Materials and methods

A total of 41 sampling sites throughout Portugal were selected taking into consideration the information given by the pulp companies, and the distribution of Eucalyptus plantations in the country (Fig 2.1). All provinces in continental Portugal were surveyed since the beginning of 2011 for symptoms and signs of attack by fungal pathogens. In addition, the companies’ nurseries were also surveyed. Field and nursery procedures involved visual assessment of seedlings/trees to detect dieback and other symptoms.

Samples collected from stems, trunk, collar and leaves of symptomatic plants were examined with a dissection microscope. When fructifications bearing spores were found, spores were spread over the surface of a water agar plate. After 24 h of incubation at 24 °C, single germinating spores were transferred to half-strength potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA). When no fructifications were observed, small pieces (approx.

2 mm × 2 mm) taken from the margin of the lesions were surface-disinfected with 2.5%

sodium hypochlorite, rinsed twice in sterile distilled water, dried on sterile filter paper, and plated onto half-strength PDA supplemented with 0.1 mg/ml of streptomycin sulphate (Sigma-Aldrich, St. Louis, MO, USA) to suppress bacterial growth. Petri dishes were incubated at 25 °C under near-ultraviolet light. Cultures were purified either by preparation of single-spore cultures or by transferring hyphal tips taken from the colony margins. Voucher specimens of each isolate were deposited in the MEAN fungal collection at Instituto Nacional de Investigacão Agrária e Veterinária – INIAV (Oeiras, Portugal).

Figure 2.1 Sampling locations for Quambalaria eucalypti in Portugal. Black circles indicate locations where Q.

eucalypti was detected.

Morphological characterization

Colony morphology was described on half-strength PDA incubated for two weeks at 25 °C ± 2 °C. Mycelium, conidiophores and conidia were examined from slide cultures prepared according to Riddell (1950) and mounted in lactic acid. One hundred measurements were made of conidia and ten of conidiophores. Conidial dimensions are reported as the 95%

confidence intervals with extreme values shown in parentheses. Means and standard deviations of conidial dimensions are also reported.

Figure 2.2 Symptoms and signs of Quambalaria eucalypti on Eucalyptus globulus: (a, b) spore mass on seedling stems, (c) white spore masses on a dead seedling, (d, e) cankers on over 10-year-old trees, (f) seedlings inoculated with Q. eucalypti isolate PE27, (g) control, non-inoculated seedlings.

Molecular phylogenetic characterization

Identification was confirmed by DNA sequence comparisons. Mycelium was scraped from the surface of a PDA culture and DNA extracted with the NucleoSpin Plant II kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions.

Instead of ground lyophilized mycelium, fresh mycelium was used and was disrupted by vortexing with approx. 50 µl glass beads (425–600 µm diam.) added to the extraction buffer (Bragança et al. 2007).

Primers ITS5 (White et al. 1990) and LR5 (Vilgalys and Hester 1990) were used to amplify part of the nuclear rDNA operon spanning the 3’ end of the 18S rRNA gene (SSU), the first

end of the 28S rRNA gene (LSU) using the protocol of Cheewangkoon et al. (2008). The amplicons were purified with NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. The ITS region was sequenced in both directions by STAB VIDA, Lda. using primers ITS1 and ITS4 (White et al. 1990). Sequences were edited with BioEdit v. 7.1.3.0 (Hall 1999) and aligned with ClustalX2 (2.0) (Larkin et al. 2007) with the parameters used by Phillips and Alves (2009).

Table 2.1 Quambalaria eucalypti isolates from Eucalyptus globulus used in the morphological and phylogenetic study.

a MEAN - fungal collection of Instituto Nacional de Investigação Agrária e Veterinária INIAV (Oeiras, Portugal).

Available sequences of the known species of Quambalaria including ex-type strains were retrieved from GenBank and included in the alignment (Tab. 2.2). Phylogenetic analyses

Isolate no. ^a Origin Year ITS GenBank accession no.

PE3/MEAN 996 Évora 2011 JX297605

PE6/MEAN 997 Lourinhã 2011 JX297603

PE27/MEAN 998 Furadouro 2010 JX297604

PE28/MEAN 999 Anadia 2011 JX297600

PE29/MEAN 1000 Anadia 2011 JX297602

PE30/ MEAN 1001 Anadia 2011 JX297601

PE52/ MEAN 1002 St Tirso 2012 JX297606

PE53/ MEAN 1003 Ponte de Lima 2012 JX297598

PE54/ MEAN 1004 Sever do Vouga 2012 JX297599

PE92/ MEAN 1005 Polvarinho, Castelo Branco 2012 Not sequenced PE93/ MEAN 1006 Calafate, Idanha Nova 2012 KR336802 PE94/ MEAN 1007 S. Teotónio, Odemira 2012 Not sequenced PE95/ MEAN 1008 S. Teotónio, Odemira 2012 Not sequenced

PE96/ MEAN 1009 S. Luís, Odemira 2012 KR336803

PE97/ MEAN 1010 S. Luís, Odemira 2012 Not sequenced

PE150/ MEAN 1011 Alfândega da Fé, Mirandela 2013 Not sequenced PE151/ MEAN 1012 Vale de Baraça, Vila Flor 2013 KR336804 PE152/ MEAN 1013 Vale de Baraça, Vila Flor 2013 KR336805 PE153/ MEAN 1014 Avidagos, Mirandela 2013 KR336806

PE154/ MEAN 1015 Vila Real 2013 KR336807

listed by their accession numbers in the tree (Fig. 2.4, Tab. 2.2), while newly generated sequences are listed by isolate number. Newly generated sequences were deposited in GenBank (Tab. 2.1) and the alignment and trees in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S17479).

