On the Nature, Symptoms and Genetic Diversity of Monilinia Isolates and Their Viruses in Western Australia

ON THE NATURE, SYMPTOMS AND GENETIC DIVERSITY OF MONILINIA ISOLATES AND THEIR VIRUSES IN WESTERN AUSTRALIA

Thesis is presented by

Thao Thi Tran

For the degree of Doctor of Philosophy at School of Veterinary and Life Sciences Murdoch University, Perth, Western Australia, Australia 2019

DECLARATION

I declare that this work is my own account of my research, and it contains as its main content work which has not previously been submitted for a degree at any tertiary education institution. To the best of my knowledge, it contains no material or work performed by others, published or unpublished without reference being made within the text.

Date: Thao Thi Tran

ABSTRACT

Monilinia species occur in stone fruit (Prunus species) production areas in many parts of the world, where they cause the serious disease brown rot. Incursion into Western Australia (W.A.) by Monilinia fructicola was declared in 1997, and along with Monilinia laxa has subsequent spread throughout the state has cost the stone fruit industry millions of dollars in lost production and the cost of fungicidal sprays. Until this study, no studies had been made in W.A. of Monilinia species identity, genetic diversity, distribution, virulence, fungicide tolerance or presence of mycoviruses. This project aimed to address these knowledge gaps.

A collection of Monilinia isolates was made across the major stone fruit production regions in W.A., and the fungal species were identified. Both species were present, but they were not evenly distributed between and within production regions, indicating the main agent of spread is people. High levels of genetic diversity have been reported for WA population, and both pathogens are not recently introduced in this state. ISSR (Inter Simple Sequence Repeat) markers were used to identify intra-specific diversity in populations, and compare modern populations with the strains that first invaded W.A.. This study revealed that the original incursive strain of M. fructicola has probably become extinct, replaced by new genotypes. Conversely the incursive strain of M. laxa remains widely distributed within the state. New strains appear to have subsequently invaded W.A. and pose ongoing serious threats to the industry.

Although commercial stone fruit growers routinely spray their crops with fungicides to control brown rot, small scale and ‘organic’ growers may not. A study was done to assay the relative tolerance to three fungicides of Monilinia isolates collected from sprayed and unsprayed trees. No resistant isolates have been found in WA. Sprayed isolates exhibited a significantly greater range of responses to fungicides than unsprayed isolates, indicating that there is strong positive selection for tolerance in the orchards that routinely apply fungicides. We found there were significant differences between M. fructicola and M. laxa isolates in average tolerance to propiconizole-based fungicides.

Assays for virulence revealed the existence of highly virulent, moderately virulent and avirulent strains of both species, and these occurred in both sprayed and unsprayed orchards.

On nutrient agar plates, M. laxa isolates presented as four distinct morphologies, while M. fructicola colonies were very similar in appearance across all isolates tested.

A survey of 28 isolates of both species revealed three mycoviruses co-infecting three M. laxa isolates and one M. fructicola isolate. The complete or partial sequences of one isolate each of all three viruses was obtained. One M. fructicola isolate was co-infected with all three viruses: Sclerotinia sclerotiorum hypovirus 2 (SsHV2, genus Hypovirus), Fusarium poae virus 1 (FPV1, genus Betapartitivirus), and Botrytis virus F (BVF, genus Mycoflexivirus). To test the influence of these viruses on fungal pathogenicity, several methods were applied to cure isolate M196 of one or more mycoviruses. Of these treatments, hyphal tip culture either alone or in combination with antibiotic treatment generated isogenic lines free of one or more mycoviruses. Morphology and virulence assays were carried out to determine how mycoviruses influence growth of the fungal host. Surprisingly, growth of fungal mycelia was promoted by the presence of three viruses when they were cultured on nutrient agar medium in vitro, but did not influence fungal virulence after inoculation to fruits of sweet cherry.

TABLE OF CONTENTS

DECLARATION...... 1

ABSTRACT ...... 2

TABLE OF CONTENTS ...... 4

LIST OF ABBREVIATIONS ...... 8

LIST OF PUBLICATIONS ...... 10

First author: ...... 10

Chapter 1. Literature review ...... 14 1.1. Stone fruit production ...... 14 1.2. Economic losses of brown rot disease ...... 15 1.3. Monilinia species ...... 15 1.3.1. Taxonomy and host range ...... 15 1.3.2. Monilinia life cycle ...... 16 1.3.3. Identification of Monilinia species...... 17 1.3.4. Geographical distribution ...... 18 1.4. Genetic diversity ...... 19 1.5. Management of brown rot disease ...... 20 1.5.1. Fungicide application and development of fungicide tolerance ...... 20 1.5.2. Potential alternative control measures to fungicides ...... 21 1.6. Mycoviruses ...... 22 1.6.1. Identification of mycoviruses ...... 23 1.6.2. Influence of mycoviruses on host fungi ...... 23 1.6.3. Mycoviruses of Monilinia ...... 24 1.7. Research gaps in Australia ...... 25 1.8. The five aims of this research project ...... 25 1.9. Notifications ...... 25

Chapter 2. Spatial distribution of Monilinia fructicola and M. laxa in stone fruit production areas in Western Australia ...... 27 Statement of contribution of the authors contributed to the work ...... 28 Abstract ...... 29 Introduction ...... 29

Material and Method ...... 31 Result and Discussion ...... 35 References ...... 39 Addendum to Chapter 2 ...... 42

Chapter 3. Genotypic structure of Monilinia populations in Western Australia two decades after incursion ...... 43 Statement of contribution of the authors contributed to the work ...... 44 Abstract ...... 45 Introduction ...... 45 Materials and methods ...... 47 Fungus collection ...... 47 Fungal isolation ...... 47 DNA extraction ...... 48 ISSR assay ...... 48 Data analysis ...... 49 Results ...... 49 M. fructicola isolates ...... 49 M. laxa isolates ...... 49 Discussion ...... 50 References ...... 53

Chapter 4. Comparisons between Monilinia fructicola and Monilinia laxa isolates on genetic diversity, virulence and colony morphology ...... 65 Abstract ...... 66 Introduction ...... 66 Materials and methods ...... 68 Fungal collection and isolations ...... 68 DNA extraction ...... 69 ISSR assay ...... 69 Virulence test ...... 70 Colony morphology assessment ...... 71 Data analysis for genetic variation...... 71 Results ...... 71 Genetic diversity ...... 71

AMOVA analysis ...... 73 Virulence test ...... 73 Colony morphology ...... 76 Discussion ...... 77 References ...... 82

Chapter 5. Monilinia fructicola and Monilinia laxa isolates from stone fruit orchards sprayed with fungicides displayed a broader range of responses to fungicides than those from unsprayed orchards ...... 85 Statement of contribution of the authors contributed to the work ...... 86 Abstract ...... 87 Introduction ...... 87 Materials and methods ...... 89 Results ...... 97 Discussion ...... 103 References ...... 106 Addendum Chapter 5 ...... 112

Chapter 6. Co-infection with three mycoviruses stimulates growth of a Monilinia fructicola isolate on nutrient medium, but does not induce hypervirulence in a natural host ...... 113 Statement of contribution of the authors contributed to the work ...... 114 Abstract ...... 115 1. Introduction ...... 115 2. Materials and Methods ...... 116 2.1. Fungus Collection and Isolation ...... 116 2.2. DNA and RNA Extraction ...... 117 2.3. cDNA Synthesis, PCR Amplification and Library Preparation for High-Throughput Sequencing ...... 117 2.4. Sequence Analysis ...... 118 2.5. Generation of Isogenic Fungal Lines Free of Mycoviruses ...... 119 2.6. Virulence on Cherry ...... 119 2.7. Mycelial Growth In Vitro ...... 120 3. Results ...... 121 3.1. Sequencing Analysis and Virus Assays ...... 121

3.2. The Presence of Mycoviruses in Other Monilinia Isolates ...... 123 3.3. Elimination of Mycoviruses ...... 124 3.4. Mycoviruses Influenced Growth In Vitro ...... 125 3.5. Influence of Mycoviruses on Virulence of M. fructicola ...... 126 4. Discussion ...... 126 References ...... 129

Chapter 7. General discussion ...... 136 Incursion ...... 137 Identity and distribution ...... 137 New incursions and genetic diversity ...... 139 Responses to fungicides ...... 141 Disease ...... 144 Viruses and virulence ...... 144 A vision of the future ...... 146

References ...... 148

LIST OF ABBREVIATIONS

ANOVA Analysis of variance BLAST Basic local alignment search tool BVF Botrytis virus F CABI Centre for Agriculture and Biosciences International CDD Conserved Domain Database cDNA Complementary DNA CHV1 Cryphonectria hypovirus 1 CHV2 Cryphonectria hypovirus 2 CHV3 Cryphonectria hypovirus 3 CHV4 Cryphonectria hypovirus 4 CP Coat protein DNA Deoxyribonucleic acid dsRNA Double-stranded RNA DPIRD Department of Primary Industries and Regional Development EC Effective concentration EDTA Ethylenediaminetetraacetic acid EPPO European and Mediterranean Plant Protection FpV1 Fusarium poae virus 1 ICTV International Committee on Taxonomy of Viruses ITS Internal transcribed spacer ISSR Inter simple sequence repeat NCBI National Center for Biotechnology Information ORF Open reading frame PCR Polymerase chain reaction PDA Potato dextrose agar PDB Potato dextrose broth PVPP Polyvinylpolypyrrolidone RAPD Randomly amplified polymorphic DNA RdRp RNA-dependent RNA polymerase RNA Ribonucleic acid RT-PCR Reverse transcription PCR

SDS Sodium dodecyl sulfate SsHV2 Sclerotinia sclerotiorum hypovirus 2 ssRNA Single-stranded RNA Tris Tris (hydroxymethyl) aminomethane UPGMA Unweighted Pair Group Method with Arithmetic Mean USDA United States Department of Agriculture UTR Untranslated region W.A. Western Australia

LIST OF PUBLICATIONS

First author:

Tran, T. T., Li, H., Nguyen, D. Q., Sivasithamparam, K., Jones, M. G. K., & Wylie, S. J. (2019). Comparisons between genetic diversity, virulence and colony morphology of Monilinia fructicola and Monilinia laxa isolates. (Journal of Plant Pathology, under consideration, May 2019)

Tran, T. T., Li, H., Nguyen, D. Q., Sivasithamparam, K., Jones, M. G. K., & Wylie, S. J. (2019). Genotypic structure of Monilinia populations in Western Australia two decades after incursion. Australasian Plant Pathology 48:167-178 https://doi.org/10.1007/s13313-019-0612-1

Tran, T. T., Li, H., Nguyen, D. Q., Sivasithamparam, K., Jones, M. G. K., & Wylie, S. J. (2019). Co-infection with three mycoviruses stimulates growth of a Monilinia fructicola isolate on nutrient medium, but does not induce hypervirulence in a natural host. Viruses, 11(1), 89; https://doi.org/10.3390/v11010089

Tran, T. T., Li, H., Nguyen, D. Q., Jones, M. G. K., Sivasithamparam, K., & Wylie, S. J. (2018). Monilinia fructicola and Monilinia laxa isolates from stone fruit orchards sprayed with fungicides displayed a broader range of responses to fungicides than those from unsprayed orchards. European Journal of Plant Pathology, 153:985-999. https://doi.org/10.1007/s10658-018-01613-x

Tran, T. T., Li, H., Nguyen, D. Q., Sivasithamparam, K., Jones, M. G. K., & Wylie, S. J. (2017). Spatial distribution of Monilinia fructicola and M. laxa in stone fruit production areas in Western Australia. Australasian Plant Pathology, 46:339-349 https://doi.org/10.1007/s13313-017-0497-9

Co-author:

Wylie, S. J., Tran, T. T., Nguyen, D. Q., Koh, S-H., Chakraborty, A., Xu, W., Jones, M. G. K., Li Hua (2019). A virome from ornamental flowers in an Australian rural town. Archives of Virology (In Press. May 2019).

LIST OF PRESENTATIONS

Presentations

1. Cellular and Molecular Fungal Biology Conference, The Power of Fungi: From Cell Biology to Ecology, held in New Hampshire, United State, 2018.

Topic: A comparison of genetic diversity and fungicide tolerance of Monilinia isolates from organic and conventional stone fruit orchards.

2. Combined Biological Sciences Meeting, held at University of Western Australia, 2017.

Topic: Distribution, genetic diversity and pathogenicity of Monilinia fructicola and M. laxa in Western Australia.

3. Murdoch Agricultural Research Symposium, held at Murdoch University, 2017.

Topic: Brown rot cause by Monilinia fructicola and M. laxa in Western Australia.

ACKNOWLEDGEMENTS

To Associate Professor Stephen John Wylie, my principle supervisor, an expert on viruses and many other fields, I would like to express my deepest gratitude to you. It is hard to find words to say my thanks to you. To say thank you for the tremendous support and your time editing papers and orienting all experiments sounds too weak to describe your real contribution to my PhD life. Thank you so much for making it both scientifically achievable and enjoyable. I will never forget all the field trips with you and other colleagues to collect samples and explore Western Australia. All your invaluable advice, guidance and care will be with me in all of my careers ahead.

To my supervisor Assoc. Prof. Li Hua, thank you so much for your help and advice on techniques in the laboratory as well as the constructive criticism on the papers we published together. I felt so thankful to have you who were always there to help me go through any issues when I first came to work in a new laboratory with many new machines and techniques.

I would like to thank Prof. Mike Jones, my supervisor, for sharing the confidence, extensive knowledge and optimistic attitude, which extremely encouraged me to complete my PhD.

I also would like to thank Prof. Krishnapillai Sivasithamparam for his encouragement, insightful advice and discussions on Monilinia fungi and other related topics.

My sincere thanks also go to everyone in the SABC and Murdoch University who rendered help to me during my PhD study. Thanks to them all, especially my husband Quang Duy Nguyen, who is also my colleague at SABC, for filling my PhD life with sweet memories of a supportive friendship and fun.

A very special thanks to my Dad, Tran Dinh Duc, my two brothers and two sisters for their love and encouragement, especially Thanh for coming all the way to Australia to care for Bich Ngoc while I was in the laboratory.

I am greatly indebted to Vietnam International Education Development (VIED) and Murdoch University for offering me a scholarship, without which this project would not have eventuated.

DEDICATION

I would like to dedicate this thesis to my dear much-loved father Tran Dinh Duc who cares nothing about scientific aspects of my PhD but ardently supported, encouraged and loved me, his little dearly-loved daughter who is always in his mind. I also delicate it to my lovely daughter Nguyen Bich Ngoc who needs to do nothing but be in my life to shine it up.

Chapter 1. Literature review

1.1. Stone fruit production Stone fruits (Prunus sp.) are important horticultural crops, the main ones being peaches, nectarines, plums, apricots, almonds and cherries. According to the USDA, the world production of peach and nectarine was 19.9 million tons, and 3.3 million tons of cherries (sweet and sour) in 2017. The top three peach and nectarine growing countries are China, Italy and Spain, and the United States, having productions of 13.5, 3.6, and 0.7 million tons, respectively (USDA, 2018). All stone fruit crops originated in areas from eastern Europe to eastern Asia (although the genus Prunus has a wider geographical range), but now they are cultivated in all inhabited continents.

The Australian stone fruit industry is dominated by peach, nectarine and cherry, with smaller scale production of plum and apricot, all of which are harvested during summer seasons. There are a mix of small-scale and larger-scale enterprises in the three major production regions - Tasmania, Victoria, and New South Wales. In terms of economic value in Australia, cherry production exceeds AUD $100 million, and peaches and nectarines about AUD $200 million (Hugh and Roger, 2014). In Western Australia (W.A.), stone fruit is mainly grown in the Darling Scarp hills to the east of the state capital city of Perth, near the southern towns of Manjimup, Dwellingup and Donnybrook, but to a lesser extent to the north of Perth near the town of Gingin. Stone fruit grown in W.A. consists of cherries, peaches, nectarines, apricots and plums, which are harvested from November to March (BA, 2006).

Australian stone fruit production not only aims to supply domestic markets but also international ones. In 2014, Australian exported 2,737 tonnes of cherry for AUD $39 million, and 8,000 tonnes peaches and nectarines to markets in Hong Kong and Singapore (Hugh and Roger, 2014). Australia also imports out of season stone fruit from the USA and New Zealand. In 2013, Australia was the seventh biggest market consuming stone fruit imported from the USA, and the fourth in terms of value (Hugh and Roger, 2014). Although, out of season imports may not affect local producers in economic terms as they are counter- seasonal, they might be sources of new pathogens and strains, including Monilinia fungi causing brown rot, the most destructive fungal disease of stone fruit.

1.2. Economic losses of brown rot disease Brown rot caused by Monilinia species in stone fruit cost about USD 1.9 billion worldwide annually. In the United States, Monilinia costs the stone fruit industry 170 million USD per year. In Europe, up to 80% of the crops can be lost to brown rot, depending on the cultivar and seasonal weather conditions (Martini and Mari, 2014; Teixidó, 2016). In China, in 2003, there was potential to lose up to 100% of fruit post-harvest from rot caused by Monilinia species (Fan et al., 2010).

In Australia, Monilinia fructicola and Monilinia laxa species can cause local yield losses of up to 100% production in nectarine and peach crops, costing the Australian stone fruit industry over $20 million per year in lost production, and $25 million in control. Although, since 2014, M. fructicola is no longer listed as a quarantine pest in European Union (EU; action directive 2014/78/UE), its distribution remain restricted to some countries and regions in EU., which impacts on Australia’s ability to market stone fruit to Europe. In 2006 and 2007, shipments of Australian peaches and nectarines were rejected on arrival in the UK because of brown rot infection (Holmes et al., 2011).

1.3. Monilinia species 1.3.1. Taxonomy and host range

Monilinia species belong to the phylum Ascomycota, class Leotiomycetes, order Helioteliales, family Sclerotiniaceae. They are pathogens mainly of the families Ericaceae and Rosaceae. The first published report on stone fruit was in 1796 (as cited in Ritchie, 2005), and placed in genus Sclerotinia, but it was reclassified as genus Monilinia in 1928 (Harrision, 1928). Since then, 35 species of Monilinia have been described, of which Monilinia fructicola (G. Winter) Honey, Monilinia fructigena (Aderhold & Ruhland) Honey, and Monilinia laxa (Aderhold & Ruhland) Honey (Lino et al., 2016), Molinia polystroma (Van Leeuwen et al., 2002) and Molinia yunnanensis (Lin et al., 2015) are well-known and common pathogens causing brown rot on Malus, Pyrus and Prunus species.

These Monilinia species are reported as having different host preferences. M. fructicola is more frequently found on nectarine and peach (P. persica) (CABI/EPPO, 2010) and M. laxa is commonly present on almond (P. dulcis), apricot (P. armeniaca), sweet cherry (P. avium), plum (P. domestica and other species) (CABI/EPPO, 1991), although both species occur on all Prunus species. M. fructicola has also been identified to occasionally cause brown rot on

apple (Terui and Harada, 1966; Batra and Harada, 1986; Duchoslavová et al., 2007; Grabke et al., 2011; Vasić et al., 2012; Martini et al., 2013; Chen et al., 2013; Pereira and May De Mio, 2018). M. laxa was also reported in apple in Belarus (Lesik, 2013) and Slovenia (Celar and Valic,1999). The occurrence of M. laxa f. sp. mali in Slovenia was first reported in 1997, when an unusual dying of apple shoots was observed. Interestingly, Sholberg et al. (2003) identified M. fructicola, based on morphology and DNA sequences on wine grapes (Vitis vinifera) from British Columbia, Canada. M. fructigena usually occurs on apple (Malus domestica), pear (Pyrus communis), quince (Cydonia oblonga), but also on plum, sweet cherry and other Prunus species (CABI/EPPO, 2000).

The form of Monilinia for which no sexual stage is described, the anamorph, is referred to as Monilia. Monilia polystroma, previously considered Monilinia fructigena, was identified in Japan in 2002 (van Leeuwen et al., 2002). This species was also isolated in Hungary (Petroczy and Palkovics, 2009), China (Zhu and Guo, 2010), Poland (Poniatowska et al., 2013), Italy (Martini et al., 2014), Switzerland (Hilber-Bodmer et al., 2012). Some authors refer to this species as Monilinia polystroma (Freimoser et al, 2016). Monilia species M. mumecola and M. yunnanensis are also reported from plum in Asia (Hu et al., 2011).

1.3.2. Monilinia life cycle

The primary inoculum sources are the overwintering mummified fruits either on the ground or on the trees, resulting in blossom blight (Holb, 2008). The infection is then spread into peduncles and the twig, resulting in twig canker. Both immature and mature fruit are infected by conidia produced from the infected tissues (Figure 1). Immature fruit are less susceptible to infection than mature ones, unless injured, as they have lower sugar content.

Figure 1. Life cycle of Monilinia species (Lino et al., 2016)

Brown rot severity depends on weather conditions. The worst outbreaks occur at warm temperatures and humidity from blossom to fruit maturity. The most critical time for control is three weeks before fruit maturity (Ritchie, 2000).

1.3.3. Identification of Monilinia species

It is difficult to distinguish Monilinia species on the field since they present similar morphologies and disease symptoms. However, when spores are cultured on nutrient agar plates, including potato dextrose agar (PDA) and V8 agar, in vitro, the growth forms are often distinctive for each species, although strain variation occurs. Colonies of M. laxa are greenish-grey, usually with a rosette growth form and dark rings between rosettes. Monilinia fructicola colonies are brown with an even margin. Monilinia fructigena colonies are white/cream with even margins (Lino et al., 2016). Growth rates of three Monilinia species are different in vitro. The highest growth rate was for M. fructicola, followed by M. fructigena and M. laxa, respectively, when they were isolated from peach and nectarine commercial orchards in China and grown on PDA in the dark at 22oC. However, lesion growth rate on peach fruit was greatest for M. laxa (Hu et al., 2011).

Distinguishing Monilinia species based on morphological characteristics in cultures grown in vitro requires isolation of pure isolates with a risk of misidentification. More definitive PCR- based assays used to distinguish Monilinia species include those that use species-specific primers (Côté et al., 2004; Gell et al., 2007), generic ITS primers (Ioos and Frey, 2000), and tef1 primers (Marin-Felix et al., 2017).

1.3.4. Geographical distribution

M. fructicola, M. fructigena, and M. laxa have different regulatory statuses depending on the national plant protection organizations. M. fructicola is recorded as a quarantine fungus in Chile, Jordan, Israel, and Norway; M. fructigena in the United States, Canada, Australia, New Zealand and Chile; and M. laxa in Jordan (EPPO global data base).

Monilinia fructicola has been reported in all continents around the world. It was first reported in Australia in 1921 (Harrision, 1928), and in New Zealand in 1927 (Byrde and Willetts, 1977). Later, it was identified in China (Zhu et al., 2005; Yin et al., 2013), Japan (Terui and Harada, 1966; Batra and Harada, 1986), Canada (Sholberg et al., 2003), the USA (Chen et al., 2013), Chile (Latorre et al., 2014), and Brazil (Pereira and May De Mio, 2018). In Switzerland in 2006, M. fructicola was identified in apricot and nectarine imported from the USA (Bosshard et al., 2006); and later it was reported elsewhere in Europe such as the Czech Republic (Duchoslavová et al., 2007), Italy (Pellegrino et al., 2009; Martini et al., 2013), Spain (De Cal et al., 2009), Germany (Grabke et al., 2011), Poland (Hilber-Bodmer et al., 2010; Poniatowska & Bielenin, 2013), Serbia (Vasić et al., 2012; Hrustić et al., 2013), Croatia (Ivíc et al., 2014), and Greece (Papavasileiou et al., 2015).

Monilinia laxa was reported later in Australia (Jenkins, 1965; Penrose 1979), and from New Zealand (Boesewinkel and Corbin, 1970), Japan (Byrde and Willet, 1977), China (Hu et al., 2011), the USA (Cox et al., 2011), and Brazil (Souza et al., 2008).

In contrast, M. fructigena occurs in Eurasia, but has not been reported in Australia, New Zealand and North America (Cox et al., 2018). It was reported from Japan, China, Korea, India, Israel, Iran (Byrde and Willet, 1977), and Europe in Switzerland (Patocchi et al., 2009), Croatia (Ivíc et al., 2014), and Belarus (Lesik, 2013). M. polystroma has been reported

on several occasions in Europe in the last eight years on apricot, peach, and apple (Hilber- Bodmer et al., 2012; Martini et al., 2014; Petróczy and Palkovics 2009; Poniatowska et al., 2013; Vasic et al., 2013). This species is likely to be of Japanese origin and is closely related to M. fructigena (van Leeuwen et al., 2002).

Of three Monilinia species that affect stone fruit and pome fruit production, Monilinia fructigena, which mainly infects pome fruit (but less commonly stone fruit) has not been officially recorded from Australia (Stansbury et al., 2000). Monilinia fructicola, previously known as Sclerotinia fructicola, was first described from stone and pome fruit in eastern Australia in 1921 (Harrison, 1928), and M. laxa (S. laxa) was first identified in 1962 (Jenkins, 1965; Penrose, 1979). Of the two species, M. fructicola is reported to be more commonly associated with brown rot disease in Australian stone fruit orchards (Kreidl et al., 2015).

In Australia, the sexual stage (apothecia) has been observed in M. fructicola, but not in M. laxa. Harrison (1928) found apothecia of M. fructicola in an apricot orchard in Pennant Hills, New South Wales, Australia. Later of the same year, the author revisited the area and found thousands of similar apothecia from mummified fruits of apricot, plums, peaches, and also apples, quinces, and pears.

In Western Australia, Monilinia is a relatively recent invader (Stansbury et al., 2000). It was first officially identified and collected in 1997 in Donnybrook and Manjimup. The Minister of Primary Industries Monty House, stated ‘… the whole of the state of Western Australia is infected with the disease brown rot (Monilinia fructicola)’ (House, 1997). During its short history in Western Australia there have been no surveys of species distribution or epidemiology published.

Knowledge of the relative distribution of the Monilinia species is important for the stone fruit industry because the different species may have different sensitivities to fungicides and differential pathologies, and for protection of export markets. Also, protecting Monilinia-free production regions is important and may be achieved by quarantine policies of each country.

1.4. Genetic diversity Studying the genetic diversity of Monilinia populations provides information for a better understanding about fungal evolution, origins of isolates and responses to control measures.

