The taxonomy and pathology of BUf!Tusp!lOeria spp., with special reference to their relevance in Eucalyptus plantations of Venezuela

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Two new Fusicocculll species from Acacia and Eucalyptus in Venezuela, based on morphology and DNA sequence data

BotlyospllOeria spp. are common endophytes 0 I’ woody plants and they a I so iIlC I ude some serious pathogens of Eucalyptlls am! Acadu species. In this sluuy, we characterise two new Botlyosphae,.ia anamorphs, isolated from £/lcahp//ls and ,iC(lciu trees in Venezuela. These fUllgi were characterised based on morphological !Calmes in culture and comparisons of DNA sequence data. The two taxa, which have been provided the names FliSicoCClIIll andilllllll and FlISicoCClIlII SII »OIIJaticlIlI/, resided in 1\vo wei 1supported clades (bootstrap values = 100 %) based on a combined data set of the internal transcribed spacers (ITS) of the rONA operon am! translation elongation factor l-a (EFl- a) gene sequences. The conidia of F. (il/dilll/II’ are ullusually large amongst BO/lyosphaeria anamorphs and peripherally resemble those of B. IIlLIllWlle am! B. lIIelonops. FusicoCClIlil s/,.O/l/ulicllIlI is characterized by large conidiomata in culture, growth at 35°C and slightly thickened conidial walls, characteristics different from most othcr FliSicocuIII/ spp. No telcol1lorph states were observed for these fungi , but DNA sequence data show that they are anamorphs of floll)’osphae,.ia.

INTRODUCTION

Species of BOli)’osphaeria Ces. & De Not have a cosmopolitan distribution and occur 011 a wiel e range of monocotyledonous, dicotyledonous and gymnosperm hosts. BOli)’osplwerio spp. inCect the stems, branches, twigs and leaves of mal1Y woody plants and they have also been found in the stems of grasses and thalli of lichens (l3arr, 1987). These fungi include 0pp0i »lunistic pathogens that gi\c rise to symptOl11s such as shoot blights, stem cankers, fruit rots. die-back <JIll! gUllllllosis (VOIl Arx, 1987; Old. 2000: Old & Davison, 20(0).
Tile taxollomy of BOfrm.\pllOeriu has been confused for many years. This is mainl y due to the f~lC t that the morphology or the teleolllorphs is very similar and these states ,1I »e rarely enc ount ered either in nature or under laboratory conditions (Jacobs & Rel1l1er, 1998; Slippers Cf ul., 2004(1). Host assoc iatiolJ has been Llsed to (Jssign n<lllles to speci es but this has led to confusion bccause somc species are host specific, while others are generalists (Jacobs & Relliler. 1998; Smith cf ul.. 200 I; Smith & Stanosz. 200 I; Crous & Palm, 1999; Slippers cf ul., 2004a).
The anamorphs of BOli) »osplwcria species are generally encoLintered in culture or on diseased plant parts. For this reaSOIl, identification of Bofr) »(}splwCI »iu spp. has commonly been based 011 conidial morphology or the anal1l 0rpilS (Jacobs & Reimer, 1998; Smith & Stanosz, 2001; Smith ef al., 2001: Phillips cf ul., 2002; Slippers cf ul.. 2004a, d).
Conidial characters considered to be L1se ful ror the taxonomic delilllitatioll or BOIi ) »ospiloeria anamorphs are size, color, septation, wall thickness allli texture, as well as the presence of microconidia and mode of con idiogenesis (Sutton, 1980; Sivallesan, 1984; Pennycook & Samuels, 1985). These characters, however, require careful interpretation, as there is substantial overlap between the characters of many species.
Thus conidial size represents a continuous character and it is also variable between isolates and may change with age or 011 substrates and hosts (Pennycook & Sal11uels, 1985; Butin, J 993; Crous & Palm, 1999; Slippers el u/., 200,ta). In recent years, analyses of DNA sequence data have contributed substantially towards resolving taxonomic questions in BOlrrosp/weriu. Nucleotide sequences of the internal transcribed spacers (ITS) have in particular beell used to resolve phylogenetic relationships between species and these have aligned with morphological characters
(Jacobs & Rel1I1cr, 199~; Dell man el u/., 2000; Zhou & Stanosz, 200 I; Phillips cI 0/., 2002; J\lves e/ a/., 2004; Slippers el u/., 2004a). Another approach to characterise BOII}’osphueriu spp. is to use comparisons of l11ultiple gene sequences and restriction fragment length polymorphisl11 (RFLP) of anonymous simple sequence rcpeat (SSR) loci to distinguish closely related species such as BOlrrvsp/weria pWTa Pennycook & Samuels and B. ribis Grossenb. & Duggar (Slippers. 2003; Slippers el 01., 2004a).
BOfJyosp/weria spp. are known to occur on various forestry and agricultural crops in Venezuela, but very little attention has been given to their identity (Ccdeiio el at. 1994, 1996; Mohali, 1997; Mohali & Encinas. 200 l: Mohali, Encinas & Mora, 2002). Lasiodip/odia IheobroJ/lae (Pat.) Griffon & Maublanc., Dip/odiu jJilleu (Desl11.) Kickx (= 5p/Jaeropsis sapinca (Fr.) Dyko & Sutton), D. IIlllli/o (Fries) Mont., and a species of Dothiorella Sacco have been identified as the disease causing agents (Cedeiio el a/., 1994,1996; Mohali, 1997; Mohali & Encinas, 2001; Mohali e/ u/., 2002; De Wet et 0/.,2003).
The aim of this study was to characterise t\\’o FlISicocclIJ1l spp. C0l1111101l1y isolated from Eucalypllls and Acacia trees in Venezuela. and which appeared to be undescribed. These fungi were thus studied based 011 morphology and a comparisoll of DNA sequence data for tbe ITS rDNA (ITS I and ITS 2) and translation elongatioll factor I-a (EF 1- a).

