Application of recombinase polymerase amplification to diagnosis of phytophthora diseases of strawberry

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Dissertations in Forestry and Natural Sciences

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND

MUSTAFA AHMAD MUNAWAR

Application of recombinase

polymerase amplification to

diagnosis of Phytophthora

diseases of strawberry

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APPLICATION OF RECOMBINASE POLYMERASE AMPLIFICATION TO

DIAGNOSIS OF PHYTOPHTHORA

DISEASES OF STRAWBERRY

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Mustafa Ahmad Munawar

APPLICATION OF RECOMBINASE POLYMERASE AMPLIFICATION TO

DIAGNOSIS OF PHYTOPHTHORA DISEASES OF STRAWBERRY

Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences

416

University of Eastern Finland Kuopio

2021

Academic dissertation

To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium SN201 in Snellmania Building at the University of Eastern Finland, Kuopio, on January,15, 2021 at 12

o’clock noon.

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Grano Oy Jyväskylä, 2021

Editors: Pertti Pasanen, Matti Vornanen, Jukka Tuomela, Matti Tedre

Distribution: University of Eastern Finland / Sales of publications www.uef.fi/kirjasto

ISBN: 978-952-61-3684-4 (nid.) ISBN: 978-952-61-3685-1 (PDF)

ISSNL: 1798-5668 ISSN: 1798-5668 ISSN: 1798-5676 (PDF)

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Author’s address: Mustafa Ahmad Munawar University of Eastern Finland

Depart. of Environmental and Biological Sciences P.O. Box 1627

70211 Kuopio, Finland

email: mustafm@student.uef.fi

Supervisors: Professor Elina Oksanen, Ph.D.

University of Eastern Finland

70211 Kuopio, Finland email:elina.oksanen@uef.fi Frank N. Martin, Ph.D.

Agricultural Research Service of United States Department of Agriculture, Salinas, USA email: frank.martin@usda.gov

Harri Kokko, M.Sc. (technical supervisor) University of Eastern Finland

70211 Kuopio, Finland email: harri.kokko@uef.fi

Reviewers: Peter Bonants, Ph.D

Wageningen University & Research

Business Unit Biointeractions & Plant Health P.O. Box 16

6700 AA Wageningen, The Netherlands email: peter.bonants@wur.nl

Hannele Lindqvist-Kreuze, PhD

Genetics Genomics and Crop Improvement International Potato Center (CIP)

Apartado 1558, Lima 12, Peru email: h.lindqvist-kreuze@cgiar.org

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Opponent: Assoc. Prof. Timo Hytönen, PhD University of Helsinki

Department of Agricultural Sciences PL 27 (Latokartanonkaari 7)

00014 Finland

email: timo.hytonen@helsinki.fi

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7 Munawar, Mustafa Ahmad

Application of recombinase polymerase amplification to diagnosis of Phytophthora diseases of strawberry

Kuopio: University of Eastern Finland, 2021 Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences 2021; 416 ISBN: 978-952-61-3684-4 (print)

ISSNL: 1798-5668 ISSN: 1798-5668

ISBN: 978-952-61-3685-1 (PDF) ISSN: 1798-5676 (PDF)

ABSTRACT

Phytophthora is an increasing threat to agriculture and natural ecosys- tems. The Irish potato famine of the mid-1800s which lead to the death of a million humans in Europe and the recent decline of oak forest in California and kauri forests in New Zealand were all caused by Phytophthora species. Similarly, strawberry crop also suffers heavy losses from Phytophthora fragariae and P. cactorum. Projects aiming at the eradication of Phytophthora have remained largely unsuccessful, however, projects aiming at the prevention of the Phytophthora spread through blocking initial introduction have the highest potential for success. The aim of this dissertation is to develop rapid detection means for strawberry Phytophthora pathogens, which can assist in stopping the spread of the pathogens, utilizing recombinase polymerase amplification (RPA) technology. RPA is a rapid isothermal nucleic acid amplification technology that enables the amplification of target nucleic acid directly from crudely macerated plant tissues, excluding the need for laborious plant DNA extraction. Being isothermal, RPA can be done with a small portable device on-site and does not require expensive thermocyclers.

In this dissertation, we have presented the devolvement of rapid real- time RPA assays with TwistAmp exo kit for P. cactorum (chapter 2) and P. fragariae (chapter 3) utilizing the Phytophthora genus specific identification marker atp9-nad9. Both assays detected as low as 10 fg Phy- tophthora genomic DNA with reaction turnaround time less than 30 minutes. The dissertation also presents a combination of RPA and PCR technologies to expedite plant diagnostics and overcome amplification

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inhibitors (chapter 4). The combination of technologies ‘RPA-PCR couple’ is about first doing an RPA (TwistAmp Liquid Basic) at slower reaction kinetics to amplify the target and some flanking region directly from crude plant macerate and then transferring RPA reaction to PCR reaction for specific and exponential amplification of the target. The RPA-PCR couple overcomes the limitations of both the traditional diagnostic method of plant DNA extraction followed by PCR, and RPA. The traditional PCR diagnostic method includes the laborious process of DNA extraction and sometimes the extraction brings PCR inhibitor into the samples. In contrast, RPA has a limitation of target size (100–200 bp optimal) and also generates background noise that sometimes compli- cate the purification of the target product. We applied the RPA-PCR couple method to Phytophthora pathogens of strawberry with the atp9- nad9 marker and found the method DNA-extraction free, reliable, spe- cific, sensitive down to 10 fg Phytophthora genomic DNA, capable of amplifying longer targets, and time-saving (compared to the traditional method). We expect the RPA-PCR couple concept may have vast applications, especially for amplifying targets from recalcitrant samples such as ancient samples, woody plants, or food.

Universal Decimal Classification: 577.113, 581.2, 582.639.13, 632.4, 634.75 CAB Thesaurus: plant pathogens; plant diseases; strawberries; Oomycetes;

Phytophthora; Phytophthora cactorum; Phytophthora fragariae; detection; di- agnosis; diagnostic techniques; assays; DNA; DNA amplification; polymerase chain reaction; strawberry red stele root rot

Yleinen suomalainen ontologia: kasvitaudit; mansikat; tyvimätä; tunnistami- nen; määritys; diagnoosi; diagnostiikka; DNA; DNA-analyysi; polymeraasiket- jureaktio

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ACKNOWLEDGEMENTS

Most of this work was carried out in the Department of Environmental and Biological Sciences at the University of Eastern Finland (UEF), Kuopio, Finland. About 80% of the bench and field work and around 30% of the writing work was completed through employment (April 2016 to Dec 2018) in the UEF project ‘Tauti voi ei!’, funded by Euroopan maaseudun kehittämisen maatalousrahasto (European Agricultural Fund for Rural Development/ EAFRD), Pohjois-Savon ELY-Keskus, while rest of the bench and writing work was completed afterward.

