisms that can host various different viromes, where

bacteriophages hosted by bacteria are the most diverse virus group (Sutela et al. 2019). However, their influence on ecological processes is largely unknown. Sutela et al further state that some studies have shown that pathogens might have a role in soil carbon cycling. Many of these viruses infect fungi and oomycetes, possibly causing disease. Hence, it could be beneficial to study some of these pathogens to get a better understanding of their abundance and effect on below- and above-ground biodiversity.

Many pathogens target specific species or types of plants (Heijden et al. 2008). Pathogens may gather under the root of the plant target, thereby preventing the offspring of the plant to grow in the same place. This may leave the spot open for colonisation by other species. However, some pathogens are less specific and target any plant not resistant to it. The pathogens killing old plants may also have a role in freeing the nutrients, so they can go back into the system.

2.1.1. Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic fungal pathogen that can infect many important plants and crops, such as oilseed rape, soy bean, and other vegetable crops (Jiang et al. 2013). It secretes cell-wall degrading enzymes and oxalic acid to kill plant cells and take up nutrients from the dead tissue

(Hegedus & Rimmer 2005). This pathogen is distributed widely across the world due to its broad host range. Mehmood et al. (2020) showed that S. sclerotiorum suppresses the presence of other fungal pathogens while enriching the presence of beneficial microbes, thus reducing the general microbial biodiversity in the soil (Mehmood et al. 2020). Hence, it is a pathogen that is beneficial to soil health, but harmful for some plants. S. sclerotiorum needs access to senescent (aged or damaged) tissue to cause an infection (Hegedus & Rimmer 2005). This implies that it can be present in soil without negatively affecting some plants.

2.1.2. Phytophthora

Phytophthora is a family of oomycetes typically infecting deciduous trees. The pathogen targets the fine roots of plants and makes them more susceptible to other infestants (Weller et al. 2002, Herrera 2015, Witzell & Cleary 2017). They spread through various types of spores and can hybridize, forming new variants that can potentially be more aggressive or have another target species. Phytophthora seems to be able to get through the plant's defense mechanisms by forming spores that contain degrading enzymes and suppressors of plant defences (Blanco & Judelson 2005).

When in its natural habitat, Phytophthora seldom causes any problems but when it is moved to a new environment it can become harmful and invasive, causing disease or even death to some plants

(Blomquist et al. 2016). Research shows that a trophically complex Phytophthora community could be important in many ecosystems.

(12)

Phytophthora spp.- have not been abundant in the cold nordic climate until recently, and some species can now be found in forests in southern Sweden (Witzell & Cleary 2017). New species not native to Sweden have recently been observed (Witzell & Cleary 2016). According to one source (Herrera 2015) the knowledge about Phytophthora in Sweden is highly limited, and it is probable that more species and variants exist that we do not yet know about. Furthermore, Phytophthora is a

hemibiotrophic pathogen, meaning it can live off both living and dead cells. Thus, disease may develop slowly and it can take a long time before an infected plant shows any symptoms. Therefore it is possible that Phytophthora exists in Uppsala, even though it has not been reported or discovered yet, and it could be beneficial to analyze soil samples for any signs of infection. Phytophthora is often undiscovered until it is already established in a forest and the trees show severe symptoms such as bleeding wounds on the trunks.

Due to climate change and rising temperatures, it is not impossible that Phytophthora could constitute a future problem in Uppsala even if it is found that it does not yet exist in or around the city (Witzell &

Cleary 2017). The symptoms of infection may be hard to detect until conditions benefiting the microbe have occurred and the host plant has already been severely harmed. Witzell & Cleary (2017)

recommend doing soil sampling to investigate the presence of the microbe.

2.2. Beneficial microbes

The microbiome is extremely important to the plants. The plants exude a big percentage of its photosynthetic product to the microbes (Bulgarelli et al. 2013, Glick 2014). It is hypothesised that plants even make a selection for certain microbes using root secretions that only the wanted beneficial microbes can utilize (Lugtenberg & Kamilova 2009, Berendsen et al. 2012, Bulgarelli et al. 2013, Glick 2014). Plants may even be able to control these microbes to a degree through mimicking quorum regulation (Berendsen et al. 2012, Bulgarelli et al. 2013). The rhizobium, the microbes living in the rhizosphere, may be linked to the genotype of the plant (Berendsen et al. 2012, Bulgarelli et al. 2013), although Bulgarelli et al. (2013) state that the link is weak and that most microbes are the same as in bulk soil. In the endosphere the link is stronger, implying increased selectivity from the plants. All of these various types of microbes would be of interest to investigate in order to better understand soil biodiversity, due to the important roles in maintaining a healthy ecosystem.

One important beneficial activity microbes may contribute to is making nutrients available to the plant.

This can be called biofertilization (Lugtenberg & Kamilova 2009). Nitrogen fixation is a well known important microbial process (Heijden et al. 2008, Lugtenberg & Kamilova 2009, Berendsen et al.

2012, Bulgarelli et al. 2013, Fierer 2017). Bacteria such as Azospirillum, found free-living in bulk soil, as well as Rhizobium and Bradyrbi, found in association to the root, are nitrogen fixing bacteria (Lugtenberg & Kamilova 2009). They can take atmospheric nitrate and make ammonium that can be utilized by the plants (Heijden et al. 2008, Bulgarelli et al. 2013). Bacteria with nitrogen metabolism can also benefit the plant (Bulgarelli et al. 2013). Through denitrification nitrate is converted into nitrite that can then be made into nitrogen oxides, which are used by plants as a signaling molecule.

Other microbes such as Bacillus, Pseudomonas, and Penicillium solubilize phosphorus and thereby make it more accessible for the plants. Mycorrhizal fungi can supply the root with nutrients, transported from the nearby soil (Heijden et al. 2008). They use their hyphae (a type of filament structure) to scavenge for nutrients. In return the fungi are supplied with photosynthetic carbon from the plants (Heijden et al. 2008, Berendsen et al. 2012, Bulgarelli et al. 2013).

