Canker and Dieback diseases or Eucalyptus in Portugal

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CANKER AND DIEBACK OF EUCALYPTUS IN PORTUGAL.

TAXONOMY AND PATHOLOGY OF ASSOCIATED FUNGI

EUGÉNIO LUÍS FRAGA DIOGO Master in Plant Production

DOCTORATE IN BIOLOGY

NOVA University Lisbon

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EUGÉNIO LUÍS FRAGA DIOGO

DOCTORATE IN BIOLOGY

MASTER IN PLANT PRODUCTION

Examination Committee:

Chair: Pedro Miguel Ribeiro Viana Baptista

Full Professor, NOVA School of Science and Technology, NOVA University Lisbon

Rapporteurs: Maria Helena Mendes da Costa Ferreira Correia de Oliveira,

Associate Professor, The School of Agriculture, University of Lisbon

Artur Jorge da Costa Peixoto Alves

Assistant Professor with Habilitation, Department of Biology, University of Aveiro

Co-Adviser: Maria Helena Pires Bragança

Assistant Researcher, National Institute for Agricultural and Veterinary Research, I.P.

Members Maria do Céu Machado Lavado da Silva

Assistant Researcher, The School of Agriculture, University of Lisbon

Catarina Isabel Antunes Gonçalves

Senior Technical Researcher, RAIZ, Forest and Paper Research Institute

Pedro Miguel Ribeiro Viana Baptista

Full Professor, NOVA School of Science and Technology, NOVA University Lisbon

José Paulo Nunes de Sousa Sampaio

Associate Professor with Habilitation, NOVA School of Science and Technology, NOVA University Lisbon

CANKER AND DIEBACK OF EUCALYPTUS IN PORTUGAL.

TAXONOMY AND PATHOLOGY OF ASSOCIATED FUNGI

NOVA University Lisbon

Adviser: Alan John Lander Phillips

Invited Principal Researcher, University of Lisbon

Co-Adviser: Maria Helena Pires Bragança

Assistant Researcher, National Institute for Agricultural and Veterinary Research, I.P.

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University Lisbon.

The NOVA School of Science and Technology and the NOVA University Lisbon have the right, perpetual and without geographical boundaries, to file and publish this dissertation through printed copies reproduced on paper or on digital form, or by any other means known or that may be invented, and to disseminate through scientific repositories and admit its copying and distribution for non-commercial, educational or research purposes, as long as credit is given to the author and editor.

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To my wife Leonor and my daughters, Ana Rita and Sofia

To my parents.

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collaboration of many people. They helped me to accomplish the tasks that I set to myself and to whom I want to express my sincere thanks.

First, I am deeply indebted to my advisor, Dr. Alan Phillips, for agreeing to guide me and for the commitment and availability that he always showed throughout the work, and for everything I learned from him.

To my dear colleague and co-supervisor Helena Bragança, for her commitment to accompanying me in the fieldwork, for sharing all the laboratory work and for negotiating the financial support that allowed this work to be carried out. Without her encouragement, especially at times when things didn't go as planned and the desire to give up was great, I would never have finished this thesis.

To my colleague Helena Machado, for her help, permanent encouragement, and advice, in particular with the work on Phytophthora.

To my best friend Marco Naia for all the support and for providencial advice.

To my master and lab colleague Leonor Cruz. In those moments of despair, your wise words helped me to get back on my feet and move forward.

To my colleague Ana Lança, with whom I share the office, thank you for your ongoing support and last-minute corrections and suggestions.

I acknowledge the financial support from the RAIZ Institute and Altri Florestal S. A.

through a protocol signed with INIAV to carry out the work that gave rise to this thesis. In particular, from the RAIZ Institute, Dr. Carlos Valente, Dra. Catarina Gonçalves and Dr. Nuno Borralho. From Altri Florestal S.A., Drª Lucília Neves, Drª Clara Araújo, Eng. Luís Leal and Engª Ana Reis.

I thank INIAV for allowing me to undertake this thesis without leaving my normal tasks, especially the Director of the Agricultural and Forestry Systems and Plant Health Unit, Drª Amélia Lopes, for her constant and permanent support and encouragement.

To Dr. Nuno Onofre and Luís Bonifácio, whose help with the statistical analysis was essential to achieve good results.

To Diana Pereira for the interesting and stimulating discussions and help with microscope work.

To the laboratory technicians: Isabel Lourenço, Florinda Medeiros always available to prepare everything I need for experiments, to Adérito Matos and Francisco Martins for greenhouse maintenance and for taking good care of the plants.

During these long years, I shared the laboratory and tasks with several masters or doctoral students. Not only their help but also the questions constituted a stimulus that directly or indirectly contributed to the result presented here. Ana Silva, Joana Neno, Joana Henrique, Sandra Veloso, thank you all.

To my wife and daughters, for all the time I was not present and, even so, they always encouraged me not to give up.

Last but not the least, to my parents, who, despite not being with me anymore and not having had the opportunity to have a higher education, knew how to transmit to me essential values and, above all, to recognize the importance of education. Without their support, sacrifice, and encouragement to study, I would not have accomplished this stage of my career.

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It always seems impossible until it's done.

Nelson Mandela

The important thing is not to stop questioning.

Curiosity has its own reason for existing.

Albert Einstein

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to their fast growth rate and adaptability to different environments. They were introduced into Portugal over 100 years ago, and presently represent 26% of the forest area. Their use is mainly for pulp and paper production, representing a very important economic activity. Until a few decades ago, they were relatively free of pests and diseases. Since the 1990s, a leaf disease caused by species of Mycosphaerella and Teratosphaeria have caused severe defoliation in young plantations in several areas in the country. Since then, other diseases, such as canker and dieback, became more frequent. This situation also occurred in other areas of the world where eucalypts are planted as exotic species. Although difficult to determine, several factors can contribute to these emerging diseases such as climate change, introduction of new pathogens or hosts shifts of pathogen from native species.

To identify the fungi associated with these diseases, a national survey was conducted and samples from diseases trees collected. The fungi associated with the symptoms were identified using classical morphological methods and biomolecular techniques and their pathogenicity tested in controlled conditions. As a result of this survey, two new pathogen of eucalypts, Quambalaria eucalypti and Phytophthora alticola, were reported for the first time in Portugal and in the Northern Hemisphere. An account of the diversity and pathogenicity of Botryosphaeriaceae species associated with eucalypts and its possible host shift with cork oak, another important species of Portuguese forests, was also investigated. During this study, a new nursery disease associated with Neopestalotiopsis species was characterized and five species of this genus described as new.

The results reported in this thesis are of paramount importance to take measures to avoid the spread and damage caused by these diseases. Among others, the most efficient measure to control fungal disease in forests is breeding for resistance. Since these are emergent diseases, they were not previously included in the breeding programs of eucalypts.

Another possible tool is biological control. Although very successful in the control of forest pests, it is a new area to be developed for fungi causing diseases in forest.

Keywords: Emergent forest diseases, Neopestalotiopsis species, Pathogenicity, Phytophthora alticola, Quambalaria eucalypti.

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o mundo devido ao seu rápido crescimento e adaptabilidade a diferentes ambientes. Foi introduzido em Portugal há mais de 100 anos e, atualmente, representa 26% da área florestal.

