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Recent Publications in this Series A.O OGHENEKARO Molecular Analysis of the Interaction between White Rot Pathogen (

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Sustainable Cropping of Reed Canary Grass for Energy Use 30/2015 Anna Knuuttila Diagnostics and Epidemiology of Aleutian Mink Disease Virus 31/2015 Jose Martin Ramos Diaz Use of Amaranth, Quinoa, Kañiwa and Lupine for the Development of Gluten-Free Extruded Snacks 32/2015 Aleksandar Klimeski Characterization of Solid Phosphorus-Retaining Materials – from Laboratory to Large-Scale Treatment of Agricultural Runoff 33/2015 Niina Idänheimo ) and Rubber Tree ( The Role of Cysteine-rich Receptor-like Protein Kinases in ROS signaling in Arabidopsis thaliana 34/2015 Susanna Keriö Terpene Analysis and Transcript Profiling of the Conifer Response to Heterobasidion annosum s.l. Infection and Hylobius abietis Feeding 35/2015 Ann-Katrin Llarena Population Genetics and Molecular Epidemiology of Campylobacter jejuni DEPARTMENT OF FOREST SCIENCES Hevea brasiliensis 1/2016 Hanna Help-Rinta-Rahko FACULTY OF AGRICULTURE AND FORESTRY The Interaction of Auxin and Cytokinin Signaling Regulates Primary Root DOCTORAL PROGRAM IN MICROBIOLOGY AND BIOTECHNOLOGY Procambial Patterning, Xylem Cell Fate and Differentiation in Arabidopsis thaliana UNIVERSITY OF HELSINKI ) 2/2016

Helsinki 2016 ISSN 2342-5423 ISBN 978-951-51-1881-3

Department of Forest Sciences Faculty of Agriculture and Forestry and Doctoral program in Microbiology and Biotechnology University of Helsinki

Molecular analysis of the interaction between white rot pathogen (Rigidoporus microporus) and rubber tree (Hevea brasiliensis)

Abbot O. Oghenekaro

ACADEMIC DISSERTATION

To be presented for public examination, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, in the lecture room LS7 (Latokartanonkaari 9, B-building) on January 15th 2016 at 12 o’clock.

Helsinki 2016

Supervisor Professor Fred O. Asiegbu Department of Forest Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Finland

Pre-examiners Professor Yu-Cheng Dai Institute of Microbiology, P.O. Box 61, Beijing Forestry University, Beijing 100083, China

Dr. Hakeem O. Shittu Department of Biology & Biotechnology, Faculty of Life Sciences, University of Benin, P.M.B 1154, Benin City, Nigeria

Opponent Assoc. Prof. Caroline Mohammed Tasmanian Institute of Agriculture, University of Tasmania, Hobart 7001, Tasmania, Australia

Custos Professor Fred O. Asiegbu Department of Forest Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Finland

Cover: Healthy rubber trees with flowing latex (left); of R. microporus on the base (top centre) and collar (lower centre) of white rot infected rubber tree; Characteristic mycelial strand of R. microporus in culture (upper right); Micrograph of R. microporus penetrating the vessel of rubber wood during saprotrophic growth (middle right); Rubber tree clone used for inoculation studies (lower right).

ISSN 2342-5423 (Print) ISSN 2342-5431 (Online) ISBN 978-951-51-1881-3 (Paperback) ISBN 978-951-51-1882-0 (PDF) https://ethesis.helsinki.fi/ Hansaprint 2015

To the memory of my father, Awhabro Okotie Oghenekaro

CONTENTS

ABBREVIATIONS ...... 6

LIST OF ORIGINAL PUBLICATIONS AND SUBMITTED MANUSCRIPTS ...... 8

ABSTRACT ...... 10

1. INTRODUCTION ...... 12 1.1 Global forestry and tree pests and diseases ...... 12 1.2 The Para rubber tree, Hevea brasiliensis ...... 12 1.3 The white rot pathogen, Rigidoporus microporus ...... 13 1.4 and molecular phylogeny of Rigidoporus microporus ...... 15 1.5 Wood lignocellulose degradation by white rot basidiomycetes ...... 16 1.5.1 Lignin degrading enzymes of wood decay fungi ...... 17 1.5.2 Polysaccharide degrading enzymes of wood decay fungi ...... 18 1.5.3 Current advances of next generation sequencing technology in furthering our understanding of fungi transcriptome ...... 19 1.6 Defence gene expression of forest trees to pests and pathogens ...... 20 2. AIMS OF THE STUDY ...... 22 3. MATERIALS AND METHODS ...... 23 4. RESULTS AND DISCUSSION ...... 24 4.1 Molecular phylogeny of Rigidoporus microporus isolates associated with rubber tree plantations ...... 24 4.1.1 Analysis of ITS sequences of Rigidoporus microporus isolates ...... 24 4.1.2 Analysis of β-tubulin sequences of Rigidoporus microporus isolates ...... 25 4.1.3 Analysis of tef1 sequences of Rigidoporus microporus isolates ...... 25 4.1.4 Analysis of combined ITS, β-tubulin and tef1 sequences of Rigidoporus microporus isolates ...... 25 4.1.5 Analysis of LSU sequences of Rigidoporus microporus isolates ...... 26 4.2 Saprotrophic growth of Rigidoporus microporus on rubber wood ...... 27

4.2.1 Mass loss of Hevea brasiliensis wood blocks due to degradation by Rigidoporus microporus isolates ...... 27 4.2.2 Effects of temperature on in vitro growth rates of Rigidoporus microporus isolates 28 4.2.3 Light microscopy of structural alterations in wood blocks of Hevea brasiliensis induced by Rigidoporus microporus isolates ...... 28 4.3 Gene expression of Rigidoporus microporus during saprotrophic growth on rubber wood ...... 29 4.3.1 General overview of the Rigidoporus microporus transcriptome ...... 29 4.3.2 Rigidoporus microporus genes encoding polysaccharide degrading enzymes during saprotrophic growth on rubber wood ...... 31 4.3.3 Rigidoporus microporus genes encoding glycolignin attacking enzymes during saprotrophic growth on rubber wood ...... 32 4.3.4 Rigidoporus microporus genes encoding enzymes involved in fatty acid and rubber tree latex degradation during saprotrophic growth on rubber wood ...... 34 4.3.5 Rigidoporus microporus ABC transporters and hydrophobins expressed during saprotrophic growth on rubber wood ...... 35 4.3.6 Rigidoporus microporus genes encoding enzymes involved in pathways related to energy metabolism during saprotrophic growth on rubber wood ...... 35 4.4 Defence gene expression of Hevea brasiliensis in response to Rigidoporus microporus infection ...... 36 4.4.1 Necrotic lesions produced in Hevea brasiliensis clones after inoculation with Rigidoporus microporus ...... 37 4.4.2 Expression of pathogenesis related proteins ...... 37 4.4.3 Expression of cell wall modification genes ...... 38 4.4.4 Expression of signal transduction genes ...... 39 4.4.5 Expression of a Myb transcription factor ...... 39 5. SUMMARY AND CONCLUSIONS ...... 40 6. FUTURE PERSPECTIVES ...... 42 ACKNOWLEDGEMENTS ...... 44 REFERENCES ...... 46

ABBREVIATIONS

ABC ATP binding cassette

CAZy carbohydrate active enzymes

CBM carbohydrate-binding module cDNA complementary DNA

CE carbohydrate esterase

DAMP damage-associated molecular pattern

DNA deoxyribonucleic acid

GH glycoside hydrolase

GT glycosyl transferase

HAMP herbivore-associated molecular pattern

HR hypersensitive response

ITS internal transcribed spacer

JA jasmonic acid

LiP lignin peroxidase

LPMO lytic polysaccharide monooxygenase

LSU large sub unit

MAPK mitogen activated protein kinase

MCO multicopper oxidase

MnP manganese peroxidase

ORF open reading frame

PAL phenylalanine ammonia lyase

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PAMP pathogen-associated molecular pattern

PL polysaccharide lyase

PR pathogenesis related

PRR pattern recognition receptor qRT-PCR quantitative real-time PCR

RNA ribonucleic acid

RNA-Seq RNA sequencing

RNS reactive nitrogen

ROS reactive oxygen species

SA salicylic acid tef1 translation elongation factor 1-α

TF transcription factor

VP versatile peroxidase

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LIST OF ORIGINAL PUBLICATIONS AND SUBMITTED MANUSCRIPTS

The doctoral thesis is based on the following publications, which are referred to in the text by their roman numerals.

I. Oghenekaro, A.O., Miettinen, O., Omorusi, V.I., Evueh, G.A., Farid M.A., Gazis, R. and Asiegbu, F.O. (2014) Molecular phylogeny of Rigidoporus microporus isolates associated with white rot disease of rubber trees (Hevea brasiliensis). Fungal Biology. 118: 495-506. DOI: http://dx.doi.org/10.1016/j.funbio.2014.04.001 II. Oghenekaro, A.O., Daniel, G. and Asiegbu, F.O. (2015) The saprotrophic wood- degrading abilities of Rigidoporus microporus. Silva Fennica. 49(4): 1320. DOI: http://dx.doi.org/10.14214/sf.1320 III. Oghenekaro, A.O., Raffaello, T., Kovalchuk, A. and Asiegbu, F.O. (2015) De novo transcriptomic assembly and profiling of Rigidoporus microporus during saprotrophic growth on rubber wood (Accepted subject to minor revision in BMC Genomics) IV. Oghenekaro, A.O., Omorusi, V.I. and Asiegbu, F.O. (2015) Defence-related gene expression of Hevea brasiliensis clones in response to the white rot pathogen, Rigidoporus microporus. Forest Pathology. (Accepted for publication)

All publications are reprinted with the kind permission of their copyright holders.

Other articles not included in this thesis:

. Kovalchuk, A., Keriö, S., Oghenekaro, A.O., Jaber, E., Raffaello, T. and Asiegbu, F.O. (2013) Antimicrobial Defenses and Resistance in Forest Trees: Challenges and Perspectives in a Genomic Era. Annual Review of Phytopathology. 51: 221-44. DOI: 10.1146/annurev-phyto-082712-102307

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Author contribution:

I. The author collected data, planned the experiment and did the laboratory work. The author analysed the data, interpreted the results and wrote the article. OM contributed to data collection, phylogenetic analysis and writing of the article. VIO contributed to data collection and experimental design. G.A.E and M.A.F contributed to data collection. RG contributed to data collection and writing of the article. FOA conceived the study and contributed to the experimental design and writing of the article.

II. The author planned the experiment and did the laboratory work. The author analysed the data, interpreted the results and wrote the article. GD contributed to wood microscopy and writing of the article. FOA conceived the study and contributed to the experimental design and writing of the article.

III. The author planned the experiment and did the laboratory work. The author analysed the data, interpreted the results and wrote the article. TR and AK contributed to data analysis and writing of the article. FOA conceived the study and contributed to the experimental design and writing of the article.

IV. The author collected data, planned the experiment and did the laboratory work. The author analysed the data, interpreted the results and wrote the article. VIO contributed to data collection and experimental design. FOA conceived the study and contributed to the experimental design and writing of the article.