I hereby sincerely thank every contribution to this work. I am extremely grateful to the Finnish Cultural Foundation and the Olvi Foundation, Finland for providing working grants for the completion of this disseration. Moreover, I am genuinely indebted to the welfare institutes of Finland, Employment and Economic Development Office (TE toimisto) and Unemployment Fund for Higher Educated Employees (ERKO), for allowing me to continue my Ph.D. as part-time during the job search period. And finally, I dedicate my dissertation to Abdul Sattar Edhi of Pakistan, though this dedication is insignificant in comparison to what he achieved.

Kuopio, January 2021 Mustafa Ahmad Munawar

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LIST OF ABBREVIATIONS

3SR self sustained sequence replication

ATP adenosine triphosphate

BHQ black hole quencher

bp base pairs

BSA bovine serum albumin

CABI Centre for Agriculture and Biosciences International

DIAPOPS detection of immobilized amplified product in a one-phase system

DNA deoxyribonucleic acid

dsDNA double stranded DNA

DTT dithiothreitol

dTTP deoxythymidine triphosphate

dUTP deoxyuridine triphosphate

ELISA enzyme-linked immunoassay

ELY-Keskus Centre for Economic Development, Transport and the Environment

EPPO European and Mediterranean Plant

Protection Organization

EVIRA Finnish Food Safety Authority

EAFRD European Agricultural Fund for Rural Development

GEB general extract buffer

HDA helicase-dependent amplification

ILVO Institute for Agricultural, Fisheries and Food Research

IO invasion oligonucleotide

ITS internal transcribed spacer

JKI Julius Kühn-Institut

LAMP loop-mediated isothermal amplification LUKE Natural Resources Institute Finland NASBA nucleic acid sequence based amplification

NCBI National Center for Biotechnology

Information

NEAR nicking enzyme amplification reaction

NTC no template control

PCR polymerase chain reaction

PVP polyvinylpyrrolidone

RCA rolling circle amplification

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11 RPA recombinase polymerase amplification

SDA strand displacement amplification SIBA strand invasion based amplification

SNP single nucleotide polymorphism

SPIA single primer isothermal amplification

SSB single-stranded DNA binding

ssDNA single stranded DNA

TE buffer Tris-EDTA buffer

TEG triethyleneglycol

THF tetrahydrofuran

TMA transcription mediated amplification

UDG uracil DNA deglycosylase

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LIST OF ORIGINAL PUBLICATIONS

This thesis is based on data presented in the following articles, referred to by the chapter numbers 2-4.

2) Munawar M., Toljamo A., Martin F. & Kokko H. 2019. Recombinase polymerase amplification assay for fast, sensitive and on-site detection of Phytophthora cactorum without DNA extraction. European Journal of Horticultural Science 84: 14-19. https://doi.org/10.17660/eJHS.2019/84.1.2 3) Munawar M., Toljamo A., Martin F., Oksanen E. & Kokko H. 2020a.

Development and evaluation of a recombinase polymerase amplification assay for rapid detection of strawberry red stele pathogen. Phyto- pathology Research 2: 1-12. https://doi.org/10.1186/s42483-020-00069-4 4) Munawar M.A., Martin F., Toljamo A., Kokko H. & Oksanen E. 2020b.

RPA-PCR couple: an approach to expedite plant diagnostics and overcome PCR inhibitors. BioTechniques 69. https://doi.org/10.2144/btn-2020- 0065

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AUTRHOR’S CONTRIBUTION

2) The design of the RPA assay was conceived by MA Munawar. MA Munawar also designed the experiments and conducted most of the experiments including Phytophthora isolations, DNA extractions, PCR assays, crude macerations, and RPA assay optimization. MA Munawar analyzed results/ data and wrote the manuscript. MA Munawar actively contributed to the collection of strawberry field plants.

3) The design of the RPA assay was conceived by MA Munawar. MA Munawar also designed the experiments and conducted most of the experiments including Phytophthora isolations, DNA extractions, PCR assays, crude macerations, RPA assay optimization, field validation, and baiting experiments. MA Munawar analyzed results/ data and wrote the manuscript. MA Munawar actively contributed to the collection of strawberry field plants and soil samples.

4) MA Munawar conceived the initial design of the experiments. MA Munawar also designed the RPA assay and SYBR Green PCR assays. MA Munawar conducted most of the experiments including optimization of the RPA-PCR couple, optimization of PCR assays, and field validation of the RPA-PCR couple. MA Munawar also analyzed results/ data and wrote the manuscript.

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1 GENERAL INTRODUCTION

1.1 PHYTOPHTHORA, AN AGGRESSIVE PLANT DE- STROYER

1.1.1 Discovery of the devastating pathogen

Phytophthora is a Greek word meaning plant destroyer. Phytophthora species have caused substantial damage to crops and natural ecosystems. The Irish potato famine of mid-1800s and the recent decline of oak forest in California and kauri forests in New Zealand were caused by a Phytophthora species. Phytophthora pathogens were unknown until the Irish potato famine in 1845 and 1846. The potato famine led to displacement and starvation of two million inhabitants of Ireland. Moreover, the spread of potato blight across Europe starved millions of people to death. Many scientists and even an expert panel of those times believed the excessive inbreeding or atmospheric influences causing the crop losses, while the idea of a fungus causing blight was widely unpopular. In 1845, David Moore, director of Royal Dublin Society's Botanic Garden in Glasnevin, Dublin, investigated the problem in potatoes and proposed a fungus behind the devastating problem.

Moore also communicated several years with Reverend Berkeley, a noted amateur mycologist, who was seeking confirmation that a fungus was causing the blight. In 1846, Berkeley’s publication formally identified the fungus as the causative agent of blight, but the idea was dismissed until more reports confirmed Berkeley’s findings. The fungus was initially given different names including Botrytis infestans and Peronospora trifurcata, but Anton de Bary in 1876 renamed the fungus as Phytophthora infestans and proposed a new genus. The genus Phytophthora was subsequently expanded with Phytophthora cactorum, Phytophthora nicotianae, Phytophthora phaseoli, and Phytophthora colocasiae until 1900 (Ribeiro, 2013).