This information assists with devising effective biosecurity strategies to keep new isolates invading, and for management strategies locally. Information about population structure has been obtained using molecular markers. Although the internal transcribed spacer region (ITS) of ribosomal RNA genes has sufficient resolution to distinguish Monilinia species, it lacks the resolution to inform on intra-species variation (van Leeuwen et al., 2002). Several studies described the use of inter simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) genetic markers in M. laxa populations in Spain (Gell et al., 2007), in Hungary (Fazekas et al., 2014), and Greece (Papavasileiou et al., 2015), and in M. fructicola populations in Spain (Villarino et al., 2012), China (Fan et al. 2010), and Greece (Papavasileiou et al., 2015). Everhart and Scherm (2015) as well as Dowling et al. (2017) provided novel markers for intraspecific characterization of M. fructicola populations in USA. These studies compared genetic diversity of isolates from different geographical regions and/or from different host species. They revealed that the majority of genetic variation is found within populations for both M. laxa and M. fructicola. For instance, genetic variation within Spanish M. laxa populations was 97% of the total genetic diversity (Gell et al., 2007), 95% within Greek M. laxa (Papavasileiou et al., 2015), and 93% within Chinese M. fructicola populations (Fan et al., 2010). Papavasileiou et al. (2015) studied genetic diversity of 145 M. fructicola isolates and 156 M. laxa isolates collected from two distinct regions of Greece, and they showed that M. laxa populations were genetically less diverse than were M. fructicola populations. There has been no study of genetic diversity within M. fructicola and M. laxa populations in Western Australia. Information about genetic structure of Monilinia species in the regions will help biosecurity to limit the spread of species and genotypes within the region.

1.5. Management of brown rot disease 1.5.1. Fungicide application and development of fungicide tolerance Brown rot is mainly controlled in stone fruit by application of synthetic fungicides from a range of chemical classes. Extensive applications of synthetic fungicides such as carbendazim and tebuconazole, have resulted in development of fungicide tolerance in Monilinia species, which is recorded widely where stone fruit is grown commercially. For instance, M. fructicola isolates resistant to benomyl (Ogawa et al., 1984), M. fructicola highly tolerant to triazole fungicides are recorded from the USA (Schnabel et al., 2004), and from other countries such as Brazil (May-De Mio et al., 2011), Greece (Malandrakis et al., 2012), Spain

(Egüen et al., 2015), and Serbia (Hrustić et al., 2017). The MfCYP51 gene from Brazilian M. fructicola isolates had a point mutation that caused a glycine to serine substitution at codon 461. This mutation was associated with tebuconazole tolerance (Lichtemberg, 2017). Another example is the development of demethylation inhibitor (DMI) fungicide tolerance of M. fructicola isolates was strongly associated with the Mona element within the promoter of the 14α-demethylase gene MfCYP51, that induces its over-expression (Luo and Schnabel, 2008) Resistance to the dicarboximide fungicide iprodione is recorded from New Zealand (Elmer and Gaunt, 1994; Sanoamuang and Gaunt, 1995) and in Western Australia (Wherrett et al., 2001). Field strains of M. fructicola resistant to both methyl benzimidazole carbamate (MBC) and demethylase inhibitor (DMI) fungicides were identified from stone fruit orchards in the Eastern United States (Chen et al., 2013). Resistance to MBC in M. fructicola was also detected in South Carolina peach orchards (Zhu et al., 2010).

The mechanism of tolerance to benzimidazole fungicides by M. fructicola from California was a representative example of fungicide tolerance mechanism which was well characterised (Ma et al., 2003). The authors sequenced the β -tubulin gene and showed that the less tolerant and highly tolerant M. fructicola isolates had single point mutations at codon 6 and codon 198, respectively.

In Australia, a widely-used demethylation inhibitor fungicide is propiconazole. Reduced sensitivity in Monilinia populations to propiconazole has been reported (Zehr et al., 1999) resulting in a serious concern about the sustainability of this fungicide in Australia (Kreidl et al., 2015). Flowers are protected from blossom blight during flowering period by weekly application, immature fruit is protected by monthly sprays, and then weekly sprays as the fruit nears maturity. However, those spraying programs are not always successful, especially when it is humid during flowering and harvesting times (Holmes et al., 2011).

1.5.2. Potential alternative control measures to fungicides

Fungicide tolerance developed from intensive application of synthetic fungicide as described above, and this has encouraged researchers to seek potential alternative methods to control brown rot. Lazar‐Baker et al. (2011) found that lemon myrtle oil exhibited strong antifungal activity against M. fructicola. Yao and Tian (2005) investigated effects of methyl jasmonate (MeJA) and the yeast Cryptococcus laurentii alone or in combination against brown rot

postharvest disease caused by M. fructicola, and concluded that MeJA in combination with C. laurentii was beneficial for controlling M. fructicola. Chitosan (CS) and oligochitosan (OCS), which effectively inhibited growth of M. fructicola (Yang et al., 2012). Recently, Fu and colleagues (2017) reported that berberine, a traditional antidiarrheal compound, strongly inhibited M. fructicola. They also found that M. fructicola did not develop tolerance to berberine after sixteen subcultures in vitro, and no leaf damage was recorded over two years of applying berberine on peach fields. Another biological agent considered as an efficacious potential or complementary application to control brown rot was Bacillus amyloliquefaciens CPA-8, which was investigated under laboratory and field conditions (Gotor-Vila et al., 2017). They highlighted that B. amyloliquefaciens CPA-8 reduced disease caused by M. laxa and M. fructicola on peach and nectarine under laboratory and field conditions.

Heat treatments have been used to control brown rot at post-harvest stage, recently. Some studies incubated in hot water at 60oC for 20 s to control M. laxa (Spadoni et al., 2014), or for 40s (Casals et al., 2010a), or for 60 s (Karabulut et al., 2010). Others used hot air instead of hot water, such as Casals at el. (2010b), who treated peaches and nacterine innoculated with M. fructicola and M. laxa at 50oC for 2 h and completely controled disease development. Another method applied to control post-harvest stone fruits was radio frequency heating (Casals et al., 2010c).

Dominant gene-based tolerance to brown rot has been identified only in peaches (Martínez- García et al., 2013). In other Prunus species, fungicides remain the main control measure, although they are expensive and have environmental and human health risks associated with their use. Sanitary control methods such as removing cankered twigs and mummified fruits are also very important because they reduce the inoculum sources for flower and fruit infections in spring and summer (Villalta et al., 2015).

1.6. Mycoviruses

Fungal viruses (mycoviruses) have been identified from fungi in all major fungal phyla, namely the Zygomycota, Chytridiomycota, Ascomycota, and Basidiomycota (Ghabrial and Suzuki, 2009; Pearson et al., 2009). Since the first formal description of mycoviruses (Hollings, 1962), more than 250 mycoviruses have been sequenced and registered in the National Center for Biotechnology Information (NCBI) database (Xie and Jiang, 2014). The

reported mycoviruses are mainly double-stranded (ds) RNA or single-stranded (ss) RNA genomes. The dsRNA mycoviruses are classified into seven families including Chrysoviridae, Endornaviridae, Megabirnaviridae, Quadriviridae, Partitiviridae, Reoviridae, and Totiviridae, and the ssRNA mycoviruses are classified into six families including Alphaflexiviridae, Barnaviridae, Gammaflexiviridae, Hypoviridae, and Narnaviridae, and Mycomononegaviridae (Pearson et al., 2009; Niu et al., 2018). There was a noval circular ssDNA mycovirus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), identified in China (Yu et al., 2010), and in New Zealand (Kraberger et al., 2013). However, more than 20% of mycoviruses remain unassigned taxa beyond the species level (Pearson et al., 2009).

1.6.1. Identification of mycoviruses

Recently, high-throughput sequencing, also known as ‘next generation’ and ‘deep’ sequencing has been used to discover novel mycoviruses. For example, Mu et al. (2018) used RNA-Seq analysis to identify fragments of 57 viruses (34 of these were proposed novel viruses) from 84 Sclerotinia sclerotiorum isolates in Australia. In 2016, Marzaron et al. found 66 undescribed mycoviruses from isolates of five plant pathogenic fungi, including Colletotrichum truncatum, Macrophomina phaseolina, Diaporthe longicolla, Rhizoctonia solani, and Sclerotinia sclerotiorum. Osaki et al. (2016) identified 16 mycoviruses co- infected a strain of Fusarium poae, the causal agent of Fusarium head blight disease in wheat, durum and barley. In another study, Ong et al. (2016) used a combination of dsRNA- enrichment and high-throughput shotgun sequencing to identify sixteen alpha- and beta- partitiviruses co-infecting two mycorrhizal Ceratobasidium isolates associated with a plant of an Australian orchid, Pterostylis sanguinea.

1.6.2. Influence of mycoviruses on host fungi

The influence of mycoviruses on their host fungi has not been well studied. Some mycoviruses influence the ability of the fungus to cause disease in the plants. These that increase virulence of host fungi are known as ‘hypovirulent’ mycoviruses. In this case, the term hypovirulent refers to the influence of the mycovirus on the virulence of its phytopathogenic fungal host towards its plant host. In one case, a hypovirulent mycovirus, Cryphonectria hypovirus 1 (CHV1) and CHV2 significantly reduced the virulence of Cryphonectria parasitica, causal agent of chestnut blight on chestnut (Nuss, 1992; Myteberi

et al., 2013). Other research into using hypovirulent mycoviruses to control plant disease have been reported with mixed success for white mold fungus Sclerotinia sclerotiorum (Xie et al., 2011, Liu et al., 2015) and white root rot fungus Rosellinia necatrix (Kanematsu et al., 2014). In contrast, other studies found several mycoviruses associated with hypervirulence and increased sporulation of the fungal host. Ahn and Lee (2001) identified a 6 kbp dsRNA that increased virulence of Nectria radicicola, the causal fungus of ginseng root rot disease. It is noteworthy what many other mycoviruses are cryptic, they appear to be neither hypervirulent nor hypovirulent, in their fungal host. Lee et al. (2014) studied the influences of four mycoviruses including FgV1 (Fusarium graminearum virus 1), FgV2, FgV3, and FgV4 on strain PH-1of Fusarium graminearum fungi, which causes disease in wheat, barley, maize, and other cereal crops. They found that FgV1 and FgV2 reduced the fungal host’s growth rate, sporulation and virulence, while FgV3 and FgV4 infections did not cause any obvious symptoms. A similar example was the case of four viruses of genus Hypovirus isolated from Cryphonectria parasitica. While Cryphonectria hypovirus 1 (CHV1) and CHV2 reduced their host fungal virulence, CHV3 and CHV4 had a latent influence on the host fungus (Hu et al., 2014). Other mycoviruses associated with latent influences in their natural hosts have been reported, including Sclerotinia sclerotiorum hypovirus 1 (SsHV1) (Xie et al., 2011), Valsa ceratosperma hypovirus 1 (VcHV1) (Yaegashi et al., 2012), Fusarium graminearum hypovirus 1 (FgHV1) (Wange et al., 2013), Phomopsis longicolla hypovirus 1 (PlHV1) (Koloniuk et al., 2014).

1.6.3. Mycoviruses of Monilinia

Our understanding of Monilinia-infecting mycoviruses is limited to one study of mycoviruses of M. fructicola (Tsai et al., 2004), which was based on the presence of dsRNA molecules and virus-like particles of nectarines and peaches from several orchards in Kumeu, Henderson, Waiuku and Hawke’s Bay regions of the North Island of New Zealand. These researchers identified from one to seven virus-like double-stranded RNA (dsRNA) species in 36 of 49 M. fructicola isolates analysed. Although not identified at the molecular level, the authors described virus-like particles resembling those of partitiviruses, totiviruses, tobraviruses and furoviruses from fungal isolates. They undertook preliminary work to identify differences in growth rates of fungal cultures in vitro, and identified no differences between those with putative viruses and those without.

1.7. Research gaps in Australia Although Monilinia species are known to have invaded Western Australia approximately 22 years ago, and the disease they induce causes major losses to the state stone fruit industry that requires application of expensive and toxic fungicides, there has been no formal study of them. The relative distribution of the two species within the main stone fruit production regions is not known. It is not known if the original invasive genotype(s) remain, or if new Monilinia genotypes have subsequently invaded. It is not known if the two Monilinia species respond differentially to the fungicides used to control them, or whether fungicide-tolerant lines exist. It is not known if there is variation in virulence within and between the two fungal species, or whether mycoviruses are present and influence virulence. Provision of answers to these questions would inform evidence-based measures to control this fungus, thereby benefiting the stone fruit industry in Western Australia and elsewhere.

1.8. The five aims of this research project

Aim 1: Study the distribution of M. fructicola and M. laxa in stone fruit production areas in Western Australia. Aim 2: Investigate the genetic structure of M. fructicola and M. laxa isolates in Western Australia collected recently and those that first invaded the state over two decades ago. Aim 3: Compare genetic diversity, pathogenicity and colony morphology of Monilinia isolates. Aim 4: Compare relative sensitivity to fungicides of M. fructicola and M. laxa isolates collected from sprayed and unsprayed orchards. Determine if there are differential responses of the two Monilinia species to fungicides. Aim 5: Identify mycoviruses from Monilinia isolates and study their influences on growth and virulence of their fungal hosts.

1.9. Notifications

1. The aims of this thesis are presented as first-author papers that are published (Aims 1, 2, 4, 5) or in press in peer-reviewed journals (aim 3). 2. Because the results chapters in thesis are mostly presented as papers, some of the tables of fungal isolates and descriptions of methods are repeated in part or full in different chapters.

3. One co-authored paper was published during the course of this project, but this was unrelated to the aims of this project, and so is not presented in this thesis. Wylie, S. J., Tran, T. T., Nguyen, D. Q., Koh, S-H., Chakraborty, A., Xu, W., Jones, M. G. K., Li Hua (2019). A virome from ornamental flowers in an Australian rural town. Archives of Virology (In Press. May 2019).

Chapter 2. Spatial distribution of Monilinia fructicola and M. laxa in stone fruit production areas in Western Australia

This chapter addressed aim 1 of the thesis and was published in 2017.

Citation: Tran, T. T., Li, H., Nguyen, D. Q., Sivasithamparam, K., Jones, M. G. K., & Wylie, S. J. (2017). Spatial distribution of Monilinia fructicola and M. laxa in stone fruit production areas in Western Australia. Australasian Plant Pathology, 46(4), 339-349.

Statement of contribution of the authors contributed to the work

Abstract In 2016 and 2017, 90 fungal isolates were collected from Prunus species exhibiting symptoms of brown rot dis-ease at 12 sites in stone fruit production areas in Western Australia. ITS region analysis showed that 49 isolates belonged to Monilinia laxa and 34 to M. fructicola, species that cause brown rot in stone fruit. The two species were spatially separated to the south of the Perth Hills region, where only M. laxa was found, and to the north of Perth Hills where only M. fructicola was found. The two species co-existed only in the Perth Hills. The implications for control and trade are discussed, as is the need to implement biosecurity guidelines to prevent mixing of the two species where currently only one exists.

Introduction Brown rot, caused by Monilinia species, is one of the most destructive diseases of stone fruit and pome fruit (genera Prunus, Malus, Pyrus, Cydonia) in many temperate countries, including Australia. Genus Monilinia (family Sclerotiniaceae) has 35 described species that infect members of families Ericaceae and Rosaceae (Lino et al. 2016). Those of interest to fruit growers are M. fructicola (Wint.) Rehm. M. laxa (Aderh and Ruh), and M. fructigena (Honey ex Whetzel). The fungi infect blossoms, twigs, leaves, and fruit. Infection of blossoms causes the floral structure to become brown and shrivelled. Cankers may develop at the base of the flower and the fungus may spread into the twig if damp weather follows flowering. Gum-oozing cankers may form on the twigs. Spores germinating on leaves cause ‘shot-hole’ symptoms (which can also be a hypersensitive reaction to Pseudomonas syringae infection in leaves). Although the fungus can exist in latent form on the fruit from fruit set, symptoms are usually evident upon fruit maturity. A rapidly spreading brown rot develops and the fungus produces grey-brown conidia. In M. laxa and M. fructigena, conidia may form in concentric rings. Affected fruits shrivel and dry to become ‘mummies’ that often remain adhered to the tree. The fungus may over-winter in cankers, and in mummies on trees or on the ground.

Monilinia species reproduce mainly by asexual conidia, which are spread by wind and water splash. Conidia may also be vectored by insects, such as carpophilus beetles. The fruiting body (apothecia) of the sexual stage (teleomorph) of M. fructicola was recorded in Australia from stone and pome fruit in the 1920s (Harrison 1928). In Europe and North

America, apothecia of M. fructicola occur rarely under natural conditions, but they can be induced to form under laboratory conditions after chilling treatment (Holtz et al. 1998; De Cal et al. 2014). The sexual stage of M. laxa has been described only rarely from Europe, and is not recorded from elsewhere (Byrde and Willetts 1977; Willetts and Harada 1984; Holtz et al. 1998). Several vegetative compatibility groups have been identified for M. fructicola, but not for M. laxa (De Cal et al. 2014). Thus, it seems that M. fructicola may have access to greater genetic variation than M. laxa because it can potentially utilise both sexual and parasexual recombination.

Its greater potential for genetic change may explain why M. fructicola is seen as the more serious pathogen in stone fruit. Brown rot is managed mainly by application of fungicides at flowering and fruiting. Resistance to fungicides containing benzimidazoles is recorded in both M. fructicola and M. laxa, where several different amino acid substitutions occur in the betatubulin gene to effect resistance, but a greater number of mutations are recorded in M. fructicola than M. laxa (Ma et al. 2003, 2005; Ma and Michailides 2005). Resistance to dicarboximide and IBS fungicides is frequently documented in M. fructicola populations but less so from M. laxa populations (Elmer and Gaunt 1993; Penrose et al. 1979; Sanoamuang and Gaunt 1995).

M. fructicola, previously known as Sclerotinia fructicola, was first described from stone and pome fruit eastern Australia in 1921 (Harrison 1928), and M. laxa (S. laxa) was first identified in 1962 (Jenkins 1965; Penrose et al. 1979). Both species cause losses of up to 100% in Australian nectarine and peach crops. M. fructigena, which mainly infect pome fruit but also stone fruit has not been recorded from Australia (Stansbury et al. 2000). Brown rot costs the Australian stone fruit industry over $20 million per year in lost production, and $25 million in control. M. fructicola is listed as a quarantine pest in the European Plant Protection Zone, which impacts on Australia’s ability to market stone fruit to Europe. In 2006 and 2007, shipments of Australian peaches and nectarines were rejected on arrival in the UK because of brown rot infection (Holmes et al. 2011). M. fructicola is absent from Australia’s main Southern Hemisphere competitor, South Africa, giving it a significant advantage in the European market (Carstens et al. 2010).

In Western Australia, Monilinia is a relatively recent invader (Stansbury et al. 2000). It was first collected by officers of the Department of Agriculture Western Australia in Donnybrook

on December 1st 1997, and in Manjimup on December 17th 1997. Its presence in Western Australia was announced in the State Government Gazette on the 30th of December that year by the Minister of Primary Industries Monty House, who stated ‘… the whole of the state of Western Australia to be infected with the disease brown rot (Monilinia fructicola)’ (House 1997). These first Monilinia isolates collected from Donnybrook and Manjimup were later shown to be M. laxa, the name by which the original collections are referred to in the State pathogen collection.

There have been no published surveys of Monilinia distribution in Western Australia, although by 1999 both species were said to be widespread in Western Australia (Biosecurity Australia 2006). Knowledge of the relative distribution of the two species is important for the stone fruit industry because it has implications for control of the pathogen, and for access to export markets.

Differentiation of species by morphological characteristics or disease symptoms is difficult in the field (Anon 2014), but when spores are cultured on potato dextrose agar (PDA) plates grown at 22 °C, the growth form is distinctive for each species. M. laxa is typically slow growing and has a rosette growth form with dark rings between rosettes. Spores are produced at about 14 days tightly appressed to agar at the margins of rosettes. M. fructicola colonies are fast growing and have an even margin. Spores appear after 7–10 days evenly over the surface of the colony (Bush et al. 2009). Molecular tests using species-specific primers (Côté et al. 2004; Gell et al. 2007a, b) or generic ITS (Ioos and Frey 2000) or tef1 (Marin-Felix et al. 2017) primers are commonly used to distinguish species (Anon 2014).

Here, we present the results of a survey of Monilinia isolates from stone fruit production areas in Western Australia in 2016 and 2017.

Material and Method Ninety samples were collected from flowers, mature fruit, twig cankers and mummified fruit of apricot (Prunus armeniaca), nectarine and peach (P. persica), sweet cherry (P. avium) and plum (P. domestica) from 12 locations in stone fruit production areas of Western Australia, including Gingin, located 80 km to the north of Perth City, the Perth Hills (within

the greater Perth metropolitan region), Donnybrook, Pemberton and Manjimup, located 200– 300 km to the south of Perth (Table 1, Fig. 1). Spores were collected from symptomatic plant tissues in a drop of water. The spores were spread on 1% sterile water agar and single spores identified under a microscope. Individual spores were transferred to PDA plates and incubated in the dark at 22 °C for 6–7 days to allow the spores to germinate and the mycelium to grow. A 5 mm2 piece of agar containing actively growing mycelium was transferred into 250 ml flasks containing 50 ml of potato dextrose broth (PDB), and incubated on an orbital shaker at 100 rpm in the dark at 22 °C. After five days on the shaker, the mycelium was collected, rinsed with distilled water and pressed between two absorbent paper towels to remove excess liquid. DNA was extracted from the mycelium as follows. Mycelium (100 mg) was ground to a fine powder using a mortar and pestle in the presence of liquid nitrogen. Powder was transferred to a 1.5 ml centrifuge tube containing 450 μl Extraction Buffer (0.1 M NaCl, 50 mM Tris pH 8.0, 0.5 mM EDTA pH 8.0, 1% SDS, 1% PVPP) and 450 μl phenol-chloroform (1–1) (pH 8.0) saturated with TE. The mixture was homogenis ed for 5 min before being centrifuged for 2 min. The aqueous phase was transferred to a new tube containing an equal vol-ume of phenol-chloroform, then mixed and centrifuged 2 min. Then, 300 μl of the aqueous phase was removed to a fresh tube containing 58 μl absolute ethanol, to which 200 mg of cellulose powder was added and mixed thoroughly. After centrifuging at top speed for 1 min, 250 μl of the supernatant was transferred to a fresh tube and 25 μl 3 M NaOAC (pH 5.2) and 625 μl absolute ethanol added. The tube was centrifuged for 20 min at 20,000 x g and the supernatant poured off. The pellet was washed in 70% ethanol. The purified pellet was dissolved in 100 μl of elution buffer (10 mM Tris, pH 8.0) and stored at -20 °C.

Primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3) and ITS4 (5′- TCCTCCGCTTATTGATATGC-3) primers (White et al. 1990) were used to identify fungal sequences in a 20 μl PCR mixture composing of 10 ul GoTaq® Green PCR Master Mix

(Promega), 1 μl of either purified fungal DNA, 0.5 μl (5 μM) of each primer and 8 μl H2O. PCR amplification was run with an initial cycle of 5 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 55 °C, 30 s at 70 °C; the last one, polymerase extension, at 72 °C for 7 min.

PCR products were purified using an AxyPrep® Mag PCR Clean-up Kit. Sequencing was done in a 10 μl volume consisting of 4 μl purified PCR product, 3.2 μM of either ITS1 or ITS4 primer, 1.5 μl 5× ABI sequence buffer, 1 μl BigDye® Terminator v3.1 Ready Reaction

Mix and 2.5 μl H2O. The reaction was carried out with an initial cycle at 96 °C for 4 min; following by 25 cycles of 10 s at 96 °C, 5 s at 55 °C, and 4 min at 60 °C. Comparison of sequences with those on the NCBI GenBank database was done using Blastn (Altschul et al. 1997) against the NCBI and Q-Bank (Fungi) databases to identify Monilinia species. Alignment of the ITS sequences was done with ClustalW (Thompson et al. 2002). Neighbor- Joining (Saitou and Nei 1987) analysis using the Maximum Composite Likelihood method (Tamura et al. 2004) was used to estimate evolutionary distance after bootstrapping with 1000 replicates. The homologous ITS region of Sclerotinia sclerotiorum TNS:F-40021 (GenBank accession AB926054) was used as an outgroup. A phylogenetic tree was constructed in Mega 7 (Kumar et al. 2016) (Figs. 1 and 2).

An attempt was made to induce formation of the sexual stage of both Monilinia species. Mummified fruits were incubated in sealed plastic containers at 4 °C in the dark for 8–12 weeks. Relative humidity was maintained at >97%. Following the chilling period, the mummies were incubated in a damp matrix at 15 °C under a 12-h photo-period for 12 weeks. They were observed every few days for apothecia formation.

