Detection, Assessment, and Management of Phytophthora species in an Urban Forest

by Mohammed Y. Khdiar (MSc)

This thesis is submitted for the degree of Doctor of Philosophy

School of Biological Sciences and Biotechnology

Murdoch University

Perth, Western Australia

2018

I

Declaration

I declare that this thesis is my own account of my research and contains as its main content work which has not previously been submitted for a degree at any tertiary education institution

Mohammed Y. Khdiar

July 2018

II

Abstract

Urban forests contribute to human well-being and environmental health and function, and this role will increase as urbanization continues to grow. Due to anthropogenic activities, urban forests are considered a suitable starting point for invasive pathogens. Members of the genus Phytophthora are important pathogens involved in tree and forest declines worldwide. To date, there are approximately 160 formally described Phytophthora species, with 50% of these species described in the last decade. There are many factors contributing to these recent descriptions, including introductions from the nursery trade, increased studies in forests and natural environments, and an increase in the number of Phytophthora scientists globally. Improved molecular tools to detect and identify Phytophthora species (as like other genera of the oomycetes) has also had a profound effect on the increased number of species that have recently been described. Within urban forests, trees are under considerable stress from polluting agents, mechanical damage, and other human activities and are often in sub-optimum health and are thus more vulnerable to pathogens such as Phytophthora.

Two hundred and thirty-six soil and root sites were sampled from declining native Australian trees in 91 parks and nature reserves in the City of Joondalup, Perth Western Australia. Metabarcoding DNA detected forty-five Phytophthora species. Phytophthora multivora was isolated the most frequently while P. cinnamomi was the fifth most frequent. Seven species P. capsici, P. sp. pecan, P. fluvialis, P. gonapodyides, P. sp. walnut, P. erythroseptica and P. fallax were isolated only once. In contrast, only P. nicotianae, P. multivora, P. boodjera and P. arenaria were isolated into the culture based on rhizosphere baiting. A range of environmental factors was examined to determine if Phytophthora communities were related to specific environmental factors. The effect of environmental factors ranged from no effect (land class, plant diversity, park type, soil type, soil unit and canopy health) to a slight effect (canopy cover and park size) on the overall Phytophthora community. When looking at the incidence of P. cinnamomi, park type was significant, while for P. multivora, soil type, soil unit, and park size were significant drivers.

III

A pathogenicity test was conducted on excised branches from 15 tree species underbark inoculated with 19 Phytophthora species. All Phytophthora species formed lesions in Eucalyptus marginata and Corymbia calophylla, while Fraxinus excelsior was resistant to 7 of the 19 Phytophthora species. Six Phytophthora species were pathogenic to all 15-tree species, whereas Phytophthora versiformis formed lesions in only three tree species. Phytophthora cinnamomi, P. pseudocryptogea, and P. citrophthora were the most pathogenic species causing the largest lesions in most of these tree species. These results show that Phytophthora species potentially play an essential role in the declining health of urban trees and indicate the importance of managing Phytophthora in urban forests.

Two chelate complexes [Zn (Val)2(bipy)] and [Ca(Val)2(bipy)] were developed and screened for their potential as fungicides to control Phytophthora species. These complexes were examined using three spectroscopic methods (X-ray crystallography, ultraviolet rays–visible spectra and Fourier-transform infrared spectroscopy). The complexes were pseudo-octahedral in structure, and both complexes had the same patterns of crystal structure symmetry. The yield of the calcium chelate and zinc chelate of basic materials were 81% and 50%, respectively. The activity of these complexes with or without the addition of phosphite against P. cinnamomi in vitro and in planta was examined. In vitro, the effect of the chemical treatments was assessed by measuring the EC50 of mycelial biomass in a liquid medium with 0-160 µg/ml (0-0.16g/l) of each complex. Phytophthora cinnamomi was highly sensitive to the Ca chelate alone and Ca chelate + phosphite, but not to the zinc chelate. Two plants species Banksia grandis and Eucalyptus marginata were sprayed with three concentrations (0, 0.25 and 0.5%) of each chemical treatment (phosphite, Ca chelate, Zn chelate and Ca chelate + phosphite) and were inoculated with P. cinnamomi together with a non-inoculated control. Ca chelate significantly reduced lesion development of P. cinnamomi compared to phosphite alone. Whilst Ca chelate (0,25%) + phosphite (0,25%) gave the highest improvement in reducing the effects of P. cinnamomi in both plant species. None of the Zn chelate applications were effective. The Ca chelate has the potential to be used as a fungicide to control Phytophthora species. These results suggest that Ca chelate may be a suitable alternative to phosphite to control Phytophthora species.

IV

Table of contents 1 Chapter One ...... 1 Literature Review ...... 1 1.1 General Introduction ...... 2 1.2 Why are urban forests important? ...... 3 1.3 Tree Decline Overview ...... 4 1.4 The role of Phytophthora in tree declines ...... 5 1.5 The effect of predisposing and inciting factors on trees decline caused by Phytophthora species ...... 6 Predisposing factors ...... 6 Inciting factors ...... 9 1.6 New technologies to study urban tree health ...... 11 High-throughput sequencing ...... 11 Remote sensing ...... 12 1.7 Management of Phytophthora species with chemical applications ...... 13 1.8 Status of knowledge of Phytophthora species associated with urban forests in Perth, Western Australia ...... 15 Perth, Western Australia ...... 15 Urban forests as an initial point of spread for Phytophthora species ...... 15 1.9 Summary ...... 16 1.10 Thesis Aims ...... 17 2 Chapter Two ...... 18 Association of Phytophthora with declining vegetation in an urban forest environment ...... 18 2.1 Abstract ...... 19 2.2 Introduction ...... 20 2.3 Materials and Methods ...... 22 Study area and sample collection ...... 22 Baiting technique ...... 24 eDNA extraction from fine roots and metabarcoding ...... 24 Accompanying data ...... 25 Data analysis ...... 26 2.4 Results ...... 27 V

Isolation by baiting ...... 27 Phytophthora species detected from fine root eDNA ...... 27 Phytophthora community ...... 29 Distribution of Phytophthora cinnamomi and Phytophthora multivora ...... 31 2.5 Discussion ...... 38 Phytophthora community ...... 38 2.6 Conclusion ...... 45 3 Chapter Three ...... 46 Phytophthora species associated with declining urban trees ...... 46 3.1 Abstract ...... 47 3.2 Introduction ...... 48 3.3 Materials and Methods ...... 50 Phytophthora isolates ...... 50 Plant material ...... 50 Inoculum production and inoculation ...... 51 Harvest and measurement ...... 52 Statistical analysis ...... 52 3.4 Results ...... 54 3.5 Discussion ...... 56 4 Chapter Four ...... 59 Synthesis, and characterization studies of Ca (II) and Zn (II) mixed-ligand complexes containing the amino acid L-valine and 2,2- bipyridine ...... 59 4.1 Abstract ...... 60 4.2 Introduction ...... 61 4.3 Materials and Methods ...... 63 Preparation of Ca chelate ...... 63 Preparation of Zn chelate ...... 63 X-ray crystallography (XRD) ...... 64 Ultraviolet rays–visible spectra ...... 64 Fourier-transform infrared spectroscopy (FTIR) ...... 64 4.4 Results ...... 64 Synthesis of Metal Complexes ...... 64 VI

Structural Characterization of Metal Complexes ...... 66 4.5 Discussion ...... 70 5 Chapter Five ...... 71 The potential control of Phytophthora cinnamomi in vitro and in planta using calcium and zinc chelate with or without phosphite ...... 71 5.1 Abstract ...... 72 5.2 Introduction ...... 73 5.3 Materials and Methods ...... 74 The effect of phosphite and new chelates on Phytophthora cinnamomi in vitro ...... 74 The effect of phosphite and new chelates on Phytophthora cinnamomi in planta ...... 75 Statistical analysis ...... 76 5.4 Results ...... 77 The effect of phosphite and new chelates on Phytophthora cinnamomi in vitro ...... 77 The effect of phosphite and new chelates on Phytophthora cinnamomi in planta ...... 80 5.5 Discussion ...... 84 5.6 Conclusion ...... 86 6 Chapter Six ...... 87 General Discussion ...... 87 6.1 Findings of Thesis ...... 88 6.2 Phytophthora community ...... 89 Urban forest pathway of Phytophthora species ...... 89 Phytophthora community and environmental filtering ...... 94 6.3 Phytophthora species is associated with urban tree declines ...... 94 Phytophthora multivora and Phytophthora cinnamomi ...... 94 Species of Phytophthora ITS Clade 6 ...... 96 Other Phytophthora species detected ...... 97 New Phytophthora species in Australia and Western Australia ...... 98 6.4 Management ...... 99 6.5 Future Research ...... 100 6.6 Conclusions ...... 101 References ...... 102 Appendix A ...... 135 VII

List of abbreviations bipy 2,2′-bipyridine

Ca Calcium

CoJ City of Joondalup

COX Mitochondrially inherited cytochrome oxidase eDNA Environmental DNA

FTIR Fourier-transform infrared spectroscopy

HTS High-throughput sequencing

ITS Internal Transcribed Spacer

K2HPO3 Potassium phosphite

NDVI Normalized Difference Vegetation Index

UHI Urban heat island

UV-Vis Ultraviolet rays–visible spectra

Val Valine

XRD X-ray crystallography

Zn Zinc

VIII

Acknowledgments

I would like to thank my three supervisors Prof. Giles Hardy, Associate Prof. Treena Burgess and Dr. Paul Barber for their guidance, enthusiasm, and encouragement throughout my time at Murdoch.

I would like to acknowledge the input of all my colleagues over the years.

I would also like to thank Diane White for advice and technical help with experiments especially for Chapter 2; Briony Williams and Chris Shaw with the collection of samples of experiment of Chapter 2; Emma Steel and Cameron McMains for help with statistical analysis in Chapter 2; Peter Scott with statistical analysis assistance in Chapter 3; Bill Dunstan and Ishan Khaliq for assistance with the collection of stems of experiment of Chapter 3; and Rajah Belhaj for technical advice with Chapter 5.

Finally, I ‘d like to thank my long-suffering family who has stoically endured the ups and downs of my successes and failures and encouraged me through the Ph.D. process.

I want to thank The Higher Committee for Education Development in Iraq for my scholarship.

IX

1 Chapter One

Literature Review

1

1.1 General Introduction

Urban forests are considered an important extension of natural ecosystems (Alvey, 2006, Dobbs et al., 2011, Dobbs et al., 2014, Stagoll et al., 2012). As the world’s population increases, urban forests will play increasingly important roles in biodiversity conservation (Alvey, 2006). Urban forests also reduce air pollutants (Nowak et al., 2006, Grote et al., 2016) and contribute to the enhancement of human health and welfare (Kuo, 2003a, Donovan et al., 2013). Unfortunately, urban forests are exposed to numerous biotic and abiotic threats which impact on their health and ecosystem functions and services.

There are many different kinds of pathogens that contribute to the destruction of urban and peri- urban forests (Poland and McCullough, 2006, Tubby and Webber, 2010). Of particular interest, and importance to the current study, are Phytophthora species. They cause a variety of diseases in numerous plant species in urban ecosystems (Jung et al., 2016, Burgess et al., 2009, Barber et al., 2013, Dale et al., 2017). In some cities, Phytophthora species are commonly associated with declining urban trees (Barber et al., 2013). These declines further impact on plant communities, the invertebrate and vertebrate fauna and microflora that depend on these plant species and communities (Garkaklis et al., 2004). However, very few studies have been undertaken in urban ecosystems on the impact of Phytophthora on the health of trees and the animals they support.

In urban forests, trees experience even more stress than in natural forests, due to planting density, poor rooting, compaction, increased UV exposure, the urban heat island effect, species planted in inappropriate environments, excess irrigation and nutrient inputs. Consequently, it is important to understand the interaction of Phytophthora species with predisposing and inciting biotic and abiotic factors.

To maintain the benefits trees provide to the urban environment, it is important to manage Phytophthora species and other plant pathogens in urban forests. New technologies such as molecular and remote sensing techniques can assist in the rapid detection and identification of Phytophthora species and to monitor their rate of spread and impact. Management can include the use of chemicals (Gonzalez et al., 2017a) such as phosphite, an important chemical commonly

2

used to control Phytophthora species and the diseases they cause (Hardy et al., 2001). Additionally, clean nursery stock, cultural techniques and improved hygiene and quarantine measures can prevent both the introduction and the spread of pathogens into and between urban forest landscapes and beyond.

1.2 Why are urban forests important?

Over half of the world’s population live in urban regions, and it is thought that the ratio will gradually increase (World Health Organization, 2014), with more than two-thirds expected by 2050 (World Health Organization, 2018), leading to more buildings and increased demands on green space. The higher temperatures experienced within urban areas compared to the neighboring rural areas is called the urban heat island (UHI) effect. The UHI is due to the increase in non-photosynthetic surfaces such as buildings, pavements, and roads, as a result of urbanization. The accumulation of these non-photosynthetic surfaces (1) absorb more radiation from the sun and reduce the level of evapotranspiration per unit area (Oke, 1987, Waffle et al., 2017), and (2) reduce wind movement at the ground level which leads to the heated air not dissipating as quickly (Oke, 1987, Waffle et al., 2017). Urban forests and urban greenspaces help mitigate the UHI effect and also improve air quality within a city (Solecki et al., 2005) through purification of the air (Rosenzweig and Solecki, 2001). They also store CO2 which helps to reduce global warming (Rosenfeld et al., 1998). Urban forests also have very important positive impacts on the physical and mental health of urban dwellers (Velarde et al., 2007, Grinde and Patil, 2009, Lafortezza et al., 2009, Paquet et al., 2013, Dinnie et al., 2013).

Urban forests also play a significant role in the maintenance of biodiversity (Balmford et al., 2001, Jim and Liu, 2001, Godefroid and Koedam, 2003, Cornelis and Hermy, 2004, Kühn et al., 2004). A survey of 15 urban parks in Flanders, Belgium illustrates this; these parks contained approximately 30, 50, 40, and 60% of the total number of native trees and shrubs, birds, butterflies, and amphibians, respectively, that were found in the country (Cornelis and Hermy, 2004). Other key benefits of urban forests include an increase in people’s feelings of happiness and comfort through urban greening (Kuo, 2001, Kuo, 2003b), the provision of multiple

3

ecosystem services (McPherson, 1994, Costanza et al., 1997), diminished energy consumption (McPherson, 1994), mitigating flooding events (Bolund and Hunhammar, 1999), and reducing the number of airborne particulates and other pollutants (Nowak, 1994, Freer-Smith et al., 2004, Escobedo et al., 2008).

1.3 Tree Decline Overview

Forest decline has been described as the gradual failure in the health of trees irrespective of the cause (Manion, 1991). In general, forest decline is “an interaction of interchangeable, specifically ordered abiotic and biotic factors that produce a gradual general deterioration, often ending in the death of trees” (Manion, 1991). Symptoms of decline include loss of foliar biomass, loss of feeder root biomass, and decreased annual growth (in height and diameter); the metabolic response to stress is the “breakdown and mobilization of nitrogenous compounds and translocation of nitrogen from stressed tissue” (Hain, 1987). All of these symptoms result in a general loss of tree vigor. In fact, declines not only affect forest trees and shrubs but also affect other organisms that depend on the forest ecosystems for their survival (e.g., for shelter and food). Tree decline is not limited to one area of the world; decline has been documented on most continents (Allen et al., 2010). The factors contributing to tree declines are numerous and there tend to be interactions between these factors. According to Manion (1991), a common complex causal relationship between many factors such as climate conditions, pathogens, and human activities are responsible for forest decline. These factors are grouped into predisposing, inciting, and contributing factors (Fig 1.1) (Manion, 1991). The loss of forests due to agriculture, horticulture, forest plantation monocultures and urban development make it essential for the need to preserve and maintain healthy trees in urban landscapes. Unfortunately, while urban forests are obviously very important for the environment and biological conservation, they are under more stress than natural ecosystems as anthropogenic activities both biotic and abiotic add additional stresses to trees that are otherwise not present in natural ecosystems.

4

Fig 1.1 The disease decline spiral from Manion (1991)

1.4 The role of Phytophthora in tree declines

One of the most important contributing (Orlikowski et al., 2011) to tree declines worldwide are diseases caused by pathogens in the genus Phytophthora. Once an invasive Phytophthora species arrives in a novel ecosystem it can cause significant losses to plant species and communities over large geographic areas. Phytophthora ramorum has destroyed huge areas of Californian forests (Werres et al., 2001, Rizzo and Garbelotto, 2003, Hansen et al., 2005), and is responsible sudden oak death epidemic in the urban environment(Garbelotto et al., 2001, Rizzo et al., 2002). Phytophthora ramorum is an introduced exotic pathogen to the USA, via the international nursery trade (Garbelotto et al., 2001, Rizzo et al., 2002).

Another introduced and invasive species is P. cinnamomi, it has an extensive host range of over 2000 plant species (Hardham 2005), and contributes to White Oak Decline (Quercus alba) in the

5

USA (McConnell and Balci, 2014). It has also destroyed huge areas of Australia’s forests and woodlands (Shearer et al., 2004). For example, in the south-west of Western Australia, out of the 6510 described plant species, approximately 41% are susceptible to P. cinnamomi (Shearer et al., 2004). In Australia, P. cinnamomi is recognized as a ‘Key threatening process to Australia’s Biodiversity’ by the Commonwealth Government’s EPBC Act 1999 (Shearer et al., 2004), because of its direct impact on plant communities and indirect effects on native fauna through the loss of food, refugia, and habitat. For example, in Australia 157 Banksia species are recorded to be a major source of food for many kinds of an animal such as honeyeaters, rodents, honey possums, pygmy possums, gliders and bats (Hackett and Goldingay, 2001, Wrigley and Fagg, 2013). Many Banksia species are threatened by Phytophthora species (Shearer et al., 2004, McCredie et al., 1985). The impacts of P. cinnamomi in urban forests are poorly understood but likely to be significant in green space where it is present.

The impact of Phytophthora diseases can have significant impacts on ecosystems, through declines in biodiversity (Garkaklis et al., 2004) and a decline in fundamental ecosystem services, such water equilibrium and river flow, modification of territorial climate patterns and carbon storage in biomass and soils (Foley et al., 2007, Alford and Richards, 1999).

1.5 The effect of predisposing and inciting factors on trees decline caused by Phytophthora species

Predisposing factors

Predisposing factors are climate or site factors that are long-term, static or slowly changing and render a tree vulnerable to a disease or disturbance (Worrall et al., 2010, Aghighi et al., 2014, Manion, 1991). Trees damaged by these factors may slowly recover (Worrall et al., 2010) if further stresses are not imposed on the system. Predisposing factors such as extreme weather and drought play an important role in increasing the susceptibility of trees to Phytophthora species (Corcobado et al., 2014).

6

Water availability

Water is a key determining factor of forest health and includes waterlogging and flooding. Too little or too much water can predispose plants to pathogens, leading to forest diseases (Doblas- Miranda et al., 2017). Waterlogging is the saturation of soil with water and can be exacerbated by poor drainage related to soil texture (De Silva et al., 1999, Bryla and Linderman, 2007), or in areas immediately adjacent to irrigation emitters (Cafe-Filho and Duniway, 1996). Waterlogging leads to low oxygen levels in soil, followed by anoxia or hypoxia and plant stress (Lambers et al., 1998, Niinemets and Valladares, 2006). There are few tree species that can tolerate long-term low oxygen concentrations and even these species produce little canopy under heavy stress conditions (Talbot et al., 1987, Kozlowski and Pallardy, 1991). Overall, soil saturation with water can predispose urban trees to infection by Phytophthora species. Corcobado et al. (2013) clearly showed how saturated soils were beneficial to P. cinnamomi, and disadvantageous to the tree leading to extensive root damage and death and decline of trees. Numerous other studies have shown tree declines linked with Phytophthora diseases are associated with waterlogging (Pratt and Heather, 1973, Ward and McKimm, 1982, Shearer and Smith, 2000, Weste, 2003). In addition, excess water assists in the dispersal of Phytophthora species (Baker and Matkin, 1978, Erwin and Ribeiro, 1996, Stanghellini et al., 1996a, Stanghellini et al., 1996b, Bush et al., 2003). Excess water through flooding can reduce the growth rate of trees in an urban area (Nash and Graves, 1993). How plants respond morphologically and physiologically to extreme flooding is a significant part of determining the susceptibility of plants to pathogens (Stolzy and Sojka, 1984).

Extreme weather

Recent climate models forecast greater fluctuations in a climate with increases in the frequency and severity of extreme events such as drought and heavy rains (Solomon, 2007, Kreyling et al., 2008). The direct harm of extreme weather events on trees appears clearly through alterations in phenology, morphology, genetic frequencies and abundance of plants (Richardson et al., 2013, Lindner et al., 2010, Lloret et al., 2004, Corcobado et al., 2014). Weather extremes will affect the growth and susceptibility of trees such as Quercus ilex to species such as P. cinnamomi

7

(Corcobado et al., 2014). Also, extreme weather events could help pathogens to increase the number of their life cycles per year (Corcobado et al., 2014).

Drought events are one of the major factors causing tree dieback, alongside high temperatures (Lloret et al., 2011). Frequent drought periods can lead to a loss of resilience by exhausting the capacity of trees to regenerate (Lloret et al., 2004), and thus may increase their susceptibility to pathogens. Indeed, the relationship between drought stress and resistance or susceptibility of plants to pathogens is complicated (Garrett et al., 2006b). Drought may increase plant susceptibility to pathogens, or under dry conditions, the success of infection may be lower (Garrett et al., 2006b, Huber and Gillespie, 1992). Irrespective, drought stress will predispose urban trees to disease development (Paoletti et al., 2001, Tubby and Webber, 2010). Together drought with Phytophthora species (e.g. P. cinnamomi) could lead to decline (Brasier, 1996). Corcobado et al. (2014) remarked that drought events increase not only the susceptibility of seedlings to infection by P. cinnamomi but also the mortality rates of these seedlings. Drought and flooding can go hand in hand; for example, P. quercina is able to cause significant damage to seedling roots under fluctuation between drought and flooding (Jung et al., 2003). Many studies reported that Phytophthora diseases developed with and after heavy rains (Choudhari et al., 2018, Das, 2009, Singh, 2002), they proved that the high peak period of disease expression was associated with heavy rainfall.

Extreme warming

Urban areas are generally warmer than adjacent rural areas, for example, the daily minimum temperature in New York City is on average ~7.2°F (~4°C) in summer months that makes it warmer than rural areas by an average 1.1°C (Rosenzweig et al., 2006). Elevated temperatures cause stress in trees and thus predisposes them to pathogens (Tubby and Webber, 2010). Elevated temperature stress can have profound health consequences for plants through wilting, leaf burn, leaf folding and abscission, and physiological responses including changes in RNA metabolism and protein synthesis, enzymes, isoenzymes, and plant growth hormones (Christiansen, 1982). These changes will surely influence susceptibility to pathogens (Chuine et al., 2000, Garrett et al., 2006a). Also, in the future, elevated temperatures are expected to have 8

a significant impact on the abundance and distribution of many tree species and also on the distribution and colonization by tree pathogens (Pautasso et al., 2012). With respect to Phytophthora species, the temperature is a determining factor for sporulation and subsequent infection of hosts by Phytophthora species (Zentmyer, 1981). For example, P. cinnamomi can infect avocado when soil temperatures range between 15 -27 °C but not at 33°C (Zentmyer, 1981). Also, optimal temperatures for sporangia producing by P. cinnamomi were between 26 °C to 30 °C while sporangia were not formed at 12 °C (Shearer, 2014).

Inciting factors

Inciting factors are short-term physiological or biological factors that can increase the susceptibility of plants to pathogens by causing severe stress (Worrall et al., 2010). Trees influenced only by inciting factors may recover rapidly (Worrall et al., 2010). Indeed, the impact of inciting factors which include defoliation and excessive salt, on urban forest decline is a central challenge in forestry and ecology. Inciting factors could enhance the occurrence of trees decline by Phytophthora species

Defoliation

Leaf defoliators have been deemed as causative factors of quick mortality in forest trees such as German oak trees (Thomas et al., 2002). The link between Phytophthora species and defoliation was examined by (Oszako et al., 2018, Jung et al., 2000). Jung et al. (2000) Indicate that there is possibly a synergistic effect of Phytophthora spp. and defoliation events on plants while Oszako et al. (2018) indicated defoliation can enhance the chances of P. plurivora to infect birch (Betula pendula).

Excessive salt

Salt can impact on tree growth by inhibiting chlorophyll synthesis through the direct effect on photochemistry reactions (Parida et al., 2004). Many studies reported that excessive salt enhances disease severity (Swiecki and MacDonald, 1991, Sulistyowati and Keane, 1992, Snapp et al., 1991). These studies proved that soil salinity enhances Phytophthora Root Rot caused by

9

P. parasitica in tomato (Swiecki and MacDonald, 1991, Snapp et al., 1991) and stem rot caused by P. citrophthora in citrus rootstocks (Sulistyowati and Keane, 1992).

Loss of mycorrhizal fungi and other microorganisms

Mycorrhizal fungi are considered one of the most important groups of microorganisms supporting the health of trees, as they help plants obtain nutrients and water through their mutualistic connection with roots of plants (Sapsford et al., 2017). Ectomycorrhizal fungi also reduce the severity of disease caused by Phytophthora species as they provide an effective barrier to Phytophthora infection (Marx, 1969, Zak, 1964, Barham et al., 1974, Branzanti et al., 1999). Watanarojanaporn et al. (2011) showed that root rot disease caused by P. nicotianae was reduced by arbuscular mycorrhiza. Similar results were found by Branzanti et al. (1999) when investigating the correlation between the severity of Q. robur decline and mycorrhizal colonization and they showed that forest decline increased when mycorrhizal colonization decreased.

In Australia, Ishaq et al. (2013, 2018) found a higher proportion of ectomycorrhizal fungi associated with soils and seedlings grown in soil collected from sites with healthy woodland E. gomphocephala (tuart) trees, and of arbuscular mycorrhizal fungi from soils and seedling roots grown in soil from sites with declining tuart trees. Scott et al. (2012) had previously found a relationship between the crown health, fine root and ectomycorrhiza density of declining tuart trees in the same sites through field exploration studies, with P. multivora recovered from declining trees at these sites and implicated in the premature decline of Eucalyptus, Agonis and Banksia (Scott et al., 2009).

There is also evidence for a link between microorganisms and Phytophthora species. Many studies describe an inverse relationship between P. cinnamomi and total microbial activity existing in soil (Broadbent and Baker, 1974, You and Sivasithamparam, 1994, Hardy and Sivasithamparam, 1991). Indeed, when there is low microbial diversity and numbers in a soil, Phytophthora species tend to be more pathogenic or the soils are more conducive to the activity of Phytophthora species as many are poor saprophytes, as has been shown for P. cinnamomi

10

(McCarren, 2006). For example, one of the reasons P. cinnamomi is so bad in WA is due to the prevalence of ancient soils which are not high in microbial activity compared to rich soils in Europe (outside Mediterranean regions).

1.6 New technologies to study urban tree health

High-throughput sequencing

One of the major challenges in studying the impact of root pathogens like Phytophthora is detecting them. With regards to Phytophthora species, various traditional methods have been employed to detect them and include different baiting techniques or isolation on Phytophthora selective agar media (Ribeiro, 1978). Until molecular techniques were available, isolates were described based on morphology that was occasionally ambiguous (Hardham, 2005, Stamps et al., 1990), as well as time-consuming (Catala Garcia, 2017).

One of the best solutions for taxonomic identification of microbial species is using DNA barcoding; the use of a standardized DNA sequence region to identify species (Valentini et al., 2009). Indeed, DNA barcoding is not a new notion. The term ‘DNA barcodes’ was first used in 1993 (Arnot et al., 1993) but the real start of DNA barcoding was in 2003 (Hebert et al., 2003). Since this time, species identification by DNA barcoding has become an essential part of biodiversity research, supplying novel insights into microbial ecology and species distribution (Anderson and Cairney, 2004, Chase and Fay, 2009). Numerous ecological studies have applied high throughput sequencing (HTS) of barcoding regions of DNA sequences to identify microbial communities in soil or on plant roots (Jumpponen and Jones, 2009, Coince et al., 2013, Burgess et al., 2017b). Several DNA regions have been used as DNA barcoding targets in different organisms, such as the 6S gene for bacteria and archaea and ITS for fungi (Schoch et al., 2012, Català et al., 2017) and Oomycetes particularly for Phytophthora species (Schoch et al., 2012, Català et al., 2017). The ITS regions are separated into two polymorphic regions (ITS1 and ITS2) by the conserved ribosomal gene 5.8S. This feature makes the ITS regions attractive targets for sequencing techniques such as HTS (Català et al., 2017). HTS provides an ideal method to describe microbial and fungal communities in environmental DNA (eDNA) with less time and cost (Coince

11

et al., 2013, Sogin et al., 2006). HTS techniques have been applied for the detection of Phytophthora species in different ecosystems (Coince et al., 2013, Vannini et al., 2013, Burgess et al., 2017a, Burgess et al., 2017b, Khaliq et al., 2018).

Remote sensing

Remote sensing is the transdisciplinary approach for the study and analysis of measurements of an object distant from any physical contact between the measuring instrument and the object (Nilsson, 1995). Remote sensing has many applications, including ecosystem classification, land- cover condition, environmental processes, landscape structure, disturbance, biodiversity and plant traits (Andrew et al., 2014). Land cover is an important target of remotely sensed image analysis. One major portion of the land cover is forest canopy and many remote sensing studies have evaluated forest canopy health and forest tree mortality. The assessment of canopy health can be positively related with both forest canopy height and diameter (Haby et al., 2010) but many other factors are associated with canopy health.