1.1.2 Biology of Phytophthora

Phytophthora is a soil-born plant pathogen and 182 Phytophthora species have been described as of May 2018 (Abad et al., 2019). Phytophthora species greatly vary in host range. For example, Phytophthora rubi only infects red raspberry, while P. cinnamomi can damage over a thousand host plant species. Phytophthora and other oomycetes have long been considered fungi due to some of their similarities with fungi. Both oomycetes and true fungi are composed of mycelium in vegetative

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phase and they both reproduce through spores and acquire nutrients through absorption. Moreover, both oomycetes and true fungi are eukaryotic and heterotrophic. Besides some similarities, several biochemical and structural differences between oomycetes and true fungi (Eumycota) have been observed with time and therefore, oomycetes are currently classified as a distinct group. Oomycetes cell wall is composed of cellulose whereas true fungi cell wall is usually composed of chitin. Oomycetes mycelium is diploid, but mycelium of true fungi is haploid or dikaryotic. In sexual reproduction, all oomycetes produce oospores, but true fungi produce zygospores, ascospores or basidiospores. Oomycetes have tubular cristae in mitochondria whereas true fungi have mitochondria with flattened cristae. Oomycetes produce motile zoospores with two types of flagella, a posterior whiplash flagellum and an anterior tinsel flagellum with hairlike projection, whereas most true fungi are non-motile except some Chytridimycota fungi that produce motile zoospores with single posterior whiplash flagellum (Rossman and Palm, 2006; Ho, 2009).

The life cycle of the genus has asexual and sexual parts. In asexual reproduction, sac-like structures called sporangia appear on dedicated hypha called sporangiophores. Sporangia can either germinate on the host surface by forming a germ tube or release of 8-32 swimming zoospores. Zoospores either actively move towards the host plant (chemotaxis) or passively disperse with the flow of water. Once a zoospore reaches the host plant surface, the zoospore encysts and germinates through a germ tube. Some Phytophthora species produce another type of asexual spores termed as chlamydospores.

Chlamydospores are relatively larger and usually globose shaped.

Chlamydospores are thick-walled and therefore survive over a long period in the decayed plant or soil (Drenth and Sendall, 2001; Heffer- Link et al., 2002).

In sexual reproduction, a large round female gametangium, termed as oogonium, is fertilized by a smaller male gametangium known as antheridium. The fertilization results in a thick-walled zygote termed as oospore. Alike chlamydospores, oospores are dormant spores and germinate once conditions are favorable. All Phytophthora species produce male and female gametangia and species are either homothallic or heterothallic. Homothallic species can quickly and abundantly produce oospores within a single culture or strain. In contrast, heterothallic species require two different strains of A1 and A2 mating types for fertilization and oospores formation. The A1 and A2 mating type strains produce mating hormones α1 and α2 respectively (Drenth and Sendall, 2001; Heffer-Link et al., 2002).

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19 1.1.3 Classification and Identification

Phytophthora is currently placed in family Peronosporaceae within the order of Peronosporales and class Oomycota. Phytophthora genus and species are detected by microscopy, culturing from infected tissue, baiting, immunoassays, PCR and isothermal nucleic acid amplification technologies. Before the era of molecular techniques, Phytophthora species were identified and classified mainly through morphological characteristics. The widely adopted classification of Waterhouse (1963) divided the genus into six groups based on the three forms of sporangia papillation and two types of antheridial attachments. Sporangia in Phytophthora species are papillate, semi-papillate, or non-papillate.

Similarly, antheridia have two kinds of attachments with oogonia:

paragynous and amphigynous arrangement. In paragynous arrangement, the antheridium is located at one side of oogonium, while in amphigynous arrangement, oogonium grows through antheridium forming a base collar of antheridium around the oogonium. Besides Waterhouse criteria, other morphological features have been utilized to distinguish Phytophthora species such as the morphology of sporangium, oogonium, and colony; the presence of chlamydospores and hyphal swellings; optimum temperature for growth; and host range. Moreover, physiological characteristics have been utilized to identify Phytophthora species such as differential growth under malachite green, temperature- growth relations, and isozyme analysis (Kroon et al., 2012). With the availability of molecular techniques, molecular phylogeny is being utilized to classify Phytophthora into clades. Based on evolutionary relationships, Phytophthora genus is currently classified into 10 clades (Blair et al., 2008; Martin et al., 2014; Yang et al., 2017a). Moreover, large scale phylogenetic analyses have uncovered close relations between Phytophthora and Downy Mildews (Thines, 2013).

1.1.4 Phytophthora Pathogens of Strawberry

Strawberry is an important economic crop all over the world and it suffers losses from Phytophthora species. Phytophthora fragariae and P.

cactorum are the commonly reported Phytophthora pathogens of strawberry. Both are among the most destructive pathogens of strawberry crop and they can cause heavy losses. P. fragariae causes red stele disease while P. cactorum causes crown rot and leather rot. These diseases are presented in detail in chapter second and third of this dissertation. Besides P. fragariae and P. cactorum, Phytophthora citricola, P.

citrophthora, P. cryptogea, P. megasperma, P. nicotianae and P. bisheria have also been isolated from strawberry plants in different countries (Abad et al.. 2008).

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P. fragariae is homothallic with paragynous or amphigynous antheridia. Antheridia length ranges from 16 to 30 µm and width ranges from 12 to 22 µm. Oogonia diameter ranges from 28 to 46 µm, often globose with a tapered base. Oospores spherical, ellipsoidal, or irregular shaped and their diameter ranges from 24 to 44 µm. Oospores are common in the host but rare in cultures. Sporangia are non-papillate and measure 28 to 56 µm in length and 27 to 49 µm in width. P. fragariae does not produce chlamydospores. P. fragariae optimum growth temperature is 18 ⁰C, while the maximum temperature for growth is 27

0C and the minimum is 3 ⁰C (Ristaino 2012; Abad et al., 2019).

P. cactorum is homothallic with paragynous antheridia (Ristaino 2012;

Abad et al., 2019). Oogonia are spherical or tapered base shaped with a diameter varying between 19 and 38 µm. Oospore diameter varies between 20 and 26 µm, and its wall is colourless with an average of 2 µm thickness. Sporangia are papillate and their shape can be ellipsoid, spherical, ovoid or obpyriform. Sporangia length ranges from 24 to 50 µm and width ranges from 19 to 36 µm. Some isolates of P. cactorum produce chlamydospores of 17–55 µm diameter. Optimum growth temperature for P. cactorum is 24 ⁰C, while the minimum temperature for growth is 4 ⁰C and the maximum is 30 ⁰C (Ristaino 2012; Abad et al., 2019).