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MATERIALS AND METHODS

Isolates alld morphological c/l(lracleri’;,atioll A survey was conducted in plantations of Elica/rp/lis IImpln’//o S.T. Blake, an unidentified Eucalyptus sp., a Eucalypllls-hybrid and Acacia IIlUlIgilllll Willd .. during 2003. Isolations were made /i’om twigs, stems and branches displaying sYJl1ptonlS or blue stain or dieback, and from dead trees. Single conidial isolates were obtained arter cultures were induced to sporulate on water agar to which sterile pine needles had been added.
For isolations, plant tissues were surface disinfcstcd with 70 % ethanol for 30 s and thereafter rinsed in sterile water for I min. Small tissue pieces (4-5 ml11) were cut t1’0l11 the plant tissue and placed on 2 ~·u ma It ex tract ,lgar (t’,,{ EA; D I rco. Dctroi t, MI, USA) and incubated at 25 « c. Cultures resembling BU/ITo.lplw(!/iu spp. were tral1sferred to water agar (WA) (2 % Biolab agar, Midrand. South Africa) with sterilized pine needles placed on the agar surface. These were incubated for 3-6 weeks at 25 uc under a combination of near-ultraviolet and cool-white nuoreseent light to induce sporulation. All isolates used in this study are maintained in the collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABl), University of Pretoria, South Africa. Reprcse ntalive iso lates have also becn deposited in the culture collec tion of the Centraalbureau voor Schil1ll11clcultures (CBS), Ulrecht, the Netherlands. Conidial morphology was studied lIsing a light microscope with ,lll Axiocam digital camera and software to analyse photographs (Carl Zei ss. Germany). Sections through some of the pycnidia and stromatal structures were made with an American Optical Freezing Microtome. Length, width; shape and color of the conidia were recorded after mounting in clear lactophenol. At least 50 conidia of each isolate of two different FliSicoCClIIl1 spp. were measured.
The growth of se lected isolates was determined by placing mycelial discs (5 111m diam) at the centres of MEA plates, with three replicate plates for each of three iso lates for each of the two morphologically different FIISicoCClIIII spp. Plates were incubated at temperatures ranging from 15-40 °C at 5 °C intervals. Two diameter l11e aS Urenlents were taken perpendicular to each other al »ler 4 d (‘or each colony, and averages computed. Colony colors were determined using the color charts of Rayner (1970).