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ABSTRACT

The white rot pathogen, Rigidoporus microporus () syn. Rigidoporus is the most destructive root pathogen in rubber plantations located in South East Asia and Africa. Most management practices for the disease have been carried out without adequate knowledge of the population genetics of the pathogen. Our primary objective in the first part of this work was to characterize isolates from rubber trees (Hevea brasiliensis) in Nigeria. The lack of global studies comparing isolates from Asia and Africa where the majority of rubber plantations are located necessitated extending our molecular phylogeny studies to include isolates from other regions of the world. We compared the Nigerian isolates with isolates from South East Asia and South/Central America. Forty isolates were analyzed by sequencing four gene regions: the nuclear ribosomal DNA ITS and LSU, β-tubulin and translation elongation factor 1- α (tef1) genes. The isolates form three distinctive clades corresponding to Africa, South East Asia and South/Central America. There was high intra-species variation within each clade, but no geographic pattern was detected among the Nigerian isolates and also among the West and Central African isolates. Our multi- gene molecular phylogenetic analysis suggests the presence of two distinctive species associated with the white rot disease. Phylogenetic analysis also placed the Rigidoporus within the Oxyporus clade of the Hymaenochataeles. This study supports the need for a thorough revision of the Rigidoporus genera within the /Hymaenochataeles. To gain insights on the saprotrophic abilities of Rigidoporus microporus isolates associated with the white rot disease of rubber trees, we performed wood decay tests of selected isolates on rubber wood blocks. We included a non-pathogenic endophytic South American isolate for comparative analysis. The structural alterations caused due to fungal growth in the rubber wood blocks was also studied. The pathogenic isolates associated with rubber trees significantly decayed the rubber wood blocks suggesting a capacity to switch to saprotrophic growth when the tree is killed. The non-pathogenic isolate had very low saprotrophic ability. Structural alterations of wood cell walls caused in the wood blocks were typical for white rot basidiomycetes. The fact that Rigidoporus microporus can switch to saprotrophic growth on dead cells of the tree led us to investigate the transcriptomic profiles of the during growth on dead rubber wood. We performed RNA-Seq de novo transcriptomic assembly which produced 25, 880 annotated unigenes. The transcriptome expressed over 400 genes encoding lignocellulose enzymes. A number of glycoside hydrolases (GHs) were highly induced in rubber wood including all nine members of GH43 genes in the transcriptome. Twenty-four manganese peroxidase encoding genes were among a large number of oxidoreductases that were significantly up-regulated in rubber wood. Several genes involved in fatty acid

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and rubber latex degradation were expressed in the transcriptome. Several ABC transporters and hydrophobin genes were also expressed in the transcriptome. Finally, pathway enrichment analysis of genes related to energy metabolism revealed some genes including alcohol dehydrogenase with potential biotechnological applications. This is the first study on the transcriptomic analysis of R. microporus on rubber wood. The data generated should serve as a useful resource for future studies. Understanding how rubber tree is able to defend itself against invading Rigidoporus microporus was an additional research goal. The expression of defence-related genes; pathogenesis related proteins (PR1, PR3, PR5, PR8 and PR9), cell wall modification genes (PAL and expansin), signal transduction genes (ACC oxidase, AOC, MAPK) and a Myb transcription factor were studied in two clones (RRIM612 and PR107) of H. brasiliensis with different levels of susceptibility. Clone RRIM612 had a higher lesion size than clone PR107 after 5 weeks post inoculation with R. microporus. A class I chitinase (PR3) was up- regulated in RRIM612 in response to the pathogen while a class IV peroxidase (PR9) was highly up- regulated in PR107. Signal transduction genes involved in ethylene and jasmonic acid signaling as well as Phenylalanine ammonia-lyase (PAL) were up-regulated almost equally in both clones. The predicted expansin like protein was up-regulated 40 fold in RRIM612 in response to the pathogen. The results obtained demonstrate the variability in defence responses in the two clones studied and provides the first set of defence genes expression profiles of the host-pathogen interaction of the white rot disease of rubber trees. This study sheds light on the phylogeny of pathogenic Rigidoporus microporus isolates associated with the white rot disease as well as evidence showing its ability to switch to saprotrophic growth. The transcriptome of the fungus during saprotrophic growth was also de novo assembled revealing a vast number of lignocellulose degrading enzymes. Finally, this work provides an overview of potential candidate defence genes in H. brasiliensis in response to infection by R. microporus.

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1. INTRODUCTION

1.1 Global forestry and tree pests and diseases

Forests which cover about 20% of the earth’s land surface and serve as a source of energy, food and industrial materials is constantly facing treats from fire, climate change and invasions by pests and pathogens. Tree pests and diseases have an important role in forest ecosystems functioning and health (Sugden et al., 2015; Teale et al., 2011). A Food and Agricultural Organization (FAO) study on Global Forest Resources Assessment (FRA) revealed that over 97.5 million hectares of forest worldwide were affected by insect pests and microbial pathogens (van Lierop 2015). Recently, there has been a surge in the introduction of pests and pathogens, influenced by human activities in planted forests worldwide (Wingfield et al., 2015). Some of these invasive species have the potential to drive some important tree species to extinction (Davies et al., 2014). Fungi belonging to the basidiomycete group are known to cause stem and root rot disease in trees in tropical forests. Important trees threatened by these pathogens include; rubber, oil palm, acacia and Eucalyptus trees (Mohammed et al., 2014). Understanding the complex relationships between tree-host and pathogens is the first step required for breeding resistant trees. Advances in genomic technologies have led to the sequencing of whole genome of forest trees especially broad-leaved trees which has led to accelerated tree breeding for disease resistance (Boshier and Buggs 2015). Advances in taxonomy and molecular studies of tree pathogens have also led to the discovery of cryptic species which has improved our understanding of these pests (Hamelin 2012). Research efforts are currently focused on the development of molecular tools to screen for resistance and susceptibility that can be integrated into breeding programs (Telford et al., 2015). Tree endophytes which has the ability to switch from mutualistic to pathogenic habits have also been recently predicted to serve as good indicators of tree health by the monitoring of changes in forest health in different ecosystems (Rajala et al., 2013; 2014; Delaye et al., 2013). Management of forest pests and diseases requires interdisciplinary collaboration between forest pathologists, landscape ecologists and endophyte scientists, as well as the combination of global, holistic and integrated management practices (Pautasso et al., 2015, Wingfield et al., 2015).

1.2 The Para rubber tree, Hevea brasiliensis

Para rubber (Hevea brasiliensis Muell. Arg.) was discovered in the Amazon forests in the 15th century by Spanish explorers (Nandris et al., 1987). It belongs to the Euphorbiaceae family and is the world’s major source of commercial natural rubber. The first rubber tree plantation outside the amazon was established in 1876 in Malaysia from seeds collected by Henry Wickham and subsequent plantations in

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Asia and Africa were established from these Wickham planting materials (Schultes 1970). The rubber tree has moved round the tropical regions of the world since the vulcanization of rubber became popular in the mid-1800s (Nandris et al., 1987). Majority of the rubber plantations in the tropics are located in tropical South East Asia and Africa because of the threat posed by the South American leaf blight (SALB) disease (Onokpise and Louime 2012). The plant was introduced into Nigeria in 1895 and is now widely cultivated in the rain forest belt in the south of the country, with small stake holders accounting for over 70% of the planted area (Shaib et al., 1997). The latex from the stem of Para rubber is useful in the manufacture of a variety of industrial products like tyres, containers, shoes and other items. Elastomers derived from natural rubber are indispensable in space, water, and ship technologies (Jacob 2006). Heavy duty tyres used in automobile and aircrafts require 100% natural rubber. (Onokpise and Louime 2012). The market value of global annual production of natural rubber in 2014 was over 25 billion USD (Rubber Statistical Bulletin 2015). Rubber seed cake is used as livestock feed for animals in developing countries (Ukpebor et al., 2007) while rubber seed oil is used as a lubricating oil (Aravind et al., 2015). Rubber is an important source of livelihood in many developing countries. Despite long-term breeding efforts, majority of cultivars in use today are those derived from seeds originating from Brazil (Warren 1987). The fact that majority of the H. brasiliensis plantations in the world originate from a single source leads to a global concern on the high genetic uniformity and narrow genetic base of the tree. The concern for stake holders and scientists is the devastating effects an invasive pathogen would have on these rubber plantations with such a high genetic uniformity (Wittenburg and Cock 2001). It also allows high possibilities of host-jump from native trees to mono-culture rubber plantations located in South East Asia and Africa.

1.3 The white rot pathogen, Rigidoporus microporus

Para rubber is affected by a wide range of pathogens that cause disease in the root, stem and leaf systems of the tree. The diseases that affect the root system in plantations are the most destructive (Mohammed et al., 2014, Geiger et al., 1986a). The most important root pathogens are the basidiomycetes (Rigidoporus microporus (Sw.) Overeem., Phellinus noxius (Corner) G. Cunn., and Ganoderma pseudoferreum (Wakef.) Overeem & B.A. Steinm. (Rao 1975). Rigidoporus microporus is the most harmful among these root pathogens. It is a well-known and serious pathogen of rubber plantations in the tropics causing the white rot disease. It also affects other tropical woody crops such as cocoa, coffee, tea, coconut, oil palm, Ceylon breadfruit and Obeche. (Nandris et al., 1987; Madushani et al., 2013; Begho and Ekpo 1987). It thrives in plantations but is less abundant in natural forests (Onokpise 2004). The fungus was first described as a pathogen of H. brasiliensis by H.N Ridley in Malaysia in 1904 (Holiday 1980).

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Though, there is no literature on specific economic loss in monetary terms, but with an assumption of 25 years as the productive life of a rubber tree, losses due to reduced latex production as a result of the white rot disease can be up to several hundred thousand dollars per hectare (Nandris et al., 1987). In Indonesia, 80,000 hectares of rubber trees were affected by the white rot disease in a survey conducted in some parts of the country (Jayasinghe 2010). A mean annual incidence of between 5 and 15% of rubber plantations surveyed in Malaysia was reported (Soepena 1993). In Nigeria, R. microporus kills up to five trees per hectare per year and is responsible for 96% of all root diseases in rubber plantations (Omorusi 2012). The fungus forms whitish rhizomorphs which can adhere to wood debris in the soil, and can grow for long distance in the absence of any wood debris to infect healthy trees (Omorusi 2012). The white rot disease starts from the roots after infection by mycelia or rhizomorphs of R. microporus on dead trees and stumps (Figure 1). It later spreads to the collar region and foliar symptoms only appear when the roots are damaged; which affects the flow of water and minerals and subsequently, the flow of latex stops. Characteristic reddish-brown basidiocarps of R. microporus are then seen on the collar region of the stem (Omorusi 2012; Ogbebor et al., 2013) (Figure 1). New fungicides have been tested to improve the control of white rot disease caused by R. microporus (Gohet et al., 1991). However, biological control of plant pathogens by microorganisms has been considered a more natural and environmentally acceptable alternative to the existing chemical treatment methods (Baker and Paulitz, 1996). Biological control methods tested in vitro include the use of species of Hypocrea and Trichoderma in the control of the pathogen (Ogbebor et al., 2015). The attack of the rubber tree by the fungus causes the lignified root tissues of the tree to decay. Rigidoporus microporus has been known for a long time to have the capability to breakdown lignin (Geiger et al., 1989). Many studies have been done on host pathogen interaction covering aspects of biochemistry, anatomy and cytology (Nicole et al., 1983; Nicole et al., 1985; Nandris et al., 1987). Growth and differentiation of mycelial strands of the fungus have been shown to depend on suitable combinations of the pH of the media and the nature of the nitrogen and carbon sources (Richard and Botton 1996). An ultrastructural and cytochemical investigation of the development of R. microporus inoculated into wood blocks was also carried out to gain better insights into the structure and role of the extracellular sheaths produced by this fungus during wood degradation. The results show that the sheaths produced may be involved in recognition mechanisms in fungal cell-wood surface interactions (Nicole et al., 1993). Geiger et al. (1986b) and Nicole (1991) have shown that the pathogen produces both lignin and cellulose degrading enzymes. The work of Galliano et al. (1990) revealed the pathogen produces laccase and manganese peroxidase used for lignin degradation. The current lack of effective methods for the control of this pathogen has led to greater interest in research on the pathobiology of the fungus among rubber producing

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countries many of which are located in developing economies like Malaysia, Indonesia, Sri Lanka, Ivory Coast, Cameroon and Nigeria.