1.2 ISOTHERMAL NUCLEIC ACID AMPLIFICATION TECHNOLOGIES

Nucleic acid amplification has become an important tool in many fields of biology since Kary Mullis developed polymerase chain reaction (PCR) in 1983 (Mullis et al., 1986). Modern PCR technology is about amplifying nucleic acid through a heat stable polymerase. The method relies on the melting/ annealing property of nucleic acid primers and template. PCR comprises cycles of three temperature distinct steps: (1)

‘denaturation’ or melting of nucleic acid at around 95 ⁰C, (2) primers

‘annealing’ with the template at about 55 ⁰C, and (3) polymerase mediated ‘extension’ of primers around 70 ⁰C. Following PCR development, several isothermal nucleic acid amplification methods have been conceived. Isothermal amplification is about amplifying nucleic acid at a constant temperature. These methods exclude the need of thermocycler and require simpler instrumentation. Like PCR, these methods rely on polymerases to amplify target, but they adopt different means to unwind double stranded DNA such as restriction enzymes, nicking enzymes, helicases, recombinase, or primers with loop forming

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21 property. Isothermal methods displace newly synthesized strands

mostly through the strand displacement activity of polymerases.

Common isothermal methods are briefly described here.

Isothermal methods of self sustained sequence replication (3SR) (Guatelli et al., 1990) and nucleic acid sequence based amplification (NASBA) (Compton, 1991) are identical methods mimicking retroviral replication. These methods recruit three enzymes: reverse transcriptase, RNase H, and RNA polymerase. In the amplification process, the first primer binds with a target RNA template and reverse transcriptase converts RNA into RNA-cDNA duplex. Following duplex formation, RNA in the duplex is degraded by RNase H. Then the second primer binds the cDNA and reverse transcriptase generates a DNA duplex.

Moreover, either one or both primers are provided with promoter sequence at 5’ ends to include promoter regions in the DNA duplex.

RNA polymerase binds double stranded promoter region in DNA duplex and transcribes copies of RNA. The RNA copies reenter amplification cycles and thereby generating millions of RNA copies.

Isothermal technique transcription mediated amplification (TMA), invented by Kacian and Fultz (1996), also mimics retroviral replication but requires only two enzymes. TMA excludes RNase H enzyme and relies on RNase activity of the reverse transcriptase enzyme (Langabeer et al., 2002).

Rolling circle amplification (RCA) technology, first developed by Fire and Xu (1995), targets single stranded circular DNA. A primer anneals to the circular DNA and a polymerase extends the primer. When polymerase extension reaches the starting point, strand displacement activity of polymerase displaces the newly formed strand. The simultaneous extension and displacement activity of polymerase continues synthesizing single stranded DNA until exhaustion the amplification system. The system generates long tandem repeats in order of kilobase or even larger.

Strand displacement amplification (SDA) method was developed by Walker et al. (1992). The original SDA method requires an initial heat denaturation of nucleic acid, and the components of polymerase with strand displacement activity, restriction enzyme HincII, and forward and reverse primers added with HincII recognition sequence (5'- GTTGAC-3') at 5’ ends. Moreover, the SDA reaction is provided with nucleotides dGTP, dCTP, TTP, and deoxyadenosine 5’-(α-thio) triphosphate. Polymerase extension of primers generates double stranded hemiphosphorothioate recognition sites which comprise one strand with deoxyadenosine 5’-(α-thio) triphosphate incorporated and the other strand with normal adenosine nucleotides. In the double

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stranded hemiphosphorothioate site, HincII only nicks the strand with adenosine, while the strand synthesized from deoxyadenosine 5’-(α- thio) triphosphate remains protected. Following nicking, polymerase extends the 3’ end of the nick and displaces the downstream strand.

Extension of nick regenerates hemiphosphorothioate recognition site for HincII and the cycle repeats itself. Another related isothermal method is the nicking enzyme amplification reaction (NEAR). NEAR was invented by Van Ness et al. (2003), and it employs nicking enzyme N.BstNBI and Vent exo-polymerase. The nicking enzymes have the inherited ability to nick one strand, thereby excluding the need for hemiphosphorothioate recognition sites. The Vent exo-polymerase is an engineered polymerase lacking 3´ to 5´ proofreading exonuclease activity. In the NEAR method, following nicking, short amplicons are released from duplexes due to their low melting temperature. The method is capable of rapidly generating copies of short amplicons.

Loop-mediated isothermal amplification (LAMP), invented by Notomi et al. (2000), is a rapid, sensitive and specific method of isothermal nucleic acid amplification. LAMP reaction is incubated at 65

0C and it requires a polymerase with strand displacement activity and four primers. The primers include an inner primer and an outer primer on both forward and reverse strands. The inner primers are designed containing both sense and antisense strand sequences, and their extension generates amplicons with the self-hybridization capability.

Following extension of an inner primer by polymerase, the inner amplicon is displaced by an outer primer’s extension. The inner amplicon once released self-hybridizes into a loop at the 5’ end, while the 3’ end is annealed by a second inner primer. The extension of primers generates dumb-bell structures which are further extended into complex cauliflower-like structures. The nucleic acid amplification in LAMP is so extensive that visible white precipitates of magnesium pyrophosphate are generated. The precipitate formation can be monitored in real time through turbidimeter (Mori et al., 2001). Besides turbidity, fluorescent intercalating dyes are also utilized to monitor LAMP amplification (Oscorbin et al., 2016). Moreover, a third pair of primers, known as loop primers, have been designed to hybridize within loops and reported to significantly reduce the amplification time of LAMP (Nagamine et al., 2002).

Helicase-dependent amplification (HDA) concept was first conceived by Vincent et al. (2004) and it is about unwinding DNA double strands through helicases, single-stranded DNA binding proteins (SSB) and accessory proteins. Following DNA unwinding, primers bind the single stranded DNA in both directions and a DNA polymerase with strand

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23 displacement activity extends the primers. Primers extension generates

two DNA duplexes which are separated again by helicases. The cycle repeats itself and generates millions of amplicons.

Single primer isothermal amplification (SPIA) is a unique method invented by Kurn et al. (2005). SPIA initiates with one chimeric primer which has a 5’ overhang RNA and a 3’ DNA portion. DNA portion of the chimeric primer hybridizes with target RNA, while reverse transcriptase extends the chimeric primer forming a cDNA strand with a 5’ end RNA overhang. The SPIA reaction is then heated to partially degrade RNA template and denature reverse transcriptase. Next, in the second phase of SPIA, RNA dependent DNA polymerase is supplied to the reaction. The polymerase utilizes the 5’ RNA portion of the cDNA to form a double-stranded cDNA with an RNA/DNA heteroduplex end.