The traditional remote sensing approach for monitoring canopy health is built on spectral indices (e.g. Normalized Difference Vegetation Index NDVI) that are linked to the symptom of interest. In general, all these technologies build on spectral reflectance and texture that is based on reflected and emitted radiation from various bodies (Nilsson, 1995, Abdulridha et al., 2016). These methods are fast, accurate and non-destructive (Graeff and Claupein, 2003). Importantly, remote sensing techniques can be used to accurately determine the presence of plant diseases and pathogen spread, and are increasingly being utilized for a wide range plant disease mapping, evaluation, and management (Nilsson, 1995, Ristaino and Gumpertz, 2000, Olsson et al., 2008, Metternicht, 2003, Wilson et al., 2012). Optical sensors are used to determine plant diseases even in the absence of visible symptoms (Pineda et al., 2017). For example, indices including bands within the visible-near infrared (V-NIR) region of the energy spectrum and multi-spectral remote sensing techniques have been used to detect diseases such as laurel wilt disease, Cercospora leaf spot, powdery mildew, leaf rust and Citrus Greening (Mahlein et al., 2010, Franke and Menz, 2007, Li et al., 2012, Sankaran et al., 2012). Results obtained from these techniques

12

were based on analysis of the spectral data from the images that were remotely captured. The monitoring of viral, bacterial and fungal infection has been described by Baron et al. (2016) and Martinelli et al. (2015), and are good illustrations of the benefits of these techniques even in the absence of visible symptoms.

With respect to Phytophthora, in the last 10 years, the number of remote sensing studies for Phytophthora species has increased especially in forests (Wilson et al., 2012). The prediction of canopy condition in Mediterranean forests and woodlands, and determination of factors associated with spatial and temporal patterns of decline in forest condition have been successfully achieved using multispectral sensors onboard satellites and manned aircraft (Evans et al., 2012, 2013), including forest areas known to be infested by P. cinnamomi and P. multivora. Indeed, monitoring the spread of Phytophthora species has been done through three techniques. Firstly, on-ground techniques such as surveys have been used to monitor the spread of Phytophthora species and the diseases they cause (Wilson et al., 2012). On-ground techniques encounter many limitations as they are expensive and need trained interpretive staff (Wilson et al., 2012). Secondly, aerial photographic surveillance has been used to map Phytophthora disease spread. These techniques can effectively evaluate huge areas rapidly and are less costly and time- consuming than ground-based survey methods (Jamieson et al., 2014). However, an archive of aerial photographs are required and adequate funds to capture photography continuously (Wilson et al., 2012). Thirdly, remote sensing imagery techniques using panchromatic, radar, microwave, and multispectral satellite imagery are able to override these obstacles. Remote sensing has been effectively used to determine the spatial pattern of Phytophthora diseases as it provides different spatial spot data (Liu et al., 2007), and can monitor temporal patterns of Phytophthora diseases so can be used to map the impact of Phytophthora dieback in natural ecosystems (Wilson et al., 2012).

1.7 Management of Phytophthora species with chemical applications

Plants defend themselves against pathogens by two kinds of defence response (i) physiological characteristics such as cell walls, and (ii) biochemical reactions such as producing toxic substances

13

(Chisholm et al., 2006, Jones and Dangl, 2006, Eshraghi et al., 2014a). Understanding plant resistance mechanisms against pathogens is a necessary step in improving disease management strategies because some control strategies are aimed at inciting plant defense responses. There are many kinds of management strategies that are used to reduce or eliminate pathogens, these include physical applications, biological applications, and chemical applications. Chemical applications are one of the most common methods used to manage diseases in agriculture and horticulture by using toxic chemical compounds to restrain pathogen activities (Agrios, 2005). The effectiveness of chemical control of pathogens is affected by a group of factors, including the mechanisms of action of the active element, and the biology of the host (Kliejunas, 2010, Rolando et al., 2017). Chemical treatments are considered effective means for control of Phytophthora species that attack plants in natural or agricultural environments (Garbelotto et al., 2002).

Phosphite is a chemical widely used to manage the spread and impact of diseases caused by oomycete plant pathogens, especially Phytophthora species (Eshraghi et al., 2014b, Hardy et al., 2001). “The term phosphite is commonly referred to as the salts of phosphorous acid and the term phosphonate is used to mean phosphite ester containing a carbon-phosphorus (C–P) bond that is chemically distinct from the labile carbon-oxygen–phosphorus (C–O–P) bond found in phosphate ester” (Thao and Yamakawa, 2009). The product of potassium phosphite (K2HPO3) is used widely against Phytophthora species to protect natural ecosystems, urban trees and orchards (McDonald et al., 2001, Hardy et al., 2001, Gonzalez et al., 2017a). Phosphite is translocated in both the xylem and the phloem through association with photo-assimilates in a source-sink relationship given that it is trapped inside the phloem (Saindrenan et al., 1988, Ouimette and Coffey, 1990, Guest and Grant, 1991, Hardy et al., 2001). Mechanisms of action of phosphite include inciting the plant defence responses and/or act on the pathogen directly by causing inhibition or death (Fenn and Coffey, 1984, Guest and Grant, 1991, Jackson et al., 2000, Stasikowski et al., 2014b).

There are other chemical applications available such as copper oxychloride, metalaxyl-M, copper sulfate pentahydrate, phosetyl-Al, propiconazole benzyl ammonium chloride, and azoxsytrobin to control Phytophthora (Garbelotto et al., 2002, Rolando et al., 2017), which are not as 14

successful as phosphite. This could be because (i) some chemicals may be powerful compounds against Phytophthora species but may be toxic to plants at the same time, (ii) some chemicals may control Phytophthora species in vitro but not in planta, (iii) some chemical applications are effective against one species of Phytophthora but other Phytophthora species can be resistant or become resistant to these chemicals, and (iv) some chemical applications are only moderately effective against Phytophthora species.

1.8 Status of knowledge of Phytophthora species associated with urban forests in Perth, Western Australia

Perth, Western Australia

Perth is the metropolis of Western Australia, situated in the south-west of the State. This city covers 5,500 km2 and is predominantly flat (Saunders et al., 2011). Perth has a Mediterranean climate that is characterized by cool rainy winter and hot dry summer (Saunders et al., 2011). Perth is one of 36 global biodiversity hotspots (Guetté et al., 2018). Patches of native plants in the urban landscape of Perth as in other areas in Australia have numerous biological and social values (Stenhouse, 2004). Unfortunately, these patches of native plants are threatened by Phytophthora species (Shearer, 1994). The study of Barber et al. (2013) revealed that there were nine Phytophthora species associated with an extensive range of declining host trees in urban areas throughout Perth.

Urban forests as an initial point of spread for Phytophthora species

Once introduced, urban landscapes can play an important role in the pathway of new Phytophthora species spreading into natural ecosystems. Many Phytophthora species have been recovered from urban environments. For example, 22% of type specimens of Phytophthora were isolated from urban environments and were described between 2001 and 2016 (Hulbert et al., 2017). The importance of nurseries for the dissemination of Phytophthora species cannot be underplayed as plant production nurseries supply material for urban and peri-urban plantings in addition to forestry, horticulture, and environmental restoration. Indeed, various Phytophthora

15

species are considered as main pathogens of nursery plants (Erwin and Ribeiro, 1996). As the nursery trade continues to grow globally, Phytophthora species continue to spread into new areas, and nurseries are one of the principal routes of pathogen movement and introduction of alien plant pathogens into natural ecosystems (Liebhold et al., 2012, Moralejo et al., 2009b, Stokstad, 2004, Wingfield et al., 2001, Coetzee et al., 2001, Parke et al., 2014, Hardy and Sivasithamparam, 1988). Hardy and Sivasithamparam (1988) found eight Phytophthora species associated with 14 nurseries in Perth, Western Australia. This indicates Phytophthora species have been problematic in nurseries for a long time in Perth.

1.9 Summary

The relationship between urban forest declines and Phytophthora species is considered important as they can result in economic and environmental losses including the loss of biodiversity and many tree species. There are many biotic and abiotic factors that can adversely impact on tree health leading to increased susceptibility of tree species to Phytophthora. However, there are gaps in our knowledge of the abiotic and biotic factors in urban or peri-urban landscapes that help predispose urban trees to Phytophthora diseases. This review has described the losses caused by Phytophthora species and has outlined some of the predisposing and inciting factors that can potentially influence the susceptibility of urban trees to Phytophthora species. It has also discussed some of the methods that can be used to rapidly identify Phytophthora species and to monitor their spread and impact across large areas, and how Phytophthora species can be managed.

16

1.10 Thesis Aims

The overall aim of this research was to detect Phytophthora species in an urban forest and to study the host range of these species and their management by using novel chemical treatments.

To achieve this aim, the specific objectives of the thesis were to:

1- utilize remote sensing to detect urban trees in decline, and use high throughput sequencing to determine what Phytophthora species were associated with these declines and to determine if there were any specific environmental factors linked to declining forest health and Phytophthora species (Chapter 2), 2- determine the potential host range of Phytophthora species associated with declining urban trees (Chapter 3), 3- synthesize and characterize Ca (II) and Zn (II) mixed-ligand complexes containing amino acid (L-Valine) and (2,2- bipyridine) (Chapter 4), and 4- determine the effectiveness of the calcium and zinc chelates with or without phosphite in vitro and in planta to control Phytophthora cinnamomi (Chapter 5).

17

2 Chapter Two

Association of Phytophthora with declining vegetation in an urban forest environment

18

2.1 Abstract

Phytophthora species are important plant pathogens causing disease and mortality of a large number of trees and shrubs in forest ecosystems worldwide. Two hundred and thirty-six soil and root sites were sampled from declining trees in 91 parks and nature reserves in the City of Joondalup, Perth Western Australia. Samples were collected from an extensive variety of declining native Australian trees and shrubs including Acacia, Allocasuarina, Banksia, Corymbia, Eucalyptus, Grevillea, Hibbertia and Xanthorrhoea. After collecting fine roots, samples were placed in plastic containers for baiting. DNA was extracted from the roots and a metabarcoding approach was followed using Phytophthora–specific primers. Eight environmental factors were considered and included the change in canopy health as determined through sequential measurements using remote sensing. Out of the 236 sites, 24 sites had one or more Phytophthora species by baiting. Whereas 168 sites contained at least one Phytophthora species by metabarcoding. Overall, forty-five Phytophthora species were detected. Phytophthora multivora was isolated the most frequently with P. cinnamomi the fifth most frequent. In contrast, seven species P. capsici, P. sp. pecan, P. fluvialis, P. gonapodyides, P. sp. walnut, P. erythroseptica and P. fallax were present only in one sample. The Phytophthora community was diverse and did not vary according to any of the measured factors.

Interestingly, P. multivora was affected by three factors (park size and soil type and soil unit) while park type had an effect on P. cinnamomi. Also, canopy health influenced the correlation between the presence of P. multivora and the presence of P. cinnamomi.

The results reinforced the potential role of Phytophthora species in the declining health of urban trees. Also, this study suggests that factors do not have a significant effect on the diversity of Phytophthora species, while some of these factors have an effect on individual species such as P. multivora.

Given that no Phytophthora species were detected at almost 30% of the sampled sites with declining trees, this study suggests that other factors may be contributing to the health of trees at some of the sampled sites.

19

2.2 Introduction

The urban forest includes trees and shrubs in urban roads, parks, woodlots, abandoned sites and residential areas (Nowak et al., 2001, Konijnendijk et al., 2006), and “it is embedded within a socially and physically complex space” (Roman et al., 2018). Recently, urban trees have received increased attention due to the critical ecosystem services they provide to human health and environmental quality, particularly as urbanization continues to grow (Nowak and Walton, 2005). However, the urban forest through their mix of native and exotic trees (Colunga-Garcia et al., 2010), proximity to transport hubs (Simberloff, 2009) and the presence of tree nurseries (Pautasso et al., 2015) can provide a pathway for invasive pathogens to establish and then move into natural ecosystems (Colunga-Garcia et al., 2010, Paap et al., 2017a).

The Phytophthora genus contains invasive pathogenic species responsible for tree diseases worldwide. Indeed, the spread of Phytophthora species is a universal source of anxiety for nature preservation due to epidemics such as sudden oak death (USA), ramorum blight (UK), Phytophthora dieback (Australia) and protea root rot (Republic of South Africa). Within the urban forest, trees are under stress from polluting agents (Freer-Smith et al., 2004), mechanical damage, climate change (Tubby and Webber, 2010) and environmental factors (e.g waterlogging, flooding and drought) (Lambers et al., 1998, Niinemets and Valladares, 2006, Lloret et al., 2011) and are thus more vulnerable to pathogens such as Phytophthora.

It can be difficult to detect changes in the health of the urban forest given that it is scattered in the urban landscape. New technologies such as remote sensing can aid in the detection of tree health in an urban forest. Remote sensing is a promising tool for mapping disease locations, infected trees (Gillis, 2014) and forest health (Liu et al., 2006) as well as landscape structure, disturbance, biodiversity and plant traits (Andrew et al., 2014). Remote sensing has been used for the mapping of Phytophthora and the distribution of the diseases they cause in different areas of the world (Hill et al., 2009, Gillis, 2014, Cardillo et al., 2018). Hill et al. (2009) conducted their study in the urban environment of Melbourne in Australia and mapped P. cinnamomi infestation through use of differences in the imagery that was captured through digital multispectral imagery

20

techniques. Gillis (2014) mapped Sudden Oak Death (caused by Phytophthora ramorum) distribution in the natural environment of California in the USA by using multi-spectral Landsat satellite imagery to determine tree mortality at the canopy scale and applied across time series to show a pattern of spread. The study of Cardillo et al. (2018) was carried out in the natural environment of Extremadura in Spain, where patterns of disease foci and their dispersal in a spatial and temporal context was described using aerial photograph records.

In recent years, numerous new Phytophthora species have been described (Yang et al. 2017) and many of these include either a type isolate and/or additional isolates from an urban or disturbed ecosystem associated with the original description (Hulbert et al., 2017, Yang et al., 2017). These species, including well known Phytophthora species, not only impact on the health of trees within the urban forest, but the urban forest can act as a pathway by which invasive Phytophthora species can enter natural ecosystems (Hulbert et al., 2017). In order to prevent the dissemination of invasive Phytophthora species early detection, accurate identification, and the ability to track pathogens back to their potential origin are essential (Park et al., 2008). Isolation and identification of Phytophthora species can be difficult and time-consuming (Khaliq et al., 2018). However, new technologies have made this much easier. High throughput sequencing /metabarcoding has been used for Phytophthora in natural ecosystems (Burgess et al., 2017b, Català et al., 2017) and to a very limited extent in urban environments (Khaliq et al., 2018).

Perth, Western Australia is the most isolated and biodiverse city in the world (Hopper and Gioia, 2004). Much of native vegetation in the region is susceptible to the invasive species P. cinnamomi. The City of Joondalup is a new development north of Perth, established on the Swan Coastal Plain in what was once a Banksia woodland. Within the City of Joondalup, a lot of native vegetation remains. Remotely sensed data taken over a four years period which enables changes in tree health to be determined was available from the City of Joondalup. The remote sensing data was ground-truthed by examining 236 declining groups of plants across 91 parks in the City’s urban forest. Rhizosphere soil and roots were taken from all of these to determine if Phytophthora species were associated with the declining plants. In this study, the Phytophthora community of the samples were examined to answer the following questions (1) what 21

Phytophthora species are associated with declining trees? (2) is the Phytophthora community influenced by land class and park type?, (3) does canopy cover and change in canopy health as determined by remote sensing predict Phytophthora infestation?, (4) is the Phytophthora community influenced by soil type and soil unit?, (5) is the Phytophthora community influenced by plant community?, (6) is the Phytophthora community affected by park size?, and (7) do the known invasive pathogens P. cinnamomi and P. multivora have any preferred associations?

2.3 Materials and Methods

Study area and sample collection

The area investigated was the City of Joondalup located approximately 26 kilometers north of central Perth, Western Australia and surrounded by Wanneroo Road and Lake Joondalup to the east, Beach Road to the south, Amala Park to the north and the Indian Ocean to the west. Joondalup covers an area of 98.9 square kilometers (38.2 sq mi). For the purposes of this research, the study area was delineated by geo-rectified multi-spectral imagery. Between 2012 and 2015, digital multi-spectral imagery (DMSI) was captured annually with a fixed-wing aircraft across all urban bushland in Joondalup, as four spectral bands of data (red, green, blue, and near infrared) at a spatial resolution of 5 m pixels. The image gap was calculated by subtracting 2015- pixel values from that of 2012. An increase in pixel values means an increase in tree health over this period, while a decrease in pixel values means a decline in health. Using GIS, 236 sites from 91 parks where evidence of tree decline was present (Fig 2.1) were established for collecting soil and root samples to determine the presence of Phytophthora species. Bulked soil and root sites were collected during summer and autumn in 2014, 2015 and 2016. Each sample was approximately 150g made up of 8-12 scoops of rhizosphere soil collected within a 5m radius from under declining trees.

22

Fig 2.1 Location of the 91 parks in the City of Joondalup, Western Australia, sampled during the study

23

Baiting technique

For each bulked soil sample, soil with fine root samples was placed in 1L containers (11.5 x 16.5 x 7.5 mm; GENFAC 111 Plastics Pty Ltd) and replicated three times for each sample. The samples were flooded with distilled water (ratio 3:1 water: soil and roots). Baits consisted of young leaves from six plant species (Quercus ilex, Q. suber, Pimelea ferruginea, Poplar sp., Scholtzia involucrata, and Hedera helix) were placed on the surface and containers were incubated at 20- 25 oC for 7 days. Leaves were monitored daily for lesions. The baits with lesions were dried on paper towels and lesions were excised from them and lesioned sections approximately 2mm x 2mm in size were placed on NARPH, a Phytophthora selective agar medium (Simamora et al., 2017) in 9 cm diameter Petri-plates. Plates were kept in the dark at room temperature and regularly examined for colonies typical of Phytophthora species. Any growth of Phytophthora was sub-cultured onto fresh NARPH plates twice and ultimately transferred onto vegetable juice agar (V8A) plates [100 ml/L filtered vegetable juice (Campbells V8 vegetable juice; Campbell Grocery products Ltd., Norfolk, UK), 900 ml/L distilled water, 0.1 g/L CaCO3, pH adjusted to 7 and 17 g Grade Agar (Becton, Dickenson and Company, Sparks, MD, USA). The soil was then allowed to dry and baited a second time to increase the chances of isolation success (Jeffers and Aldwinckle, 1987, Davison and Tay, 2005). eDNA extraction from fine roots and metabarcoding

Fine roots from each sample were air-dried, and 60-80 g was ground to a fine powder using the TissueLyser LT (Qiagen). After each sample, the grinding tubes were cleaned by detergent (Pyroneg) then rinsed in an acid bath for 5 minutes (HCl 0.4 mM), then rinsed with water and finally sprayed with ethanol 70% and allowed to air dry. All ground samples were stored frozen. Free water was used to controls. DNA extractions were performed using the Mo-Bio PowerPlant DNA isolation kit (Carlsbad, CA) following the manufacturer’s protocol. Amplicon pyrosequencing and clustering were conducted as described previously (Burgess et al., 2017b, Khaliq et al., 2018).

24

Accompanying data

Accompanying data source generally indicates the spatial scale of the data that include five categorical variables (park type, land class, soil type, soil unit and plant community) and three continuous variables (park size, canopy cover and canopy health)

(1) Park type; Source: City of Joondalup (CoJ) internal GIS layer Units; Foreshore (22), Natural Areas (101), Park (40) and Thoroughfare (5). All combined = 168.

(2) Land class: Source: CoJ internal GIS layer Units: Open Space (32), Conservation area (118), Neighborhood parks (13) and Thoroughfare (5). All combined = 168.

(3) Soil type; Source: http://www2.landgate.wa.gov.au/ows/wmspublic? Description: DAFWA- 033 Soil Landscape Mapping Western Australia - Best available; Quindalup soil (46) Karrakatta soil (88), and Spearwood (34). All combined = 168.

(4) Soil unit; Source: http://www2.landgate.wa.gov.au/ows/wmspublic? Description : DAFWA- 033 Soil Landscape Mapping Western Australia - Best available : 211Qu_Q2 (10), 211Qu_Q3 (7) 211Qu_Q4 (11) 211Qu_Qp (9), 211Qu_Qs (3), 211Qu_Qu (6), 211Sp_cSp (10), 211Sp_Kls (20), 211Sp_Ky (68), 211Sp_LS1 (8), and 211Sp_S7 (16). All combined = 168.

(5) Plant community; based on recorded hosts taken at time of the collection of the bulked samples; 1= Myrtaceae (32), 2= Proteaceae (22), 3= Fabaceae (27), 4= Myrtaceae and Proteaceae (43), 5= Myrtaceae, Proteaceae and Fabaceae (18), 6= Myrtaceae and Fabaceae (21), 7= Proteaceae and Fabaceae (17), 8= Myrtaceae, Proteaceae and Xanthorrhoeaceae (17), 9= Casuarinaceae (15), 10= Casuarinaceae and Myrtaceae (16), 11= Casuarinaceae and Proteaceae (17), and 12= other familias (11).

(6) Park size; Source: CoJ internal GIS layer Units: square meters; Description: calculated from polygon layers. Ranged in size from 486.89 m2 to 1079298.87 m2. High park size was observed at sites with size > 0.5 standard deviation above the mean, medium at those +/- <= 0.5 standard deviation from the mean, and low at > 0.5 standard deviation below the mean.

25

(7) Canopy cover; Source: data were collected by using digital multispectral imagery (standard deviation of plant cell density (PCD = IR/Red) from pixels within 5 m of the sample point) ranged for 170592.25 to 49.25m. High canopy cover was observed at sites with canopy cover > 0.5 standard deviation above the mean, medium at those +/- <= 0.5 standard deviation from the mean, and low at > 0.5 standard deviation below the mean.

(8) Canopy health; Source: data were collected using digital multispectral imagery (standard deviation of plant cell density (PCD = IR/Red) from pixels of the sample point). The images were taken in 2012 and 2015, and ranged from -17.4 to +19.5, a negative value means a decrease in canopy health a positive value means an increase in canopy health.

Data analysis

An analysis was carried out to obtain any correlation between the 8 environmental factors at the sites and the Phytophthora community for sites where Phytophthora was detected. The Phytophthora community was down-sampled to the same number of reads (1000 reads per sample) using the rrarefy function in the Vegan package for R (Oksanen et al., 2007). Data of environmental factors obtained from different sources was merged with rarefactions in Excel (Microsoft). Then PERMANOVA was used to test the relationship between environmental factors and Phytophthora community (Anderson, 2005). PERMANOVA was best suited to the task because the environmental variables were stored as different types of data (categorical and continuous) and they did not fit the assumptions of normality. After that, Chao dissimilarity matrix (Chao et al., 2005) was created and the distances between points were calculated using NMDS that assigns all of the samples to x- and y- coordinates that places them close to similar samples and at a distance from dissimilar samples, these coordinates are then plotted on an ordination plot. After this has been completed, ellipses were drawn around the sample points that fell into the categories which had an effect on the Phytophthora community according to the PERMANOVA analysis: park size and canopy cover. The software attempted to draw an ellipse that encompassed 95% of the variation in each category. These ellipses show how the samples within each category were distributed. Finally, an ANOVA test was conducted to test

26

the effect of environmental conditions on the distribution of the two most common, known Phytophthora pathogens; P. cinnamomi and P. multivora.

2.4 Results

Isolation by baiting

Four species of Phytophthora (P. nicotianae, P. multivora, P. boodjera and P. arenaria) were isolated by baiting from 24 out of 236 sites at 18 out of 91 parks sampled (Table 2.1).

Phytophthora species detected from fine root eDNA

In this study, three 454 runs gave a total of 197,927 reads which corresponded to 45 Phytophthora phylotypes (Table 2.1). The mean number of Phytophthora phylotypes per sample was 5.8 (range 1 - 21). Phytophthora species were detected in 168 of the 236 sites which corresponded to 69 of the 91 parks.

Out of 236 sites, Phytophthora multivora was detected at 127 sites (63,649 reads) from 52 parks and was the most widely distributed species, followed by P. pseudocryptogea at 75 sites (14329 reads), P. arenaria at 97 sites (13,221 reads), P. amnicola 88 sites (12,384 reads), P. cinnamomi 78 sites (11,623 reads) and P. nicotianae 78 sites (8262 reads). Of the 45 phytotypes, 7 phylotypes (P. capsici, P. sp. pecan, P. fluvialis, P. gonapodyides, P. sp. walnut, P. erythroseptica and P. fallax) were rare accounting for less than 100 of reads at one site only.

Of the 45 phylotypes, 36 corresponded to described species and four are ‘designated’ but undescribed species (P. aff. meadii, P. sp. pecan, P. sp. walnut and P. sp. kelmania) and three were identified as potential new taxa (P. sp. nov. 1D, P. sp. nov. 2B and P. sp. nov. 8C) in clades (2 and 8). Eight species (P. aff. meadii, P. sp. nov. 1D, P. sp. nov. 8C, P. sp. nov. 2B, P. sp. walnut, P. sp. pecan, P. capsici, P. gonapodyides) were never previously detected in Western Australia by metabarcoding, and five species (P. fallax, P. frigida, P. sp. nov. 11A, P. sp. nov. 2A and P. pachypleura) were never isolated in Western Australia. Nineteen of described species were recorded for the first time in an urban forest of Western Australia (Table 2.1). Phylotype

27

identification was unequal across the Phytophthora clades, as 15 phylotypes reside in Clade 6, while only two phylotypes were identified from Clade 11. Table 2.1 Records of 45 Phytophthora species in the City of Joondalup urban forest in Western Australia

No First Phytophthora No of recorded Clade of Reads Rarefied1 Isolates2 Habitat5 Status6 spp. Parks in urban sites forest P. cactorum 1 2 2 165 13 AU 2014 I P. nicotianae 1 42 78 8262 550 5(4) NHU 2004 I P. sp. nov. 1D4 2 6 8 7667 287 P. aff. meadi4 2 8 10 187 27 I P. sp. nov. 2A3 2 2 2 433 16 P. sp. nov. 2B4 2 16 21 8912 303 NH I P. capensis 2 14 22 6359 251 A I P. capsic4 2 1 1 92 12 AU 2015 I P. citrophthora 2 18 28 3113 169 N N P. elongata 2 6 6 105 2 H N? P. frigida3 2 4 4 43 10 HNU 1985 I P. multivora 2 52 127 63649 3251 15(11) I P. pachypleura3 2 13 15 2268 83 N P. arenaria 4 50 97 13221 783 3(2) N P. boodjera 4 19 24 4049 263 1(1) N N P. palmivora 4 13 15 461 41 NU 2011 I P. sp. pecan4 6 1 1 2 0 HU 2011 I P. amnicola 6 48 88 12384 750 P. bilorbang 6 9 13 962 81 N N P. crassamura 6 11 15 6225 224 N I P. fluvialis 6 1 1 68 5 HN I P. gonapodyides4 6 1 1 9 1 N N P. gregata 6 13 15 300 31 N I P. inundata 6 11 17 405 30 HNU 2015 N P. Kwongon 6 24 34 4006 188 HNU 2011 I P. lacustris 6 2 2 19 3 NU 2010 N P. litoralis 6 6 10 852 35 NU 1995 I P. moyootj 6 11 15 2602 132 N 2011 N P. rosacearum 6 9 13 1332 67 N N P. sp. walnut4 6 1 1 32 2 NU 2015 N? P. thermophila 6 37 68 6667 432 HN I P. cambivora 7 5 5 1329 67 NU 1995 N P. cinnamomi 7 40 78 11623 707 HN I P. fragariae 7 6 8 342 14 HNU ? I P. niederhauserii 7 2 2 276 20 A I 28

No First Phytophthora No of recorded Clade of Reads Rarefied1 Isolates2 Habitat5 Status6 spp. Parks in urban sites forest P. cryptogea 8 3 3 189 12 HNU 2012 I P. drechsleri 8 2 2 27 0 AU 2015 I P. erythroseptica 8 1 1 19 2 NAH I P. pseudocryptogea 8 37 75 14329 924 A I P. sp. kelmania 8 7 8 1832 232 HNU 2016 N? P. sp. nov. 8C4 9 2 2 162 10 AU 2016 I P. constricta 9 7 10 500 20 P. fallax3 9 1 1 26 3 N N P. versiformis 11 16 17 8120 298 N N P. sp. nov. 11A 3 11 3 3 4302 65 NU 2011 N Sum 197927 10416 24 (18) 1 Rarefied read no. 2 the number of isolates recovered by baiting form sites(parks) 3never isolated in Western Australia 4never previously detected in WA by metabarcoding 5 Known associations: A, annual crops; H, perennial crops (including forestry); N, native ecosystems. U, urban forest (including trade nurseries) 6 Status of species: I, introduced; N, native; N? putatively native

Phytophthora community

The relationships between Phytophthora community and eight factors (canopy cover, park size, soil type, soil unit, park type, land class, plant community and canopy health) were analysed. There was a significant, but weak (R2<0.05) correlation between two factors, canopy cover and park size, and the Phytophthora community (Table 2.2). The remaining factors were not related to Phytophthora communities (Table 2.2). Ordination plot results showed canopy cover and park size were negatively correlated to the diversity of the Phytophthora community. The diversity of species within the Phytophthora community decreased relatively when the canopy cover increased, as shown by the decreasing size of the ellipse encompassing the distribution of sites within each class of coverage (Fig 2.2a). The findings also revealed the Phytophthora community composition was more diverse in the small parks then decreased as park size increased (Fig 2.2b).

29

Table 2.2: The results of PERMANOVA analysis indicate that there is a significant relationship (Pr < 0.05) between the canopy cover and size of the sampled park and the Phytophthora community found there. Low F-Model values suggest that these significant relationships only have a small impact. The six other measured variables did not have a significant impact.

2 Variable DF Sum of Squares F-Model R Pr (>F)

Canopy Cover (m2) 1 0.692 2.416 0.014 0.015

Park Size (m2) 2 0.599 2.09 0.012 0.044

Soil Type 2 0.799 1.395 0.016 0.136

Soil Unit 10 3.069 1.19 0.062 0.159

Park Type 3 1.011 1.177 0.021 0.285

Land Class 3 0.874 1.017 0.018 0.437

Plant Community 11 0.254 0.888 0.005 0.538

Canopy Health (%) 1 0.181 0.634 0.004 0.759

Residuals 146 41.821 - 0.848 -

30

Fig. 2.2 While overlap was high, (a) the canopy cover (F-Model = 2.416, Pr = 0.015) and (b) park size (F-Model = 2.090, Pr = 0.044) both measured in square meters, were negatively correlated to the diversity of the Phytophthora community found there.