Figure 1: Rigidoporus microporus infection cycle in a rubber tree. Arrows indicate possible infection routes (Omorusi 2012)

1.4 Taxonomy and molecular phylogeny of Rigidoporus microporus

Rigidoporus is a genus of basidiomycetes with about 40 described species. All members of the genus are known to be (bracket fungi) and wood-inhabiting white-rotters. It is a highly polyphyletic genus and most species belong to the Polyporales, but a distinct clade of species is found

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within the Oxyporus clade in the Hymenochaetales (Larsson et al., 2006; Miettinen and Larsson 2011). This clade within the Hymenochaetales contains parasitic species that are of economic importance to numerous hardwood trees (Lindner 2006). The taxonomy of R. microporus is complex. R. microporus and R. lignosus are considered synonyms and both names have in the past been used to describe the fungus. The current accepted name is R. microporus (MycoBank). The source of the type specimen for R. microporus is Boletus microporus Sw. 1788 collected from West Indies while that of R. lignosus is missing in literature and may not exist in any culture collection (Ryvarden 1976). This makes the taxonomy of the R. microporus isolates associated with rubber tree plantations problematic. Culture morphology studies of Asian isolates (Kaewchai et al., 2010) and African isolates (Richard and Button 1996) reported similar morphological characteristics which include a characteristic marginal rhizomorphic strand produced in media. There is thus a need for a taxonomic study at the molecular level of isolates from the rubber producing regions of central Africa and South East Asia; to check for the presence of cryptic species and to show if the isolates from the two continents are conspecific. The identification of wood decay fungi was previously based on morphology, substrate utilization, and reproductive structures. This method is complicated because the fruiting bodies of wood decay fungi are frequently absent or difficult to detect in culture and also often show wide ranges of variability in physiological characteristics, appearances and abilities (Hibbett and Donoghue 2001). The morphological descriptions are used to identify cultivars; however, this approach lacks objectivity and reliability (Moukhamedov et al., 1994). The advances in molecular technology appear to offer a rapid method to identify fungi based on more objective evaluations (Diehl et al., 2004).

1.5 Wood lignocellulose degradation by white rot basidiomycetes

Forest ecosystems need a continuous supply of nutrients. Wood degrading fungi play a major role in the recycling of nutrients through the decomposition of organic matter in forest ecosystems. Successful short-term carbon cycling in forest ecosystems requires degradation of all components of wood lignocellulose by microorganisms, and the basidiomycota fungi play an important role (Lundell et al., 2014). White rot degraded wood is mostly yellow to whitish in colour and may contain deposits of manganese. Majority of the white rot decay fungi belong to the order Polyporales which colonizes wood trucks by forming resupinate basidiocarps (Lundell et al., 2014; Hori et al., 2013). The most abundant source of terrestrial carbon is lignocellulose (Cellulose, hemicellulose, pectin and lignin) which is obtained from both dead and living (Lundell et al., 2014). Plant biomass is composed of about 40-50% cellulose and 15-30% hemicellulose and lignin depending on the plant species (Eriksson et al., 1990). Cellulose is composed of β- 1,4-glycosidic d-glucopyranoses joined by hydrogen bonding and embedded

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within hemicellulose chains composed mostly of arabinoglucuronoxylan and galactoglucomannan. The amorphous, phenylpropanoid polymer lignin forms a matrix around the cellulose and hemicellulose chains (Bidlack et al., 1992; Hori et al., 2014). The lignin content of plant biomass is degraded first by white rot fungi to create access to the more structured carbohydrate cellulose and hemicellulose polymers (Lynd et al., 2002). A recent comparative genomics study has shown that the evolution of the ability to degrade lignin by white rot fungi coincided with the decline in large organic coal deposits from the carboniferous era, about 290 million years ago (Floudas et al., 2012).

1.5.1 Lignin degrading enzymes of wood decay fungi

Lignin degrading enzymes are mostly oxidoreductases produced by white rot fungi. The two major classes of oxidoreductases implicated in wood lignin-modification are laccases and the class II peroxidases (Lundell et al., 2010; Hammel and Cullen 2008; Hatakka and Hammel 2010). Laccases (benzenediol:oxygen oxidoreductases) are polyphenol oxidizing multicopper oxidases which oxidize a broad range of reducing substrates, preferably phenolic compounds. Laccases catalyze oxidation of phenolic compounds and also non-phenolic compounds in the presence of charge-transfer mediators. (Hatakka 2001; Hoegger et al., 2006; Eggert et al., 1996). Laccases are present in multiple copies in most genomes of white rot basidiomycetes sequenced except in the model white rot species, Phanerochaetae chrysosporium. Massive expansion of laccase genes has occurred in the white rot basidiomycetes while the genomes of brown rot fungi contained fewer laccase genes. (Floudas et al., 2012; Riley et al., 2014; Olson et al., 2012; Fernandez-Fueyo et al., 2012; Morin et al., 2012; Eastwood et al., 2011). The class II peroxidases employed by white rot fungi in lignin degradation are the heme-containing peroxidases [lignin peroxidase (LiP), versatile peroxidase (VP) and manganese peroxidase (MnP)] which can act on high-redox lignin-like aromatic substrate molecules (Hatakka 2001; Hammel and Cullen 2008; Hofrichter et al., 2010;

Ruiz-Duenas and Martinez 2009). In general, heme-peroxidases use H2O2 as the oxidant to act on substrate molecules with the release of two electrons producing two molecules of water (Dunford 1991). LiP was the first peroxidase discovered in white rot basidiomycete from P. chrysosporium (Tien and Kirk 1983). LiP has the ability to directly oxidize dimeric lignin compounds. Electron oxidation of the lignin molecule results in the formation of a cation radical intermediate which cleaves to form veratraldehyde and other aromatic derivatives (Lundell et al., 1993a). It has been found in a limited number of white rot fungi; Trametes versicolor, Phanerochaete chrysosporium, Phanerochaete sordida, Phlebia radiata, Phlebia tremellosa and Bjerkandera adusta (Johansson and Nyman 1996; Cullen 1997; Tien and Kirk 1983; Johansson and Nyman 1993; Moilanen et al., 1996; Lundell et al., 1993b). MnP acts on phenols, lignin and

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organopollutants by the oxidation of Mn2+ ions to Mn3+. The chelated Mn3+ act as a diffusible charge transfer mediator attacking larger biopolymers including lignin and other xenobiotic substrates (Hatakaa 2001; Hatakka et al., 2003; Husain 2009). MnP has been further structurally characterized into long-MnPs and short-MnPs. MnP has received more attention as sources of ligninolylic enzymes due to its presence in most white rot basidiomycetes (Lundell et al., 2010; Lankinen et al., 2005; Choi et al., 2014). VP was more recently discovered in Pleurotus and Bjerkandera species and can perform both functions of LiP and MnP. (Mester and Field 1998; Camero et al., 1999). Phylogenomic analysis has revealed that the first MnP enzyme evolved in the lineage. This was followed by several gene duplication events to produce both short and long MnPs. VPs emerged from MnPs by protein surface radical centre tryptophan. Modification of Mn binding site in VP resulted into LiP (Ruiz-Duenas et al., 2013). Other lignin modifying oxidoreductases found in white rot basidiomycetes include; aldo/keto reductase, alcohol oxidase, copper radical oxidase, superoxide dismutase and NADP oxidoreductase (Olson et al., 2012; Riley et al., 2014).

1.5.2 Polysaccharide degrading enzymes of wood decay fungi

Cellulose and hemicellulose are degraded by the carbohydrate active enzymes (Cazymes). The Cazymes are classified into four classes - glycoside hydrolases (GHs), glycosyltransferases (GTs), polysaccharide lyases (PLs) and carbohydrate esterases (CEs) based on their structural or functional domains (www.cazy.org). GHs, PLs and CEs start the breakdown of polysaccharides while GTs catalyse the formation of glycosidic linkages (Lundell et al., 2014). Cellulose and hemicellulose breakdown is carried out by several GHs (endoglucanases, lytic polysaccharide monooxygenases – LPMO (formerly classified as GH61), xylanases, xylosidases, α-arabinofuranosidases,) and CEs (acetyl xylan esterases and glucoronoyl esterases). Enzymes involved in pectin breakdown include galactoronases, rhamnosidases and glucoronyl hydrolases (Zhao et al., 2013). Endoglucanases act on the β-1, 4-glycosidic bonds in non- crystalline cellulose (Lundell 2014), while LPMO act on crystalline cellulose (Vaaje-Kolstad et al., 2010). Xylanases breakdown xylans in hemicellulose while the xylooligosaccharides formed is degraded to monosacharides by xylosidases (van den Brink and de Vries 2001; Shallom and Shoham 2003). Acetyl xylan esterases, glucoronoyl esterases and α-arabinofuranosidases complete the degradation of hemicellulose by acting on side chains (Couturier et al., 2012). Laccases and the class II modifying peroxidases have been included in the CAZy database (www.cazy.org) as auxiliary activity (AA) families (Levasseur et al., 2013).

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1.5.3 Current advances of next generation sequencing technology in furthering our understanding of fungi transcriptome

The advancement of next generation sequencing has resulted in the sequencing of the genome of many white rot basidiomycetes to enable comparative genomic and transcriptomic analysis. Insights on the lignocellulose degrading enzyme machinery of many white rot fungi as well as comparative analysis to infer evolutionary histories of genes are also available (Fernadez-Fueyo et al., 2012; Hori et al., 2013; Martinez et al., 2009a; Morin et al., 2012; Floudas et al., 2012; Riley et al., 2014; Olson et al., 2012; Eastwood et al., 2011; Kohler et al., 2015). The first white rot fungus genome sequenced was P. chrysosporium; revealing a repertoire of lignocellulose degrading genes used for wood degradation. The genome contained more than 200 carbohydrate active enzymes, 5 multicopper oxidases (no laccase) and 16 class II peroxidases (Martinez et al., 2004). Recent genome sequencing of another model white rot fungus Pycnoporus cinnabarinus revealed a vast array of glycoside hydrolases, 15 LPMOs, 33 carbohydrate- binding modules (CBM) but limited pectinolytic enzymes (Levasseur et al., 2014). Transcriptomic studies on P. chrysosporium and other white rot fungi has provided information on the specific groups of lignocellulolytic genes encoding enzymes that white rot fungi may employ in degrading the different polymers found in plant biomass. In particular, transcripts of GH43 and LPMOs (formerly GH61) were highly induced in wood substrates. In Heterobasidion irregulare, several multicopper oxidases and manganese peroxidases were induced on pine wood (Wymelenberg et al., 2009; Raffaello et al., 2014; Olson et al., 2012; Yakovlev et al., 2013). Almost nothing is however known about the diversity of transcripts used by the rubber pathogen during saprotrophic growth on dead wood. Comparative genomics of basidiomycetes has provided insights on the evolutionary relationship between white rot and brown rot fungi. Genome comparative studies (Martinez et al., 2009a; Eastwood et al., 2011) supports an evolutionary shift from white rot to brown rot with much reduced lignin modifying capacity for the later. However, recent similar comparative studies of 33 genomes (Riley et al., 2014) has shown that, the separation of the white rot/brown rot based on lignocellulose degradation ability might not be holistic, but may overlap between the two decay types. The recent sharp decrease in the price of genome sequencing and availability of advanced comparative genomics and transcriptomic tools would lead to better insights on our understanding of fungi lifestyles. This would additionally provide clues on how lignocellulolytic enzymes produced by white rot fungi can be exploited for potential applications in bioenergy and biofuel processing.