The heteroduplex is the crucial structure for amplifying DNA copies. In the third phase of SPIA, the reaction is supplemented with another chimeric primer, RNase H and DNA polymerase with strand displacement activity. RNase H cleaves the RNA part in the heteroduplex leaving space for the second chimeric primer to hybridize.

The second chimeric primer is designed to fully hybridize with duplex.

Extension of the primer with DNA polymerase displaces the downstream strand and regenerates a double strand DNA structure with an RNA/DNA heteroduplex end. The RNA in the heteroduplex is cleaved again by RNase H and a new primer hybridizes to repeat extension and strand displacement steps. SPIA can amplify both RNA and DNA molecules.

Recombinase polymerase amplification (RPA) technology was invented Piepenburg et al. (2006), and as its name indicates, it is about amplification through recombinase and polymerase enzymes.

Recombinase enzymes bind with long primers of 30 to 35 bases length, forming nucleoprotein complexes. The nucleoprotein complexes scan nucleic acid for homologous sequences and the strand exchange activity results in D-loop structures which is further stabilized by SSB protein gp32. In D-loop structures, recombinase dissociates from primers and allows polymerase to extend the primers. Extension of primers by polymerase with strand displacement activity in both directions leads to exponential amplification of the target nucleic acid.

The global size of the isothermal amplification technologies market was recorded at 1.6 billion USD for the year 2016 (Li et al., 2019; Grand View Research, 2017). The major technologies contributed isothermal nucleic acid market include TMA (16.84%), LAMP (14.45%), HDA (12.08%), SDA (10.37%), NEAR (9.11%), NASBA (7.87%), SPIA (7.40%), RPA (7.08%), and RCA (6.52%), while rest of the isothermal technologies

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made 8.28% share. Although RPA is not a widely adopted method yet, it is rapidly occupying the market. Around four hundred peer reviewed RPA articles have been published.

1.3 RECOMBINASE POLYMERASE AMPLIFICATION

1.3.1 RPA Reaction Insight

In RPA, recombinase enzyme T4 UvsX cooperatively binds with forward and reverse RPA primers in the presence of ATPs and forms nucleoprotein complexes. A nucleoprotein complex scans nucleic acid to find a homologous sequence for strand exchange activity. Once a homologous sequence is found it is displaced by the nucleoprotein complex, forming an intermediate D-loop structure. The displaced strands in the loop are covered with single-stranded binding proteins (SSB proteins) T4 gp32, stabilizing the D-loop structure. The ATP hydrolysis leads to spontaneous disassembly of the T4 UvsX-primer complex, allowing polymerase to bind and extend the 3’ end of the RPA primer. The cyclic bidirectional extension of RPA primers results in exponential DNA amplification of nucleic acid. Figure 1 represents molecular events of RPA. RPA utilizes the large fragment of Bacillus subtilis Pol I (Bsu) DNA polymerase and the polymerase has inherent strand displacing property. The RPA reaction is also supplied by ATP regeneration system comprising of creatine kinase, phosphocreatine and ATP. Robustness of the amplification is supported by crowding agent (Carbowax20M), and recombinase loading factor or recombination mediator protein T4 UvsY (Piepenburg et al., 2006). The loading factor supports UvsX recombinase in displacing T4 gp32 from single stranded DNA (ssDNA) and promotes UvsX-ssDNA assembly (Pant et al., 2008;

Xu et al., 2010). In typical RPA reactions, Piepenburg et al. (2006) included 300 nM primer each, 900 ng/µl gp32, 120 ng/µl UvsX, 30 ng/µl UvsY, 30 ng/µl Bsu, 200 µM dNTP each, 5% Carbowax20M, 2 mM Dithiothreitol (DTT) as enzymes stabilizer, 50 mM Tris (pH 7.9) as DNA stabilizer and solubilizer, 100 mM potassium acetate as another DNA stabilizer and solubilizer, 3 mM ATP, 50 mM phosphocreatine, 100 ng/µl creatine kinase, and 14 mM magnesium acetate as enzymes cofactors and reaction starter (Li et al., 2019).

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1.3.2 RPA technology development

Amplification of nucleic acid through recombinase enzyme is dated back to 1993 when Zarling et al. presented the amplification of double stranded DNA (dsDNA) through RecA protein and DNA polymerase.

Zarling utilized ATPɣS instead of ATP which is an inhibitor for recombinase-primer complex disassembly. Although the failure of spontaneous disassembly of the nucleoprotein complex hampers DNA amplification, it favours hybridization of detection probe with target sequences. Later, in 2007, Armes and Stemple replaced ATPɣS with ATP and developed an exponential method of DNA amplification know as RPA.

1.3.3 Kits formats

RPA is proprietary of TwistDx Inc. (United Kingdom) which sell a range of TwistAmp^TM research purpose kits in lyophilised (freeze-dried reaction pellets) and liquid forms. The freeze-dried kits are convenient to use and stable at ambient temperature for days. In contrast liquid kits are a bit laborious but more flexible to adjust reaction components for special needs. The currently available freeze-dried kits include TwistAmp Basic, TwistAmp exo and TwistAmp nfo, while liquid kits offered by TwistDx website include TwistAmp Liquid Basic and TwistAmp Liquid exo. TwistDx website also provides supporting documentation. Most of the RPA technical details described in this dissertation, unless mentioned, have been extracted from manufacturer manuals provided under the heading of ‘RPA assay design’ in the website (TwistDx a).

The TwistAmp Basic and TwistAmp Liquid Basic kits are straightforward endpoint RPA reaction mix provided with all components except primers and template. The end-product is detected by electrophoresis and prior to doing electrophoresis, RPA reactions are cleaned through phenol/ chloroform method or standard PCR cleaning kits.

TwistAmp exo and TwistAmp Liquid exo kits are provided with an additional feature of real-time monitoring. Beside template and primers, these kits require the addition of a special probe. The probe, TwistAmp exo probe, comprises of 46-52 nucleotide bases. The 3’ end of the probe is added with C3-spacer, phosphate, biotin-TEG or amine to block polymerase from extending the probe. In the probe, one nucleotide is replaced with a tetrahydrofuran (THF) base which is an abasic nucleotide equivalent also known as dSpacer. The THF base is located at least 15 nucleotide bases from 3’ end and 30 bases from 5’ end of the

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29 against one of the reverse primers. Then the optimal forward primer can

be screened against all the reverse primers to find the most efficient primer pair. In the second phase of primer screening, random minor modifications are applied to the optimal primer pair to achieve further efficiency. The modifications are about redesigning primers with minor relocation and length alterations.

