Persoonia 28, 2012: 1–13 RESEARCH ARTICLE www.ingentaconnect.com/content/nhn/pimj http://dx.doi.org/10.3767/003158512X626155 Fungal trunk pathogens associated with wood decay of almond trees on Mallorca (Spain) D. Gramaje1, C. Agustí-Brisach1, A. Pérez-Sierra1, E. Moralejo2, D. Olmo3, L. Mostert4, U. Damm5, J. Armengol1 Key words Abstract Severe decline of almond trees has recently been observed in several orchards on the island of Mal- lorca (Balearic Islands, western Mediterranean Sea). However, the identity of the causal agents has not yet been almond dieback investigated. Between August 2008 and June 2010, wood samples from branches of almond trees showing internal Botryosphaeriaceae necroses and brown to black vascular streaking were collected in the Llevant region on the island of Mallorca. Collophora Several fungal species were subsequently isolated from the margin between healthy and symptomatic tissue. Five Eutypa lata species of Botryosphaeriaceae (namely Botryosphaeria dothidea, Diplodia olivarum, D. seriata, Neofusicoccum Phaeoacremonium australe and N. parvum), Eutypa lata, Phaeoacremonium iranianum and Phomopsis amygdali were identified Phomopsis amygdali based on morphology, culture characteristics and DNA sequence comparisons. Neofusicoccum parvum was the Prunus dulcis dominant species, followed by E. lata, D. olivarum and N. australe. First reports from almond include D. olivarum and Pm. iranianum. Two species are newly described, namely Collophora hispanica sp. nov. and Phaeoacremonium amygdalinum sp. nov. Article info Received: 18 September 2011; Accepted: 6 January 2012; Published: 16 January 2012. INTRODUCTION sphaeriaceae (Aplosporella, Botryosphaeria, Diplodia, Dothio- rella, Lasiodiplodia, Macrophomina, Neofusicoccum, Spen- Almond (Prunus dulcis) is a common crop cultivated in many cermartinsia and Sphaeropsis), Calosphaeriaceae (Calo- Mediterranean countries as well as in California (USA), South sphaeria and Jattaea), Coniochaetaceae (genus Coniochaeta), Africa, and some countries in South America and Australasia. Diaporthaceae (Phomopsis), Diatrypaceae (Cryptovalsa, Dia- According to the Food and Agriculture Organization (FAO 2010), trype, Eutypa and Eutypella), Herpotrichiellaceae (Phaeomoniel- Spain is the second largest almond producer after California, la), Montagnulaceae (Paraconiothyrium), Togniniaceae (Phaeoa- accounting for 11.9 % of the world’s almond production, which cremonium) and species of the genus Collophora have been yielded 2.31 million tons in 2009. In Spain, almonds are grown reported on Prunus trees. A list of fungal trunk pathogens iso- in the south-eastern regions and on the Balearic Islands (west- lated from Prunus spp. and their worldwide distribution is shown ern Mediterranean Sea), an important region with 23 432 ha in Table 1. While several fungal species belonging to a number of this crop cultivated in 2007 (4.7 % of the Spanish almond of genera are well-recognised pathogens of Prunus trees, the cultivation) (INE 2011). causal agent of the severe decline of almond trees on the island In summer 2008, severe decline of almond trees was noticed of Mallorca is still unknown. Therefore, the objective of this study in several orchards on the island of Mallorca (Balearic Islands). was to determine the aetiology of trunk diseases associated Disease symptoms included rapid collapse of branches during with wood necroses of almond trees in this region of Spain. mid-summer, chlorosis of leaves, which suddenly wilted and died, as well as bud and shoot dieback. Internal wood symp- MATERIALS AND METHODS toms ranged from brown to black vascular streaking, visible in cross sections as spots or circular discolouration of the xylem Sampling and fungal isolation tissue. Additionally, wedge-shaped necroses were frequently A field survey was conducted on almond trees (local cultivars observed. Some affected trees in the orchard died within a few Vivot, Pons, Negre and Totsolet) in the Llevant region on the weeks of showing first symptoms. island of Mallorca (Balearic Islands, western Mediterranean Different studies have shown that Prunus represents a rich Sea) between August 2008 and June 2010 (Table 2). Wood catch-crop for many fungal trunk pathogens. Species of Botryo- samples were collected from branches of almond trees with dieback symptoms, including dead shoots, cankers, internal 1 Instituto Agroforestal Mediterráneo, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain; wood necrosis, black vascular streaking or discoloured tissues corresponding author e-mail: [email protected]. (Fig. 1). 2 Departamento de Biología (Área Botánica), Universitat de les Illes Balears, Wood segments were cut from the affected branches, washed Carretera Valldemossa km 7,5, 07122 Palma de Mallorca, Spain. 