Pathogenicity tests

To confirm the pathogenicity of the Q. eucalypti isolates, four-month-old seedlings of E.

globulus were inoculated. To satisfy Koch's postulates, the fungus was re-isolated from symptomatic plants following the methods described above.

To test differences in susceptibility of eucalypts and/or different pathogenicity of Portuguese Q. eucalypti, two full-sib families of E. globulus and two isolates of Q. eucalypti (PE3 and PE27) were tested, with 15 replicate seedlings per treatment (including the control consisting of plants sprayed with sterile water). The plants were germinated in a mixture of peat and styrofoam (80%:20%) in 120 ml Optim 40 plastic containers (40 plants per container) and allowed to grow for 4 months in the greenhouse. When the plants reached approximately 15 cm height, 15 uniform plants were selected and placed in separate trays. Plants were inoculated by spraying with a spore suspension (2.5 × 10⁵ conidia/mL) prepared from fresh cultures of Q. eucalypti. Each tray was then covered with a plastic bag and maintained in a controlled environment chamber at 24 °C during the day and 20 °C at night, with 14 h of light per day. Bags were removed after 2 days and the plants were watered daily, fertilized every other day, and monitored visually for three weeks.

Disease severity was evaluated 12 days after inoculation on two median leaves of each plant using a diagrammatic scale comprising eight levels of severity (0.4; 1; 2; 4; 8; 16; 32 and 49% of leaf area affected) as developed by Andrade et al. (2005) for determination of Q.

eucalypti severity. Since the disease severity data were ordinal, the non-parametric Kruskal-Wallis and post-hoc multiple comparisons tests were used to determine the significance of differences between treatment means. All statistical analyses were made with Statistica 6.1 (StatSoft, Tulsa, OK, USA).

Table 2.2 Isolates used for DNA sequence comparison.

Species Isolate no.¹ Host Origin Collector ITS ²

Quambalaria.

eucalypti

BRIP48414 Eucalyptus grandis Moggill, QLD, Australia

G. S. Pegg EF444821

BRIP48507 E. grandis Moggill, QLD, Australia

G. S. Pegg EF444822

BRIP48367 Corymbia torelliana

× C. citriodora subsp. variegata

Walkamin, QLD, Australia

G. S. Pegg EF444823

Species Isolate no.¹ Host Origin Collector ITS ² Quambalaria

eucalypti

BRIP48416 E. dunnii Casino, NSW, Australia

G. S. Pegg EF444824

BRIP48494 E. dunnii Dyraaba, NSW, Australia

A. J. Carnegie EF444827

BRIP48518 E. dunnii Moggill, QLD, Australia

G. S. Pegg EF444841

BRIP48520 E. grandis Moggill, QLD, Australia

G. S. Pegg EF444842

BRIP48497 E. grandis Nana Glen, NSW, Australia

A. J. Carnegie EF444843

BRIP48498 E. grandis Kew, NSW, Australia

A. J. Carnegie EF444844

CBS 118844 T CMW 1101

E. grandis South Africa M.J. Wingfield DQ317625

UY1718 Myrceugenia glaucescens

Urugay C. A. Pérez EU439923

CMW11678 E. grandis South Africa M.J. Wingfield DQ317626 Q. coyrecup WAC12951 C. ficifolia Western Australia T. Paap DQ823430

DQ82343T Western Australia T. Paap DQ823431

WAC12949 C. calophylla Western Australia T. Paap DQ823432 WAC12948 C. calophylla Western Australia T. Paap DQ823433 Q. cyanescens AUMC 6293 air of citrus

plantation

Egipt Zeinab

Soliman

JQ425376

WAC12953 T C. ficifolia Western Australia T. Paap DQ317622

QY229 red kojic rice China ? HM013823

Q. pitereka BRIP48346 C. citriodora subsp.

citriodora

Davies Creek, QLD, Australia

G. S. Pegg EF444845

BRIP48387 C. citriodora subsp.

variegata

Beaudesert, QLD, Australia

G. S. Pegg EF444846

DAR19773 T C. exima NSW, Australia Walker DQ823423 QP26 C. citriodora subsp.

variegata

Queensland, Australia

G.S. Pegg DQ823424

Q. simpsonii CBS 124772 Eucalyptus tintinnans

Australia B.A.

Summerell

GQ303290

Species Isolate no.¹ Host Origin Collector ITS ²

Q. simpsonii CBS 124773 T Eucalyptus sp. Thailand R.

Cheewangkoo n

GQ303291

Microstroma juglandis

R.B. 2042 Juglans regia Germany R. Bauer DQ317634

Entyloma calendulae

ML 1223 Calendula officinalis AY081023

Tilletiopsis washingtonensis

CBS 359.86 Japan M. Yamazaki AB025689

1 T = ex-type strain; ² internal transcribed spacers and intervening 5.8S nrDNA