(13)

The microbiome is able to modulate plants to an extent (Berendsen et al. 2012). Some microbes can produce phytohormones, substances that are similar to the plants’ own hormones (Lugtenberg &

Kamilova 2009, Bulgarelli et al. 2013). These sometimes are a factor in promoting plant growth, for example auxin which is involved in some plant growth processes. Microbes associated with plants have been detected producing auxin in response to tryptophan expulsion from the root (Glick 2014).

However, auxin might sometimes only affect a part of the plant (Lugtenberg & Kamilova 2009), and it is generally unknown how much these processes actually contribute to plant growth (Bulgarelli et al.

2013). Another phytohormone is ethylene, derived from ACC exuded from the plant (Bulgarelli et al.

2013, Glick 2014). The hormone is important in many plant growth processes, but under stress, production of the hormone is induced and may have detrimental effects on plant growth and even harm the plant. Microorganisms with ACC deaminase, for example found in certain Pseudomonas strains, break down the ACC as part of their metabolism and therefore reduce the level of ethylene produced during stress, in turn reducing the negative effects (Lugtenberg & Kamilova 2009, Bulgarelli et al. 2013, Glick 2014).

2.2.1. Soil pathogen suppressiveness

One of the most important indirect growth-promoting effects microbes have on plants is induction of soil pathogen suppressiveness (Weller et al. 2002, Heijden et al. 2008, Lugtenberg & Kamilova 2009, Berendsen et al. 2012, Lobmann et al. 2016). In some soils, called suppressive soils, plants will not get infected despite there being pathogens in the environment. In contrast, in conductive soils the plants are susceptible to infection. This may in some cases be due to abiotic factors such as the chemical and/or physical properties of the soil (Lobmann et al. 2016), but in many cases it is the microbiome that is responsible for the suppressiveness (Weller et al. 2002, Heijden et al. 2008, Lugtenberg & Kamilova 2009, Berendsen et al. 2012, Bulgarelli et al. 2013, Lobmann et al. 2016).

This biotic suppression may stem from several different biological activities. Suppressiveness is also sometimes transferable, meaning a percentage of suppressive soil mixed with conductive soil will produce more suppressive soil (Weller et al. 2002, Lugtenberg & Kamilova 2009, Berendsen et al.

2012).

Another important distinction is between general (or nonspecific) suppressiveness and specific

suppressiveness (Weller et al. 2002, Lugtenberg & Kamilova 2009, Berendsen et al. 2012, Lobmann et al. 2016). General suppressiveness suppresses several pathogens, whereas specific suppressiveness suppresses a single pathogen but often to a greater extent. Long-standing suppression continues even when the host plant has been removed, whereas induced suppression occurs after a monoculture has grown in the same soil and exposed to disease over an extended period of time (Weller et al. 2002).

Induced suppressive microbes are more favoured when a pathogen is present, possibly due to stimuli from the plant (Berendsen et al. 2012).

One of the most common ways microbes suppress pathogens is by competing for nutrients and niches (Weller et al. 2002, Lugtenberg & Kamilova 2009, Berendsen et al. 2012, Lobmann et al. 2016, Fierer 2017). Soil is often starved of resources necessary for microbes, so a non-pathogenic microbe with higher competitiveness can in starved soil prevent the pathogen from establishing itself (Weller et al.

2002, Lugtenberg & Kamilova 2009, Berendsen et al. 2012, Lobmann et al. 2016, Fierer 2017).

Suppression-inducing organisms such as strains of Pseudomonas and Bacillus commonly contain antibiotic and antifungal secondary metabolites, used to get a competitive advantage over the pathogen (Lugtenberg & Kamilova 2009).

(14)

Another way bacterial strains compete is through production of siderophores (Weller et al. 2002, Lugtenberg & Kamilova 2009). These are used to bind and take in iron more effectively. Siderophores are thought to be used by microbes, such as Pseudomonas, to outcompete the pathogens in control of the iron (Weller et al. 2002). Yet another way microbes may gain a competitive advantage is by signal interfering (Lugtenberg & Kamilova 2009). By breaking down the signal molecules used by pathogens in quorum regulation some suppressive microbes get a competitive advantage, but also may prevent the pathogen from organizing an infection in the first place.

Some plant-associated microbes can induce systemic resistance in the host (Weller et al. 2002, Heijden et al. 2008, Lugtenberg & Kamilova 2009, Berendsen et al. 2012, Bulgarelli et al. 2013, Lobmann et al. 2016). They activate the plant’s own immune system, readying the plant to repel the pathogen.

Pseudomonas and Bacillus are known species that this phenomenon occurs in (Lugtenberg &

Kamilova 2009). Other suppressive microbes directly antagonise the pathogens. They may predate on or parasitise the pathogens, thereby outright killing or inhibiting them (Lugtenberg & Kamilova 2009, Lobmann et al. 2016).

Many sources highlight the complexity of the microbiome (Weller et al. 2002, Lugtenberg &

Kamilova 2009, Berendsen et al. 2012, Lobmann et al. 2016, Fierer 2017). The effects of the microbiome are usually not caused by only one microbe, rather it is the combined community of microbes that is the cause of an effect. Some microbes may only be suppressive or beneficial under certain conditions (Lugtenberg & Kamilova 2009, Fierer 2017), and there are interspecies variations between microbes (Weller et al. 2002, Berendsen et al. 2012, Lobmann et al. 2016, Fierer 2017).

Some strains may have beneficial properties, while others are completely neutral. There are even cases where non-pathogenic strains of normally pathogenic species, such as F. oxysporum, can be

suppressive, especially toward pathogenic strains of the same species (Weller et al. 2002).