Distribution of Phytophthora cinnamomi and Phytophthora multivora

Distribution Phytophthora cinnamomi

Park type had a significant effect on the presence (11,623 reads) of P. cinnamomi (Table 2.3). The results showed that foreshore parks had the lowest numbers of P. cinnamomi detected, while land designated as parks and thoroughfares were had highest numbers of P. cinnamomi (Fig 2.3).

31

Table 2.3: ANOVA results indicate that there was a significant correlation (P-value < 0.05) between the number of reads of P. cinnamomi and the park type.

Variable DF Sum of squares Statistic P-value

Park Type 3 421.043 3.499 0.019

Canopy Health (%) 1 144.627 3.605 0.060

Soil Unit 9 518.889 1.437 0.184

Canopy Cover (m2) * 2 42.530 1.060 0.305

Soil Type 2 68.123 0.849 0.431

Land Class 3 77.342 0.643 0.589

Park Size (m2) * 2 1.159 0.029 0.865

Plant Community 11 0.179 0.638 0.727

**Log transformed

**Square-root transformed

32

Figure 2.3: The number of reads of P. cinnamomi varied significantly (P-value = 0.019) between sampled parks with different land type designations. Alone in a group, foreshore parks had the lowest numbers of observed P. cinnamomi. P. cinnamomi in parks designated as natural areas had high variability, but low overall counts, which differentiated them from all other park types. Land designated as parks and thoroughfares were similar to one another in the amount of observed P. cinnamomi, though different from all others. (the result can explain by median line and significant letters)

The correlation between Phytophthora cinnamomi with Phytophthora multivora

There was a significant correlation (P-value < 0.05) between the presence of P. cinnamomi and the presence of P. multivora in varying sites (Table 2.4). The presence of P. cinnamomi with P. multivora varied significantly (P-value = 0.029) with canopy health at the sampled park (Fig 2.4). In parks with an unhealthy canopy, P. cinnamomi populations fell as P. multivora populations increased, while in all others, P. cinnamomi increased along with P. multivora.

33

Table 2.4: ANOVA results indicate that there is a significant correlation (P-value < 0.05) between the number of reads of P. cinnamomi and the presence of P. multivora within sites with varying canopy health, other environmental factors don’t have an effect on this correlation (not shown here)

Variable DF Sum of squares Statistic P-value

Canopy Health 1 195.754 4.880 0.029

P. multivora (reads)** 1 0.680 0.002 0.967

P. cinnamomi (reads) ** 1 430.088 0.002 0.964

**Log transformed

**Square-root transformed

34

Figure 2.4: The success of P. cinnamomi in competition with P. multivora varies significantly (P-value = 0.029) by the health of the canopy at the sampled parks. Health was measured by percent change in canopy cover between 2012 and 2015, where increasing health is more than ½ standard deviation above the mean, healthy is +/- ½ standard deviation from the mean and unhealthy is more than ½ standard deviation below the mean. In parks with an unhealthy canopy, P. cinnamomi populations fell as P. multivora populations increased, while in all others, P. cinnamomi presence along with P. multivora

35

Distribution Phytophthora multivora

There was a significant correlation between soil type, soil unit, and park type and the presence of P. multivora (Table 2.5). Within the three soil types, there was no significant difference in the presence of P. multivora between Karrakatta and Quindalup soils (Fig 2.5), whereas the Spearwood soil had significantly higher read counts of P. multivora than the other soils. There was also a correlation between P. multivora and park size, as when the park size increased the number of P. multivora reads increased (Fig 2.6).

Table 2.5: ANOVA results indicate that there was a significant correlation (P-value < 0.05) between the number of reads of P. multivora and the dominant soil type and park size.

Variable DF Sum of squares Statistic P-value

Soil Type 2 3564035.456 8.659 0

Soil Unit 11 6682143.849 3.608 0

Park size (m2) * 1 1666494.158 8.097 0.005

Park Type 3 1183001.095 1.916 0.130

Land Class 3 426590.867 0.691 0.559

Canopy Cover (m2) * 2 61465.574 0.299 0.586

Canopy Health (%) 2 56887.777 0.276 0.600

Plant Community 11 0.222 0.741 0.672 *Log transformed

**Square-root transformed

36

Figure 2.5: The presence and number of reads of P. multivora at sampled parks varied significantly (P = 0.05) with soil types sites with Spearwood soil had significantly higher read counts of P. multivora than sites with Karrakatta and Quindalup South soil types, which were similar to each other. (the top and bottom of the blue box represent 75% and 25% of the population, respectively; the bold horizontal line is the median, and letters which are the same are not significantly (P>0.05) different from each other)

37

Figure 2.6: As the size of the sampled park increases, so does the number of reads of P. multivora (R = 0.069, P-value = 0.005). Park size has been log transformed for this figure.

2.5 Discussion

Phytophthora community

The current study highlights the power of using high-resolution multispectral imagery together with metabarcoding to monitor tree health. In Joondalup, there was no structure within the Phytophthora community, the Phytophthora community was diverse and did not vary according to any of the measured environmental factors.

1) Barcoding vs baiting – low priority discussion The metabarcoding results showed the high abundance of Phytophthora species within vegetation across sites in the urban forest of the City of Joondalup in Western Australia. In contrast, a low number of Phytophthora species were obtained by the baiting technique. This difference could be due to the specificity of the bait leaves used to only those Phytophthora species. Erwin and Ribeiro (1996) mention that there is a relationship between Phytophthora species and the type leaves of bait. Or some of Phytophthora 38

species cannot produce spores or may spore of some species can't swim and reach leaves of plants. (II)c or (III) season which baiting was conducted is inappropriate as Balci and Halmschlager (2003) mention that baiting assays require to be carried out at various times of the year to get a reliable understanding of what Phytophthora species are present at a site

2) What Phytophthora species are associated with declining trees? In this study, 19 species that were found previously in the urban forest of Western Australia (including one designated species) were recorded. Apart from P. cinnamomi, since 1985 18 species of Phytophthora were found in urban forests of Western Australia (including one designated species). Indeed, most of them were discovered in this decade (14 species). Some of these species were recorded in other environments, for example, P. citrophthora (detected at 28 sites from 18 parks) was recorded in the agriculture environment in 1923 and in the urban forest in 2015 and is considered a pathogen to citrus and one of the species associated with nursery plants imported into WA (Davison et al., 2006). Also, P. cactorum (detected at 2 sites) is a pathogen for more than 200 plant species within different plant families (Chen et al., 2018) and was recorded in the agriculture environment of Western Australia in 2006 and in the urban forest in 2014. Whilst, P. litoralis (detected at 10 sites) and P. boodjera (detected at 24 sites) were recorded in natural ecosystems in 2006 and in the urban forest in 2011. Interestingly, some species were recorded first in the agriculture environment, then in the urban forest and then in the natural ecosystem. For example, P. thermophila (detected at 68 sites) was recorded in the agriculture environment in 1980 and in an urban environment in 1995, then it was recorded three years later in the natural ecosystem. Another example, P. niederhauserii (detected at 2 sites) that has been recovered from several plant species in many countries (Abad et al., 2014) was recorded in the agriculture environment and urban environment in 2002 and 2012, respectively. After that, P. niederhauserii was recorded in the natural ecosystem in 2014. The invasions of these species to new environments are via a ‘‘bridgehead effect’ phenomenon (Lombaert et al., 2010).

39

Twenty-six species (including three putative new species and four designated species) were detected for the first time associated with urban trees in Western Australia. This is not to say, of course, that these species did not exist in urban ecosystems before, but it may be because of the limited number of studies that were conducted in the urban forest including nurseries compared with studies in natural areas. Alternatively, it may be due to some studies being restricted to a few sites in a small area. Some of these species are known agricultural crop pathogens. For example, P. erythroseptica a potato pathogen (Goss, 1949), was recorded in one site. Phytophthora fragariae a known strawberry pathogen was recorded at seven sites. The spread of agricultural crop pathogens into the urban forest may provide additional evidence that the urban forest through human activity is an important pathway of invasive pathogens. it is expected that these species may appear in native ecosystems after a few years. Also, of interest is that there were other species associated with plants in natural ecosystems. For examples, P. arenaria was detected at 97 sites and P. amnicola was detected at 88 sites. This suggests high spillover into the urban forest. Of importance, is that although many of these species have global distributions, eight of them had never previously been recorded in Western Australia and four species were not reported before in Australia. These results support evidence from previous observations (Jung et al., 2016, Themann et al., 2002, Jung and Blaschke, 2004, Hulbert et al., 2017, Paap et al., 2017a) which indicate that through plants being distributed via the nursery trade, urban landscapes could play an important role in the pathway of new Phytophthora species into natural ecosystems and other environments. It is notable to say by the bridgehead effect scenario, urban environments are an important source of spread Phytophthora species into natural ecosystems through many paths including plant materials, aquatic sources, human activities, and equipment.

3) Is the Phytophthora community influenced by land class or type?

It was expected that there would be a relationship between park type (foreshore, natural areas, park, and thoroughfare) and Phytophthora community. We had two alternate theories; (i) that the watering and fertilizer additions to managed parks and the large areas of grass as opposed to trees would result in lower Phytophthora diversity, as agricultural systems with annual plants

40

have a specific set of Phytophthora species and very few species known from grasslands. Phytophthora species are distinguished as some of the main pathogens on thousands of woody plants (trees and shrubs) and crop species across the world (Jung et al., 2018), thus the presence and diversity should be highest near woody plants areas than grass areas. (ii) The lower human traffic and interactions with conservation areas should result in low Phytophthora diversity in natural parklands. It is well known that more disturbed areas have more species. The urban environment (e.g. parks and gardens) in most situations is very disturbed (Barber et al., 2013, Moffatt et al., 2004) resulting from human activity (Niemelä et al., 2002, Alaruikka et al., 2002). The study of Redondo (2018) highlights the positive influence of human activity on the spread of the Phytophthora community. Indeed, human activity plays a critical function in the spread of plant pathogens by creating novel transmission pathways Also, the low diversity of within Phytophthora community in all sites due to the diversity of plant species was low too (most trees in this area belong five families and most of them represent main hosts of particular species of Phytophthora). Indeed, the results in the present study demonstrate the importance of biotic factors, susceptible hosts, and human activity to explain the Phytophthora community within an urban forest. These outcomes were compatible with previous studies that indicate the significant effect of these factors on the interpretation of species distribution especially for invasive species (Gallardo et al., 2015, Heikkinen et al., 2007, Hernandez-Lambrano et al., 2018).

There was no association between Phytophthora community and park type. This was unexpected. The most likely explanation is that although watered parks were sampled (30 sites) and thoroughfares. Regardless of where the samples were taken, they were taken from vegetation. Thus, the actual site within the park where the samples were taken was more similar between parks types than would be expected from their description.

4) Do canopy cover and canopy health as determined by remote sensing predict Phytophthora infestation?

Canopy cover has an effect not only on the composition of soil microbial communities but also the diversity of a community (Delgado‐Baquerizo et al., 2018). The canopy cover is the measurement of vegetation health whether they are getting sicker or not. The present study 41

showed there was no correlation between canopy cover and Phytophthora communities. A possible explanation for this might be that the effect of canopy cover on the diversity within the Phytophthora community seems to decrease due to other effects such as human activity or possibly climate effects. Pickett et al. (2001) mention that the different linkages (e.g. environmental and physical elements) of urban ecosystems are worked within the human ecosystem frame.

5) Is the Phytophthora community influenced by soil type and soil unit?

Soil type is the main factor affecting the diversity of the microbial community in the rhizosphere (da Silva et al., 2003, Salles et al., 2004). Indeed, soil type influences both the pathogens and host. Tamm et al. (2010) indicated that soil type has an effect on soil-borne pathogens as well as on plant resistance to airborne pathogens.

In the present study, the results showed relationship between soil type and Phytophthora communities. It is difficult to explain this result, but it might be related to water availability. As Barber et al. (2013) pointed out, most areas of the urban forest of Perth are irrigated and with compacted soil. These conditions are likely to promote diversity within the Phytophthora community.

6) Is the Phytophthora community affected by park size?

Unfortunately, few studies have reported on the relationship of study area size on pathogen diversity or distribution. However, the study of Kearney et al. (2018) mentions that the size of an area plays an important role to mitigate the impacts of different threats. A good example to explain this is that the small protected areas of Australia are more likely to suffer from impacts of different threats such as climate change (Kearney et al., 2018) thus plants in these areas are more likely to suffer from pathogens. In the present study, there was a weak correlation between park size and Phytophthora diversity, and the park size did not influence the diversity of the Phytophthora community.

42

7) Is the Phytophthora community influenced by plant community?

Plant community did not influence the diversity of the Phytophthora community. This was a little unexpected, but actually, plants species were quite similar across the parks studied, and many of the known Phytophthora species have documented broad host ranges.

This is supported by Chapter 3 where 19 species of Phytophthora (Chapter 3) were screened for pathogenicity against 15 plant species, and the majority of plant species appear to have high susceptibility to these Phytophthora species. Other strong evidence is that the hosts where Phytophthora was most commonly detected in the current study belong to the Myrtaceae and Proteaceae. Within the Myrtaceae, Eucalyptus and Corymbia species are the dominant trees throughout WA, and most of these trees (e.g. Corymbia calophylla provides food and shelter to many fauna and flora in WA (Cooper et al., 2003). Phytophthora species are associated with many species of Eucalyptus (Linde et al., 1994b, Linde et al., 1994a, Maseko et al., 2007, Scott et al., 2009, Burgess et al., 2009) and Corymbia (Burgess et al., 2009, Barber et al., 2013). The Proteaceae was another family recorded to be associated with Phytophthora community in this study. This family has many valuable genera such as Banksia species that are the endemic urban trees within Western Australia. Within the Banksia genus, many species are associated with Phytophthora species (Shearer and Dillon, 1996, Scott et al., 2009, Rea et al., 2011a, Burgess et al., 2009). The prominence of this outcome confirms that Australian natural plants still suffer significant risks to species belonging to the genus Phytophthora.

8) Do the known invasive pathogens P. cinnamomi and P. multivora have any preferred associations

The distribution of P. multivora and P. cinnamomi was studied to understand the relationship between environmental factors and their spatial distribution patterns. In this study, P. multivora was found to be the most widely distributed species, while P. cinnamomi that is listed as one of the 100 worst invasive alien species (Lowe et al., 2000) and considered one of the pathogens with the widest global distribution (Burgess et al., 2017a) was the fourth most common species isolated. Of interest is the number of reads of P. cinnamomi declined as the number of P.

43

multivora reads increased with unhealthy canopy cover. Therefore, it can be assumed that P. multivora may displace P. cinnamomi in future. A likely reason for this assumption is that P. multivora appears to be a good saprophyte as well as a strong pathogen, whilst P. cinnamomi is a poor saprophyte and a strong pathogen. However, the saprophytic ability of P. multivora is still to be ascertained.

Except for the park type, no effect of the environmental factors on P. cinnamomi was found and this was an unforeseen result. However, a possible explanation for these results could be that P. cinnamomi has been present in southwest Western Australia since the early 1900s or earlier and is now widely distributed.

Another notable finding of the current study is the relationship between the distribution of P. multivora and soil type. The Spearwood soil which is a siliceous yellow sand, and weakly acidic with some iron oxide (Parker and Mee, 1982), had the highest number of P. multivora (P> 0.5) reads. There was no significant difference between the Quindalup soil that is a calcareous whitish beach sand (contain 8-36% calcium carbonate) and the Karrakatta soil that is derived from calcareous beach sand (contain 50-70% calcium carbonate and much of it has been leached to form secondary calcite layers at greater depth) (Bessell-Browne, 1990). The reasonable interpretation of the link between P. multivora and soil type might be related to soil properties (Hernandez-Lambrano et al., 2018) such as concentrations of calcium carbonate. Many studies reported that calcium carbonate can affect Phytophthora species and their diseases (Campanella et al., 2002, Duvenhage and Kotzé, 1991, Duvenhage et al., 1992). However, P. multivora is tolerant of soils containing calcium carbonate.

Also of interest, was the increasing number of P. multivora reads with increasing size of the parks. This relationship may partly be explained by an increase in park size leads to an increased number of host trees to P. multivora. Augspurger (1990), and Saetre (1999) revealed that hosts can influence pathogens in the forest soil directly by supplying living host tissue, or indirectly by providing ecological conditions that impact their reproductive activity.

44

2.6 Conclusion

Phytophthora species had a strong relationship with tree decline in the City of Joondalup’s urban forest. It is critical to understand the dynamics of the diversity and abundance of Phytophthora species in urban forests and factors which influence their impact and spread. The number of species detected by metabarcoding of eDNA using genus-specific primers in the Joondalup urban forest, especially the new species provides evidence that urban forests are an important pathway for Phytophthora spread. Human activity is likely the main factor influencing the diversity of the Phytophthora community followed by hosts.

45

3 Chapter Three

Phytophthora species associated with declining urban trees

46

3.1 Abstract

The nursery trade and by extension the urban environment is seen as an important pathway for the introduction of many pathogens, including Phytophthora into new ecosystems. Worldwide Phytophthora species cause significant diseases in a wide range of plants in agricultural, horticultural and forest ecosystems. We have detected many different Phytophthora species from dying and declining trees in the urban and peri-urban environment. The species recovered include those commonly found in natural ecosystems, but also other species better known for associations with agricultural crops. For many of these species, little is known about their host range. Therefore, the aim of this study was to examine the host range of 19 Phytophthora species, including many newly described species, against 15 trees species commonly used in urban plantings. Briefly, excised branches of the 15-tree species were under-bark inoculated with each of the nineteen Phytophthora species. These were incubated in the dark at 25°C and lesions were measured 8 days after inoculation. All Phytophthora species formed lesions in Eucalyptus marginata and Corymbia calophylla. Fraxinus excelsior was resistant to 7 of the 19 Phytophthora species. Six Phytophthora species were pathogenic to all 15-tree species, whereas P. versiformis formed lesions in only three tree species. Phytophthora cinnamomi, P. pseudocryptogea, and P. citrophthora were the most pathogenic species causing the largest lesions in most of these trees species. It is likely many of these Phytophthora species have a negative impact on the health of urban forests where they are present.

47

3.2 Introduction

Urban forests have important and increasing values in the urban environment (Tyrväinen and Miettinen, 2000). Nowak et al. (2013) define urban forests as a mix of native tree species that existed prior to development and exotic species introduced by residents or other means. Preserving urban forests is an important goal because (i) together with rivers and hills they are considered recreational places (Shearer and Tippett, 1989), (ii) urban forests are amongst the principal providers of ecosystem services in urban areas (Alvey, 2006, Dobbs et al., 2011, Dobbs et al., 2014, Stagoll et al., 2012, Paap et al., 2017a), and (iii) urban forests also reduce the so- called ‘heat island’ effect. An urban heat island (UHI) is an urban area having a significantly warmer environment than adjacent rural areas due to the lack of vegetation and expanding development due to human activities (Alcoforado and Matzarakis, 2010, Dimoudi et al., 2013, Maloley, 2010). Unfortunately, there are many factors that cause the decline of urban trees including water stress, heat stress, airborne pollution, construction, pests and diseases (Wang et al., 2011, Aukema et al., 2011, Grasso et al., 2012, Jacobi et al., 2013, Barber et al., 2013).

Globalization has increased the frequency and number of detected bioinvasions (Hulme, 2009). Plants-for-planting, either via nursery trade or direct to consumers through internet trade, is seen as the major pathway by which pathogens are spread globally (Moralejo et al., 2009b, Jung et al., 2016, O'Hanlon et al., 2016, Hulbert et al., 2017, Brasier, 2008). Internet trade increases the number of plants sold, their sources and buyers worldwide (Humair et al., 2015), and thus increases the risk of spread pathogens. The use of fungicides can mask symptoms, and soil borne pathogens, such as many Phytophthora species, can be present in the attached soil. This pathway is a greater problem for countries with land borders or those with lax border biosecurity (Eschen et al., 2015).

The urban forest contains a complex mix of native and exotic plant species, often watered during dry periods. Urban forests are frequently the first-place new outbreaks of pathogens are observed. In particular, exotic plants in the urban forest can act as sentinels for early detection of invasive pests and pathogens (Paap et al., 2017a, Colunga-Garcia et al., 2010). These invasive

48

species can then move through the urban and peri-urban environment into natural ecosystems. As such the urban environment becomes a stepping stone to natural ecosystems. This has been described as the ‘bridgehead’ effect (Lombaert et al., 2010). In fact, the invasion that moves through a bridgehead has significant implications both for invasion theory (i.e. the amount and nature of evolutionary transformation(s) involved in extensive-scale invasions) and for continuing efforts to control invasions by exotic organisms (i.e. increased wakefulness against invasive bridgehead populations) (Lombaert et al., 2010).

Members of the genus Phytophthora are important pathogens involved in forest declines worldwide (Jung et al., 2005, Scott et al., 2009, Hansen, 2008). To date, there are approximately 160 formally described species, with 50% of those described in the last decade (Scott et al., 2013, Burgess et al., 2017b). There are many factors contributing to these recent descriptions, including introductions from the nursery trade, increased studies in forests and natural environments, and an increase in the number of Phytophthora scientists globally (Érsek and Ribeiro, 2010, Rahman et al., 2015).

There is clear evidence for a bridgehead effect with Phytophthora introductions (Hulbert et al., 2017). Out of the 100 species described since 2001, a quarter of the descriptions were based on a type isolate recovered from an urban environment. There are more than 60 Phytophthora species present in Western Australia most of these species have been described in the last decade (Barber et al., 2013) and many of these have been recovered from the urban environment (Barber et al., 2013). However, a few species are recognized for their significant detrimental influence on urban forests, including one of the world’s most destructive invasive species, P. cinnamomi, a pathogen with a wide host range of native and exotic plants in Western Australia (Shearer et al., 2007). To evaluate the risk and potential impact of some of these Phytophthora species on urban forest trees in Western Australia, fifteen tree species were investigated for their susceptibility to 19 Phytophthora species. Most of these Phytophthora species have been detected in the urban environment in Western Australia but little is known about their pathogenicity to urban trees.

49

3.3 Materials and Methods

Phytophthora isolates

Isolates were obtained from the Centre for Phytophthora Science and Management (CPSM) culture collection and were previously identified with several isolated from samples collected from diseased urban trees (Simamora et al., 2017, Barber et al., 2013). All isolates have been allocated GenBank numbers (Table 3.1). Isolates were grown on V8 agar (V8A: 0.1 g CaCO3, 100 mL V8 juice, and 17 g agar in 900 mL distilled water) and incubated at a constant temperature (20oC) for 7 days.

Plant material

Green branches (approximately 1-year-old) between 0.5-1.5cm in diameter were collected in April 2016 from trees species considered common in the urban environment of Perth; Agonis flexuosa, Banksia sessilis, Callistemon sp. ‘Kings Park Special’, Corymbia calophylla, Eucalyptus gomphocephala, E. marginata, Ficus macrocarpa, Fraxinus excelsior, Magnolia grandiflora, Melaleuca sp., Metrosideros excelsa, Olea europaea, Plantanus orientalis, Pyrus ussuriensis and Viburnum tinus. These Branches were provided by Ellenby Tree Farm. Branches of trees were cut into approximately 70 cm lengths, placed into moist hessian bags in insulated ice boxes, transported to the laboratory and inoculated within 6-12 hours of the harvest. In the laboratory, the branches were clipped into 30 cm lengths and smaller side branches were trimmed as close to the branch as possible. The branch ends were immediately dipped in melted wax in order to prevent desiccation.

50

Table 3.2. Plant species used for pathogenicity testing in the current study

Plant species Origin Family Common Name Kind

Agonis flexuosa Western Australia Myrtaceae peppermint Tree Banksia sessilis Western Australia Proteaceae parrot bush Shrub Callistemon sp. Western Australia Myrtaceae bottlebrush Tree or shrub Corymbia calophylla Western Australia Myrtaceae marri Tree Eucalyptus gomphocephala Western Australia Myrtaceae tuart Tree Eucalyptus marginata Western Australia Myrtaceae jarrah Tree Ficus microcarpa China Moraceae Chinese banyan Tree Fraxinus excelsior Norway Oleaceae ash Tree Magnolia grandiflora United States Magnoliaceae bull bay Tree Melaleuca sp. Western Australia Myrtaceae - Tree Metrosideros excelsa New Zealand Myrtaceae New Zealand Tree Christmas tree

Olea europaea Mediterranean area Olive Tree

Plantanus orientalis Balkans area Platanaceae Old World sycamore Tree

Pyrus ussuriensis Korea, Japan, and Russia Rosaceae Ussurian pear Tree

Viburnum tinus Mediterranean area Adoxaceae laurustinus Tree

Inoculum production and inoculation

Prior to inoculation, the branches were surface sterilised with 70% ethanol and inoculated using a method described previously (O'Gara et al., 1996). Briefly, a sterile scalpel was used to make a small incision under the epidermis to the cambium. Then a 5-mm diam. V8 agar (50 ml of cleared

Campbell’s Australia V8 juice, 0.05g CaCO3, 8.5g Gibco agar and 450ml distilled water) disc colonized for 7 days with a Phytophthora species was placed mycelial face down onto the cut branch, this was then covered with a piece of moist cotton wool and sealed with Parafilm©(Meanasha Wisconsin) to prevent desiccation. A non-colonised V8 agar disc was used 51

for the control inoculations. There were 10 replicate branches per isolate and one replicate of each treatment-host combination was placed together in a separate plastic bag lined with a moist paper towel to prevent desiccation and incubated at 25 +/- 2°C in the dark for 8 days.

Harvest and measurement

Eight days after inoculation for each branch the visible lesion was measured if present, after which the outer bark was gently removed from the branch beyond the visible lesion to determine if lesions extended beyond the visible lesion, if so the lesion length beyond the visible lesion was also measured. For the analysis of lesion lengths, the 20mm length at the inoculation point was subtracted from the measurements. For each isolate, a 1 cm sample of branch from the lesion front was plated onto NARH (1000 deionised water, CMA 17g, nilstat 1ml, ampicillin 0.1g, rifadin 0.5ml and hymexazol 0.05g) a Phytophthora selective medium (Simamora et al., 2017) to confirm the lesions were caused by the Phytophthora species used to inoculate the branch.

Statistical analysis

An ANOVA test (GenStat 64-bit Release 18.1) was used to identify statistical significance of the length of lesion caused by the Phytophthora species and the main interactions between tree species and Phytophthora species. A “heatmap” R matrix (SanDisk_SecureAccessV3.0_QSG) was used to show differences in lesion areas for each Phytophthora species in each host. To meet the chromatic representation map R matrix hypotheses, controls were excluded from the matrix.

52

Table 3.2 Phytophthora species used for pathogenicity testing in the current study

2

1

Isolate Genbank Known Clade Host

number no. pathogen

species

atedin WA

Phytophthora

Isol Detectedin WA

P. amnicola 6 VHS 19503 JX069841 Patersonia sp. + ● P. arenaria 4 CBS127950 HQ013219 Multiple species + ●

P. boodjera 4 PAB 11-56 KC748460 Eucalyptus spp. + ●

P. cambivora 7 MUCC808 MG182637 Multiple species + ●

P. capensis 2 P 1822 GU191325 Multiple species + ●

P. capsici 2 MUCC809 KT984951 Multiple species +

P. cinnamomi 7 MP 94-48 JX113294 Multiple species + ●

P. citrophthora 2 VHS 33174 MG182636 Multiple species + ●

P. crassamura 6 DDS 3432 HQ012949 Multiple species + ●

P. drechsleri 8 MUCC810 MG182638 Multiple species + ●

P. frigida 2 CMW 19428 KX011270 Eucalyptus smithii + ● P. multivora MG182632 ● 2 PAB 11-76 Multiple species + PAB 14-01 MG182634 P. nicotianae 1 PAB 12-23 KC748453 Multiple species + ● PAB 10-104 KC748453 P. niederhauserii 7 PAB 13-29 MG182635 Multiple species + ●

P. palmivora 4 PAB 11-107 KC748462 Multiple species + ●

P. pseudocryptogea 8 VHS 16118 KP288376 Multiple species + ●

P. ‘walnut’ 6 IMI 389735 AF541910 Unknown ?

P. thermophila 6 PAB 13-09 MG182633 Multiple species + ●

P. versiformis 11 TP 13-42 KX011278 Corymbia calophylla + ● 1 denotes species which have been isolated from Western Australia.

2 denotes species which have been detected though metabarcoding studies but have not been isolated into the culture.

53

3.4 Results

Lesion measurements produced by Phytophthora species on the tree branches ranged from 0 to 108.5 mm across the trials. All 19 Phytophthora species caused lesions in excised branches in at least three plant species by 8 days after inoculation. No lesions were produced in the control branches and Phytophthora was not isolated from the control inoculations. Phytophthora cinnamomi, P. pseudocryptogea and P. citrophthora were the three most aggressive species for all the host species (pooled mean lesions > 23 mm) (Appendix A). The least aggressive species was P. versiformis (1.01mm), and it produced lesions in only three of the 15 plant species tested (Fig 3.1).

Four Phytophthora species (P. versiformis, P. cambivora, P. boodjera and P. frigida) were slightly pathogenic (mean lesions > 3 mm < 9 mm); 10 Phytophthora species (P. thermophila, P. crassamura, P. multivora isolate PAB 11-76, P. amnicola, P. palmivora, P. nicotianae isolate PAB 10-104, P. 'walnut', P. capensis, P. niederhauserii and P. nicotianae isolate PAB 12-23 were moderately pathogenic (mean lesions >9.5 <18mm); three species (P. multivora isolate PAB 14- 01, P. drechsleri, and P. capsici) were strongly pathogenic (mean lesion lengths > 18 mm < 23mm); and three species (P. cinnamomi, P. pseudocryptogea and P. citrophthora) were strongly pathogenic (mean lesion lengths > 23 mm) (Appendix A).