É utilizado principalmente para a produção de celulose e papel, representando uma atividade económica muito importante. Até algumas décadas atrás, estava relativamente isento de pragas e doenças. Desde a década de 1990, uma doença foliar causada por espécies de Mycosphaerella e Teratosphaeria provoca desfolha severa em plantações jovens em diversas áreas do país. Desde então, outro tipo de sintomas, tais como cancros e morte tornaram-se mais frequentes. Essa situação também ocorreu em outras áreas do mundo onde o eucalipto é plantado como espécie exótica. Embora difícil de determinar, vários fatores podem contribuir para essas doenças emergentes, como as alterações climáticas, introdução de novos organismos patogénicos ou mudanças de hospedeiros com origem em hospedeiros nativos.

Para identificar os fungos associados a essas doenças, foi realizada uma prospeção em todo o país e colhidas amostras de árvores doentes. Os fungos associados aos sintomas foram identificados usando métodos morfológicos clássicos e técnicas biomoleculares e a sua patogenicidade testada em condições controladas. Como resultado deste levantamento, dois novos fungos patogénicos em eucalipto, Quambalaria eucalypti e Phytophthora alticola, foram reportados pela primeira vez em Portugal e no Hemisfério Norte. Foi também investigada a diversidade e patogenicidade de espécies de Botryosphaeriaceae associadas ao eucalipto e a sua possível mudança de hospedeiro com sobreiro, outra importante espécie da floresta portuguesa. Durante este estudo, uma nova doença em viveiros, causado por espécies do género Neopestalotiopsis foi caracterizada e cinco novas espécies deste género foram descritas.

Os resultados relatados nesta tese são de suma importância para decidir que medidas tomar para evitar a propagação e os danos causados por essas doenças. Entre outras, a medida mais eficiente para controlar doenças fúngicas em florestas é o melhoramento de variedades resistentes. Por serem doenças emergentes, não foram incluídas nos programas de melhoramento de eucalipto existentes. Outra ferramenta possível é o controle biológico.

Usado com sucesso sobretudo no controle de pragas florestais, é uma nova área a ser desenvolvida para fungos causadores de doenças na floresta.

Palavras-chave: Quambalaria eucalypti, Phytophthora alticola, Neopestalotiopsis spp., Patogenicidade, Doenças florestais emergentes.

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Acknowledgments ... ix

Abstract ... xiii

Resumo ...xv

List of Figures ... xxi

List of Tables ... xxiii

List of abbreviations ... xxv

1. General Introduction... 1

1.1. Prologue ... 1

1.2. Botany of eucalypts ... 1

1.3. Importance of eucalypts plantations ... 2

1.4. Introduction and expansion of eucalypts in Portugal ... 2

1.5. Most important canker and dieback diseases in eucalypt plantations ... 4

Pink disease ... 5

Chrysoporthe canker ... 6

Teratosphaeria canker ... 7

Botryosphaeria canker ... 8

Quambalaria canker ... 9

Other canker diseases ... 10

1.6. Root rots diseases ... 10

Armillaria root rot ... 11

Phytophthora root rot ... 11

1.7. Eucalypts diseases in Portugal with emphasis on canker and dieback diseases ... 12

1.8. Aim of this work ... 13

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1.9. References ... 14

2. Quambalaria eucalypti first reported from Portugal and Europe ... 27

Abstract... 27

2.1. Introduction ... 27

2.2. Materials and methods ... 28

Sampling and isolation ... 28

Morphological characterization ... 29

Molecular phylogenetic characterization ... 30

Pathogenicity tests ... 32

2.3. Results ... 34

Survey and Symptoms... 34

Fungal morphology ... 34

Phylogenetic analysis ... 34

2.4. Discussion ... 37

3. Identity and pathogenicity of Botryosphaeriaceae species on cork oak and eucalypts .. 41

Abstract... 41

3.2. Material and Methods ... 43

Morphology ... 45

DNA extraction, PCR amplification and sequencing ... 46

3.3. Results ... 65

Isolation and morphology ... 65

Phylogeny ... 66

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4.2. Material and methods ... 84

Survey ... 84

Isolations ... 85

Identification ... 85

4.3. Results ... 92

Survey ... 92

Morphology ... 94

5. Five new species of Neopestalotiopsis associated with diseased Eucalyptus spp. in Portugal ... 107

5.1. Abstract... 107

5.3. Materials and Methods ... 108

Morphology ... 108

5.4. Results ... 118

Symptoms, fungal isolation, and identification ... 118

Taxonomy ... 121

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6. General discussion and future prospect ... 139

6.1. Achievements ... 139

6.2. Disease origins ... 140

6.3. Disease management ... 141

6.4. Futures prospects ... 143

Appendix I ... 151

Results of statistical analysis of pathogenicity assays, first trial ... 151

Results of statistical analysis of pathogenicity assays, second trial ... 151

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Figure 1.2 Specie's relative composition of the Portuguese forest. ... 4

Figure 2.1 Sampling locations for Quambalaria eucalypti in Portugal. ... 29

Figure 2.2 Symptoms of Quambalaria eucalypti on Eucalyptus globulus. ... 30

Figure 2.3 Morphology of Quambalaria eucalypti. . ... 35

Figure 2.4 One of two most parsimonious trees resulting from the alignment of 625 characters of the ITS rDNA region. ... 36

Figure 2.5 Pathogenicity of Quambalaria eucalypti on Eucalyptus globulus... 37

Figure 3.1 Map of Portugal showing the municipalities where eucalypts and cork oak stands were surveyed. ... 44

Figure 3.2 Cork oak with symptoms of decline. ... 45

Figure 3.3 Eucalypts with symptoms of decline and branch dead. ... 46

Figure 3.4 Phylogenetic tree of Botryosphaeria species resulting from a maximum likelihood analysis of combined ITS and tef1 datasets. ... 67

Figure 3.5 Phylogenetic tree of Neofusicoccum species resulting from a maximum likelihood analysis using combined ITS and tef1 gene region datasets. ... 69

Figure 3.6 Phylogenetic tree of Diplodia species resulting from a maximum likelihood analysis of combined ITS and tef1 datasets. ... 69

Figure 3.7 Phylogenetic tree of Dothiorella species resulting from a maximum likelihood analysis of the ITS dataset. ... 70

Figure 4.1 Symptoms of root rot and dieback associated with Phytophthora spp. on Eucalyptus globulus. ... 92

Figure 4.2 Locations where Phytophthora on Eucalyptus globulus was detected. ... 93

Figure 4.3 Morphological characteristics of Phytophthora cinnamomi (MEAN 1271). ... 94

Figure 4.4 Morphological characteristics of Phytophthora alticola (MEAN 1269). ... 95

Figure 4.5 Phylogram generated from maximum likelihood analysis based ITS sequence alignment for selected species of Phytophthora. ... 97

Figure 5.1 Symptoms caused by species of Neopestalotiopsis on Eucalyptus spp. ... 119

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Figure 5.2 - Consensus phylogram of 1000 trees resulting from a RAxML analysis of the (ITS + tub2 + tef1) alignment of the analysed Neopestalotiopsis sequences. ... 120 Figure 5.3 Neopestalotiopsis eucalyptorum (Holotype LISE 96318, ex-type culture CBS 147684