3 Laboratori de Sanitat Vegetal, Millora Agrària, Conselleria d’Agricultura, under running tap water, surface-disinfected for 1 min in a 1.5 % Medi Ambient i Territori, Govern Balear, C/d’Eusebi Estada 145, 07008 sodium hypochlorite solution, and washed twice with sterile Palma de Mallorca, Spain. distilled water. Small pieces from the margin between healthy 4 Department of Plant Pathology, University of Stellenbosch, Private Bag and discoloured or decayed wood tissue were plated on malt X1, Stellenbosch 7602, South Africa. extract agar (MEA, 2 % malt extract, Oxoid Ltd., England; 1.5 % 5 CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. agar, Difco, USA) supplemented with 0.5 g/L of streptomycin © 2012 Nationaal Herbarium Nederland & Centraalbureau voor Schimmelcultures You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 2 Persoonia – Volume 28, 2012 7) 7) 0 0 0 0 2 2 al. ( al. ( el (1941) ppers et ppers et Reference Agarwal et al. (1992)McAlpine (1902)Damm et al. (2007b)Inderbitzin et al. (2010)Pusey et al. (1995)Farr et al. (1989)Damm et al. (2007a)Sutton (1980)Laundon (1973)Damm et al. (2007a)Gure et al. (2005)Wollenweber & HochapfDamm et al. (2007a)Inderbitzin et al. (2010)Pusey et al. (1995)Damm et al. (2007a), SliDamm et al. (2007a)Farr et al. (1989)Wollenweber (1941)Inderbitzin et al. (2010)Damm et al. (2007a)Pusey et al. (1995)Farr et al. (1989)Inderbitzin et al. (2010)Damm et al. (2007a)Slippers et al. (2007)Damm et al. (2007a), SliInderbitzin et al. (2010)Farr et al. (1989)Damm et al. (2007a)Damm et al. (2007a)Farr et al. (1989) Damm et al. (2008a)Damm et al. (2008a)Damm et al. (2008a) Damm et al. (2010)Popushoi (1971)Popushoi (1971)Popushoi (1971)Damm et al. (2010) Smit et al. (1996)Tuset & Portilla (1989) Uecker (1988) Diogo et al. (2010) Trouillas et al. (2010)Damm et al. (2009)Trouillas et al. (2010) a c eri m A Country India Australia South Africa USA Worldwide USA South Africa Unknown New Zealand South Africa Ethiopia France South Africa USA Worldwide South Africa South Africa USA Europe, North USA South Africa Worldwide USA USA South Africa South Africa South Africa USA USA South Africa South Africa USA South Africa South Africa South Africa South Africa Moldavia Moldavia Moldavia South Africa South Africa Worldwide Worldwide Portugal USA South Africa USA a n ci ali s P. bution. Prunus spp. P. domestica P. armeniaca P. persica var. nucipersica P. dulcis P. persica Prunus spp. P. persica P. armeniaca, P. persica P. persica P. persica P. africana P. laurocerasus P. armeniaca P. dulcis P. persica P. persica, P. salicina P. persica var. nucipersica Prunus spp. P. armeniaca, Prunus spp. P. dulcis P. salicina P. persica Prunus spp. P. dulcis P. armeniaca, P. persica P. dulcis P. salicina P. dulcis Prunus spp. P. persica, P. salicina P. persica var. nucipersica, Prunus spp. P. armeniaca P. persica var. nucipersica P. salicina P. salicina P. armeniaca, P. avium P. avium, P. domestica P. domestica P. armeniaca, P. salicina P. salicina P. dulcis, P. persica P. dulcis, Prunus spp. P. dulcis P. armeniaca Prunus spp. P. armeniaca stri di e d wi d orl w al trunk pathogens isolated from Prunus spp. and their Species Aplosporella indica, A. phyllanthina A. pruni A. prunicola Botryosphaeria dothidea Diplodia africana D. mutila D. pinea D. rosulata D. roumeguerei var. santonensis D. seriata Dothiorella sarmentorum Lasiodiplodia plurivora L. theobromae Macrophomina phaseolina Neofusicoccum australe N. mediterraneum, N. nonquaesitum, N. parvum N. ribis N. vitifusiforme Spencermartinsia viticola Sphaeropsis peckii Calosphaeria africana Jattaea mookgoponga J. prunicola Coniochaeta africana Ca. ambigua Ca. calva Ca. ligniaria Ca. prunicola, Ca. velutina Phomopsis ambigua Ps. amygdali Ps. mali, Ps. padina, Ps. parabolica, Ps. perniciosa, Ps. pruni, Ps. prunorum, Ps. stipata, Ps. ribetejana Ps. theicola, Phomopsis sp. Cryptovalsa ampelina Diatrype oregonensis g n e List of fuTable 1 Family Botryosphaeriacea Calosphaeriaceae Coniochaetaceae Diaporthaceae Diatrypaceae D. Gramaje et al.: Fungal trunk pathogens on almond trees 3 b) sulphate (MEAS) (Sigma-Aldrich, St. Louis, MO, USA). Plates 8 00 were incubated at 25 °C in the dark for 14 to 21 d, and all 2 al. ( colonies were transferred to 2 % potato-dextrose agar (PDA; et Biokar-Diagnostics, Zac de Ther, France). Single spore colonies m were derived prior to morphological and molecular identification 7) m 99 Da using the serial dilution method (Dhingra & Sinclair 1995) and Munkvold & Marois (1994)Carter (1957)Carter (1982)English & Davis (1965)Grove (1935), Ellis & Ellis (1 Damm et al. (2010)Damm et al. (2010) Damm et al. (2008c)Damm et al. (2008c)Damm et al. (2008c) Mostert et al. (2003, 2006), Hausner et al. (1992)Damm et al. (2008b) Damm et al. (2008b) Damm et al. (2008b)Rumbos (1986)Hawksworth et al. (1976)Damm et al. (2008b)Damm et al. (2008b) Damm et al. (2008b)Damm et al. (2008b) asMSaolistrnnpoodeddererde cpir cndip hetoco isnoinun eeliob don1 fani gh5aBteaei loec %ndmtdarc yallgoee otilr s dyssp2 cpeoph5eonnho °rr atl2ouCoief llgi a%rucsiytaonai owcladtuneiaseot,ai t rcnoeed nun r eaw el atsanuaegctdrrr are -ei Urb8sc i( eVd0hwWde a °el nAirCgrbate;hi y ficiB nta et Piw meod1rhk ie.bti5iahslnal r aidmspa-tDees iL1odd i a2( ncwo2g rhn0ynit o0hopcv6 sohsi) tloatoi.ec tlnsorIsniy.)