Table 1. Monilinia isolates collected from 12 sites in Western Australia, 2016–2017

Isolate Region Site Prunus species Tissue type Time of Monilinia Accession code collection species No. M0 Donnybrook 1 Nectarine (P. persica) Fruit 01/2017 M. laxa KY781237 M1 Pemberton 7 Plum (P. domestica) Mummy 03/2016 M. laxa KY781238 M1.1 Perth Hills 10 Peach (P. persica) Mummy 05/2016 M. laxa KY781239 M1.2 Perth Hills 11 Nectarine (P. persica) Fruit 11/2016 M. laxa KY781245 M101 Gingin 3 Peach (P. persica) Fruit 12/2016 M. fructicola KY781236 M102 Gingin 3 Nectarine (P. persica) Fruit 12/2016 M. fructicola KY781214 M103 Gingin 3 Peach (P. persica) Fruit 12/2016 M. fructicola KY781215 M104 Gingin 3 Nectarine (P. persica) Fruit 12/2016 M. fructicola KY781216 M105 Gingin 3 Peach (P. persica) Fruit 12/2016 M. fructicola KY781217 M106 Gingin 3 Peach (P. persica) Fruit 12/2016 M. fructicola KY781218 M107 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781219 M109 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781220 M110 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781221 M111 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781222 M112 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. laxa KY781276 M113 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781223 M114 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781224 M115 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781225 M116 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781226 M117 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781227 M118 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781228

M119 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781229 M120 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781230 M121 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781231 M122 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781232 M123 Perth Hills 9 Nectarine (P. persica) Fruit 12/2016 M. laxa KY781277 M124 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781233 M125 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781234 M126 Perth Hills 9 Apricot (P. armeniaca) Fruit 12/2016 M. fructicola KY781235 M128 Pemberton 7 Nectarine (P. persica) Fruit 01/2017 M. laxa KY781278 M131 Pemberton 8 Peach (P. persica) Fruit 01/2017 M. laxa KY781279 M133 Pemberton 8 Nectarine (P. persica) Fruit 01/2017 M. laxa KY781280 M136 Pemberton 8 Peach (P. persica) Fruit 01/2017 M. laxa KY781281 M139 Donnybrook 1 Apricot (P. armeniaca) Fruit 01/2017 M. laxa KY781282 M140 Donnybrook 1 Apricot (P. armeniaca) Fruit 01/2017 M. laxa KY781283 M141 Donnybrook 1 Apricot (P. armeniaca) Fruit 01/2017 M. laxa KY781284 M19 Perth Hills 12 Peach (P. persica) Mummy 04/2016 M. fructicola KY781202 M20 Perth Hills 12 Peach (P. persica) Mummy 04/2016 M. laxa KY781244 M22 Perth Hills 10 Peach (P. persica) Mummy 05/2016 M. laxa KY781245 M24 Perth Hills 10 Apricot (P. armeniaca) Mummy 05/2016 M. laxa KY781246 M25 Pemberton 7 Apricot (P. armeniaca) Mummy 08/2016 M. laxa KY781247 M27 Peerabeelup 6 Plum (P. domestica) Mummy 09/2016 M. laxa KY781248 M28 Peerabeelup 6 Peach (P. persica) Mummy 09/2016 M. laxa KY781249 M29 Peerabeelup 6 Peach (P. persica) Mummy 09/2016 M. laxa KY781250 M30 Peerabeelup 6 Plum (P. domestica) Mummy 09/2016 M. laxa KY781251 M31 Peerabeelup 6 Peach (P. persica) Mummy 09/2016 M. laxa KY781252 M32 Peerabeelup 6 Peach (P. persica) Mummy 09/2016 M. laxa KY781253 M33 Peerabeelup 6 Peach (P. persica) Mummy 09/2016 M. laxa KY781254 M34 Peerabeelup 6 Peach (P. persica) Mummy 09/2016 M. laxa KY781255 M35 Peerabeelup 6 Peach (P. persica) Mummy 09/2016 M. laxa KY781256 M36 Nannup 5 Apricot (P. armeniaca) Mummy 09/2016 M. laxa KY781257 M37 Nannup 5 Peach (P. persica) Mummy 09/2016 M. laxa KY781258 M40 Pemberton 7 Plum (P. domestica) Mummy 09/2016 M. laxa KY781259 M41 Pemberton 7 Apricot (P. armeniaca) Mummy 09/2016 M. laxa KY781260 M42 Pemberton 8 Peach (P. persica) Mummy 09/2016 M. laxa KY781261 M43 Manjimup 4 Plum (P. domestica) Mummy 09/2016 M. laxa KY781262 M44 Pemberton 7 Plum (P. domestica) Mummy 09/2016 M. laxa KY781263 M5 Pemberton 7 Apricot (P. armeniaca) Mummy 03/2016 M. laxa KY781240 M50 Pemberton 8 Plum (P. domestica) Mummy 09/2016 M. laxa KY781264 M51 Perth Hills 9 Peach (P. persica) Mummy 09/2016 M. laxa KY781265 M52 Perth Hills 9 Peach (P. persica) Mummy 09/2016 M. laxa KY781266 M53 Perth Hills 9 Peach (P. persica) Mummy 09/2016 M. laxa KY781267 M6 Pemberton 7 Peach (P. persica) Mummy 03/2016 M. laxa KY781241 M60 Pemberton 8 Peach (P. persica) Flower 11/2016 M. laxa KY781268 M61 Pemberton 7 Cherry (P. avium) Flower 11/2016 M. laxa KY781269 M65 Pemberton 8 Plum (P. domestica) Flower 11/2016 M. laxa KY781270 M67 Pemberton 8 Plum (P. domestica) Flower 11/2016 M. laxa KY781271 M68 Pemberton 8 Plum (P. domestica) Flower 11/2016 M. laxa KY781272 M7 Pemberton 7 Apricot (P. armeniaca) Mummy 03/2016 M. laxa KY781242 M75 Gingin 2 Nectarine (P. persica) Fruit 11/2016 M. fructicola KY781203 M81 Perth Hills 11 Nectarine (P. persica) Fruit 12/2016 M. laxa KY781273

M82 Perth Hills 11 Nectarine (P. persica) Fruit 12/2016 M. laxa KY781274 M84 Perth Hills 11 Nectarine (P. persica) Fruit 12/2016 M. laxa KY781275 M86 Gingin 2 Peach (P. persica) Fruit 12/2016 M. fructicola KY781204 M87 Gingin 2 Peach (P. persica) Fruit 12/2016 M. fructicola KY781205 M88 Gingin 2 Peach (P. persica) Fruit 12/2016 M. fructicola KY781206 M89 Gingin 2 Peach (P. persica) Fruit 12/2016 M. fructicola KY781207 M9 Pemberton 7 Cherry (P. avium) Mummy 03/2016 M. laxa KY781243 M90 Gingin 3 Peach (P. persica) Fruit 12/2016 M. fructicola KY781208 M91 Gingin 3 Peach (P. persica) Fruit 12/2016 M. fructicola KY781209 M92 Gingin 3 Peach (P. persica) Fruit 12/2016 M. fructicola KY781210 M93 Gingin 3 Peach (P. persica) Fruit 12/2016 M. fructicola KY781211 M98 Gingin 3 Peach (P. persica) Fruit 12/2016 M. fructicola KY781212

Fig. 1 Map showing the location of collection sites in Western Australia, and the identity of Monilinia isolates collected at each site

Result and Discussion Of the 90 samples collected, ITS sequence analysis con-firmed 49 isolates were of M. laxa, 34 were of M. fructicola, and seven were of Botrytis cinerea. The M. laxa and M. fructicola

ITS sequences matched 100% against ITS sequences from verified M. laxa and M. fructicola isolates listed on Q-Bank (Fungi).

Surprisingly, distribution of the Monilinia species was not uniform. All the M. fructicola isolates were from the Perth Hills and Gingin to the north of Perth, while all the M. laxa isolates were from the Perth Hills and sites to the south of Perth. Both species co-existed in the Perth Hills (Table 1, Fig. 1).

The sexual stage of isolates of M. fructicola and M. laxa were not observed after treatment by chilling. Others have found it impossible to induce apothecia of M. laxa, but possible to induce them in M. fructicola (Willetts and Harada 1984).

Both species were identified from highly managed commercial stone fruit orchards where fungicide is sprayed regularly to control brown rot, and from one or a few trees in home gardens where little or no fungicide was applied. Both species were identified from peach, nectarine and apricot, but only M. laxa was identified from plums and cherry. Both plums and cherries are susceptible to M. fructicola (Adaskaveg et al. 2000; Luo and Michailides 2001), and we explain its absence in the samples collected in this study by the limited collections of these species, and their collections being only from the southern production areas where M. fructicola was not identified.

The differential distribution of the two Monilinia species in WA reflects their relatively recent introduction to the State, and their places of introduction. The results of this study indicate that Monilinia species were introduced to WA on at least two separate occasions. M. laxa may have been the first species to be introduced, and this occurred in the southern production region. The largest local market for stone fruit is Perth city, and transport of infected fruit from the south to the city may have introduced the pathogen to the Perth Hills orchards. The introduction of M. fructicola to the Perth Hills and Gingin regions may have occurred when the WA market was opened to stone fruit grown in eastern Australian, New Zealand and North America where M. fructicola is endemic (Biosecurity Australia 2003, 2006).

Of the two Monilinia species detected in WA, M. fructicola is considered to be the most destructive pathogen because it has a reported greater capacity for genetic by sexual

recombination and by anastomosis. Thus, M. fructicola has the greater potential to develop resistance to fungicides and to overcome genetic barriers to infection in host plants.

Currently the southern stone fruit production regions of Western Australia appear to be free of M. fructicola, and it is important that they remain so. Protecting the southern production regions from M. fructicola may be achieved by restricting movement to the south of fruit and germplasm sourced from the Perth Hills and areas to the north. M. fructicola is also a quarantine pathogen in Europe, and its absence in the southern production areas provides those growers with export opportunities.

Similarly, preventing the spread of M. laxa to the northern production regions is important. Maintaining a single species of Monilinia in the Gingin production area will simplify control of brown rot there.

The most problematic region is the Perth Hills where the two species co-exist. Although brown rot caused by the two Monilinia species is controlled in essentially the same way, the two species exhibit differences in biology and pathogenicity, which may have implications for effective control (Hrustić et al. 2012).

M. laxa is reported to be a more serious pathogen on apri-cot, especially infecting flowers and on mature cherry fruit (Holb 2008). M. laxa occasionally infects pome fruits, notably quince (Cydonia oblonga). Optimal temperature for disease development is 24°C, although infection and symptom devel-opment occur in a very wide temperature range (4-30°C). M. laxa causes serious losses to chilled fruit post-harvest (Holb 2008).

M. fructicola is the cause of serious disease on peach and nectarine, but it also causes losses on cherries and plums (Wilson and Ogawa 1979). An important difference between the two species is that the mycelia of M. fructicola can remain latent on very young fruits until the beginning of fruit ripening when the symptoms of the disease become clearly evident (Batra 1991; Emery et al. 2000). Another difference is that temperatures below 10°C are unfavorable for infection establishment of M. fructicola (Luo and Michailides 2001), which has implications for cold storage of fruit.

Fig. 2 Phylogenetic tree of ITS regions of isolates of Monilinia laxa and M. fructicola collected in Western Australia. The evolutionary relationship was inferred using the Neighbor-Joining method, and the percentage of replicate trees in which the associated taxa clustered together in the M. fructicola bootstrap test (1000 replicates) is shown next to the branches. The homologous region of an isolate of Sclerotinia sclerotiorum was used as the outgroup

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Addendum to Chapter 2 Regarding the claim that the southern stone fruit production regions of western Australia appear to be free of M. fructicola, and the northern areas appear to be free of M. laxa, we add the cautionary note that this statement was based on the limited number of samples collected during the early course of this study, and that future collections may reveal more mixing of species than is currently apparent.

Chapter 3. Genotypic structure of Monilinia populations in Western Australia two decades after incursion

This chapter addressed aim 2 of the thesis and was published in Australasian Plant Pathology in 2019.

Citation:

Statement of contribution of the authors contributed to the work

Abstract In 1997, Monilinia fructicola and Monilinia laxa, fungi causing brown rot disease in stone fruit (Prunus species), were identified from Western Australia for the first time. Up until then, Monilinia were quarantine species, and importation of stone fruits to W.A. was prohibited. After Monilinia was identified in W.A., importation of stone fruit from sources outside W.A. was progressively permitted. Today, Monilinia is present in all stone fruit production regions in W.A. The aim of this study was to determine if the genotypes responsible for the first incursion subsequently spread, or if new genotypes have since become established. ISSR markers were used to identify the genotype of isolates collected during the initial incursion event in 1997, and compare them with isolates collected subsequently. Eight M. fructicola genotypes were identified, including a monotypic one on a fresh peach imported from the USA. M. fructicola isolates collected during the initial incursion in 1997 and an isolate from cherry collected in South Australia in the same year were all of the same genotype, suggesting fruit or germplasm from S.A. as the source of the W.A. incursion. However, this incursion genotype appears not have persisted, with different genotypes subsequently becoming widely or locally established. Four genotypes of M. laxa were identified. In contrast to M. fructicola, the 1997 incursion genotype of M. laxa has become widely established in W.A., infecting both stone fruits and pome fruits.

Introduction Monilinia fungi are ascomycetous pathogens of some cultivated members of the Rosaceae. Although about 35 species are described (Batra 1991), only M. laxa and M. fructicola cause brown rot mainly in Prunus (stone fruit), but can also be minor pathogens in Malus (apple), Pyrus (pear) and Cydonia (quince). Monilinia fructigena and the anamorph Monilia polystroma, are mainly pathogens of apple, pear and quince. They may also cause canker in wood, and blossom blight of flowers. M. fructigena is reported from Europe, and it is a quarantine disease for Canada, the United States, Australia and New Zealand. M. laxa is described from Europe, the Americas, Japan, and Australasia. M. fructicola is reported from the Americas, Japan, Australasia, Greece and Spain, and it is a quarantine pathogen for some European countries and the UK (De Cal et al. 2009). Monilia polystroma is a disease mainly of apple, and it has been

described from Japan (Leeuwen et al. 2002), China (Zhu and Guo 2010), and Hungary (Petroczy and Palkovics 2009).

Monilinia fructicola and M. laxa are reported from all Australian states and territories where their hosts are grown (Holmes 2011). All Monilinia species were quarantine diseases in Western Australia (W.A.) prior to identification in December 1997 from stone fruit trees at Balingup, Beelerup, Brookhampton, Carmel, Donnybook, Manjimup and Mount Barker, all located 200- 300 km to the south of Perth (House 1997; AMRiN 2015). In 2001, Monilinia was identified from Bickley, Dwellingup, and Pickering Brook, all located within 100 km of Perth. The source of the original incursion is unknown, but Monilinia may have been introduced to the region inadvertently in infected bud wood and grafted trees imported by the fruit industry or research stations, or on stone fruits smuggled into the state by members of the public. Since the initial incursion, Monilinia has spread to all commercial stone fruit production regions in the state. Our previous study reported differential distribution of the two Monilinia species in W.A.; M. fructicola occupied the region from Perth to the Gingin area to the north of Perth, while M. laxa occupied the Perth region and areas to the south of Perth (Tran et al. 2017).

Monilinia species are not easy to distinguish by disease symptoms on plant tissue. Thus, diagnosis to the species level often requires consideration of a combination of characteristics, including colony morphology in vitro (Lane 2002) and DNA sequence characteristics (Cote et al. 2004; Gell et al. 2007a). Studying the genetic structure of Monilinia populations provides information on sources of genetic variation, evolution, pathogenicity and epidemiology. The internal transcribed spacer region (ITS) of rDNA has sufficient resolution (5-13 nucleotide substitutions) to distinguish Monilinia species, but it provides little information on intra-specific variation (van Leeuwen et al. 2002). To study intra-specific genetic variation, inter simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) markers have been used in M. laxa populations in Spain (Gell et al. 2007b), Greece (Papavasileiou et al. 2015), and Hungary (Fazekas et al. 2014), and in M. fructicola populations in Spain (Villarino et al. 2012), Greece (Papavasileiou et al. 2015) and China (Fan et al. 2010).

In the approximately two-decade history of Monilinia in Western Australia, there has been no study of the genetic structure of M. fructicola and M. laxa populations in this region. Here, we investigate the genotypes of the M. fructicola and M. laxa ‘incursion isolates’ collected from W.A. in 1997 and 2001, and compare them with more than 160 isolates collected subsequently from the stone fruit production regions in the state. Knowledge of the genetic diversity of these serious pathogens will inform biosecurity measures that aim to limit dispersal of species and genotypes within the region.

Materials and methods Fungus collection

Historical isolates of M. fructicola (10 isolates) and M. laxa (8 isolates) were provided by the Department of Primary Industries and Regional Development (DPIRD). DRIRD isolates were collected between 1997 and 2013 from stone fruit orchards in W.A. and South Australia, grown on millet seed, and stored as lyophilized cultures in airtight vials. During the period 2016 to 2018, the authors collected 72 M. fructicola and 75 M. laxa isolates from 147 Prunus flowers, mature fruits and twig cankers showing symptoms of Monilinia infection across all the major stone fruit production regions of Western Australia. One M. fructicola isolate from a symptomatic fresh peach fruit imported from the USA was collected from a Perth supermarket in July 2017 (Table 1, Fig 1).

Fungal isolation Conidia were collected from symptomatic plant tissues in a drop of sterile water. The spores were spread on 1% sterile water agar and single spores were identified under a microscope. Only one single spore was picked from each sample and transferred to potato dextrose agar (PDA) or V8 agar plates and incubated in the dark at 22oC for 6-7 days. A five mm3 cube of agar containing actively growing hyphae was excised from the edge of the mycelium and transferred to potato dextrose broth (PDB) or V8 liquid media, incubated on an orbital shaker in the dark at 22oC. After 7 days shaking on those liquid media, mycelium was harvested for DNA extraction, or stored in 15% glycerol at -70oC.

DNA extraction

The mycelial mass was collected, rinsed with distilled water and pressed between two absorbent paper towels to remove excess liquid. Mycelium was frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. Powdered mycelium was transferred to a 1.5ml centrifuge tube containing 450 l extraction buffer (0.1M NaCl, 50mM Tris pH8.0, 0.5mM EDTA pH8.0, 1% SDS, 1% PVPP) and 450 l phenol-chloroform (1:1) saturated with TE (pH 8.0). The mixture was homogenised on a vortexer for 5 min before being centrifuged at room temperature for 2 min. The aqueous phase was transferred to a new tube containing an equal volume of phenol-chloroform, mixed and centrifuged 2 min. Then, 300 l of the aqueous phase was removed to a fresh tube containing 58 l absolute ethanol, to which 200 mg of cellulose powder (CF11, Whatman) was added and mixed. After centrifuging at high speed for 1 min, 250 l of the supernatant was transferred to a fresh tube and 25 l 3M NaOAC (pH 5.2) and 625 l absolute ethanol added. The tube was centrifuged for 20 min at maximum speed (20,000 x g) and the supernatant was removed. The DNA pellet was washed in 70% ethanol. The DNA was dissolved in 100 l of elution buffer (10 mM Tris, pH 8.0) and stored at -20oC. Species identification was done by comparison of ITS sequences, as described (Tran et al. 2017).

ISSR assay

Six ISSR primers reported to generate reproducible polymorphic banding patterns from M. fructicola DNA (Villarino et al. 2012) were used to measure genetic diversity of 83 M. fructicola and 86 M. laxa isolates. Primers used were Mf-2 (AC)8 (Lim et al., 2004), Mf-5 (AAC)5, Mf-7

(AAG)8, Mf-8 (AG)8C, Mf-9 (GACA)4, Mf-13-Mf (GAG GGT GGC GGC GGT TCT) (Ma and Michailides 2003). Amplification was done in a 20 l PCR mixture composing 10 l GoTaq®

Green PCR Master Mix (Promega), 1 l of purified fungal DNA (approx. 300 ng), 8 l H2O, and 1 l primer. PCR amplification was done after an initial denaturation cycle of 3 min at 95oC. Annealing temperatures differed depending on the primer used. Amplification occurred over 40 cycles of 1 min at 94oC in all cases, then 1 min at 50oC (for Mf-5, Mf-7, Mf-13-Mf), or 1 min at 45oC (for Mf-8), or 1 min at 36oC (for Mf-9), or 1 min at 52oC (for Mf-2), followed in all cases by 1 min at 72oC. There was a final incubation at 72oC for 10 min. PCR products were separated

on 3% agarose gels stained with SYBR™ safe stain (Thermo Fisher). All ISSR experiments were done twice. Data analysis

PCR products ranged in size from approximately 0.2 - 2.0 kb, and each clear band (allele) was recorded as either present (1) or absent (0) for each isolate. Where bands were very faint and/or were not reproducible between amplifications, they were ignored. ISSR data was analysed using GenAlEx6.5 software (Peakall and Smouse, 2012). Dendrograms were generated using Maximum Composite Likelihood model (Tamura et al. 2004) with the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) (Sneath and Sokal, 1973).

Results M. fructicola isolates

The six ISSR markers generated 57 polymorphic alleles for the 82 M. fructicola isolates tested (Fig S1). UPGMA analysis of banding patterns showed that M. fructicola isolates fell into eight groups, referred to henceforth as genotypes F1 to F8. Genotype F8 comprised all six M. fructicola isolates collected from Donnybrook in 1997, and one isolate from South Australia. Genotype F7 was represented by one isolate collected from a peach imported from the USA in 2017. Genotype F6 consisted of two isolates collected in 2001 from Bickley and Gidgegannup. These sites are located in the Perth Hills region. Genotype F5 was present at Roleystone and Karragullen, both in the Perth Hills region. Genotype F4 appears to be confined to Kirup, located about 200 km south of Perth. Monotypic genotypes F2 and F3 were represented by isolates from Karragullen (2012) and from Gingin (2017), respectively. Genotype F1 isolates were collected 2016-2017 from the Perth Hills region and Gingin (Fig 2). M. laxa isolates

The six ISSR markers generated 36 polymorphic alleles for the 83 M. laxa isolates tested, and these clustered into four groups, referred to as genotypes L1 to L4 (Fig S2). One M. laxa isolate, WAC9458, was identified from the 1997 Donnybrook collection, confirming that both species were present when Monilinia was first discovered in the state. The incursion isolate belongs to genotype L1, which remains a dominant and widespread genotype in the region today. Genotype

L1 occurred in most sampling regions in the current study, from Mount Barker (~350 km south of Perth) to the Perth Hills region. Two L1 isolates, WAC10037 and WAC13636, were unusual in that they were collected from non-Prunus hosts, apple and pear, respectively. In contrast, genotype L2 occurred only at Mullaylyup. Genotype L3 was represented by only one isolate collected in Dwellingup in 2001. Genotype L4 contained two isolates collected in 2001 and 2004, one from apple (Fig 3).

Discussion

Although Monilinia (previously named Sclerotinia) was recorded first in New South Wales from stone fruit in 1921 (Harrison 1928), Western Australia remained free of it until M. fructicola and M. laxa were recorded from Balingup, Beelerup, Brookhampton, Carmel, Donnybrook, Manjimup and Mount Barker in 1997. Unfortunately, only the 1997 isolates from Donnybrook were kept in the DPIRD collection. This apparently wide distribution of Monilinia in the southern production region in 1997 indicates either that the fungus was present in the region for some time before 1997 and spread between sites, or that it was simultaneously introduced into the state at different sites.

The means by which Monilinia was introduced into W.A. is not known, but our data suggests there was only one genotype present at incursion sites in 1997 (at Donnybrook), and its source may have been South Australia. It may have arrived in infected stone fruit smuggled into W.A., or it came in via germplasm imported by growers or a nursery, or to a stone fruit research station, such as the one located at Manjimup.

The incursion genotype of M. fructicola present at Donnybrook in 1997 (genotype F8) was not identified in isolates collected subsequently. It may have been eliminated in efforts to eliminate this fungus after the incursion was revealed, or it may still be present in the region but it rare and/or wasn’t collected in this study. Genotype F6, collected in 2001 from the Perth Hills area clearly represents an incursion distinct from the Donnybrook one from four years earlier. Genotype F6 was not identified from recent collections in the Perth Hills, indicating it too may be rare or extinct. In contrast, the incursion genotype of M. laxa (L1) is widely distributed in

W.A. today, infecting both stone fruits and apple. Genotype L2 appears to be locally distributed at one site, and genotypes L3 and L4 were identified from isolates collected in 2001 and 2004. More extensive collections of isolates may reveal other genotypes.

Some genotypes of both Monilinia species occurred widely, notably genotypes F1 and L1, while others (e.g. F4 and L2) appeared to be locally restricted. This indicates that wind dispersal of spores is not a primary means of spread of Monilinia between sites. Although there have been several studies on natural dispersal of spores of Monilinia species, including M. laxa (Wilson and Baker 1946), M. fructicola (Kable 1965), M. fructigena (Banoon et al. 2009), and M. vaccinii- corymbosi (Cox and Scherm 2001), there remain questions about spore dissemination and its relation to environmental factors. Short distance dispersal occurs when conidia (vegetative spores) are spread to flowers, wood and fruit of surrounding trees by wind, rain splash, insects, etc (Holb 2008). Ascospores (sexual spores), which have not been identified in W.A., are spread in spring to flowers by the same means. Kable (1965) studied M. fructicola conidia dispersal through the air in peach orchards in New South Wales, and found spores travelled only up to 100 m under natural conditions. In M. vaccinii-corymbosi, conidia traveled only 20 m in wind, while ascospores traveled 30 m in the same conditions (Cox and Scherm 2001). Dispersal over longer distances is facilitated by humans when they transport infected fruit and germplasm.

Four scenarios in isolation or in combination may account for current distribution patterns of Monilinia genotypes in W.A.: i. Germplasm (bud wood, grafted seedlings) infected with Monilinia isolates of one genotypes was imported to one location, for example to establish a new orchard, and this genotype remains isolated at that site. ii. Infected germplasm was imported by a commercial nursery or research facility, and grafted trees were distributed widely from there to orchards in different regions. The isolates of this genotype become widespread. iii. Fresh stone fruit was first imported from the eastern states of Australia from 2001, from New Zealand in 2004 (Biosecurity Australia 2006), and from the USA in 2013 (Boothroyd 2013). Imported fruits infected with Monilinia isolates may become established in W.A. if they are able to infect local Prunus trees.

iv. Novel Monilinia genotypes evolve from existing genotypes in situ as a result of recombination (during meiosis), selection (e.g. fungicide application) and genetic drift.

Scenarios i and ii seem to explain the current genotype distribution patterns. Scenario i may explain locally-distributed genotypes, e.g. F4 and L2 that each occurred at only one site, whereas scenario ii may explain the more widely distributed genotypes. e.g. F1 and L1 which consisted of isolates from several sites.

There is no evidence that scenarios iii and iv have occurred in W.A. The discovery of a M. fructicola isolate on an imported peach from the USA is evidence that new Monilinia genotypes are transported into the state on imported fruit, but evidence that isolates arriving on fruit establish in the state is lacking at present. Plant pathogens such as viruses infecting plant propagules are known to ‘hitchhike’ across the globe in traded fresh produce and become established in new areas when they are planted (Wylie et al. 2014). However, in the case of Monilinia-infected fruits, orchardists are well aware of the risks of bringing imported fruit onto their orchards. Infected imported fruit would need to be discarded in or very near stone fruit trees for infection to take place. In the case of isolates from American-sourced fruit, it seems less likely that American isolates could become established than isolates from Southern Hemisphere sources because Prunus trees are dormant when American stone fruit imports occur. We consider scenario iii a minor, yet possible, route to establishment of new genotypes.

Regarding scenario iv, the rate of genome change of Monilinia has not been studied, so the role of mutation and selection in generating new genotypes in W.A. is unknown. Recombination through meiosis may not be possible because sexual reproduction has not been observed in Monilinia in Western Australia.

The current study did not identify pathogenicity or fungicide tolerance characteristics of the genotypes identified. Recent studies have been undertaken into the pathogenicity and responses to fungicides of members of the genotypes for which live cultures exist (Tran et al., 2018). This information will enable a more targeted approach to fungicide application, enabling growers to

select fungicides based on knowledge of the fungicide tolerance of genotypes present on their properties.

Although Monilinia is firmly established in all stone fruit production regions in W.A. today, it should remain a priority for biosecurity authorities and the stone fruit industry to prevent new genotypes entering the state. New genotypes could bring with them new pathogenicities and fungicide tolerances, which will negatively impact the industry.