= MEAN 1308). ... 121 Figure 5.4 Neopestalotiopsis hispanica (Holotype LISE 96319, ex-type culture CBS 147686

= MEAN 1310). ... 123 Figure 5.5 Neopestalotiopsis iberica (Holotype LISE 96320, ex-type culture MEAN 1313

= CBS 147688). ... 124 Figure 5.6 Neopestalotiopsis longiappendiculata (Holotype LISE 96321, ex-type culture CBS 147690 = MEAN 1315). ... 127 Figure 5.7 Neopestalotiopsis lusitanica (Holotype LISE 96322, ex-type culture CBS 147692

= MEAN 1317). . ... 129

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and phylogenetic study. ... 31 Table 3.1 Isolates used in morphological studies, sequence analysis and pathogenicity tests ... 49 Table 3.2 Isolates used in the phylogenetic analysis ... 53 Table 3.3 Statistics and substitution models used in the phylogenetic analysis. ... 66 Table 3.4 Results of the pathogenicity tests 40 days after inoculation. ... 71 Table 4.1 Phytophthora species and isolates used in this study, and GenBank ITS accession numbers. ... 89 Table 4.2 Mean length (cm) ± standard error (SE) of the external lesion and percentage of reisolations from branches inoculated with P. alticola and P. cinnamomi. ... 96 Table 4.3 Mean length (cm) of new roots, height (cm) of stem, dry height (g) of new roots and stems ± standard error (SE) and percentage of reisolations from seedlings grown in soil infested with P. alticola (MEAN 1269) and P. cinnamomi (MEAN 1271). ... 96 Table 5.1 - Isolates used in this study. ... 111 Table 5.2 Morphological comparison of Neopestalotiopsis species related to this study. ... 125

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act Partial Actin Gene

AIC Akaike Information Criterion ANOVA Analysis of Variance

BI Bayesian Inference

BLAST Basic Alignment Search Tool

BRIP Queensland Plant Pathology Herbarium, Brisbane, Australia cal Calmodulin Partial Gene

CBS Culture Collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands

CERC Culture Collection of China Eucalypt Research Centre, Chinese Academy of Forestry, ZhanJiang, GuangDong Province, China

CFCC China Forestry Culture Collection Center, Research Institute of Forest Ecology, Environment and Protection, Beijing, China

CGMCC China General Microbial Culture Collection

CGMCC China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

CI Consistency Index

CMW Culture Collection of M.J. Wingfield, at Forestry and Agricultural Biotechnology Institute

COAD Coleção Octávio Almeida Drummond, Universidade Federal de Viçosa, Brazil Cox1 Cytochrome C Oxidase I Gene

CPA Carrot Piece Agar

DAR Plant Pathology Herbarium, Orange Agricultural Institute, Departmento of Primary Industries, Orange, New SouthWales, Australia

diam. Diameter

DNA Deoxyribonucleic Acid

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e.g. exempli gratia (latin meaning “for example”) et al. et alii (latin meaning “and others”)

gapdh Glyceraldehyde-3-phosphate dehydrogenase gene GDP Gross Domestic Product

gs Glutamine Synthase Gene

GTR General Time Reversible model of nucleotide substitution

GZCC Guizhou Academy of Agricultural Sciences Culture Collection, GuiZhou, China HGUP Plant Pathology Herbarium of Guizhou University, China

HI Homoplasy Index

HKY Hasegawa-Kishino-Yano model of nucleotide substitution HSP90 Heat Shock Protein 90

ICNF Instituto da Conservação da Natureza e Floresta

IMI Culture Collection of CABI Europe UK Centre, Egham, UK INIAV Instituto Nacional de Investigação Agrária e Veterinária, I. P.

ITS internal transcribed spacers and intervening 5.8S nrDNA

JZB Culture Collection of Institute of Plant and Environment Protection, Beijin Academy of Agriculture and Forestry Sciences, China

KUMCC Kunming Institute of Botany Culture Collection, Yunnan, China LISE Hebarium from ex-Estação Agronómica Nacional

LSD Least Significant Difference

LSU Large Subunit ribosomal Ribonucleic Acid MCL Maximum Composite Likelihood

MCMC Markov Chain Monte Carlo Method

MEAN Fungal Collection at INIAV, Oeiras, Portugal

MFLUCC Mae Fah Luang University Culture Collection, Chiang Rai, Thailand;

ML Maximum Likelihood

MLB Maximum Likelihood Bootstrap Support

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NJ Neighbour–Joining nrDNA Nuclear Ribosomal DNA

PAB Paul Barber, in Murdoch University Culture Collection PCR Polymerase Chain Reaction

PDA Potato Dextrose Agar ppm Parts Per Million

RAIZ Forest and Paper Research Institute RAxML Random Accelerated Maximum Likelihood RI Retention Index

rpb1 DNA-directed RNA Polymerase II, Subunit 1 Gene s.l. Sensu lato

SNP Single Nucleotide Polymorphism

SSU Small Subunit Ribosomal Ribonucleic Acid Taq Thermus aquaticus DNA polymerase TBE Tris/Borate/EDTA buffer solution

tef1 Partial Translation Elongation Factor 1-α gene TL Tree length

tub2 Partial β-tubulin Gene

URM Culture Collection of the Universidade Federal de Pernambuco, Brazil UV Ultraviolet

V8 Vegetable Juice Agar

VHS Vegetation Health Service Collection, Department of Parks and Wildlife, Perth, Australia

WA Water agar

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WAC Department of Agriculture, Western Australia Plant Pathogen Collection, South Perth, Western Australia

WPC World Phytophthora Genetic Resource Collection, University of California, Riverside

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1. General Introduction

1.1. Prologue

Diseases caused by plant pathogenic fungi impose a serious constraint to eucalypts productivity worldwide (Keane et al. 2000). Where eucalypts were introduced as exotic plantations particularly in the tropics and Southern Hemisphere, they remained free of most pests and diseases for decades (Wingfield et al. 2013). One explanation is the enemy escape hypothesis, that has been used to explain the success of biological invasion. This theory says that an exotic species has more success in introduced areas because is free of their natural enemies (Keane and Crawley 2002; Mitchell and Power 2003). But this situation has changed in the last decades with the increase in the number of reports of new diseases in eucalypts plantations around the world (Su-See 1999; Wingfield et al. 2008, 2015; Armengol et al. 2012;

Burgess and Wingfield 2017). Several factors could explain this increase: the globalization with more people and commodities moving around the globe increasing the dispersion of native pests and diseases (Wingfield et al. 2010; Stenlid et al. 2011), and host jumps of pathogens from native hosts (Wingfield 2003; Slippers et al. 2005). Austropuccinia psidii is a good example of a pathogen of Myrtaceae in South America that jumps to non-native Eucalyptus species in Brazil and has rapidly spread to other regions of the world, including Australia, where it is threatening the numerous species of endemic Myrtaceae (Alfenas et al. 2003; Pegg et al. 2017; Stewart et al. 2018; Fernandez Winzer et al. 2019; Wingfield et al. 2020). Global climate changes can also play a role in this increase of pests and diseases creating favourable conditions for pests in areas where they could not survive previously and making mild pathogens became more aggressive (Garrett et al. 2006; Pautasso et al. 2012; Booth 2013;

Prospero et al. 2021).