2.2.1.1. Pseudomonas

One of the commonly mentioned plant-beneficial bacteria is Pseudomonas spp. (Heijden et al. 2008, Berendsen et al. 2012, Bulgarelli et al. 2013, Glick 2014). Pseudomonas is mentioned by several articles as producing important secondary metabolites and several strains are suspected to be key players in suppressiveness (Weller et al. 2002, Lugtenberg & Kamilova 2009, Berendsen et al. 2012, Bulgarelli et al. 2013). Pseudomonas is also known for its high affinity siderophores, allowing for competitiveness for iron (Weller et al. 2002). Pseudomonas chlororaphis MA342 has been found able to suppress several disease pathogens in cereals, but it failed in suppressing highly localized pathogens (Hermans et al. 2019). This may imply it being part of the general suppressiveness. The article also concludes that the suppressive effect continues even after plants are removed, implying long-standing suppression. Weller et al. (2002) mention Pseudomonas fluorescens strains producing 2,4-DAPG, inhibiting many plant pathogens. Pseudomonas fluorescens strains may induce systemic resistance in plants and is mentioned as one of the best known root colonizers (Lugtenberg & Kamilova 2009), and the group contains bacteria that solubilize phosphorus which can play a role in biofertilization

(Heijden et al. 2008, Bulgarelli et al. 2013). P. chlororaphis 6G5 and P. putida GR12-2 strains have been found to produce ACC deaminase (Bulgarelli et al. 2013).

(15)

3. Sampling of soil

The methodology for soil sampling depends on the composition of the soil. The soil in and around Uppsala has a high content of fine clay, which might complicate the sampling process. Since clay binds to extracellular DNA (eDNA), it is important to separate the intact cells from the bound eDNA in the clay before DNA extraction (Högfors-Rönnholm et al. 2018). Otherwise the DNA sequencing will not be able to differentiate between the living bacterial cells and remnant species no longer present, which will result in a skewed picture of the biodiversity in the sample. Högfors-Rönnholm et al. (2018) have written a guide on how to sample soil with high clay content. They suggest that a sodium phosphate buffer could be used for the release of bacterial cells and eDNA from the soil.

An important factor to account for is how the sample is taken. What species is found in a sample highly depends on where it is taken. One of the reasons that many diverse species inhabit soil is the many microhabitats it consists of (Fierer 2017). Therefore, the samples have to be taken in similar environments to get an accurate picture of the abundance and distribution of species.

4. Extraction of nucleic acids from soil

There are two general categories of methods for extracting DNA from soil: direct and indirect (Miller et al. 1999). In a direct extraction method, the cells are lysed in the soil as opposed to an indirect method where the cells are separated from the soil particles before the lysis (Parachin et al. 2010). The direct methods will often give a higher DNA yield than indirect methods, sometimes up to 100 times higher (Delmont et al. 2011). However, the direct methods have a higher rate of contamination with humic and fulvic acids, metal ions, and salts and therefore require an additional purification step (Narayan et al. 2016). A direct method also causes DNA shearing which makes the extracted DNA fragments shorter (Delmont et al. 2011, Narayan et al. 2016).

Separation of cells from soil particles is accomplished in two steps. The first step is dispersion where the cells are parted from soil aggregates and the second step is separation where the cells are separated from the soil by centrifugation (Ehlers et al. 2008, Liu J et al. 2010).

4.1. Dispersion

The dispersion method can be physical, chemical, enzymatic, or a combination. Common physical dispersion methods are ultrasonic treatment, with for example an ultrasonic probe or an ultrasonic bath, homogenization and shaking the sample together with glass beads (Lindahl & Bakken 1995, Liu J et al. 2010). These methods use physical force to detach the cells from the soil particles which can cause cell damage, especially the methods that use sonic force (Liu J et al. 2010). The chemical dispersants can be detergents and are often used in combination with a physical dispersion method (Lindahl & Bakken 1995). Enzymatic dispersion releases the cells by breaking down the soil particle structures with enzymes such as cellulase or lipase (Böckelmann et al. 2003, Liu J et al. 2010).

However, the enzymatic method was not as effective as other methods when tested and compared, and the execution was both complicated and expensive (Liu J et al. 2010).

The separation step is usually performed using the different properties of the components in the dispersed sample, such as sedimentation rates and buoyant densities. The preferred methods are the ones based on buoyant density, such as nycodenz density gradient centrifugation (NDGC) or sucrose

(16)

density gradient centrifugation (SDGC), since these are shown to give a higher yield and purity of the sample (Liu J et al. 2010).

4.2. Lysis

There are a lot of different methods for lysing cells. The methods can be divided into mechanical and non-mechanical where the latter can be divided into physical, chemical, and biological (Shehadul Islam et al. 2017).

The mechanical methods include bead milling which is commonly used in laboratories due to the efficiency being very high. Using this method, the cells are lysed by being mixed together with small beads at high speeds. The force of the beads hitting the cells breaks their cell membranes. Mechanical methods have a high efficiency and can be used to lyse many different cells. However, due to heat being generated, degrading of the cellular products can occur and the methods are often expensive (Shehadul Islam et al. 2017). Physical non-mechanical methods use heat, pressure, or sound to disrupt the cell membrane. These methods are either expensive or not suitable for all cell types (Shehadul Islam et al. 2017).

Chemical non-mechanical methods use buffers that rupture the cell membrane by changing the pH, sometimes together with detergents that dissolve the membrane proteins and disrupt the cell membrane. One of these methods is detergent lysis where the interactions between the hydrophobic and hydrophilic molecules in the cell membrane are disrupted by a detergent. This causes the cell membrane to break down. To use this method on bacterial cells, the cell wall needs to be broken down first. This can be done with lysozymes that destroy bonds in the peptidoglycan layer. A common lysis agent is SDS because it is very strong and works on most cell types (Shehadul Islam et al. 2017).