Six Phytophthora species (P. pseudocryptogea, P. citrophthora, P. drechsleri, P. capensis, P. nicotianae isolate PAB 10-104 and P. multivora isolate PAB 11-76) caused lesions in all 15 hosts tested (Fig 3.1). For the individual plant species, P. cinnamomi recorded the longest lesions in A. flexuosa (mean 108.5mm) and in E. marginata (mean 105.6mm) (Fig 3.2). Phytophthora multivora isolate PAB 14-01 produced the second-longest lesions (mean 76.4mm) in A. flexuosa. Overall, E. marginata was the most susceptible species (overall mean 50.83mm) and all Phytophthora species tested caused lesions in this host (Fig 3.1). Fraxinus excelsior was the most resistant plant species to the Phytophthora species tested (overall mean 2.6mm) followed by O. europaea (overall mean 2.56mm) (Appendix A).

54

Figure 3.1 A heatmap showing the pathogenicity of Phytophthora species (21 isolates from 19 species) in fifteen plant species. White squares represent no lesions and dark red the longest lesions

55

Figure 3.2 Examples of lesion development on Eucalyptus marginata branches inoculated with (a) Phytophthora cinnamomi, (b) P. multivora, (c) P. pseudocryptogea, (d) P. citrophthora, (e) P. capsici, (f) P. drechsleri, (g) P. capensis (h), and P. versiformis compared to (i) the non-inoculated control

3.5 Discussion

Through the ‘bridgehead’ effect, urban environments are considered important sources for the spread of Phytophthora species into natural ecosystems. Therefore, a detection program for invasive Phytophthora species within the urban forest, as well as an evaluation of their risk and possible influence on urban forest trees, is valuable. In the present study, all 19 Phytophthora species caused lesions in at least three hosts, many of which have been isolated from diseased trees (e.g. P. versiformis), but not previously shown to cause disease, or have only been tested with few hosts in pathogenicity trials (Croeser et al., 2018). Phytophthora capsici, P. capensis, and P. ‘walnut’, not previously recorded in WA, appear to be potential threats to urban trees in WA given their moderate to strong pathogenicity.

Phytophthora cinnamomi was the most aggressive of the Phytophthora species screened and it was pathogenic on 14 of the 15 hosts tested, with only V. tinus exhibiting no lesions. This is not

56

surprising as P. cinnamomi is the most widespread species in natural ecosystems in Australia (Burgess et al., 2017a) where it has a wide host range and causes destructive disease in native vegetation (Cahill et al., 2008). It is also listed as a ‘Key threatening process to Australia’s biodiversity’ by the Australian Government’s Environmental Protection and Biodiversity Conservation Act 1999. This result for V. tinus is somewhat unexpected because V. tinus has been recorded as a susceptible species to P. cinnamomi (Moralejo et al., 2009a). This outcome could be because there are differences between individuals/provenances of V. tinus in susceptibility to P. cinnamomi. In the present study, all branches used to screen pathogenicity in V. tinus was from one individual plant. Phytophthora cinnamomi produced the longest lesions in Agonis flexuosa and in Eucalyptus marginata. This was corroborated by other studies which mention P. cinnamomi as a pathogen to at least 14% of the plant species present in the E. marginata forest (Davison and Shearer, 1989, Anderson et al., 2010, Belhaj et al., 2018) and to 40% of approximately 6000 native plant species in southwestern Australia (Shearer et al., 2004).

The second most aggressive species was P. pseudocryptogea. This is a recently described heterothallic species in Clade 8 (Safaiefarahani et al., 2015), and is now known to be part of a species complex, but prior to molecular tools was previously known as P. cryptogea. Phytophthora citrophthora is known to cause disease in a range of different trees (Pettersson et al., 2017), and in the present study was the third most aggressive species across all of the hosts tested. Phytophthora multivora has previously been recovered from A. flexuosa together with E. marginata and many other plant species (Scott et al., 2009), and in the present study after P. cinnamomi produced the second-longest lesions in A. flexuosa. It was reported as the most commonly isolated Phytophthora species from diseased urban trees by Barber et al. (2013). In the current study, there was significant variation in the pathogenicity between the two isolates of P. multivora to the different plant species. These results are similar to those reported by Croeser et al. (2018). This variation in pathogenicity between the P. multivora isolates may be because they have been present in the south-west of Western Australia for a long time, leading to diversification, and differences in host range (Scott, 2011).

57

Phytophthora versiformis has only been recovered from the rhizosphere of declining C. calophylla (Paap et al., 2017b), but is only a weak pathogen to this species (Croeser et al., 2018). In the present study, P. versiformis produced lesions in three host species; on two (Metrosideros excelsa and Eucalyptus marginata) the lesion did not differ significantly to the non-inoculated wounded controls, and in its only known host C. calophylla, the lesions were small but significant.

The present study indicates that all Phytophthora species screened produced lesions in Eucalyptus marginata, and most of these species have been recovered from Eucalyptus species (Keane et al., 2000, Scott et al., 2009, Burgess et al., 2009, Shea et al., 1982, Oh et al., 2013, Maseko et al., 2001).

Fraxinus excelsior (ash) is a tree native to the Britain Isles and the majority of Europe (Thomas, 2016) and four Phytophthora spp. (Phytophthora cactorum, P. plurivora, P. lacustris and P. gonapodyides) are implicated in predisposing Fraxinus excelsior to infection by Hymenoscyphus fraxineus – the causal agent of ash decline ash decline (Orlikowski et al., 2011). Fraxinus excelsior was the most resistant plant species to the 19 Phytophthora species screened in the present study. This could be due to the host being resistant to the Phytophthora species tested in the present study as few common northern hemisphere species were included.

Both native and exotic woody plant species common in the urban forest in Perth were susceptible to a wide range of Phytophthora species including those already introduced to Western Australia and a few species considered likely to be introduced given they have already been detected in eastern Australia. Vigilance toward disease detection within the urban forest can assist in the early detection of invasive pathogens, including Phytophthora. However, the additional tests would be needed to determine if similar results would be obtained from whole-plant root inoculation studies.

58

4 Chapter Four

Synthesis, and characterization studies of Ca (II) and Zn (II) mixed- ligand complexes containing the amino acid L-valine and 2,2- bipyridine

59

4.1 Abstract

Two new mixed ligand complexes of transition metals were manufactured by reaction of Zn(II) ion or Ca(II) ion with 2,2′-bipyridine as a primary ligand and L-valine as a secondary ligand. The ligands and their metal complexes were examined utilizing X-ray crystallography, ultraviolet rays– visible spectra and Fourier-transform infrared spectroscopy. The mixed ligand complexes were described with formulae as Zn (Val)2(bipy) and Ca(Val)2(bipy). The metal complexes had a pseudo-octahedral structure, and both complexes have the same patterns of structure indicating crystal structure symmetry. However, the yield of Zn chelate was at 50% of the basic materials and the yield of Ca chelate was 81% of basic materials.

60

4.2 Introduction

The study of ligand complexes has become increasingly important with respect to their biological activity toward pathogens (Eshwika et al., 2004, Mrinalinil and Manihar Singh, 2012, Girgaonkar and Shirodkar, 2012, Sanap and Patil, 2013). In general, mixed ligand complexes have been extensively used as antimicrobial (Shebl et al., 2017, Chai et al., 2017, Taghizadeh et al., 2017) and anticancer agents (Van Rijt and Sadler, 2009). Recently, the importance of bioinorganic chemistry has grown globally, especially when European Union countries picked "Bio- coordination Chemistry" as one of the seven priority research fields in 1991 (Shimazaki et al., 2009).

L-Valine is one of the 20 proteinogenic amino acids with the chemical formula

HO2CCH(NH2)CH(CH3) (Canpolat et al., 2004, Aliyu and Isyaku, 2010). It is classified as a non-polar branched-chain amino acid and is widely present in human food sources such as cottage cheese, fish, poultry, peanuts, sesame seeds, and lentils (GDR et al., 1984). However, it is seldom present at a ratio exceeding 10 %. It can be obtained from alanine via adding two methyls (CH3) groups to the α-carbon atom (Aliyu and Isyaku, 2010, Shimazaki et al., 2009). The properties and structure of valine are shown in Figure 4.1 and Table 4.1, respectively.

Figure 4.1 structure of valine

61

Table 4.1 the different properties of valine

Compound molecular Density Molar mass Solubility Acidity (pKa) Physical state Melting formula g/cm3 g mol-1 point °C

Soluble 2.32 (carboxyl) White crystalline 298 Valine C5H11NO2 1.316 117.15 in water 9.62 (amino) powder decomp

With regards to the structure of ligand complexes, the insertion of a second competing ligand such as 1,10-phenanthroline, 2,2-bipyridine diminishes the dimensions of the metal complex structure. The 2,2′-bipyridine ligand (Figure 4.2) has been widely used as a metal chelating ligand especially when combined with other transition metals owing to its following properties (1) strong redox stability and ease of functionalization, and (2) neutrality that can constitute charged complexes with metal cations and form symmetrical and asymmetrical isomers (Kaes et al., 2000). The properties of 2,2′-bipyridine are shown in Table 4.2.

Figure 4.2 structure of 2,2′-bipyridine

Table 4.2 the different properties of 2,2′-bipyridine

Compound molecular Density Molar mass Solubility Acidity Physical state Melting formula g/cm3 g mol-1 (pKa) point °C

2,2′- Slightly Soluble White crystalline bipyridine C10H8N2 1.316 156.18 in water 9.67 powder 70-73

Another part of complexes (Ca chelate or Zn chelate) represents transition metal ions such as Fe, Co, Ni, Cu, Zn, Ca and Cd that play a vital role in living systems, such as in enzymes or in carriers in a macrocyclic ligand field environment. Bio-coordination chemistry studies into the role of

62

transition metal ions in living systems are now of great interest to chemists (Fenton, 2001). Bio- coordination chemistry can improve our understanding about living systems and the use of these metal ions to create and/or prepare different metal complexes can have a range of applications in our daily life. For many years, the use of bio-coordination chemistry to produce fungicides was surprisingly neglected and currently is still in its early stages. The specific objective of this study was to synthesize new ligand complexes that can potentially use as fungicides against Phytophthora species and to investigate of their physical and chemical structural characteristics.

4.3 Materials and Methods

Preparation of Ca chelate

The Ca chelate was made using a common method of making mixed ligands metal complexes (Bhattacharya et al., 2003, Burger, 1973). Briefly, a solution of 2,2′-bipyridine (0.156 g, 1 m.mol) in aqueous ethanol (1:1:5 ml) and solution of L-Valine (0,234, 2 m.mol) in aqueous ethanol (1:1:5 ml) containing sodium hydroxide (0.08, 2mmol) were added simultaneously to a solution of

CaCl2.6H2O (1 m.mol) in aqueous ethanol (1:1:10 ml) in the stoichiometric ratio [2Val: Ca: bipy]. The solution was stirred constantly at room temperature for 4 hours then allowed to stand overnight. The crystallized product was filtrated off and washed with aqueous ethanol.

Preparation of Zn chelate

The Zn chelate was made using the same method of making Ca chelate (Bhattacharya et al., 2003, Burger, 1973). Briefly, a solution of 2,2′-bipyridine (0.156 g, 1 m.mol) in aqueous ethanol (1:1:5 ml) and solution of L-Valine (0,234, 2 m.mol) in aqueous ethanol (1:1:5 ml) containing sodium hydroxide (0.08, 2mmol) were added simultaneously to solution of ZnCl2 (1 m.mol) in aqueous ethanol (1:1:10 ml) in the stoichiometric ratio [2Val: Zn: bipy] (Figure 4.3). The solution was stirred constantly at room temperature for 4 hours then allowed to stand overnight. The crystallized product was filtrated off and washed with aqueous ethanol.

63

X-ray crystallography (XRD)

The crystallographic features of the synthesized complexes were studied by X-ray diffraction (XRD) analysis via a GBC EMMA diffractometer with CuKα radiation (=0.154nm). The diffraction angle value (2θ) in the range (20° - 70°) was scanned at 2°/min with a step size of 0.02°. The analysis was carried out with beam acceleration at operating voltage and current of 35 kV and 28 mA, respectively.

Ultraviolet rays–visible spectra

The absorption spectra of the synthesized complexes were recorded within the wavelength range of 250 – 850 nm using a UV-vis PerkinElmer spectrometer. A halogen lamp was used as a light source coupled with a diffraction grating and photodiode detector. Light intensity calibration was performed by recording a baseline spectrum for a quartz tube filled in water. This calibration process eases the suppression of residual noise.

Fourier-transform infrared spectroscopy (FTIR)

The infrared reflectance spectra of the synthesized complexes were obtained using a “reflected off” type of Perkin Elmer Spectrum 100 FTIR spectrometer in a wavenumber range of 400 – 4000 cm-1. The samples were placed on a diamond crystal surface area and a pressure the arm was positioned and locked at a force of 100 N in order to confirm the sample was touching evenly onto the crystal surface. Background correction was made before the collection of each spectrum.

4.4 Results

Synthesis of Metal Complexes

In aerobic conditions, the complexes were made through interacting the metal salts with the corresponding ligands using a ratio of (1 MCl2:1 2,2-bipyridine:2 sodium valinate) (Fig 4.3).

64

The mixed ligand metal complexes synthesis can be expressed in the following equations:

1. 2Val H +2NaOH 2Val- Na+ + H2O

2. 2Val- Na+ + phen + MCl2 [M (Val)2(bipy)] + 4H2O + Na Cl

M= Ca (II), Zn (II)

Where bipy is 2,2-bipyridine and Val H is amino acid L-valine

Figure 4.3 Schematic representation of the preparation of the complexes [M (Val)2(bipy)]

The physicochemical properties, formulae weights and the melting points of the chemicals are listed in Table 4.3. Both complexes were non-hygroscopic, stable at room temperature and white in color for both solid and liquid states. The results observed in this investigation suggest that the ligands acid L-valine and 2,2-bipyridine coordinate with either Ca (II) or Zn (II) form octahedral geometry. However, the yield of Zn chelate was at 50% of basic materials. Whereas, the yield of Ca chelate was at 81% of the basic materials and is considered a good yield rate.

65

Table 4.3 The physicochemical properties of the calcium and zinc chelate complexes

Molecular Compounds Physical state % yield Weight

Ca[(Val)2(bipy)] 453.80 White crystalline powder 81.39

Zn[(Val)2(bipy)] 428.50 White crystalline powder 55.55

Structural Characterization of Metal Complexes

XRD Analysis

The crystalline structures of the Zn and Ca chelate complexes were determined by X-ray diffraction (XRD) (Fig 4.4). The XRD results reported reflection faces at (77), (72), (89), (123), (143), (75), (100) and (99) nm for the Ca-complex, and (73), (75), (108), (80), (130), (103), (86) and (96) nm for the Zn-complex.

Both samples had the same patterns of structure indicating crystal structure symmetry. For example, the main peak of the Ca-chelate was located at (2θ = 32.86) which is very close to that of the Zn-chelate located at (2θ = 32.9). The principal diffraction peaks of both the Ca-chelate and Zn-chelate were placed in a wide range of temperatures between 25-50 °C. The intense and sharp peaks indicate the improved crystalline quality of the complexes.

66

Fig 4.4 X-ray crystallography data of the calcium and zinc chelate complexes Ultraviolet rays-visible spectra (UV-Vis)

Recorded absorption spectra of the calcium and zinc chelate complexes can be utilized to confirm their structure and to provide evidence that the electronic transitions occur. In both mixed ligand complexes (Zn chelate and Ca chelate), the absorption spectra displayed an absorption band around 280 nm (Fig 4.5) which could be attributed to (n→ π*) transition. Also, both complexes did not show any d-d transitions because of their weakness.

Fig 4.5 Electronic spectral data of the Zn and Ca chelates 67

Fourier-transform infrared spectroscopy (FTIR)

The Zn and Ca complexes were also examined by FTIR analysis. This technique can predict the coordination of the complexes to their corresponding ligands. The most important IR band (peak) for both the Zn and Ca chelates was observed at a wavelength of 1565 cm-1 (Fig. 4.6a and b) which reflects a vibrational mode for the  (C=N) group of 2,2-bipyridine. This vibrational mode suggests that 2,2-bipyridine is similar to 1,10-phenanthroline, and is coordinated to the metal centers (Jawetz, 2007, Fayad et al., 2013). Another strong band for both chelates was observed around 3317 cm-1 and corresponds to the vibrational mode of (N-H) of the amine group. Two bands at 1480 cm-1 and 1364 cm-1 were related to  (OCO) symmetry, which indicates the coordination of the carboxylic group to the central metal ion (Nakamoto, 1996, Nishida et al., 2009, Fayad et al., 2012). Moreover, other bands with low intensities in the spectra of both complexes were observed in the ranges of 610-542 cm-1 and 472-401 cm-1 are due to metal-nitrogen  (M-N) and metal–oxygen  (M-O) stretching vibrations, respectively (Nishida et al., 2009, Fayad et al., 2012, Fayad et al., 2013).

68

Fig 4.6a Fourier-transform infrared spectroscopy data of the Ca chelate

Fig 4.6b Fourier-transform infrared spectroscopy data of the Zn complex

69

4.5 Discussion

Ligand complexes represent a novel group of candidates with potential as fungicides to control a range of plant pathogens including Phytophthora species. The XRD results showed similar patterns for both complexes indicating a similar crystal structure for both of them. This finding is consistent with that of Shahedi et al. (2017) who indicated that similar complex patterns lead to similar crystal structures. The FT-IR results confirmed the presence of carboxylic and amine groups as well as metal-nitrogen (M-N) and metal–oxygen (M-O) in the metal complexes (Nishida et al., 2009, Fayad et al., 2012). Whilst, the UV-Vis results confirmed the presence of an aromatic ring. This result agrees with the findings of Sanap and Patil (2013). The yield of the Zn chelate was at 50% of basic materials whilst the yield of the Ca chelate was better at 81% of basic materials. The higher yield of Ca chelate makes it more suitable than the Zn chelate as a potential fungicide. These results were consistent with numerous studies (Kumar et al., 2018, Sevgi et al., 2018, Liu et al., 2018, Pervaiz et al., 2018) that indicate the good biological activity of mixed ligand complexes against pathogenic microorganisms in both animals and plants.

70

5 Chapter Five

The potential control of Phytophthora cinnamomi in vitro and in planta using calcium and zinc chelate with or without phosphite

71

5.1 Abstract

Members of the genus Phytophthora cause significant economic losses in crops, as well as environmental damage to forests and natural ecosystems worldwide. Currently, phosphite is the most effective chemical for disease management but excessive phosphite concentrations can result in phytotoxicity in plants. Also, some Phytophthora species have tolerance to phosphite. Here two newly developed chelates were tested for their in vitro and in planta efficacy against Phytophthora cinnamomi. The Ca chelate, Zn chelate, phosphite and a combination of Ca chelate and phosphite at different concentrations were compared. In vitro, the effect of the chemical treatments was estimated by measuring the EC50 of the mycelial biomass in liquid media with 0- 160 µg/ml of the chemical treatments. Phytophthora cinnamomi was highly sensitive to Ca chelate alone and a combination of Ca chelate and phosphite. Three concentrations (0, 0.25 and 0.5%) of each chemical treatment (phosphite, Ca chelate, Zn chelate) and Ca chelate + phosphite were applied as a foliar application to two plant species (Banksia grandis and Eucalyptus marginata), which were then either non-inoculated or inoculated with P. cinnamomi. All non- inoculated control plants remained healthy, while significant root damage and reduction of root dry weights was observed for inoculated control plants. Phosphite reduced lesion development of P. cinnamomi compared to the control. However, control of P. cinnamomi following the application of Ca chelate was significantly greater to that obtained with phosphite. The Ca chelate + phosphite had the largest reduction on lesion development in both plant species than all other treatment combinations. None of the Zn chelate applications were effective. Ca chelate has the potential to be developed as a fungicide to control Phytophthora species.

72

5.2 Introduction

Phytophthora is a genus of plant-damaging oomycetes and cause significant economic losses as well as environmental damage. Phytophthora cinnamomi is one of the most invasive species of Phytophthora disseminated worldwide causing serious disease and mortality to ornamental and native plants, in agriculture, horticulture and forestry (Burgess et al., 2017a).

Although controlling any soilborne plant pathogen is difficult, methods of control for Phytophthora diseases include biological, chemical and cultural methods (Ahmed et al., 1999). Biological control of Phytophthora diseases has limited use and is mainly applicable to agriculture and horticulture (Smith et al., 1990, Tran et al., 2007, Xiao et al., 2002), but not natural ecosystems. Cultural techniques including drainage, sanitation, and clean plant stock, can reduce the impact and severity of Phytophthora diseases but cannot result in total control (Strömberg and Brishammar, 1991).

Chemical control is most commonly used due to its speediness, high efficiency and cost- effectiveness (Gisi, 2002). Phosphite is the most effective systemic fungicide to control many plant diseases caused by Phytophthora species (Hardy et al., 2001, Gómez-Merino and Trejo-

2– Téllez, 2015). Phosphite is the anionic form of phosphonic acid (HPO3 ) and can be applied as a soil drench, foliar spray or by trunk injection (Guest and Grant, 1991, Hardy et al., 2001), although its efficacy as a soil drench is limited. It acts both directly on the pathogen and indirectly through stimulation of host defence (Jackson et al., 2000). Excessive phosphite concentrations can result in phytotoxicity in horticultural crops (Seymour et al., 1994) and native species (Tynan et al., 2001, Barrett et al., 2004). Also, some Phytophthora species have tolerance to phosphite (Veena et al., 2010). Other fungicides such as metalaxyl, fosetyl-Al, mefenoxam, dimethomorph, and cymoxanil have been used against oomycete pathogens; however, many species of Phytophthora have developed resistance to these fungicides (Thomidis and Elena, 2001, Parra and Ristaino, 2001). Additionally, these fungicides do not stimulate plant defense to Phytophthora species, so they do not persist and therefore need frequent applications. Many of them are also considered to be toxic to humans. Consequently, there is a need to develop new fungicides to ensure on-

73

going protection of plants to disease and to reduce the risk of exposure of toxic chemicals to humans.

The advancement in the field of bioinorganic chemistry has highlighted the potential role of transition metal complexes as chemical treatments to microorganisms. Indeed, mixed transition ligand complexes are now widely used in therapeutic fields, and have been used as antimicrobial (Shebl et al., 2017, Chai et al., 2017, Taghizadeh et al., 2017), and anticancer agents (Van Rijt and Sadler, 2009). With respect to plant pathogens, few studies have used transition metal complexes to control pathogens and the diseases they cause(Liu et al., 2018, Smaili et al., 2017). In Chapter 4, the synthesis and characterization of Ca (II) and Zn (II) mixed-ligand complexes containing the amino acid L-valine and 2,2- bipyridine was described. The aim of the current study was to evaluate in vitro and in planta the efficacy of these compounds for the control of Phytophthora species, compared to phosphite.

5.3 Materials and Methods

The effect of phosphite and new chelates on Phytophthora cinnamomi in vitro

Calcium and zinc chelate were prepared as described in Chapter 4. Ribeiro’s modified medium (RMM) (Ribeiro, 1978) with 0.35 mM phosphate and the Zn and Ca chelate were used for liquid media. The pH of the medium was adjusted to 6.4 (using KOH) and autoclaved before addition of phosphite, Zn chelate or Ca chelate. Media were dispensed into 9 cm diameter Petri-dishes (25 ml of liquid Ribeiro’s modified medium).

The P. cinnamomi isolate MP94-48 was used in this study. Briefly, the isolate was grown on V8 agar plates for 7 days in the dark at 25°C, after which 5 mm diameter plugs were transferred to the RMM plates containing the different phosphite, Zn chelate and Ca chelate treatments. The P. cinnamomi isolate was grown in liquid RMM at an initial pH of 6.4 with and without the addition of 0.03 M MES (2-(N-morpholino)ethanesulfonic acid) and at different concentrations (0, 0.005, 0.40, 0.8 and 0.16, g/l) of phosphite or the chelate complexes. Inoculated plates (3 replications)

74

were sealed with Parafilm® and incubated in the dark at 25°C without shaking. Mycelial growth was assessed by measuring the mycelial biomass dry weight after 7 days. Briefly, the mycelia were lifted out of the liquid, blotted dry with filter paper and dried in an oven at 70°C for one day before weighing. The EC50 values were computed from plots of the percent inhibition at different phosphite and Zn chelate or Ca chelate concentrations compared to growth in the RMM without treatments.

The effect of phosphite and new chelates on Phytophthora cinnamomi in planta

Two P. cinnamomi susceptible woody plant species (Banksia grandis and Eucalyptus marginata) (McDougall et al., 2001, Tynan et al., 2001) native to the south-west of Western Australia were used as hosts. Seeds were obtained from Nindethana Seed Service Pty Ltd. (1007 Chester Pass Rd, King River WA 6330) and germinated in trays in the glasshouse. After germination, plants were transferred to individual pots (Garden City Plastics, Canning Vale, WA, 1.5L pot). Pasteurised washed river sand was the growth medium. The sand was steam pasteurised for at least two hours at 65 °C. A sterile 10 mL plastic tube was pressed into the sand in each pot to retain the space for later insertion of the inoculum minimizing any root damage.

After 3 months, fourteen pots of each plant species were sprayed with one of six treatments (I) control pots spayed only with water, (II) 0.25% phosphite (iii) 0.5% phosphite (IV) 0.25% Ca chelate, (V) 0.5% Ca chelate, (VI) Ca chelate (0.125%) + phosphite (0.125%) (VII) Ca chelate (0.25%) + phosphite (0.25%). There were an additional two treatments for E. marginata (VIII) 0.25% Zn chelate and (IX) 0.5% Zn chelate. The plants were sprayed to run-off two days prior to inoculation and the container substrate was covered with plastic to prevent the chemicals from reaching the soil.

Phytophthora cinnamomi isolate MP94-48 was used and vermiculite inoculation was prepared as described previously (Belhaj et al., 2018). After infecting an apple by P cinnamomi, inoculated into 500 mL Erlenmeyer flask has vermiculite substrate (100mL vermiculite, 4 g millet [Panicum miliaceum] seeds and 80mL V8 broth). The inoculation was inserted 48 hours after the foliage was sprayed with the chemical treatments. At the time of inoculation, the plastic tubes were 75

removed and 15 g of vermiculite {prepared as described by Belhaj et al (2018)} (1% of the weight of sand in a pot) was inserted into the holes. Half the pots of each spray treatment were inoculated with P. cinnamomi inoculum, sterile vermiculite was used for the non-inoculated controls. The pots were then flooded for 24 hours so that water reached the surface of the soil. The flooding was repeated every two weeks. Pots were arranged in a randomized complete block design in an evaporatively cooled glasshouse at Murdoch University. The trial was conducted in 2017 between February and July; after inoculation, the mean maximum and minimum temperatures were 26.6oC and 15.6oC, respectively. Plants were watered daily with deionized water and fertilized weekly with 2g/5L of Thrive (Yates Company, Australia, Ingredients: Nitrogen, phosphorus, potassium and trace elements). The experiment was repeated once.

Assessment of host plants at harvest and re-isolation of Phytophthora were conducted 8 weeks after inoculation and by using a method were described by Belhaj et al. (2018). The roots and shoots were harvested, washed and dried then the weight of them were measured. The root damage was rated 1 to 4 (1= no damage, 4= severe root damage)

Statistical analysis

Both the in vitro and in planta experiments were conducted twice; the results between each experiment were similar and so the data were combined. ANOVA was used to analyse the variance and treatment means. The significance level of data analysis was 0.05. Statistical calculations were performed using Excel 2016 (analysistoolPak-VBA).

76

5.4 Results

The effect of phosphite and new chelates on Phytophthora cinnamomi in vitro

The mycelial growth of P. cinnamomi was significantly greater for all chemical treatments when grown in the liquid media with the addition of MES (2-(N-morpholino)ethanesulfonic acid ) buffer than without MES (Fig 5.1, Table 5.1). At 0.005 g/l of all chemical treatments (with and without MES buffer) the mycelial growth did not differ statistically from the control. For the Ca chelate, Ca chelate + phosphite treatments at 0.04, 0.08, and 0.16 g/l as well as phosphite at 0.16 with and without MES, and phosphite at 0.08 without MES, mycelial growth differed statistically to the control. In contrast, mycelial growth in all concentrations of Zn chelate with and without MES buffer in liquid media did not differ to the control. The Ca chelate + phosphite reduced mycelial growth of P. cinnamomi more than any of the other treatments tested, followed by Ca chelate (Table 5.1).

77

Table 5.1. Dry weights (±SE) and EC50 values of Phytophthora cinnamomi grown in liquid Ribeiro’s modified medium in the presence of different chemical treatments with and without the addition of 0.03 M MES. For each column, values with the same letter are not significantly different (P≤ 0.05).

With MES Without MES

1 Dry weight Dry weight Treatments Conc. g/l EC50 EC50 (µg) (µg)

Control 0 10.04±0.26 d 100 2.14+0.21 d 100

Phosphite 0.005 10.00±0.45 d 100 2.14+0.08 d 100

Phosphite 0.04 9.85±0.24 d 99 1.90+0.12 d 87

Phosphite 0.08 9.42±0.21 d 96 1.57+0.21c 73

Phosphite 0.16 6.71±0.49c 71 1.38+0.33b 67

Ca chelate 0.005 9.90±0.39 d 99 2.09+0.53 d 100

Ca chelate 0.04 6.09±0.46c 62 1.42+0.33b 67

Ca chelate 0.08 5.52±0.09b 59 1.28+0.14b 60

Ca chelate 0.16 4.90±0.09b 49 1.04+0.25a 47

Zn chelate 0.005 10.00±0.08 d 100 2.14+0.08 d 100

Zn chelate 0.04 9.95±0.20 d 100 2.00+0.08 d 93

Zn chelate 0.08 9.90±0.25 d 99 2.00+0.21 d 93

Zn chelate 0.16 9.47±0.50 d 94 1.85+0.16 d 87

Ca chelate + Phosphite 0.005 10.04±0.26 d 100 2.14+0.08 d 100

Ca chelate + Phosphite 0.04 5.93+0.43c 60 1.41+0.02b 67

Ca chelate + Phosphite 0.08 4.52+0.15b 46 1.21+0.09c 73

Ca chelate + Phosphite 0.16 3.83+0.21a 39 1.00+0.02a 47

1 For the Ca chelate + phosphite mix this is the total concentration as half that amount of each chemical was added

78

A

B

Figure 5.1. Colony growth of Phytophthora cinnamomi grown in liquid Ribeiro’s modified medium in the presence of Ca chelate with Phosphite (a) with the addition of 0.03 M MES (b) and without the addition of 0.03 M MES the concentration of the chemical of g/l

79

The effect of phosphite and new chelates on Phytophthora cinnamomi in planta

All non-treated and non-inoculated (positive control) plants of B. grandis (Fig. 5.2e) and E. marginata (Fig. 5.3e) remained healthy for the duration of the trial, and no P. cinnamomi was isolated from them. The highest root damage and the largest reduction of root dry weight were recorded in the inoculated unsprayed (negative control) plants (Fig. 5.2a, 5.3a). In the presence of P. cinnamomi, two untreated plants of E. marginata and three untreated plants of B. grandis and five plants of E. marginata treated with Zn chelate died, and P. cinnamomi was reisolated from them.