The situation in Portugal seems to follow the trends observed in other areas where eucalypts are planted as an exotic species. Although introduced more than a century ago, only in the last decades has the incidence and severity of pests and diseases caused significant damages, which has raised the concern of industry. The present work is a response to those concerns and aims to increase the knowledge of diseases causing canker and dieback on eucalypts plantations in Portugal.

1.2. Botany of eucalypts

The plants commonly known as eucalypts belong to three genera in the family Myrtaceae (Angophora, Corymbia and Eucalyptus) (Nicolle 2019). These three genera comprise about 800 species mostly native to Australia with a few species native to Indonesia, East Timor, Philippines, and Papua New Guinea (Chippendale 1988; Hill and Johnson 1995).

Eucalyptus deglupta and E. urophylla are examples of a few species not recorded in Australia (FAO 1979).

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The genus Eucalyptus was introduced by Charles Louis L’Héritier de Brutelle, working at the British Museum from a specimen collected by the botanist David Nelson, during the third voyage of James Cook to Australia as E. obliqua (Brooker 2002).

The eucalypts vary from trees attaining 70 m with a unique trunk to small trees with multiple trunks (mallee) and small shrubs (Rejmánek and Richardson 2011). They are adapted to a wide range of environments from sea level to the alpine treeline and from high rainfall regions to semi-arid zones, from the tropics to latitudes as high as 44 degrees south (Potts 2004; Whitehead and Beadle 2004).

Although about 800 species of eucalypts are known, only a few are cultivated outside its native range. Eucalyptus globulus (Tasmanian blue gum) is probably the most cultivated species of eucalypt on temperate regions. The other three most commonly cultivated eucalypts species are E. camaldulensis (river red gum), E. grandis (flooded gum), and E.

tereticornis (forest red gum) planted in tropical and subtropical regions (Rejmánek and Richardson 2011). In colder areas where frost is common, usually the fast-growing species planted is E. nitens (shining gum) (Tibbits and Hodge 2003; Leslie et al. 2012).

1.3. Importance of eucalypts plantations

The eucalypts were introduced in Europe in the 18^th century. First as a botanical curiosity in botanical gardens and private arboreta. As the potential uses of eucalypts was recognized, the botanical gardens in Europe became the secondary dispersal centres of eucalypts to other countries. Later in the 19^th century eucalypts were introduced directly from Australia to many other countries in the world (Turnbull 2000).

Initially, eucalypts were planted for many different uses. As fast-growing species, they were used for windbreaks, land reclamation, and leaf-oil production, but mainly for fuel wood, timber, and charcoal (Potts 2004). However, it was the demand from the paper and pulp industry that caused the great expansion of exotic eucalypts plantations in the second half of the 20^th century (Potts 2004).

Due to their suitable wood properties for many uses including pulp and paper, fast growth and adaptability to different edaphoclimatic conditions, ability to grow in poor soils, eucalypts are one the most widely planted hardwood forest trees cultivated in over 100 countries around the world and occupying an area of over 20 million ha of planted forest (Iglesias-Trabado and Wilstermann 2008).

1.4. Introduction and expansion of eucalypts in Portugal

In Portugal, eucalypts were introduced in the middle of the 19^th century, probably in 1854 (Coutinho 1886) or 1859 (Pimentel 1884). At first, they were planted in botanic gardens, by collectors and used as amenity trees and for their medicinal proprieties. Due to a chronic lack of wood, the appearance of a species of fast-growing trees with slender trunks, like eucalypts, became an effective way to provide wood for use on farms and as fuel (Alves et al.

2007). From the beginning, Eucalyptus globulus proved to be the most suitable species, better

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adapted to the growing conditions of the country. In 1884 a monograph of E. globulus was published to promote the planting of this species (Pimentel 1884). A few years later, E.

globulus was already the most common species in forests, while the other species were planted only for ornamental purposes (Coutinho 1886, 1887). In 1920, Jaime de Magalhães Lima described his observations of about eighty eucalypt species and also mentions that, if the goal is to “produce the maximum volume of wood in a given time and space should not hesitate: plant the globulus” (Lima 1920).

Some of these trees are still standing. The tallest tree in Europe is a E. diversicolor, known as “Karri Knight”, with 73 m high in Vale de Canas, Coimbra. Its age is calculated to be about 140 years old, about the time the first eucalypts were planted in Portugal. According to the site Monumental Trees in the top ten tallest trees of Europe are also included an E.

globulus with 63.1 m and an E. viminalis, with 62.3 m in the same woodland (https://www.monumentaltrees.com).

The expansion of eucalypts in Portugal was slow during the 19^th century and the first half of the 20^thcentury. The first industrial plantation of eucalypts was done in 1870 destined to be used for railway slippers (Pimentel 1884). The exponential growth of the area planted with eucalypts occurred with the development of the paper and pulp industry.

Portugal was pioneer in the use of eucalypts in the pulp and paper industry. In 1925 “The Caima Estate Timber & Wood Pulp Company” replaced pines with eucalypts as raw material for pulp production and was the first in the word to use eucalypts for paper production.

(https://www.urbex.nl/companhia-de-celulose-do-caima). But the breakthrough was achieved in 1957, with the introduction of the sulphate method known as “kraft” also in Portugal, by the “Companhia Portuguesa de Celulose” in its factory at Cacia (http://en.thenavigatorcompany.com/Institutional/History). Since then, E. globulus has been the species most used for pulp production not only in Portugal but all over the world (Turnbull 1999; Iglesias-Trabado and Wilstermann 2008). Nowadays eucalypt forests in Portugal are almost entirely composed of E. globulus destinated for the paper and pulp industry. The industry demand makes the plantations of eucalypts economically attractive, and the planted area have grown exponentially and is still growing (Figure 1.1).

According to the last forest inventory, in 2015 eucalypts plantations occupy 845,000 ha, around 26% of the continental forest and have shown a systematic increase over the last 50 years (Fig. 1.2). The second and third most represented species are cork oak (Quercus suber) and maritime pine (Pinus pinaster) (ICNF 2019).

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Figure 1.1 Evolution of the area of eucalypts plantation in Portugal Source: Alves et al 2007, ICNF 2019.

Eucalypt plantations are mainly destined for the pulp and paper industry. This activity has a very important socio-economic value since it accounts for 2.15% of the GDP (gross domestic product), 4.5 % of national exports and for 12,290 direct jobs (Jordão 2019).

Figure 1.2 Specie's relative composition of the Portuguese forest, Source: ICNF, 2019.

1.5. Most important canker and dieback diseases in eucalypt plantations

The term canker refers to a symptom characterized by localized necrotic lesions of bark and cambium, often sunken. They can occur on branches or trunks of trees (Old and Davison

0 100 200 300 400 500 600 700 800 900

1920 1940 1960 1980 2000 2020

Thousand hectares

Years

26,2%

22,3%

22,1%

10,8%

5,9%

6,0%

1,6% 2,5% 1,5% 1,1%

Eucalypt Cork oak Maritime pine Holm oak Other hardwoods Stone pine Other softwoods Other oaks Chestnut Others

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2000; Agrios 2005). Cankers can be classified as non-infectious (abiotic), caused by abiotic factors like frost, sunscald, or mechanical damage or infectious (biotic), caused by bacteria virus or fungi. Most cankers are caused by fungi, basidiomycetes and more frequently ascomycetes (Blanchard and Tattar 1981).