Several assays use SDS as a lysis agent at 65 °C (Narayan et al. 2016, Cheng et al. 2016). However, one study (Ahmed et al. 2014) states that heat is applied to enhance the lysis, and incubate the sample at 37 °C for 10 to 60 minutes after adding the SDS. They do however add another lysis agent

afterwards and incubate the mixture at 65 °C.

Biological non-mechanical lysis methods use enzymes such as lysozymes or proteases to disrupt the cells and need to be used together with detergents when lysing bacteria. Common enzymes used to lyse bacterial cells are lysozymes and proteinase K. Lysozymes have an optimal working temperature at 35 ℃ (Shehadul Islam et al. 2017), and while proteinase K can work at similar temperatures, its optimal working temperature is higher (Ware et al. 2020). Proteinase K is very efficient for lysing gram negative bacteria but not as much for gram positive bacteria (Ahmed et al. 2014).

Many of these methods can be used on both macro- and microscale. Another approach for microscale cell lysis is microfluidics, where volumes on a nano- to picoliters scale are used in microchannels (Shehadul Islam et al. 2017). Microfluidics can be used to combine different operations into one device. For example, cell lysis, PCR, and capillary electrophoretic analysis has been performed by one device (Khandurina et al. 2000). The microfluidics cell lysis methods are divided into mechanical, thermal, chemical, optical, acoustic, and electrical lysis. Most of these methods have a fast lysis time but the equipment or reagents are often expensive (Shehadul Islam et al. 2017)

(17)

5. Identifying microbial species

There are many potential methods available for identification of a species. The most promising

methods will be summarized more extensively below. Less promising methods will only be mentioned in passing. For a more in-depth description of these methods see Appendix 3. All investigated methods are summarized briefly in Table 1.

Table 1: comparison of different methods for identification.

Method Reaction

Temperature [℃]

Time required

Number of Primers

Visual Confirmation Target

LAMP 60 - 65 1 hour

(amplification) 4 Fluorescence dyes and probes,

turbidimetry ssDNA

RPA 22 - 45

(optimal: 37 - 42) 20 minutes

(amplification) 2 Fluorescence probes, Lateral

flow strip dsDNA

SPIA 60 - 65 (95*) 2-4 hours

(assay)

1** Fluorescence dyes and probes DNA/RNA

TMA 42 4 hours

(assay) 2 Fluorescence probes,

hybridization protection assay RNA

NASBA 41 (95*) 1 hour

(assay) 2 Fluorescence RNA

QBRR 37 (100*) 30 minutes

(amplification) 0 Gel electrophoresis,

Secondary probe RNA/DNA

***

HDA 37 / 60 - 65 100 minutes

(amplification) 2 Fluorescence,

Lateral flow strip dsDNA

RCA 30 - 65 90 minutes

(amplification)

2 Fluorescence dsDNA

(circular)

SDA 37-40 (95*) 2-3 hours

(amplification) 2 (4) Fluorescence ssDNA

Metagenomics - Several days

(assay) - Sequencing eDNA

ISH 46**** 2-3 hours

(hybridization)

- fluorescent, chromogenic, and radioactive

RNA

* Initial denaturing heating step, ** chimeric,

*** Complicated target molecule, **** More effective at higher temperatures

(18)

5.1. Loop-mediated isothermal amplification (LAMP)

LAMP is a DNA amplification method performed under isothermal conditions (Notomi et al. 2000, Macuhova et al. 2010). Incubation occurs at 60 - 65 ℃, and results are usually obtained within an hour. Termination of the reaction is accomplished by inactivating the polymerases, which is done by raising the temperature to 80 ℃ for 10 min. Some of the major advantages of LAMP are that the method is performable by untrained persons, cheap to perform, gives results relatively quickly, results can be visually confirmed, and it can be performed and analyzed in the field (Nikolskaia et al. 2017, Gandasegui et al. 2018, Blaser et al. 2018, Schenkel et al. 2019, Kamber et al. 2020, Barr et al. 2021).

Some results even show LAMP to be more sensitive than PCR, meaning lower concentrations of the target DNA needs to be present in the sample for it to be detected (Liu A et al. 2012, Zhao et al. 2016, Wang L et al. 2020).

Four primers are involved in LAMP: forward inner primer (FIP), backward inner primer (BIP), B3, and F3 (Notomi et al. 2000). All of these are specific to the gene that is to be studied and can be designed for example with the program PrimerExplorer, found at:

https://primerexplorer.jp/e/v4_manual/index.html(Zatti et al. 2019).

The LAMP reaction starts with the

annealing of the forward inner primer (FIP) (Notomi et al. 2000). This is used to extend a new complementary strand (Strand 3) which displaces the original

complementary strand. The forward outer primer (F3 / FOP) anneals. When it is extended, strand 3 is displaced. Strand 3 contains self-complementary sequences which lead to the formation of a loop structure. Backward inner primer (BIP) thereafter anneals to Strand 3 and is extended to create a new strand (Strand 4).

This strand is then displaced by the

annealing and extending of backward outer primer (B3 / BOP), which frees up Strand 4. Strand 4 in turn contains the

self-complementary sequences of Strand 3 as well as self-complementary sequences on the other end of the strand, leading to loop structures on both ends of the strand. The resulting structure contains several initiation sites from where further amplification can occur.

A reaction product resulting from LAMP can

be confirmed visually. This can be accomplished by adding SYBR Green I to the mixture, which changes color from orange to green if the amplification has occurred (Notomi et al. 2000, Zatti et al.