-- period (Philips TDL18W/33) (Slippers et al. 2004). Isolates were examined weekly for formation of pycnidia and conidia in order to record their morphology (size, shape, colour, presence or Worldwide Worldwide Worldwide Worldwide Worldwide South Africa South Africa South Africa South Africa South Africa South Africa Canada South Africa South Africa South Africa Greece Tunisia South Africa South Africa South Africa South Africa aSthbiens ceDeni acitte ris yo pdfa isfcfieecpautealt tabona ddsie scdtei nlolg nwu aimsllho s rstprphueocctloiuegrsei c)o.a rl ecvheanr agcetenresr ao wf tihtheinir Libertella anamorph (Glawe & Rogers 1984), the morphological identification of Eutypa lata was only tentative using characters a such as conidial size and shape, and colony characters on PDA. n ci ali Morphological characters used to distinguish Phaeoacremonium s P. species included conidiophore morphology, phialide type and a, shape, size of hyphal warts and conidial size and shape. Colony c cina persi ca characters and pigment production on MEA, PDA and oatmeal avium armeniaca dulcis, P. salicina virginiana avium, P. dulcis, P. salicina, P. spinosa salicina armeniaca persica persica, P. persica var. nucipersica, P. salipersica, P. salicina armeniaca pennsylvanica persica, P. salicina salicina armeniaca avium armeniaca armeniaca armeniaca, P. persica, P. persica var. nuci persica, P. persica var. nucipersica dulcis, P. persica, P. persica var. nucipersi aaCotostbmanhiopglffefoo.ei tc nocaretcl2 eglrsoarrrooo0ud unl(p1n 4s0l rOtiaah9ifcdo 9dawtfAo7oi )itiaccoo er6pk;ieo a nnmr.)ia6c n .,cos2n 0C aaiTdufpd d ltwoobsghlaei yaclyua ek,co o e onbt ifnmeeoacxotnlhyeudstfariim d emc- l gmwtcaaitrureoiehotueicorn apdac 2etrwneolhey r;5sou tadcoc 1h ctnwlc °lrraa2 hiCe ctioeehvfe.as t5n renyerrcw e sa(pdtroMgD-e ch 2pin panrEataa oeieincwdgmnAeo rudinaeika,r,smb or aatPaepn;ait ogsndeieeDDtg nead detwbmiA drf dacaa oae m ealloadse.rflln tn e,etd 2se aetddFd2i 0rtwsn ipi 4r1Ouo8o daesr 0mn no°onArai)zCa edcct .n(eh( l oeu lSTpdCiy enn)caol N ra 1iti pn (onditeAcC6ohrdsuineea n;er psd shdosN e .hdaeaw ueiocraadnnsteent eprd ccarntekedeeeiol--,.t P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. 2009) incubated at 24 °C. Species of Phomopsis were identified based on morphology s oniae, ana, ospf ocrounlaidtiioan f owramse edn ihna pnyccendi dbiya a(mvaenn dNiniegk e2r %k eWt Aa l.c u2l0tu0r5e)s. wThiteh oide mort afric sterilised pine needles and incubating them at 25 °C under Eutypa lata Eutypella prunastri Phaeomoniella dura, Pa. effusa, Pa. prunicola, Pa. zymP. tardicola Paraconiothyrium africanum Pc. brasilense Pc. variabile Phaeoacremonium aleophilum Pm. australiense, Pm. fuscum, Pm. griseorubrum, Pm. Pm. prunicolum, Pm. viticola Pm. iranianum, Pm. pallidum, Pm. subulatum, Togninia Tg. griseo-olivacea Pm. parasiticum Pm. scolyti Collophora africana, Co. capensis, Co. paarla, Co. pallida Co. rubra mwpcdnwMluaw1mdwiuhaeel2iseeee.faiimfcca irta0rerrnege(erreeerr02k rgoontrn es,Uom0lcd ssi tmyfi nNhf0saccVe o ictnfelo7oopicfiiiof akadle su)pptonirRotug leedn ieil ewcofnorhias ton de e(bn nhct.yorCDt d dy)uweiemCfnbe o oM lwrnweiositsairftrnun erh e)eli ptsaot c r icr hror(.oetalluen,ve1uaul r n abynaJ 9m1lcdwtdt actaeio7tc2Nyiveeiieotpoccf0ti iaint ahhn nk ahlep)ctogas .gosennarpyo uu cm nCd)ci htnfMNrqir,ho nd s oaeitcunweorirEi ,tkramwad eeo ndiaoAamtsig,depea ihl nnn tlet.reen pa h daeaifanETrlrtlusel lsa ina uu dcoh n3N gtd smelwedeserg0iir pexkty. scoi iMie5 tsnsopsmI ohwwrstiepatynlnhmn eaoeet tect h8 ihimilSadraitooncan 0heesinMttsnet aeie ei (eu dpvme dDm.d Zm(sre hxdD e 9ia 1ui gtabwpacm5rId0msieyCererette0iokae hrnrmer) e A0aslne. gfad pcelr tSrt Sl udtioestaezosu ihritmg6 xa.rp smree (gchaun–seP1 s ei tmccmevl 4 houon0otDwec0uioyusrt0lntniX oe criitl 0°nenbeoe ureeMiCegdgn×sssyr-t with 3 °C intervals (Collophora) or 5–40 °C with 5 °C intervals e ea e (Phaeoacremonium), also including 37 °C. Radial growth was c a Herpotrichiella Montagnulace Togniniaceae Unknown m2iCn4e et nha°Ctesr ue c(r Cu(eClodtuBl lraoSefpt) ceh, oroU lrl8tear ec)d.ct iIhoastnto, 2lToa5fht ete°hsC eN o eC(fP tBnhhoeSavr-elKeaolnN asdAcpsrWe.ecm Fieousnn iwguaemlr )eB oidored 2piv owesrkist eiatdyt 4 Persoonia – Volume 28, 2012 Table 2 Names, accession numbers, and collection details of isolates studied. Species Accession no.1 Location Collector GenBank accessions2 Botryosphaeria dothidea Bdo-1 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 – JN183856 – JF503990 – Collophora hispanica Col-1, CBS 128566 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 – – JN808850 JN808839 JN808843 Col-3, CBS 128567 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 – – JN808851 JN808840 JN808844 Col-4, CBS 128568 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 – – JN808852 JN808841 JN808845 Col-5, CBS 128569 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 – – JN808853 JN808842 JN808846 Diplodia olivarum Dol-1 Sant Llorenç, Mallorca, Spain J. Armengol, 2009 – JN183857 – JF693916 – Dol-2 Sant Llorenç, Mallorca, Spain J. Armengol, 2009 – JN183858 – JF693917 – Dol-3 Sant Llorenç, Mallorca, Spain J. Armengol, 2009 – JN183859 – JF693918 – Dol-4 Sant Llorenç, Mallorca, Spain J. Armengol, 2009 Dol-5 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Dol-6 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Dol-7 