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DNA isolatioll and amplificatioll

DNA was extracted from iso lates of unkIlowll identity (Table I) using tile technique described by Slippers el ill. (2004a). The quantification of nucleic acids was made using a spectrophotometer with a radio of absorbance at 260 11111 and 280 11111 . The DNA extraction was used as template to amplify part of the nuclear rRNA operon in PCR reactions using tile primers ITS 1 and lTS4 (White et 01. 1990). The amplified fragments included the 3′ elld of the small subunit (SSU) rRNA gene, the internal transcribed spacer ITS (ITSI), the complete 5.8S rRNA gene, the second region
ITS2 and tbe 5′ end of the large subunit (LSU) rRNA gene. A part of the EF I-a was amplified using the primers EFI-728F and EFI-986R (Carbone et ill., 1999). The PCR reaction mi xture contained 0.02 U roq DNA poiYlllerase (Roc he Molecular Bioc hemicals, Mannheim, Germany), I X PCR buffer containing MgCI2 (Roche Molecular Biochemicals, Al ameda, CA), 0.4 mM of each dNTPs, 0.2 pM or each primer and 20-25 ng/~I of DNA templ ate and made up to a final volu/lle of 25 ~d with Sabax water. Standard PCR reaction cycles were followed with primer annealing at 58 0c. Due to difficulties in amplifying the EF I-a region for some isolates it was necessary to vary the PCR annealing temperature between 52 to GO °C for this region. PCR al11plicons were separated on 1.5 % (w/v) agarose gels, stained with ethidiul11 bromide and visualized under UV light. The sizes of the PCR amplicons were estimated using DNA molecular weight l11arker XIV (100 bp ladder) (Roche Molecular Biochemicals, Mannheim, Germany).

Sequence analysis

A total of twenty-seven isolates were used in the phylogenetic analysis (Table I). All the sequences used are frol11 isolates maintained in the CMW anu CBS culture collection. BLAST searches were done to determine whether any related sequences are present in GcnBank, but none were found that were more closely related to the test isolates than those chosen for comparison here. The trees were rooted to sequence data of an isolate of a Biol1ecl,.iu sp., which \\as included as an outgroLip taxon ill the analysis of 30 ingroup taxa.
All PCR amplicons were purified prior to sequcncing using High Purc peR Product Purification Kit (Roche Molecular Biochemicals. Almeda, California. USA) following the manufacturer’s specifications. The PCR products were sequenced ill both directions using the primers ITSI, ITS4 and EF l-n8F, EF I -98GR. Sequencing reactions were perfonned using ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Applied BioSystenls, Foster City, CA) as recommended by the manu facturer and run 011 an ABI PRlSM 3 100 automated sequencer (Perkin-Elmer Applied BioSysteOls, Foster City. CA).

Acknowledgements 
Preface 
Chapter 1 The taxonomy and pathology of BUf!Tusp!lOeria spp., with special reference to their relevance in Eucalyptus plantations of Venezuela.
Chapter 2 Two new Fusicoccum species from Acacia and Eucal)pfUS in Venezuela, based on morphology and DNA sequence data.
Chapter 3 Identification of Boliyusp!lUcria species from EUC(/{)jJfUS, Acacia and Pillus in Venezuela.
Chapter 4 Diversity and host association of the tropical tree endophyte Lasiodipludia fheobromae revealed using SSR markers.
Chapter 5 Genetic diversity amongst isolates of BofIY0:’jJ!weria ribis and B. parva In South America and Hawaii.
Chapter 6 Pathogenicity of Botryo:,p!weria species on commercial ElIcal)pfUS clones in Venezuela.
Summary 
Opsomming 
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