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1.6 Defence gene expression of forest trees to pests and pathogens

Insect pests and microbial pathogens pose severe threat in tree plantations. There has been an unprecedented rise in the number of pathogens introduced in commercially planted forests. This problem is predicted to increase based on effects of climate change and movement of humans as a result of the growth of international trade. Commercial plantations are experiencing a big surge in the influx of new pests and diseases or more virulent strains of existing pests and pathogens (Santini et al., 2013; Klapwijk et al., 2013; Wingfield et al., 2015, Payn et al., 2015). Clonally propagated trees like H. brasiliensis and Eucalyptus face the greatest threats as a single insect or pathogen can completely wipe out a single clonal genotype in plantations (Naidoo et al., 2014). Forest trees like all plants defend themselves against various harmful microbes like fungi, oomycete, bacteria and virus. Forest trees possess both preformed barriers and induced resistance mechanisms that are employed to ward off pests and pathogens (Kovalchuk et al., 2013a). Preformed defence mechanisms vary with the type of invading microbe. There are varied responses to biotrophs, hemibiotrophs or necrotrophs. Biotrophs feed on the living host tissues and cause symptoms of disease. Hemibiotrophs has both biotrophic and necrotrophic feeding habit in its life cycle, while the necrotrophs actively kill host cell tissues before feeding on it (Garcia-Guzman et al., 2014; Naidoo et al., 2014). Preformed structural barriers such as the bark, pectin and lignified cell walls are used as the first line of defense against an invading pathogen. Preformed defence may also include antimicrobial peptides and toxic secondary metabolites (Pearce 1996; Kovalchuk et al., 2013a). Inducible defence response is normally employed by forest trees when the preformed barrier is breached by the invading microbe leading to an activation of the innate immunity of the plant. Recognition of the pathogen is followed by signaling and subsequent induction of defence related proteins and other molecules (Jones and Dangl 2006; Dodds and Rathjen 2010). Figure 2 shows a simplified schematic diagram representing induced responses in host cells to pests and pathogens. Pathogenesis related (PR) proteins, (PR1-PR17), which may have anti-microbial properties, are induced in plants in response to microbial attack. Several important PR proteins that have been studied in tree-pathogen interaction systems include PR2, 3, 4 and 8 which are chitinases that attack fungal cell wall. PR5 are thaumatin-like proteins, while PR 9 consists of lignin-forming peroxidases (van Loon and van Strien 1999; van Loon 2009, Veluthakka and Dasgupta 2010). Plants use cell wall modification genes to reinforce cell walls to prevent pathogen attack. Examples of these genes include the α and β expansins (Sampredo and Cosgrove 2005). Phenylalanine ammonia lyase (PAL) signaling gene has also been implicated in cell wall modification (Koutaniemi et al., 2007). In order to increase the intensity of defence signals, various signaling hormones are activated. These signaling molecules include; mitogen-activated kinase (MAPK), salicyclic acid (SA), jasmonic acid (JA) and ethylene (Thancher et al., 2005; Rodriguez

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et al., 2010; Naidoo et al., 2013). Equally important are various transcription factors that activate pathogenesis-related genes during induced defence response in trees.

Figure 2: Simplified schematic diagram representing induced responses in host cells to pests and pathogens. Step 1-PRRs in the plasma membrane recognize PAMPs, HAMPs or DAMPs from pest or pathogen attack. Alternative recognition is the detection of avirulence (Avr) genes by R genes. Step 2-ROS and RNS which contribute to the formation of HR are produced. Step 3-Perception of pathogen by MAPK and other hormone signaling cascades occurs resulting in amplification of defence signal. Perception may also include activation of nucleotidyl cyclase leading to an increase in cytosolic calcium levels. Calcium binds to calmodulin leading to the regulation of NO and H2O, compounds essential for HR. Step 4-Secondary metabolites stimulate production of volatile compounds to alert neighbouring cells of the pathogen attack. Step 5-TFs which activate PR genes are produced. PR genes may have anti-bacterial, antifungal or anti-insect activity. Abbreviations: PRRs-pattern recognition receptors; PAMPs-pathogen-associated molecular patterns; HAMPs-herbivore-associated molecular patterns; DAMPs-damage associated molecular patterns; ROS-reactive oxygen species; RNS-reactive nitrogen species; HR- hypersensitive response; MAPK-mitogen- activated protein kinase; TFs-transcription factors; PR-pathogenesis related (Naidoo et al., 2014) The genomes of a number of tree genera like Populus, Eucalytus, Picea and Pinus have been sequenced. This has allowed for the study and characterization of candidate defence genes. More recently, the genome of the economically important rubber tree, H. brasiliensis was sequenced (Rahman et al., 2013). Until now, there is paucity of information on the kinds of genes engaged by living rubber tree during active defence responses against the invading R. microporus. Therefore, the availability of sequenced tree genomes including that of the rubber tree creates the possibilities of studying gene expression and regulation during pest or pathogen challenge. This would allow for a better understanding of molecular mechanisms and epigenetic regulation involved in host-pathogen interactions. The knowledge acquired from these can be used to select candidate defence genes and molecular markers for marker assisted breeding of commercially planted trees.

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2. AIMS OF THE STUDY

The general aim of the study was to investigate the molecular interaction between Rigidoporus microporus and Hevea brasiliensis

Our specific aims in this study were as follows;

I. To characterize and compare R. microporus isolates from rubber plantations in Nigeria and other geographic regions to see if they belong to one species or represent independent lineages. II. To study the saprotrophic wood decay abilities of isolates of R. microporus pathogenic on H. brasiliensis by comparing with non-pathogenic and non-host isolates. III. To investigate the transcript profile of R. microporus during saprotrophic growth on dead H. brasiliensis wood. IV. To document expression profiles of defence-related genes in two clones of H. brasiliensis inoculated with R. microporus.

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3. MATERIALS AND METHODS

The materials, methods, plant materials and fungal strains used in this study are summarized in Table 1 and Table 2.

Detailed description is available in the original publications.

Table 1: Materials and methods used in this study Materials and methods Publications Analysis of differentially expressed genes III cDNA synthesis III, IV Data filtering, de novo assembly and annotation of RNA-Seq data III DNA extraction I Fungal growth rate assay II Fungal strain and growth conditions I, II, III, IV Microscopy II PCR amplification of ITS, LSU, β-tubulin and tef1 genes I qRT-PCR and data analysis III, IV RNA extraction III, IV Sequencing and phylogenetic analysis I Wood decay test II

Table 2: Plant materials and fungi strains used for saprotrophic and pathogenicity studies Plant materials and Fungal strains Publications Hevea brasiliensis clone PR107 IV H. brasiliensis clone RRIM612 IV Rigidoporus microporus ED310 Nigeria II, III, IV R. microporus M13 Malaysia II R. microporus MS564b Peru II R. microporus MUCL45064 Cuba II

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4. RESULTS AND DISCUSSION

4.1 Molecular phylogeny of Rigidoporus microporus isolates associated with rubber tree plantations

Our aim was to get an insight on the taxonomy of the isolates causing the white rot disease in rubber plantations located in Asia and Africa. We sequenced and analyzed 4 different gene regions (ITS, LSU, β- tubulin and tef1) of 40 isolates of Rigidoporus microporus isolated mainly from rubber plantations in Asia and Africa and some found on other hosts in different regions of the world. Furthermore, comparative phylogenetic analysis of the Rigidoporus genus was carried out with other members of the Polyporales and Hymenochaetales. We generated 83 new sequences (38 ITS rDNA, 5 nLSU rDNA, 19 β-tubulin and 21 tef1 genes) in our study (I; Table 1).

4.1.1 Analysis of ITS sequences of Rigidoporus microporus isolates

The closest genus to the Rigidoporus species in the GenBank was Oxyporus, so Oxyporus corticola was selected as the out-group in our ITS analysis of the R. microporus isolates. The closest species to R. microporus, R. ulmarius was also added to the analysis. Three separate clades were observed in the ITS phylogram [(Clade I (Africa), Clade II (Asia) and Clade III (South/Central America)] with strong bootstrap values (I; Figure 1). The bootstrap values separating the African and Asia isolates were lower than that separating the South/Central America isolates. The pathogenic isolates (Africa and Asia) causing white rot in rubber trees were separated clearly from the other saprotrophic and endophytic isolates from South/Central America isolates using ITS sequences. Addition of other genes in the analysis is expected to produce a clearer picture of the difference between the pathogenic species from Africa and Asia. Differences in symptoms on the host of the white rot disease in Asia and Africa were attributed to environmental factors (Riggenbach 1960). The ITS phylogeny also revealed that, the Africa and Asian isolates did not show any geographic pattern in their distribution with respect to countries within the clades (I; Figure 1). The Cuban isolate separated from the Brazilian and Peruvian isolates, but they all clustered together in Clade III.

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4.1.2 Analysis of β-tubulin sequences of Rigidoporus microporus isolates

The β-tubulin gene was sequenced for selected isolates from Clade I (Africa) and Clade II (Asia) which were not clearly resolved by ITS phylogeny. The β-tubulin coding region has more intra-specific variability in basidiomycetes than the ITS region (Thon and Royce 1999). The β-tubulin phylogram separated the isolates into their respective clades I and II with high bootstrap values for both maximum parsimony and maximum likelihood analysis (I; Figure 2). The β-tubulin phylogram showed some intra- specific variation between the isolates in each clade. However, there was no clear separation with respect to bootstrap values within the clades. In Clade I, the isolates collected from four different locations in Nigeria clustered together and the Cameroon isolates were nested within the Nigerian isolates. The β- tubulin gene analysis also suggested gene flow within the Nigerian and Cameroon isolates. Interestingly, isolates collected from non-H. brasiliensis hosts in Africa and Asia clustered with those collected from H. brasiliensis (I; Figure 2), suggesting possibilities of a possible host-jump from native trees to rubber trees. The pathogen had earlier been reported to be pathogenic on an indigenous African woody species, Triplochiton scleroxylon (Begho and Ekpo 1987).

4.1.3 Analysis of tef1 sequences of Rigidoporus microporus isolates

Additionally, a third gene, the tef1 was sequenced for selected isolates from Clade I (Africa) and Clade II (Asia) to support the results gotten from β-tubulin analysis. The tef1 phylogram was similar to that of the β-tubulin, clearly separating the isolates into Clade I (Africa) and Clade II (Asia) (I; Figure 3). The isolate from non-H. brasiliensis host also clustered among those isolated from rubber trees (I; Figure 3). Intra-specific variations within the clades were observed in the phylogram, with the Cameroon isolates clustering together, but with low bootstrap support. The low bootstrap support within the clades suggests the presence of a high level of intra-species variation. These results were in agreement to other studies. Kaewchai et al. (2009) using inter simple sequence repeat (ISSR) markers discovered a very high level of genetic variation among R. microporus isolates collected from rubber trees in different regions in Thailand.

4.1.4 Analysis of combined ITS, β-tubulin and tef1 sequences of Rigidoporus microporus isolates

Combining the sequences of several gene regions in phylogenetic analysis improves resolution of phylogenetic trees. In order to further support our phylogenetic analysis of the separation of Clade I (Africa)

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and Clade II (Asia), we performed homogeneity test to check if the 3 gene regions (ITS, β-tubulin and tef1) could be combined for phylogenetic analysis. Homogeneity test showed that the genes were congruent and could be combined. The topography of the phylogram of the combined genes was similar to that of the β- tubulin and tef1 analysis (I; Figure 4). However, there were more intra-specific variations within the clades with slightly increased bootstrap support at the nodes. The isolates from non-H. brasiensis host also clustered with those collected from diseased rubber trees (I; Figure 4). Our data suggest that it seems very unlikely that the R. microporus causing disease in rubber plantations in Africa and Asia originated in South America, rather the pathogen may have jumped host from native trees into H. brasiliensis. This would not be surprising as host shifts through evolutionary processes have been reported for several basidiomycete pathogens (Yang 2011). Fomitoporia mediterranean, the vine yard pathogen jumped hosts from native trees to grapevines and Citrus (Elena et al., 2006). F. capensis, native to South Africa was also reported to jump host to grapevines (Cloete et al., 2013). Further studies would be targeted at the development of microsatellite markers to trace the precise origin of the pathogen. This can improve our knowledge of the spread of the disease and how to manage it in rubber plantations.