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Diplodia seriata Dse-1 Sant Llorenç, Mallorca, Spain J. Armengol, 2009 – JN183860 – JN183850 – Dse-2 Sant Llorenç, Mallorca, Spain J. Armengol, 2009 – JN183861 – JN183851 – Dse-3 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 – JN183862 – JN183852 – Dse-4 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Eutypa lata Ela-1 Sant Llorenç, Mallorca, Spain J. Armengol, 2009 – – – JN183853 – Ela-2 Sant Llorenç, Mallorca, Spain J. Armengol, 2009 – – – JN183854 – Ela-3 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 – – – JN183855 – Ela-4 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Ela-5 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Ela-6 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Ela-7 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Neofusicoccum australe Nau-1 Sant Llorenç, Mallorca, Spain J. Armengol, 2009 – JN191293 – JF421449 – Nau-2 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 – JN191294 – JF421450 – Nau-3 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 – JN191295 – JF421451 – Nau-4 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Nau-5 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Nau-6 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Neofusicoccum parvum Npa-1 Sant Llorenç, Mallorca, Spain J. Armengol, 2009 – JN191296 – JF330779 – Npa-2 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 – JN191297 – JF330780 – Npa-3 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 – JN191298 – JF330781 – Npa-4 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Npa-5 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Npa-6 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Npa-7 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Npa-8 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Npa-9 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Npa-10 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Npa-11 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Npa-12 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Npa-13 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 Phaeoacremonium amygdalinum Psp-1 Sant Llorenç, Mallorca, Spain D. Gramaje, 2009 JN191301 JN191305 – – – Psp-2 Sant Llorenç, Mallorca, Spain D. Gramaje, 2009 JN191302 JN191306 – – – Psp-3, CBS 128570 Sant Llorenç, Mallorca, Spain D. Gramaje, 2009 JN191303 JN191307 – – – Psp-4 Sant Llorenç, Mallorca, Spain D. Gramaje, 2009 JN191304 JN191308 – – – Phaeoacremonium iranianum Pir-1 Sant Llorenç, Mallorca, Spain D. Gramaje, 2009 JN191300 JN191299 – – – Phomopsis amygdali Pam-1 Sant Llorenç, Mallorca, Spain J. Armengol, 2010 – – – JF693919 – 1 CBS: Culture collection of the Centraalbureau voor Schimmelcultures, Fungal Diversity Centre, Utrecht, The Netherlands. 2 GenBank accessions correspond to ACT, BT, EF, ITS and GAPDH sequences. DNA isolation analysis of partial β-tubulin gene (BT) sequences amplified Fungal mycelium and conidia from pure cultures grown on PDA using primers Bt2a and Bt2b (Glass & Donaldson 1995). For for 2 wk at 25 °C in the dark were scraped and mechanically species of Phaeoacremonium, ± 600 bp of the 5’ end of the disrupted by grinding to a fine powder in liquid nitrogen with a BT and ± 300 bp of the 5’ end of the actin (ACT) genes were mortar and pestle. Total genomic DNA was extracted with the amplified as described by Mostert et al. (2006) using primer E.Z.N.A. Plant Miniprep Kit (Omega Bio-tek, Norcross, GA) sets T1 (O’Donnell & Cigelnik 1997) and Bt2b, and ACT-512F following the manufacturer’s instructions. DNA was visualised and ACT-783R (Carbone & Kohn 1999), respectively. For Collo- on 0.7 % agarose gels stained with ethidium bromide and the phora spp., the ITS region was amplified using the primer pairs DNA aliquots were stored at -20 °C. ITS1-F (Gardes & Bruns 1993) and ITS4. Additionally, a 200- bp intron of the glyceraldehyde-3-phosphate dehydrogenase Molecular identification and phylogenetic analysis (GAPDH) and a partial sequence of the translation elongation Morphological identifications of Botryosphaeriaceae spp., dia- factor 1α (EF-1α) were amplified and sequenced using the primer pairs GDF1 and GDR1 (Guerber et al. 2003) and EF1- trypaceous fungi and Phomopsis spp. were confirmed by se- 728F and EF1-986R (Carbone & Kohn 1999). quence analysis of the internal transcribed spacer (ITS) nrDNA region using the primers ITS1 and ITS4 (White et al. 1990). PCR products were purified with the High Pure PCR Product Species in the Botryosphaeriaceae were also confirmed by Purification Kit (Roche Diagnostics, Germany) and sequenced D. Gramaje et al.: Fungal trunk pathogens on almond trees 5 a b d c e f g h Fig. 1 Disease symptoms on almond trees on the island of Mallorca associated with fungal trunk pathogens. a, b. Dieback and wilting of branches; c–h. internal symptoms visible when transversal and longitudinal cuts were made in branches used for fungal isolation: black spots and dark brown to black streak- ing of the xylem tissue (d, h), circular (c, g) or sectorial necrosis (f), and wood discoloration (e). in both directions by Macrogen