Cankers are also in three types: annual, perennial of diffuse. Annual cankers usually attack weakened trees. In annual cankers the fungus is established during the dormant season of the host. During the following growing season, the host is able to produce callus tissue and wound wood sufficient to prevent further spread (Blanchard and Tattar 1981). Perennial cankers, also known as target-shaped cankers, are more important because the fungus survives in the host causing infection year after year. As the host responds with callus formation, the wounds usually have layers of concentric rings of callus tissues given the appearance of a target. These cankers rarely kill the trees but reduce the volume of usable wood and makes trees more susceptible to wind damage (Tainter and Baker 1996).

Diffuse cankers are the most dangerous because the invading fungus grows so quickly that the host does not have time to respond. These cankers usually girdle the infected tree, and the outcome is death of the tree (Blanchard and Tattar 1981; Tainter and Baker 1996).

Canker incidence is usually associated with environmental stress, particularly drought (Old and Davison 2000). It can cause reduced tree growth rates, tree mortality, and may destroy forests if infection is widespread (Yuan 1998). In eucalypts in particular, cankers can reduce the quantity and quality of wood, whether it is intended for timber or pulp production (Gezahgne et al. 2003; Aylward et al. 2019).

Pink disease

Pink disease is caused by the basidiomycete Erythricium salmonicolor (Berk. & Broome) Burds (syn. Corticium salmonicolor Berk. & Broome, Pellicularia salmonicolor (Berk. &

Broome), Botryobasidium salmonicolor (Berk. & Broome) Venkatar., Phanerochaete salmonicolor (Berk. & Broome) Jülich, Upasia salmonicolor (Berk. & Broome) Harsojo-Tjokr., Necator decretus Massee). The disease in named for the pink to salmon-coloured corticioid basidiocarps formed on branches or trunk with advanced infections (Old and Davison 2000).

The fungus has a very wide host range including forest trees, fruit trees and ornamental species (Farr and Rossman 2022). It affects many tropical forest trees such as Eucalyptus and Acacias (Old et al. 2003) and economically important crops such as coffee, tea, cocoa and rubber (Old et al. 2000). Pink disease is a serious problem of Eucalyptus in India and Brazil (Ciesla et al. 1996). The fungus has a worldwide distribution in the tropics and subtropics and cause most damage in areas with high rainfall or with frequent mist. The spores are windborne and are able to invade healthy intact bark (Old et al. 2003). Cankers with bark cracks are formed on the infected branches or trunks girdling the branch or trunk resulting in and death of the branches above the cankers (Old and Davison 2000). In India Seth et al. (1978) noted that E. tereticornis epicormic branches may form new shoots when the main trunk is infected,

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but in E. grandis only a few branches are formed and do not develop and affected plants usually die.

Chrysoporthe canker

Chrysoporthe canker (previously known as Cryphonectria canker) is caused by several species of the genus Chrysoporthe and other fungi in the Cryphonectriaceae family. The disease has been attributed to Cryphonectria cubensis (Bruner) Hodges (syn. Cryptosporella eugeniae Nutman & F.M. Roberts, Diaporthe cubensis Bruner, Endothia eugeniae (Nutman &

F.M. Roberts) J. Reid & C. Booth), first described as Diaporthe cubensis Bruner on several Eucalyptus species in Cuba (Bruner 1917). Based on morphological and phylogenetic evidence, Cryphonectria cubensis was transferred to a new genus, Chrysoporthe and segregated in three species, Ch. cubensis (Bruner) Hodges, Ch. austroafricana Gryzenh. & M.J. Wingf and Ch.

deuterocubensis Gryzenh. & M.J. Wingf (Gryzenhout et al. 2004; van der Merwe et al. 2010).

Other species were recently described also affecting Eucalyptus, including Ch. doradensis Gryzenh. & M.J. Wingf in Ecuador (Gryzenhout et al. 2005), Ch. zambiensis Chungu, Gryzenh.

& Jol. Roux from Zambia (Chungu et al. 2010), and Ch. puriensis M.E.S. Oliv., T.P.F. Soar. &

M.A. Ferr in Brazil (Oliveira et al. 2021).

Species of other genera in Cryphonectriaceae also associated with Eucalyptus canker include Amphilogia gyrosa (Berk. & Broome) Gryzenh., H.F. Glen & M.J. Wingf., Celoporthe eucalypti S.F. Chen, Gryzenh., M.J. Wingf. & X.D. Zhou, C. guangdongensis S.F. Chen, Gryzenh., M.J. Wingf. & X.D. Zhou, C. syzygii S.F. Chen, Gryzenh., M.J. Wingf. & X.D. Zhou and C. cerciana W. Wang, Q.L. Liu & S.F. Che in China (Chen et al. 2011; Wang et al. 2018), C. dispersa Nakab., Gryzenh., Jol. Roux & M.J. Wingf., C. fontana M. Verm., Gryzenh. & Jol. Roux and C. woodiana M. Verm., Gryzenh. & Jol. Roux in South Africa (Vermeulen et al. 2013), Parvosmorbus eucalypti W. Wang & S.F. Chen, and Parvosmorbus guangdongensis W. Wang & S.F. Chen, (Wang et al. 2020) in China, Holocryphya eucalypti (M. Venter & M.J. Wingf.) Gryzenh. & M.J.

Wingf. (syn. Cryphonectria eucalypti M. Venter & M. J. Wingf.), Holocryphia mzansi S.F. Chen

& Jol. Roux in South Africa (Chen et al. 2013), Microthia havanensis (Bruner) Gryzenh. & M.J.

Wingf. (syn. Endothia havanensis Bruner, Cryphonectria havanensis (Bruner) M.E. Barr) (Venter et al. 2002; Gryzenhout et al. 2006a), Cryptometrion aestuescens Gryzenh. & M.J.

Wingf. in Indonesia (Gryzenhout et al. 2010) and Myrtoporthe bodenii Abdul in Malaysia (Rauf et al. 2020).

Hosts of the Cryphonectriaceae include more than 100 tree species in over 26 families of 16 orders. Population genetic studies have shown that the species of Chrysoporthe infecting Eucalyptus are reported mainly from native host belonging to the families Melastomataceae, and Myrtaceae (Myrtales) native of the areas where eucalypts are planted as exotics and had undergone host jumps into Eucalyptus and not introduced in these areas, as initially thought.

(Wingfield 2003; Gryzenhout et al. 2006b; Wang et al. 2020)

This disease is widespread in tropical and sub-tropical regions where eucalypts are planted. It has been reported from Africa (Venter et al. 2002; Roux et al. 2005; Nakabonge et

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al. 2006; Chungu et al. 2019), Southeast Asia (Sharma et al. 1985; Gryzenhout et al. 2004; van der Merwe et al. 2010; Fan et al. 2013), South and Central America (Bruner 1917; Auer 1996;

Gryzenhout et al. 2006b), Florida, Hawaii and Puerto Rico (Hodges et al. 1979) and Australia (Davison and Coates 1991; Venter et al. 2002; Pegg et al. 2010).