4.1.5 Analysis of LSU sequences of Rigidoporus microporus isolates

Current classification of the Rigidoporus genus placed it in the order Polyporales. However, previous taxonomic studies showed that some species of Rigidoporus clustered together with the Oxyporus clade in the Hymenochaetales (Larsson et al., 2006; Miettinen and Larsson 2011). In order to resolve the taxonomic grouping of the pathogenic isolates used in this study, we sequenced and analyzed the LSU gene region for selected isolates. The LSU gene is reported to have a better classification accuracy at the genus level compared to ITS and functional protein coding region genes (Porras-Alfaro et al., 2014). We analyzed 5 sequences of R. microporus from our study and 33 sequences of members of the Polyporales and Hymenochaetales. The isolates from Clade I (Africa) and Clade II (Asia) clustered together, while the South American isolate from Brazil formed a distinctive group (I; Figure 5). Interestingly, the R. microporus isolates also clustered together closer to Oxyporus (Hymenochatales) than other members of Polyporales (I; Figure 5). Similar cultural and morphological characteristics had been reported for R. microporus and some species of Oxyporus and R. microporus was combined to Oxyporus (Roy and De 1998). Our data revealed that some species of the Rigidoporus genus share close sequence similarities to the Hymenochaetales which suggests that a thorough revision of the Rigidoporus genus within the Polyporales/Hymaenochataeles is needed.

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4.2 Saprotrophic growth of Rigidoporus microporus on rubber wood

Rigidoporus microporus produces rhizomorphs which can spread several meters in the soil. The rhizomorphs are used as a means of infecting and deriving nutrients from the host. These rhizomorphs have also been noticed to colonize dead rubber wood debris in the soil for very long periods (Nandris et al., 1988). This observation points to a capacity for saprotrophic growth on wood. Based on our hypothesis that R. microporus can equally live as a saprotroph on dead trees, we performed wood decay and light microscopy test on selected isolates. We added two isolates from clade III (South/central America) in our previous study (I); the Cuban isolate obtained from a non-H. brasiliensis host and an isolate from Peru isolated from the sapwood of healthy H. brasiliensis in the natural forest and identified to have an endophytic habit (Martin et al., 2015). The isolates used were; one pathogenic isolate each from Africa (ED310 Nigeria) and Asia (M13 Malaysia), the Cuban isolate (MUCL45064) and the endophytic isolate from Peru (MS564b). The mass loss and structural alterations of rubber wood blocks (after 3 and 6 months) due to saprotrophic growth of the isolates were analysed.

4.2.1 Mass loss of Hevea brasiliensis wood blocks due to degradation by Rigidoporus microporus isolates

The rubber wood blocks were densely colonized with the characteristic mycelial strand of Rigidoporus microporus (Richard and Button 1996) on 3 of the isolates except for the endophytic isolate which showed very faint colonization. The isolate MUCL45064 had the highest wood decay capacity; degrading 19.3% and 27% of the wood after 3 and 6 months respectively. This was followed by the ED310 and M13 pathogenic isolates with decay capacities of 13.7% and 21.2%, and 11.3% and 15.7% after 3 and 6 months respectively. The endophytic isolate had a very low decay ability; 4.3% after 6 months (II; Figure 1). The values for mass loss in rubber wood blocks in this study were comparable to those reported from similar studies with the blue stain fungus, Lasiodiplodia theobromae on rubber wood (Encinas and Daniel 1996; 1997). An interesting observation was that the Cuban isolate from a non-H. brasiliensis host had the highest wood decay ability. At this point, we cannot speculate if the saprotrophic ability of this isolate equates to a potential to be pathogenic on rubber trees if it undergoes a host-jump. Evidence from our previous study (I) suggests the possibility of R. microporus to have jumped from native trees into rubber plantations distributed in South East Asia and Africa. Further support for this is provided by recent studies on the discovery of R. microporus for the first time on diseased nobilis in Sri Lanka (Madushani et al., 2013). The absence of the characteristic mycelial strand in the non-pathogenic isolate further supports its endophytic habit. There was no significant difference between the mass loss caused by the pathogenic

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isolates ED310 and M13 after 3 months (p > 0.05). There was also no significant difference in mass loss between the Peruvian endophytic isolate and the control (sterile agar) after both 3 and 6 months (p > 0.05) (II; Figure 1). 4.2.2 Effects of temperature on in vitro growth rates of Rigidoporus microporus isolates

We also investigated the effects of temperature on the growth rate of the isolates in malt extract media. This was done to support the differences observed with the wood decay capabilities. The isolates (MUCL45064, MS564b) grew at all the temperatures tested while the pathogenic isolates (ED310, M13) did not grow at all after 6 days at 15 °C. Isolate M13 had the highest hyphae growth length (78.3mm) after 6 days (II; Figure 2). The interaction of temperature and isolate on hyphae length was tested using a two- way ANOVA (p ≤ 0.05). Results indicated a significant interaction (F = 103.26, p = 0.001). Based on this, we decided not to consider the individual effects of temperature and isolate on hyphae length. Nevertheless, increased growth rate at increased temperature was observed except for the endophytic isolate which had a faster growth rate at 25 °C (52.1 mm) than at 30 °C (41 mm) after 6 days (II; Figure 2). This data suggest that the isolates used in this study and collectively named as R. microporus (Mycobank, GenBank) are likely different species as our previous study (I) suggested.

4.2.3 Light microscopy of structural alterations in wood blocks of Hevea brasiliensis induced by Rigidoporus microporus isolates

In order to confirm the mass loss results, we carried out light microscopy to visualize structural changes in rubber wood blocks due to saprotrophic growth by Rigidoporus microporus. Characteristic simultaneous preferential degradation and cell wall thinning typical for white rot basidiomycetes were observed for all 4 isolates. Signs of delignification were prominent in the ray cells by the differential staining changes from red to pink, indicating a gradual loss of lignin towards the fibers (II; Figure 3A, C). Delignification was accompanied by cell wall thinning (II; Figure 3A). Delignification of the middle lamella also occurred which resulted into fiber separation (II; Figure 3B). Aniline blue staining showed hyphae of the isolates in the vessels and ray cells (II; Figure 3D, E, F). Direct hyphal penetration through the rays, vessels and other cellular components was observed for all the isolates except the endophytic isolate (MS564b) which only spread in fibers (II; Figure 3F). The differential pink and red safranin staining (Srebotnik and Messner 1994) observed in the decayed rubber wood was an evidence that delignification by the fungus occurred. There was widespread distribution of fungal hyphae in the vessels, fibres and tracheids as seen in other basidiomycete decayed wood (Schwarze et al., 2004; Schwarze et al., 2007). This

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was most prominent and commonly observed with isolate ED310. The decay patterns observed were similar to those seen in structural alterations of wood by other white rot basidiomycetes like Heterobasidion annosum (Daniel et al., 1998), Phanerochaete chrysosporium (Koyani and Rajput 2014) and Schizophyllum commune (Padhiar and Albert 2012). This study further shed lights on the potential contribution of R. microporus to carbon cycling through saprotrophic abilities on dead trees killed by the fungus. This knowledge would be vital in developing control measures for the pathogen in rubber plantations.

4.3 Gene expression of Rigidoporus microporus during saprotrophic growth on rubber wood

Besides, its lifestyle as a pathogen on rubber trees, our previous study (II) confirmed that Rigidoporus microporus has the ability to switch to saprotrophic growth on dead wood. It is therefore expected that the fungus would possess a range of enzymes that are utilized in lignocellulose degradation. Such enzymes could have potential applications in bioenergy processing and utilization. On the other hand, the rubber tree, Hevea brasiliensis is economically important because of the natural rubber (latex) it produces. Natural rubber (cis-1, 4-isoprene) is a secondary organic compound produced by the tree after wounding on the stem. This latex is indispensable in the production of a variety of industrial materials which includes tyre for aircrafts and heavy duty machinery. Ability of the pathogen to survive on the root and collar of the tree may depend on its ability to degrade and metabolize natural rubber; therefore R. microporus may possess relevant genes that code for enzymes that can degrade natural rubber. Furthermore, there is almost no information on the interaction of this pathogen on the rubber tree at the genomic and transcriptomic level. With the above information, we decided to perform RNA-Seq de no transcriptomic assembly and profiling of R. microporus during saprotrophic growth on rubber wood. We sequenced the libraries created using the Illumina HiSeqTM 2000, assembled and analysed genes expressed by comparing the transcriptomes of the fungus grown on wood and in the absence of wood material. The RNA-Seq data was validated by quantitative real-time PCR (qRT-PCR) for 23 selected genes of interest. The qRT-PCR transcript profiles for all the genes tested were consistent with the RNA-Seq data (III; Figure 8).

4.3.1 General overview of the Rigidoporus microporus transcriptome

Three replicates each of the two experimental conditions; with or without wood were separately sequenced: W (R. microporus growing on rubber wood blocks) and C (R. microporus in the absence of rubber wood). Each of the six samples produced over 40 million RNA-Seq raw reads (90bp) using the Illumina HiSeqTM 2000 which was considered a very good coverage. In total, 266.6 million raw reads

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were generated, which gave rise to 251.1 million clean reads and 22.6 billion clean nucleotides after cleaning and quality checks (III; Table 1). Assembly of the clean reads was done using Trinity sequence clustering program. Assembly of the clean reads resulted into 34, 518 unigenes with an average length of 2179 bp. Two sets of unigenes were assembled. Distinct clusters (26,447) with the prefix CL are cluster unigenes; the same cluster contains similar unigenes (>70% similarity). Distinct singletons (8,701) with the prefix Unigene are unigenes from a single gene (III; Table 2). Unigenes having lengths in the range of ≥ 3000bp represented the highest number in the assembly (III; Figure 1). The quality of the sequencing was further checked by mapping the clean reads to the assembled unigenes. Mapping coverage for all samples were greater than 95 % (III; Table S1). Functional annotation of unigenes was done using five different databases [NR/NT (genBank), Swiss-Prot, KEGG, COG and GO]. This ensured a high percentage of annotated unigenes. Protein coding prediction of open reading frames (ORF) of unigenes resulted in 26, 663 unigenes, out of which 25, 880 (74.98%) unigenes were functionally annotated (III; Table S2). The number of annotated unigenes compared favourably with the gene models annotated in other basidiomycetes fungi; white rotters: Fomitiporia mediterranea, 11,738; Heterobasidion irregulare, 13,328; Phanerochaetae chrysosporium, 13,602 and Trametis versicolor, 14,530 (Ohm et al., 2014) and brown rotter: Postia placenta, 17,173 (Martinez et al., 2009a). The number of unigenes annotated with each database is shown in III; Table S3. The R. microporus transcriptome showed closest similarities with the Fomitiporia mediterranea genome (51.6% of hits). The sequence homology of blast results of the transcriptome against NCBI non-reductant (Nr) database is shown in III; Figure S1. Cluster of orthologous groups (COG) and Gene Ontology (GO) classification of the unigenes are provided on III; Figure 2 and III; Figure 3 respectively. Differentially expressed genes of the two transcriptomes (W and C) were analysed using edgeR package in R programming. A total of 2996 genes were significantly up-regulated while 2128 genes were significantly down-regulated in rubber wood at a cut cut-off of FDR < 0.05 and log2FC > 2 (III; Fig 4). The list of all differentially up-regulated genes (2996) and all differentially down-regulated genes (2128) are shown in (III; Table S4) and (III; Table S5) respectively. We increased the stringency of differentially expressed genes up to FDR < 0.001 and log2FC > 4 and still obtained a high number of differentially expressed genes, 392 up-regulated and 228 down-regulated (III; Fig 4). A subset of the most-highly up- regulated and down-regulated genes with functional annotation was compiled in (III; Table 3) and (III; Table 4) respectively. There were over 400 lignocellulose degrading enzyme genes differentially expressed in the transcriptome.