RPA works optimally at a temperature range of 37 – 42 ⁰C. At a higher temperature, enzymes present in the reaction increasingly lose activity leading to suboptimal performance of the system. In contrast, at a lower temperature such as ambient temperature, amplification is accomplished at slower rates, and due to the limited energy clock of the reaction, less product is generated. TwistAmp exo kit, designed for rapid reaction, is particularly more vulnerable to lower temperatures, while TwistAmp Basic and TwistAmp nfo kits are comparatively more resistant to ambient temperatures.

Currently available TwistAMP kits support amplification of small amplicons, ideally of 100-200 bases. The recommended reaction conditions are suitable for most RPA assays, but the performance of some assays can be improved by altering incubation temperature, time of mixing, and the concentration of magnesium acetate (MgOAc), primers and probe. Lowering incubation temperature within the range supports the amplification of longer amplicon. Time of mixing after the reaction initiation can vary between three to seven minutes, but the standard is four minutes. Delay in mixing favors amplification of longer or slowly amplifying targets. MgOAc should vary from 12-30mM, while optimal concentration is 14mM. MgOAc plays the role of RPA reaction starter and lowering its concentration slows RPA reaction kinetics and vice versa. Therefore, longer amplicons can benefit from lowering MgOAc concentration. Similarly, each primer concentration should be from 150-600 nM, while the probe concentration between 50 – 150 nM.

For basic kits, the recommended concentration for each primer is 480 nM, while for exo and nfo type of kits, each primer concentration is recommended at 420 nM and probe at 120 nM. Altering proportion of forward and reverse primer can benefit some RPA assays, however the total oligonucleotide concentration should be within range of 750- 2000nM. Lowering oligonucleotide concentration also promotes amplification of the longer targets.

1.3.5 Pros and cons

RPA is rapid with reaction turnaround time less than 30 minutes. RPA is also tolerant to amplification inhibitors. RPA can amplify target from simple plant tissue macerates, excluding the need of laborious plant

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30

DNA extraction. RPA does not require a thermocycler and it can be done with portable inexpensive instruments at field sites or remote areas. Most of the RPA assays designed have opted for RPA exo and nfo kits, while some assays were also designed with RPA basic kits. The RPA exo kits (TwistAmp exo or TwistAmp Liquid exo) assays are real time and require an instrument with incubator and fluorescence reader such as T8-ISO and T16-ISO from Axxin, Australia, or AmpliFire Isothermal Fluorometer from Agdia, Inc. It is notable that RPA exo kits assays can also be done with any flexible PCR instrument such as Mx3000P QPCR System (Agilent, Germany). RPA basics kits (TwistAmp Basic or TwistAmp Liquid Basic) and RPA nfo kit (TwistAmp nfo) assays only require a tube strip incubator. RPA basics kits assays require post-incubation cleaning and gel electrophoresis of the end product, while TwistAmp nfo kit assays need post-incubation dilution of the end- product and detection of end product through Lateral Flow strips. In contrast to close tube detection of RPA exo kits, basic and nfo kits tubes are opened after incubation and this poses a risk of carryover contamination (Yang et al., 2017b). The carryover contamination for TwistAmp nfo kit can be prevented by using ‘U-Star Disposable Nucleic Acid Lateral Flow Detection Units’ (TwistDx Inc.). Another way to combat carryover contamination is replacement of dTTP with dUTP, and utilizing uracil DNA deglycosylase (UDG) enzyme and UDG inhibitor (Piepenburg et al., 2013). Moreover, the contamination risk can be minimized by physical separation of pre and post amplification laboratories, utilization of positive displacement pipettors or filter tips, disposing contaminated tips and tubes in acid or bleach, and inclusion of no template controls (NTCs).

Although RPA assays have been often reported specific, some researchers have observed RPA non-specificity in their RPA assays (Patel et al., 2016; Yang et al., 2017b; Moore and Jaykus, 2017). RPA assays have been reported tolerating 5 mismatches (El Wahed et al., 2013), 7 mismatches (Daher et al., 2015), and even 9 mismatches (Boyle et al., 2013) with target sites, and therefore it is recommended to design RPA assay from unique target markers or markers with at least 36%

difference in respect to closely-related species (Daher et al., 2015).

Another limitation of RPA is target marker length. RPA cannot efficiently amplify long target such 500 bp due to the competing background noise and the limited energy available, and designing sensitive assays requires short nucleic acid targets, ideally of 100-200 bp size. This means RPA technology is not suitable for amplifying long targets, especially when in low quantity, and long amplicons are desired in different applications such as Sanger sequencing. Moreover, due to

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31 background noise, purification of target RPA amplicons is not always

possible through routine PCR purification kits and an additional step of gel extraction is sometimes needed prior to purification. Regarding sensitivity of RPA technology in comparison to that of PCR, reports are contradicting. Comparative studies have found RPA less (Rosser et al., 2015; Londoño et al., 2016; Howson et al., 2017, Wang et al., 2017), equally (Euler et al., 2013; Shahin et al., 2018; Xing et al., 2017, Bentahir et al., 2018), and even more sensitive (Mohandas and Bhat, 2020) when amplifying targets from extracted nucleic acid.

1.3.6 Application to plant disease diagnostics

Several RPA assays have been developed for plant pathogens including Phytophthora. Miles et al. (2015) developed Phytophthora genus-specific assay through the mitochondrial region of trnM-trnP-trnM. Miles et al.

(2015) also developed species-specific assays for Phytophthora ramorum and P. kernoviae from the mitochondrial intergenic spacer between atp9 and nad9. Utilizing the atp9-nad9, Rojas et al. (2017) designed species- specific RPA assays for Phytophthora sojae and Phytophthora sansomeana.

Si Ammour et al. (2017) designed an RPA assay for Phytophthora infestans through the internal transcribed spacer 2 (ITS2) region.

Moreover, this dissertation also includes atp9-nad9 based species- specific RPA assays for Phytophthora cactorum (Munawar et al., 2019) and P. fragariae (Munawar et al., 2020a). Regarding plant pathogenic fungi, RPA assays have been developed for detection of Gaeumannomyces avenae, Magnaporthiopsis poae, Ophiosphaerella korrae (Karakkat et al., 2018), Macrophomina phaseolina (Burkhardt et al., 2018), Fusarium oxysporum f. sp. fragariae (Burkhardt et al., 2019), Fusarium oxysporum f.

sp. conglutinans, Fusarium oxysporum f. sp. lycopersici, and Botrytis cinerea (Lau et al., 2016).