Segregation of Chrysoporthe cubensis into three cryptic species with non-overlapping geographical distribution and the discovery that these species occur on native Myrtaceae and Melastomataceae in the areas where eucalypts are planted as exotic plants lead to the conclusion that these fungi are native and have undergone a host shift from native Myrtales to Eucalyptus (Seixas et al. 2004; Gryzenhout et al. 2006b; Mausse-Sitoe et al. 2016; Granados et al. 2020; Wang et al. 2020).

Chrysoporthe cubensis is thought to be native to south and central America whereas its sibling species Ch. austroafricana and Ch. deuterocubensis are likely native species in southern Africa and Asia respectively (Gryzenhout et al. 2004; van der Merwe et al. 2010; Rauf et al.

2020).

Typical symptoms are the cracking and swelling of the bark at the bases of infected trees (Roux et al. 2005). On trees less than one year old, usually the canker girdles the tree causing his dead. Older trees may react by forming callus at the site of infection, leading to bulging of the outer layer of bark. This layer is eventually shed resulting in a canker (Conradie et al. 1990).

Cankers can occur at the bases of the trunks or are found higher up, especially in areas where the climate favours disease development (Hodges et al. 1979). In India gummosis was observed in older cankers on Eucalyptus grandis but not on E. tereticornis (Sharma et al. 1985).

Occurrence and severity of Chrysoporthe canker on eucalypts are influenced by environmental conditions, with most damage occurring in regions where the average annual temperatures are above 23 °C and rainfall exceeds 1200 mm per annum (Hodges et al. 1979;

Alfenas et al. 1982; Sharma et al. 1985; Heerden and Wingfield 2002).

This disease is one of the most severe canker diseases of eucalypts (Old and Davison 2000). The damage results from reduced growth rate, tree mortality and reduced coppicing (Barnard et al. 1987; Old et al. 2003). For pulp production, trunks with canker yield less pulp and the cankers affect the bleaching (Foelkel et al. 1976; Ferrari et al. 1984; de Souza et al.

2010). The physical and mechanical properties of the wood are also adversely affected (Dahali et al. 2021).

Teratosphaeria canker

Teratosphaeria canker (previously known as Coniothyrium canker) is caused by two species, Teratosphaeria zuluensis (syn. Coniothyrium zuluense M.J. Wingf., Crous & T.A. Cout., Colletogloeopsis zuluensis (M.J. Wingf., Crous & T.A. Cout.) M.-N. Cortinas, M.J. Wingf. & Crous [as 'zuluense'], Readeriella zuluensis (M.J. Wingf., Crous & T.A. Cout.) Crous & U. Braun), Kirramyces zuluensis (M.J. Wingf., Crous & T.A. Cout.) Andjic & M.J. Wingf., and T. gauchensis (M.-N. Cortinas, Crous & M.J. Wingf.) M.J. Wingf. & Crous (syn. Colletogloeopsis gauchensis M.-N. Cortinas, Crous & M.J. Wingf., Readeriella gauchensis (M.-N. Cortinas, Crous & M.J.

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Wingf.) Crous & U. Braun, Kirramyces gauchensis (M.-N. Cortinas, Crous & M.J. Wingf.) Andjic, M.-N. Cortinas & M.J. Wingf.).

This disease was first observed in South Africa in 1988 but the causal agent was characterized and described later as Coniothyrium zuluense M.J. Wingf., Crous & T.A. Cout.

(Wingfield et al. 1996). Teratosphaeria gauchensis was described in 2006 in Uruguay as Colletogloeopsis gauchensis M.-N. Cortinas, Crous & M.J. Wingf. (Cortinas et al. 2006a).

Although causing similar symptoms, these two species have a very different geographical distribution. In Africa, T. zuluensis is present in South Africa (Wingfield et al. 1996), Mozambique (Maússe-Sitoe et al. 2016), Malawi (Roux et al. 2005), Uganda (Jimu et al. 2014) and Zambia (Muimba-Kankolongo et al. 2009). In Asia it occurs in China (Cortinas et al. 2006b), Thailand (Van Zyl et al. 2002) and Vietnam (Gezahgne et al. 2003). Outside these continents, it has been reported from Mexico (Roux et al. 2002) and was recently detected for first time in Paraguay, South America (Silva et al. 2020). Teratosphaeria gauchensis is present in Zimbabwe (Jimu et al. 2015), Uganda (Jimu et al. 2014) Kenya (Machua et al. 2016) and Ethiopia (Gezahgne et al. 2005) in Africa, Argentina (Gezahgne et al. 2003), and Uruguay (Simeto et al. 2020) in South America, Hawaii (Cortinas et al. 2004) and continues to spread, having been recently detected in Portugal (Silva et al. 2015) and Italy (Vitale et al. 2019).

These two species are known only from Eucalyptus spp. (Old et al. 2003). Teratosphaeria gauchensis seems to have a wider host range than T. zuluensis, having been reported from seven Eucalyptus species namely, E. camaldulensis, E. grandis, E. globulus, E. maidenii, E.

paniculate, E. propinqua, and E. tereticornis. In contrast T. zuluensis has been isolated from only four species namely, E. camaldulensis, E. grandis, E. cloeziana, and E. urophylla (Aylward et al. 2019). Recently it was also reported on a hybrid of E. camaldulensis × E. viminalis in Italy (Vitale et al. 2019).

The first signs of infection are small discrete necrotic lesion on the green bark (Wingfield et al. 1996). These lesions expand, becoming elliptical, and the dead bark covering them typically cracks, giving a “cat-eye” appearance (Cortinas et al. 2006a). Twigs and branches are also affected (Vitale et al. 2019). The fungus invades the cambium resulting on the formation of kino pockets. Severely affected stem tends to produce epicormic shoots around the cankers, usually associated with tree top death (Old et al. 2003). These cankers seldom kill the trees but decrease the wood quality making it useless for construction (Gezahgne et al. 2003).

The cost and quality of pulping are also negatively affected because the cankers hinder the debarking process (Aylward et al. 2019).

Botryosphaeria canker

Botryosphaeria canker was attributed to Botryosphaeria ribis Grossenb. & Duggarand Botryosphaeria dothidea (Moug.) Ces. & De Not. (Old and Davison 2000). With the changes on the taxonomy, the genus Botryosphaeria was segregated in many different genera and new species described. Botryosphaeria ribis was reassigned to a new genus as Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips (Crous et al. 2006).

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According to the most recent outline of fungi, the family Botryosphaeriaceae comprises 22 genera (Wijayawardene et al. 2020). This family includes a large number of species of plant pathogens and endophytes having a wide host range of mainly woody species and occur in temperate, mediterranean and tropical climates worldwide (Slippers and Wingfield 2007;

Phillips et al. 2013; Jami et al. 2017; Zlatković et al. 2018). Most species behave as latent pathogens causing disease when the host is subjected to stress caused by environmental factors like drought, flooding, high temperatures or by other pests or pathogens (Slippers and Wingfield 2007; Slippers et al. 2009; Barradas et al. 2018).