Inc., Sequencing Center (Seoul, quences (BT and ACT) together with the reference sequences South Korea). Sequences were edited using the Sequencher (Mostert et al. 2006, Damm et al. 2008b, Essakhi et al. 2008, software v. 4.7 (Gene Codes Corporation, Ann Arbor, MI). Graham et al. 2009, Gramaje et al. 2009b) and the outgroup The Collophora sequences (ITS, GAPDH, EF-1α) were added to taxa, Pleurostomophora richardsiae (ACT: AY579271, BT: reference sequences (Damm et al. 2010) and the outgroup (Ca- AY579334) and Wuestneia molokaiensis (ACT: AY579272, dophora luteo-olivacea CBS 141.41, ITS: GU128588, GAPDH: BT: AY579335) obtained from GenBank were aligned using JN808849, EF-1α: JN808856). The multi-locus alignment was MAFFT sequence alignment program v. 6 (Katoh & Toh 2010) manually adjusted using Sequence Alignment Editor v. 2.0a11 followed by manual adjustments of the alignments in Sequence (Rambaut 2002). To determine whether the three sequence Alignment Editor v. 2.0a11. The BT and ACT alignments were datasets were congruent and combinable, tree topologies of concatenated. A partition homogeneity test was conducted 70 % reciprocal Neighbour-Joining bootstrap with Maximum in PAUP (Phylo genetic Analysis Using Parsimony) v. 4.0b10 Likelihood distances (10 000 replicates) with substitution mod- (Swofford 2003). The congruence between the ACT and TUB els determined separately for each partition using Modeltest datasets were tested at 1 000 replicates. Phylogenetic analy- v. 3.5 (Posada & Crandall 1998) were compared visually ses of all aligned sequence data were performed with PAUP. (Mason-Gamer & Kellogg 1996). The Phaeoacremonium se- Alignment gaps were treated as missing data and all characters 6 Persoonia – Volume 28, 2012 were unordered and of equal weight. Any ties were broken assigned to three different species: Botryosphaeria dothidea, randomly when encountered. All characters were unordered Neofusicoccum parvum and Neofusicoccum sp. and of equal weight. The second group of isolates was characterised by white to Maximum parsimony analysis was performed for the combined reddish cream, slow growing mycelium, turning red to blood Phaeoacremonium dataset using the heuristic search option colour with age. Isolates formed a red pigment that coloured with 10 random simple taxon additions and tree bisection and the colony and surrounding medium. Conidiophores on hy- reconstruction (TBR) as the branch-swapping algorithm with phae were reduced to conidiogenous cells. Sporulation was the option of saving no more than 10 trees with a score greater abundant, with hyaline, 1-celled conidia aggregated in masses than or equal to 5 (Harrison & Langdale 2006). The maximum around the hyphae. All morphological characters observed were parsimony analysis for the combined Collophora dataset was consistent with the description of Collophora spp. (Damm et performed using the heuristic search option with 100 random al. 2010). However, the isolates could not be assigned to one sequence additions and TBR without further restrictions for of the known species. tree saving. Branches of zero length were collapsed and all The third fungal group was characterised by having white to multiple, equally parsimonious trees were saved. The robust- white-cream cottony slow-growing mycelium on PDA lacking ness of the trees obtained was evaluated by 1 000 bootstrap fruiting structures after an incubation time of 3 wk in the dark. replications (Hillis & Bull 1993). Tree length (TL), consistency With age, some cultures developed a grey pigment. After 3–4 index (CI), retention index (RI) and rescaled consistency index wk under continuous fluorescent light, small black pycnidia (RC) were calculated. were formed on the pine needles. Conidia developing in the Sequences derived in this study were lodged at GenBank, the fruiting bodies were filiform and mostly curved in shape, which alignments in TreeBASE (www.treebase.org/), and taxonomic corresponds to descriptions of species in the Diatrypaceae novelties in MycoBank (www.MycoBank.org; Crous et al. 2004). family (Glawe & Rogers 1984). GenBank accession numbers of the strains collected during The fourth fungal group was characterised by pale to medium this study are listed in Table 2. Additional GAPDH and EF- brown flat slow-growing cultures on MEA. Different types of 1α sequences were generated for strains CBS 141.41 (see phialides that were variable in size and shape were observed above), CBS 120878 (JN808847, JN808854) and CBS 120873 in the aerial mycelium, and either discrete or integrated in (JN808848, JN808855). conidiophores. Sporulation was abundant and conidia hyaline and aseptate. All morphological characters corresponded to the genus Phaeoacremonium (Mostert et al. 2006). RESULTS The last fungal group was characterised by white, cottony, Morphological identification and characterisation slow-growing, raised mycelium, with margins becoming pale Based on their appearance in culture, the isolates obtained in brown with age. Dark brown, eustromatic pycnidia released a this study, could be assigned to five main fungal groups (Table mucilaginous light-cream drop containing