(19)

Another method of visualizing the results is via turbidimetry, when a white magnesium pyrophosphate precipitate will form if there has been a positive reaction (Zatti et al. 2019). Further methods for identification, in these cases via fluorescence, include adding Eva Green, SYTO, calcein,

fluorescent-labeled probes, quencher-labeled primers, dye-labeled primers, magnesium-colorimetric titration (hydroxynaphthol blue), or pH sensitive dyes (Zatti et al. 2019). Any one of these can be added to visualize whether or not a reaction has occurred. Malachite green can also be used, which will change from being translucent to a green/blue color for positive results (Kamber et al. 2020).

The heat necessary to perform LAMP can be provided by simple means, without instruments, by utilizing a reaction between CaO and water (LaBarre et al. 2011). This is the same reaction used in many ready-to-eat meals and hand warmers, and is readily available to purchase in stores. The resulting reaction needs to be stabilized to maintain the desired temperature for the duration of the amplification. This can be done by insulation with an engineered phase change material (EPCM). The EPCM used by LaBarre et al. (2011) had a melting point around 65 ℃, which would then maintain the temperature at this point while it changed phase. It was provided by Renewable Alternatives, Inc. The EPCM consisted of a fat product, which could be produced from beef tallow, palm oil, coconut oil, or soybean oil. Commercially available CaO sufficed for the reaction.

LaBarre et al. (2011) also proposed a kit device to be used in the field to easily perform LAMP. This kit, dubbed NINA Heater (NINA stands for non-instrumental nucleic acid), uses a two-chambered thermos to perform the reaction. In the bottom chamber they performed the CaO-water reaction, while the upper chamber contained the EPCM along with an aluminium material to improve the heat

transfer. The EPCM had the reaction wells for the LAMP reaction integrated into its structure. Using this kit LAMP could be performed easily in a low-resource setting.

5.2. Recombinase polymerase amplification (RPA)

Recombinase polymerase amplification (RPA) is an isothermal amplification method used for identifying microbes (McCoy et al. 2020). RPA is selective, easy to execute and does not require a denaturation step (Lobato & O’Sullivan 2018). It is also very sensitive because it can detect as low as 1 copy of the target nucleic acid. Three key proteins are involved in RPA: recombinase, recombinase loading factor, and single-stranded binding protein (Li et al. 2018). The method is suitable for

multiplexing, meaning several tasks can be run in parallel (White 2003), and is targeting RNA or DNA (Lobato & O’Sullivan 2018). Lobato & O’Sullivan (2018) state that RPA can be performed at

temperatures between 22 - 45 ℃, but is usually optimized between 37 - 42 °C. The authors also claim that the incubation time for amplifying DNA to noticeable levels normally is 20 minutes. However, it can be done in less time. DNA needs to be extracted before application of RPA, which can be done with commercially available kits or other novel methods (Wang X et al. 2020, Wang X et al. 2020).

Kits are also available for the RPA reaction itself (Wang T-M & Yang 2019, McCoy et al. 2020).

The process is described in Figure 4 below. In the initial step of a RPA procedure, recombinase proteins are attached to a reverse primer and a forward primer when a crowding agent and ATP are present (Lobato & O’Sullivan 2018). This results in two recombinase-primer complexes that search through DNA after a homologous sequence. The complexes invade DNA, after locating a homologous sequence, which results in a D-loop structure (Zhang et al. 2020). One of the DNA strands hybridizes with the reverse primer and the other strand hybridizes with the forward primer while single stranded binding proteins stabilize both strands to avoid the expulsion of the primers (Lobato & O’Sullivan

(20)

2018, Zhang et al. 2020). The crowding agent is an important factor in this step due to its ability to avert spontaneous recombinase-primer dismantlement when single stranded binding proteins are present (Lobato & O’Sullivan 2018). In the last step, the recombinase is dismantled and DNA polymerase binds to the 3’ end of the reverse and forward primer. DNA polymerase extends the primers with dNTPs, creating amplicons. This procedure results in an exponential amplification when repeated.

Two primers are needed for RPA (Daher et al. 2016). The length of the primers is an important factor to keep in mind when designing them, and primers used for RPA are usually between 30 - 35

nucleotides (Bentahir et al. 2018, Peng et al. 2019, Zhang et al. 2020). Shorter primers than this can be used, but can decrease speed and sensitivity (Li et al. 2018). Normally, the guanine-cytosine content is 40 - 60 percent of the primers (Zhang et al. 2020). Another important factor to consider when

designing primers is to prevent repeats of guanines at the 5’ end, but these boost performance at the 3’

end (Lobato & O’Sullivan 2018, Zhang et al. 2020). In addition, cytidines also boost performance at the 3’ end. Secondary structures such as hairpins ought to be avoided (Zhang et al. 2020), as well as sequences that encourage primer-primer interactions (Lobato & O’Sullivan 2018).

The reagents needed for RPA are in a lyophilized pellet and can be stored for half a year at room temperature (Lobato & O’Sullivan 2018). A rehydration buffer, primers, probes, magnesium acetate, and DNA template are all the components needed for the lyophilized pellet to initiate amplification (McCoy et al. 2020). Initiation occurs when the magnesium acetate is added (Daher et al. 2016).

Normally, the primers and probes are not mixed in with the lyophilized pellet at first, but McCoy et al.

(2020) attempted a preformulated kit with primers and probes already mixed in. This reduces the risk of contamination, as well as increasing the simplicity of using RPA in the field. The conclusion of this

(21)

commercially available kits. Reagents for RPA are available in freeze-dried, liquid, and pellet form for purchase, as well as kits for performing RPA that cater to both RNA and DNA (Daher et al. 2016, Li et al. 2018).

RPA is more resistant to inhibition than PCR, but high levels of polysaccharides may reduce the amplification (Miles et al. 2014). The author speculates that this inhibition would be reduced by dilution, but this would then reduce sensitivity.