There was no significant difference in root dry weight and root damage of plants treated with Zn chelate at all concentrations compared to the negative control (inoculated with P. cinnamomi) plants (Table 5.2). There were significant improvements in root dry weight and root damage of plants treated with Ca chelate, phosphite and Ca chelate + phosphite when compared to the negative control plants (Table 5.2, Figs. 5.2, 5.3).

At 0.25%, the root dry weights and root damage score of both plant species inoculated with P. cinnamomi and treated with phosphite only (Figs. 5.2b, 5.3b) did not differ (P>0.05) to the negative control plants (Figs. 5.2a, 5.3a). Ca chelate (Figs 5.2c, 5.3c) significantly reduced the detrimental effect of P. cinnamomi on the root dry weights and root damage scores of plants at the same concentration (0.25 %) (Table 5.2).

At 0.5% Ca chelate (Figs. 5.2g, 5.3g), phosphite (Figs. 5.2f, 5.3f) and Ca chelate + phosphite (Figs. 5.2h, 5.3h) applied to both plant species showed significant differences in dry weights and root damage scores compared with negative control plants (Figs. 5.2a, 5.3a). Ca chelate + phosphite at 0.5 % gave the greatest improvement in reducing the effects of P. cinnamomi followed by Ca chelate alone (Table 5.2).

80

Table 5.2: The effect of chemical treatments on Phytophthora cinnamomi root rot development on (a) Eucalyptus marginata and (b) Banksia grandis in planta. For each column, values with the same letter are not significantly different (data for each host analysed separately) (P≤ 0.05)

Root Root Dry Shoot Dry Total Dry Treatment Conc.1% Inoc.2 Damage Weight (g) Weight (g) Weight (g) Score

(a) Eucalyptus marginata

Ca chelate 0.25 + 0.71±0.29b 0.87±0.07d 3.67±0.41b 4.54±0.44b

Ca chelate 0.5 + 0.57±0.30b 0.93±0.13c 3.78±0.51b 4.71±0.57b

Phosphite 0.25 + 3.00±0.22de 0.57±0.02f 2.17±0.18e 2.44±0.18e

Phosphite 0.5 + 1.71±0.36c 0.65±0.07e 3.05±0.27c 3.70±0.23c

Ca chelate + Phosphite 0.25 + 2.71±0.29d 0.62±0.12e 2.58±0.50d 3.20±0.54d

Ca chelate + Phosphite 0.5 + 0.29±0.18ab 0.99±0.10b 4.01±0.62a 5.00±0.63 a

Zn chelate 0.25 + 3.00±0.32de 0.52±0.05g 1.80±0.24 f 2.32±0.29e

Zn chelate 0.5 + 3.40±0.24e 0.47±0.03g 1.60±0.19f 2.07±0.17f

Negative control 0 + 3.14±0.26de 0.50±0.07f 1.86±0.24 e 2.36±0.26e

Ca chelate 0.25 - 0.00±0.00 a 1.04±0.11a 4.30±0.36 a 5.34±0.42 a

Ca chelate 0.5 - 0.00±0.00 a 1.9±0.15a 4.50±0.45 a 5.64±0.52 a

Phosphite 0.25 - 0.00±0.00 a 1.04±0.09a 4.27±0.32 a 5.31±0.27 a

Phosphite 0.5 - 0.00±0.00 a 1.04±0.11a 4.23±0.40 a 5.27±0.44 a

Ca chelate + Phosphite 0.25 - 0.00±0.00 a 1.05±0.07a 4.27±0.31 a 5.34±0.34a

Ca chelate + Phosphite 0.5 - 0.00±0.00 a 1.05±0.14a 4.27±0.31 a 5.32±0.38 a

Zn chelate 0.25 - 0.00±0.00 a 0.99+0.22b 3.28±0.27c 4.27±0.46b

Zn chelate 0.5 - 0.00±0.00 a 0.93±0.22c 1.82±0.24f 2.75±0.26e

Positive control 0 - 0.00±0.00a 1.08±0.12a 4.27±0.42a 5.35±0.51 a

(b) Banksia grandis

Ca chelate 0.25 + 1.14±0.34b 2.23±0.24c 8.93±0.12d 11.16±0.29d

Ca chelate 0.5 + 1.00±0.31b 2.50±0.33b 9.69±0.27c 12.19±0.58c

Phosphite 0.25 + 2.71±0.18d 1.35±0.19d 8.01±0.17f 9.36±0.27e

Phosphite 0.5 + 1.86±0.14c 2.09±0.21c 8.89±0.25d 10.98±0.32d

Ca chelate + Phosphite 0.25 + 2.57±0.20d 1.41±0.23d 8.29±0.21e 9.70±0.33e

81

Root Root Dry Shoot Dry Total Dry Treatment Conc.1% Inoc.2 Damage Weight (g) Weight (g) Weight (g) Score

Ca chelate + Phosphite 0.5 + 0.71±0.29a 2.79±0.23b 10.04±0.22 b 12.83±0.30b

Negative control 0 + 2.86±0.26e 1.32±0.12d 7.73±0.29f 9.05±0.37f

Ca chelate 0.25 - 0.00±0.00a 3.70±0.26a 10.45±0.25 a 14.15±0.50 a

Ca chelate 0.5 - 0.00±0.00a 3.75±0.31a 10.50±0.29 a 14.25±0.56 a

Phosphite 0.25 - 0.00±0.00a 3.34±0.29a 10.24±0.27 a 13.58±0.45 a

Phosphite 0.5 - 0.00±0.00a 3.35±0.32a 10.70±0.34 a 14.05±0.53 a

Ca chelate + Phosphite 0.25 - 0.00±0.00a 3.41±0.22a 10.40±0.19 a 13.81±0.34 a

a a a Ca chelate + Phosphite 0.5 - 0.00±0.00a 3.51±0.23 10.50±0.29 14.01±0.39

Positive control 0 - 0.00±0.00a 3.34±0.25a 10.44±0.31 a 13.78±0.33 a

1concentration of the chemical. For the Ca chelate + phosphite mix this is the total concentration as half that amount of each was added

2inoculations with Phytophthora cinnamomi

82

Fig 5.2: The effect of chemical treatments on Phytophthora cinnamomi in Banksia grandis

83

Fig 5. 3: The effect of chemical treatments on Phytophthora cinnamomi in Eucalyptus marginata

5.5 Discussion

The results from the in vitro experiment correspond with those obtained from the in planta glasshouse experiment. Phytophthora cinnamomi caused damage to plant roots within 8 weeks and its damage was reduced significantly by all the chemical treatments except for the Zn chelate. The Ca chelate was more effective than phosphite in reducing the damage of P. cinnamomi to plants. However, when Ca chelate was applied together with phosphite, there was a synergistic

84

effect with a greater negative effect on P. cinnamomi than when either treatment was applied alone. The effect of the chemicals on P. cinnamomi varied significantly between concentrations.

The Zn chelate did not control P. cinnamomi in vitro or in planta, and it also caused damage in the non-inoculated control plants of E. marginata and made them more susceptible to P. cinnamomi. At 0.5% Zn chelate root damage was higher and root weights were less than in the negative control (P. cinnamomi infected) plants. At high concentrations, Zn can detrimentally impact on plant growth, photosynthetic activity, water relationships, and metabolism (Apel and Hirt, 2004).

Phosphite is a systemic fungicide applied to manage oomycete pathogens and in particular Phytophthora species (Hardy et al., 2001, Shearer and Fairman, 2007, Stasikowski et al., 2014a). The results of the effects of phosphite to control P. cinnamomi in both experiments agreed with previous studies, where phosphite reduces lesions through the stimulation of plant defense responses and/or inhibition of pathogen growth (Fenn and Coffey, 1984, Guest and Grant, 1991, Jackson et al., 2000, Stasikowski et al., 2014c). The results of the present study show that 0.5% phosphite effectively reduced root damage by P. cinnamomi, but not to the same degree as the Ca chelate at 0.5 %, 0.25%alone and 0.25% phosphite and 0.25% Ca chelate combined. A possible explanation for the significant effect of Ca chelate might be that calcium ions stimulate plant defenses (Stab and Ebel, 1987, Sugimoto et al., 2008), and may inhibit the growth of Phytophthora species by suppression of sporangia formation (Messenger et al., 2000, Serrano et al., 2012), or a combination of both (Sugimoto et al., 2008). All plants sprayed with 0.25% Ca chelate + 0.25% phosphite gave the highest level of resistance to P. cinnamomi. Likewise, Ca chelate + phosphite had the highest effect on P. cinnamomi in vitro. This may be due to synergistic effects of Ca and phosphite. Future studies should also examine 0.5% Ca chelate and 0.5% phosphite combined to determine if the control of P. cinnamomi in planta is further improved.

85

5.6 Conclusion

This study provides strong evidence on the potential efficacy of the Ca chelate to be used as an alternative to phosphite or in conjunction with phosphite to control P. cinnamomi and the diseases it causes. However, there are a number of challenges to overcome before the Ca chelate can be used effectively against Phytophthora species. These challenges include testing the efficacy of Ca chelate on other Phytophthora species, testing the efficacy of Ca chelate and its persistence on a wider range of plant species particularly with native plants, testing the effectiveness of Ca chelate under field conditions, determining if Ca chelate has any effect on the metabolism of Phytophthora species, and determining the mode of action of the Ca chelate in plants.

86

6 Chapter Six

General Discussion

87

6.1 Findings of Thesis

This thesis has demonstrated that numerous Phytophthora species potentially contribute to urban forest tree declines in WA. It has also highlighted some of the management tools available including new chemical treatments. The major findings include:

I. High throughput sequencing was shown to be a powerful tool in elucidating the presence of Phytophthora species associated with declining trees. Forty-Five Phytophthora phylotypes were detected from 168 out of 236 sites within the 91 parks sampled, all samples collected from sites that consist of declining groups of plants. In contrast, rhizosphere baiting only isolated four Phytophthora species from 24 sites. II. Remote sensing by using high-resolution multispectral imagery is a potentially useful tool to monitor tree health. III. There was no structuring of the Phytophthora community based on site, soil type, park type or any of the other, including the remote plant healthy parameters measured. It appears that there were no factors limiting the diversity of Phytophthora species in the urban environment. IV. The distribution of P. multivora was influenced by three environmental factors (park size and soil type and unit) while park type had an effect on the distribution of P. cinnamomi. Also, although canopy health was not correlated with Phytophthora communities, it was correlated with the presence of P. multivora and of P. cinnamomi. V. The 19 Phytophthora species screened for pathogenicity were able to cause lesions in at least three of the 15 plant species tested. There were clear differences in the ability of the different Phytophthora species to cause lesions in the different plant species. Phytophthora cinnamomi, P. pseudocryptogea, and P. citrophthora caused significant lesions in the plant species they infected. VI. Two novel chemical ligands [Zn (Val)2(bipy)] and [Ca(Val)2(bipy)] were synthesized, and their physical and chemical structures validated. VII. The Ca chelate was shown to be more efficient than phosphite both in vitro and in planta. The Ca chelate (0.5% and 0.25%), phosphite (0.5%) and the Ca chelate (0.25%) + phosphite

88

(0.25%) combined were able to control disease expression of P. cinnamomi when applied as a foliar application to two plant species. The Ca chelate was more effective than phosphite in controlling P. cinnamomi. Therefore, the Ca chelate is a potential alternative to phosphite for the control of Phytophthora species.

6.2 Phytophthora community

In the present study, forty-five Phytophthora phylotypes were detected from 169 of 236 sites across 69 of 91 urban forest parks sampled in association with different tree species exhibiting decline symptoms based on high-resolution multispectral imagery (Table 2.3). What stands out from this study is the Phytophthora species richness across the urban forest sites sampled, which included Phytophthora species from eight clades. This provides strong evidence for urban forests potentially being a conduit of invasive Phytophthora species into natural environments. Although not documented in this study, it also highlights the role human activities play in this conduit, as in urban areas human activities are numerous and varied. These results compare favorably with the 49 Phytophthora species known to occur within Western Australia prior to this study. So, finding all these species within a relatively small urban forest was very interesting. Previously, 25 of these Phytophthora species were isolated from urban forests, 21 from agricultural crops (7 from annual crops and pastures and 14 from perennial and forest crops) and 25 from natural environments (Table 6.1).

Urban forest pathway of Phytophthora species

Urban forests are expected to grow in value to society as their importance in heightening peoples’ sense of well-being and their contributions to ecosystem services and functions become more appreciated. However, urban forests may represent a major pathway associated with the spread of pathogens to ecosystems outside urban environments. Although the current study did not examine the population structure of Phytophthora species in natural ecosystems adjacent to the sampled park sites, it does provide evidence for the potential dispersion of Phytophthora species from urban to natural ecosystems. Some studies indicated that when an area contained a particular invasive community, it could be a source of spread to new areas

89

(Miller et al., 2005, Kolbe et al., 2004, Downie, 2002, Floerl et al., 2009, Hanfling et al., 2002), and this phenomenon is called the bridgehead effect (Lombaert et al., 2010). In the bridgehead effect scenario, urban environments are an important source of spread of Phytophthora species into natural ecosystems via two major pathways: movement of plants and human activities. For example, Phytophthora ramorum was first found in the urban environment (Rizzo et al., 2002, Grunwald et al., 2012) and then spread into other environments where nursery grown plants had been out-planted (Herrero et al., 2006). Redondo (2018) showed that Phytophthora communities were higher in urban forests than in natural forests, and human activities were pathways during the invasion process. Hernandez-Lambrano et al. (2018) demonstrated that human-altered areas are a common reservoir of soil pathogens and may increase the risk of dispersal into natural or semi-natural areas. Also, human activities would potentially make the site more conducive to disease development. For example, soil compaction associated with increased foot traffic could result in changes in the soil moisture status that would favor sporulation and infection of susceptible hosts. Given these scenarios, in multiple forms, urban forests represent an important pathway for Phytophthora dispersal into the wider environment. However, further investigations are required to prove that urban areas are a more likely bridgehead for Phytophthora species than natural areas.

When these results are combined with previous results for Phytophthora species detected in Western Australia (Table 6.1), 63 species (including species recorded in this study) from 9 clades have been detected in Western Australia. Of the 63 species, 56 are recorded in the urban forest. This result somewhat reflects the work of Hulbert et al. (2017) who estimated that "approximately 110 species will be described based on type specimens found in urban environments". Surprisingly, 7 species (P. asparagi, P. parvispora, P. syringae, P. aquimorbida, P. insolita, P. baylanboodja and P. condilina) previously isolated in the Western Australian urban forest (Burgess et al., 2017b) were not recorded in the current study, while 26 species (including three putatively new species and four designated species) were detected for the first time in association with urban trees in Western Australia. This does not mean that these species were not present in urban ecosystems before but may reflect that few studies that have been

90

conducted in the urban forests including nurseries, or that have been restricted to small areas. Indeed, most of these species were recovered in environments elsewhere in Western Australia. Another interesting finding from this study is that of the 63 species, 56% are introduced species and 30% are considered native species in Western Australia. Overall, the results support the consensus that globally many Phytophthora species are invasive plant pathogens that are potential spread and via the nursery trade and that urban forest and landscapes should be considered as the primary pathway for Phytophthora species invasions, particularly introduced species. Finally, this study supports the idea of concentration monitoring efforts in urban environments because this will enhance the early detection and prompt response needed to inhibit the inadvertent spread of Phytophthora species into natural and agricultural ecosystems.

Table 6.1. The first date of recovery of Phytophthora species from different environments in Western Australia. Most records have been confirmed by molecular data, but a few older records are based on morphology (see footnotes). In the current study, species were detected by metabarcoding of eDNA and some based on morphology

agriculture agriculture This urban and (annual (perennial) natural Phytophthora species1 Clade2 Status3 study4 nursery5 crops and and ecosystem5 pasture)5 forestry5

P. cactorum 1 I + 2014 2006

P. clandestina 1 I 1985

P. nicotianae 1 I + 2004 1989 2004

P. aff meadii 2 I ++

P. capensis 2 I +

P. capsici 2 I ++

P. citricola7 2 I + 1992

P. citrophthora 2 I + 2015 1923

P. frigida 2 I +

P. multivora 2 I + 1985 1986

P. pachypleura/plurivora 2 I +

P. palmivora 4 I + 2011

P. asparagi 6 I 2016 2007

P. bilorbang 6 I + 2012

91

agriculture agriculture This urban and (annual (perennial) natural Phytophthora species1 Clade2 Status3 study4 nursery5 crops and and ecosystem5 pasture)5 forestry5

P. chlamydospora 6 I 1995

P. crassamura 6 I + 2016 1992

P. gonapodyides 6 I ++

P. inundata 6 I + 2011 2005 1986

P. lacustris 6 I + 1995

P. megasperma7 6 I 1995

P. ornamentata 6 I 2016

P. sp. personii 6 I 2005

P. cambivora6 7 I + 1991

P. cinnamomi 7 I + ? 1960 1967

P. niederhauserii 7 I + 2012 2002 2014

P. parvispora 7 I 2017

P. cryptogea7 8 I + 2015 ?

P. drechsleri6 8 I + ?

P. erythroseptica 8 I + 1992

P. hibernalis6 8 I ?

P. porri6 8 I ?

P. sp kelmania 8 I + 2016

P. syringae 8 I 2015

P. aquimorbida 9 I 2017

P. insolita 9 I 2004

P. boodjera 4 I/N + 2011 2006

P. sp. pecan 4 I/N ++

P. baylanboodja 6 I/N 2011

P. condilina 6 I/N 2008

P. gibbosa 6 I/N 2009

P. gregata 6 I/N + 2015 1982 1996

92

agriculture agriculture This urban and (annual (perennial) natural Phytophthora species1 Clade2 Status3 study4 nursery5 crops and and ecosystem5 pasture)5 forestry5

P. rosacearum 6 I/N + 2015 1989

P. sp. walnut 6 I/N ++

P. pseudocryptogea 8 I/N + 2016 1979 1986

P. sp. AUS1D 1 N ++

P. elongata 2 N + 1989

P. sp. AUS2A 2 N +

P. sp. AUS2B 2 N ++

P. arenaria 4 N + 1986

P. amnicola 6 N + 1995

P. cooljarloo 6 N 1996

P. fluvialis 6 N + 1997

P. kwongonina 6 N + 2010 1993

P. litoralis 6 N + 2011 2006

P. moyootj 6 N + 2006

P. pseudorosacearum 6 N 1998

P. thermophila 6 N + 1995 1980 1998

P. sp. AUS8C 8 N ++

P. constricta 9 N + 1992

P. fallax 9 N +

P. gondwanense8 10 N 1996

P. sp. AUS11A 11 N +

P. versiformis 11 N + 2011 2011

Total no. species 45 26 7 14 26

1Phytophthora species including several taxa with designated names that are not yet described

2Phylogenetic Clade based on phylogeny of all known Phytophthora species

3Known status of species in Western Australia I = introduced, N = native, N? = putatively native

4+ species detected in the current study using high throughput sequencing, ++ species detected in the current study for the first time in Western Australia 93

5 Date of the first record for Western Australia within Australian databases.? denotes species reported within a database without a date attached to the record. Records for natural ecosystems in WA come from Vegetation Health Service (VHS), Department of Biodiversity, Conservation and Attractions. Records for urban environment come from VHS, CPSM, and PAB

6 These identities have not been confirmed by sequencing

7P. citricola, P. cryptogea, and P. megasperma were names commonly assigned to species prior to molecular re- evaluation, these have not been detected since and earlier reports are probably incorrect

8P. gondwanense reported as P. boehmeriae by D’Souza et al. (1997)

Phytophthora community and environmental filtering

Tree decline symptoms associated with Phytophthora are frequently associated with more than one species (Perez‐Sierra et al., 2013, Scanu et al., 2015, Gonzalez et al., 2017b). However, only a few studies have examined the diversity of Phytophthora species in a plant community (Parke et al., 2014, Vannini et al., 2013, Sims et al., 2015). Indeed, compared with determining community structure in natural ecosystems, evaluating Phytophthora community structure in an urban forest and the environmental factors which influence this community is challenging. This could be because, in urban forests, human activities are diverse and constant, which in turn introduce and change the biotic and abiotic factors that predispose urban forests to the introduction, establishment, and spread of new pathogens (Hernandez-Lambrano et al., 2018, Redondo, 2018). In this study, Phytophthora species were widespread and there appeared to be no environmental filtering. Many studies have reported that Phytophthora can be spread long- distances through moving infested soil by animals or humans along preferred pathways (Cushman and Meentemeyer, 2008, Webber and Rose, 2008).

6.3 Phytophthora species is associated with urban tree declines

Phytophthora multivora and Phytophthora cinnamomi

Phytophthora multivora was the most frequently detected species in this study. These results agree with those of Barber et al. (2013) who found P. multivora to be the most commonly isolated species in the City of Perth, Western Australia. Phytophthora multivora was described in 2009, where it was recovered from a wide range of native plants in Western Australia (Scott et al.,

94

2009). It has been associated with several hosts over a wide geographical range, including: Australia (Scott et al., 2009), New Zealand (Scott and Williams, 2014), South Africa (Nagel et al., 2015), Canada, Korea, Japan and a number of European countries (Mrazkova et al., 2011, Jung et al., 2016). It has commonly been assumed that P. multivora like P. cinnamomi, will impact multiple plant systems including agriculture, forestry and natural ecosystems (Scott and Williams, 2014). In the future, more work on the host range and pathogenicity of P. multivora should be conducted on native plant species to determine its potential to impact these plant species and communities.

Phytophthora cinnamomi was the fifth most frequently detected species in the City of Joondalup’s urban forest; however, it caused the longest lesions when underbark inoculated into excised stems of 14 of the 15-plant species commonly planted in the urban environment. Phytophthora cinnamomi is an invasive soil-borne species that is responsible for many serious tree diseases worldwide (Burgess et al., 2017a, Sena et al., 2018). In Australia, P. cinnamomi has been linked to tree declines across a range of forest ecosystems. For example, in South West Western Australia 2284 out of 6510 described plant species are listed as susceptible to P. cinnamomi, and 800 species are ranked as very susceptible to P. cinnamomi (Shearer et al., 2004). These results are very interesting because 20 years ago P. cinnamomi was the most frequently isolated Phytophthora species in the Perth environment. P. multivora which was also introduced to WA, has in the last decade been isolated very frequently, particularly in the urban environment. Its host range is also increasing, and it appears that it is possible it could be displacing P. cinnamomi. Potentially, if P. multivora is a good saprophyte as well as a strong pathogen, it could be outcompeting P. cinnamomi which is a poor saprophyte and a strong pathogen. Alternatively, P. multivora was introduced later into Western Australia via the nursery trade and is now displacing P. cinnamomi for the reasons given above in the urban areas where it was first and most widely introduced. Further work is required to establish if P. multivora is a good saprophyte and to establish if it is indeed displacing P. cinnamomi. For example, baiting techniques may be more conducive for P. multivora than P. cinnamomi.

95

Species of Phytophthora ITS Clade 6

This study also highlights that species within the Phytophthora ITS Clade 6 have a strong association with forest trees as evidenced by the 14 species from this clade that were isolated in this study. Similar observations have been made for this clade and their association with urban forest trees and riparian ecosystems by (Jung et al., 2011). Based on phylogenetic analysis, Clade 6 is divided into three sub-clades that include many species (Jung et al., 2011, Crous et al., 2012, Kroon et al., 2012, Nagel et al., 2013). Clade 6 has 20 described species and a number of other designated taxa (Burgess et al., 2018). It includes a number of aquatic species that feature a highly effective ecological strategy "as rapid colonisers of leaf litter and opportunistic pathogens of woody plants under flooded conditions" (Jung et al., 2011), as well as species that are aggressive soilborne pathogens of agricultural and horticultural crops such as P. rosacearum and P. megasperma (Jung et al., 2011).

Three species in sub-clade I (P. inundata, P. kwongonina, P. sp. walnut and P. rosacearum) were detected in this study. These species form non-papillate sporangia and are considered homothallic except P. inundata which is partially heterothallic (Brasier et al., 2003) and P. sp. walnut which is sterile in culture (Burgess et al., 2018). Phytophthora inundata was recorded for the first time in 1986 in Western Australian associated with declining Xanthorrhoea preissii (Stukely et al., 2007). In the present study, P. inundata was detected from 11 sites collected from declining urban trees, whilst P. kwongonina was detected from 24 sites.

Ten species in sub clade II (P. amnicola, P. bilorbang, P. crassamura, P. fluvialis, P. gonapodyides, P. gregata, P. lacustris, P. litoralis, P. moyootj and P. thermophila) were detected in this study. All these species form non-papillate sporangia and are considered homothallic except for P. litoralis, P. gonapodyides and P. fluvialis which are heterothallic (Table 6.2). Phytophthora amnicola was recently described from Western Australia (Crous et al., 2012), and was the fourth most frequently isolated species in the present study and produced lesions on 13 of the 15 plant species screened. It is possibly an emerging multi-host pathogen, so the further study is required to confirm its host range and pathogenicity.

96

Table 6.2: Some characteristics of Phytophthora taxa from ITS Clade 6 detected in the present study.

Phytophthora Clade Sub- Sex1 Chlamyd- Hyphal Antheridia Sporangia species clade ospores swellings

P. amnicola 6 2 Ho Absent Present non-papillate

P. bilorbang 6 2 Ho Absent Present non-papillate

P. crassamura 6 2 Ho Absent Present Paragynous non-papillate

P. fluvialis 6 2 He Absent Absent non-papillate

P. gonapodyides 6 2 He Absent Absent Amphigynous non-papillate

P. gregata 6 2 Ho Absent Absent non-papillate

P. inundata 6 1 He Absent Absent Amphigynous non-papillate

P. kwongonina 6 1 Ho Absent Absent Paragynous non-papillate

P. lacustris 6 2 ST Absent Absent non-papillate

P. litoralis 6 2 He Present Present non-papillate

P. moyootj 6 2 Absent Absent non-papillate

P. rosacearum 6 2,1 Ho Absent Present Paragynous non-papillate

P. sp. walnut 6 1 ST Absent Absent non-papillate

P. thermophila 6 2 ST Present Present Paragynous non-papillate

1 Ho= homothallic, ST=sterile, He=Heterothallic

Other Phytophthora species detected

Phytophthora arenaria from Clade 4 was the second most frequently isolated species and produced lesions in seven of the 15 plant species screened in the under-bark experiment. This homothallic species was originally isolated from declining Banksia species in south-west Western Australia (Rea et al., 2011b). Another important species is P. pseudocryptogea that was recovered from more than 10% of sites surveyed across four states in Australia (Tasmania, Victoria, New South Wales and Western Australia) (Burgess et al., 2017b). In the current study, P. pseudocryptogea was the second most frequently isolated species and was recovered from 75 sites across 37 parks and produced lesions in all 15-plant species tested in the underbark

97

inoculation trial and was the second most aggressive species after P. cinnamomi. Also, P. citrophthora produced lesions in all plants tested and was the third most aggressive species in the underbark pathogenicity trial. This outcome is noteworthy since this species was not recovered in natural ecosystems in previous investigations (Table 3.1) (Burgess et al., 2017b). All these species need more work to define their host range and pathogenicity in the future.

New Phytophthora species in Australia and Western Australia

In this study, three previously unrecorded phylotypes (P. sp. nov. 1D, P. sp. nov. 2B and P. sp. nov. 8C) representing putative new species were isolated, and they did not match any known species in the phylogenetic analysis (Table 6.1). Also, eight Phytophthora species (P. aff. meadii, P. sp. nov. 1D, P. sp. nov. 8C, P. sp. nov. 2B, P. sp. walnut, P. sp. pecan, P. capsici, and P. gonapodyides) have not previously been detected as well as five species (P. fallax, P. frigida, P. sp. nov. 11A, P. sp. nov. 2A and P. pachypleura) have not been isolated previously in Western Australia. All of these species need further study especially as they were associated with declining urban trees and especially as some of them (P. capsici and P. sp. walnut) were moderate to strongly pathogenic in the current study.

A major limitation of the current study was the method of pathogenicity testing as it used excised stems and under-bark inoculation hence by-passing a living host, and any potential defence responses associated with wounding and inserting the inoculum into the wound. A more appropriate method in future would be to undertake a pot trial with a range of putatively susceptible plant species and apply the inoculum to the soil and stimulate sporangial production and zoospore release. Although this excised stem and under-bark method has been used previously and shown to correlate well with trials conducted on living plants and soil inoculation with E. marginata and E. gomphocelphala (Hüberli et al., 2002, Scott, 2011). Consequently, there is still a need to conduct controlled trials with multiple isolates of these pathogens these pathogens on a range of hosts using soil infestation as a source of inoculation. Such studies are required to really understand their importance of these species as pathogens of trees.