only one character- 2). The first group was characterised by dark green or grey to istic spore type, usually ovoid-ellipsoidal with one obtuse and dark grey fast-growing mycelium on PDA. Some isolates pro- one acute end. These morphological characteristics resembled those of Phomopsis species (van Niekerk et al. 2005). duced a yellow pigment after 3 days that diffuses into the agar. With age, most of these cultures developed single or grouped, Botryosphaeriaceae spp. were the most common fungi isolated black, globose fruiting bodies (pycnidia) on the surface of pine from symptomatic almond wood from Mallorca Island, followed needles on WA releasing either pigmented or hyaline conidia. by Eutypa lata, Phaeoacremonium spp., Collophora hispanica Based on descriptions of species of Botryosphaeriaceae (van and Phomopsis amygdali (Table 2). Of the Botryosphaeriaceae Niekerk et al. 2004, Phillips 2006) and comparison with previ- species isolated, N. parvum was the most abundant species ously identified isolates from Spain, fungal cultures with pig- (13 strains). In contrast, B. dothidea was the least abundant, mented conidia were assigned to two species: Diplodia seriata with only one strain. Other species of Botryosphaeriaceae were and Diplodia sp. Fungal cultures with hyaline conidia were also frequently isolated, and included D. olivarum (7 strains), 10 changes Col-5/CBS128569 72 Col-4/CBS128568 T Co. hispanica 100 Col-3/CBS128567 Col-1/CBS128566 100 CBS 120873 T 100 Co. rubra CBS 121441 100 CBS 120872 T Co. africana CBS 120879 100 CBS 120877 T Co. paarla CBS 120878 CBS 141.41 Cadophora luteo-olivacea Fig. 2 One of two most parsimonious trees obtained from heuristic searches of ITS, GAPDH and EF-1α gene sequences of Collophora species. Bootstrap support (1 000 replicates) above 70 % are shown at the nodes. Cadophora luteo-olivacea CBS 141.41 was used as outgroup. Ex-type strains for each species are indicated with a ‘T’ after the strain number. D. Gramaje et al.: Fungal trunk pathogens on almond trees 7 97 CCBBSS110113703685T Pm. viticola 100100 CBS101737 CBS114991 Pm. angustius 97 CBS114992T CBS112949T 90 100 CBS114993 T. austroafricana 96 CBCSB1S111518469T94 Pm. theobromatis STE U6104TPm. pallidum CBS110703 69 100 CBS100397 T. minima 98 CBS246.91T CBS117114 100 100 CBS101357T Pm. iranianum Pir-1 CBS123033TPm. tuscanum CBS1120212 100 CBS211.97 T. fraxinopennsylvanica 98 98 CBS101585T 100 ICMP17037T Pm. occidentale 97 CBS123036T Pm. hungaricum CBS110156T 100 CBS114512 Pm. novae-zealandiae 65 CBS110157 ICMP16987 100100 ICMP17038 Pm. globosum 100 100 ICMP16988T 100 ICMP17421TPm. armeniacum CBS777.83T T. argentinensis 84 100 SSTTEE UU55996687T Pm. prunicolum STE U6177T. africana STE U5966TT. griseo-olivacea Psp-1 100 Psp-4 Psp-3/CBS128570T Pm. amygdalinum Psp-2 CBS113593 100 CBS112585 Pm. scolyti CBS113597T 100 CBS566.97 Pm. griseorubrum CBS111657T 95 100 92 10C0BCSB1S1101632578T4TPmP. mam. ssutebloudlaatmumense 100 CBS113587 64 100 CBS113589T Pm. australiense CBS113592 CBS110573TPm. tardiscrescens CBS110034T 92 CBS729.97 Pm. alvesii 93 100 CBS113590 91 CCBBSS419182.09446T T. rubrigena CBS514.82 100 CBS860.73T T. parasitica CBS113585 Pm2 94 87 98 100 PPmm45T Pm. cinereum 100 Pm8T Pm. hispanicum 100 CBS117115 T. vibratilis 100 CBS391.71T 100 CBS13273 Pm. inflatipes CBS166.75 CBS110119 100 CBS113595 Pm. venezuelense 100 CBS651.85T STE U5969TPm. fuscum CBS110118 100 CBS109479T T. krajdenii 88 CBS110368 100 CBS337.9T Pm. sphinctrophorum CBS694.88 100 CBS123034T Pm. sicilianum CBS123035 CBS270.33 Pleurostomophora richardsiae CBS114877Weustneia molokaiensis 10 changes Fig. 3 One of 90 most parsimonious trees obtained from heuristic searches of a combined alignment of the TB and ACT gene sequences. Bootstrap support (1 000 replicates) above 60 % are shown at the nodes. Pleurostomophora richardsiae and Wuestneia molokaiensis were used as outgroup. Ex-type strains for each species are indicated with a ‘T’ after the strain number. Thickened lines indicate branches present on strict consensus tree. N. australe (6 strains) and D. seriata (4 strains). Diatrypaceae, 121484 (GenBank EU650670) and N. parvum CBS 110301 represented by E. lata, were also frequently isolated in this (GenBank AY259098). Diplodia sp. isolates showed 100 % study (7 strains), while only one strain of Ps. amygdali was identity with isolates previously described as D. olivarum (Gen- collected. While Pm. iranianum was infrequently isolated (one Bank GQ923873, GQ923874; Lazzizera et al. 2008a), while strain), the novel species Pm. amygdalinum and Co. hispanica Neofusicoccum sp. isolates showed 99 % identity with isolates were collected several times (4 strains each). previously identified as Neofusicoccum australe CBS 115185 Species of Botryosphaeriaceae, Eutypa lata and Ps. amyg- (GenBank FJ150696) and CBS 119046 (GenBank DQ299244). dali isolates were mostly isolated from circular (Fig. 1c, g) or The ITS sequences of the second group of isolates were sectorial necrosis (Fig. 1f), and wood discoloration (Fig. 1e). 