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4.3.2 Rigidoporus microporus genes encoding polysaccharide degrading enzymes during saprotrophic growth on rubber wood

Rigidoporus microporus is a white rot and members of the Polyporales are known to be active wood lignocellulose degraders (Hori et al., 2013). All 4 classes [glycoside hydrolases (GHs), glycosyltransferases (GTs), polysaccharide lyases (PLs) and carbohydrate esterases (CEs)] of carbohydrate active enzymes in the database (http://www.cazy.org) were differentially expressed in the transcriptome. The transcriptome produced 225 differentially expressed GHs in 33 families and 105 of these were up- regulated during saprotrophic growth on rubber wood. The highest numbers of expressed transcripts were observed in GH7, GH13 and GH16 families with 36, 23, 18 and 15 transcripts respectively. All GH12, GH28, GH30, GH35, GH39, GH43, GH51, GH53, GH78, GH79 and GH88 genes were up-regulated in rubber wood (III; Table S6, Figure 5), while all GH3, GH17, GH23, GH27, GH32, GH37, GH38 and GH72 genes were down-regulated in rubber wood (III; Table S6). GH1, GH7, GH12, GH55 and GH61 transcripts were highly expressed in the transcriptome. GH12 and GH55 contain bond cleaving endoglucanases. The bond cleaving endoglucanases are known to attack β-1, 4-glycosidic bonds in non- crystalline cellulose (Medie et al., 2012). It is important to note here that all GH12 transcripts were up- regulated in rubber wood. GH61 family which is now re-classified in the auxiliary activity family (AA9) lytic polysaccharide monooxygenase –LPMO in the CAZy database (http://www.cazy.org) is implicated in crystalline cellulose degradation (Levasseur et al., 2013). Our data agrees with other transcriptomic studies for Phanerochaete chrysosporium and Heterobasidion irregulare (Wymelenberg et al., 2011; Raffaello et al., 2014) where several GH61 transcripts were up-regulated in wood. Seven GH family unigenes [GH7 (CL2079.Contig4), GH10 (Unigene4693), GH61 (CL374.Contig4), GH71 (CL94.Contig1), GH43 (CL114.Contig6), GH43 (CL114.Contig2), GH61 (CL374.Contig3)] were up-regulated more than 16 fold in rubber wood; out of which GH7 (CL2079.Contig4) and GH10 (Unigene4693) were up-regulated more than 71 and 69 fold respectively (III; Figure 5, Table S6). Hemicellulose breakdown is complex and requires backbone cleaving and de-branching due to the presence of acetyl groups and covalent cross- linkages (Martinez et al., 2009b). GH10, GH39, GH43, GH51, CE1 and CE15 genes implicated in hemicellulose degradation were strongly up-regulated in rubber wood. Xylanases important for xylan breakdown are found in the GH10 and GH11 families (van den Brink and de Vries 2001). Xylooligosaccharides produced from the action of xylanases on xylan is degraded by xylosidases seen in GH3 and GH39 (De Vries and Visser 2001). An important GH family gene implicated in hemicellulose degradation is GH43. It consists of a variety of enzymes that cleave glycosidic linkages of hemicellulose (Floudas et al., 2012). Interestingly, all 9 of the GH43 and all GH39 transcripts were up-regulated in rubber wood. Hemicellulose breakdown is also accomplished with the activities of α- arabinofuranosidases, seen

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in GH51 and GH54 families (Couturier et al., 2012). Carbohydrate esterases involved in hemicellulose breakdown include CE1 (acetyl xylan esterases) and CE15 (glucoronoyl esterases). GH51, GH54, CE1 and CE15 are involved in the latter stages of hemicellulose degradation where they cleave complex branching (Couturier et al., 2012). All GH28, GH78 and GH88 transcripts which have been implicated in pectin degradation (Martens-Uzunova and Schaap 2009) were up-regulated in rubber wood. The pectin genes up- regulated in the transcriptome suggests its importance during saprotrophic growth and might also be relevant for the pathogenic lifestyle of the fungus. Zhao et al. (2013) had earlier reported the important role of GH28 family in pectin degradation for pathogenic fungi. Eleven families of 37 GTs were differentially expressed in the transcriptome (III; Table S6). Unigene2737 representing GT1 was up-regulated 96 fold in rubber wood (III; Figure S2A, Table S6). Two families of 8 PLs and five families of 28 CEs were differentially expressed in the transcriptome. Four PLs were up-regulated more than 4 fold, while 11 CEs were up-regulated more than 8 fold in rubber wood (III; Table S6, Figure S2B, and Figure S2C). Chitin deacetylase (CE4) and Pectin esterases (CE8) highly expressed in the transcriptome have previously been identified in genomes of some white rot polypores (Hori et al., 2013). A carbohydrate esterase family 7 (CE7) transcript (CL2913.Contig1) was up-regulated 575 fold in rubber wood (III; Figure S2B). This transcript was also in the top 3 most up-regulated gene with functional annotation in the transcriptome (III; Table 3). The CE7 transcript (CL2913.Contig1) shared closest similarity (54%) with a CE7 (Cephalosporin esterase) of the white rot fungus, Fomitiporia mediterranea (GenBank: EJD07867.1). This gene has been widely studied and characterized in ascomycetes (Politino et al., 1997) but not in basidiomycetes. Further study and characterization of this gene should provide some insights on its function in basidiomycetes. The number of unigenes in the transcriptome that are involved in glycan biosynthesis and metabolism is shown in III; Figure S3.

4.3.3 Rigidoporus microporus genes encoding glycolignin attacking enzymes during saprotrophic growth on rubber wood

The lignin component of wood forms a matrix around the more structural cellulose and hemicellulose units, and thus is usually degraded first to expose the other components. Laccases [multicopper oxidases (MCO)] and the class II peroxidases (lignin, versatile and manganese peroxidases) are the major oxidoreductase lignin-modifying enzymes employed by white rot basidiomycetes in lignin degradation (Hatakka 2001). Several laccases and a large number of manganese peroxidases were expressed in the transcriptome (III; Table S7). Other oxidoreductase encoding genes differentially expressed in the transcriptome include; aldo/keto reductase, NADP oxidoreductase, alcohol oxidase, aryl-alcohol oxidase, copper radical oxidase, superoxide dismutase, cellobiose dehydrogenase and NADH:ubiquinone

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oxidoreductase (III; Table S7). Seven multicopper oxidases (5 laccases and 2 ferroxidases) encoding genes were up-regulated more than 4 fold in rubber wood (III; Figure 6A). Twenty-six manganese peroxidases (MnP1, MnP2 and MnP3) encoding genes were differentially expressed in the transcriptome. Twenty-four of these were up-regulated in the presence of the rubber wood. All 22 MnP3 genes and the single MnP2 gene were all up-regulated in rubber wood (III; Figure 6B, Table S7). Ten MnP3 genes were up-regulated more than 4-fold (III; Figure 6B, Table S7). The single MnP2 gene (CL4964.Contig4) was up-regulated 195 fold in rubber wood (III; Figure 6B, Table S7). The MnP2 unigene (CL4964.Contig4) was in the list of the top 12 most up-regulated genes with functional annotation in the transcriptome (III; Table 3). The number of laccases and manganese peroxidase transcripts highly expressed in rubber wood suggests potential relevance of these enzymes in lignocellulose degradation of rubber wood. Previous study had earlier shown that R. microporus produces laccases and manganese peroxidases which act synergistically suggesting they may have complementary role in lignin degradation (Galliano et al., 1990; 1991). The single MnP2 found to be highly expressed in rubber wood shared closest similarity (75%) with the MnP2 of the white rot fungus, Fomitiporia mediterranea (GenBank: EJD02610.1). The number of significantly up-regulated MnPs transcript in this study is significantly high compared with other white rot transcriptomic studies using wood as substrate. Transcriptomic studies of Heterobasidion irregulare growing on a gradient of bark, heartwood and sapwood using microarray, showed that a total of 5 MnPs and 3 multicopper oxidases were significantly up-regulated (Raffaello et al., 2014). Another RNA-Seq transcriptomic study of the model white rot basidiomycete, Pycnoporus cinnabarinus growing on birch wood expressed only 3 laccases and 1 MnP (Levasseur et al., 2014). The differences observed with these other studies could be due to a number of reasons. Firstly, differences could be due to certain technical advantages of deep sequencing RNA-Seq technology (higher increased sensitivity, better discrimination of transcripts and ability to detect new gene models) compared to microarray (Westermann et al., 2012; Kohler and Tisserant 2014). Secondly, differences could be due to the fact that, previous reports have indicated that the number and variety of enzymes employed in lignocellulose degradation is quite diverse and depends on substrate, lifestyle and fungal species (Floudas et al., 2012; Kohler et al., 2015). Eight aldo/keto reductase genes were up-regulated more than 4 fold in rubber wood (III; Table S7, Figure 6C). Unigenes; NADP oxidoreductase (CL4346.Contig2) and alcohol oxidase (CL4203.Contig3) were up-regulated more than 30 fold in rubber wood (III; Figure 6D). Eleven transcripts of yteT-domain oxidoreductase genes were specifically induced only in rubber wood and 5 of these genes were up-regulated more than 30 fold (III; Figure S4, Table S7). The yteT-domain oxidoreductase has been implicated in the degradation of type I rhamnogalacturonan derived from plant cell walls (Ochiai et al., 2007).

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4.3.4 Rigidoporus microporus genes encoding enzymes involved in fatty acid and rubber tree latex degradation during saprotrophic growth on rubber wood

Rigidoporus microporus characteristic reddish brown basidiocarps are often seen growing on the roots and stems of a decaying rubber tree. Natural rubber latex (cis-1, 4-isoprene) is located in the laticifer tubes of the phloem in H. brasiliensis. The flow of latex is actually a defence response to wounding on the stem of the tree. This is a process exploited for tapping and collection of rubber latex by cutting the bark of the tree. Survival of R. microporus evidenced by the basidiocarps seen even on partly decayed trees might require the capacity to degrade latex and survive in the latex rich environment on the rubber tree. Ability to degrade natural rubber latex would also mean, the pathogen could probably have the capacity to degrade other fatty acid metabolites. The degradation pathway for latex has been proposed using rubber latex degrading bacteria Gordonia polyisoprenivorans (Hiessl et al., 2012). A significantly large number of unigenes directly implicated in the rubber latex degradation pathway were expressed in the R. microporus transcriptome. KEGG Pathway enrichment analysis revealed the number of expressed unigenes in the transcriptome involved in fatty acid biosynthesis, elongation and degradation. Genes implicated in natural rubber latex (cis-1, 4-isoprene) degradation were also found to be induced in the transcriptome (III; Table 5, Figure S5). Acyl-CoA synthetase, Enoyl-CoA hydratase, 3-Hydroxyacyl-CoA dehydrogenase and acyl- CoA acetyltransferase transcripts involved in rubber latex degradation pathway were also detected in the fatty acid metabolism pathways (III; Table 5). Acyl-CoA acetyltransferase releases acetyl-CoA into the citric acid cycle (Hiessl et al., 2012). Acyl-CoA synthetase, enoyl-CoA hydratase and acyl-CoA acetyltransferase transcripts were highly induced in both conditions (C and W) used in this study suggesting constitutive expression of some of the genes. The total number of unigenes in the transcriptome involved in other lipid metabolism pathways is shown in III (Figure S5). Our study shows that R. microporus possess the potential abilities for rubber latex degradation based on the number of unigenes expressed. The constitutive expression of these unigenes in both experimental conditions also point to the potential abilities of the fungus to degrade and metabolize rubber tree latex. This is further supported by the induction of several genes in the transcriptome that are implicated in fatty acid degradation. This ability might be crucial for the pathogen to survive on the living H. brasiliensis tree. Considering the molecular evidence for a possible host shift from other trees to H. brasiliensis and the evolution of the pathogen in absence of H. brasiliensis (I), the pathogen might have acquired genes that can effectively metabolize fatty acid secondary metabolites produced by the tree. This ability might be responsible for its survival in the tree with the presence of latex.