98

6.4 Management

The presence of Phytophthora species in an urban forest represents a critical challenge to managers as they are linked to declines in urban forest health, and in turn will impact biodiversity conservation and other ecosystem functions. A comprehensive and integrated management program is recommended for the control of Phytophthora in the urban environment as no single treatment or approach can be guaranteed to control the diseases this genus causes. Chemical treatments represent an essential part of a management program, especially in urban landscapes where human activities, many of which have no concept of hygiene or quarantine and how pathogens move in landscapes, have spread Phytophthora species indiscriminately. Therefore, chemical treatments are frequently the only option available. Phosphite is the most efficient systemic fungicide currently available to manage Phytophthora species. It acts both directly on the pathogen and indirectly through stimulation of host resistance. However, if applied incorrectly excessive phosphite can cause phytotoxicity in horticultural crops (Seymour et al., 1994) and native species (Tynan et al., 2001, Barrett et al., 2004). Also, some Phytophthora species are tolerant to phosphite (Veena et al., 2010). Additionally, other fungicides that do not stimulate plant defense to Phytophthora species such as metalaxyl, fosetyl-Al, mefenoxam, dimethomorph, and cymoxanil do not persist and need frequent applications. Applications in long-lived trees are problematic due to the cost of the frequent applications needed and to the high likelihood or fungicide resistance occurring. Resistance to these chemicals has been shown for metalaxyl in P. infestans (Cohen and Reuveni, 1983), mefenoxam in P. capsici (Parra and Ristaino, 2001) and dimethomorph and cymoxanil in P. cactorum (Thomidis and Elena, 2001).

In the current study, a new chemical formulation was developed and successfully used to control P. cinnamomi. In vitro and in planta, the calcium chelate was shown to control P. cinnamomi more effectively than phosphite. Ca chelate (0.25%) combined with phosphite (0.25%) was also shown to reduce the pathogenicity of P. cinnamomi in planta, more effectively than when applied singly. Consequently, the Ca chelate is potentially a viable alternative to phosphite for the control of P. cinnamomi and potentially other Phytophthora species. The mode of action of Ca chelate on Phytophthora species is still not clear and requires further detailed study. However, calcium

99

ions are known to act on Phytophthora species by indirectly stimulating plant defenser (Stab and Ebel, 1987, Sugimoto et al., 2008), or directly by inhibiting sporangia formation (Messenger et al., 2000, Serrano et al., 2012) and restraining the action of pyrophosphatase by Phytophthora species (Sysuev et al., 1978) or by both indirect and direct mechanisms together (Sugimoto et al., 2008).

Indeed, there are many questions pertaining to the Ca chelate that need to be answered, including how frequently it would need to be applied to mature trees, can it be injected into trunks, does it work directly and indirectly, and are there any human/environmental safety issues associated with its use? Based on the elements used in the formulation of the Ca chelate, it is likely safe for the environment and humans, but this needs to be confirmed.

6.5 Future Research

The results from this study can be used to frame future studies around Phytophthora species and their role in urban forests and their possible spread into other ecosystems. For example, the impact of the species that were detected in this study needs to be understood by soil infestation studies under controlled glasshouse conditions to determine the host range and pathogenicity of the new Phytophthora species (Belhaj et al., 2018). Such studies will contribute to a better understanding of the potential effects of these Phytophthora species on plant health and biodiversity in urban forests. There is also a need to determine the life cycles including potential saprophytic ability of these new species or other recently described species Whether species can survive in association with intolerant hosts and how long they can persist in a soil in the absence of hosts are important characteristics that need additional study. A number of these Phytophthora species were isolated from the same rhizosphere sites as each other. For example, 21 species were detected together, so an obvious question is how these different species interact with each other and what impact do they have on tree health. A number of studies have mentioned that the occurrence of Phytophthora diseases needs more than one species of Phytophthora present (Perez‐Sierra et al., 2013, Scanu et al., 2015, Redondo, 2018). This increases the importance of studying the Phytophthora community in its entirety.

100

The high-resolution multispectral imagery was shown to be a useful tool to determine remotely where trees were in decline. Given that no Phytophthora species were detected from about 30% of the sampled sites with declining trees suggests that a better understanding of the biotic and abiotic factors that affect tree health in urban forests in WA is needed. The potential complex of factors that contribute to the declining health of trees, may in part help explain why the there was no correlation between the canopy health remote sensing data and the presence of Phytophthora communities in the soil. As indicated that all samples collected from sites that consist of declining groups of plants. So, for more understanding, samples from sites that did not consist of declining groups of plants should be collected and be compared with these results.

Future work could include determining if high-resolution multispectral imagery can be used to detect changes in hosts that indicate whether Phytophthora is present or not in the host. It is possible that the spectral signatures might differ between different host species and Phytophthora species, further controlled glasshouse trials are needed to determine if high- resolution multispectral imagery as a tool can be used more strategically to inform managers. The role of urban forests as a pathway to other areas suggests that the research should be targeted towards populated areas (Hulbert et al., 2017). The new Ca chelate chemical developed in this study should be tested in more detail in a range of different hosts to check if this chemical works (i) on different Phytophthora species, (ii) across different host (genera, families), (iii) if different hosts can take more or less of the chemical without showing phytotoxicity symptoms, and (iv) determine the persistence of the Ca chelate in planta and the duration of its effectiveness before reapplication is required.

6.6 Conclusions

Although more work is required as described above, Phytophthora species have the potential to impact many plants in the City of Joondalup’s urban forest in Western Australia. A diverse population of Phytophthora species were found in soils associated with about 70% of the sites where declining trees were identified by remote sensing. The new Ca chelate developed during this study has potential as a future oomycete fungicide as it was shown to control P. cinnamomi on two hosts 101

References

Abad, Z. G., Abad, J. A., Cacciola, S. O., Pane, A., Faedda, R., Moralejo, E., Pérez-Sierra, A., Abad- Campos, P., Alvarez-Bernaola, L. A. & Bakonyi, J. 2014. Phytophthora niederhauserii sp. nov., a polyphagous species associated with ornamentals, fruit trees and native plants in 13 countries. Mycologia, 106, 431-447.

Abdulridha, J., Ehsani, R. & De Castro, A. 2016. Detection and differentiation between Laurel Wilt disease, Phytophthora disease, and salinity damage using a hyperspectral sensing technique. Agriculture, 6, 56-69.

Aghighi, S., Fontanini, L., Yeoh, P., Hardy, G. E. S. J., Burgess, T. & Scott, J. 2014. A conceptual model to describe the decline of European blackberry (Rubus anglocandicans), a weed of national significance in Australia. Plant Disease, 98, 580-589.

Agrios, G. N. 2005. Plant Pathology. 5th eds. Department of Plant Pathology. The University of Florida. The United States of America.

Ahmed, S. A., Pérez‐Sánchez, C., Egea, C. & Candela, M. 1999. Evaluation of Trichoderma harzianum for controlling root rot caused by Phytophthora capsici in pepper plants. Plant Pathology, 48, 58-65.

Alaruikka, D., Kotze, D. J., Matveinen, K. & Niemelä, J. 2002. Carabid beetle and spider assemblages along a forested urban-rural gradient in southern Finland. Journal of Insect Conservation, 6, 195-206.

Alcoforado, M. J. & Matzarakis, A. 2010. Urban climate and planning in different climatic zones. Geographicalia, 5-39.

Alford, R. A. & Richards, S. J. 1999. Global amphibian declines: a problem in applied ecology. Annual Review of Ecology and Systematics, 30, 133-165.

Aliyu, H. & Isyaku, S. 2010. Spectroscopic and potentiometric studies of N-(2-Hydroxybenzyl)-L- α-Valine Cobalt (II) Complex. Biokemistri, 22, 91-97.

Allen, C. D., Macalady, A. K., Chenchouni, H., Bachelet, D., Mcdowell, N., Vennetier, M., Kitzberger, T., Rigling, A., Breshears, D. D. & Hogg, E. T. 2010. A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests. Forest Ecology and Management, 259, 660-684.

102

Alvey, A. A. 2006. Promoting and preserving biodiversity in the urban forest. Urban Forestry & Urban Greening, 5, 195-201.

Anderson, I. C. & Cairney, J. W. 2004. Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques. Environmental Microbiology, 6, 769-779.

Anderson, M. J. 2005. PERMANOVA: a FORTRAN computer program for permutational multivariate analysis of variance. Department of Statistics, University of Auckland, New Zealand.

Anderson, P., Brundrett, M., Grierson, P. & Robinson, R. 2010. Impact of severe forest dieback caused by Phytophthora cinnamomi on macrofungal diversity in the northern jarrah forest of Western Australia. Forest Ecology and Management, 259, 1033-1040.

Andrew, M. E., Wulder, M. A. & Nelson, T. A. 2014. Potential contributions of remote sensing to ecosystem service assessments. Progress in Physical Geography, 38, 328-353.

Apel, K. & Hirt, H. 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annual Review of Plant Biology, 55, 373-399.

Arnot, D. E., Roper, C. & Bayoumi, R. A. 1993. Digital codes from hypervariable tandemly repeated DNA sequences in the Plasmodium falciparum circumsporozoite gene can genetically barcode isolates. Molecular and Biochemical Parasitology, 61, 15-24.

Augspurger, C. 1990. Spatial patterns of damping-off disease during seedling recruitment in tropical forests. Pests, pathogens and plant communities., 131-144.

Aukema, J. E., Leung, B., Kovacs, K., Chivers, C., Britton, K. O., Englin, J., Frankel, S. J., Haight, R. G., Holmes, T. P. & Liebhold, A. M. 2011. Economic impacts of non-native forest insects in the continental United States. PLoS One, 6, e24587.

Baker, K. F. & Matkin, O. 1978. Detection and control of pathogens in water. Ornamentals Northwest Newsletter, 2, 12-14.

Balmford, A., Moore, J. L., Brooks, T., Burgess, N., Hansen, L. A., Williams, P. & Rahbek, C. 2001. Conservation conflicts across Africa. Science, 291, 2616-2619.

Balci, Y. & Halmschlager, E. 2003. Phytophthora species in oak ecosystems in Turkey and their association with declining oak trees. Plant Pathology, 52, 694-702.

103

Barber, P., Paap, T., Burgess, T., Dunstan, W. & Hardy, G. E. S. J. 2013. A diverse range of Phytophthora species are associated with dying urban trees. Urban Forestry & Urban Greening, 12, 569-575.

Barham, R. O., Marx, D. H. & Ruehle, J. 1974. Infection of ectomycorrhizal and nonmycorrhizal roots of Shortleaf pine by nematodes and Phytophthora cinnamomi. Phytopathology, 64, 1260-1264.

Baron, M., Pineda, M. & Pérez-Bueno, M. L. 2016. Picturing pathogen infection in plants. Journal of Natural Science, 71, 355-368.

Barrett, S., Shearer, B. & Hardy, G. S. J. 2004. Phytotoxicity in relation to in planta concentration of the fungicide phosphite in nine Western Australian native species. Australasian Plant Pathology, 33, 521-528.

Belhaj, R., Mccomb, J., Burgess, T. & Hardy, G. S. J. 2018. Pathogenicity of 21 newly described Phytophthora species against seven Western Australian native plant species. Plant Pathology, 67, 1140-1149.

Bessell-Browne, J. 1990. Kings Park soil survey. Department of Agriculture and Food, Western Australia. 103,1-12.

Bhattacharya, P. K., Lawson, H. J. & Barton, J. K. 2003. 1H NMR studies of nickel (II) complexes bound to oligonucleotides: a novel technique for distinguishing the binding locations of metal complexes in DNA. Inorganic Chemistry, 42, 8811-8817.

Bolund, P. & Hunhammar, S. 1999. Ecosystem services in urban areas. Ecological Economics, 29, 293-301.

Branzanti, M. B., Rocca, E. & Pisi, A. 1999. Effect of ectomycorrhizal fungi on chestnut ink disease. Mycorrhiza, 9, 103-109.

Brasier, C. 2008. The biosecurity threat to the UK and global environment from international trade in plants. Plant Pathology, 57, 792-808.

Brasier, C. M. 1996. Phytophthora cinnamomi and oak decline in southern Europe. Environmental constraints including climate change. Annales des Sciences Forestieres, 53, 347-358.

Brasier, C. M., Sanchez-Hernandez, E. & Kirk, S. A. 2003. Phytophthora inundata sp. nov., a part heterothallic pathogen of trees and shrubs in wet or flooded soils. Mycological Research, 107, 477-484. 104

Broadbent, P. & Baker, K. F. 1974. Behaviour of Phytophthora cinnamomi in soils suppressive and conducive to root rot. Australian Journal of Agricultural Research, 25, 121-137.

Bryla, D. R. & Linderman, R. G. 2007. Implications of irrigation method and amount of water application on Phytophthora and Pythium infection and severity of root rot in highbush blueberry. HortScience, 42, 1463-1467.

Burger, K. 1973. Coordination Chemistry: Experimental Methods, Butterworth, London. Burgess, T., Simamora, A., White, D., Wiliams, B., Schwager, M., Stukely, M. & Hardy, G. S. J. 2018. New species from Phytophthora Clade 6a: evidence for recent radiation. Persoonia- Molecular Phylogeny and Evolution of Fungi, 41, 1-17.

Burgess, T. I., Scott, J. K., McDougall, K. L., Stukely, M. J., Crane, C., Dunstan, W. A., Brigg, F., Andjic, V., White, D. & Rudman, T. 2017a. Current and projected global distribution of Phytophthora cinnamomi, one of the world's worst plant pathogens. Global Change Biology, 23, 1661-1674.

Burgess, T. I., Webster, J. L., Ciampini, J. A., White, D., Hardy, G. E. S. & Stukely, M. J. 2009. Re- evaluation of Phytophthora species isolated during 30 years of vegetation health surveys in Western Australia using molecular techniques. Plant Disease, 93, 215-223.

Burgess, T. I., White, D., McDougall, K. M., Garnas, J., Dunstan, W. A., Català, S., Carnegie, A. J., Worboys, S., Cahill, D. & Vettraino, Stukely, M.J.C., Liew, E.C.Y., Paap, T., Bose, T., Migliorini, D., Williams, B., Brigg, F., Crane, C., Rudman, T. and Hardy, G.E.S.J. A.-M. 2017b. Distribution and diversity of Phytophthora across Australia. Pacific Conservation Biology, 23, 150-162.

Bush, E. A., Hong, C. & Stromberg, E. L. 2003. Fluctuations of Phytophthora and Pythium spp. in components of a recycling irrigation system. Plant Disease, 87, 1500-1506.

Cafe-Filho, A. & Duniway, J. 1996. Effect of location of drip irrigation emitters and position of Phytophthora capsici infections in roots on Phytophthora root rot of pepper. Phytopathology, 86, 1364-1369.

Cahill, D. M., Rookes, J. E., Wilson, B. A., Gibson, L. & Mc Dougall, K. L. 2008. Phytophthora cinnamomi and Australia’s biodiversity: impacts, predictions and progress towards control. Australian Journal of Botany, 56, 279-310.

Campanella, V., Ippolito, A. & Nigro, F. 2002. Activity of calcium salts in controlling Phytophthora root rot of citrus. Crop Protection, 21, 751-756.

105

Canpolat, E., Kaya, M. & Gür, S. 2004. Synthesis, characterization of some Co (III) complexes with vic-dioxime ligands and their antimicrobial properties. Turkish Journal of Chemistry, 28, 235-242.

Cardillo, E., Acedo, A. & Abad, E. 2018. Topographic effects on dispersal patterns of Phytophthora cinnamomi at a stand scale in a Spanish heathland. PloS One, 13, e0195060.

Català, S. 2017. Development of DNA massive sequencing techniques and Real-Time PCR for the detection, identification and quantitation of Phytophthora spp. in environmental samples. (Doctoral dissertation), Valencia University

Català, S., Berbegal, M., Pérez‐Sierra, A. & Abad‐Campos, P. 2017. Metabarcoding and development of new real‐time specific assays reveal Phytophthora species diversity in holm oak forests in eastern Spain. Plant Pathology, 66, 115-123.

Chai, L.-Q., Zhang, K.-Y., Tang, L.-J., Zhang, J.-Y. & Zhang, H.-S. 2017. Two mono-and dinuclear Ni (II) complexes constructed from quinazoline-type ligands: Synthesis, X-ray structures, spectroscopic, electrochemical, thermal, and antimicrobial studies. Polyhedron, 130, 100- 107.

Chao, A., Chazdon, R. L., Colwell, R. K. & Shen, T. J. 2005. A new statistical approach for assessing similarity of species composition with incidence and abundance data. Ecology Letters, 8, 148-159.

Chase, M. W. & Fay, M. F. 2009. Barcoding of plants and fungi. Science, 325, 682-683.

Chen, X.-R., Huang, S.-X., Zhang, Y., Sheng, G.-L., Zhang, B.-Y., Li, Q.-Y., Zhu, F. & Xu, J.-Y. 2018. Transcription profiling and identification of infection-related genes in Phytophthora cactorum. Molecular Genetics and Genomics, 293, 541-555.

Chisholm, S. T., Coaker, G., Day, B. & Staskawicz, B. J. 2006. Host-microbe interactions: shaping the evolution of the plant immune response. Cell, 124, 803-814.

Choudhari, R., Gade, R., Lad, R. & Bangar, S. 2018. Epidemiological relations to Phytophthora spp. causing citrus gummosis in Nagpur mandarin. International Journal of Current Microbiology and Applied Sciences, 418-430.

Christiansen, M. N. 1982. Breeding plants for less favorable environments. New York:Wiley.

Chuine, I., Cambon, G. & Comtois, P. 2000. Scaling phenology from the local to the regional level: advances from species‐specific phenological models. Global Change Biology, 6, 943-952. 106

Coetzee, M., Wingfield, B. D., Harrington, T. C., Steimel, J., Coutinho, T. A. & Wingfield, M. J. 2001. The root rot fungus Armillaria mellea introduced into South Africa by early Dutch settlers. Molecular Ecology, 10, 387-396.

Cohen, Y. & Reuveni, M. 1983. Occurrence of metalaxyl-resistant isolates of Phytophthora infestans in potato fields in Israel. Phytopathology, 73, 925-927.

Coince, A., Cael, O., Bach, C., Lengelle, J., Cruaud, C., Gavory, F., Morin, E., Murat, C., Marcais, B. & Buee, M. 2013. Below-ground fine-scale distribution and soil versus fine root detection of fungal and soil oomycete communities in a French beech forest. Fungal Ecology, 6, 223- 235.

Colunga-Garcia, M., Magarey, R. A., Haack, R. A., Gage, S. H. & Qi, J. 2010. Enhancing early detection of exotic pests in agricultural and forest ecosystems using an urban‐gradient framework. Ecological Applications, 20, 303-310.

Corcobado, T., Cubera, E., Juárez, E., Moreno, G. & Solla, A. 2014. Drought events determine performance of Quercus ilex seedlings and increase their susceptibility to Phytophthora cinnamomi. Agricultural and Forest Meteorology, 192, 1-8.

Corcobado, T., Cubera, E., Moreno, G. & Solla, A. 2013. Quercus ilex forests are influenced by annual variations in water table, soil water deficit and fine root loss caused by Phytophthora cinnamomi. Agricultural and Forest Meteorology, 169, 92-99.

Cornelis, J. & Hermy, M. 2004. Biodiversity relationships in urban and suburban parks in Flanders. Landscape and Urban Planning, 69, 385-401.

Costanza, R., D'arge, R., De Groot, R., Farber, S., Grasso, M., Hannon, B., Limburg, K., Naeem, S., O'neill, R. V. & Paruelo, J. 1997. The value of the world's ecosystem services and natural capital. Nature, 387, 253.

Croeser, L., Paap, T., C. Calver, M., E. Andrew, M., Hardy, G. & Burgess, T. 2018. Field survey, isolation, identification and pathogenicity of Phytophthora species associated with a Mediterranean-type tree species. Forest Pathology, 48, e12424.

Crous, P., Summerell, B., Shivas, R., Burgess, T., Decock, C., Dreyer, L., Granke, L., Guest, D., Hardy, G. S. J. & Hausbeck, M. 2012. Fungal Planet description sheets: 107–127. Persoonia: Molecular Phylogeny and Evolution of Fungi, 28, 138.

Cushman, J. H. & Meentemeyer, R. K. 2008. Multi‐scale patterns of human activity and the incidence of an exotic forest pathogen. Journal of Ecology, 96, 766-776. 107

D’souza, N., Webster, J. & Tay, F. 1997. Phytophthora boehmeriae isolated for the first time in Western Australia. Australasian Plant Pathology, 26, 204-204.

Da Silva, K. R. A., Salles, J. F., Seldin, L. & Van Elsas, J. D. 2003. Application of a novel Paenibacillus- specific PCR-DGGE method and sequence analysis to assess the diversity of Paenibacillus spp. in the maize rhizosphere. Journal of Microbiological Methods, 54, 213-231.

Dale, A., Feau, N., Ponchart, J., Bilodeau, G., Berube, J. & Hamelin, R. Urban activities influence on Phytophthora species diversity in British Columbia, Canada. Proceedings of the sudden oak death sixth science symposium. Gen. Tech. Rep. GTR-PSW-255. Albany, CA: US Department of Agriculture, Forest Service, Pacific Southwest Research Station: 31-32., 2017. 31-32.

Das, A. 2009. Fungal disease in citrus and its management: Integrated management in citrus. National Reseacrh Center for Citrus Bulletin, Nagpur, 53-57.

Davison, E., Drenth, A., Kumar, S., Mack, S., Mackie, A. & Mckirdy, S. 2006. Pathogens associated with nursery plants imported into Western Australia. Australasian Plant Pathology, 35, 473-475.

Davison, E. & Shearer, B. 1989. Phytophthora spp. in indigenous forests in Australia. New Zealand Journal of Forestry Science, 19, 277-89.

Davison, E. & Tay, F. 2005. How many soil samples are needed to show that Phytophthora is absent from sites in the south-west of Western Australia? Australasian Plant Pathology, 34, 293-297.

De Silva, A., Patterson, K., Rothrock, C. & Mcnew, R. 1999. Phytophthora root rot of blueberry increases with frequency of flooding. HortScience, 34, 693-695.

Delgado‐Baquerizo, M., Fry, E. L., Eldridge, D. J., De Vries, F. T., Manning, P., Hamonts, K., Kattge, J., Boenisch, G., Singh, B. K. & Bardgett, R. D. 2018. Plant attributes explain the distribution of soil microbial communities in two contrasting regions of the globe. New Phytologist, 219, 574-587.

Dimoudi, A., Kantzioura, A., Zoras, S., Pallas, C. & Kosmopoulos, P. 2013. Investigation of urban microclimate parameters in an urban center. Energy and Buildings, 64, 1-9.

Dinnie, E., Brown, K. M. & Morris, S. 2013. Community, cooperation and conflict: Negotiating the social well-being benefits of urban greenspace experiences. Landscape and Urban Planning, 112, 1-9. 108

Dobbs, C., Escobedo, F. J. & Zipperer, W. C. 2011. A framework for developing urban forest ecosystem services and goods indicators. Landscape and Urban Planning, 99, 196-206.

Dobbs, C., Kendal, D. & Nitschke, C. R. 2014. Multiple ecosystem services and disservices of the urban forest establishing their connections with landscape structure and sociodemographics. Ecological Indicators, 43, 44-55.

Doblas-Miranda, E., Alonso, R., Arnan, X., Bermejo, V., Brotons, L., De Las Heras, J., Estiarte, M., Hódar, J., Llorens, P. & Lloret, F. 2017. A review of the combination among global change factors in forests, shrublands and pastures of the Mediterranean Region: Beyond drought effects. Global and Planetary Change, 148, 42-54.

Donovan, G. H., Butry, D. T., Michael, Y. L., Prestemon, J. P., Liebhold, A. M., Gatziolis, D. & Mao, M. Y. 2013. The relationship between trees and human health: evidence from the spread of the emerald ash borer. American Journal of Preventive Medicine, 44, 139-145.

Downie, D. 2002. Locating the sources of an invasive pest, grape phylloxera, using a mitochondrial DNA gene genealogy. Molecular Ecology, 11, 2013-2026.

Duvenhage, J. & Kotzé, J. 1991. The influence of calcium on saprophytic growth and pathogenicity of Phytophthora cinnamomi and on resistance of avocado to root rot. South African Avocado Growers’ Association Yearbook, 14, 13-14.

Duvenhage, J., Kotzé, J. & Maas, E. M. 1992. The influence of nitrogen and calcium on mycelial growth and disease severity of Phytophthora cinnamomi and the effect of calcium on resistance of avocado to root rot. South African Avocado Growers’ Association Yearbook, 15, 12-14.

Érsek, T. & Ribeiro, O. 2010. Mini review article: an annotated list of new Phytophthora species described post 1996. Acta Phytopathologica et Entomologica Hungarica, 45, 251-266.

Erwin, D. C. & Ribeiro, O. K. 1996. Phytophthora diseases worldwide, American Phytopathological Society (APS Press).Eschen, R., Britton, K., Brockerhoff, E., Burgess, T., Dalley, V., Epanchin-Niell, R., Gupta, K., Hardy, G., Huang, Y. & Kenis, M. 2015. International variation in phytosanitary legislation and regulations governing importation of plants for planting. Environmental Science & Policy, 51, 228-237.

Escobedo, F. J., Wagner, J. E., Nowak, D. J., De La Maza, C. L., Rodriguez, M. & Crane, D. E. 2008. Analyzing the cost effectiveness of Santiago, Chile's policy of using urban forests to improve air quality. Journal of environmental management, 86, 148-157.

109

Eshraghi, L., Anderson, J. P., Aryamanesh, N., Mccomb, J. A., Shearer, B. & Hardy, G. E. S. J. 2014a. Defence signalling pathways involved in plant resistance and phosphite-mediated control of Phytophthora cinnamomi. Plant Molecular Biology Reporter, 32, 342-356.

Eshraghi, L., Anderson, J. P., Aryamanesh, N., Mccomb, J. A., Shearer, B. & Hardy, G. E. S. J. 2014b. Suppression of the auxin response pathway enhances susceptibility to Phytophthora cinnamomi while phosphite-mediated resistance stimulates the auxin signalling pathway. BMC Plant Biology, 14, 68.

Eshwika, A., Coyle, B., Devereux, M., Mccann, M. & Kavanagh, K. 2004. Metal complexes of 1, 10- phenanthroline-5, 6-dione alter the susceptibility of the yeast Candida albicans to Amphotericin B and Miconazole. Biometals, 17, 415-422.

Evans, B., Stone, C. & Barber, P. A. 2013. Linking a decade of forest decline in the south-west of Western Australia to bioclimatic change. Australian Forestry, 76, 164-172.

Evans, B. J., Lyons, T., Barber, P. A., Stone, C. & Hardy, G. E. S. J. 2012. Enhancing a eucalypt crown condition indicator driven by high spatial and spectral resolution remote sensing imagery. Journal of Applied Remote Sensing, 6, 063605.

Fayad, N., Al-Noor, T. H. & Ghanim, F. 2012. Synthesis, characterization and antibacterial activity of mixed ligand complexes of some metals with 1-nitroso-2-naphthol and L- phenylalanine. Synthesis, 2, 18-29.

Fayad, N. K., Al-Noor, T. H., Mahmood, A. A. & Malih, I. K. 2013. Synthesis, characterization, and antibacterial Studies of Mn (II), Fe (II), Co (II), Ni (II), Cu (II) and Cd (II) mixed-ligand complexes containing amino acid (L-Valine) and(1, 10-phenanthroline). Synthesis, 3, 66- 72.

Fenn, M. A. & Coffey, M. 1984. Studies on the in vitro and in vivo antifungal activity of fosetyl-Al and phosphorous acid. Phytopathology, 74, 606- 611.

Fenton, D. E. 2001. Biocoordination chemistry, Oxford,UK, Oxford University Press. Floerl, O., Inglis, G., Dey, K. & Smith, A. 2009. The importance of transport hubs in stepping‐stone invasions. Journal of Applied Ecology, 46, 37-45.

Foley, J. A., Asner, G. P., Costa, M. H., Coe, M. T., Defries, R., Gibbs, H. K., Howard, E. A., Olson, S., Patz, J. & Ramankutty, N. 2007. Amazonia revealed: forest degradation and loss of ecosystem goods and services in the Amazon Basin. Frontiers in Ecology and the Environment, 5, 25-32.

110

Franke, J. & Menz, G. 2007. Multi-temporal wheat disease detection by multi-spectral remote sensing. Precision Agriculture, 8, 161-172.

Freer-Smith, P., El-Khatib, A. & Taylor, G. 2004. Capture of particulate pollution by trees: a comparison of species typical of semi-arid areas (Ficus nitida and Eucalyptus globulus) with European and North American species. Water, Air, and Soil Pollution, 155, 173-187.

Gallardo, B., Zieritz, A. & Aldridge, D. C. 2015. The importance of the human footprint in shaping the global distribution of terrestrial, freshwater and marine invaders. PLoS One, 10, e0125801.

Garbelotto, M., Rizzo, D. M. & Marais, L. 2002. Phytophthora ramorum and sudden oak death in California: IV. Preliminary studies on chemical control. In: Standiford, Richard B., et al, tech. editor. Proceedings of the Fifth Symposium on Oak Woodlands: Oaks in California's Challenging Landscape. Gen. Tech. Rep. PSW-GTR-184, Albany, CA: Pacific Southwest Research Station, Forest Service, US Department of Agriculture, 184, 811-818. Garbelotto, M., Svihra, P. & Rizzo, D. 2001. New pests and diseases: Sudden oak death syndrome fells 3 oak species. California Agriculture, 55, 9-19.

Garkaklis, M., Calver, M., Wilson, B. & Hardy, G. E. S. J. Habitat alteration caused by an introduced plant disease, Phytophthora cinnamomi: a significant threat to the conservation of Australian forest fauna. 2004. Royal Zoological Society of New South Wales, 899-913.

Garrett, K., Dendy, S., Frank, E., Rouse, M. & Travers, S. 2006a. Climate change effects on plant disease: genomes to ecosystems. Annual Review of Phytopathology, 44, 489-509.

Garrett, K. A., Dendy, S. P., Frank, E. E., Rouse, M. N. & Travers, S. E. 2006b. Climate change effects on plant disease: genomes to ecosystems. Annual Review of Phytopathology, 44, 489- 509.

Gdr, H. B., Sharon, N. & Australia, E. W. 1984. Nomenclature and symbolism for amino acids and peptides. Pure and Applied Chemistry, 56, 595-624.