End point detection or real-time detection can be used to identify the RPA amplicons (Lobato &

O’Sullivan 2018). Real-time detection occurs during the amplification and end point detection occurs after the amplification. Lobato & O’Sullivan (2018) suggests that some available end point detection strategies include lateral flow, fluorophore-modified reverse primers, and bridge flocculation. A real-time detection strategy is using fluorescent probes and visualizing with a fluorimeter (Lobato &

O’Sullivan 2018, Peng et al. 2019). The dyes SYBR green or Eva Green can also be used for real-time detection (Lobato & O’Sullivan 2018). However, these two dyes can cause false-positives because they can not distinguish between primer-dimer formations and amplicons.

TwistDx™ have developed a commercially available easy to use kit for RPA (Craw & Balachandran 2012, Miles et al. 2014, Goux et al. 2018, Lei et al. 2019, TwistDx). The kit has been shown to be able to detect different pathogens such as fungi and Phytophthora. The kit consists of a lyophilized pellet and contains all reagents needed including a detection probe, with exception of rehydration buffer and primers (Miles et al. 2014). GEB2 was found to be a good buffer, combining ease of use and success rate. The authors also found that most extraction buffers worked as rehydration buffers.

5.3. Other methods

Other methods available for identification of species include in situ hybridization (see A3.1.), metagenomics (see A3.2.), SPIA (see A3.3.), TMA (see A3.4.), NASBA (see A3.5.), QBRR (see A3.6.), HDA (see A3.7.), RCA (see A3.8.), and SDA (see A3.9.). See Appendix 3 for a more detailed description of these.

6. Design of primers

A conserved region of a genome is a region that has stayed mostly the same throughout evolution and therefore is similar amongst closely related species. To make a primer, a unique and conserved sequence in the species must be targeted. These regions are often genes, since the function of a gene often disappears if the gene sequence changes. Many microbes can be detected through the 16S rRNA gene or internal transcribed spacer regions (ITS) (Clark & Hirsch 2008, Yang Y et al. 2017, Kissling et al. 2018, Orgiazzi et al. 2018, Edge et al. 2020, Xing et al. 2020). Internal transcribed spacer regions are transcribed to RNA and then used as spacers between the two subunits of the ribosomes. Although in this context it is more important that they are conserved enough to be used to identify microbes. For some organisms such as fungi and oomycetes, internal transcribed spacer regions are better targets (Miles et al. 2014, Lei et al. 2019). Many primers may still have to be tested to ensure they find the target and that they are not non-specific (Winkworth et al. 2020).

(22)

Miles et al. (2014) used internal transcribed spacers to detect Phytophthora. The spacers inside the mitochondrial DNA are preferred due to the shorter length of the mitochondrial genome. Traditionally the cox1-2 spacer region is used. The authors however used the trnM-trnP-trnM locus as a marker to detect the genus and atp9-nad9 to distinguish between species within the genus.

Winkworth et al. (2020) also designed primers for detection of Phytophthora. Several subspecies were aligned to find conserved regions. These potential target region primers were designed using

PrimerExplorer v5 (https://primerexplorer.jp/e/). Primers were tested, only saving those that detected the microbe. Using the Basic Local Alignment Search Tool on NCBI the non specific primers were also removed.

The National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/) is an organization that maintains databases (the NCBI et al.). The important databases in this context are the ones storing sequenced genomes. These can be used to find sequences for target design. Some of the genomes have been annotated, meaning the sequences containing genes have been identified. There are sequenced genomes for many Phytophthora strains in NCBI, however only some are annotated, complicating primer design since it is harder to find conserved regions. Pseudomonas

(NZ_LT907842.1) and Sclerotinia sclerotiorum (NC_035155.1) are well annotated (Cuomo et al.

2017, Varghese & Submissions 2020).

7. Detection and visualization

The resulting amplicons from the amplification can be detected in several ways to see if the soil sample contained the species of interest. Various detection methods for identifying the amplicons and visualizing the result are mentioned below.

7.1. Lateral flow

Lateral flow is an end point detection method that is commonly used and from which the result can be observed by the naked eye when using a lateral flow strip (Lobato &

O’Sullivan 2018, Peng et al. 2019). The labeled amplicons created from amplification can be detected by antibodies in the lateral flow strip (Lobato & O’Sullivan 2018). Lateral flow strips consist of a sample pad that is in a tris-buffered saline solution (Zhang et al. 2020), conjugate release pad, detection zone, test line, control line, and an absorbent pad (Koczula & Gallotta 2016). A solution containing the labeled amplicons mixed with phosphate buffered saline (Zhang et al. 2020) is added onto the sample pad and moves to the conjugate release pad (Koczula & Gallotta 2016). The conjugate release pad has antibodies united with dyed particles that bind to the amplicons, which migrate to the detection zone (Koczula & Gallotta 2016). The detection zone consists of the test line and control line. Koczula &

Gallotta (2016) mention that the biological components that

(23)

bound amplicons are captured by the biological components in the test line, a positive result is seen and the excess is captured by the control line (see Figure 5) (Koczula & Gallotta 2016, Lobato &

O’Sullivan 2018). A negative result is obtained if only the control line responds (Koczula & Gallotta 2016). The authors state that the absorbent pad is used for stopping the solution from flowing back as well as making sure that the solution flows by capillary force.

The following is an example of how the labeled amplicons used in a lateral flow strip are produced when using RPA. A probe between 46-52 nucleotides and nfo endonuclease IV are added to the RPA process (Lobato & O’Sullivan 2018, Zhang et al. 2020). One of the primers normally used in the process is now labeled with biotin and is in the reverse direction while the other primer used is unlabeled and in the same forward direction as the probe. The probe has an antigenic label at the 5’

end, for instance FITC (Zhang et al. 2020), and a blocking group at the 3’ end that prevents DNA polymerase elongation (Lobato & O’Sullivan 2018). A dSpacer is also found in the probe. It is an abasic site, meaning it has no base, and the dSpacer replaces one nucleotide in the probe

complementary to one nucleotide in the target sequence (Lee et al. 2015, Lobato & O’Sullivan 2018).