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4.3.5 Rigidoporus microporus ABC transporters and hydrophobins expressed during saprotrophic growth on rubber wood

The ATP binding cassette (ABC) transporters and hydrophobin genes differentially expressed in the transcriptome were also compiled and analysed. ABC transporters have various predicted functions including the transport of materials across biological membranes (Kovalchuk et al. 2013b). ABC transporters belonging to eight families (ABC-A, ABC-B, ABC-C, ABC-D, ABC-E, ABC-F, ABC-G and ABC-I) were differentially expressed in the transcriptome. Families ABC-B and ABC-G were more highly expressed in the transcriptome. Three genes [ABC-G (CL3640.Contig9, CL3640.Contig8); and ABC-B (CL893.Contig27)] were up-regulated more than 5 fold in rubber wood (I; Table S8). Studies have shown that the ABC family ABC-G may play important roles in host-pathogen interactions involving white rot basidiomycetes. A specific ABC-G transporter was shown to exhibit monoterpene resistance in the pine pathogen Grosmannia clavigera (Wang et al., 2013). Another ABC-G transporter in the wood decay fungus Phlebiopsis gigantea was implicated in resistance to terpenes produced by conifers (Hori et al., 2014). Another interesting protein are hydrophobins, which are surface active proteins implicated in different stages of fungal life cycle, pathogenicity factors and biological control mechanisms (Karlsson et al., 2007; Mgbeahuruike et al., 2012). Twenty-two hydrophobin encoding genes were differentially expressed in the R. microporus transcriptome. The 22 hydrophobin transcripts expressed in the transcriptome is comparable to 16 transcripts expressed in microarray studies of Heterobasidion sp. on pine wood (Mgbeahuruike et al., 2013). Unigenes representing trihydrophobins (CL3523.Contig2, Unigene2377) and hydrophobin 2 (CL996.Contig2, Unigene3334) genes were up-regulated more than 2.5 fold in rubber wood (I; Table S9). A fungal hydrophobin (CL2382.Contig2) was down-regulated 145 fold in rubber wood and is in the list of top 6 most down-regulated genes with functional annotation in the transcriptome (I; Table S9, Table 4).

4.3.6 Rigidoporus microporus genes encoding enzymes involved in pathways related to energy metabolism during saprotrophic growth on rubber wood

White rot basidiomycetes are active wood degraders and thus would express transcripts involved in various pathways related to carbohydrate metabolism. Lastly, in our transcriptome analysis, we further elucidated the mechanisms involved in wood degradation and utilization of the degradation products by R. microporus by analyzing genes affected during carbohydrate metabolism. We analyzed genes in the transcriptome that encodes enzymes involved in the Glycolysis/Gluconeogenesis and Citric acid (TCA) pathways using KEGG pathway enrichment analysis with cut-off for significantly expressed genes set at; FDR < 0.001 and Fold Change > 2. Two genes coding for enzymes involved in the early stages of glycolysis

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(phosphoglucomutase (EC:5.4.2.2) and glucose-6-phosphate isomerase (EC:5.3.1.9) were down-regulated while fructose-1,6-bisphosphatase I (EC:3.1.3.11) was up-regulated in rubber wood (I; Figure 7). The higher induction of these transcripts in the control might be due to more resource allocation due to the absence of lignocellulose substrates in the control. Isocitrate dehydrogenase (NAD+) (EC:1.1.1.41) was up-regulated more than 10.5 fold, while acetyl-CoA synthetase (EC:6.2.1.1) was up-regulated more than 3 fold in rubber wood (I; Figure 7). Isocitrate dehydrogenase (NAD+) converts isocitrate to 2-oxoglutarate while acetyl-CoA synthetase synthesizes acetyl-CoA for the citric acid cycle. Finally, alcohol dehydrogenase (NADP+) (EC:1.1.1.2) was up-regulated more than 3 fold in rubber wood (I; Figure 7). Alcohol dehydrogenase (NADP+) interconverts acetaldehyde to ethanol in a reversible reaction. The up- regulation of alcohol dehydrogenase in the R. microporus suggests a possible exploitation of this step for bioethanol production or the direct production of ethanol from lignocellulose biomass. Basidiomycete mushrooms that produce alcohol dehydrogenase have been exploited in mushroom fermentation to produce alcoholic wines (Okamura et al., 2000; 2001). The alcohol dehydrogenase producing basidiomycete, Flammulina velutipes was reported to convert D-glucose to ethanol at an efficiency of 88% during ethanol fermentation (Mizuno et al., 2009). The total number of unigenes in the transcriptome involved in other pathways of carbohydrate metabolism is shown in I (Figure S6).

4.4 Defence gene expression of Hevea brasiliensis in response to Rigidoporus microporus infection

There are presently no studies on defence gene expression profiles of Hevea brasiliensis after inoculation with the white rot pathogen, Rigidoporus microporus. In order to get an overview of candidate defence genes in H. brasiliensis, we artificially inoculated two clones (RRIM612 and PR107) with different levels of susceptibilities with the fungus. With the use of molecular tools and availability of the draft genome sequence of H. brasiliensis (Rahman et al., 2013), we studied gene expression profiles of pathogenesis related proteins (PR1, PR3, PR5, PR8 and PR9), cell wall modification genes (PAL and expansin), signal transduction genes (ACC oxidase, AOC, MAPK) and a Myb transcription factor. H. brasiliensis is mainly propagated through the use of clones derived by budding or grafting and the variability between clones may lead to significant differences in relative transcript abundance. Gene expression profiles in grafted or budded clones are influenced greatly by the root stock as reported in grafted apple clones (Jenson et al., 2012). Some candidate defence genes of interest were also discovered in this study.

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4.4.1 Necrotic lesions produced in Hevea brasiliensis clones after inoculation with Rigidoporus microporus

Strong necrotic lesions were observed in both clones after 5 weeks (IV; Figure 1A and B). Significant differences (P < 0.05) were observed between inoculated and mock-inoculated for both clones (IV; Figure 1A). The necrotic lesion produced in RRIM612 was higher than that in PR107 (IV; Figure 1B), so we decided to classify the clones as RRIM612 (Highly susceptible) and PR107 (Least susceptible).

4.4.2 Expression of pathogenesis related proteinss

The eukaryotic translation initiation factor (eIF3) described previously for gene expression studies in H. brasiliensis (Li et al., 2011), was used as the internal reference gene for normalization (IV; Figure 2). The pathogenesis related proteins are induced in pathological or related situations which include wounding (van Loon et al., 2006). Expression of PR1 varied widely for the two clones. The treatment was down-regulated 10 fold in RRIM612 (P < 0.001) while the expression in PR107 had contrasting results as there was no significant difference in the transcript fold change for both treatment and control (IV; Figure 3A). The PR1 group of proteins is distinct and highly conserved, however, they are not known to have a specific type of defence function in plants, neither are they related to other proteins (Van Loon and Van Strien 1999). It seems the PR1 protein might not be a pre-condition for inducing resistance, but rather acts as a contributor to the protective state of the plant (Van Loon 1997). Expression of PR3 (a putative class I chitinase) was highly up-regulated in the treatment over control in both clones. The PR3 expression results were similar to results obtained in transcriptomic studies of two Eucalyptus glandis clones challenged with the stem canker pathogen, Chrysoporthe austroafricana where several PR3 chitinases transcripts were up- regulated in both clones (Mangwanda et al., 2015). The transcript fold change was significantly up- regulated 43 times in RRIM612 (P < 0.05) and 30 times in PR107 (P < 0.001) (IV; Figure 3B). The PR5, thaumatin-like protein family was up-regulated in both clones in response to the pathogen; Up-regulated 11 times in response to treatment over control in both clones. A significant difference at P < 0.01 for RRIM612 and P < 0.05 for PR107 was observed (IV; Figure 3C). PR5 proteins have the potential for crop improvement and biotechnological applications because of their established antifungal properties (Liu et al., 2010). Studies of pathogenesis-related proteins in Eucalyptus glandis revealed the up-regulation of both PR3 and PR5 in response to the phytohormones, methyl jasmonate and salicyclic acid respectively (Naidoo et al., 2013). Expression of PR8 (an endochitinase) also varied between the two clones. It was significantly down-regulated 3-fold in RRIM612 (P < 0.05), but there was no significant difference between treatment and control for clone PR107 (IV; Figure 3D). PR9 (peroxidases) had two contrasting transcript fold change

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level for the clones studied. There was no significant difference between treatment and control for RRIM612 while transcript fold change was up-regulated 9 times in the treatment for PR107 at a significance level of P < 0.05 (IV; Figure 3E). Although, there has been no studies using modern molecular tools like qRT-PCR and genome resources to study gene expression of H. brasiliensis when challenged with R. microporus, Geiger et al. (1989), using in vitro enzymatic assays reported the production of peroxidase in tissues of H. brasiliensis inoculated with R. microporus. Peroxidases are known to play an active role in cell wall lignification; it is therefore possible that increased transcript abundance of peroxidase in clone PR107 may have contributed to its low susceptibility to R. microporus infection. Other authors have reported similar results. The peroxidase activity in two clones of Eucalyptus glandis challenged with Chrysoporthe austroafricana varied with respect to the clones; the clone with the lesser necrotic lesion having a higher peroxidase activity according to RNA-seq transcriptome data (Mangwanda et al., 2015). A PR10 (Ribonuclease-like protein) from Jatropha curcus, a woody tree also of the Euphorbiaceae family was highly expressed in response to salicyclic acid, methyl jasmonate and the pathogen, Macrophomina phaseolina (Agarwal et al., 2013).

4.4.3 Expression of cell wall modification genes

Cell wall enhancement is an induced defence response used by trees to ward off pathogens. Phenylalanine ammonia-lyase (PAL) was significantly up-regulated in both clones; 6.7 times (P < 0.01) in RRIM612 and 6.3 times in PR107 (P < 0.05) (IV; Figure 4A). The PAL gene is involved in the monolignol biosynthesis pathway where the synthesis of lignin occurs (Carocha et al., 2015). PAL gene was up- regulated in both clones in response to the pathogen; an indication of enhanced lignification as a systemic acquired resistance against the pathogen. Five PAL genes in Populus trichocarpa were shown to have similar transcript abundance and were involved in stem wood forming tissue using RNA-Seq technology (Shi et al., 2013). The PAL gene is also implicated in the biosynthetic pathway of salicyclic acid (SA) signal transduction as SA-dependent response is significantly increased by PAL activity (Arnerup et al., 2013). Expansins are implicated in cell wall modification but their exact role remains unknown (Sampedro and Cosgrove 2005). A B3 expansin gene was highly up-regulated in the treatments for both clones. There was higher up-regulation (40 times) in RRIM612 than in PR107 (19 times). Significant difference in fold change of treatment over control for the clones was observed at P < 0.01 and P < 0.05 for RRIM612 and PR107 respectively (IV; Figure 4B). Microarray gene expression analysis in tissues of poplar revealed a rich repertoire of expansins in active tissues (cambium, xylem, and phloem) compared to the dormant tissues (Geisler-Lee et al., 2006).

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4.4.4 Expression of signal transduction genes

Genes involved in ethylene biosynthesis (1-aminocyclopropane-1-carboxylate oxidase-ACC oxidase) and jasmonic acid metabolic pathways (Allene oxide cyclase-AOC) were significantly up- regulated in treatment over control. ACC oxidase was up-regulated 8.2 times in RRIM612 (P < 0.05) and 7.9 times in PR107 (P < 0.01) (IV; Figure 5A). AOC was up-regulated 4.9 times in both clones; RRIM612 (P < 0.05) and PR107 (P < 0.01) (IV; Figure 5B). The response in the signal transduction genes, ACC oxidase and AOC were comparable for both clones studied. Three ACC oxidase genes (HbACO1, HbACO2 and HbACO1) were shown to be up-regulated in response to ethylene and wounding in bark of H. brasiliensis (Kuswanhadi et al., 2010). Genes involved in wounding and ethylene/jasmonic acid pathways have also been investigated in previous experiments on H. brasiliensis. Signal transduction genes MAPK, ETR, EIN were induced by wounding, while ethylene and jasmonic acid regulated a chitinase and a Myb transcription factor (Duan et al., 2010). An elicitor mitogen-activated protein kinase (MAPK) was significantly up-regulated 3.5 fold in treatment in PR107 (P < 0.05), but there was no significant fold change difference between treatment and control for RRIM612 (IV; Figure 5C). Gene expression studies in four H. brasiliensis clones in response to drought also showed varied expression levels of a MAPK gene (Luke et al., 2015). The genetic characteristic of the clone might dictate the role of the elicitor-induced resistance to restrict infection by specific fungal pathogens.