96 % identical to those of Co. africana STE-U 6113 (GenBank Phaeoacremonium and Collophora spp. were isolated from GQ154570) and Co. capensis STE-U 6341 (GenBank GQ154574), black spots and dark brown to black streaking of the xylem while the EF sequences of these isolates were 91 % identical tissue (Fig. 1d, h). to that of Co. rubra STE-U 6198 (GenBank GQ154642). The ITS sequences of the Diatrypaceae isolates from this study had Molecular identification and phylogenetic analyses 99–100 % identity with isolates previously identified as Eutypa To confirm the identification based on morphology, BLASTn lata (GenBank AY462541, AY662394). Regarding Phaeoacr- searches in GenBank showed that ITS sequences of Botryo- emonium isolate Pir-1 from Mallorca Island, BT sequences sphaeriaceae isolates had 99–100 % identity with isolates of D. had 99–100 % identity with Pm. iranianum isolates (GenBank seriata CBS 121485 (GenBank EU650671), B. dothidea CBS EU128077, FJ872406). The ACT sequences of the remaining 8 Persoonia – Volume 28, 2012 Phaeoacremonium isolates were 87 % identical to those of topologies were retained (length = 318 steps, CI = 0.921, RI = Pm. pallidum STE-U 6104 (GenBank EU128103) and Pm. viti- 0.907, RC = 0.836) of which one is shown in Fig. 2. Isolates of cola STE-U 6180 (GenBank EU128094), while the BT se- Col-1, Col-3, Col-4 and Col-5 from almond trees on the island quences were 81 % identical to those of Pm. theobromatis CBS of Mallorca form a distinct clade (100 % bootstrap support) sis- 111586 (GenBank DQ173132) and Pm. viticola CBS 100947 ter to Co. rubra. Sequences of the type strains of Co. africana (GenBank DQ173134). A BLASTn search showed that the ITS (CBS 120872) and Co. capensis (CBS 120879) as well as Co. sequence of isolate Pam-1 had 100 % identity with isolates paarla (CBS 120877) and Co. pallida (CBS 120878) are each previously identified as Ps. amygdali (GenBank AF102996, present in well-supported clades (100 % bootstrap support) AF102997). Sequences of three representative isolates of each with no or little variability. species derived in this study were lodged in GenBank (Table 2). The partition homogeneity test of the BT and ACT alignments of Phylogenetic analysis was performed only with genera of un- Phaeoacremonium gave a P-value of 0.05 indicating that the known species, Collophora and Phaeoacremonium (Fig. 2, 3). datasets were congruent and could be combined. The com- The ITS, GAPDH and EF-1α sequence datasets of the genus bined sequence dataset consisted of 78 isolates including the Collophora did not show any conflicts in tree topology for the outgroup and had 918 characters, of which 510 characters were 70 % reciprocal bootstrap trees, which allowed us to combine parsimony-informative, 126 parsimony-uninformative and 282 them. The combined sequence dataset consisted of 11 isolates constant. Ninety equally most parsimonious trees were retained including the outgroup and had 1 097 included characters, (length = 2 690 steps, CI = 0.465, RI = 0.823, RC = 0.383). A tree of which 133 characters were parsimony-informative, 130 that closely resembled the strict consensus tree was chosen parsimony-uninformative and 834 constant. After a heuristic and is presented in Fig. 3. The strain Pir-1, grouped inside the search, two equally most parsimonious trees with identical Pm. iranianum clade with 100 % bootstrap support. Four strains a c d b e f g h k i j l m n p o q r Fig. 4 Collophora hispanica. a. Colony on MEA that is stained red by the pigment exuded by the fungus; b. conidioma on pine needle; c–h. conidiogenous cells and conidia on hyphal cells; i, j. conidiophores formed in conidiomata on pine needles; k, l. microcyclic conidiation (indicated by arrows) in conidia from conidiomata (k) and from hyphal cells (l); m. conidia formed in conidiomata after 4 wk; n, o. endoconidia; p, q. conidia formed on hyphal cells after 4 wk; r. conidia formed on hyphal cells after 2 wk. a, b: DIC, c–r: DM. — Scale bars: a = 1 mm; b = 100 µm; c = 5 µm; scale bar for c applies to c–r. D. Gramaje et al.: Fungal trunk pathogens on almond trees 9 (Psp 1–4) grouped together in a monophyletic clade with 100 % CBS H-20518 holotype, culture ex-type CBS 128568 = Col-4; Mallorca, bootstrap support, basal to the clades containing T. minima and Sant Llorenç del Cardassar, isolated from branches of Prunus dulcis trees, Pm. novae-zealandiae, with no other closely related species. June 2010, J. Armengol, Col-1 herb, CBS H-20516, culture Col-1 = CBS 128566; Mallorca, Sant Llorenç del Cardassar, isolated from branches of Prunus dulcis trees, June 2010, J. Armengol, Col-3 herb, CBS H-20517, Taxonomy culture Col-3, CBS 128567; Mallorca, Sant Llorenç del Cardassar, isolated Based on the DNA sequence analyses and morphological from branches of Prunus dulcis trees, June 2010, J. Armengol, Col-5 herb, characters, two species, one species each of Collophora and CBS H-20519, culture Col-5, CBS 128569. Phaeoacremonium, proved distinct from all known species, and Notes — The phylogeny of the combined sequence dataset are newly described below. showed that Co. hispanica does not group with any of the known species. Colonies of Co. hispanica resemble those of Collophora hispanica D. Gramaje, J. Armengol & Damm, Co. africana and Co. rubra in forming red pigments that stain sp. nov. — MycoBank MB561926; Fig. 4 the colony and surrounding medium. Conidia formed in the mycelium are often slightly curved like those of Co. rubra. Un- Etymology. Named after Spain where this fungus was first collected. like both of those species, discrete phialides are common and Vegetative hyphae hyaline, smooth-walled, septate, branched, endoconidia are formed similar to Co. pallida and Co. paarla. 