In a recent review of worldwide occurrence of Botryosphaeriaceae, 58 species were accounted only on Eucalyptus spp. belonging to 11 genera namely Botryosphaeria, Cophinforma, Diplodia, Dothiorella, Endomelanconiopsis, Lasiodiplodia, Macrophomina, Neofusicoccum, Neoscytalidium, Pseudofusicoccum, and Sphaeropsis (Batista et al. 2021, Data available at: https://mdr-bot-cesam-ua.shinyapps.io/bot_database). On this database, 58 species of eucalypts are mentioned as hosts of Botryosphaeriaceae species. Most of the records are from species used in plantations worldwide or commercially exploited in their native areas, namely Eucalyptus camaldulensis, E. dunnii, E. globulus, E. grandis, E. marginata, E. nitens, E. pellita, E. saligna, E. tereticornis, E. urophylla, and E. viminalis.

Disease symptoms typically caused by Botryosphaeriaceae include tree dieback, stem canker, branch canker and twig blight (Slippers and Wingfield 2007; Li et al. 2018). Cankers on the stems are often elongated with a lens shape and bark cracking. Kino exudations are frequent (Old and Davison 2000).

Quambalaria canker

Quambalaria is a genus that includes endophytes and pathogenic species (de Beer et al.

2006; Crous et al. 2019). Several species are associated with Eucalyptus diseases. Quambalaria eucalypti (M.J. Wingf., Crous & W.J. Swart) J.A. Simpson (syn. Sporothrix eucalypti M.J. Wingf., Crous & W.J. Swart) causes shoot and leaf blight on Eucalyptus species in several southern hemisphere countries including South Africa (Wingfield et al. 1993), Brazil (Alfenas et al. 2001), Uruguay (Bettucci et al. 1999) and Australia (Pegg et al. 2008) and was also found in Myrceugenia glaucescens, a native tree in Uruguay (Pérez et al. 2008).

Although found and described for the first time in South Africa as Sporotrix eucalypti Wingfield, Crous & Swart (Wingfield et al. 1993), this species seems to be native from Australia, where it was found more than a decade later (Carnegie 2007b; Pegg et al. 2008).

Quambalaria pitereka (J. Walker & Bertus) J.A. Simpson (syn. Ramularia pitereka J. Walker &

Bertus, Sporothrix pitereka (J. Walker & Berterus) U. Braun) causes similar symptoms but is specific to Corymbia spp. (Crous et al. 2019) and has a narrower distribution, having been reported from Australia and China (Zhou et al. 2008; Pegg et al. 2011). The most aggressive species is Q. coyrecup Paap, causing stem cankers on Corymbia calophylla (marri) that lead to severe decline of native populations of this species (Paap 2006; Paap et al. 2008; Hossain 2020).

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Although reported as pathogenic in grapevine (Vitis vinifera) in Iran (Narmani and Arzanlou 2019), and isolated from withered grapes in Italy Q. cyanescens (≡ Sporothrix cyanescens de Hoog & G.A. de Vries, Cerinosterus cyanescens (de Hoog & G.A. de Vries) R.T.

Moore, Fugomyces cyanescens (de Hoog & G.A. de Vries) Sigler (de Hoog & G.A. de Vries) Z.W.

de Beer, Begerow & R. Bauer) is not considered a primary pathogen of eucalypts (Crous et al.

2019). It has also been reported as an endophyte on Birch (Betula pendula) in Russia (Antropova et al. 2014), pomegranate (Punica granatum) (Vahedi-Darmiyan et al. 2017), cherry (Prunus avium) and sour cherry (Prunus cerasus) in Iran (Abdollahi Aghdam and Fotouhifar 2016) and was found associated with bark beetles in the Netherlands, Turkey, Syria, Bulgaria, Hungary and Spain (Kolarík et al. 2006).

Quambalaria pusilla (U. Braun & Crous) J.A. Simpson (syn. Sporothrix pusilla U. Braun &

Crous, Q. simpsonii Cheewangkoon & Crous) was recorded from E. camaldulensis in Thailand as well as E. tintinnans in the Northern Territory of Australia (Cheewangkoon et al. 2009).

Other species recently isolated and described from Eucalyptus spp. include Q. tasmaniae Crous, associated with leaf spots of Eucalyptus sp. in Tasmania, Australia and Q. rugosae Crous, in Kangaroo Island, Australia (Crous et al. 2019).

Other canker diseases

Several other fungi are isolated from eucalypts canker but usually they are opportunistic pathogens causing symptoms on weakened trees. In general, they cause little damage and, in many cases, the geographical distribution is limited (Old and Davison 2000).

Seiridium eucalypti Nag Raj has been reported only from south Australia and Tasmania (Yuan and Old 1995; Ciesla et al. 1996; Yuan and Mohammed 1997a; Yuan et al. 1999).

Another species, Seiridium papillatum Z. Q. Yuan was described from cankers on Eucalyptus delegatensis in Tasmania (Yuan and Mohammed 1997b) but inoculations on E. globulus and E. nitens showed that it is weakly pathogenic (Yuan et al. 1999).

Cytospora eucalypticola Van der Westh is frequently found on eucalypt cankers but is considered a very weak pathogen (Old and Davison 2000). In pathogenicity trials it did not cause significant symptoms (Yuan et al. 1999; Alonso et al. 2005).

Biscogniauxia mediterranea (De Not.) is the cause of charcoal disease of cork oak and can also infect eucalypts. In both hosts, this fungus has very low degree of aggressiveness and only attacks weakened trees.

Endothia gyrosa is generally considered to be opportunistic and favoured by stress (drought or severe defoliation by insects). However, evidence from eucalypt-growing areas, such as South Africa and Tasmania suggests it is a serious pathogen on clones of various Eucalyptus species (Ciesla et al. 1996; Yuan et al. 1999).

1.6. Root rots diseases

Root rots are caused by soil pathogens, mostly fungi and oomycetes. Usually, the above ground symptoms are common to all root rot pathogen including poor growth, sparse foliage,

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branch tip dieback and, in severe cases, death of the tree (Blanchard and Tattar 1981). The most important pathogens causing root rot of eucalypts are oomycetes of the genera Phytophthora and Pythium and basidiomycetes in the genus Armillaria (Kile 2000; Shearer and Smith 2000).

Armillaria root rot

Woody root rots of eucalypts can be caused by at least 12 genera of Basidiomycota but the genus Armillaria is the most important acting as primary or secondary pathogens (Kile 2000). Eight species of Armillaria have been reported from eucalypts, including A. fumosa Kile

& Watling, A. hinnulea Kile & Watling, A. limonea (G. Stev.) Boesew., A. luteobubalina Watling

& Kile, A. mellea (Vahl: Fr.) P. Kumm, A. montagnei (Singer) Herink, A. novae-zelandiae (G.

Stev.) Herink, and Desarmillaria tabescens (Scop.) R.A. Koch & Aime (syn. Armillaria tabescens (Scop.) Emel) (Farr and Rossman 2022). The species causing most damage in eucalypts, either in their native range or in exotic plantations are A. mellea with a worldwide distribution and A. luteobubalina, restricted to Australia (Robinson 2016; Coetzee et al. 2018). These species have a wide host range of tree species, including forest trees, fruit trees and amenity trees (Old et al. 2000). Armillaria mellea has been recorded from more than 2,000 host species and A. luteobubalina from 96 (Farr and Rossman 2022).

Symptoms are characterized by growing patches of dying or dead trees, with the more affected trees in the centre. The first symptoms are paler green foliage and small and sparse leaves. The disease evolves to a general dieback and severely affected trees are susceptible to windthrow (Kile 1983, 2000; Robinson 2016). The fungus causes root, collar, and lower stem rot. White mycelial sheets or fans of mycelium developed through the inner collar bark (Kile 2000).