The probe hybridizes to the target (Zhang et al. 2020), which leads to a split of the dSpacer by the nfo endonuclease IV (Lobato & O’Sullivan 2018). This results in the release of the blocking group, followed by the alteration of the probe to a primer instead (Lobato & O’Sullivan 2018). The newly created primer and the other primers produce double labeled amplicons by DNA polymerase. These amplicons can be detected by antibodies in a lateral flow strip.

7.2. Turbidimetry

Another detection method is using turbidimetry (Zatti et al. 2019). It is a measurement on how cloudy a solution is by analyzing the amount of light scatter (Marmer & Hurtubise 1996). The instrument used for this is a photometer or a spectrophotometer. The authors mention that the reagents are added into different chambers and centrifuged, followed by the measurement of the control and sample cuvettes after the reagents have mixed. A detector collects the light beam and measures the absorption signal.

7.3. Hybridization protection assay

One strategy for detecting amplicons is by using a hybridization protection assay (Langabeer et al.

2002, Hofmann et al. 2005). In the hybridization protection assay, the amplicons are detected by using sequence specific oligonucleotide probes with a chemiluminescent acridinium ester tag (Langabeer et al. 2002). The probes hybridize with the amplicons and a luminometer is used for measuring the signal from the resulting probe-amplicon complexes.

7.4. Fluorescence

Fluorescence is commonly used when wanting to visualize results and involves the emission of light by a fluorophore (Marshall & Johnsen 2017). The fluorophore absorbs light with shorter wavelengths.

To detect amplicons, primers or probes with fluorescence can be used (Kurata et al. 2001).

Fluorometer, laser scanner (Lobato & O’Sullivan 2018), and fluorescence spectrometer (Kurata et al.

2001) are instruments that can be used for obtaining the results. Different dyes can also be used, such as Eva Green or SYBR green (Barreda-García et al. 2018, Lobato & O’Sullivan 2018, Zatti et al.

2019), and can be visualized by using a white lamp or an UV lamp (Zatti et al. 2019).

(24)

7.5. Bridge flocculation

Bridge flocculation assay can also be used to detect amplicons (Lobato & O’Sullivan 2018).

Flocculation is a term that describes components that are clustered together (Masojídek & Torzillo 2014). The first step is the addition of a bead solution to the amplicons (Lobato & O’Sullivan 2018).

Thereafter, the beads are rinsed with ethanol and added in a buffer with low pH for re-suspension. The beads are still flocculated if the result is positive and conversely a negative result indicates that they are not.

7.6. Gel electrophoresis

Gel electrophoresis is another method used for visualizing the results (Pfeffer et al. 1995). DNA is negatively charged, which this method makes use of (Carter & Shieh 2010a). The authors state that the DNA is loaded into a well, followed by migration through a gel toward the positively charged side.

Smaller DNA fragments migrate faster over the gel, so this method detects fragments based on their size. The fragments are compared and determined with the help of a DNA ladder, which uses known fragments as a reference point. Gel electrophoresis methods are however cumbersome and time consuming (Wang J et al. 2016, Yang Q et al. 2018, Yang Q et al. 2020).

8. Database and interface - tracking the species

A database would require an easy to use input system that takes in all the metadata, as stated in the background section (Bahls 2014, Kissling et al. 2018, Baker et al. 2019). Standardizing the metadata may be an important part of constructing a database (Kissling et al. 2018, Baker et al. 2019). By making the data uniform it becomes more comparable. One suggestion for such a standardization is using three dimensions in building a useful database: space, time, and taxa (Kissling et al. 2018). In other words the database must contain when and where each sample was taken (coordinates) and what species was detected. For each of the dimensions the article states that there needs to be an extent, a resolution, and a unit. The unit is important for comparing measurements, but in this project the unit should be uniform. With resolution the author means the accuracy of the measurement. In spatial terms this can refer to the study area, in temporal periodicity, and in taxa the taxonomic level (for example species, subspecies, etc.) detected. In taxa the extent is the amount of species detected, in temporal and spatial dimensions it is the timeframe and area covered.

For an example of a database concerning soil, see the LUCAS project (Land Use/Cover Area frame statistical Survey Soil) (Orgiazzi et al. 2018). The project investigates topsoil and has built up a database of several properties (originally physicochemical, but now microbial data is also measured) from sites across the entire European Union. The authors state that a map could be a powerful tool to visualize the data. Kissling et al. (2018) also suggest using maps to visualize the results, as well as graphs and tables.

The study will also need to evaluate the accuracy of the reports. Statistical methods are therefore needed (Kissling et al. 2018, Baker et al. 2019). Earlier user history might be used to evaluate the data too (Baker et al. 2019). Users that submit bad data would automatically be less trusted by the system.

However, this might also lead to concerns surrounding privacy.

(25)

9. Discussion

9.1. Selection of species of interest - Phytophthora

As stated earlier, in 1.2. and 2.2.1, the biodiversity mostly affects soil health through complex interactions involving several parties. In most cases it is hard to pin down a single responsible

organism. The device may only be able to identify key species and will at first most likely not be able to differentiate specific strains. This focus is due to technical limitations in application of the

identification methods to citizen science in the field, rather than any single species being especially important. The results from this study could be used as a foundation for further studies. The device could at a later date be adapted to identify other species or identify organisms on a lower taxonomic level such as subspecies or strain level.

As the device to be designed is meant for a field study and meant for citizen science, no cumbersome and expensive detection equipment is recommended. It may therefore only be possible to detect the existence of a species, as measuring the amount may require more advanced analysis equipment.