References AMRiN (2015) Occurrence record: WAC - WAC9462 Monilinia. Department of Agriculture and Food - Western Australia. http://amrin.ala.org.au/occurrences/e656aaee-e0c4-4330-ab2d- eb15023f876a;jsessionid=4F6F18291AFA044E7FCB9C2AFFF8A7DC Bannon F, Gort G, van Leeuwen G, Holb I, Jeger M (2009) Diurnal patterns in dispersal of Monilinia fructigena conidia in an apple orchard in relation to weather factors. Agric For Meteorol 149:518-525. Batra LR (1991) World species of Monilinia (Fungi): their ecology, biosystematics and control Mycologia Memoir No 16, 246 pp. Biosecurity Australia (2006) Final Report: Pest risk analysis for stone fruit from New Zealand into Western Australia. Biosecurity Australia, Agriculture, Fishery and Forestry - Australia (AFFA). Doi: org/10.1093/eurrag/29.3.329. Boothroyd A (2013) Imported US stone fruit approved for Aussie supermarkets. Food and Beverage. 29 July. https://foodmag.com.au/imported-us-stone-fruit-approved-for-aussie- supermarkets/ Accessed 11 May 2018. Cote MJ, Tardif MC, Meldrum AJ (2004) Identification of Monilinia fructigena, M.fructicola, M.laxa, and Monilia polystroma on inoculated and naturally infected fruit using multiplex PCR. Plant Dis 88:1219-1225. Cox KD, Scherm H (2001) Gradients of primary and secondary infection by Monilinia vaccinii- corymbosi from point sources of ascospores and conidia. Plant Dis 85:955-959. De Cal A, Gell I, Usall J, Vinas I, Melgarejo P (2009) First report of brown rot caused by Monilinia fructicola in peach orchards in Ebro Valley, Spain. Plant Dis 93:763-763.

Fazekas M, Madar A, Sipiczki M, Miklós I, Holb IJ (2014) Genetic diversity in Monilinia laxa populations in stone fruit species in Hungary. World J Microbiol Biotechnol 30:1879-1892. Gell I, Cubero J, Melgarejo P (2007a) Two different PCR approaches for universal diagnosis of brown rot and identification of Monilinia spp. in stone fruit trees. J Appl Microbiol 103:2629-2637. Gell I, Larena I, Melgarejo P (2007b) Genetic diversity in Monilinia laxa populations in peach orchards in Spain. J Phytopathol 155:549-556. Harrison TH (1928) Brown rot of fruits and associated diseases in Australia. I. History of the disease and determination of the causal organism. J Proc R Soc NSW 52:99-148. doi: 10.2307/3754152 Holb IJ (2008) Monitoring conidial density of Monilinia fructigena in the air in relation to brown rot development in integrated and organic apple orchards. Eur J Plant Pathol 120:397. Holmes R, Kreidl S, Villalta O, Gouk C (2011) Through chain approach for managing brown rot in summerfruit and canning fruit. Project code MT08039. Biosciences Research Division, Department of Primary Industries, Victoria, Australia House M (1997) AG401. Plant Diseases Act 1914. Government Gazette, W.A.. Government Printer, State Law Publisher 235:7507 Lane CR (2002) A synoptic key for differentiation of Monilinia fructicola, M. fructigena and M. Iaxa, based on examination of cultural characters. Bulletin OEPP 32:489-493. Lim S, Notley-McRobb L, Lim M, Carter DA (2004) A comparison of nature and abundance of microsatellite in 14 fungal genomes. Fungal Genet Biol 41:1025–1036. Ma Z, Luo Y, Michailides TJ (2003) Nested PCR assays for detection of Monilinia fructicola in stone fruit orchards and Botryosphaeria dothidea from pistachios in California. J Phytopathol 151:312–322. Papavasileiou A, Karaoglanidis GS, Michailides TJ (2015) Intraspecific diversity of Monilinia fructicola and M. laxa populations from blossoms and fruit of different hosts in Greece. Plant Dis 99:1353-1359. Peakall R, Smouse PE (2012) GenAlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research – an update. Bioinformatics 28:2537-2539. Petróczy M, Palkovics L (2009) First report of Monilia polystroma on apple in Hungary. Eur J Plant Pathol 125:343-347.

Sneath PHA, Sokal RR (1973) Numerical Taxonomy. Freeman, San Francisco. Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Nat Acad Sci (USA) 101:11030-11035. Tran TT, Li H, Nguyen DQ, Sivasithamparam K, Jones MGK, Wylie SJ (2017) Spatial distribution of Monilinia fructicola and M. laxa in stone fruit production areas in Western Australia. Aust Plant Pathol 46:339-349 doi:10.1007/s13313-017-0497-9 Tran TT, Li H, Nguyen, DQ, Jones, MGK, Sivasithamparam K, Wylie SJ (2018) Monilinia fructicola and Monilinia laxa isolates from stone fruit orchards sprayed with fungicides displayed a broader range of responses to fungicides than those from unsprayed orchards. Eur J Plant Pathol https://doi.org/10.1007/s10658-018-01613-x Van Leeuwen GC, Yen RPB, Holb IJ, Jeger MJ (2002) Distinction of the Asiatic brown rot fungus Monilia polystroma sp. nov. from M. fructigena. Mycol Res 106:444-451. Villarino M, Larena I, Martinez F, Melgarejo P, De Cal A (2012) Analysis of genetic diversity in Monilinia fructicola from the Ebro Valley in Spain using ISSR and RAPD markers. Eur J Plant Pathol 132:511-24. Wilson EE, Baker GA (1946) Some aspects of the aerial dissemination of spores, with special reference to conidia of Sclerotinia laxa. J Agric Res 72:301. Wylie SJ, Li H, Saqib M, Jones MGK (2014) The global trade in fresh produce and the vagility of plant viruses: A case study in garlic. PloS One, 9:p.e105044. Zhu XQ, Guo LY (2010) First report of brown rot on plum caused by Monilia polystroma in China. Plant Dis 94:478-478.

Figure captions

Figure 1. Map of study sites showing approximate distances between them.

Genotype F1

Genotype F2 Genotype F3

Genotype F4

Genotype F5

Genotype F6

Genotype F7

Genotype F8 57

Figure 2. Evolutionary relationships of 82 Monilinia fructicola isolates inferred using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) (Sneath and Sokal 1973). The optimal tree with a sum of branch lengths of 5.735 is shown. Evolutionary distances were computed using Maximum Composite Likelihood (Tamura et al. 2004). The overall mean distance was 0.404. There were 57 positions in the final dataset. Isolate name, collection sites, collection year and hosts are given, where known.

Genotype L1

Genotype L2

Genotype L3 Genotype L4 59

Figure 3. Evolutionary relationships of 83 Monilinia laxa isolates inferred using Unweighted Pair Group Method with Arithmetic Mean (UPGMA) (Sneath and Sokal 1973). The optimal tree with the sum of branch lengths of 5.039 is shown. The evolutionary distances were computed using Maximum Composite Likelihood (Tamura et al. 2004). The overall mean distance was 0.333 with 36 positions in the final dataset. Isolate name, collection sites, collection year and hosts are given, where known. SWH = South-west highway.

Supplementary Figures

1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20

(a) Primer Mf-2, M. fructicola species. Lane 1: 100 bp DNA ladder, lanes 2-20: M19, M75, M76, M86, M87, M88, M90, M91, M92, M94, M101, M102, M104, M105, M106, M107, M109, M110, M111.

1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20

(b) Primer Mf-7, M. fructicola species. Lane 1: 100 bp DNA ladder, lanes 2-20: M19, M75, M76, M86, M87, M88, M90, M91, M92, M94, M101, M102, M104, M105, M106, M107, M109, M110, M111.

1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20

(c) Primer Mf-8, M. fructicola species. Lane 1: 100 bp DNA ladder, lanes 2-20: M19, M75, M76, M86, M87, M88, M90, M91, M92, M94, M101, M102, M104, M105, M106, M107, M109, M110, M111.

1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20

(d) Mf-9, M. fructicola species. Lane 1: 100 bp DNA ladder, lanes 2-20: M19, M75, M76, M86, M87, M88, M90, M91, M92, M94, M101, M102, M104, M105, M106, M107, M109, M110, M111.

Fig. S1: Electrophoretic patterns of the ISSR markers Mf-2 (a), Mf-7 (b), Mf-8 (c), Mf-9 (d) for 19 M. fructicola isolates.

1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 21 22 23 24 25

(a) Primer Mf-2, M. laxa species. Lane 1 and 25: 100 bp DNA ladder, lanes 2-24: M1, M1-1, M5, M6, M7, M9, M112, M20, M22, M23, M24, M25, M27, M28, M29, M30, M31, M32, M33, M34, M35, M36, M37.

1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 21 22 23 24 25

(b) Primer Mf-7, M. laxa species. Lane 1 and 25: 100 bp DNA ladder, lanes 2-24: M1, M1-1, M5, M6, M7, M9, M112, M20, M22, M23, M24, M25, M27, M28, M29, M30, M31, M32, M33, M34, M35, M36, M37.

1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 21 22 23 24 25

(c) Primer Mf-8, M. laxa species. Lane 1 and 25: 100 bp DNA ladder, lanes 2-24: M1, M1-1, M5, M6, M7, M9, M112, M20, M22, M23, M24, M25, M27, M28, M29, M30, M31, M32, M33, M34, M35, M36, M37.

1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 21 22 23 24 25

(d) Primer Mf-9, M. laxa species. Lane 1 and 25: 100 bp DNA ladder, lanes 2-24: M1, M1-1, M5, M6, M7, M9, M112, M20, M22, M23, M24, M25, M27, M28, M29, M30, M31, M32, M33, M34, M35, M36, M37.

Figure S2: Electrophoretic patterns of the ISSR markers Mf-2 (a), Mf-7 (b), Mf-8 (c), Mf-9 (d) for 23 M. laxa isolates.

Chapter 4. Comparisons between Monilinia fructicola and Monilinia laxa isolates on genetic diversity, virulence and colony morphology

This chapter addressed aim 3 of the thesis and it is under the review of the Journal of Plant Pathology.

Abstract Monilinia fructicola and M. laxa, causing brown rot in stone fruit (Prunus species), were first detected in Western Australia in 1997. Our previous studies present that since then, there are still only these two species exist in the stone fruit production areas in the State, and they differentially respond to different fungicides used to control brown rot. This research aims to compare these two species in terms of genetic diversity, morphology, and virulence. Genetic diversity of 68 M. laxa and 66 M. fructicola isolates collected in 2016-2017 from five populations was measured using six ISSR markers. The genetic diversity within M. fructicola was higher than that of M. laxa, having Shannon’s diversity indices of 0.50 and 0.41, respectively. Most genetic variation was within populations for both M. fructicola (77%) and M. laxa (76%), with the rest of the variation between geographically separated populations. Genotype did not correlate with virulence of isolates as measured by symptom severity index (SSI) on inoculated plum fruit as well as the morphology on potato dextrose agar (PDA) medium. M. fructicola isolates with wide genetic variation exhibited closely similar SSIs and only one morphology on PDA, whereas M. laxa isolates exhibited a broad range of SSIs and four different colony morphologies.

Introduction Monilinia fungi are ascomycetous pathogens causing diseases known as brown rot in fruits, canker in wood, and blossom blight of flowers of some cultivated members of family Rosaceae. Although 35 species are described, six species are pathogens of pome and stone fruit: Monilinia fructicola (G. Winter) Honey, including Monilinia fructigena (Aderhold & Ruhland) Honey, Monilinia laxa (Aderhold & Ruhland) Honey (Lino et al. 2016), Monilinia polystroma G.C.M. van Leeuwen (Johnston et al., 2014), M. yunnanensis (Hu et al. 2011), and M. mumecola (Zhu et al., 2016).

M. fructigena has been reported from Europe, and it is a quarantine pest for Canada, the United States, Australia and New Zealand. M. laxa is described from Europe, the Americas, Japan, and Australasia. M. fructicola is reported from the Americas, Japan, Australasia. M. polystroma was detected also in China (Zhu and Guo, 2010) on mummified plums (Prunus aitianli). In Europe, the presence of M. polystroma was first reported on apple leaf petioles and pedicels in Hungary

(Petroczy & Palkovics, 2009), and subsequently on fruits of apple and peach in the Czech Republic (Anonymous, 2011); apricot in Switzerland (Hilber-Bodmer et al. 2012); apple in Serbia (Vasic et al., 2012); apple, peach and plum in Poland (Poniatowska et al. 2013; 2016); peach in Italy (Martini et al. 2014).

The six species infecting stone and pome fruit are not easy to distinguish by disease symptoms. Thus, diagnosis to the species level often requires consideration of a combination of characteristics, including colony morphology in vitro (Lane 2002) and DNA sequence characteristics (Cote et al. 2004; Gell et al. 2007b). Studying the genetic structure of Monilinia populations provides information on sources of genetic variation, evolution, pathogenicity, and the means of spread. The internal transcribed spacer region (ITS) of rDNA has sufficient resolution (5-13 nucleotide substitutions) to distinguish these four Monilinia species, but it provides little information on intra-specific variation (van Leeuwen et al. 2002). To study intraspecific genetic variation, inter simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) markers have been used in M. laxa populations in Spain (Gell et al. 2007a), Greece (Papavasileiou et al. 2015), and Hungary (Fazekas et al. 2014), and in M. fructicola populations in Spain (Villarino et al. 2012), Greece (Papavasileiou et al. 2015) and China (Fan et al. 2010). These studies compared genetic diversity of isolates from different geographical regions and/or from different host species (Papavasileiou et al. 2015).

M. laxa and M. fructicola are reported from all Australian states and territories where their hosts are grown (Holmes 2011). M. laxa was first identified in Western Australia in December 1997 from stone fruit orchards located in the Donnybook and Manjimup regions, located 200 and 300 km south, respectively, of the state capital city of Perth (AMRiN 2015). Western Australia is a region isolated the north and east by sparsely occupied desert, and the west and south by oceans, so it is certain that Monilinia was introduced by people. Since the initial outbreak in Western Australia, Monilinia has spread to commercial stone fruit orchards further south and east, and northwards to the Perth and Gingin orchard regions. The first M. laxa isolate was collected from the Perth Hills orchard region in 2001 (AMRiN 2015). The Monilinia species responsible for the Perth outbreak was not recorded. Our previous study was the first publication recorded the identification of M. fructicola in Western Australia. It also found distribution of these two

Monilinia species in W.A. is not homogeneous (Tran et al. 2017). M. laxa and M. fructicola co- existed in the Perth Hills stone fruit production region. They were, however, spatially isolated to the north of Perth Hills where only M. fructicola was found, and to the south of the Perth Hills region, where only M. laxa was found.

Although there have been several studies about natural dispersal of conidia (vegetative spores) of Monilinia species, including M. laxa (Wilson and Baker 1946), M. fructicola (Kable 1965), M. fructigena (Banoon et al. 2009), and M. vaccinii-corymbosi (Cox and Scherm, 2001), accurate information about modes of Monilinia spore dissemination and its relation to environmental factors are still unclear. Kable (1965) studied M. fructicola conidia (asexual spores) dispersal in the air to peaches in seven orchards in New South Wales, Australia, and found that they travel about 100 m under natural wind conditions. Similarly, ascospores (sexual spores) are dispersed naturally only for short distances. M. vaccinii-corymbosi ascospores disseminated 30 m in wind, while conidia disseminated 20 m under the same conditions (Cox and Scherm 2001). Humans are usually involved in dispersal over longer distances through trade in fruit and germplasm.

Here, we investigate correlations between genetic diversity of M. fructicola and M. laxa populations collected from different locations and virulence and colony morphology.

Materials and methods Fungal collection and isolations

A total of 134 samples were collected from flowers, mature fruits, twig cankers and mummified fruits from major stone fruit production regions of Western Australia. The populations were located at Gingin (~80 km to the north of Perth City), the Perth Hills (within the greater Perth metropolitan region), Pemberton and Mullalyup regions (~300 km to the south of Perth City) (Table S1).

Conidia were collected from symptomatic plant tissues in a drop of water. The spores were spread on 1% sterile water agar and single spores were identified under a microscope. Individual spores were transferred to potato dextrose agar (PDA) or V8 agar plates and incubated in the

dark at 22oC for 6-7 days. A five mm3 cube of agar containing actively growing hyphae was excised from the edge of the mycelium and transferred to a 250ml flask containing 50 ml of potato dextrose broth (PDB) or V8 liquid media, and this was incubated on an orbital shaker at 100 rpm in the dark at 22oC.

DNA extraction

After five days on a shaker, the mycelial mass was collected, rinsed with distilled water and pressed between two absorbent paper towels to remove excess liquid. Mycelium (100 mg) was frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. Powdered mycelium was transferred to a 1.5ml centrifuge tube containing 450 l Extraction Buffer (0.1M NaCl, 50mM Tris pH8.0, 0.5mM EDTA pH8.0, 1% SDS, 1% PVPP) and 450 l phenol- chloroform (1:1) saturated with TE (pH 8.0). The mixture was homogenized for 5 min before being centrifuged at room temperature for 2 min. The aqueous phase was transferred to a new tube containing an equal volume of phenol-chloroform, mixed and centrifuged 2 min. Then, 300 l of the aqueous phase was removed to a fresh tube containing 58 l absolute ethanol, to which 200 mg of cellulose powder (cf11, Whatman) was added and mixed. After centrifuging at high speed for 1 min, 250 l of the supernatant was transferred to a fresh tube and 25 l 3 M NaOAC (pH 5.2) and 625 l absolute ethanol added. The tube was centrifuged for 20 min at 20,000 x g and the supernatant was removed. The DNA pellet was washed in 70% ethanol. The DNA was dissolved in 100 l of elution buffer (10 mM Tris, pH 8.0) and stored at -20oC. Species identification was done by comparison of ITS sequences, as described (Tran et al. 2017).

ISSR assay

Six ISSR primers reported to generate reproducible polymorphic DNA product patterns from M. fructicola isolates (Villarino et al. 2012) were used to amplify DNA of 68 M. laxa and 66 M. fructicola isolates. Primers used were Mf-2 (AC)8 (Lim et al. 2004), Mf-5 (AAC)5, Mf-7

(AAG)8, Mf-8 (AG)8C, Mf-9 (GACA)4, Mf-13-Mf (GAG GGT GGC GGC GGT TCT) (Ma and Michailides 2003). Amplification was done in a 20 l PCR mixture composing 10 l GoTaq®

Green PCR Master Mix (Promega), 1 l of purified fungal DNA (approx. 300 ng), 8 l H2O, and

1 l primer. PCR amplification was done after an initial denaturation cycle of 3 min at 95oC. Annealing temperatures differed depending on the primer used. Amplification occurred over 40 cycles of 1 min at 94oC in all cases, then 1 min at 50oC (for Mf-5, Mf-7, Mf-13-Mf), or 1 min at 45oC (for Mf-8), or 1 min at 36oC (for Mf-9), or 1 min at 52oC (for Mf-2), followed in all cases by 1 min at 72oC. There was a final incubation at 72oC for 10 min. PCR products were separated on 3% agarose gels stained with SYBR®safe. Reactions were done twice.

Virulence test

Eleven M. laxa and 14 M. fructicola isolates were randomly chosen to study virulence on fruit (Table S1). The fruit used was Japanese plum (P. salicina) cv Fortune. Experiments were done in a completely randomised design with three replicates per treatment and six fruit per replicate. After washing with water and drying, fruits were wounded by a single puncture with a sterile needle. A 5mm diameter potato dextrose agar (PDA) plug with actively growing hyphae was placed on the wound site. Controls consisted of wounded fruit inoculated with PDA plugs containing no fungi. Inoculated fruits were incubated in the dark at 20oC at 100% humidity (Yin et al. 2015). From 36-hours post-inoculation (pi), the diameter of the fungal lesion on the fruit surface was measured every 12 hours until the lesion covered the whole fruit.

Virulence development was based on Symptom Severity Index (SSI), which was recorded according to the mean lesion growth rate. SSI 0, no lesion growth SSI 1, lesion growth 1-5 mm per day SSI 2, lesion growth 6-10 mm per day SSI 3, lesion growth 11-20 mm per day SSI 4, lesion growth >20mm per day

The raw growth rate values (mm/day) were compared using the t-test in SPSS. The differences in growth rates of isolates were analysed assuming a null hypothesis - that there was no difference between the growth rates of the isolates. If the P-value was ≤ 0.05, the null hypothesis was rejected.

Colony morphology assessment

Eleven M. laxa and 14 M. fructicola isolates used in virulence test were chosen for morphology experiment. Each isolate was subcultured onto three PDA plates, incubated in the dark at 25oC. After 7 days, the diameter of each colony was measured and the average colony growth of M. laxa and M. fructicola was compared using an independent-sample T test on SPSS.

Data analysis for genetic variation

PCR products ranged in size from approximately 0.2 - 2.0 kb, and each clear band (allele) was recorded as being either present (1) or absent (0) for each isolate. Where bands were very faint and/or were not reproducible between two amplification reactions, they were ignored. ISSR data was analysed using GenAlEx v6.5 software (Peakall and Smouse 2012). Nei’s genetic identity (Nei 1978) was calculated to establish degrees of identity between isolates and populations. Genetic diversity of each population was determined by Shannon’s diversity index (Shannon and Weaver 1949). GenAIEx v6.5 was used for analysis of molecular variance (AMOVA) to examine the source of observed genetic variation.

Results

Of 134 isolates collected, 68 isolates were identified as M. laxa and 66 isolates as M. fructicola based on ITS sequence analysis. Only populations with more than 20 isolates were used to analyse the genetic structure based on ISSR data. Thus, there were three M. fructicola populations that together consisted of 66 isolates, and three M. laxa populations consisting of 68 isolates were used to estimate genetic diversity (Table S1).

Genetic diversity

The six ISSR primers generated 79 alleles for both species. The average polymorphic loci (P = 70.92%) and expected heterozygosity (He = 0.28) of M. laxa was lower than those of M. fructicola, which were P = 84.38% and He = 0.34, respectively (Table 1), indicating that the M.

fructicola isolates were genetically more diverse than the M. laxa isolates. This was confirmed by Shannon’s diversity (I) indices of 0.41 for M. laxa and 0.50 for M. fructicola (Table 1). Table 1. Genetic diversity within populations of M. laxa and M. fructicola

Species Population Location N Na Ne I He P (%) M. fructicola 1F Gingin 20 1.84 1.59 0.49 0.34 84.38 2F Perth Hills, 24 1.84 1.59 0.49 0.34 84.38 Orchard A

3F Perth Hills, 22 1.81 1.61 0.50 0.34 84.38 Orchard B

Average 33 1.92 1.68 0.50 0.34 84.38 M. laxa 1L Mullalyup 25 1.62 1.44 0.36 0.25 61.70 2L Pemberton 22 1.81 1.59 0.47 0.32 80.85 3L Perth Hills, 21 1.70 1.50 0.40 0.28 70.21 Orchard A Average 22.67 1.71 1.51 0.41 0.28 70.92 N = number of isolates; Na = number of observed alleles; P = percentage of polymorphic loci; Ne = number of effective alleles = 1 / (p2+ q2); I = Shannon's information index = -1* (p * Ln (p) + q * Ln(q)); He = expected heterozygosity = 2 * p * q, where p = band frequency and q = 1 – p.

Among M. laxa populations, the highest genetic identity was between populations 2L (Pemberton) and 3L (Perth Hills) (Nei’s genetic identity was 0.96) located ~300 km apart. This suggests that M. laxa populations located at Pemberton and Perth Hill have a more recent common ancestor (a common source) than other populations. In contrast, the lowest genetic identity (0.81) was between populations 1L and 2L (Mullalyup and Pemberton, respectively) located only ~100 km apart, indicating they originated from different sources (Table 2).

Table 2. Nei’s genetic identities between populations of M. laxa and of M. fructicola

M. fructicola Population Pop 1F Pop 2F Pop 3F Pop 1F 1.00 Pop 2F 0.95 1.00 Pop 3F 0.7 0.79 1.00 M. laxa Population Pop 1L Pop 2L Pop 3L Pop 1L 1.00 Pop 2L 0.81 1.00 Pop 3L 0.84 0.96 1.00

Similarly, two M. fructicola populations,1F and 2F, located ~100km apart, were more closely related (Nei’s genetic identity of 0.95) than two populations 2F and 3F located only 2 km apart (Nei’s genetic identity of 0.79) (Table 2). Thus, the physical distance between populations was not closely correlated to genetic identity between them, suggesting that genetic information may not be shared by natural means (e.g. windborne spores) between populations.

AMOVA analysis

Analysis of molecular variance (AMOVA) indicated that most genetic variation was within populations for M. laxa and M. fructicola, 76% (P value = 0.001) and 77% (P value = 0.001), respectively, with the remaining variation due to differences between populations (Table 3). These values were similar for both species, revealing that geographic separation influenced genetic variation of M. laxa populations (24%) and M. fructicola populations (23%) at the same level.

Table 3. Analysis of molecular variance (AMOVA) of M. laxa and M. fructicola populations

M. laxa M. fructicola Source of Df SS MS % P-value Df SS MS % P-value variation Between groups 2 81.41 40.71 24 0.001 2 72.23 31.12 23 0.001

Within groups 65 322.2 4.96 76 63 298.7 4.74 77

Total 67 403.7 65 371

Df is degrees of freedom associated with the respective sources of variance; Df (total) = n – 1 where n is number of samples; SS is the sum of squares that corresponds to the sources of variation; MS is the Mean Squares corresponding to the partitions of the total variance. The MS is defined as SS/Df.

Virulence test

Virulence was measured on plum fruit as the rate of lesion expansion per day. There was considerable variation in lesion growth between isolates and between species. Overall, M. laxa lesions expanded at a slower rate than M. fructicola lesions, the means for the species being 11.37 mm and 18.60 mm per day, respectively.

The range of lesion expansion rates for individual M. laxa isolates (P value = 4.38e-09) was greater than for M. fructicola isolates (P value = 0.999) (Table 4). M. laxa isolates induced a wider range of symptoms on plum fruit, as measured by symptom severity index (SSI), than did M. fructicola isolates (Figure 1, Table 5).

Table 4. Variation in the growth rates of lesions caused by Monilinia isolates on Fortune plum fruit.

M. laxa M. fructicola Source of variation SS Df MS F P value SS Df MS F P value Between groups 1077.98 10 107.8 9.7 4.38E-09 50.53 13 3.89 0.15 0.999

Within groups 611.25 55 11.11 1820.7 70 26.01 Total 1689.23 65 1871.3 83

Df, degrees of freedom associated with the respective sources of variance; SS, sum of squares that corresponds to the three sources of variation; MS is the Mean Squares corresponding to the partitions of the total variance. The MS is defined as SS/Df.

Genetic diversity, as determined by six ISSR markers, did not correlate with pathogenicity, as measured by SSIs. M. fructicola isolates were more genetically diversity (I = 0.50) than M. laxa isolates (I = 0.41), but M. laxa isolates exhibited greater variance in pathogenicity. For example, M. laxa lesions induced by infection with isolates M1, M25, and M41 grew very quickly and produced conidia quickly (SSI 3 and 4). In contrast, lesions of the slowest growing M. laxa isolates (M22, M128, M133) grew less than 1 mm per day and they did not produce conidia during the course of the experiment (SSI 1). In contrast, all the M. fructicola isolates tested had SSIs of 3 and 4 and all isolates produced conidia 3.5 to 4 days after inoculation (Table 5).