4.4.5 Expression of a Myb transcription factor

The expression of a Myb transcription factor also varied between the two clones. A fold change of 3.4 was observed for RRIM612 (P < 0.05), while there was no significant fold change difference between treatment and control for PR107 (IV; Figure 6). A Myb transcription factor, down regulated in tapping panel dryness (TPD) in H. brasiliensis, suppressed cell induced death in transgenic tobacco and could suppress stress induced cell death in H. brasiliensis (Peng et al., 2011). Genome wide studies of WRKY transcription factor genes in the rubber tree genome identified 81 WRKY genes for which 74 were expressed in at least one of the tissues studied (Li et al., 2014). Rubber latex biosynthesis is a defence response to wounding and transcription factors may play a role in its regulation.

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5. SUMMARY AND CONCLUSIONS

In our first study we applied molecular phylogenetics to study the phylogeny of Rigidoporus microporus isolates with emphasis on isolates associated with rubber tree plantations in Asia and Africa. R. microporus causes the white rot disease of rubber trees in plantations. The disease is not common in South America, the centre of origin of the rubber tree; due to the devastating effects of the South American leaf blight (SALB). Majority of the rubber plantations supplying 99% of total world natural rubber demand are located in South East Asia and Africa where the disease is a major problem. Most of the plantations are exotic, set up with seeds gotten from Brazil and other South American countries. We conducted global studies comparing isolates from the two rubber producing continents of Asia and Africa to test for the existence of cryptic species and conspecificity. Our multi-gene molecular phylogenetic analysis suggests the presence of two distinctive species associated with the white rot disease. Isolates collected from Non- H. brasiliensis host clustered with those isolated from H. brasiliensis. It is therefore very likely that, the white rot fungus jumped host from native trees to H. brasiliensis. The pathogen may have evolved in the absence of rubber trees and thus became pathogenic in rubber plantations. The high intra-species variation observed within the African clade also points to a continuous gene flow among these populations due to material exchange that exist between the neighbouring African countries as well as nationally within each country. Phylogenetic analysis of the Rigidoporus genus in this study also supports the need for a thorough revision of the Rigidoporus genus within orders of the Polyporales and Hymaenochataeles. Rigidoporus microporus rhizomorphs have been observed to persist in the soil on wood debris for long periods. This suggests that apart from its lifestyle as a pathogen, it can also live saprophytically on dead wood. These observations led us to investigate the wood degrading properties of the fungus during saprotrophic growth on rubber wood. Isolates selected for the study showed varying saprotrophic abilities with the pathogenic isolates associated with rubber trees displaying significantly high wood decay capacity after 6 months. The results proved that the fungus has the potential to switch to a saprotrophic habit by degrading dead wood lignocellulose. Light microscopy revealed structural alterations typical for white rot basidiomycetes; that were as a result of the saprotrophic growth of the fungus on rubber wood. RNA-Seq de novo transcriptomic assembly and profiling of R. microporus was done to elucidate the genes expressed by the fungus during saprotrophic growth on rubber wood. Our emphasis was on genes encoding lignocellulose degrading enzymes, which might have potential applications in bioenergy processing. The transcriptome expressed over 400 lignocellulose degrading genes with a very high number of genes encoding manganese peroxidases highly expressed in rubber wood. We also explored the possibility of the fungus to degrade natural rubber latex produced by rubber trees. Interestingly, a large

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number of unigenes involved not only in rubber latex degradation, but in fatty acid degradation in general were constitutively expressed in the transcriptome. R. microporus evolved in the absence of H. brasiliensis and might have acquired genes that can effectively metabolize and degrade fatty acid related secondary metabolites. Pathway enrichment analysis revealed up-regulation in rubber wood of important enzymes like alcohol dehydrogenase with potential biotechnological applications. Validation of 23 transcripts in the RNA-Seq data by quantitative real-time PCR also confirmed the reproducibility of the results from the RNA-Seq data. In the last project, we took advantage of the availability of the draft genome sequence of H. brasiliensis to study host defence response during interaction with the rubber tree pathogen. We studied defence gene expression in two clones of H. brasiliensis with different levels of susceptibility. The results suggest that the genetic characteristics of H. brasiliensis clones may play a crucial role in defence gene expression. Understanding the white rot disease host pathogen interaction in rubber trees should involve a comparative analysis of both resistant and susceptible clones. The study provides the first overview of candidate defence-related genes that are expressed during the host-pathogen interaction of the white rot disease of rubber trees. The precise role of these genes and how they are regulated during pathogen infection by R. microporus merits further research.

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6. FUTURE PERSPECTIVES

The knowledge that the isolates of Rigidoporus microporus originating from Africa and Asia are different species should imperatively have implications for developing management strategies for the disease in these regions. This would help in management and quarantine procedures. A large scale phylogenetic study of Polyporales/Hymaenochataeles in the light of additional morphological and molecular data is encouraged. An interesting observation in the saprotrophic degrading abilities of the R. microporus isolates in the study was the endophytic habit exhibited by an isolate collected from healthy H. brasiliensis trees in the natural forest in Peru. It virtually could not degrade rubber wood blocks for a period of six months. Why was this isolate not able to degrade rubber wood? Could this low saprotrophic ability be as a result of co- evolution with H. brasiliensis? What are the genes present in the pathogenic isolates but absent in the endophytic isolate? These are very vital questions and we can hopefully get further insights on possible answers to these questions by genome and transcriptomic comparative analysis of these isolates. The R. microporus transcriptome had a really good coverage (34, 518 unigenes). This work has provided a vast amount of genetic resources for future studies on this fungus. It opens up a lot of information to explore with the transcriptome. Manganese peroxidases together with laccases are receiving a lot of research attention as potential sources of ligninolytic enzymes. A large number of these genes that can be further explored were highly expressed in rubber wood. The MnP2 transcript that was up-regulated 71 times in rubber wood share closest similarity (75%) to the MnP2 of Fomitiporia mediterranea. Characterization and evolutionary analysis of this gene would be interesting to see how different it is from other MnP2 expressed by other white rot polypores. The cephalosporin esterase gene not previously characterized in basidiomycetes would be another interesting gene to explore. The number of lignocellulose degrading enzyme encoding gene transcripts (426) expressed in the transcriptome makes the need to sequence the genome of the fungus even paramount. Finally, this study provides the first overview of potential candidate defence-related genes in H. brasiliensis against the white rot pathogen. Characterization and identification of such genes are crucial in understanding and developing control measures based on selection of resistant molecular markers against the pathogen. Considering the known fact that some up-regulated genes may eventually not be translated; proteomic studies should be conducted to further confirm and shed more light on gene expression levels of selected candidate genes. The availability of the genome sequence of H. brasiliensis was a mile stone and would pave the way for more studies on host-pathogen interactions. Using RNA-Seq technology to study the response of the host to the pathogen would certainly provide a more global transcriptomic overview of

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other novel genes. Clones originating from different rubber growing regions can be screened for resistance for the disease so as to discover traits that can be used for marker-assisted breeding.

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ACKNOWLEDGEMENTS

This work was conducted at the Department of Forest Sciences, Faculty of Agriculture and Forestry at the University of Helsinki. I would like to thank the Delta state government of Nigeria for awarding me a post-graduate scholarship which enabled me to start this doctoral program. I would also like to acknowledge financial support in the form of travel grants from the University of Helsinki which assisted me in my field trips to Nigeria. I will forever be grateful to my supervisor, Professor Fred O. Asiegbu who gave me the opportunity to study for a PhD in his research group. His thorough supervision, support, enthusiasm and his ability to always suggest the right solutions to problems encountered on this journey paved the way for the successful completion of my program. My academic growth during my PhD studies was influenced by some excellent researchers. I would like to thank Docent Risto Kasanen for the invaluable knowledge on forestry derived from him over the years. I would also like to acknowledge and thank Dr. Otto Miettinen for introducing me to the world of molecular phylogeny and for his collaboration. Special thanks also to Tuomo Niemela and Viacheslav Spirin from the Finnish Botanical museum of natural history for sharing their expertise with me. My deepest gratitude to Dr. Dmitry Schigel for his criticism and constructive suggestions which helped to develop my PhD research. My special thanks also to Professor Geoffrey Daniel and post-doc Jong Sik Kim for the microscopy experience during my lab visit to SLU, Sweden. I also thank my other collaborators, Dr. Romina Gazis and Dr. Mohd Farid. I appreciate the efforts of my colleagues in the forest pathology (MPAT) group; Andriy, Tommaso, Emad and Susanna who I collaborated with during my PhD studies. I would like to thank my pre-examiners, Professor Yu-Cheng Dai and Dr. Hakeem O. Shittu for their constructive comments and suggestions which helped to improve this thesis. Majority of the data generated for this work were from the Rubber Research Institute, Nigeria (RRIN) were I had a lot of help from many persons. I would like to thank Dr. Victor Omorusi of the Plant Protection Division of RRIN for facilitating the official documentation to use facilities at RRIN. Special thanks also to Grace Evueh. I appreciate your help and collaboration. I appreciate the efforts of Samson Igbinobaro and Saint Akhainemhen in helping to generate rubber tree clones used for my pathogenicity studies. Special thanks also to Kensington Orumwense who kept the research environment lively with very useful discussions. I also want to appreciate my early mentors in my formative years as a Mycologist/Plant pathologist; Professors John Okhuoya and Omon Isikhuemhen and Drs. Omorefosa Osemwegie and Emmanuel Akpaja. I appreciate your tutoring over the years. I also thank my colleagues and fellow diaspora

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PhD researchers; Oghenemise Abirhire, Henry Oamen and Charles Eghweree. The many and long phone calls kept me going. I really appreciate. I have been privileged for many years to study for my PhD in the best research group one can ever hope for; the forest pathology group (MPAT). The MPAT group is a close-knit family and an international research group which has provided priceless support in various forms in the laboratory, seminars, dinners and personal interactions. I want to express my unreserved appreciation to colleagues in the MPAT group; Andriy, Hui, Tommaso, Emad, Riikka, Eeva, Nicolas, Hongxin, Susanna, Jiayao, Ximena, Zhen, Mukrimin, Tiia, Bikash and Mengxia. I also would like to thank former members of the MPAT group; Anthony, Julia and Hsiao-Che for the interaction and experience shared. I also had the opportunity to know some persons in other research groups: Milton, Mustafa, Kashif and Rafique. Thanks for the many laughs and encouragement. Thanks also to my office mate, Milla Niemi for the constructive discussions and encouragement. I sincerely thank the administrative staff; Jukka and Sofia and technical staff; Marjut for their help in ensuring a smooth sailing of my program. I have some wonderful friends in Finland. Some I knew back in Nigeria and others I met here: Pedro Orumwense, you have been more like a brother to me from way back up to this day. I appreciate your friendship. Boris Tupek, my Slovakian friend. Thanks for all the laughs and discussions about life. Onyeka Unadike, John Ovie, Matthew Adeboye, Nosakhare Osamwonyi, Eric Uwe, Raphael Oshevire, Adeyanju Alade and Nicholas Addy. Thanks for making my stay in Finland exciting. Special thanks also to Anthony Akhagba for the phone discussions on sports and music which took my mind off research for a while. Finally, I would like to thank my wonderful family. My loving mother, who has kept me going with her constant phone calls and prayers. My brothers and sisters for their constant support, care and never ending love. I love you all.

Helsinki, December 2015

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