RPA assays have also been developed for bacterial plant pathogens including Candidatus Phytoplasma pruni (Villamor and Eastwell, 2019), Candidatus Liberibacter asiaticus (Ghosh et al., 2018; Qian et al., 2018), Pectobacterium species (Ahmed et al., 2018), Xanthomonas gardneri, X.

euvesicatoria, X. perforans (Strayer-Scherer et al., 2018), Agrobacterium species (Fuller et al., 2017), Candidatus Phytoplasma mali (Valasevich and Schneider, 2017), Candidatus Phytoplasma oryzae (Wambua et al., 2017), Pseudomonas syringae (Lau et al., 2016; Lau et al., 2017), and Liberibacter (Kalyebi et al., 2015). RPA assays designed for plant viruses include cucumber green mottle mosaic virus (Zeng et al., 2019), apple stem grooving virus (Kim et al., 2018), rice black-streaked dwarf virus (Zhao et al., 2019), sugarcane yellow leaf virus (Feng et al., 2018), citrus yellow mosaic virus (Kumar et al., 2018), Yam mosaic virus (Silva et al.,

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32

2015), Yam mild mosaic virus (Silva et al., 2018), rose rosette virus (Babu et al., 2017), grapevine red blotch virus (Li et al., 2017), tomato chlorotic dwarf viroid (Hammond and Zhang, 2016), begomoviruses (Londoño et al., 2016), potato virus Y, potyvirus, wheat dwarf virus (Glais and Jacquot, 2015), plum pox virus (Zhang et al., 2014), and little cherry virus 2 (Mekuria et al., 2014).

Agdia licensed TwistDx’s patented RPA technique for plant pathogen detection. Agdia, Inc. also develop and sell RPA assays for different plant pathogens. Agdia supplies RPA kits with trade name of AmplifyRP. The ready-to-use AmplifyRP products include assays for little cherry virus 2, banana bunchy top virus, Fusarium oxysporum f.sp.

vasinfectum, grapevine red blotch-associated virus, Candidatus Liberibacter asiaticus, plum pox virus, tomato chlorotic dwarf viroid, Clavibacter michiganensis subsp. michiganensis, Clavibacter michiganensis subsp. nebraskensis, Dickeya species, Fusarium odoratissimum, grapevine leafroll-associated virus 3, grapevine pinot gris virus, hop stunt viroid, Phytoplasma solani Bois Noir, Candidatus Phytoplasma vitis Flavescence dorée, tobacco rattle virus, Xylella fastidiosa, and Clavibacter michiganensis subsp. Sepedonicus.

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35

2 Recombinase polymerase ampli- fication assay for fast, sensitive and on-site detection of Phy-

tophthora cactorum without DNA extraction

Mustafa Munawar, Anna Toljamo, Frank Martin and Harri Kokko

European Journal of Horticultural Science issue 84-1 (February 2019) (invited article) https://doi.org/10.17660/eJHS.2019/84.1.2

Reproduced with permission from the publisher.

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SUMMARY

Crown rot, caused by Phytophthora cactorum, is an increasing problem for the strawberry crop in Europe. Most of the assays available for the detection P. cactorum are either laborious or inadequate for on-site testing. Recombinase Polymerase Amplification (RPA) is an attractive alternative for rapid detection of pathogens from plant material. We have developed an RPA assay for P. cactorum using the intergenic mi- tochondrial DNA spacer between atp9 and nad9. The assay is specific, and sensitive with a lower limit of detection with purified DNA of 10 fg.

Moreover, the assay is DNA extraction-free, and requires only two minutes of tissue maceration prior to running the RPA assay.

SIGNIFICANCE OF THIS STUDY What is already known on this subject?

• Crown rot caused by Phytophthora cactorum has been damaging strawberry crops in Europe for more than half a century. For the detection of P. cactorum, the current recommended technique is Polymerase Chain Reaction (PCR). TaqMan PCR assays based on ras- related protein gene Ypt1, and atp9-nad9 marker have been developed for P. cactorum. PCR is a time-consuming method requiring DNA extraction, and is not suitable for on-site testing.

What are the new findings?

• Using the Recombinase Polymerase Amplification (RPA) technology, we have developed an ultra-sensitive, specific, and on-site assay for detection of P. cactorum and diagnosis of crown rot. The assay enables detection of P. cactorum directly from crudely macerated crown tissue and eliminates the need of time-consuming DNA extraction.

What is the expected impact on horticulture?

• For nurseries, the assay will assist production and maintenance of healthy plant stocks, and may serve as a certificate of P. cactorum absence in plant material being sold.

• For farmers, the assay can serve as pre-screen of propagation material before planting into fields.

• For diagnostic laboratories, the assay will accelerate the diagnosis process for the pathogen.

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37 INTRODUCTION

Phytophthora cactorum has a broad host range comprising more than 154 genera of vascular plants and exhibits genetic variation depending on host and geographic location (Phytophthora Database, 2006). P. cactorum is one of the primary causative agents of the soil-borne disease of strawberry crown rot all over the world. It infects the crown and causes necrosis and death of infected plants (Maas, 1998; Eikemo et al., 2004).

European and Mediterranean Plant Protection Organization (OEPP/EPPO, 1994) allows up to 1% presence of crown rot disease in certified plant production fields (Stensvand et al., 1999). This pathogen can also cause symptomless latent infections of strawberry plants, leading to transport of infected propagation material across Europe and contributing to the spread of disease (Harris and Stickels, 1981; Santos et al., 2002).

In Europe, strawberry crown rot was first reported in Germany during 1952 (Deutschmann, 1954). Since about 1960, the disease has significantly damaged the strawberry crop in Europe (Maas, 1998). In Northern Europe, crown rot was first detected in Sweden in 1988 (Stensvand et al., 1999). In Finland, P. cactorum was first isolated in 1990 from crown rot of an infected strawberry plant (Parikka, 1991). We have noticed up to 50% loss in fields of Eastern Finland in summer 2016. In Norway, crown rot was first detected in 1992 (Stensvand and Semb, 1995). A survey in 1999 reported presence of crown rot in several strawberry cultivating regions of Norway despite the country strictly bans import of strawberry plants since 1986 (Stensvand et al., 1999).

Regarding Southern Europe, a study found five fields having crown rot between 2000 and 2001 in Huelva city of Spain (Santos et al., 2002).

Beside Europe, strawberry crown rot has also been reported in the USA, Japan, South Africa, Australia and New Zealand (Eikemo et al., 2004). In eastern North America, crown rot was reported for the first time in 1988 (Wilcox, 1989). Similarly, P. cactorum is considered as the most common agent of strawberry crown rot in Florida, United States (Marin et al., 2018). In Japan, outbreaks of strawberry crown rot in 1978 were caused by Phytophthora nicotianae and P. cactorum (Li et al., 2013).