Table 5. Mean lesion growth, symptom severity indices and conidia development of M. laxa and M. fructicola isolates on plum fruit. Species Isolate Mean growth Symptom Days till conidia rate (mm/day) severity index development (SSI) M. laxa M1 21.50 4 3.5 M112 6.22 2 5.5 M25 19.72 3 3.5 M22 0.61 1 No conidia produced M133 0.39 1 No conidia produced M65 7.52 2 5 M50 16.81 3 4.5 M128 0.26 1 No conidia produced M41 19.09 3 3 M82 17.85 3 3.5 M61 15.09 3 4.5 Mean 11.37 M. fructicola M19 16.94 3 3.5 M75 18.46 3 3.5 M76 20.02 4 3.5 M111 17.28 3 3.5 M87 15.59 3 3.5 M105 19.04 3 3.5 M88 19.43 3 4.5 M91 15.98 3 4.5 M90 20.46 4 3.5 M94 20.52 4 3.5 M119 18.72 3 4.5 M120 20.05 4 3.5 M118 20.06 4 3.5 M121 17.94 3 3.5 Mean 18.60

M1, M. laxa M41, M. laxa M25, M. laxa M82, M. laxa

M22, M. laxa M112, M. laxa M50, M. laxa M61, M. laxa

M91, M. fructicola M19, M. fructicola M94, M. fructicola M120, M.fructicola

Figure 1. Symptoms induced on Japanese plum cv Fortune by eight isolates of M. laxa and four isolates of M. fructicola 5-days post-inoculation. Symptom Severity Indices (SSI) are given in parentheses. SSI describes growth rate of the lesion over 5 days: SSI0 = no lesion growth, SSI1 = 1-5 mm/day, SSI2 = 6-10 mm/day, SSI3 = 11-20 mm/day, SSI4 = >20 mm/day. Colony morphology

On PDA medium, M. fructicola grows twice as fast as M. laxa does, having the average of colony diameters of 76 and 35mm, respectively, after 7 days of inoculation (Table 6). Also, on PDA, M. fructicola starts to generate abundant conidia spores just after 2 days of inoculation while M. laxa does not produce spores on PDA media. M. laxa presents four different morphologies of the colony on PDA media (Figure 2a, b, c, d), while M. fructicola exhibits only one colony morphology (Figure 2e).

Table 6. Comparison on colony growth of M. laxa and M. fructicola

Species N Mean (mm) Std. Deviation P-value M. laxa 33 35 8.2 0.000 M. fructicola 42 76 3.9

(a) M41, M. laxa (b) M61, M. laxa (c) M50, M. laxa

(d) M112, M. laxa (e) M120, M. fructicola Figure 2. Colony morphology of M. laxa and M. fructicola after 10 days inoculated on PDA media, incubated in the dark at 25oC.

Discussion Although Monilinia was recorded first in the Australian state of New South Wales from stone fruit in 1921 (Harrison 1928), Western Australia remained free of it until it was first recorded there in 1997. Because the approximate time of invasion is known, there exists an opportunity to study the variance of genetic, virulence and colony morphology present at distinct regions in the state. The study region is geographically isolated from other populations of Monilinia by deserts

to the north and east, and by oceans to the south and west. Sexual and asexual Monilinia spores apparently do not disperse over long distances (Wilson and Baker 1946; Kable 1965; Banoon et al. 2009, Cox and Scherm 2001), so it seems unlikely that the pathogen arrived in Western Australia by natural means.

Recombination could be one of the potential sources of genetic variation in Monilinia populations; the sexual stages of M. laxa and M. fructicola have not been recorded in Western Australia (Tran et al. 2017). Therefore, all the genotypic variation of Monilinia present in the region is likely to the result of the inadvertent introduction by people of new genotypes from other regions. The natural rate of genetic change in vegetatively reproducing populations of Monilinia species is unknown. In some cases, the genetic diversity of Monilinia populations may broadly reflect the time they have been confined to a region. For example, M. fructicola populations from China and California share Shannon’s diversity indices of about 0.50, similar to populations in Western Australia. Monilinia was officially recorded in China in 2003 (but was probably present for far longer) (Fan et al. 2010), and in California it was first recorded in 1936 (Hewitt and Leach 1939). In Greece, where M. fructicola was first recored in 2012, the Shannon’s diversity index of isolates was only 0.24 (Papavasileiou et al. 2015).

The source of the original isolates of M. laxa and M. fructicola that invaded Western Australia is unknown. Because international imports of stone fruit were banned until 2004, other eastern Australian sources of Monilinia are the most likely source. Infected fruit may have arrived illegally from the east by road or air. Another possible source is germplasm imported from eastern Australia and overseas by a state government breeding program that was located in the Manjimup region of Western Australia, the region where Monilinia was first identified. After the initial invasion, subsequent introductions of new Monilinia genotypes may have occurred when import restrictions to Western Australia were lifted for eastern Australian stone fruit in 2001, for New Zealand stone fruit from 2004 (Biosecurity Australia 2006), and for North American stone fruit from 2013 (Boothroyd 2013).

A surprising finding was the much broader variation in virulence presented by M. laxa compared to the relative uniformity of virulence in M. fructicola. Symptom severity indices ranged from 1

to 4 for the M. laxa isolates tested, but only 3 to 4 for M. fructicola isolates. Some M. laxa isolates that developed fast growing lesions, such as M41 and M25, produced much larger spore masses than any M. fructicola isolate did (Fig 2). Similarly, M. laxa isolates exhibited four different colony morphologies on PDA media, but only one for M. fructicola isolates. This study was carried out at 22oC. Others showed that M. laxa isolates produced more conidia at 10oC, and fewer at 30oC, than did M. fructicola isolates, whereas there was no difference in the spore production between both fungal species at 20oC (Angeli et al. 2017).

Maintaining the relatively low genetic diversity of both Monilinia species should be a priority for the stone fruit industry in Western Australia. By reducing the genotypic diversity present, opportunities for populations to evolve resistance to fungicides are reduced, as is the evolution of new severe pathotypes. Similarly, preventing incursion of both species into stone fruit production areas in which they are currently absent should be a high priority.

Supplementary Files

Table S1. Monilinia isolates used in this study

Monilinia Population Region Isolate code Prunus species Collection species time M. fructicola 1F Gingin M75* nectarine 2016 M76* peach 2016 M86 peach 2016 M87* peach 2016 M88* peach 2016 M90* peach 2016 M91* peach 2016 M92 peach 2016 M94* peach 2016 M95 nectarine 2016 M96 nectarine 2016 M97 nectarine 2016 M98 nectarine 2016 M99 nectarine 2016 M100 nectarine 2016 M101 peach 2016 M102 nectarine 2016 M104 nectarine 2016 M105* peach 2016 M106 peach 2016 2F Perth Hills, M107 apricot 2016 Orchard A M109 apricot 2016 M110 apricot 2016

M111* apricot 2016 M113 apricot 2016 M114 apricot 2016 M115 apricot 2016 M116 apricot 2016 M117 apricot 2016 M118* apricot 2016 M119* apricot 2016 M120* apricot 2016 M121* apricot 2016 M122 apricot 2016 M124 apricot 2016 M125 apricot 2016 M126 apricot 2017 M143 nectarine 2017 M144 nectarine 2017 M145 nectarine 2017 M146 nectarine 2017 M147 nectarine 2017 M149 nectarine 2017 M151 nectarine 2017 3F Perth Hills, M152 nectarine 2017 Orchard B M153 nectarine 2017 M155 nectarine 2017 M158 peach 2017 M159 peach 2017 M161 peach 2017 M162 peach 2017 M170 peach 2017 M171 peach 2017 M172 peach 2017 M173 peach 2017 M174 peach 2017 M175 peach 2017 M176 peach 2017 M177 peach 2017 M178 peach 2017 M180 peach 2017 M181 peach 2017 M183 peach 2017 M184 peach 2017 M186 peach 2017 M19 peach 2016 M. laxa 1L Mullalyup M197 peach 2017 M198 peach 2017 M199 peach 2017 M200 peach 2017 M201 peach 2017 M202 peach 2017 M203 peach 2017 M204 peach 2017 M205 peach 2017 M207 peach 2017

M208 peach 2017 M209 peach 2017 M210 peach 2017 M211 peach 2017 M212 peach 2017 M213 peach 2017 M215 peach 2017 M216 peach 2017 M217 peach 2017 M218 peach 2017 M219 peach 2017 M220 peach 2017 M222 peach 2017 M223 peach 2017 M224 peach 2017 2L Pemberton M1* plum 2016 M1-1 plum 2016 M128* nectarine 2017 M131 peach 2017 M133* nectarine 2017 M136 peach 2017 M25* apricot 2016 M40 Pplum 2016 M41* apricot 2016 M42 peach 2016 M43 peach 2016 M44 plum 2016 M5 apricot 2016 M50* plum 2016 M6 peach 2016 M60 peach 2016 M61* cherry 2016 M65* plum 2016 M67 plum 2016 M68 plum 2016 M7 apricot 2016 M9 cherry 2016 3L Perth Hill, M187 apricot 2017 orchard A M188 apricot 2017 M189 apricot 2017 M190 apricot 2017 M191 apricot 2017 M192 apricot 2017 M193 apricot 2017 M194 apricot 2017 M195 apricot 2017 M112* apricot 2016 M123 nectarine 2016 M20 peach 2016 M22* peach 2016 M23 peach 2016 M24 apricot 2016 M51 peach 2016 M52 peach 2016 M53 peach 2016

M81 nectarine 2016 M82* nectarine 2016 M84 nectarine 2016 The symbol * refers the isolates used in virulence and morphology tests.

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Fazekas, M., Madar, A., Sipiczki, M., Miklós, I., and Holb, I. J. 2014. Genetic diversity in Monilinia laxa populations in stone fruit species in Hungary. World Journal of Microbiology Biotechnology 30, 1879-1892. Gell, I., Larena, I., and Melgarejo, P. 2007a. Genetic diversity in Monilinia laxa populations in peach orchards in Spain. Journal of Phytopathology 155, 549-556. Gell, I., Cubero, J., and Melgarejo, P. 2007b. Two different PCR approaches for universal diagnosis of brown rot and identification of Monilinia spp. in stone fruit trees. Journal of Applied Microbiology 103, 2629-2637. Harrison, T. H. 1928. Brown rot of fruits and associated diseases in Australia. I. History of the disease and determination of the causal organism. Journal of the Proceedings of the Royal Society of New South Wales 52, 99-148. Hewitt, W. B. and Leach, L. D. 1939. Brown rot Sclerotinias occurring in California and their distribution on stone fruits. Phytopathology 29, 337–351. Holmes, R., Kreidl, S., Villalta, O., and Gouk, C. 2011. Through chain approach for managing brown rot in summerfruit and canning fruit. Project code MT08039. Biosciences Research Division, Department of Primary Industries, Victoria, Australia Kable, P. F. 1965. Air dispersal of conidia of Monilinia fructicola in peach orchards. Animal Production Science 5, 166-171. Lane, C. R. 2002. A synoptic key for differentiation of Monilinia fructicola, M. fructigena and M. Iaxa, based on examination of cultural characters. Bulletin OEPP 32, 489-493. Lim, S., Notley-McRobb, L., Lim, M., and Carter, D. A. 2004. A comparison of nature and abundance of microsatellite in 14 fungal genomes. Fungal Genetics and Biology 41,1025– 1036. Ma, Z., Luo, Y., and Michailide, T. J. 2003. Nested PCR assays for detection of Monilinia fructicola in stone fruit orchards and Botryosphaeria dothidea from pistachios in California. Journal of Phytopathology 151, 312–322. Nei, M. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89, 583–590. Papavasileiou, A., Karaoglanidis, G. S., and Michailides, T. J. 2015. Intraspecific diversity of Monilinia fructicola and M. laxa populations from blossoms and fruit of different hosts in Greece. Plant Disease 99, 1353-1359.

Peakall, R. and Smouse, P. E. 2012. GenAlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research – an update. Bioinformatics 28, 2537-2539. Petróczy, M., & Palkovics, L. (2009). First report of Monilia polystroma on apple in Hungary. European journal of plant pathology, 125(2), 343-347. Poniatowska, A., Michalecka, M., & Bielenin, A. (2013). Characteristic of Monilinia spp. fungi causing brown rot of pome and stone fruits in Poland. European journal of plant pathology, 135(4), 855-865. Shannon, C. E. and Weaver, W. 1949. The mathematical theory of information. Urbana, IL. Illinois University Press. Tamura, K., Nei, M., and Kumar, S. 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences (USA) 101, 11030-11035. Tran, T. T., Li, H., Nguyen, D. Q., Sivasithamparam, K., Jones, M. G. K., and Wylie, S. J. 2017. Spatial distribution of Monilinia fructicola and M. laxa in stone fruit production areas in Western Australia. Australasian Plant Pathology doi:10.1007/s13313-017-0497-9 Van Leeuwen, G. C., Yen, R. P. B., Holb, I. J., and Jeger, M. J. 2002. Distinction of the Asiatic brown rot fungus Monilia polystroma sp. nov. from M. fructigena. Mycological Research 106, 444-451. Villarino, M., Larena, I., Martinez, F., Melgarejo, P., and De Cal, A. 2012. Analysis of genetic diversity in Monilinia fructicola from the Ebro Valley in Spain using ISSR and RAPD markers. European Journal of Plant Pathology 132, 511-24. Wilson, E. E. and Baker, G. A. 1946. Some aspects of the aerial dissemination of spores, with special reference to conidia of Sclerotinia laxa. Journal of Agricultural Research 72, 301.

Chapter 5. Monilinia fructicola and Monilinia laxa isolates from stone fruit orchards sprayed with fungicides displayed a broader range of responses to fungicides than those from unsprayed orchards

This chapter addressed aim 4 of the thesis and was published in European Journal of Plant Pathology 2018.

Citation:

Statement of contribution of the authors contributed to the work

Abstract Monilinia fructicola and Monilinia laxa are causal agents of brown rot, the most serious fungal disease of stone fruit (Prunus species). The disease is controlled primarily by applying fungicides. It was hypothesised that Monilinia isolates exposed to a regime of fungicidal sprays would exhibit greater tolerance to those compounds than isolates that had not been subjected at all to such fungicide sprays. Sixty-six M. fructicola and 52 M. laxa isolates were collected from fungicide-sprayed and unsprayed commercial and domestic orchards. The fungicides propiconazole, iprodione, and a mixture of fluopyram and trifloxystrobin were used regularly on all the sprayed orchards tested, and these were used to challenge all Monilinia isolates in vitro. We found that isolates col-lected from sprayed orchards were on average more tolerant to the fungicides, as measured by effective concentration of fungicide reducing mycelial growth by

50% (EC50). This trend was evident for both fungal species tested, but it was statistically significant only for M. fructicola. Monilinia laxa isolates were on average more tolerant to propiconazole than were M. fructicola isolates irrespective of orchard type, while average responses to iprodione and fluopyram + trifloxystrobin were similar for both species. Although tolerant and sensitive isolates were identified under both sprayed and unsprayed regimes, there was a greater range of responses to all three fungicides by isolates from sprayed orchards. Isolates with tolerance to two fungicides were not exclusively from sprayed orchards, but occurred more frequently there.

Introduction

Brown rot of stone fruit, caused by Monilinia fructicola and Monilinia laxa, was officially first identified in the state of Western Australia (W.A.) in 1997 (AMRiN 2015). Today, there are still these two Monilinia species in all stone fruit production regions in the state (Tran et al. 2017). However, a study of the genetic variation of Monilinia in the state revealed there are on-going incursions of new genotypes, probably on imported fruit and germplasm (Tran et al. 2018). The sexual stage of the fungus (apothecia) has not been recorded in W.A., so genetic diversity is primarily from incursions, but somatic mutation may be a minor factor.

In common with other parts in the world where Monilinia species are major pathogens, brown rot in the commercial stone fruit orchards in W.A. has been managed mainly by fungicide application, although there exist a few fungicide-free orchards. The decision not to spray for brown rot is largely a lifestyle choice based on a desire to avoid pesticides in food.

The fungicides that act against Monilinia contribute significantly to increasing yield and quality of stone fruit (Holb and Schnabel 2005; Rungjindamai et al. 2014a). A risk associated with prolonged use of any fungicide is the potential to inadvertently select fungal genotypes that overcome the activity of the active fungicidal com-pound (Malandrakis et al. 2013). The degree of risk of developing fungicidal tolerance is dependent on the mode of action of the chemical, how and when it is used in the orchard, the presence of other fungicides, and the evolutionary potential of the fungus to overcome the fungicide (Shaw 2000). For example, south-eastern USA isolates of M. fructigena derived from conidia that were tolerant to the demethylation inhibitor triazole propiconazole were more likely to also become tolerant to the quinone outside inhibitor fungicide azoxystrobin, than were the propiconazole-sensitive isolates (Luo and Schnabel 2008a). Similarly, cultures treated with non-lethal doses of the fungicide thiophanate-methyl more rapidly developed tolerance to propiconazole (Luo and Schnabel 2008a). Tolerance to propiconazole is strongly associated with the Mona element within the promoter of the 14α- demethylase gene MfCYP51, that induces its over-expression (Luo and Schnabel 2008b). Monilinia fructicola has also developed resistance to dicarboxamides in New Zealand (Elmer and Gaunt 1994) and Australia (Wherrett et al. 2001), and benzimidazoles in the USA (Zehr et al. 1991).

Non-chemical control of brown rot includes removal of the over-wintering stage in mummified fruit after harvest, application of Bordeaux mixture (lime and cop-per) during winter, and application of bacteria (Pratella et al. 1993) and other fungi (Epicoccum nigrum, Peni- cillium frequentans) (Cal et al. 1988; Larena et al. 2005; Holb and Kunz 2013; Rungjindamai et al. 2014b). Post-harvest application of fungicides to stored fruit is prac-ticed in some countries, but prohibited in others (Larena et al. 2005). Non-fungicidal control of Monilinia post-harvest is reported by dipping in peracetic acid, chlorine dioxide (Mari et al. 1999), copper and sulfur (McLaren and Fraser 2000), and treatment with Bacillus subtilis (Pusey and Wilson 1984).

Fungicide tolerance in the field is recorded widely where stone fruit is grown commercially. Monilinia fructicola and M. laxa isolates highly tolerant to propiconazole are recorded from the USA (Ogawa et al. 1984; Ma et al. 2003, 2005; Schnabel et al. 2004), Greece (Malandrakis et al. 2012), Brazil (May-De Mio et al. 2011), Spain (Egüen et al. 2015), and Serbia (Hrustić et al. 2018). Resistance to other fungicides is also recorded, including the dicarboximide fun-gicide iprodione, for which resistance is recorded from New Zealand (Elmer and Gaunt 1994; Sanoamuang and Gaunt 1995), and in Western Australia (Wherrett et al. 2001).

Field strategies to delay the build-up of fungicidal resistance involve applying mixtures of fungicides from different groups, using each fungicide infrequently, alternating fungicides of different groups, measuring fungicide resistance levels, applying fungicides only when the fungus present is susceptible to those fungicides, and applying fungicides only at certain stages of crop developmental (Gindrat and Forrer 1985; Holb et al. 2013). Recent research with Septoria leaf blotch and grapevine powdery mildew has shown that application of mixtures of fungicides is more effective at delaying build-up of tolerance than is alternate application of the same fungicides (Elderfield et al. 2018).

A range of site-specific and multi-site fungicides with different modes of action are used to control brown rot in W.A.. Despite development of resistance to propiconazole (demethylation inhibitor - DMI) around the world, it is still used regularly to control Monilinia in W.A. Other widely used fungicides include iprodione (class dicarboximide) and fluopyram+trifloxystrobin (Succinate dehydrogenator inhibitor (SDI)/Quinone outside inhibitor (QoI) classes). The objective of this study was to compare sensitivity to these three fungicides by Monilinia isolates collected from orchards that are either regularly sprayed, or are not sprayed at all with these three commercial fungicides to determine if there were patterns of tolerance development.

Materials and methods

Fungi

Monilinia isolates, including 66 of M. fructicola and 52 of M. laxa, were collected with permission of land owners from four stone fruit production regions in W.A. in 2016, 2017, and 2018. Samples of flowers, twig cankers, mature fruit and mummified fruit of nectarine and peach (Prunus persica), apricot (Prunus armeniaca), plum (Prunus domestica), and sweet cherry (Prunus avium) samples exhibited symptoms of brown rot. They were collected from six orchards, including one near the town of Gingin (~80 km to the north of Perth City), three in the Perth Hills region (within the greater Perth metropolitan region), one close to the town of Mullalyup, and one about 10 km from the town of Pemberton (~200 and 300 km, respectively, to the south of Perth City) (Table 1, Fig. 1).

Table 1 Monilinia fructicola and Monilinia laxa isolates used in this study, collection sites, fungicide application status, and EC50 values of individual isolates to propiconazole, iprodione, and fluopyram + trifloxystrobin

Monilinia species Orchard code Spray regime Region Isolate code Tolerance to fungicide (EC50) (mg/L)

Propiconazole Iprodione Fluopyram + Trifloxystrobin

M. fructicola 1F Sprayed Gingin M75 0.024 0.341 0.035 M76 0.013 1.604 0.301 M86 0.015 0.301 0.046 M87 0.016 0.413 0.413 M88 0.028 0.080 0.015 M90 0.024 0.161 0.030 M91 0.019 0.083 0.023 M92 0.016 0.296 0.028 M94 0.017 0.180 0.079 M95 0.020 0.116 0.003 M96 0.017 0.159 0.002 M97 0.027 0.872 0.004 M98 0.019 0.182 0.016 M99 0.018 0.076 0.003 M100 0.020 0.335 0.048 M101 0.028 0.298 0.057 M102 0.019 0.064 0.010 M104 0.019 0.188 0.046 M105 0.023 0.118 0.020 M106 0.022 0.225 0.031 Mean 0.020 0.341 0.061 2F Unsprayed Perth Hills M109 0.016 0.146 0.022 M110 0.016 0.131 0.024 M111 0.016 0.127 0.054 M113 0.015 0.216 0.002

M114 0.013 0.346 0.028 M115 0.022 0.267 0.032 M116 0.015 0.382 0.033 M117 0.013 0.096 0.035 M118 0.020 0.101 0.024 M119 0.010 0.105 0.038 M120 0.020 0.073 0.002 M121 0.017 0.096 0.005 M122 0.023 0.101 0.001 M124 0.017 0.124 0.000 M125 0.015 0.175 0.000 M126 0.010 0.083 0.031 M143 0.019 0.086 0.019 M144 0.018 0.285 0.005 M145 0.018 0.211 0.038 M146 0.017 0.135 0.046 M147 0.021 0.285 0.057 M149 0.024 0.176 0.054 M151 0.019 0.146 0.012 Mean 0.017 0.131 0.024 3F Sprayed Perth Hills M152 0.017 1.920 0.194 M153 0.017 1.705 0.077 M155 0.039 0.443 0.056 M158 0.033 1.075 0.075 M159 0.046 1.100 0.081 M161 0.026 0.582 0.037 M162 0.025 0.592 0.092 M170 0.016 1.641 0.038 M171 0.022 0.052 0.223 M172 0.016 0.200 0.041 M173 0.017 0.102 0.040 M174 0.021 0.400 0.035 M175 0.019 0.881 0.030 M176 0.019 0.366 0.043 M177 0.031 0.698 0.073 M178 0.016 0.318 0.033 M180 0.016 0.180 0.039 M181 0.017 0.213 0.037 M183 0.020 0.149 0.040 M184 0.020 0.035 0.023 M186 0.026 0.084 0.012 M15 0.029 0.520 0.045 M19 0.014 0.464 0.194 Mean 0.023 0.597 0.068 M. laxa 1 L Sprayed Mullalyup M197 0.034 0.567 0.205 M198 0.021 0.572 0.856 M199 0.003 0.201 0.016 M200 0.001 0.169 0.022 M201 0.055 0.646 0.006 M202 0.011 0.175 0.005

M203 0.017 0.640 0.022 M204 0.072 0.456 0.022 M205 0.029 4.249 0.387 M207 0.048 0.102 0.028 M208 0.813 0.189 0.021 M209 0.028 0.664 0.028 M210 0.961 0.112 0.033 M211 0.046 0.210 0.006 M212 0.031 0.251 0.020 M213 0.035 0.259 0.022 M215 0.031 0.630 0.008 M216 0.022 0.325 0.016 M217 0.079 0.274 0.010 M218 0.073 0.017 0.006 M219 0.021 0.722 0.005 M220 0.023 0.220 0.332 M222 0.047 0.507 0.387 M223 0.029 0.285 0.025 M224 0.034 0.567 0.205 Mean 0.021 0.572 0.856 2 L Unsprayed Pemberton M1 0.019 0.539 0.060 M1–1 0.045 0.551 0.033 M128 0.057 0.042 0.044 M133 0.010 0.096 0.079 M25 0.024 0.398 0.033 M40 – 0.169 0.021 M41 0.140 0.768 0.010 M42 0.000 0.337 0.012 M44 0.013 0.010 0.047 M5 0.000 0.016 0.044 M50 – 0.133 0.032 M6 0.277 1.053 0.027 M60 0.033 0.301 0.033 M61 0.069 0.210 0.042 M67 0.010 0.204 0.033 M7 0.054 1.602 0.026 M9 0.058 0.301 0.031 Mean 0.057 0.418 0.036 3 L Unsprayed Perth Hills M187 0.018 0.331 0.020 M188 0.011 0.422 0.055 M189 0.023 0.589 0.050 M192 0.004 0.473 0.087 M194 0.047 0.336 0.024 M195 0.010 0.699 0.040 M112 0.050 0.629 0.036 M123 0.027 0.420 0.087 M20 0.024 0.574 0.055 M23 0.013 0.524 0.000 M51 0.009 0.374 0.036 M52 0.025 0.661 0.037

M81 0.056 0.605 0.001 M82 0.019 0.606 0.035 Mean 0.024 0.517 0.040

Gingin (Orchard 1F)

Orchard 3F 80km

1km 1km Perth Hills Orchard 2F Orchard 3L 1km

200km

Mullalyup (Orchard 1L)

100km

Pemberton (Orchard 2L)

Figure 1. Map showing approximate distances between orchards where Monilinia isolates were collected in Western Australia. Each orchard was given a code, F for orchards from which Monilinia fructicola isolates were collected, L is for orchards from which Monilinia laxa isolates were collected.