In Australia A. luteobubalina can cause mortality in undisturbed native forests but the damages are more important in lodged areas or plantations (Podger et al. 1978; Kile 1983).

Outside Australia, more than 30 species of Eucalyptus have been recorded as hosts of Armillaria with A. mellea and Desarmillaria tabescens as the most important pathogens (Guillaumin et al. 1993; Kile 2000; Farr and Rossman 2022).

Phytophthora root rot

Species of the genus Phytophthora are the most devastating plant pathogen in the world, destroying crops, forests and threating natural ecosystems. (Hardham 2001; Hansen 2015; Jung et al. 2018). Over 30 species of Phytophthora have been reported from Eucalyptus spp. (Burgess et al. 2021; Farr and Rossman 2022). One of the most dangerous is Phytophthora cinnamomi Rands that has caused extensive destruction on native populations of Eucalyptus in Australia also destroying other endemic species threatening the biodiversity of important areas such as the jarrah forest (dominated by Eucalyptus marginata) in the south-west of Australia (Newhook and Podger 1972; Podger 1972; Davison and Shearer 1989).

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In plantations, either in Australia or elsewhere, P. cinnamomi causes little damage, presumably because the most planted eucalypt species are tolerant (Wingfield and Knox- Davies 1980a; Linde et al. 1994; Shearer and Smith 2000; Nagel et al. 2013). Despite being the most aggressive species, several studies have shown that not all species of Eucalyptus are susceptible to P. cinnamomi. The Eucalyptus species most susceptible to P. cinnamomi belong to the Monocalyptus subgenus while the species from the Symphyomyrtus, to which E.

globulus belongs, are highly tolerant or resistant (Podger 1972b; Marks et al. 1981; Tippett et al. 1985; O’Gara et al. 2005).

Several other Phytophthora species have reported or recently described from plantations in several countries around the world. In South Africa P. alticola Maseko, Cout. &

M.J. Wingf. on E. macarthurii, E. dunni and E. badjensis and P. frigida Maseko, Coutinho & M.J Wingf., on E. dunnii, and E. smithii (Maseko et al. 2007), P. cinnamomi on E. fraxinoides and E.

fastigata (Wingfield and Knox-Davies 1980b), P. nicotianae on E. fastigata, E. elata, E.

macarthurii, E. nitens, E. dunii and E. smithii (Maseko et al. 2001). In Australia, P. boogera was reported associated with damping-off of seedlings of several Eucalyptus species including E.

marginata (Simamora et al. 2015) and P. cinnamomi on several species and hybrids with E.

pilularis being the most affected (Carnegie 2007a). In New Zealand, two new species were recently described, P. captiosa M. A. Dick & Dobbie on E. salignae and E. botryoides and P.

fallax Dobbie & M. A. Dick on E. delegatensis, E. regnans, E. fastigata, and E. nitens. Disease symptoms caused by these species are confined to the canopy (Dick et al. 2006).

Pythium species are mostly associated with damping-off of seedlings (Shearer and Smith 2000). The only report of plantation diseases was Pythium splendens causing root and collar rot of E. grandis in South Africa (Linde et al. 1994).

Soilborne species of Phytophthora invade the host through the roots and the mycelia can invade de cortex, progress to the collar and lower trunk (Hardham 2001). This cause fine root losses, root and collar rots and bleeding bark cankers (Jung et al. 2018). The initial symptoms may be cryptic and difficult to identify and can be confused with drought, often exhibiting as secondary symptoms of wilt and drought dieback resulting from root and vascular necrosis (Scott and Williams 2014). It may also include chlorosis of the leaves as well as gum exudation through the cankers on the tree collar. As the disease progresses, the trees usually wilt and die due to girdling (Maseko et al. 2001). Infected tree may die suddenly, with brown leaves attached, but death usually take several years (Weste and Marks 1987; Barber et al. 2013).

1.7. Eucalypts diseases in Portugal with emphasis on canker and dieback diseases

Until the 1990s there were only sporadic cases of disease in eucalypt plantations without serious outbreaks (Branco 2007). Disease surveys on eucalypt plantations have identified Botryosphaeria dothidea (Moug.: Fr.) Ces. & De Not (as Botryosphaeria

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berengeriana De Not.) (Azevedo and Santos 1955; Azevedo 1971; Sampaio 1975) causing stem canker but with a limited distribution (Algarve, Estremadura and Ribatejo). Root rots, caused by Armillaria mellea and Dematophora necatrix R. Hartig (as Rosellinia necatrix (Hart.) Berl.) have also been reported. Dematophora necatrix was found at only one site and it did not cause symptoms on standing trees but caused the rot of 90% of the stumps, thus preventing sprouting (Azevedo 1971, 1979; Sampaio 1975). This causes serious damage on coppiced stands.

Other fungi reported in the same studies, but considered of minor importance, included Botrytis cinerea, occasionally causing leaf blight in young plants, but the most damage is caused in nurseries, Biscogniauxia mediterranea (De Not.) Kuntze (as Hypoxylon mediterraneum (De Not.) Ces. & De Not.), also a weak pathogenic species affecting stressed trees either by drought, fire, or pests and Cytospora australiae Speg., also a weak pathogen frequently found associated with other canker causing fungi.

Since the 1990s Mycosphaerella leaf disease (MLD) has been found causing severe defoliation in young plantations. An in-depth study has shown that this disease is caused by several species of Teratosphaeria and Mycosphaerella (Silva et al. 2009, 2012; Branco et al.

2014; Silva 2015).

1.8. Aim of this work

The main losses observed in the eucalypt stands in Portugal are caused by insects. So far, the diseases that has caused significant damage is leaf disease and, sporadically Botryosphaeria canker. However, in the last decades, besides leaf defoliation, tree cankers, dieback and decline became more frequent and with climate change it is expected that they will become even more serious in the future. Since the number of pests and diseases has increased in the last decades, it is of paramount importance to identify the causal agents of these diseases to establish management measures to minimize the damage they may cause.

To accomplish this objective, a protocol was established between RAIZ, Altri Florestal, S. A.

and INIAV aiming to identify the main causal agents of fungal canker and dieback diseases. A survey was conducted in all major areas occupied by eucalypts all over the country. Although root rots usually cause wilting or a general decline, in some cases the first symptoms can be a dieback. As several cases were detected during the survey, we also include root rots in this study. After the survey, both companies have kept a permanent surveillance on the plantations and samples are regularly send to the INIAV laboratory for diagnosis. These samples include a disease of unknown cause that was found in a nursery, and this was also included in the study.

Some of the results of the ongoing work are reported in this thesis. Chapter 1 includes a general introduction about eucalypts, underlining the botany, economic importance, expansion, and the most important fungal diseases causing canker and dieback on eucalypts plantations worldwide and in Portugal. Chapters 2 to 4 address the identity and pathogenicity

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of the most important diseases found in plantations. In chapter 5, new species of Neopestalotiopsis are described, associated with a new nursery disease.

1.9. References

Abdollahi Aghdam S, Fotouhifar KB (2016) New reports of endophytic fungi associated with cherry (Prunus avium) and sour cherry (Prunus cerasus) trees in Iran. Mycol Iran 3:75–

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