There are already isothermic devices for detecting microbe presence inside plant material, but detecting pathogens in soil would give an earlier warning before they establish infection.

Phytophthora is one potential target. It has recently been detected in southern Sweden and is

speculated to spread northward. As we do not know if Phytophthora spp. has spread to Uppsala yet it could be an interesting target for the study. Especially since sources state that it is more pathogenic toward plants when spread to new environments and that it is spreading northward. As it accumulates around plants, testing the soil could give a warning before infection symptoms occur. Phytophthora may be of interest to the public as it has been known to infect trees in parks and gardens. Simply proving the presence of the pathogen would be helpful in tracking the spread in Sweden.

Since S. sclerotiorum , another potential pathogenic target, is widely spread and infects crops, it could be of interest for the general public to know if it is present in their soil. A farmer could for example then know if their crops are at risk of being infected. However, since not all straints are pathogenic and some pathogens only seemingly are pathogenic to certain plants, simply proving the species existence does not guarantee infection.

The beneficial microbes, such as Pseudomonas and mycorrhizal fungi, are interesting and extremely important for plant life. Plants obviously benefit from the microbiome since they are willing to sacrifice much of their photosynthetic product in exchange for microbial services. Plants may even be selecting for specific microbes to fulfill certain needs, but the interactions often require more parties.

Simply proving the presence may not prove the benefits since the root associated microbes often are the same as in bulk soil. What benefits for example Pseudomonas gives varies drastically depending on subspecies and strain. With the constraints of the project this may be hard to detect.

Therefore we propose using the project to search for pathogens like Phytophthora. Although the primer design may be more complicated due to not all variants having annotated genomes, it fits the format of the study the best. Simply proving whether or not the species exists is of interest since it is not usually native to Uppsala.

(26)

9.2. Selection of amplification method - RPA

While metagenomics is a very good method for investigating the biodiversity in a sample it has several factors that makes it less suitable for a citizen science application, in the way sought after by this project. It would have been very good if it was usable, as metagenomics is capable of reconstructing the total genome in a sample, of both known and unknown species. However, the requirements for a metagenomic analysis means it is less usable in the context of this project. The first factor is

extraction, where the quality must be assessed, requiring a spectrophotometer. Then it requires equipment such as a vortex and centrifuge. All of these are expensive and quite large pieces of equipment, meaning they are unsuitable to a citizen science project with limited resources and that would be mostly carried out in a field setting. Similarly, the sequencing requires some kind of sequencer. While there are devices available today that are portable, all the sequencing technologies are still expensive when considering the likely quite limited budgets any participant would have available. There is also the issue of interpretation, as the resulting data might require a trained person to get useful information out of it. Overall, metagenomics is a great tool for investigating biodiversity, just not in a citizen science project performed in the field.

In situ hybridization is not of particular interest to us. This method takes a lot of time to perform, at temperatures that are harder to maintain in a field setting. It also involves more steps in the process than many of the other methods, which makes it potentially harder to perform, especially for untrained persons. One advantage the method does have however is that cells do not need to be lysed before it can be performed.

As opposed to this, many isothermal amplification methods do not require extensive training nor relatively speaking expensive equipment, and would likely be suitable for a citizen science application.

Providing citizen scientists with the necessary reagents and a way to obtain samples would allow them to quickly and easily identify whether a given species is present in an environment.

RPA is the main method of interest. While many of the isothermal amplification methods are

potentially applicable to citizen science, RPA is of particular interest due to its characteristics. Namely, it is performable at temperatures that facilitate amplification at room or body temperature. The lack of denaturation step also simplifies the application, as this eliminates the need of an initial heating step to a higher temperature, which some other methods require. The fact that it only requires two primers is also a positive aspect, as primer design can be complicated and potentially expensive. Thus having the smallest number of primers possible is preferable. RPA is also a very sensitive method, being able to amplify very small concentrations of the target. It is also able to amplify either DNA or RNA, depending on the needs of the specific amplification. The reagents may be stored in a pellet,

simplifying use in the field. Lastly there is the short reaction time, which makes it more suitable for a field application, since the logistics of spending a lot of time at a sampling site might be detrimental to a method’s practicality. There is the issue of price, as RPA kits are quite expensive when bought from the manufacturer, but this is an issue that applies for all isothermal amplification methods unless reagents are bought separately. In most cases this may not be possible since the methods are intellectual properties of companies.

LAMP has some good properties, but might be unsuitable as compared to RPA. The temperature required is harder to maintain in a field setting, and although this problem has been investigated and solved by other authors, the solution requires the construction of a more complex device involving

References

Related documents

The increasing availability of data and attention to services has increased the understanding of the contribution of services to innovation and productivity in

Generella styrmedel kan ha varit mindre verksamma än man har trott De generella styrmedlen, till skillnad från de specifika styrmedlen, har kommit att användas i större

Parallellmarknader innebär dock inte en drivkraft för en grön omställning Ökad andel direktförsäljning räddar många lokala producenter och kan tyckas utgöra en drivkraft

Närmare 90 procent av de statliga medlen (intäkter och utgifter) för näringslivets klimatomställning går till generella styrmedel, det vill säga styrmedel som påverkar

I dag uppgår denna del av befolkningen till knappt 4 200 personer och år 2030 beräknas det finnas drygt 4 800 personer i Gällivare kommun som är 65 år eller äldre i

Generell rådgivning, såsom det är definierat i den här rapporten, har flera likheter med utbildning. Dessa likheter är speciellt tydliga inom starta- och drivasegmentet, vilket

Den förbättrade tillgängligheten berör framför allt boende i områden med en mycket hög eller hög tillgänglighet till tätorter, men även antalet personer med längre än

Detta projekt utvecklar policymixen för strategin Smart industri (Näringsdepartementet, 2016a). En av anledningarna till en stark avgränsning är att analysen bygger på djupa