The orchards from which Monilinia were collected were given codes to preserve identities. Orchard 1F was a commercial sprayed orchard of about 40 ha producing peaches and nectarines. It was located close to Gingin. Orchard 2F was a small unsprayed domestic orchard located in the Perth Hills region consisting of about 20 apricot, nectarine and plum trees. Orchard 3F was a commercial sprayed orchard of about 30 ha located in the Perth Hills region, producing peaches, nectarines and plums. Orchard 1 L was a commercial sprayed orchard of 45 ha producing peaches and nectarines, and located near Mullalyup. Orchard 2 L was a semi-commercial unsprayed orchard of about 3 ha located near Pemberton. It produced cherries, plums, peaches and apricots. Orchard 3 L was a small unsprayed domestic orchard of about 0.5 ha located in the Perth Hills region. It produced apricots and nectarines.

The orchardists from each of the sprayed commercial orchards sampled indicated that they applied propiconazole to trees at flowering time, iprodione 3-4 days before harvesting or as a postharvest dip, and fluopyram + trifloxystrobin about 10 days before har-vesting. Propiconizole and iprodione-based sprays had been applied for several years on the sprayed orchards, while the fluopyram + trifloxystrobin has been applied only from 2015 when it became commercially available. The owners of the unsprayed properties claimed never to have applied fungicides in their orchards.

Conidia spores were collected from symptomatic plant tissue in a drop of water, which was then spread on 1% water agar. Single spores were identified under a microscope, transferred to potato dextrose agar (PDA) or V8 vegetable juice agar plates, and incubated at 25°C in the dark for 7 days. For each sample, a single culture (derived from one spore) was used in the current study. Mycelia were stored in 15% glycerol at -80°C for future use.

DNA extraction

A 4-mm2 piece of agar containing actively growing mycelium was excised from the edge of the fungal colonies and transferred to a 250-ml flask containing 100 ml of potato dextrose broth (PDB) or V8 liquid media. This flask was incubated on an orbital shaker at 100 rpm in the dark at 25 °C until there was 100 mg of mycelium for DNA extraction.

After 5 days on the shaker, the mycelium was harvested, rinsed with distilled water and pressed between two absorbent paper towels to remove excess liquid. DNA was extracted by grinding 100 mg mycelium to a fine powder in liquid nitrogen. The powdered mycelium was transferred to a 1.5-ml centrifuge tube containing 450 μl Extraction Buffer (0.1 M NaCl, 50 mM Tris pH 8.0, 0.5 mM EDTA pH 8.0, 1% SDS, 1% PVPP) and 450 μl phenol-chloroform (1:1) saturated with TE (pH 8.0). After mixing on a vortexer for 5 min, centrifuging at room temperature for 2 min, the aqueous phase was transferred to a new tube containing an equal volume of phenol-chloroform (pH 8.0), mixed and centrifuged for 2 min. Then, 300 μl of the aqueous phase was removed to a fresh tube containing 58 μl absolute ethanol. To this tube, 200 mg of cellulose powder (Cf11, Whatman) was added, mixed and centrifuged at high speed for 1

min. After that, 250 μl of the supernatant was transferred to a fresh tube and 25 μl 3 M NaOAC (pH 5.2) and 625 μl absolute ethanol added. The tube was centrifuged for 20 min at maximum speed, then the supernatant was removed, leaving the pellet containing nucleic acids, which was then rinsed in 70% ethanol. Nucleic acids were dissolved in 100 μl of elution buffer (10 mM Tris, pH 8.0) and stored at -20°C.

Fungus identification

Species were identified by analysis of ITS sequences (Tran et al. 2017). Briefly, amplicons generated using primers ITS1 (5′- TCCGTAGGTGAACCTGCGG-3) and ITS4 (5′- TCCTCCGCTTATTGATATGC-3) (White et al. 1990) were sequenced using BigDye® Terminator v3.1 Ready Reaction Mix. The sequences were used to identify Monilinia species after comparison with those on the NCBI GenBank database.

Fungicide challenge

Sixty-six M. fructicola and 52 M. laxa isolates were used in the fungicide challenge (Table 1). Fungicides used were propiconazole supplied as Cracker Jack (Imtrade) which contains 550 g/L propiconazole, iprodione supplied as Rovral (FMC Australia) which contained 250 g/L iprodione, and fluopyram + trifloxystrobin supplied as Luna Sensation (Bayer) which contains fluopyram + trifloxystrobin, each at 250 g/L.

Six concentrations of each fungicide (calculated as mg/L active ingredient) were used in a preliminary experiment to determine the range of tolerances of Monilinia isolates of both species. Concentrations used were 0.015, 0.03, 0.06, 0.12, 0.24, and 0.48 mg/L active ingredient. The fungicide was added to potato dextrose agar (PDA) medium after it was autoclaved and cooled to 60 °C. A block of agar (4 × 4 mm) containing fungal mycelia cut from the growing edge of each actively growing mother culture was placed in the centre of an agar plate and incubated in the dark at 25 °C. The colony diameter was measured after six days. This preliminary study revealed the approximate concentration by which >50% of isolates grew at 50% of the rate of isolates growing on fungicide-free medium. Then, fungicide concentrations

that were higher and lower were chosen to challenge isolates in a larger-scale experiment. The three concentrations chosen for each fungicide were: 0.0075, 0.015, and 0.03 mg/L for propiconazole; 0.03, 0.06, and 0.12 mg/L for iprodione; and 0.03, 0.06, and

0.12 mg/L for fluopyram + trifloxystrobin. The control treatment was PDA without fungicides. For each experiment, four plates (three plates with fungicide and one control plate) were used for each fungicide concentration/isolate combination. Each experiment was repeated three times. The plates were inoculated and grown as described above. After each plate was incubated for the same time period under the same conditions, the colony diameter was recorded by measuring the widest diameter of each colony.

Data analysis

The mean mycelial growth rate for each isolate was calculated for each fungicide treatment and compared with the control. EC50 (effective concentration of fungicide concentration that reduced mycelial growth by 50%) was calculated by estimating the growth reduction as a percentage of the control. The logarithm of this figure was used to calculate linear regression using log10(fungicide concentration) = a.log10 (% reduction in growth) + b, where a = slope and b = ^([log (50) − b]/a) intercept. EC50 was estimated using the formula EC50 = 10 10 (Mair et al. 2016). The

EC50 of less-sensitive isolates was determined by extrapolation of the measurement.

For comparing the mean EC50 between orchards, data was checked to determine if there was a normal distribution. To determine this, a test of normality was done using Shapiro-Wilk analysis (because the number of samples per orchard was less than 50). Abnormal data was transformed into normality using Log10 transformation on SPSS. The tests of homogeneity of variance were used to check if variances across different orchards are equal or unequal so that the appropriate method was employed for further analysis. One-way ANOVA (analysis of variance) was done to analyse of there was significant difference in EC50 data among different orchards. Where there was a significant difference, a multiple comparison between the EC50 values of different orchards was performed using a post-hoc test with the Dunnett T3 method (in case of equal

variances), or the Tukey method (in case of unequal variances), at a significance level of 0.05 on SPSS software v.24.

Individual isolates within orchards were ranked ac-cording to response to each fungicide by comparing its individual EC50 to the mean EC50 for all the isolates of that species for each fungicide. Thus, the relative ranking of fungicide sensitivity was based on deviation from the mean sensitivity (EC50) value. Where sensitivity was less than the mean, the isolate was ranked as sensitive or very sensitive. Where tolerance was greater than the mean, it was ranked as tolerant or very tolerant. The rank for each isolate was determined as follows: i Very sensitive. Individual EC50 less than 0.5× the mean EC50 for the species vs fungicide. ii Sensitive. Individual EC50 between 0.5× and 0.99× the mean EC50 for the species vs fungicide. iii Tolerant. Individual EC50 between 1.0× to 1.5× the mean EC50 for the species vs fungicide. iv Very tolerant. Individual EC50 is greater than 1.5× the mean EC50 for the species vs fungicide.

Results

Fungicide sensitivities for individual isolates of M. fructicola and M. laxa collected from sprayed and unsprayed trees were tested against three widely used fungicides (Table 1). On average, M. laxa isolates from both orchard types (sprayed and unsprayed) were about three times more tolerant to propiconazole than were M. fructicola isolates, whereas the two species did not show significant differences in response to the other two fungicides tested.

Irrespective of orchard type, the overall mean EC50 re-sponse of 66 M. fructicola isolates to propiconazole was 0.02 mg/L, to iprodione was 0.356 mg/L, and to fluopyram + trifloxystrobin was 0.051 mg/L. For 52 M. laxa isolates, the overall mean EC50 to propiconazole was 0.062 mg/L, 0.501 mg/L to iprodione, and 0.059 mg/L to fluopyram + trifloxystrobin (Table 2).

Table 2. Summary of the EC50 mean values for Monilinia fructicola and M. laxa Species Orchards Sprayed Propiconazole Iprodione Fluopyram & Trifloxystrobin regimes Average Variance Range Average Variance Range Average Variance Range (μg/ml) (μg/ml) (μg/ml) M. 1F Sprayed 0.020 1.87E-05 0.013- 0.341 0.126 0.064- 0.061 0.011 0.002- fructicola 0.028 1.604 0.413

2F Unsprayed 0.017 1.37E-05 0.010- 0.131 0.008 0.073- 0.024 0.0003 0.000- 0.024 0.383 0.057

3F Sprayed 0.023 6.74E-05 0.014- 0.597 0.322 0.035- 0.068 0.025 0.012- 0.046 1.920 0.223

Mean 0.020 0.356 0.051

M. laxa 1L Sprayed 0.104 0.057 0.001- 0.571 0.717 0.017- 0.100 0.039 0.005- 0.961 4.249 0.856

2L Unsprayed 0.057 0.006 3.75E- 0.418 0.174 0.010- 0.036 0.0003 0.010- 08- 1.602 0.079 0.277 3L Unsprayed 0.024 0.0003 0.0036- 0.517 0.015 0.331- 0.040 0.001 E-06- 0.0560 0.699 0.087

Mean 0.062 0.501 0.059

P value * 0.002 0.083 0.814

* Independent sample test for equality of EC50 mean between a group of 66 M. fructicola and 54 M. laxa isolates. These data sets were grouped by the species, regardless of the cultivar methods. P value < 0.05 indicates the means

of EC50 between M. fructicola and M. laxa were significantly different.

Sprayed isolates were on average more tolerant to all three fungicides than unsprayed isolates. Mean fungicide tolerances of M. fructicola isolates collected from sprayed orchards 1F and 3F were significantly greater than those collected from unsprayed orchard 2F (Table 3). Monilinia laxa isolates from sprayed orchard 1 L showed greater tolerance to all fungicides than those isolates from unsprayed orchards 2 L and 3 L, even though this was not statistically significant (Table 3).

Table 3 Statistical analysis of EC50 values of Monilinia fructicola and Monilinia laxa populations to the fungicides propiconazole, iprodione, and fluopyram + trifloxystrobin

Fungicide Orcharda Average Test of normalityb Test of ANOVAd Multiple comparison e EC50 homogeneity (mg/L) of variancec

P value Normal P value Equal P value Significant distribution variances difference (a = 0.05) assumed between orchards

M. fructicola Propiconazole 1F 0.020 0.849 Yes 0.138 Yes 0.004 Yes a 2F 0.017 0.314 Yes b 3F 0.023 0.090 Yes a,c Average 0.020 Iprodione 1F 0.341 0.275 Yes 0.009 No 0.004 Yes a, b 2F 0.131 0.195 Yes a 3F 0.597 0.651 Yes b Average 0.356 Fluopyram + 1F 0.061 0.357 Yes 0.004 No 0.001 Yes a, b Trifloxystrobi n 2F 0.024 0.060 Yes a 3F 0.068 0.067 Yes b Average 0.051 M. laxa Propiconazole 1 L 0.104 0.003 No 0.005 No 0.206 No a 2 L 0.057 0.000 No a 3 L 0.024 0.645 Yes a Average 0.062 Iprodione 1 L 0.571 0.104 Yes 0.007 No 0.073 No a 2 L 0.418 0.269 Yes a 3 L 0.517 0.159 Yes a Average 0.501 Fluopyram + 1 L 0.100 0.003 No 0.016 No 0.499 No a Trifloxystrobi n 2 L 0.036 0.232 Yes a 3 L 0.040 0.000 No a Average 0.059 1 (1) Tests of normality were carried out on SPSS based on Shapiro-Wilk analysis as the number of samples per population was less than 50. P value > 0.05 means the data distribution is normal. (2) Because the number of samples in each population in the current study was unequal, the test of homogeneity of variance was done to check the equal variance of the data. If P value in this test < 0.05, the equal variances are not assumed, and the Dunnett T3 method will be chosen for multiple comparison. If P value > 0.05, the equal variances are assumed, and the Tukey method will be used to analyse the multiple comparison in Post hoc test. (3) If ANOVA shows the P value < 0.05, there are the differences in the EC50 mean among the populations.

(4) Multiple comparison was fulfilled on SPSS using the Post hoc test of Dunnett T3 method where equal variances are not assumed, and using Tukey method when the variances are equal. The different lowercase

letter in one multiple comparison reveals the significant differences in the EC50 mean between the pair of population compared. Monilinia fructicola isolates from sprayed orchards showed greater variance in response to two of the fungicides than those from unsprayed orchards. The variance in M. fructicola response to iprodione was 15 and 40 times greater amongst isolates on sprayed orchards (1F, 0.126; 3F, 0.322) than it was amongst isolates from the unsprayed orchard 2F (0.008). Similarly, variation of responses to fluopyram+trifloxystrobin by M. fructicola isolates from the two sprayed orchards was three and eight times (0.011 and 0.025) greater than those from the unsprayed orchard (0.003) (Table 2). In contrast, the range of responses of M. fructicola isolates to propiconazole was relatively even, irrespective of the spray regime (sprayed 1F 1.87E-05, unsprayed 2F 1.37E-05, sprayed 3F 6.74E-05).

For M. laxa isolates, a similar pattern occurred, al-though the variance in response to iprodione and fluopyram + trifloxystrobin was generally greater than that observed for M. fructicola isolates. There was a greater variation in response to fungicides among isolates from sprayed orchards than those from unsprayed orchards. The variance in response to propiconizole from sprayed orchard 1 L (0.057) was 10 and 190 times greater than it was in M. laxa isolates from unsprayed orchards 2 L and 3 L (0.006 and 0.0003), respectively. For iprodione, there was 4 and 48 times greater variance between M. laxa isolates in the sprayed orchard than there was from those collected at the unsprayed orchard.

Similarly, for fluopyram+trifloxystrobin there was 37 to 130 times greater variance in response by sprayed M. laxa isolates than by isolates that hadn’t been ex-posed to fungicide spray (Table 2).

In sprayed orchards 1F and 3F, 45 and 48% of M. fructicola isolates, respectively, were ranked as tolerant or very tolerant to propiconazole, but in the unsprayed orchard, only 17% were tolerant and none were very tolerant. A similar pattern was apparent for the other two fungicides. On the unsprayed orchard, no M. fructicola isolates were tolerant or very tolerant to iprodione or

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fluopyram + trifloxystrobin, whereas isolates were tolerant and very tolerant to both fungicides on both sprayed orchards (Table 4).

Table 4. Susceptibility/tolerance of Monilinia fructicola and Monilinia laxa isolates to three fungicides at sprayed and un-sprayed stone fruit orchards. Susceptibility and tolerance rankings were assigned by calculating variation from the mean EC50 for the species on the orchard versus response to the fungicide

Very susceptible Susceptible Tolerant Very tolerant Fungicide Orchard (%) (%) (%) (%)

M. fructicola Propiconizole 1F sprayed 0 55 45 0 2F unsprayed 5 78 17 0 3F sprayed 0 50 32 18 Iprodione 1F sprayed 45 45 0 10 2F unsprayed 70 30 0 0 3F sprayed 27 9 23 41 Fluopyram + Trifloxystrobin 1F sprayed 45 35 5 15 2F unsprayed 78 22 0 0 3F sprayed 23 32 18 27 M. laxa Propiconizole 11L sprayed 44 36 12 18 22L unsprayed 47 33 7 13 33L unsprayed 79 21 0 0 Iprodione 11L sprayed 36 24 32 8 22L unsprayed 47 24 12 17 33L unsprayed 0 43 57 0 Fluopyram + Trifloxystrobin 1 L sprayed 72 8 0 20 2 L unsprayed 31 63 6 0 3 L unsprayed 29 57 14 0

There were also differences in fungicide tolerances between the two sprayed orchards. A greater proportion of highly tolerant M. fructicola isolates for the three fungicides tested occurred at sprayed orchard 3F than occurred at sprayed orchard 1F. Notably, on 3F, 45% of isolates were tolerant or highly tolerant to fluopyram + trifloxystrobin, whereas on 1F, only 20% of isolates were tolerant or highly tolerant (Table 4).

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Interestingly, M. laxa isolates from one of the un-sprayed orchards (2 L) responded in a similar way to propiconizole as did isolates from sprayed orchards (Table 4). In orchard 2 L, 20% of isolates were tolerant or very tolerant to propiconizole, and from sprayed orchards 1 L and 2 L, 30 and 20%, respectively, of isolates were tolerant or very tolerant. Similarly, responses to iprodione were similar at orchards 1 L and 2 L. However, on unsprayed orchard 3 L, no isolates were tolerant or very tolerant to propiconizole, but more than half the 3 L isolates were tolerant to iprodione. Some isolates from unsprayed orchards 2 L and 3 L were tolerant to fluopyram + trifloxystrobin, but none were very tolerant, as were 20% of isolates from sprayed orchard 1 L (Table 4). Although orchards 2F and 3F were located only about 1 km apart, it appears that isolates tolerant or very tolerant to iprodione or fluopyram and trifloxystrobin did not naturally travel from sprayed orchard 3 L, where 64 and 45% of isolates were tolerant or very tolerant, to unsprayed orchard 2 L, where all isolates were susceptible.

Multiple fungicide susceptibility occurred on all or-chards, but multiple fungicide tolerance occurred mainly on sprayed orchards. For both Monilinia species from sprayed and unsprayed orchards, there were isolates sensitive or very sensitive to all three fungicides tested. On the other hand, isolates very tolerant to one of the fungicides tested occurred mainly on sprayed orchards. Isolates tolerant to two fungicides occurred uncommonly, and no isolates were tolerant to all three fungicides.

For instance, isolate M153 (M. fructicola sprayed or-chard 3F) was ranked as very tolerant to iprodione, but it was sensitive to the other fungicides. Similarly, isolate M171 (M. fructicola sprayed orchard 3F) was very tolerant to fluopyram+trifloxystrobin, but sensitive to the two other fungicides. A few isolates were tolerant to two fungicides; isolate M152 was very tolerant to both iprodione and fluopyram+trifloxystrobin.

For M. laxa isolates, isolates that were sensitive to all three fungicides occurred on sprayed orchard 1 L (e.g. M199, M218) and on both unsprayed orchards (e.g. M128, M44, M187). From sprayed M. laxa orchard 1 L, isolate M205 was very tolerant to both iprodione and fluopyram+trifloxystrobin, whereas isolates M208 and M210 were very tolerant to propiconizole

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but sensitive to the other two fungicides (Table 1). Isolate M7 from unsprayed orchard 2 L was very tolerant of iprodione (Table 1).

Discussion

A comparison of fungicide sensitivity was made be-tween Monilinia isolates collected from stone fruit or-chards that were either regularly sprayed with the three different fungicides tested, and those that were not ex-posed to fungicides at any time. When the mean responses were calculated, it was shown that on average M. fructicola and M. laxa isolates from sprayed orchards exhibited greater tolerance to the three fungicides tested than did those that had not been sprayed. Resistance to fungicides has been widely reported for M. fructicola (Luo and Schnabel 2008b; Ma and Michailides 2005; Egüen et al. 2015), and M. laxa (Egüen et al. 2016; Hrustić et al. 2018).

The variation of sensitivity to the three fungicides tested was greatest amongst M. fructicola and M. laxa isolates from sprayed orchards, revealing that fungicide application may increase the range of responses to fun-gicides. The current study also showed that M. laxa isolates were more tolerant to iprodione than were M. fructicola isolates. This is in contrast to a recent study of fungicide sensitivity in both Monilinia species in Serbia (Hrustić et al. 2018). There, isolates of M. fructicola were more tolerant than M. laxa isolates to iprodione and other fungicides. In addition, these researchers found that M. laxa isolates displayed little variability in sensitivity to fungicides, whereas M. fructicola isolates displayed a wide range of sensi-tivities. Our study showed that M. laxa isolates were more variable than M. fructicola isolates.

On average, M. laxa isolates were more tolerant to propiconizole than were M. fructicola isolates, although most research into tolerance to this fungicide has been done with M. fructicola (Cox et al. 2007; Zhu et al. 2012; Hrustić et al. 2018), not M. laxa. Propiconizole-tolerant M. fructicola isolates contain the promoter element Mona that deactivates demethylation-inhibiting triazole fungicides (Chen et al. 2017). Mona-like elements have not been identified in M. laxa. No triazole-tolerant isolates were found among 788 M. laxa isolates collected from 121 conventional stone fruit orchards in Spain (Egüen et al. 2016). A study of 10 M. laxa isolates

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from Serbia also failed to identify resistance to triazole fungicides (Hrustić et al. 2018). The presence of M. laxa isolates with high EC50 values to propiconazole in the study area warrants further investigation into the mechanism and development of triazole tolerance in M. laxa in W.A. This finding highlights the importance of identifying the distribution of Monilinia species and their fungicide tolerances before applying fungicides in orchards.

The isolates were ranked as being sensitive or tolerant to the fungicides tested. The ranking system used was a relative one, based on mean the EC50 of each Monilinia species to each fungicide used in this experiment. Therefore, the isolates ranked as resistant here may be considered susceptible under situations where mean fungicide tolerance is higher. Fungicide concentrations used in our in vitro experiments were less than those recommended for field application. The iprodione concentration range used here was 0.03-0.12 mg/L, while the field application rate recommended by the manufacture was 1.0 mg/L. For propiconazole, 0.0075-0.03 mg/L was used, while the manufacturer’s recommended field rate was 0.15 mg/L. Similarly, the fluopyram + trifloxystrobin concentration used in our experiments was 0.03-0.12 mg/L, whereas the manufacturer’s recommended field rate was 0.3 mg/L. Other researchers used a range of fungicide concentrations in in vitro studies to discriminate sensitive from tolerant isolates, and ours were generally similar to those used by other researchers. An exception was Thomidis et al. (2009), who used 500 mg/L iprodione in agar plate-based tests to discriminate tolerant and susceptible M. laxa isolates. This is a 500-fold greater concentration than that recommended for field application, and per-haps not surprisingly, no M. laxa isolates grew at this concentration. Another group used 0.3 mg/L iprodione to discriminate tolerant and sensitive isolates of M. fructicola (Egüen et al. 2015). Cox and colleagues (Cox et al. 2007) used 0.3 mg/L propiconizole to discriminate M. fructicola isolates, while Zehr and col-leagues (Zehr et al. 1999) used 0.03 mg/L. To our knowledge, no in vitro experiments have been reported using Luna Sensation (fluopyram + trifloxystrobin) to test the sensitivity of Monilinia species. Tests of fluopyram alone against Botrytis cinerea complex isolates from strawberry and raspberry found they could discriminate sensitive from tolerant isolates using fluopyram concentrations of 0.02- 0.2 mg/L (Weber et al. 2015).

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Adaskaveg et al. (2011) considered the risk of generating highly tolerant strains of a range of fungi to propiconizole and fluopyram + trifloxystrobin to be high, but medium to iprodione. Our study confirms that tolerance developed very quickly to fluopyram + trifloxystrobin, which has been used since 2015 in the region. The presence of fluopyram + trifloxystrobin tolerance in M. laxa on two unsprayed orchards, but not for M. fructicola on one unsprayed orchard, may indicate that M. laxa is inherently the more tolerant of the two species, but more research is required to confirm this.

To our knowledge, there are no previous studies comparing fungicide sensitivities of Monilinia isolates (or other fungal species) in both sprayed and unsprayed fruit orchards. A study of vineyards that either had a history of spray with two DMI fungicides (not propiconizole) or had never been sprayed with them found that Uncinula necator (powdery mildew of grapes) isolates from unsprayed vineyards had lower EC50 values (0.07-0.08 mg/L) than did those from sprayed vineyards (0.19-0.83 mg/L) (Savocchia et al. 2004). Our findings generally reflected this response across the three fungicides tested. Previous studies re-ported build-up of tolerance by Monilinia species on sprayed stone fruit orchards to propiconizole (Cox et al. 2007; Schnabel et al. 2004) and iprodione (Yoshimura et al. 2004), but none reported tolerance to the mixture of fluopyram and trifloxystrobin. Surprisingly, we identified some fluopyram + trifloxystrobin- tolerant and very tolerant isolates from both sprayed and unsprayed orchards. It is not known whether the tolerant isolates from unsprayed orchards represent natural tolerance to these fungicides in the population, or if tolerant lines were introduced to unsprayed orchards from nearby sprayed orchards. Although orchards 2F and 3F were located only about 1 km apart, there was little evidence that tolerant isolates from sprayed orchard 3F were present at unsprayed orchard 2F. No tolerance to iprodione or fluopyram + trifloxystrobin was present in the unsprayed orchard 2F. Monilinia conidia are reported to travel only short distances. Monilinia fructicola conidia in peach orchards in New South Wales travelled only 100 m under natural conditions (Kable 1965). Monilinia vaccinii-corymbosi conidia spores travelled only 20 m in wind, while ascospores of the same species travelled 30 m under the same conditions (Cox and Scherm 2001).

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Selection against fungicide-sensitive genotypes in the sprayed orchards should generate a population structure where tolerant genotypes are more prevalent than sensitive genotypes. In general, this was the case, but some mixing occurred: sensitive isolates existed in sprayed orchards, and relatively tolerant genotypes existed in unsprayed orchards. Although genetic diversity of populations on the sprayed and unsprayed or-chards was not studied here, the range of responses to fungicides is indicative of a genetically diverse population (Fazekas et al. 2014). Genetic diversity provides the population with flexibility to adapt to threats, such as application of fungicides. Knowledge about the genetic base of Monilinia populations is critically important in devising effective control strategies.

Traditionally, conventional orchardists have tried to remove wild or unmanaged stone fruit trees growing close to their orchards because these were seen as potential sources of Monilinia. Conflicts between conventional and organic farming systems have sometimes centred around organic farms being perceived as a source of pathogens that infect conventional crops (Parker and Munroe 2007). Our results indicate it is unlikely that isolates sourced from unsprayed stone fruit orchards present a risk to commercial orchards, at least for the three fungicides tested. The isolates from fungicide-free orchards were more sensitive to fungicides than those from sprayed orchards. Therefore, isolates sourced from unsprayed trees pose a lower risk to growers who spray than do isolates from other sprayed orchards.