Gillis, T. 2014. Use of remotely sensed imagery to map Sudden Oak Death (Phytophthora ramorum) in the Santa Cruz Mountains, University of Southern California.

Girgaonkar, M. & Shirodkar, S. 2012. synthesis, characterization and biological studies of Cu (II) and Ni (II) complexes with new bidentate schiff’s base ligands as 4-hydroxy-3-(1-

111

(arylimino) ethyl)(arylimino) ethyl) Chromen-2-one. Research Journal of Recent Sciences, 1, 110-116.

Gisi, U. 2002. Chemical Control of Downy Mildews. In: P. T. N. Spencer-Phillips U. Gisi and A. Lebeda (ed.) Advances in Downy Mildew Research. 119-159. Dordrecht: Springer Netherlands.

Godefroid, S. & Koedam, N. 2003. Distribution pattern of the flora in a peri-urban forest: an effect of the city–forest ecotone. Landscape and Urban Planning, 65, 169-185.

Gómez-Merino, F. C. & Trejo-Téllez, L. I. 2015. Biostimulant activity of phosphite in horticulture. Scientia Horticulturae, 196, 82-90.

Gonzalez, M., Caetano, P. & Sánchez, M. 2017a. Testing systemic fungicides for control of Phytophthora oak root disease. Forest Pathology, 47(4), 1-3.

Gonzalez, M., Perez‐Sierra, A., Serrano, M. & Sánchez, M. 2017b. Two Phytophthora species causing decline of wild olive (Olea europaea subsp. europaea var. sylvestris). Plant Pathology, 66, 941-948.

Goss, R. W. 1949. Pink rot of potatoes caused by Phytophthora erythroseptica Pethyb. University of Nebraska, Collage Agricultural, Agricultural Experiment Station. Research Bulletins. 160. Lincoln, Nebraska., 1-27.

Graeff, S. & Claupein, W. 2003. Quantifying nitrogen status of corn (Zea mays L.) in the field by reflectance measurements. European Journal of Agronomy, 19, 611-618.

Grasso, F., Marini, M., Vitale, A., Firrao, G. & Granata, G. 2012. Canker and dieback on Platanus x acerifolia caused by Diaporthe scabra. Forest Pathology, 42, 510-513.

Grinde, B. & Patil, G. G. 2009. Biophilia: does visual contact with nature impact on health and well-being? International Journal of Environmental Research and Public Health, 6, 2332- 2343.

Grote, R., Samson, R., Alonso, R., Amorim, J. H., Cariñanos, P., Churkina, G., Fares, S., Thiec, D. L., Niinemets, Ü. & Mikkelsen, T. N. 2016. Functional traits of urban trees: air pollution mitigation potential. Frontiers in Ecology and the Environment. 14, 543-550.

Grunwald, N. J., Garbelotto, M., Goss, E. M., Heungens, K. & Prospero, S. 2012. Emergence of the sudden oak death pathogen Phytophthora ramorum. Trends in Microbiology, 20, 131- 138. 112

Guest, D. & Grant, B. 1991. The complex action of phosphonates as antifungal agents. Biological Reviews, 66, 159-187.

Guetté, A., Godet, L., Juigner, M. & Robin, M. 2018. Worldwide increase in Artificial Light At Night around protected areas and within biodiversity hotspots. Biological Conservation, 223, 97-103.

Haby, N., Tunn, Y. & Cameron, J. 2010. Application of QuickBird and aerial imagery to detect Pinus radiata in remnant vegetation. Austral Ecology, 35, 624-635.

Hackett, D. J. & Goldingay, R. L. 2001. Pollination of Banksia spp. by non-flying mammals in north- eastern New South Wales. Australian Journal of Botany, 49, 637-644.

Hain, F. P. 1987. Interactions of insects, trees and air pollutants. Tree Physiology, 3, 93-102.

Hanfling, B., Carvalho, G. R. & Brandl, R. 2002. mt-DNA sequences and possible invasion pathways of the Chinese mitten crab. Marine Ecology Progress Series, 238, 307-310.

Hansen, E., Parke, J. & Sutton, W. 2005. Susceptibility of Oregon forest trees and shrubs to Phytophthora ramorum: a comparison of artificial inoculation and natural infection. Plant Disease, 89, 63-70.

Hansen, E. M. 2008. Alien forest pathogens: Phytophthora species are changing world forests. Boreal Environment Research, 13, 33-41.

Hansen, E. M., Goheen, D. J., Jules, E. S. & Ullian, B. 2000. Managing Port-Orford-cedar and the introduced pathogen Phytophthora lateralis. Plant Disease, 84, 4-14.

Hardham, A. R. 2005. Phytophthora cinnamomi. Molecular Plant Pathology, 6, 589-604.

Hardy, G. E. S. J., Barrett, S. & Shearer, B. 2001. The future of phosphite as a fungicide to control the soilborne plant pathogen Phytophthora cinnamomi in natural ecosystems. Australasian Plant Pathology, 30, 133-139.

Hardy, G. E. S. J. & Sivasithamparam, K. 1988. Phytophthora spp. associated with container-grown plants in nurseries in Western Australia. Plant Disease, 72, 435-437.

Hardy, G. E. S. J. & Sivasithamparam, K. 1991. Suppression of Phytophthora root rot by a composted Eucalyptus bark mix. Australian Journal of Botany, 39, 153-159.

113

Hebert, P. D., Ratnasingham, S. & De Waard, J. R. 2003. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London B: Biological Sciences, 270, S96-S99.

Heikkinen, R. K., Luoto, M., Virkkala, R., Pearson, R. G. & Körber, J. H. 2007. Biotic interactions improve prediction of boreal bird distributions at macro‐scales. Global Ecology and Biogeography, 16, 754-763.

Hernandez-Lambrano, R. E., Gonzalez-Moreno, P. & Sanchez-Agudo, J. A. 2018. Environmental factors associated with the spatial distribution of invasive plant pathogens in the Iberian Peninsula: The case of Phytophthora cinnamomi Rands. Forest Ecology and Management, 419, 101-109.

Herrero, M., Toppe, B., Klemsdal, S. & Stensvand, A. 2006. First report of Phytophthora ramorum in ornamental plants in Norway. Plant Disease, 90, 1458-1458.

Hill, R., Wilson, B., Rookes, J. & Cahill, D. 2009. Use of high resolution digital multi-spectral imagery to assess the distribution of disease caused by Phytophthora cinnamomi on heathland at Anglesea, Victoria. Australasian Plant Pathology, 38, 110-119.

Hopper, S. D. & Gioia, P. 2004. The southwest Australian floristic region: evolution and conservation of a global hot spot of biodiversity. Annual Review of Ecology, Evolution, and Systematics, 35, 623-650.

Huber, L. & Gillespie, T. 1992. Modeling leaf wetness in relation to plant disease epidemiology. Annual Review of Phytopathology, 30, 553-577.

Hüberli, D., Tommerup, I. C., Colver, M. C., Colquhounc, I. J. & Hardy, G. E. S. J. 2002. Temperature and inoculation method influence disease phenotypes and mortality of Eucalyptus marginata clonal lines inoculated with Phytophthora cinnamomi. Australasian Plant Pathology, 31, 107-118.

Hulbert, J. M., Agne, M. C., Burgess, T. I., Roets, F. & Wingfield, M. J. 2017. Urban environments provide opportunities for early detections of Phytophthora invasions. Biological Invasions, 19, 3629-3644.

Hulme, P. E. 2009. Trade, transport and trouble: managing invasive species pathways in an era of globalization. Journal of Epplied ecology, 46, 10-18.

Humair, F., Humair, L., Kuhn, F. & Kueffer, C. 2015. E‐commerce trade in invasive plants. Conservation Biology, 29, 1658-1665. 114

Ishaq, L., Barber, P. A., Hardy, G. E. S. J., Calver, M. & Dell, B. 2013. Seedling mycorrhizal type and soil chemistry are related to canopy condition of Eucalyptus gomphocephala. Mycorrhiza, 23, 359-371.

Ishaq, L., Barber, P. A., Hardy, G. E. S. J. & Dell, B. 2018. Diversity of fungi associated with roots of Eucalyptus gomphocephala seedlings grown in soil from healthy and declining sites. Australasian Plant Pathology, 1-8.

Jackson, T., Burgess, T., Colquhoun, I. & Hardy, G. S. 2000. Action of the fungicide phosphite on Eucalyptus marginata inoculated with Phytophthora cinnamomi. Plant Pathology, 49, 147-154.

Jacobi, W., Koski, R. & Negron, J. 2013. Dutch elm disease pathogen transmission by the banded elm bark beetle Scolytus schevyrewi. Forest Pathology, 43, 232-237.

Jamieson, A., Bassett, I., Hill, L., Hill, S., Davis, A., Waipara, N., Hough, E. & Horner, I. 2014. Aerial surveillance to detect kauri dieback in New Zealand. New Zealand Plant Protection, 67, 60-65.

Jawetz, M. 2007. Adelberg’s Medical microbiology. Antibacterial and antifungal chemotherapy (Prentice-Hall International Inc).

Jeffers, S. & Aldwinckle, H. 1987. Enhancing detection of Phytophthora catorum in naturally infested soil. Phytopathology, 77, 1475-1482.

Jim, C. & Liu, H. 2001. Species diversity of three major urban forest types in Guangzhou City, China. Forest Ecology and Management, 146, 99-114.

Jones, J. D. & Dangl, J. L. 2006. The plant immune system. Nature, 444, 323.

Jules, E. S., Kauffman, M. J., Ritts, W. D. & Carroll, A. L. 2002. Spread of an invasive pathogen over a variable landscape: a nonnative root rot on Port Orford cedar. Ecology, 83, 3167-3181.

Jumpponen, A. & Jones, K. 2009. Massively parallel 454 sequencing indicates hyperdiverse fungal communities in temperate Quercus macrocarpa phyllosphere. New Phytologist, 184, 438- 448.

Jung, T., Blaschke, H. & Ojlwakf, W. 2003. Effect of environmental constraints on Phytophthora- mediated oak decline in Central Europe. Phytophthora in Forests and Natural Ecosystems, 89.

115

Jung, T., Blaschke, H. & Oßwald, W. 2000. Involvement of soilborne Phytophthora species in Central European oak decline and the effect of site factors on the disease. Plant Pathology, 49, 706-718.

Jung, T. & Blaschke, M. 2004. Phytophthora root and collar rot of alders in Bavaria: distribution, modes of spread and possible management strategies. Plant Pathology, 53, 197-208.

Jung, T., Hudler, G. W., Jensen-Tracy, S., Griffiths, H., Fleischmann, F. & Osswald, W. 2005. Involvement of Phytophthora species in the decline of European beech in Europe and the USA. Mycologist, 19, 159-166.

Jung, T., Orlikowski, L., Henricot, B., Abad‐Campos, P., Aday, A., Aguín Casal, O., Bakonyi, J., Cacciola, S., Cech, T. & Chavarriaga, D. 2016. Widespread Phytophthora infestations in European nurseries put forest, semi‐natural and horticultural ecosystems at high risk of Phytophthora diseases. Forest Pathology, 46, 134-163.

Jung, T., Pérez-Sierra, A., Durán, A., Jung, M. H., Balci, Y. & Scanu, B. 2018. Canker and decline diseases caused by soil-and airborne Phytophthora species in forests and woodlands. Persoonia, 40, 182-220.

Jung, T., Stukely, M., Hardy, G. S. J., White, D., Paap, T., Dunstan, W. & Burgess, T. 2011. Multiple new Phytophthora species from ITS Clade 6 associated with natural ecosystems in Australia: evolutionary and ecological implications. Persoonia: Molecular Phylogeny and Evolution of Fungi, 26, 13.

Kaes, C., Katz, A. & Hosseini, M. W. 2000. Bipyridine: the most widely used ligand. A review of molecules comprising at least two 2, 2 ‘-bipyridine units. Chemical Reviews, 100, 3553- 3590.

Keane, P., Kile, G., Podger, F. & Brown, B. (eds.) 2000. Diseases of eucalypts caused by soilborne species of Phytophthora and Pythium, CSIRO Publishing: eds.Shearer, BL, Smith, IW,pp.259-291.

Kearney, S. G., Adams, V. M., Fuller, R. A., Possingham, H. P. & Watson, J. E. 2018. Estimating the benefit of well-managed protected areas for threatened species conservation. Oryx, 1-9..

Khaliq, I., Hardy, G. E. S. J., White, D. & Burgess, T. I. 2018. eDNA from roots: a robust tool for determining Phytophthora communities in natural ecosystems. FEMS microbiology ecology, 94, fiy048.

116

Kliejunas, J. T. 2010. Sudden oak death and Phytophthora ramorum: a summary of the literature. Gen. Tech. Rep. PSW-GTR-234. Albany, CA: US Department of Agriculture, Forest Service, Pacific Southwest Edition 2010, 181 p, 234 Kolbe, J. J., Glor, R. E., Schettino, L. R., Lara, A. C., Larson, A. & Losos, J. B. 2004. Genetic variation increases during biological invasion by a Cuban lizard. Nature, 431, 177.

Konijnendijk, C. C., Ricard, R. M., Kenney, A. & Randrup, T. B. 2006. Defining urban forestry–A comparative perspective of North America and Europe. Urban forestry & Urban Greening, 4, 93-103.

Kozlowski, T. T. & Pallardy, J. 1991. The Physiological Ecology of Woody Plants. San Diego: Academic Press.

Kreyling, J., Beierkuhnlein, C., Elmer, M., Pritsch, K., Radovski, M., Schloter, M., Wöllecke, J. & Jentsch, A. 2008. Soil biotic processes remain remarkably stable after 100-year extreme weather events in experimental grassland and heath. Plant and Soil, 308, 175.

Kroon, L., Brouwer, H., De Cock, A. & Govers, F. 2012. The Phytophthora genus anno 2012. Phytopathology, 102, 348-364.

Kühn, I., Brandl, R. & Klotz, S. 2004. The flora of German cities is naturally species rich. Evolutionary Ecology Research, 6, 749-764.

Kumar, S. S., Biju, S. & Sadasivan, V. 2018. Synthesis, structure characterization and biological studies on a new aromatic hydrazone, 5-(2-(1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro- 1H-pyrazol-4-yl) hydrazono)-2, 2-dimethyl-1, 3-dioxane-4, 6-dione, and its transition metal complexes. Journal of Molecular Structure, 1156, 201-209.

Kuo, F. E. 2001. Coping with poverty: Impacts of environment and attention in the inner city. Environment and Behavior, 33, 5-34.

Kuo, F. E. 2003a. The role of arboriculture in a healthy social ecology. Journal of Arboriculture, 29, 148-155.

Kuo, F. E. 2003b. Social aspects of urban forestry: the role of arboriculture in a healthy social ecology. Journal of Arboriculture, 29,148-155.

Lafortezza, R., Carrus, G., Sanesi, G. & Davies, C. 2009. Benefits and well-being perceived by people visiting green spaces in periods of heat stress. Urban Forestry & Urban Greening, 8, 97-108. 117

Lambers, H., Chapin Iii, F. S. & Pons, T. L. 1998. Photosynthesis, respiration, and long-distance transport: Plant physiological ecology. 10-153. (pp.7-21), Springer. New York. USA.

Li, X., Lee, W. S., Li, M., Ehsani, R., Mishra, A. R., Yang, C. & Mangan, R. L. 2012. Spectral difference analysis and airborne imaging classification for citrus greening infected trees. Computers and Electronics in Agriculture, 83, 32-46.

Liebhold, A. M., Brockerhoff, E. G., Garrett, L. J., Parke, J. L. & Britton, K. O. 2012. Live plant imports: the major pathway for forest insect and pathogen invasions of the US. Frontiers in Ecology and the Environment, 10, 135-143.

Linde, C., Kemp, G. & Wingfield, M. 1994a. Diseases of pines and eucalypts in South Africa associated with Pythium and Phytophthora species. South African Forestry Journal, 169, 25-32.

Linde, C., Kemp, G. & Wingfleld, M. 1994b. Pythium and Phytophthora species associated with eucalypts and pines in South Africa. European Journal of Forest Pathology, 24, 345-356.

Lindner, M., Maroschek, M., Netherer, S., Kremer, A., Barbati, A., Garcia-Gonzalo, J., Seidl, R., Delzon, S., Corona, P. & Kolström, M. 2010. Climate change impacts, adaptive capacity, and vulnerability of European forest ecosystems. Forest Ecology and Management, 259, 698-709.

Liu, D., Kelly, M. & Gong, P. 2006. A spatial–temporal approach to monitoring forest disease spread using multi-temporal high spatial resolution imagery. Remote Sensing of Environment, 101, 167-180.

Liu, D., Kelly, M., Gong, P. & Guo, Q. 2007. Characterizing spatial–temporal tree mortality patterns associated with a new forest disease. Forest Ecology and Management, 253, 220- 231.

Liu, W., Qin, Y., Liu, S., Xing, R., Yu, H., Chen, X., Li, K. & Li, P. 2018. C-coordinated O-carboxymethyl chitosan metal complexes: Synthesis, characterization and antifungal efficacy. International Journal of Biological Macromolecules, 106, 68-77.

Lloret, F., Keeling, E. G. & Sala, A. 2011. Components of tree resilience: effects of successive low‐ growth episodes in old ponderosa pine forests. Oikos, 120, 1909-1920. 118

Lloret, F., Siscart, D. & Dalmases, C. 2004. Canopy recovery after drought dieback in holm‐oak Mediterranean forests of Catalonia (NE Spain). Global Change Biology, 10, 2092-2099.

Lombaert, E., Guillemaud, T., Cornuet, J.-M., Malausa, T., Facon, B. & Estoup, A. 2010. Bridgehead effect in the worldwide invasion of the biocontrol harlequin ladybird. PloS One, 5, e9743.

Lowe, S., Browne, M., Boudjelas, S. & De Poorter, M. 2000. 100 of the world's worst invasive alien species: a selection from the global invasive species database, Invasive Species Specialist Group Auckland. 12. Mahlein, A.-K., Steiner, U., Dehne, H.-W. & Oerke, E.-C. 2010. Spectral signatures of sugar beet leaves for the detection and differentiation of diseases. Precision Agriculture, 11, 413- 431.

Maloley, M. Thermal Remote Sensing of Urban Heat Islands: Greater Toronto Area. Masters Abstracts International, 2010. 1-82.

Manion, P. D. 1991. Tree disease concepts, United States of America, Prentice Hall Career and Technology., second ed. Martinelli, F., Scalenghe, R., Davino, S., Panno, S., Scuderi, G., Ruisi, P., Villa, P., Stroppiana, D., Boschetti, M. & Goulart, L. R. 2015. Advanced methods of plant disease detection. A review. Agronomy for Sustainable Development, 35, 1-25.

Marx, D. H. 1969. The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to pathogenic infections. I. Antagonism of mycorrhizal fungi to root pathogenic fungi and soil bacteria. Phytopathology, 59, 153-163.

Maseko, B., Burgess, T., Coutinho, T. & Wingfield, M. 2001. First report of Phytophthora nicotianae associated with Eucalyptus die‐back in South Africa. Plant Pathology, 50, 413- 413.

Maseko, B., Burgess, T. I., Coutinho, T. A. & Wingfield, M. J. 2007. Two new Phytophthora species from South African Eucalyptus plantations. Mycological Research, 111, 1321-1338.

McCarren, K. 2006. Saprophytic ability and the contribution of chlamydospores and oospores to the survival of Phytophthora cinnamomi. Murdoch University

McConnell, M. & Balci, Y. 2014. Phytophthora cinnamomi as a contributor to white oak decline in mid-Atlantic United States forests. Plant Disease, 98, 319-327.

119

McCredie, T., Dixon, K. W. & Sivasithamparam, K. 1985. Variability in the resistance of Banksia Lf species to Phytophthora cinnamomi Rands. Australian Journal of Botany, 33, 629-637.

McDonald, A. E., Grant, B. R. & Plaxton, W. C. 2001. Phosphite (phosphorous acid): its relevance in the environment and agriculture and influence on plant phosphate starvation response. Journal of Plant Nutrition, 24, 1505-1519.

McDougall, K., Hardy, G. S. J. & Hobbs, R. J. 2001. Additions to the host range of Phytophthora cinnamomi in the jarrah (Eucalyptus marginata) forest of Western Australia. Australian Journal of Botany, 49, 193-198.

McPherson, E. G. 1994. Energy-saving potential of trees in Chicago. Chicago’s urban forest ecosystem: results of the Chicago Urban Forest Climate Project. Gen. Tech. Rep. NE-186. Radnor [Newtown Square], PA: US Department of Agriculture, Forest Service, Northeastern Research Station, 95-113.

Messenger, B., Menge, J. & Pond, E. 2000. Effects of gypsum on zoospores and sporangia of Phytophthora cinnamomi in field soil. Plant Disease, 84, 617-621.

Metternicht, G. 2003. Vegetation indices derived from high-resolution airborne videography for precision crop management. International Journal of Remote Sensing, 24, 2855-2877.

Miller, N., Estoup, A., Toepfer, S., Bourguet, D., Lapchin, L., Derridj, S., Kim, K. S., Reynaud, P., Furlan, L. & Guillemaud, T. 2005. Multiple transatlantic introductions of the western corn rootworm. Science, 310, 992-992.

Moffatt, S., Mclachlan, S. & Kenkel, N. 2004. Impacts of land use on riparian forest along an urban–rural gradient in southern Manitoba. Plant Ecology, 174, 119-135.

Moralejo, E., García‐Muñoz, J. & Descals, E. 2009a. Susceptibility of Iberian trees to Phytophthora ramorum and P. cinnamomi. Plant Pathology, 58, 271-283.

Moralejo, E., Pérez‐Sierra, A., Álvarez, L., Belbahri, L., Lefort, F. & Descals, E. 2009b. Multiple alien Phytophthora taxa discovered on diseased ornamental plants in Spain. Plant Pathology, 58, 100-110.

Mrazkova, M., Cerny, K., Tomsovsky, M. & Strnadova, V. 2011. Phytophthora plurivora T. Jung & TI Burgess and other Phytophthora species causing important diseases of ericaceous plants in the Czech Republic. Plant Protection Science, 47, 13-19.

120

Mrinalinil, L. & Manihar Singh, A. 2012. Mixed ligand Co (III) complexes with 1-amidino-O-methyl urea and amino acids. Research Journal of Chemical Sciences. 2, 45-49.

Nagel, J. H., Gryzenhout, M., Slippers, B., Wingfield, M. J., Hardy, G. E. S. J., Stukely, M. J. & Burgess, T. I. 2013. Characterization of Phytophthora hybrids from ITS clade 6 associated with riparian ecosystems in South Africa and Australia. Fungal Biology, 117, 329-347.

Nagel, J. H., Slippers, B., Wingfield, M. J. & Gryzenhout, M. 2015. Multiple Phytophthora species associated with a single riparian ecosystem in South Africa. Mycologia, 107, 915-925.

Nakamoto, K. 1996. Infrared spectra of inorganic and coordination compounds, J.Wiley and Sons, New York. 4thed.Nash, L. J. & Graves, W. R. 1993. Drought and flood stress effects on plant development and leaf water relations of five taxa of trees native to bottomland habitats. Journal of the American Society for Horticultural Science, 118, 845-850.

Niemelä, J., Kotze, D. J., Venn, S., Penev, L., Stoyanov, I., Spence, J., Hartley, D. & De Oca, E. M. 2002. Carabid beetle assemblages (Coleoptera, Carabidae) across urban-rural gradients: an international comparison. Landscape Ecology, 17, 387-401.

Niinemets, Ü. & Valladares, F. 2006. Tolerance to shade, drought, and waterlogging of temperate Northern Hemisphere trees and shrubs. Ecological Monographs, 76, 521-547.

Nilsson, H. 1995. Remote sensing and image analysis in plant pathology. Annual Review of Phytopathology, 33, 489-528.

Nishida, Y., Niinuma, A. & Abe, K. 2009. Uptake of V IV ion of vanadocene dichloride by apo- transferrin investigated by ESR spectroscopy. Inorganic Chemistry Communications, 12, 198-200.

Nowak, D. 1994. Air Pollution Removal by Chicago's Urban Forest. In Chicago’s Urban Forest Ecosystem: Results of the Chicago Urban Forest Climate Project. General Technical Report NE-186. E.G. McPherson, D.J. Nowak and R.A. Rowntree (eds). U.S. Department of Agriculture, Forest Service, Northeastern Forest Experiment Station, Radnor, PA, , 186, 83–95.

Nowak, D. J., Crane, D. E. & Stevens, J. C. 2006. Air pollution removal by urban trees and shrubs in the United States. Urban forestry & Urban Greening, 4, 115-123.

Nowak, D. J., Noble, M. H., Sisinni, S. M. & Dwyer, J. F. 2001. People and trees: assessing the US urban forest resource. Journal of Forestry, 99, 37-42.

121

Nowak, D. J., Robert Iii, E., Bodine, A. R., Greenfield, E. J., Ellis, A., Endreny, T. A., Yang, Y., Zhou, T. & Henry, R. 2013. Assessing urban forest effects and values: Toronto's urban forest. pp. 1-59.

Nowak, D. J. & Walton, J. T. 2005. Projected urban growth (2000–2050) and its estimated impact on the US forest resource. Journal of Forestry, 103, 383-389.

O'Gara, E., Hardy, G. S. J. & Mccomb, J. 1996. The ability of Phytophthora cinnamomi to infect through unwounded and wounded periderm tissue of Eucalyptus matginata. Plant Pathology, 45, 955-963.

O'Hanlon, R., Choiseul, J., Corrigan, M., Catarame, T. & Destefanis, M. 2016. Diversity and detections of Phytophthora species from trade and non‐trade environments in Ireland. EPPO Bulletin, 46, 594-602.

Oh, E., Gryzenhout, M., Wingfield, B. D., Wingfield, M. J. & Burgess, T. I. 2013. Surveys of soil and water reveal a goldmine of Phytophthora diversity in South African natural ecosystems. IMA Fungus, 4, 123-131.

Oke, T. 1987. Boundary layer climates. 2nd. Methuen, 289p, 548.

Oksanen, J., Kindt, R., Legendre, P., O’hara, B., Stevens, M. H. H., Oksanen, M. J. & Suggests, M. 2007. The vegan package. Community ecology package, 10, 631-637.

Olsson, H., Schneider, W. & Koukal, T. 2008. 1240 H. Olsson et al. International Journal of Remote Sensing, 29, 1239-1242.

Orlikowski, L., Ptaszek, M., Rodziewicz, A., Nechwatal, J., Thinggaard, K. & Jung, T. 2011. Phytophthora root and collar rot of mature Fraxinus excelsior in forest stands in Poland and Denmark. Forest Pathology, 41, 510-519.

Oszako, T., Voitka, D., Tkaczyk, M., Keča, N., Belbahri, L. & Nowakowska, J. A. 2018. Assessment of interactions between defoliation and Phytophthora plurivora stem infections of birch seedlings. The Forestry Chronicle, 94, 140-146.

Ouimette, D. & Coffey, M. 1990. Symplastic entry and phloem translocation of phosphonate. Pesticide Biochemistry and Physiology, 38, 18-25.

Paap, T., Burgess, T. I. & Wingfield, M. J. 2017a. Urban trees: bridge-heads for forest pest invasions and sentinels for early detection. Biological Invasions, 19, 3515-3526.

122

Paap, T., Croeser, L., White, D., Aghighi, S., Barber, P., Hardy, G. S. J. & Burgess, T. 2017b. Phytophthora versiformis sp. nov., a new species from Australia related to P. quercina. Australasian Plant Pathology, 1-10.

Paoletti, E., Danti, R. & Strati, S. 2001. Pre-and post-inoculation water stress affects Sphaeropsis sapinea canker length in Pinus halepensis seedlings. Forest Pathology, 31, 209-218.

Paquet, C., Orschulok, T. P., Coffee, N. T., Howard, N. J., Hugo, G., Taylor, A. W., Adams, R. J. & Daniel, M. 2013. Are accessibility and characteristics of public open spaces associated with a better cardiometabolic health? Landscape and Urban Planning, 118, 70-78.

Parida, A. K., Das, A. & Mittra, B. 2004. Effects of salt on growth, ion accumulation, photosynthesis and leaf anatomy of the mangrove, Bruguiera parviflora. Trees, 18, 167-174.

Park, J., Park, B., Veeraraghavan, N., Jung, K., Lee, Y.-H., Blair, J. E., Geiser, D. M., Isard, S., Mansfield, M. A. & Nikolaeva, E. 2008. Phytophthora database: a forensic database supporting the identification and monitoring of Phytophthora. Plant Disease, 92, 966-972.

Parke, J. L., Knaus, B. J., Fieland, V. J., Lewis, C. & Grünwald, N. J. 2014. Phytophthora community structure analyses in Oregon nurseries inform systems approaches to disease management. Phytopathology, 104, 1052-1062.

Parker, W. F. & Mee, B. J. 1982. Survival of Salmonella adelaide and fecal coliforms in coarse sands of the swan costal plain, Western Australia. Applied and Environmental Microbiology, 43, 981-986.

Parra, G. & Ristaino, J. B. 2001. Resistance to mefenoxam and metalaxyl among field isolates of Phytophthora capsici causing Phytophthora blight of bell pepper. Plant Disease, 85, 1069- 1075.

Pautasso, M., Döring, T. F., Garbelotto, M., Pellis, L. & Jeger, M. J. 2012. Impacts of climate change on plant diseases—opinions and trends. European Journal of Plant Pathology, 133, 295- 313.

Pautasso, M., Schlegel, M. & Holdenrieder, O. 2015. Forest health in a changing world. Microbial Ecology, 69, 826-842.

Perez‐Sierra, A., Lopez‐Garcia, C., Leon, M., Garcia‐Jimenez, J., Abad‐Campos, P. & Jung, T. 2013. Previously unrecorded low‐temperature Phytophthora species associated with Quercus decline in a Mediterranean forest in eastern Spain. Forest Pathology, 43, 331-339.