Several methods have been developed for detection of Phytophthora, including microscopy, baiting, immunological assays and polymerase chain reaction (PCR) (Martin et al., 2012). Microscopic identification of Phytophthora on species level is complicated. Some Phytophthora species have overlapping morphological characteristics, while other species exhibit intraspecific morphological variations (Erwin and Ribeiro, 1996).

The bait tests are time-consuming and require up to five weeks for

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certain Phytophthora species (Bonants et al., 1997). Moreover, the immunological assays like enzyme-linked immunoassay (ELISA) lack specificity. The commonly available genus specific ELISAs for Phytophthora detection have been reported to cross react with some Pythium spp. (Bulluck et al., 2006; Kox et al., 2007). Similarly, the few Phytophthora species level ELISAs developed also encountered false positivity with the non-target Phytophthora species (Avila et al., 2009).

The PCR, especially TaqMan real time PCR, is considered as a gold standard in diagnostics, however a few of the PCR assays developed for identification of Phytophthora species have also been reported to cross react with non-target Phytophthora species (Hughes et al., 2006; Bilodeau et al., 2007; Schena et al., 2008; Martin et al., 2009). The cross reactivity probably resulted due to the level of interspecific variability present in loci targeted by these assays.

TaqMan PCR assays have been developed specifically for P. cactorum detection. Li et al. (2013) developed TaqMan assay using single copy gene, ras-related protein gene Ypt1 and recorded detection limit as 1 pg.

Recently a suitable, multicopy and non-cross reacting intergenic DNA marker, the mitochondrial spacer between atp9 and nad9, has been reported for Phytophthora detection and identification at a species level.

Through atp9-nad9, TaqMan PCR assays have been developed for 13 Phytophthora species, including P. cactorum. The lower limit of detection of those TaqMan assays were reported around 100 fg (Bilodeau et al., 2014; Miles et al., 2017).

Although PCR is adequately sensitive and specific for Phytophthora detection, it is not suitable for on-site testing due to the need for DNA extraction and use of a thermocycler. Moreover, PCR also can be inhibited by some substances extracted along the plant nucleic acid (Schrader et al., 2012). To address these problems, isothermal assays like Recombinase Polymerase Amplification (RPA) are promising alternatives. RPA is a rapid, sensitive and specific method of isothermal nucleic acid amplification. RPA utilizes longer primers, recombinase enzyme, single-stranded DNA binding (SSB) protein and strand displacing polymerase enzyme for amplifying nucleic acid (Piepenburg et al., 2006). RPA is also more tolerant to inhibitors and from a crude maceration of the plant tissue the target nucleic acid can be amplified (Mekuria et al., 2014; Miles et al., 2015).

RPA assays for few Phytophthora species have been developed targeting the mitochondrial spacer between atp9 and nad9 (Miles et al., 2015; Rojas et al., 2017). In these assays, the forward primer and the common probe was placed in the genus-conserved region of atp9, while the reverse primer was in the intergenic spacer atp9-nad9. Following the

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39 TwistDx Inc., UK recommendations, a different design for the assay was

adopted in this study. Only the forward primer was positioned in the genus-conserved region of atp9, while the reverse primer and the overlapping probe were placed the intergenic spacer atp9-nad9. Selecting both the probe and reverse primer from the intergenic region may increase the assay specificity, and also reduce template length, hence shortening time of the assay. This kind of strategy may also prove useful for design of assays for additional Phytophthora species for multiplexing.

So, this study aims to develop RPA assay for detection of P. cactorum and presents a different approach to design atp9-nad9 based species level assays for Phytophthora detection.

MATERIALS AND METHODS Phytophthora isolation

Strawberry plants of different cultivars from fields and imported plant trade were collected from Finland. Then crowns were dissected vertically, and crown tissue pieces were surfaced sterilized by single dip in 70% ethanol. Then crown pieces were left for drying on filter paper for two to three hours. Later the pieces were placed on Phytophthora selective agar plates and plates were incubated on room temperature and inspected every 48 h for possible Phytophthora growth. The Phytophthora selective agar was composed of corn meal agar supplemented with 250 mg L^-1 ampicillin, 25 mg L^-1 benomyl, 10 mg L^-1 pimaricin, 10 mg L^-1 rifampicin and 50 mg L^-1 hymexazol as described by Drenth and Sendall (2001). Corn meal agar (product no. 42347), ampicillin and benomyl were purchased from Sigma-Aldrich, pimaricin (natamycin) from Molekula, Germany, rifampicin from Duchefa Biochemie Netherlands, and hymexazol from Alfa Aesar.

DNA extraction and sequencing

P. cactorum isolates originated from strawberry fields, ten were isolated from the Savo region of Finland and fifteen were from Poland. The agar plugs containing hyphae were added to petri dishes filled with peptone glucose broth at room temperature. After 7–10 days growth, the hyphae were separated from the agar plug and washed three times with sterile water. The hyphae were lyophilized overnight and then powdered by small plastic pestle in a round bottom 2-mL Eppendorf tube. DNA was extracted from up to 50 mg freeze-dry hyphae or up to 200 mg wet hyphae using E.Z.N.A. Fungal DNA Mini Kit from Omega Bio-tek, Georgia, US.

Application of recombinase polymerase amplification to diagnosis of phytophthora diseases of strawberry

Dissertations in Forestry and Natural Sciences

MUSTAFA AHMAD MUNAWAR

Application of recombinase

polymerase amplification to

diagnosis of Phytophthora

diseases of strawberry

APPLICATION OF RECOMBINASE POLYMERASE AMPLIFICATION TO

DIAGNOSIS OF PHYTOPHTHORA

DISEASES OF STRAWBERRY

Mustafa Ahmad Munawar

APPLICATION OF RECOMBINASE POLYMERASE AMPLIFICATION TO

DIAGNOSIS OF PHYTOPHTHORA DISEASES OF STRAWBERRY

ABSTRACT

ACKNOWLEDGEMENTS

LIST OF ABBREVIATIONS

LIST OF ORIGINAL PUBLICATIONS

AUTRHOR’S CONTRIBUTION

CONTENTS

1 GENERAL INTRODUCTION

1.1 PHYTOPHTHORA, AN AGGRESSIVE PLANT DE- STROYER

1.2 ISOTHERMAL NUCLEIC ACID AMPLIFICATION TECHNOLOGIES

1.3 RECOMBINASE POLYMERASE AMPLIFICATION

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2 Recombinase polymerase ampli- fication assay for fast, sensitive and on-site detection of Phy-

tophthora cactorum without DNA extraction

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