The relative importance of the two Monilinia species to production losses in the study area is unknown, and this is a subject of ongoing study. Knowledge of relative sensitivities of each Monilinia species to each fungicide, and of the sensitivities of individual isolates within orchards, is of immense value to producers who aim to control the disease economically and with minimal environmental cost.

References

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Savocchia, S., Stummer, B. E., Wicks, T. J., Van Heeswijck, R., & Scott, E. S. (2004). Reduced sensitivity of Uncinula necator to sterol demethylation inhibiting fungicides in southern Australian vineyards. Australasian Plant Pathology, 33, 465–473. Schnabel, G., Bryson, P. K., Bridges, W. C., & Brannen, P. M. (2004). Reduced sensitivity in Monilinia fructicola to propiconazole in Georgia and implications for disease man-agement. Plant Disease, 88, 1000–1004. Shaw, M. W. (2000). Models of the effects of dose heterogeneity and escape on selection pressure for pesticide resistance. Phytopathology, 90, 333–339. Thomidis, T., Michailides, T., & Exadaktylou, E. (2009). Contribution of pathogens to peach fruit rot in northern Greece and their sensitivity to iprodione, carbendazim, thiophanate-methyl and tebuconazole fungicides. Journal of Phytopathology, 155, 194–200. Tran, T. T., Li, H., Nguyen, D. Q., Sivasithamparam, K., Jones, M. G. K., & Wylie, S. J. (2017). Spatial distribution of Monilinia fructicola and M. laxa in stonefruit production areas in Western Australia. Australasian Plant Pathology, 46, 339– 349. Tran, T. T., Li, Hua, Nguyen, D. Q., Sivasithamparam, K., Jones, M. G. K., Wylie, S. J. (2018) Genetic and pathogenic diver-sity of Monilinia fructicola and M. laxa isolates in Western Australia. (Under review). Weber, R. W., Entrop, A. P., Goertz, A., & Mehl, A. (2015). Status of sensitivity of northern German Botrytis populations to the new SDHI fungicide fluopyram prior to its release as a commercial fungicide. Journal of Plant Diseases and Protection, 122, 81–90. Wherrett, A. D., Sivasithamparam, K., & Kumar, S. (2001). Detection of possible systemic fungicide resistance in Western Australian Monilinia populations. Phytopathology, 91, S95.

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Addendum Chapter 5

Propiconazole tolerant isolates do not always contain a Mona element as implied in the paper. There were some reported without Mona in the north-eastern USA (Villani and Cox, 2011), and there are some resistant isolates reported in Brazil without Mona (Lichtemberg, 2015).

Lichtemberg, P.D.S.F., 2015. Dynamics and stability of resistance to tebuconazole in Monilinia fructicola populations from Brazilian peach orchards. https://acervodigital.ufpr.br/handle/1884/37675?show=full Villani, S.M. and Cox, K.D., 2011. Characterizing fenbuconazole and propiconazole sensitivity and prevalence of ‘Mona’in isolates of Monilinia fructicola from New York. Plant disease, 95(7), pp.828-834.

Correction: Schnabel et al. 2004 in their study included orchards never exposed to fungicides.

Note: There is a possible relationship between application timing and sensitivity to triazoles in M. laxa (triazoles at bloom, SDHI and Qois early preharvest, iprodione late preharvest) that was not discussed in the paper.

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Chapter 6. Co-infection with three mycoviruses stimulates growth of a Monilinia fructicola isolate on nutrient medium, but does not induce hypervirulence in a natural host

This chapter addressed aim 5 of the thesis and was published in Viruses in January 2019.

Citation:

Tran, T. T., Li, H., Nguyen, D. Q., Jones, M. G. K., & Wylie, S. J. (2019). Co-infection with three mycoviruses stimulates growth of a Monilinia fructicola isolate on nutrient medium, but does not induce hypervirulence in a natural host. Viruses, 11(1), 89; doi:10.3390/v11010089

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Statement of contribution of the authors contributed to the work

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Abstract Monilinia fructicola and Monilinia laxa are the most destructive fungal species infecting stone fruit (Prunus species). High-throughput cDNA sequencing of M. laxa and M. fructicola isolates collected from stone fruit orchards revealed that 14% of isolates were infected with one or more of three mycoviruses: Sclerotinia sclerotiorum hypovirus 2 (SsHV2, genus Hypovirus), Fusarium poae virus 1 (FPV1, genus Betapartitivirus), and Botrytis virus F (BVF, genus Mycoflexivirus). Isolate M196 of M. fructicola was co-infected with all three viruses, and this isolate was studied further. Several methods were applied to cure M196 of one or more mycoviruses. Of these treatments, hyphal tip culture either alone or in combination with antibiotic treatment generated isogenic lines free of one or more mycoviruses. When isogenic fungal lines were cultured on nutrient agar medium in vitro, the triple mycovirus-infected parent isolate M196 grew 10% faster than any of the virus-cured isogenic lines. BVF had a slight inhibitory effect on growth, and FPV1 did not influence growth. Surprisingly, after inoculation to fruits of sweet cherry, there were no significance differences in disease progression between isogenic lines, suggesting that these mycoviruses did not influence the virulence of M. fructicola on a natural host.

1. Introduction

Mycoviruses are viruses that infect fungi. They have been identified from all major fungal phyla, namely the Zygomycota, Chytridiomycota, Ascomycota, and Basidiomycota [1,2]. Since mycoviruses were first described [3], the partial or complete genomes of more than 250 mycoviruses have been sequenced [4]. Fungi can be multiply infected with closely-related and distantly-related viruses [5]. Most mycoviruses have double-stranded (ds) or single-stranded (ss) RNA genomes, and some groups do not encode a coat protein.

The influence that mycoviruses have on the ecology of their hosts is not well studied. Some mycoviruses reduce the ability of the fungal host to cause disease in plants. These are known as hypovirulent mycoviruses, and they have potential as biological control agents. The most well- known are Cryphonectria hypoviruses 1 and 2 (CHV1, CHV2), which significantly decreased

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the virulence of the fungus Cryphonectria parasitica, the causal agent of chestnut blight [6,7]. Other hypovirulent mycoviruses reduce the pathogenicity of white mold fungus Sclerotinia sclerotiorum [8,9] and white root rot fungus Rosellinia necatrix [10]. In contrast, several mycoviruses are associated with hypervirulence, described as a higher level of virulence or sporulation in their fungal hosts [11–13]. Other mycoviruses are reported to be associated with latent infections [8,14–16].

Monilinia fructicola and M. laxa were first recorded in Western Australian stone fruit production regions in 1997 [17,18], and they have since spread to all other stone fruit production regions in the state [19]. They incur costs in control (fungicides, gathering and destroying mummified fruit) and crop losses. Very little is known about mycoviruses that infect Monilinia species. Tsai, et al. [20] identified seven virus-like double-stranded RNA species in 36 of 49 M. fructicola isolates infecting nectarine and peach orchards in New Zealand. Although not characterized genetically, the authors described virus-like particles resembling those of partitiviruses, totiviruses, tobraviruses and furoviruses. They identified no differences in host growth rates between isolates with and without virus infection. In this current study, we identified three mycoviruses infecting a collection of M. fructicola and M. laxa isolates, and undertook to determine if these viruses influenced growth rates of infected fungal cultures in vitro, and the virulence of the pathogen on a natural host.

2. Materials and Methods 2.1. Fungus Collection and Isolation

Eighteen M. laxa and ten M. fructicola isolates were collected from symptomatic flowers, twig cankers and fruits from stone fruit orchards in Western Australia (Table S1). Conidia were collected in a drop of water, then spread on 1% water agar media to separate single spores using a microscope. Individual spores were transferred to V8 agar medium (V8 juice 200 mL, distilled water 800 mL, agar 15 g) and incubated in the dark at 22oC. After a week, a 5x5 mm square of agar containing actively-growing hyphae was excised from the edge of the mycelium and transferred to V8 liquid medium, and placed on a shaker at 100 rpm in the dark at 22oC. After about a week, DNA and RNA were extracted for further studies.

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2.2. DNA and RNA Extraction

Nucleic acids were extracted from 100 mg of mycelium, which was frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. The powdered mycelium was transferred into a 1.5 mL centrifuge tube containing 450 l extraction buffer (0.1 M NaCl, 50 mM Tris (hydroxymethyl) aminomethane (Tris) pH8.0, 0.5 mM Ethylenediaminetetraacetic acid (EDTA) pH8.0, 1% Sodium dodecyl sulfate (SDS), 1% Polyvinylpolypyrrolidone (PVPP) and 450 l phenol-chloroform (50:50) saturated with Tris-EDTA buffer (pH 8.0). The mixture was homogenized before being centrifuged at room temperature for 2 min. After that, 400 l of the aqueous phase was transferred to a new tube containing 400 l of phenol-chloroform, then mixed and centrifuged for 2 min. Then, 300 l of the aqueous phase containing nucleic acids was removed to a new tube containing 58 l of absolute ethanol to which 200 mg of cellulose powder (CF11, Whatman) was added and mixed. After centrifuging at high speed for 1 min, the material was separated into two phases, the DNA-containing supernatant and the pellet containing RNA. The supernatant (250 l) was transferred to a fresh tube and 20 l 3M NaOAC pH5.2 and 625 l absolute ethanol was added to precipitate DNA. The pellet containing RNA was washed three times with 750 l application buffer (0.1 mM NaCl, 50 mM Tris pH8.0, 0.5 mM EDTA pH8.0, absolute ethanol). The RNA pellet was eluted in 450 l elution buffer (0.1 mM NaCl, 50 mM Tris pH8.0, 0.5 mM EDTA pH8.0). The RNA solution (450 l) was removed and precipitated after incubation at 20 C in a new tube containing 1 mL absolute ethanol and 45 l 3M NaOAC pH5.2 for RNA collection.

DNA was used to identify the fungal species based on the comparison of internal-transcribed spacer (ITS) region sequences of ribosomal RNA genes, as previously described [19]. RNA was used for virus identification in M. fructicola isolate M196 by high-throughput sequencing. 2.3. cDNA Synthesis, PCR Amplification and Library Preparation for High-Throughput Sequencing

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cDNA synthesis was carried out in 20 l volume containing the following components: 4 l 5 GoScript™ Buffer; 2 l 0.1 mM DDT; 1 l 10 mM deoxynucleotides; 1 l GoScript™ reverse transcriptase (Promega Corporation, Sydney, Australia); 1 l 10 mM Tris EDTA; 8 l water; 1 l RNA, and 1 l random primer adaptor (5'-CGTACAGTTAGCAGGCNNNNNNNNNNNN- 3', where N is any nucleotide, annealed to the complement of the adaptor sequence added to cDNA molecules). The mixture was incubated at 25oC for 10 min; 42oC for 60 min; and 72oC for 15 min. cDNA was amplified by PCR using primer 5'-CGTACAGTTAGCAGGC-3', which annealed to the complement of the adaptor sequence added to cDNA molecules, in 20 l volume of 10 l 2x GoTaq®Green Master Mix (Promega Corporation, Sydney, Australia); 4 l cDNA products; 5 l water; 1 l barcode primer. The reaction was carried out with an initial cycle of 5 min at 94oC; 40 cycles of 10 min at 94oC, 20 s at 45oC and 30 s at 72oC, an extension cycle of 5 min at 72oC, and a final cycle of 5 min at 37oC. The library was purified, quantified, and paired- end sequenced on an Illumina MiSeq platform at the Australian Genome Research Facility.

2.4. Sequence Analysis

The sequences obtained were analyzed as previously described [21]. Briefly, in CLC Genomics Workbench (Qiagen, Sydney) the reads were trimmed using a quality score of 0.05 and of ambiguous bases, then of primer sequences and indices after assigning them to source bins. De novo assembly was carried out on the trimmed reads to form contigs >300 nt in length. Assembly parameters were word size of 40 and bubble size of 50. Contigs were compared to sequences lodged in GenBank (National Center for Biotechnology Information, NCBI) databases (https://blast.ncbi.nlm.nih.gov) using Blastn and Blastx [22] to identify virus-like sequences. Overlapping contigs were joined together when possible, and missing sequences were determined after designing specific primers to span the gaps (Table S2). Depth of sequencing was determined by mapping raw reads back to consensus sequences using the ‘map to reference’ function in Geneious v9.1.7 (Biomatters, Auckland, New Zealand) [23]. Annotation of the genomes was done manually in Geneious v9.1.7 after comparison with related sequences, and amino acid sequences were deduced from the nucleotide sequences of open reading frames (ORF).

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Specific primers (Table S3) were designed from consensus sequences and used to confirm the presence of these three mycoviruses in other fungal isolates. When an amplicon was detected, both strands were sequenced using the Sanger method to confirm presence of the virus.

2.5. Generation of Isogenic Fungal Lines Free of Mycoviruses

Four methods were tested alone or in combination to eliminate viruses from fungal isolates: (1). Cold treatment. Fungal mycelium was stored in 30% glycerol at 80oC for two years before being recovered on V8 liquid medium. (2). Temperature shock. Fungal mycelium was stored in 30% glycerol at 80oC for two years was heated to 30oC for 30 s, incubated in liquid nitrogen for 45 s, then heated again to 30oC for 45 s. Mycelium was then recovered on V8 liquid medium. (3). Hyphal tipping. Hyphal tips were harvested from the edges of rapidly-growing colonies, and sub-cultured to a fresh water agar plate. (4). Antibiotic treatment. Antibiotics were added singly to water agar when the autoclaved media had cooled to approximately 50oC. Antibiotic concentrations used were 12.5 mg/L cycloheximide, 100 mg/L kanamycin, and 250 mg/L streptomycin. Hyphal tips were inoculated to plates and cultures incubated in the dark at 22oC for six days before hyphal tips were harvested from it and the process repeated. Each line was treated this way five times before it was tested for the presence of all three mycoviruses by RT-PCR using species-specific primers. Species- specific primers were designed from high-throughput sequences (Table S3).

Virus-free lines were maintained in culture for up to six months before their virus-free status was reconfirmed by RT-PCR with species-specific primers. These virus-free lines were used for subsequent growth rate and virulence experiments.

2.6. Virulence on Cherry

Isolate M196 (triple virus-infected) and isogenic M196 lines free of at least one virus (M196-1, M196-4, M196-6) (Table 1) were grown on V8 agar medium without antibiotics at 22oC for six days before a 2x2 mm square of mycelium was harvested from the margin of the colony. This

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was placed on the surface of a washed and dry cherry fruit (Prunus avium) of cultivar Bing. Inoculated fruits were incubated in 100% humidity in the dark at room temperature (17–20oC) for seven days. Each fungal isolate was inoculated to 36 cherry fruits; each fruit was treated as a replication. At the end of the incubation period, the widest extent of the fungal lesion was measured using a compass and ruler. The entire experiment was repeated twice.

Table 1. Presence (+) or absence (-) of mycoviruses from isogenic lines of M. fructicola M196 after treatment. The antibiotic treatments were combined with hyphal tipping.

Cycloheximid Kanamyci Streptomyci Mycoviruses a 80oC Temperature Hyphal e n n Storage Shock Tipping SsHV2 + + - - - - FpV1 + + - + + + BVF + + - + - -

Number of lines obtained b 6 1 1 2 1 1 Name of line M196 M196-1 M196-4 M196-6 a SsHV2, Sclerotinia sclerotiorum hypovirus 2; FpV1, Fusarium poae virus 1; BVF, Botrytis virus F. b Number of isogenic lines showing the given pattern out of six treated lines sub-cultured from parent isolate M196. c Name and source of line used in subsequent experiments.

2.7. Mycelial Growth In Vitro

Four virus-free and virus-infected isogenic lines (M196, M196-1, M196-4, M196-6) were grown on V8 agar plates at 22oC for six days. Each isolate was inoculated into 36 V8 plates (three replications 12 plates per replication). After six days, the maximum diameter of each colony was measured and differences in morphology recorded. The entire experiment was repeated twice.

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To determine the differences between treatments, a one-way analysis of variance (ANOVA) was done at a significance level of 0.05 using SPSS version 24.

3. Results 3.1. Sequencing Analysis and Virus Assays

A MiSeq sequencing run created 13,264,058 reads of 100 nt. Barcode (index) sequences were used to assign reads to samples. After screening for quality and trimming the barcode and primer sequences, de novo assembly resulted in 37,146 contigs within the size ranges of 300 to 5385 nucleotides. Blastn and Blastx analysis of the contigs revealed 20 that shared nucleotide or amino acid identities with viral sequences. Where two or more contigs mapped to the same virus, gaps in the sequence were filled by RT-PCR so that the sequences of complete or almost complete genomes were determined (Table S2). Three previously-identified mycoviruses infected M. fructicola M196 (Table S1). RT-PCR assays using species-specific primers (Table S3) with appropriate controls were used to confirm the presence of the viruses and to check for their presence in isogenic lines over time. Isogenic lines of isolate M196 lacking one, two or three viruses were maintained in culture for 26 weeks before in vitro and in planta growth rates were measured.

3.1.1. Sclerotinia sclerotiorum Hypovirus 2

A contiguous virus-like sequence of 13,535 nucleotides was obtained from M. fructicola isolate M196. The nucleotide and deduced amino acid sequences shared the greatest identities with the replicase gene of three previously described isolates of Sclerotinia sclerotiorum hypovirus 2 (SsHV2) (genus Hypovirus, family Hypoviridae) classified as a ssRNA virus. Alignment of the new sequence revealed that it shared 85–89% pairwise nucleotide and 93– 95% amino acid identities with SsHV2 isolates 5427 from New Zealand (KF525367) [24], isolate SsHV2 from the USA (KF898354) [25], and isolate SX247 from China (KJ561218) [13]. The demarcation criterion for hypovirus species is less than 50% pairwise nucleotide identity over the complete genome [26]. Although identity with Rosellinia necatrix hypovirus 2 is 54%, identities with other SsHV2 isolates is far higher, and so we propose this to be a

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member of species Sclerotinia sclerotiorum hypovirus 2. It is designated Sclerotinia sclerotiorum hypovirus 2 isolate Monilinia-TNS. SsHV2 was identified only in Monilinia M196.

The sequence of SsHV2-Monilinia-TNS is estimated to represent 93% of the complete genome. The SsHV2-Monilinia-TNS genome comprises one large ORF, which is incomplete at the 50end. The ORF extended from nucleotide 1–13,205 where it was terminated by an opal stop codon (UGA). The ORF is followed by a 3’ untranslated region (UTR) of 333 nucleotides, present from nucleotides 13,206 to 13,535. The conserved RdRp core motifs V and VI (S/TG x3 T x3 NS/T x22 GDD) (where x is any amino acid residue) [27] was present as TG x3 T x3 DS x38 GDD. No poly(A) tail region was detected. The SsHV-2 sequence was assigned GenBank accession MH665657.

3.1.2. Fusarium poae Virus 1

Two contigs representing the complete genomic segments of a bipartite virus were identified from M. fructicola M196. The sequence shared the highest sequence identity with published isolates of Fusarium poae virus 1 (FpV1) (genus Partitivirus, family Partitviridae), a double- stranded RNA virus. One segment (RNA1) encodes the replicase (RdRp) and the other (RNA2) encodes the coat protein. Comparison of the 2100 nucleotide sequence of RNA1 revealed that it shared 90% pairwise identity with RNA1 of two FpV1 isolates: A11 from Slovakia (AF047013) and 240374 from Japan (LC150606). The deduced amino acid sequence of the RdRp segment shared 92% identity with the RdRps of these two isolates. The ORF of RNA1 extended from nucleotide 54-2084 where it was terminated by an opal stop codon, encoding an RdRp-like protein of 689 amino acid residues with an estimated molecular weight of 79.9 kDa. The conserved core RdRp motifs V and VI [28] were present as SG x3 T x3 DS x29 GDD. The 5'UTR extended from nucleotide 1 to 53 and the 3'UTR from nucleotide 2085 to 2160.

The RNA2 segment, encoding the coat protein (CP) gene, was 2093 nucleotides in length. The single ORF encoding a protein deduced to be 654 amino acid residues in length, with an estimated mass of 72.6 kDa. Surprisingly, a partial RdRp-like motif (T x2 DS x27 GDD) was

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present with the CP ORF. The deduced amino acid sequence of ORF1 of RNA2 shared greatest identity (85–86%) with CPs of the two other FpV1 isolates, and the nucleotide identity was 83– 84% with them. The 5'UTR extended from 1 to 102 nt, and the 3'UTR from 2017 to 2093 nt.

The levels of identity of this new virus with isolates of FpV1 were above the species demarcation of partitiviruses recommended by the ICTV [29] of <40% amino acid identity between the RdRps, and so the isolate is proposed here as a member of species Fusarium poae virus 1. The isolate from M196 was designated Fusarium poae virus 1 isolate Monilinia-TNS. FpV1- Monilinia-TNS RNA1 was assigned GenBank accession MH665658, and FpV1-Monilinia-TNS RNA2 assigned GenBank accession MH665659.

3.1.3. Botrytis Virus F

Contigs representing partial genomic sequences of a virus were detected from M. fructicola M196. The deduced amino acid sequences of fragments each shared 90–95% identity to those of an isolate of Botrytis virus F (BVF) (accession NP068550) (genus Mycoflexivirus, family Gammaflexiviridae), a ssRNA virus.

Together, the three fragments were estimated to represent about 30% of the BVF complete genome, designated Botrytis virus F isolate Monilinia-TNS. One of the fragments held the core RdRp motifs V and VI as SG x3 T x3 NT x21 GDD. The three contigs of BVF-Monilinia-TNS were assigned GenBank accessions MH665660, MH665661, and MH665662.

3.2. The Presence of Mycoviruses in Other Monilinia Isolates

Eighteen M. laxa isolates and nine M. fructicola isolates were screened using SsHV2, FpV1 and BVF-specific primers (Table S3). Three M. laxa isolates harbored one or more of these viruses, including M82 (Fusarium poae virus 1), M84 (Fusarium poae virus 1 and Botrytis virus F), and M140 (Fusarium poae virus 1) (Table S1), but none of the other nine M. fructicola isolates tested held these viruses.

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3.3. Elimination of Mycoviruses

The cold treatment and temperature shock methods failed to eliminate any mycoviruses, and the temperature shock method resulted in the death of most cultures (Table 1). On the other hand, hyphal tipping with and without antibiotics was effective at curing M. fructicola M196 of one or more of the three mycoviruses. It is unclear whether any of the antibiotics played a significant role in eliminating the viruses because tipping alone without antibiotics generated isogenic line M196-1 that was free of all three mycoviruses. Treatment with 12.5 mL/L cycloheximide eliminated SsHV2 from two isogenic lines, one of which, M196-4, was used in subsequent experiments. Treatment with kanamycin (100 mg/L), or with 250 mg/L streptomycin, eliminated both SsHV2 and BVF, but not the partitivirus FpV1 (Table 2). All isogenic lines were assayed by RT-PCR using virus-specific primers (Table S3, Figure S1) before and after subsequent experiments, and these tests confirmed the maintenance of their virus status.

Table 2. Summary of colony diameter on V8 medium and lesion diameter on cherry of isogenic M. fructicola lines.

Colony Diameter on V8 Lesion Diameter on Cherry Medium Fruit

Range Mean Varianc Range Mean Line Treatment a Viruses present b e Variance (mm) (mm) (mm) (mm)

SsHV2; FpV1; M196 None BVF 68–80 73.8 11.8 3–21 8.7 26.5 M196-1 Hyphal tipping None 56–70 66.4 12 3–22 9.6 33.7 M196-4 Cycloheximide FpV1; BVF 55–70 62.9 16.9 3–21 8.9 29.5 M196-6 Kanamycin FpV1 60–71 66.0 11.5 3–25 10.9 33.4

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1 a Antibiotic treatments were combined with hyphal tipping; b SsHV2, Sclerotinia sclerotiorum hypovirus 2; FpV1, Fusarium poae virus 1; BVF, Botrytis virus F.

3.4. Mycoviruses Influenced Growth In Vitro

Triple-infected parent isolate M196 grew significantly faster (p < 0.05) than lines lacking one or more viruses. After six days in culture, the mean diameter of colonies of isolate M196 was 73.8 mm, while virus-cured lines M196-1, M196-4, and M196-6 were less: 66.4, 62.9, and 66.0 mm, respectively (Table 2).

Where SsHV2 was absent but FpV1 and BVF were present (M196-4), colony growth was suppressed, suggesting that SsHV2 enhanced growth. There was no significant difference (P > 0.05) in growth between virus-free line M196-1 and FpV1-infected M196-6, indicating that FpV1 had no influence on growth. Where FpV1 and BVF co-occurred (M196-4), mycelial growth was suppressed compared to virus-free line M196-1. Although FpV1 alone had no apparent effect on growth, in combination with BVF it suppressed growth. This result could be interpreted as BVF alone suppressing growth and FpV1 remaining latent or as a synergistic suppressive influence of both viruses (Tables 2–4, Figure S2).

Table 3. p-values of paired comparisons between M. fructicola isogenic lines of colony diameter on V8 medium and lesion diameter on cherry fruits.

Cherry Line a V8 Medium Fruit M196 M196-1 M196-4 M196 M196-1 M196-4 M196-1 2.20e 13 - 0.5 - M196-4 4.30e 19 1.50e 04 - 0.8 0.6 - M196-6 1.60e 14 0.6 0.0007 0.09 0.4 0.1

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p-value < 0.05 indicates a significant difference between means. a Isolate M196 contains SsHV2-Monilinia-TNS, FpV1-Monilinia-TNS, BVF-Monilinia-TNS, isogenic M196- 1 line is virus-free, M196-4 line contains FpV1-Monilinia-TNS, BVF-Monilinia-TNS, M196-6-Monilinia-TNS line carries FpV1-Monilinia-TNS.

Table 4. Possible effects on mycelial growth by each mycovirus.

Virus V8 Medium Cherry Fruit

SsHV2-Monilinia-TNS Increase mycelial growth No effect on lesion FpV1-Monilinia-TNS No effect on mycelial growth No effect on lesion BVF-Monilinia-TNS Decreases mycelial growth No effect on lesion

3.5. Influence of Mycoviruses on Virulence of M. fructicola

When cherry fruits were inoculated with the four isogenic lines of M196, there was no significant difference (p > 0.05) in the diameters of the resulting lesions. Although mean lesion size was lowest for M196 (8.7 mm), and virus-cured lines grew faster (9.6, 8.9, and 10.9 mm for M196-1, M196-4, and M196-6, respectively) (Table 3), they were not significantly different (p > 0.05) (Table 3). Mock inoculated fruits used as controls never became infected with Monilinia or other pathogens during the course of the experiment.