123

Pervaiz, M., Ahmad, I., Yousaf, M., Kirn, S., Munawar, A., Saeed, Z., Adnan, A., Gulzar, T., Kamal, T. & Ahmad, A. 2018. Synthesis, spectral and antimicrobial studies of amino acid derivative Schiff base metal (Co, Mn, Cu, and Cd) complexes. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. https://doi.org/10.1016/j.saa.2018.05.057

Pettersson, M., Frampton, J., Rönnberg, J., Shew, H., Benson, D., Kohlway, W., Escanferla, M. & Cubeta, M. 2017. Increased diversity of Phytophthora species in Fraser fir Christmas tree plantations in the Southern Appalachians. Scandinavian Journal of Forest Research, 32, 412-420.

Pickett, S. T., Cadenasso, M. L., Grove, J. M., Nilon, C. H., Pouyat, R. V., Zipperer, W. C. & Costanza, R. 2001. Urban ecological systems: linking terrestrial ecological, physical, and socioeconomic components of metropolitan areas. Annual Review of Ecology and Systematics, 32, 127-157.

Pineda, M., Pérez-Bueno, M. L., Paredes, V. & Barón, M. 2017. Use of multicolour fluorescence imaging for diagnosis of bacterial and fungal infection on zucchini by implementing machine learning. Functional plant biology, 44, 563-572. Poland, T. M. & Mccullough, D. G. 2006. Emerald ash borer: invasion of the urban forest and the threat to North America’s ash resource. Journal of Forestry, 104, 118-124.

Pratt, B. & Heather, W. 1973. The origin and distribution of Phytophthora cinnamomi Rands in Australian native plant communities and the significance of its association with particular plant species. Australian Journal of Biological Sciences, 26, 559-574.

Puno, V., Laurence, M., Guest, D. & Liew, E. 2015. Detection of Phytophthora multivora in the Wollemi Pine site and pathogenicity to Wollemia nobilis. Australasian Plant Pathology, 44, 205-215.

Rahman, M. Z., Uematsu, S., Suga, H. & Kageyama, K. 2015. Diversity of Phytophthora species newly reported from Japanese horticultural production. Mycoscience, 56, 443-459.

Rea, A., Burgess, T., Hardy, G. E. S. J., Stukely, M. & Jung, T. 2011a. Two novel and potentially endemic species of Phytophthora associated with episodic dieback of Kwongan vegetation in the south‐west of Western Australia. Plant Pathology, 60, 1055-1068.

Rea, A., Burgess, T., Hardy, G. S. J., Stukely, M. & Jung, T. 2011b. Two novel and potentially endemic species of Phytophthora associated with episodic dieback of Kwongan vegetation in the south‐west of Western Australia. Plant Pathology, 60, 1055-1068.

124

Redondo, M. A. 2018. Invasion biology of forest Phytophthora species in Sweden. Swedish University of Agricultural Sciences (Doctoral dissertation).

Ribeiro, O. K. 1978. A source book of the genus Phytophthora, Vaduz,Liechtenstein, J. Cramer. Richardson, A. D., Keenan, T. F., Migliavacca, M., Ryu, Y., Sonnentag, O. & Toomey, M. 2013. Climate change, phenology, and phenological control of vegetation feedbacks to the climate system. Agricultural and Forest Meteorology, 169, 156-173.

Ristaino, J. B. & Gumpertz, M. L. 2000. New frontiers in the study of dispersal and spatial analysis of epidemics caused by species in the genus Phytophthora. Annual Review of Phytopathology, 38, 541-576.

Rizzo, D., Garbelotto, M., Davidson, J., Slaughter, G. & Koike, S. 2002. Phytophthora ramorum as the cause of extensive mortality of Quercus spp. and Lithocarpus densiflorus in California. Plant disease, 86, 205-214.

Rizzo, D. M. & Garbelotto, M. 2003. Sudden oak death: endangering California and Oregon forest ecosystems. Frontiers in Ecology and the Environment, 1, 197-204.

Rolando, C., Dick, M., Gardner, J., Bader, M. & Williams, N. 2017. Chemical control of two Phytophthora species infecting the canopy of Monterey pine (Pinus radiata). Forest Pathology, 47.

Roman, L. A., Pearsall, H., Eisenman, T. S., Conway, T. M., Fahey, R. T., Landry, S., Vogt, J., Van Doorn, N. S., Grove, J. M. & Locke, D. H. 2018. Human and biophysical legacies shape contemporary urban forests: A literature synthesis. Urban Forestry & Urban Greening, 31, 157-168.

Rosenfeld, A. H., Akbari, H., Romm, J. J. & Pomerantz, M. 1998. Cool communities: strategies for heat island mitigation and smog reduction. Energy and Buildings, 28, 51-62.

Rosenzweig, C. & Solecki, W. 2001. Climate change and a global city: an assessment of the Metropolitan East Coast Region. Columbia earth institute, New York, PP 210.

Rosenzweig, C., Solecki, W. & Slosberg, R. 2006. Mitigating New York City’s heat island with urban forestry, living roofs, and light surfaces. A report to the New York State Energy Research and Development Authority.

Saetre, P. 1999. Spatial patterns of ground vegetation, soil microbial biomass and activity in a mixed spruce‐birch stand. Ecography, 22, 183-192. 125

Safaiefarahani, B., Mostowfizadeh-Ghalamfarsa, R., Hardy, G. S. J. & Burgess, T. 2015. Re- evaluation of the Phytophthora cryptogea species complex and the description of a new species, Phytophthora pseudocryptogea sp. nov. Mycological Progress, 14, 108.

Saindrenan, P., Barchietto, T., Avelino, J. & Bompeix, G. 1988. Effects of phosphite on phytoalexin accumulation in leaves of cowpea infected with Phytophthora cryptogea. Physiological and Molecular Plant Pathology, 32, 425-435.

Salles, J. F., Van Veen, J. A. & Van Elsas, J. D. 2004. Multivariate analyses of Burkholderia species in soil: effect of crop and land use history. Applied and Environmental Microbiology, 70, 4012-4020.

Sanap, S. & Patil, R. 2013. Synthesis, characterisation and biological activity of chiral mixed ligand Ni (II) complexes. Research Journal of Pharmacutical Sciences, 2, 1-10.

Sankaran, S., Ehsani, R., Inch, S. A. & Ploetz, R. C. 2012. Evaluation of visible-near infrared reflectance spectra of avocado leaves as a non-destructive sensing tool for detection of laurel wilt. Plant disease, 96, 1683-1689

Sapsford, S. J., Paap, T., Hardy, G. E. S. J. & Burgess, T. I. 2017. The ‘chicken or the egg’: which comes first, forest tree decline or loss of mycorrhizae? Plant Ecology, 218,1093-1106.

Saunders, S., Dade, E. & Van Niel, K. An Urban Forest Effects (UFORE) model study of the integrated effects of vegetation on local air pollution in the Western Suburbs of Perth, WA. 19th International Congress on Modelling and Simulation, 2011. 1824-1830.

Scanu, B., Linaldeddu, B. T., Deidda, A. & Jung, T. 2015. Diversity of Phytophthora species from declining Mediterranean maquis vegetation, including two new species, Phytophthora crassamura and P. ornamentata sp. nov. PloS One, 10, e0143234.

Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L., Levesque, C. A., Chen, W., Bolchacova, E., Voigt, K. & Crous, P. W. 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences, 109, 6241-6246.

Scott, P. 2011. The role of Phytophthora multivora in Eucalyptus gomphocephala (tuart) woodland decline. Doctoral dissertation, Murdoch University.

Scott, P., Burgess, T. & Hardy, G. 2013. Globalization and Phytophthora, CAB International, Wallingford, UK.

126

Scott, P., Jung, T., Shearer, B., Barber, P., Calver, M. & Hardy, G. E. S. J. 2012. Pathogenicity of Phytophthora multivora to Eucalyptus gomphocephala and Eucalyptus marginata. Forest Pathology, 42, 289-298.

Scott, P. & Williams, N. 2014. Phytophthora diseases in New Zealand forests. NZ Journal of Forestry, 59, 15.

Scott, P. M., Burgess, T., Barber, P., Shearer, B., Stukely, M., Hardy, G. E. S. J. & Jung, T. 2009. Phytophthora multivora sp. nov., a new species recovered from declining Eucalyptus, Banksia, Agonis and other plant species in Western Australia. Persoonia-Molecular Phylogeny and Evolution of Fungi, 22, 1-13.

Sena, K., Crocker, E., Vincelli, P. & Barton, C. 2018. Phytophthora cinnamomi as a driver of forest change: Implications for conservation and management. Forest Ecology and Management, 409, 799-807.

Serrano, M. S., De Vita, P., Fernández-Rebollo, P. & Hernández, M. E. S. 2012. Calcium fertilizers induce soil suppressiveness to Phytophthora cinnamomi root rot of Quercus ilex. European Journal of Plant Pathology, 132, 271-279.

Sevgi, F., Bagkesici, U., Kursunlu, A. N. & Guler, E. 2018. Fe (III), Co (II), Ni (II), Cu (II) and Zn (II) complexes of schiff bases based-on glycine and phenylalanine: Synthesis, magnetic/thermal properties and antimicrobial activity. Journal of Molecular Structure, 1154, 256-260.

Seymour, N., Thompson, J. & Fiske, M. 1994. Phytotoxicity of fosetyl Al and phosphonic acid to maize during production of vescicular-arbuscular mycorrhizal inoculum. Plant Disease, 78, 441-446.

Shahedi, Z., Jafari, M. R. & Zolanvari, A. A. 2017. Synthesis of ZnQ 2, CaQ 2, and CdQ 2 for application in OLED: optical, thermal, and electrical characterizations. Journal of Materials Science: Materials in Electronics, 28, 7313-7319.

Shea, S., Shearer, B. & Tippett, J. 1982. Recovery of Phytophthora cinnamomi Rands from vertical roots of jarrah (Eucalyptus marginata Sm)[Western Australia]. Australasian Plant Pathology (Australia), 11, 25-28.

Shearer, B. 1994. The major plant pathogens occurring in native ecosystems of. Journal of the Royal Society of Western Australia, 77, 113-122.

127

Shearer, B. 2014. Time course studies of temperature and soil depth mediated sporangium production by Phytophthora cinnamomi. Australasian Plant Pathology, 43, 235-244.

Shearer, B., Crane, C., Barrett, S. & Cochrane, A. 2007. Phytophthora cinnamomi invasion, a major threatening process to conservation of flora diversity in the South-west Botanical Province of Western Australia. Australian Journal of Botany, 55, 225-238.

Shearer, B., Crane, C. & Cochrane, A. 2004. Quantification of the susceptibility of the native flora of the South-West Botanical Province, Western Australia, to Phytophthora cinnamomi. Australian Journal of Botany, 52, 435-443.

Shearer, B. & Dillon, M. 1996. Impact and disease centre characteristics of Phytophthora cinnamomi infestations of Banksia woodlands on the Swan Coastal Plain, Western Australia. Australian Journal of Botany, 44, 79-90.

Shearer, B. & Fairman, R. 2007. A stem injection of phosphite protects Banksia species and Eucalyptus marginata from Phytophthora cinnamomi for at least four years. Australasian Plant Pathology, 36, 78-86.

Shearer, B. & Smith, I. 2000. Diseases of eucalypts caused by soilborne species of Phytophthora and Pythium. Diseases and pathogens of eucalypts, 259-291. CSIRO, Collingwood, Australia Shearer, B. & Tippett, J. T. 1989. Jarrah dieback: the dynamics and management of Phytophthora cinnamomi in the jarrah (Eucalyptus marginata) forest of south-western Australia, Department of Conservation and Land Management Perth. Bulletin 3, 1-75. Shebl, M., Adly, O. M., Abdelrhman, E. M. & El-Shetary, B. 2017. Binary and ternary copper (II) complexes of a new Schiff base ligand derived from 4-acetyl-5, 6-diphenyl-3 (2H)- pyridazinone: Synthesis, spectral, thermal, antimicrobial and antitumor studies. Journal of Molecular Structure, 1145, 329-338.

Shimazaki, Y., Takani, M. & Yamauchi, O. 2009. Metal complexes of amino acids and amino acid side chain groups. Structures and properties. Dalton Transactions, 7854-7869.

Simamora, A. V., Paap, T., Howard, K., Stukely, M. J., Hardy, G. E. S. J. & Burgess, T. I. 2017. Phytophthora contamination in a nursery and Its potential dispersal into the natural environment. Plant Disease, 102, 132-139.

Simberloff, D. 2009. The role of propagule pressure in biological invasions. Annual Review of Ecology, Evolution, and Systematics, 40, 81-102.

128

Sims, L. L., Sutton, W., Reeser, P. & Hansen, E. M. 2015. The Phytophthora species assemblage and diversity in riparian alder ecosystems of western Oregon, USA. Mycologia, 107, 889- 902.

Singh, S. 2002. Citrus in India: Hi Tech Citrus Management. National Reseacrh Center for Citrus Bulletin, Nagpur, 278-303.

Smaili, A., Rifai, L. A., Esserti, S., Koussa, T., Bentiss, F., Guesmi, S., Laachir, A. & Faize, M. 2017. Copper complexes of the 1, 3, 4-thiadiazole derivatives modulate antioxidant defense responses and resistance in tomato plants against fungal and bacterial diseases. Pesticide Biochemistry and Physiology, 143, 26-32. Smith, V., Wilcox, W. & Harman, G. 1990. Potential for biological control of Phytophthora root and crown rots of apple by Trichoderma and Gliocladium spp. Phytopathology, 80, 880- 885.

Snapp, S., Shennan, C. & Van Bruggen, A. 1991. Effects of salinity on severity of infection by Phytophthora parasitica Dast., ion concentrations and growth of tomato, Lycopersicon esculentum Mill. New Phytologist, 119, 275-284.

Sogin, M. L., Morrison, H. G., Huber, J. A., Welch, D. M., Huse, S. M., Neal, P. R., Arrieta, J. M. & Herndl, G. J. 2006. Microbial diversity in the deep sea and the underexplored “rare biosphere”. Proceedings of the National Academy of Sciences, 103, 12115-12120.

Solecki, W. D., Rosenzweig, C., Parshall, L., Pope, G., Clark, M., Cox, J. & Wiencke, M. 2005. Mitigation of the heat island effect in urban New Jersey. Global Environmental Change Part B: Environmental Hazards, 6, 39-49.

Solomon, S. 2007. Climate change 2007-the physical science basis: Working group I contribution to the fourth assessment report of the IPCC, Cambridge University Press. 4. Stab, M. R. & Ebel, J. 1987. Effects of Ca2+ on phytoalexin induction by fungal elicitor in soybean cells. Archives of Biochemistry and Biophysics, 257, 416-423.

Stagoll, K., Lindenmayer, D. B., Knight, E., Fischer, J. & Manning, A. D. 2012. Large trees are keystone structures in urban parks. Conservation Letters, 5, 115-122.

Stamps, D. J., Waterhouse, G., Newhook, F. & Hall, G. 1990. Revised tabular key to the species of Phytophthora, CAB-International.Stanghellini, M., Kim, D., Rasmussen, S. & Rorabaugh, P. 1996a. Control of root rot of peppers caused by Phytophthora capsici with a nonionic surfactant. Plant disease, 80, 1113-1116.

129

Stanghellini, M., Rasmussen, S., Kim, D. & Rorabaugh, P. 1996b. Efficacy of nonionic surfactants in the control of zoospore spread of Pythium aphanidermatum in a recirculating hydroponic system. Plant Disease, 80, 422-428.

Stasikowski, P., Clark, D., Mccomb, J., O’brien, P. & Hardy, G. E. S. J. 2014a. A direct chemical method for the rapid, sensitive and cost effective detection of phosphite in plant material. Australasian Plant Pathology, 43, 115-121.

Stasikowski, P., Mccomb, J., Scott, P., Paap, T., O’ Brien, P. & Hardy, G. E. S. J. 2014b. Calcium sulphate soil treatments augment the survival of phosphite-sprayed Banksia leptophylla infected with Phytophthora cinnamomi. Australasian Plant Pathology, 43, 369-379.

Stasikowski, P., Mccomb, J., Scott, P., Paap, T., O’brien, P. & Hardy, G. S. J. 2014c. Calcium sulphate soil treatments augment the survival of phosphite-sprayed Banksia leptophylla infected with Phytophthora cinnamomi. Australasian Plant Pathology, 43, 369-379.

Stenhouse, R. N. 2004. Fragmentation and internal disturbance of native vegetation reserves in the Perth metropolitan area, Western Australia. Landscape and Urban Planning, 68, 389- 401.

Stokstad, E. 2004. Nurseries may have shipped sudden oak death pathogen nationwide. American Association for the Advancement of Science.

Stolzy, L. & Sojka, R. 1984. Effects of flooding on plant disease. Flooding and plant growth, 221- 264.

Strömberg, A. & Brishammar, S. 1991. Induction of systemic resistance in potato (Solanum tuberosum L.) plants to late blight by local treatment with Phytophthora infestans (Mont.) de Bary, Phytophthora cryptogea Pethyb. & laff. or dipotassium phosphate. Potato Research, 34, 219-225.

Stukely, M., Webster, J., Ciampini, J., Brown, E., Dunstan, W., Hardy, G. E. S. J., Woodman, G., Davison, E. & Tay, F. 2007. Phytophthora inundata from native vegetation in Western Australia. Australasian Plant Pathology, 36, 606-608.

Sugimoto, T., Watanabe, K., Yoshida, S., Aino, M., Irie, K., Matoh, T. & Biggs, A. 2008. Select calcium compounds reduce the severity of Phytophthora stem rot of soybean. Plant Disease, 92, 1559-1565.

130

Sulistyowati, L. & Keane, P. 1992. Effect of soil salinity and water content on stem rot caused by Phytophthora citrophthora and accumulation of phytoalexin in citrus rootstocks. Phytopathology, 82, 771-777.

Swiecki, T. & Macdonald, J. 1991. Soil salinity enhances phytophthora root rot of tomato but hinders asexual reproduction by Phytophthora parasitic. Journal of the American Society for Horticultural Science, 116, 471-477.

Sysuev, V., Konoshenko, G., Terekhova, V., Okorokov, L. & D'iakov, I. 1978. Phosphohydrolases of the phytopathogenic fungus Phytophthora infestans (Mont) de Bary. Biokhimiia (Moscow, Russia), 43, 1790-1795.

Taghizadeh, L., Montazerozohori, M., Masoudiasl, A., Joohari, S. & White, J. 2017. New tetrahedral zinc halide Schiff base complexes: Synthesis, crystal structure, theoretical, 3D Hirshfeld surface analyses, antimicrobial and thermal studies. Materials Science and Engineering: C, 77, 229-244.

Talbot, R. J., Etherington, J. R. & Bryant, J. A. 1987. Comparative studies of plant growth and distribution in relation to waterlogging. New Phytologist, 105, 563-574.

Tamm, L., Thürig, B., Bruns, C., Fuchs, J. G., Köpke, U., Laustela, M., Leifert, C., Mahlberg, N., Nietlispach, B. & Schmidt, C. 2010. Soil type, management history, and soil amendments influence the development of soil-borne (Rhizoctonia solani, Pythium ultimum) and air- borne (Phytophthora infestans, Hyaloperonospora parasitica) diseases. European Journal of Plant Pathology, 127, 465-481.

Thao, H. T. B. & Yamakawa, T. 2009. Phosphite (phosphorous acid): Fungicide, fertilizer or bio‐ stimulator? Soil Science & Plant Nutrition, 55, 228-234.

Themann, K., Werres, S., Lüttmann, R. & Diener, H.-A. 2002. Observations of Phytophthora spp. in water recirculation systems in commercial hardy ornamental nursery stock. European Journal of Plant Pathology, 108, 337-343.

Thomas, F., Blank, R. & Hartmann, G. 2002. Abiotic and biotic factors and their interactions as causes of oak decline in Central Europe. Forest Pathology, 32, 277-307.

Thomas, P. A. 2016. Biological Flora of the British Isles: Fraxinus excelsior. Journal of Ecology, 104, 1158-1209.

Thomidis, T. & Elena, K. 2001. Effects of Metalaxyl, Fosetyl‐Al, Dimethomorph and Cymoxanil on Phytophthora cactorum of Peach Tree. Journal of Phytopathology, 149, 97-101. 131

Tran, H., Ficke, A., Asiimwe, T., Höfte, M. & Raaijmakers, J. M. 2007. Role of the cyclic lipopeptide massetolide A in biological control of Phytophthora infestans and in colonization of tomato plants by Pseudomonas fluorescens. New Phytologist, 175, 731-742.

Tubby, K. & Webber, J. 2010. Pests and diseases threatening urban trees under a changing climate. Forestry: An International Journal of Forest Research, 83, 451-459.

Tynan, K., Wilkinson, C., Holmes, J., Dell, B., Colquhoun, I., Mccomb, J. & Hardy, G. E. S. J. 2001. The long-term ability of phosphite to control Phytophthora cinnamomi in two native plant communities of Western Australia. Australian Journal of Botany, 49, 761-770.

Tyrväinen, L. & Miettinen, A. 2000. Property prices and urban forest amenities. Journal of Environmental Economics and Management, 39, 205-223.

Valentini, A., Pompanon, F. & Taberlet, P. 2009. DNA barcoding for ecologists. Trends in Ecology & Evolution, 24, 110-117.

Van Rijt, S. H. & Sadler, P. J. 2009. Current applications and future potential for bioinorganic chemistry in the development of anticancer drugs. Drug Discovery Today, 14, 1089-1097.

Vannini, A., Bruni, N., Tomassini, A., Franceschini, S. & Vettraino, A. M. 2013. Pyrosequencing of environmental soil samples reveals biodiversity of the Phytophthora resident community in chestnut forests. FEMS microbiology ecology, 85, 433-442.

Veena, S., Anandaraj, M. & Sarma, Y. 2010. Variability in the sensitivity of Phytophthora capsici isolates to potassium phosphonate. Indian Phytopathology, 63, 71.

Velarde, M. D., Fry, G. & Tveit, M. 2007. Health effects of viewing landscapes–Landscape types in environmental psychology. Urban Forestry & Urban Greening, 6, 199-212.

Vettraino, A., Barzanti, G., Bianco, M., Ragazzi, A., Capretti, P., Paoletti, E., Luisi, N., Anselmi, N. & Vannini, A. 2002. Occurrence of Phytophthora species in oak stands in Italy and their association with declining oak trees. Forest Pathology, 32, 19-28.

Waffle, A. D., Corry, R. C., Gillespie, T. J. & Brown, R. D. 2017. Urban heat islands as agricultural opportunities: An innovative approach. Landscape and Urban Planning, 161, 103-114.

Wang, H., Ouyang, Z., Chen, W., Wang, X., Zheng, H. & Ren, Y. 2011. Water, heat, and airborne pollutants effects on transpiration of urban trees. Environmental pollution, 159, 2127- 2137.

132

Ward, B. & Mckimm, R. 1982. A preliminary survey of severe dieback in the coastal, mixed eucalypt forests of east Gippsland [Victoria]. Forestry Technical Papers Forests Commission Victoria.

Watanarojanaporn, N., Boonkerd, N., Wongkaew, S., Prommanop, P. & Teaumroong, N. 2011. Selection of arbuscular mycorrhizal fungi for citrus growth promotion and Phytophthora suppression. Scientia horticulturae, 128, 423-433.

Webber, J. & Rose, J. Dissemination of aerial and root infecting Phytophthoras by human vectors. Proceedings of the sudden oak death 3rd science symposium. Gen. Tech. Rep. PSW-GTR- 214. Albany, CA: US Department of Agriculture, Forest Service, Pacific Southwest Research Station, 2008. 195-198.

Werres, S., Marwitz, R., De Cock, A. W., Bonants, P. J., De Weerdt, M., Themann, K., Ilieva, E. & Baayen, R. P. 2001. Phytophthora ramorum sp. nov., a new pathogen on Rhododendron and Viburnum. Mycological Research, 105, 1155-1165.

Weste, G. 2003. The dieback cycle in Victorian forests: a 30-year study of changes caused by Phytophthora cinnamomi in Victorian open forests, woodlands and heathlands. Australasian Plant Pathology, 32, 247-256.

Wilson, B. A., Zdunic, K., Kinloch, J. & Behn, G. 2012. Use of remote sensing to map occurrence and spread of Phytophthora cinnamomi in Banksia woodlands on the Gnangara Groundwater System, Western Australia. Australian Journal of Botany, 60, 495-505.

Wingfield, M. J., Slippers, B., Roux, J. & Wingfield, B. D. 2001. Worldwide Movement of Exotic Forest Fungi, Especially in the Tropics and the Southern Hemisphere: This article examines the impact of fungal pathogens introduced in plantation forestry. AIBS Bulletin, 51, 134- 140.

World Health Organizatio 2018. Urban Population Prospects.

World Health Organization 2014. Urban Population Growth. Retrieved January 20, 2016.

Worrall, J. J., Marchetti, S. B., Egeland, L., Mask, R. A., Eager, T. & Howell, B. 2010. Effects and etiology of sudden aspen decline in southwestern Colorado, USA. Forest Ecology and Management, 260, 638-648.

Wrigley, J. W. & Fagg, M. 2013. Australian native plants: cultivation, use in landscaping and propagation, Reed New Holland.

133

Xiao, K., Kinkel, L. L. & Samac, D. A. 2002. Biological control of Phytophthora root rots on alfalfa and soybean with Streptomyces. Biological Control, 23, 285-295.

Yang, X., Tyler, B. M. & Hong, C. 2017. An expanded phylogeny for the genus Phytophthora. IMA fungus, 8, 355-398.

You, M. & Sivasithamparam, K. 1994. Hydrolysis of fluorescein diacetate in an avocado plantation mulch suppressive to Phytophthora cinnamomi and its relationship with certain biotic and abiotic factors. Soil Biology and Biochemistry, 26, 1355-1361.

Zak, B. 1964. Role of mycorrhizae in root disease. Annual Review of Phytopathology, 2, 377-392.

Zentmyer, G. 1981. The effect of temperature on growth and pathogenesis of Phytophthora cinnamomiand on growthof its avocado host, Ecology and Epidemiology,71, 925-928. IS thiS the COrreCT journal name

134

Appendix A

Mean lesion lengths of Phytophthora species (21 isolates from 19 species) in fifteen plant species

mean

0

1.01 3.97 4.57 4.63 5.13 9.81

10.19 11.21 11.86 12.25 12.87 14.61 15.43 16.61 16.37 18.89 19.51 21.50 23.32 23.95 26.63

Phytophthora

0 0 0 0 0 0 4 0 0 2

1.2 2.7 5.4 1.3 1.6 2.5 1.9 1.3 5.6 2.2 8.1 3.5

2.06

Fraxinusraywoodii

0 0 0 0 0 4

4.5 4.9 2.1 0.9 4.1 0.9 2.5 3.9 4.8 3.4 3.3 2.1 1.3 4.1 1.5 5.4

2.56

aeuropaea

Ole

0 0 0 0 3

1.5 1.7 1.6 0.4 9.2 1.1 1.3 3.9 4.8 7.7 0.8 4.3 2.5 8.8 2.2 5.8 9.1

3.32

Melalecua sp. Melalecua

0 0 4 0 8 6 2 4

0.7 8.5 5.6 1.1 3.8 3.9 2.3 3.1 7.8 8.3 4.2 2.5

11.7 11.9 4.73

Pyrus ussuriensis Pyrus

0 0 0 0 0 0

1.7 5.6 2.8 3.6 0.2 1.3 6.5 1.4 2.9 1.3 7.7 8.5 7.7

10.4 10.5 10.5 3.93 Plantanus orientalis Plantanus

135

0 3 0

12

0.2 2.1 2.4 1.2 0.3 0.2 4.3 7.9 8.5 4.2 4.4 7.2 7.7

10.5 10.3 15.8 11.8 17.6 6.27

Metrosideros excelsa Metrosideros

0 0 0 0 0 0 3 0 3 0 0

10

2.4 3.5 6.3 2.1 4.7 5.5

23.9 21.3 19.8 16.5 5.81

Banksia sessilis Banksia

0 0 0 0 2 0 6

48

8.8 4.4 7.3 8.1 7.7 9.7

21.5 11.5 20.9 14.3 31.9 11.3 13.2 40.4

12.71

Eucalyptus Eucalyptus gomphocephala

0 7

17 10

9.9 3.5 6.6 7.2

21.2 22.2 22.5 16.5 10.2 18.7 11.7 15.3 13.2 17.2 13.4 23.5 34.9 32.8

15.93

Corymbia calophylla Corymbia

0 0 0 0 0 1

10 11 10 50

1.5 1.6 6.1 6.2 6.8 4.6 2.8 5.5 3.8

14.3 10.3 74.1

10.46

Callistemon sp. Callistemon

7

0 0 0 0 0 0 0 0 0 0

29

1.7 7.5 4.

18.4 13.1 15.9 24.9 28.3 52.2 46.4 32.3

13.07 Viburnum tinus Viburnum

136

0 0 0 0 0 0 5 0

17

6.2 8.6 4.8

17.8 25.3 11.2 29.4 25.8 26.7 36.7 25.5 38.7 20.3

14.24

Ficusmacrocarpa

0 0

19 37 41 38

5.7 5.3

11.2 21.5 15.2 17.3 19.5 39.4 30.5 30.5 40.5 48.7 36.5 56.8 30.8 38.2

27.74

Magnolia grandiflora Magnolia

0 0 9 0

14 64 15 67

4.2 3.3 7.8

17.7 13.9 51.5 17.1 10.3 16.5 63.4 76.4 27.6 30.7

108.5 29.42

Agonisflexuosa

0 5

36 50 60

23.8 19.7 29.5 39.4 34.6 61.3 57.3 48.9 71.1 48.3 55.7 65.1 50.9 66.5 66.4 72.3

105.6 50.83

Eucalyptus marginata Eucalyptus

1 2

- -

1 2

- -

ocryptogea

control P.versiformis P.boodjera P.cambivora P.frigida P.arenaria P.thermophila P.crassamura P.multivora P.amnicola P.palmivora P.nicotianae P.'walnut' P.capensis P.niederhauserii P.nicotianae P.multivora P.drechsleri P.capsici P.citrophthora P.pseud P.cinnamomi mean Host Plant species Plant

137