4. Discussion

The M. fructicola and M. laxa isolates tested were not widely infected with mycoviruses. Of the 18 M. laxa isolates and 10 M. fructicola isolates tested, only three M. laxa and one M. fructicola isolate were infected with one or more of three mycoviruses. This overall infection rate of 14% is far below that reported in the only other study of mycoviruses from Monilinia [20]. That study reported 76% of M. fructicola isolates tested were infected with at least one

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mycovirus. This report was based on RNA profiles and visualization of virus-like particles, not by sequence analysis [20]. Visualization of RNA species by electrophoresis or virus particles by TEM may not be sensitive enough to detect very low-titer viruses or those lacking a coat protein, and it provides only clues to identity based on genome size and/or virion shape and size. A shotgun sequencing approach, as was used here, should be capable of detecting all RNA-based viruses present, and the resulting sequence provide evidence for taxonomic placement.

The genome sequences of SsHV2 and FpV1 obtained using this approach were complete or almost complete, while that of BVF was partial. The incomplete genome may be a function of the low titer of BVF relative to the other two viruses, but it is possibly a function of differential RNA extraction efficiencies of the extraction procedure (cellulose-based) used.

Monilinia fructicola isolate M196 was infected with three mycoviruses, all of which were originally identified from other host genera, and none had previously been identified from Australia. Their presence in Australia probably reflects anthropogenic international translocations of mycovirus-infected M. fructicola isolates infecting Prunus fruit and germplasm, although the other known hosts of these mycoviruses, Sclerotinia sclerotiorum, Fusarium poae and Botrytis cinerea, all occur in Australia and may carry these viruses. Notably, M. fructicola and other known fungal hosts of the three viruses identified all have international distribution, they are all serious plant pathogens, and they are all members of the family Sclerotiniaceae [30].

Until now, SsHV2 has been identified in S. sclerotiorum in China, New Zealand and the USA [8,25], but not from Monilinia and not from Australia. Two groups have shown SsHV2 induces hypovirulence in its host. A study with isogenic lines of S. sclerotiorum infected with SsHV2 and an endornavirus showed that the presence of these mycoviruses reduced mycelial pigmentation and sclerotia formation in vitro. SsHV2 induced hypovirulence of S. sclerotiorum on lettuce and soybean, delaying production of sclerotia and reducing their numbers, but its influences on mycelial growth rate or virulence were not recorded [25]. Hu et al. [13] reported SsHV2 was associated with hypovirulence of S. sclerotiorum on canola (Brassica napus) in

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China. In contrast, our studies with SsHV2 infecting a M. fructicola line (co-infected with with FpV-1 and BVF) did not indicate hypovirulence. Our studies suggest SsHV2 enhanced Monilinia mycelial growth in vitro, it did not visibly change pigmentation or conidia production, and it did not appear to influence virulence on cherry. Unfortunately, we were unable to generate a line containing only SsHV2 to confirm these findings.

This is the first report of the betapartitvirus FpV1 being identified in a host beyond Fusarium poae, and its first report from Australia [29]. In F. poae [31] and here in M. fructicola, FpV1 does not seem to induce abnormal morphology or changes to virulence, and this is a common observance for partitiviruses generally [1]. Known exceptions are Aspergillus fumigatus partitivirus-1 (AfuPV-1) which induced abnormal aconidial sectors and a light pigmentation phenotype in Aspergillus fumigatus [28], and Sclerotinia sclerotiorum partitivirus-1 (SsPV1) that induced a hypovirulence phenotype in Sclerotinia sclerotiorum after damaging cell organelles [8].

Botrytis virus F is the sole species in genus Mycoflexivirus, family Gammaflexiviridae, a group closely aligned to other fungus-infecting viruses in the Deltaflexiviridae and to plant-infecting viruses in the Alphaflexiviridae and Betaflexiviridae [32]. BVF was the first virus identified from Botrytis cinerea, on strawberries in New Zealand [33]. It was also identified from a fungus associated with grapes in South Africa [34]. Our results suggest that BVF slightly reduced M. fructicola growth in vitro. That BVF may negatively impact growth of M. fructicola is of potential interest, given that both B. cinerea and M. fructicola are important plant pathogens, together affecting over 200 plant species [35,36]. The possibility that BVF induces hypovirulence should be studied further.

Investigation of the influence of mycoviruses on the growth of M. fructicola is not a trivial exercise, involving ‘curing’ parent fungal isolates of viruses to obtain isogenic lines [37]. Culturing fungi with cycloheximide is used widely to eliminate RNA mycoviruses [38]; however, this approach is not always successful. Incubation of Aspergillus niger cultures infected with a number of virus-like particles on cycloheximide failed to eliminate any of the viruses [39,40]. It was unclear if the antibiotics used in our experiments had any influence on

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virus elimination because hyphal tip culture alone without antibiotics effectively cured line M196-1 of all three viruses.

A limitation of our experiment is that conclusions of hypervirulence are based on linear colony growth measurements, not on measurements of total biomass, metabolism, or fecundity. It is unclear how the triple-infection by viruses might stimulate M. fructicola growth in vitro, but not affect virulence on fruit. In another study, growth of a chryso-like-virus-infected strain of Alternaria alternate in vitro was severely restricted, while virulence against fruit was enhanced [41]. The agar plate and the fruit are very different environments. On the plate, the complex cellular interactions between the fungal cells and plant cells is lacking [42–44]. We can only speculate that infection by one or more of these viruses, probably SsHV1, provided a means by which M. fructicola could metabolize nutrients more efficiently on the plate than could the virus-free isogenic line, but the mechanism for this is unknown.

Supplementary Materials: The following are available online at http://www.mdpi.com/1999- 4915/11/1/89/s1, Figure S1: Presence and absence of mycoviruses in isogenic fungal lines treated to remove mycoviruses; Figure S2. Comparison of typical plates of four isogenic lines of M. fructicola isolate M196 inoculated on V8 media after 5-days incubation in the dark at 25 C; Table S1. Monilinia isolates from Western Australia and their mycovirus-infection status; Table S2. Primers used to fill the gaps of the virus sequences; Table S3: Species-specfic primers used to reconfirm the presences of mycoviruses in fungal hosts.

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Addendum Chapter 6

Note: No hyphal tip colony is genetically identical to the next, especially in Monilinia fructicola, a fungus that has irregular numbers of nuclei in their cells (15 to 30). Each nucleus in the fungal colony may be unique in some way. Thus, differences attributed to viruses may possibly be also attributed to natural genetic variation within the fungus.

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Chapter 7. General discussion

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The first formal description of brown rot fungus of stone fruit was published in Europe over 220 years ago (Persoon, 1796). Today, brown rot disease caused primarily by M. laxa, M. fructigena and to a lesser extent by Monilia polystroma (Van Leeuwen et al., 2002) is responsible for severe losses in commercial and domestic crops of Prunus species (family Rosaceae), including Prunus persica (peach and nectarine), P. armeniaca (apricot), P. domestica (plum), P. avium (sweet cherry) and P. cerasus (sour cherry), P. serotina (black cherry), P. amygdalus (almond) and some ornamental species. These pathogens have a narrow host range, mainly being confined to Prunus, but occasionally also affecting Malus, Pyrus, Rosa, Chaenomeles and Cydonia species (family Rosaceae), and even Vitis vinifera (family Vitaceae) (Petroczy et al., 2012). Thus, it is likely that the (mainly) stone fruit-infecting Monilinia species co-evolved with their hosts over millennia in the regions of Eurasia, Africa and the Americas where species within Prunus evolved (Bortiri et al., 2001; Lino et al., 2016).

Incursion

Until recently, Western Australia was one of very few stone fruit production regions in the world free of Monilinia, so it was a terrible blow to the industry when M. fructicola and M. laxa were discovered there for the first time in 1997. At that time, an enormous effort was made by the Western Australian State Government authorities to eliminate the fungus from the incursion sites at Donnybrook and Manjimup in the south-west. This effort failed, and subsequently the fungus was detected throughout the region. In the intervening two decades, very little research had been done to understand the pathogen in its new environment, other than the collection of a few isolates by the W.A. Department of Agriculture, now known as Department of Primary Industries and Regional Development (DPIRD). Therefore, the overall aim of this research project was to fill some of the gaps in understanding about the biology of M. fructicola and M. laxa in W.A., until then the only species thought to be present.

Identity and distribution

One of the first activities of this project was to make a collection of Monilinia isolates from infected stone fruit trees across all of the main commercial production areas in W.A. Analysis of

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these samples revealed that two species of Monilinia were present in W.A., M. fructicola and M. laxa. Surprisingly, the two species were differentially distributed; M. fructicola being the sole species in sites surveyed to the north of Perth city, and M. laxa being the sole species at sites surveyed in the south. The southern region was where the M. fructicola incursion was first reported.

The northern stone fruit production region of Gingin records an average annual minimum temperature of 13.6oC, and average annual maximum of 27.9oC. The southern region of Donnybrook records an average annual minimum of 9.8oC, and average annual maximum of 23.1oC (BoM, 2018). We initially considered that average temperature preferences may account for the observed distribution pattern. Recent findings by a group from Brazil (Angeli et al., 2017) seemed to support this hypothesis. They compared conidial germination and lesion formation of both Monilinia species under a range of temperature regimes. The optimum temperature for M. fructicola conidial germination was 24.5oC, while for M. laxa it was 19.8oC. These authors also showed that M. fructicola isolates produced significantly more conidia at 30oC than did those of M. laxa, whereas M. laxa isolates produced significantly more conidia at 10oC than did M. fructicola. Similarly, the optimum temperature for lesion development was greater for M. fructicola than for M. laxa. These findings indicated that M. fructicola preferred slightly warmer conditions and M. laxa preferred slightly cooler conditions, and that these temperature preferences may explain the distribution pattern of Monilinia species in Western Australia.

However, a subsequent collection of Monilinia isolates from two peach orchards only about 1 km apart at Kirup, located in the southern region 18 km from Donnybrook, put this hypothesis in doubt. On one orchard, only M. laxa was detected, and on the other, only M. fructicola was detected. This situation raised two important points. It revealed that M. fructicola was capable of surviving in the relatively cool conditions of the southern region, and therefore was not restricted to the northern region by temperature constraints alone. The other point was that natural spread by conidia was likely to be less than one km. If wind-borne conidia were able to travel one km, both species would probably exist on both orchards. Although spores of some fungi travel thousands of kilometres on air currents (Nagarajan and Singh, 1990), previous studies with Monilinia showed conidia could travel naturally only tens to hundreds of metres (Kable, 1965;

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Cox and Scherm, 2001). Together, these findings indicated that distribution of the two Monilinia species within Western Australia was unlikely to be a product of natural long-range distribution of spores by the wind with subsequent selection by climatic factors. Instead, the Monilinia distribution pattern probably reflects inadvertent transport and placement of infected trees, tools, fruit bins, pallets, etcetera, by humans.

New incursions and genetic diversity

Following the first report of incursion of Monilinia into W.A, fruit retailers lobbied the W.A. State Government to relax the laws that prohibited stone fruit from being imported into the State. Changes to the regulations governing importations of stone fruit to W.A. since 1997 have progressively allowed different species of stone fruit to enter the State from various eastern States of Australia (2001 - 2004), from New Zealand (2003 - 2006) (Biosecurity Australia, 2006) and from the USA (2013) (Biosecurity Australia, 2010; Boothroyd, 2013).

Imports have clearly disadvantaged local stone fruit growers in the market place, because they have on occasion been forced to dump fruit as retailers sourced cheaper supplies from elsewhere (Sparvell, 2016). However, it is unclear that whether imported fruit was a route by which new Monilinia genotypes could ‘hitch hike’ into W.A. and become established on Prunus there. The basis of adaptation to environment, including fungicide application, is genetic diversity. In order to visualise the diversity of genotypes existing in the state, we required a method of determining intra-specific diversity. The ITS region of the nuclear ribosomal repeat unit provided sufficient phylogenetic inference to distinguish the species M. fructicola from M. laxa (Nilsson et al., 2008), but the resolution of this approach was too low to identify variability within each species. We used six ISSR markers on isolates of both species that had been reported to identify genotypic variation in M. fructicola (Luo and Schnabel, 2008; Fan et al., 2010). ISSR markers are polymorphic, multi-allelic, co-dominant and robust because they are PCR-based (Gobbin et al., 2003). We also tested RAPD markers to assess genetic diversity, but these gave less reproducible banding patterns so we discontinued this. We obtained cultures in sealed glass vials of the incursive Monilinia isolates collected by the Department of Agriculture from 1997 onwards as the fungus spread north from Donnybrook. Many of the isolates in this collection were not viable, yet some were and DNA from these and several of the dead ones was able to be

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purified and amplified for ISSR analysis. When compared with recently-collected isolates, it was clear that the incursive isolates of M. fructicola collected in 1997 were all of the same genotype, indicative of a single incursion of this species at the time. Further, an isolate of M. fructicola collected from South Australia in the same year was of the same genotype, pointing to South Australia as the possible source of the W.A. incursion. Surprisingly, the incursive M. fructicola genotype was not detected amongst the recent collection of isolates from numerous locations, but another six genotypes (haplotypes) were present. Notably, ten genotypes of M. fructicola were reported from Europe a decade after incursion (Jansch et al., 2011). If the original W.A. incursive isolate was no longer present, what was the origin of the other six genotypes? The original sources of these apparently recent invasive genotypes were not determined, but a M. fructicola-infected peach imported from the USA showed that imported fruit could be a source of new isolates into W.A. Similarly, stone fruits in Switzerland that were infected with M. fructicola were imported from the USA (Bosshard et al., 2006). Incursion of M. fructicola into Europe occurred at approximately the same time as incursion into W.A. Analysis of European isolates using five SSR markers suggested that there were two invasion events, both originating from the USA.

It is unclear how imported fruit isolates destined for eating within a few days of sale could become established on Prunus trees in commercial orchards. Clearly, more research into how fruit importation may facilitate invasion by new Monilinia genotypes is urgently required. The first report of M. fructicola from Hungary was on fresh peaches imported from Italy and Spain in 2005 (Petroczy and Palkovics, 2006), although its establishment there had not been established by 2012 (Petroczy et al., 2012), revealing that presence alone does not predicate establishment. The importation of Prunus budwood as germplasm is another potential route by which Monilinia could enter the State, and one we did not investigate. Little information is available on the source, amount and frequency of budwood importations by nurseries and growers, and yet this is potentially a more probable entry route than fresh fruit because the ultimate destination of budwood is orchards.

In contrast to the apparent extinction in W.A. of the incursive M. fructicola genotype from Donnybrook, the incursive M. laxa genotype, originally misidentified as M. fructicola, was

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widespread in the State. There were also several other genotypes of M. laxa present in our current isolate collection, presumably entering W.A. through the same (unknown) route(s) by which M. fructicola entered.

Responses to fungicides

Before Monilinia arrived in W.A., fungicides were not routinely applied to commercial and domestic Prunus trees. Other fungal pathogens of minor importance occurred in W.A., including Venturia carohila causing fruit freckle and Taphrina deformans causing leaf curl (Hoffmann et al., 2005). These were controlled by application of fungicides as required. After brown rot became established in W.A., all commercial growers were recommended to routinely apply fungicides to protect their crops. In years where there was high rainfall in spring and summer, uncontrolled brown rot could destroy all or most of the crop. Nevertheless, a few isolated semi- commercial ‘organic’ stone fruit orchards and many home gardens still exist where fungicides are not sprayed on trees. The reason for not spraying is often given as perceived risk of the chemicals to human health. In these small-scale enterprises, some measure of control of brown rot is achieved by manual removal of affected fruit to prevent spread by conidia. There are no previous studies comparing fungicide sensitivities of Monilinia isolates (or other fungal species) in both sprayed and unsprayed fruit orchards. Research in vineyards that either had a history of spray with two DMI fungicides or had never been sprayed with them showed that Uncinula necator (powdery mildew of grapes) isolates from unsprayed vineyards had lower EC50 values than did those from sprayed vineyards (Savocchia et al., 2004). An aim of this project, therefore, was to compare the tolerances to fungicides of Monilinia isolates collected from sprayed and unsprayed trees. Isolates were collected from unsprayed properties, and tolerances to three commonly-applied fungicides were compared with isolates collected from sprayed properties. It wasn’t surprising that unsprayed isolates were generally more susceptible (had lower EC50) to the three fungicides than were the sprayed isolates, although tolerant and susceptible genotypes occurred at both sprayed and unsprayed sites. The original source of all W.A. Monilinia genotypes, irrespective of the fungicide regime they currently find themselves in in W.A., is likely to be from imported fruit or budwood that has been subject to fungicidal sprays. Thus, the differential frequencies of susceptible genotypes on unsprayed trees and tolerant genotypes on

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sprayed trees appears to be a function of relatively recent (22 years or less) selection in W.A. If this hypothesis is correct, two conditions should exist: i. Isolates displaying a range of responses to fungicides (genotypes) exist at sprayed sites. (We showed this to be the case). ii. Maintenance of fungicidal tolerance is costly to the fungus. When fungicides are no longer applied, as at unsprayed sites, selection favours susceptible genotypes, which become more prevalent. We hypothesise that three possible factors may support the existence of susceptible genotypes under sprayed regimes. They are: i. There is inefficient and incomplete spray coverage and susceptible isolates escape exposure. ii. The mycelium penetrates Prunus tissue where fungicide cannot access it, and susceptible isolates thereby escape exposure. iii. Conidia of susceptible isolates are more tolerant to fungicides than is the mycelium, and spores germinate after the fungicide has broken down.

Thus, tolerant and susceptible genotypes under different fungicide regimes may persist by different mechanisms. Investigating these hypothesises is a potential area of future research.

Regardless of spray regime, we found that M. fructicola isolates were on average more susceptible to propiconazole than were M. laxa isolates, while average responses to iprodione and fluopyram + trifloxystrobin were similar for both species. Monilinia fructicola isolates displayed a smaller range of sensitivities than did M. laxa isolates, and this was in contrast to findings of Hrustić et al. (2018) who highlighted that Serbian M. fructicola isolates displayed a far greater range of sensitivities than did M. laxa isolates. These workers also found that M. fructicola were more tolerant to iprodione and other fungicides than M. laxa isolates were, which was not our experience.

It is unknown if sexual reproduction occurs in Monilinia populations in W.A.. Sexual reproduction occurs rarely in M. laxa, and is observed more often in M. fructicola, notably in California (Michaillides et al., 2007) and possibly Europe (Jansch et al., 2011). California

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experiences a Mediterranean-type climate similar to that of the stone fruit production regions of W.A., and so it is possible sexual reproduction occurs there. We did not mount a search for physical (apothecia) or molecular evidence of sexual reproduction of M. fructicola in W.A., although it is important to know if it occurs there. Its importance centres around genetic variation, the raw material of adaptation to fungicides and other stressors. In other places, M. fructicola isolates have developed tolerance to a number of fungicide groups, including the benzimidazole carbamates (Ma et al., 2003; Lim et al., 2006), the DMIs including propiconizole (Schnabel et al., 2004; Luo and Schnabel, 2008) and respiration inhibitors such as azoxystrobin (Amiri et al., 2010). Ongoing incursions of isolates combined with sexual activity could recombine fungicide tolerance genes into single isolates, creating very problematic strains with multiple fungicide tolerances. Although some evidence of multiple fungicide tolerances was detected by our experiments, it was not common.

Knowledge of the fungicide response spectrum of Monilinia isolates within an orchard or a region is important in devising control strategies. Our studies suggested that application of fungicides did not eliminate Monilinia from orchards, either because of tolerance to the fungicide, or because of escape/avoidance from the fungicide as discussed above. Thus, strategies are required to manage the strains present, and prevent new strains from establishing that may have different fungicide tolerances and different pathogenicities. The availability of cheap and rapid assays to identify tolerances to certain groups of fungicides would be an extremely valuable resource for growers, but as yet they do not exist for Monilinia, even though the basis of tolerance to several fungicidal groups is known. For such tests to be PCR-based, a detailed knowledge of the genetic basis of tolerance would need to be determined (Lendemann et al., 2015).

A frequently-voiced criticism by commercial stone fruit growers of ‘organic’ (no spray) growers and home gardeners located nearby is that the spores carried from infected fruit on these properties will contaminate their orchards. We showed that although this may happen where untreated trees are located directly adjacent, isolates from unsprayed trees are more likely to be susceptible to the fungicides sprayed routinely by commercial orchardists. However, this

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scenario is not definitive as some tolerant isolates were present on unsprayed orchards, and vice versa.

Disease

Besides fungicide tolerance, virulence and pathogenicity are other genetically-controlled traits that are of great interest to growers. Virulence and pathogenicity are terms that are often used interchangeably. Virulence describes the ability of the organism to cause infection and multiply within the host, while pathogenicity describes the extent of damage caused by the organism. Proteins controlling virulence of M. laxa isolates from apricot and apple showed that host preference to apple but not apricot may be controlled by acetolactate synthase and other mycelial proteins (Bregar et al., 2012). Within isolates, pathogenicity was stable, but between them it varied widely, indicative of a genetic basis to pathogenicity. When single-spore-derived colonies were grown on nutrient agar plates, there was a wide range of morphologies displayed by the colonies. Subsequent inoculations of isolates to plum fruit indicated there was considerable diversity in pathogenicity between isolates of both Monilinia species. The small number of ISSR markers we employed to gauge genetic diversity did not correlate with closely with pathogenicity, indicating they were not linked with pathogenicity-related genes.

Viruses and virulence

Fungal virulence is modulated in some fungi by the presence of mycoviruses. Until this study, no virulent viruses had been identified from Monilinia species. Three mycoviruses were described after testing a subset of the Monilinia isolates collected, showing that both M. fructicola and M. laxa are hosts of viruses. All three viruses found in this study had all been described previously from related fungi in family Sclerotineaceae. The method we used to isolate the RNA before sequencing was based on the affinity of dsRNA to cellulose powder in the presence of low concentrations of ethanol. However, previous experience in our laboratory had shown that the method was suitable for extracting all large RNA (ssRNA and dsRNA) and DNA molecules, and its main value was that smaller RNA groups such as ribosomal and transfer RNA were largely eliminated. Two of the three viruses identified has ssRNA genomes, so we think the apparently low numbers of viruses present was not because the extraction method used eliminated ssRNA

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viral genomes. DNA-based mycovirus are apparently uncommon; there being only one identified so far (Yu et al., 2010), and none were detected.

Isolate M196 of M. fructicola was co-infected with all three viruses. Curing it of one or more mycoviruses was achieved in some cases. Isogenic virus-cured fungal lines were then compared with the parent virus-infected M. fructicola M196 in term of mycelial growth in vitro and virulence on cherry fruits. The results presented that the virus-infected isolate grew faster than virus-cured isogenic lines, indicating all that three mycoviruses together enhanced the fungal growth. However, each virus exposed a different influence on the fungal host when they are alone in the fungal host. For instance, SsHV2 increased fungal growth while BVF had a slight inhibitory effect, and FPV1 did not influence growth. In contrast to on artificient nutrient media, on cherry fruits, these mycoviruses did not influence virulence of M. fructicola.

This is the first time mycoviruses have been identified to infect Monilinia. Noticeably, the original fungal hosts of these mycoviruses were of Sclerotinia sclerotiorum, Fusarium poae and Botrytis cinerea, which are member of famly Sclerotiniaceae where M. fructicola belongs to. Those fungi were found to be infected by the described mycoviruses outside of Australia, revealing that these mycoviruses may come and infecte Monilinia in Austraia through international transfort of mycovirus-infected fungal pathogen. For example, SsHV2 was identified to be associated with hypovirulence of S. sclerotiorum on canola (Brassica napus) in China (Hu et al., 2014), FpV1 identified in Japan (Osaki et al., 2016), BVF was the first virus identified from a Botrytis cinerea isolate collected on strawberries in New Zealand (Howitt et al., 2001).

Only 14% of both Western Australian Monilinia species tested were infected with one, two, or three mycoviruses. This is in contrast with the other study on M. fructicola isolates collected in New Zealand, which showed 76% of M. fructicola isolates infected with at last one mycovirues (Tsai, 2004). The influences of those mycoviruses on M. fructicola were of interest. SsHV2 in our study seemed to have a stimulated effect of mycelial growth of M. fructicola, in contrast with SsHV2 that induced hypovirulence of S. sclerotiorum on canola in China (Hu et al., 2014). Unfortunately, we were unable to generate a line containing only SsHV2 to confirm these

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findings. It is probable that interactions of SsVH-2 with the other two viruses co-infecting M196 influenced the observed effects on the host. In M. fructicola, similar as in F. poae (Cho et al., 2013), FpV1 doesn’t appear to induce abnormal morphology or changes to virulence, and this is a common observance for partitiviruses. BVF was the first virus identified from Botrytis cinerea, from an isolate collected on strawberries in New Zealand (Howitt et al., 2001), and from B. cinerea associated with grape leaves in South Africa (Al Rwahnih et al., 2011) with no further study of its influence on the Botrytis host. This is its first report from Australia on Monilinia although B. cinerea is widespread there (Isenegger et al., 2008). There is some evidence that the presence of BVF slightly reduced M. fructicola growth in vitro. That BVF may negatively impact growth of M. fructicola is of interest because both B. cinerea and M. fructicola are important plant pathogens. The possibility that BVF induces hypovirulence should be studied further.

A vision of the future

Pathogens of agriculture and horticulture threaten the food and feed supply that sustains lives and stabilises civilisations. Globalisation of the food supply has immense potential benefits in minimising the risk of food shortages, but can also facilitate movement of pathogens beyond their centres of diversity. Despite the best efforts of international biosecurity authorities to stop pathogens from crossing borders, they do so, as was the case of two Monilinia species in Western Australia. There has been no official report about the yield loss caused by brown rot in the State; research should be done to quantify this in future.

Environment, including climatic conditions influence the prevalence and severity of brown rot. Controlling brown rot in the field will continue to be very challenging when humidity is high during fruit development and harvesting periods. It is probable that global warming will make controlling Monilinia even more problematic and expensive in future. Monilinia is clearly capable of developing tolerance to fungicides applied to control it, and so new fungicides are required in the short term. In the longer term, non-fungicidal control strategies are needed to manage the viability of the stone fruit industry. We did not investigate the efficacy of biological control agents in controlling brown rot, but one potential bacterial control agent was

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serendipitously identified during the course of this project. A number of biological control agents of Monilinia are reported (Bonaterra et al., 2003; Larena et al., 2005; Yanez-Mendizabal et al., 2012). Monilinia strains will undoubtedly develop tolerances to these biological agents too, but we see this as the most potentially valuable area of further research into these pathogens. We envisage biological control agents and other non-chemical approaches becoming more important in the control of brown rot, especially post-harvest. Therefore, we encourage future research in controlling Monilinia in fruit crops towards the novel strategies.

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