1–3.5 μm wide, lacking chlamydospores. Sporulation abundant, Few conidiomata were formed on pine needles, but not on agar conidia formed on hyphal cells, occasionally in hyphae (endo- medium. The conidia in these conidiomata formed on conidio- conidia) and in conidiomata or due to microcyclic conidiation. phores similar to those of other Collophora species such as Conidiophores on hyphae reduced to conidiogenous cells. Co. africana, however no wall structures were observed. Conidiogenous cells enteroblastic, hyaline, mainly intercalary, reduced to collarettes formed directly on hyphal cells or on short Collophora africana Damm & Crous, Persoonia 24: 65. necks; necks cylindrical, 0.5–2 μm long, 0.5–1.5 μm wide; dis- 2010 crete phialides often observed, cylindrical to ampulliform, 4.5–8 × 1–2 μm; collarettes cylindrical to narrowly funnel shaped, very = Collophora capensis Damm & Crous, Persoonia 24: 67. 2010. thin-walled, 0.5 μm long, opening 0.5 μm wide, inconspicuous, periclinal thickening sometimes visible. Conidia aggregated Collophora paarla Damm & Crous, Persoonia 24: 67. 2010 in masses around the hyphae, hyaline, 1-celled, cylindrical, = Collophora pallida Damm & Crous, Persoonia 24: 69. 2010. sometimes obovate, often slightly bent, with both ends obtuse or with a papillate apex, smooth-walled, (2.5–)3.5–5(–6.5) Notes — Collophora africana and Co. capensis were regard- × (1–)1.5(–2) μm, av. ± SD = 4.3 ± 0.7 × 1.5 ± 0.2 μm, L/W ed as distinct taxa based on differences in conidial mor phology ratio = 2.8, after 4 wk many clavate, limoniform, subsphaerical (on hyphae and in conidiomata) and differences in cardinal or irregularly inflated conidia observed that are often > 2 μm temperature requirements for growth. Furthermore, Co. pal- wide. Microcyclic conidiation occurs occasionally, with conidia lida and Co. paarla were differentiated based on their conidial developing into mother cells, becoming > 7 μm long, 2–3 μm morphology, width of vegetative hyphae, and exudates formed wide, and sometimes septate, with a short neck or with a mere in culture (Damm et al. 2010). However, the phylogeny of the opening with a minute collarette at one end. Endoconidia unise- multigene sequence dataset of Collophora spp. generated in riate within hyphae, hyaline, 1-celled, cylindrical, slightly bent, this study (Fig. 2), only supports two species, namely Co. afri- with both ends obtuse, same size as conidia formed on hyphal cana and Co. paarla, suggesting that the observed variation cells. Conidiomata occasionally formed on pine needles in 2–4 (Damm et al. 2010) was not informative at species level. wk. Conidiophores hyaline to slightly reddish, smooth-walled, septate, branched. Conidiogenous cells enteroblastic, hyaline Phaeoacremonium amygdalinum D. Gramaje, J. Armengol & to slightly reddish, smooth-walled, cylindrical to ampulliform, L. Mostert, sp. nov. — MycoBank MB561925; Fig. 5 conidiogenous loci formed terminal and intercalary, immediately below the septum (acropleurogenously), 3–6 × 1–3 μm, col- Etymology. Named after the host it was isolated from, almond (Prunus larettes sometimes visible, ≤ 0.5 μm long, opening minute ≤ 0.5 dulcis), which is in Greek amygdali (Αμυγδαλή). μm wide, periclinal thickening not observed. Conidia hyaline or Aerial structures in vitro on MEA: Mycelium consisting of reddish, 1-celled, cylindrical, sometimes obovate, often slightly branched, septate hyphae that occurs singly or in bundles of bent, with both ends obtuse or with a papillate apex, smooth- up to 10; hyphae tuberculate with warts up to 2 µm diam, ver- walled, (2.5–)3–5(–7) × 1–2(–3.5) μm, av. ± SD = 4.2 ± 1.0 × ruculose to smooth, medium brown to pale brown and 2–3.5 µm 1.7 ± 0.5 μm, L/W ratio = 2.5. wide. Conidiophores mostly short, usually unbranched, arising Culture characteristics — Colonies reaching a radius of from aerial or submerged hyphae, erect to flexuous, up to 5-sep- 2–2.5 mm after 2 wk at 24 °C in the dark on MEA. Minimum tate, medium brown to pale brown, verruculose on the lower temperature for growth < 6 °C, optimum 18 °C, maximum 30 °C. part, (12–)15.5–40(–55) (av. = 29) µm long and 1.5–3 (av. = Colonies on PDA flat, moist, entire to undulate margin, coral, 2.1) µm wide. Phialides terminal or lateral, often polyphialidic, livid red to vinaceous with white to pale vinaceous margin, with smooth to verruculose, hyaline, collarettes, 1.5–2.5 µm long, little pale vinaceous to vinaceous aerial mycelium in the centre; 1–1.5 µm wide; type I phialides mostly cylindrical, occasionally reverse same colours, turning dark vinaceous with age, medium widened at the base, (3–)3.5–7.5(–10) × 1–2 (av. = 6 × 1.5) around culture vinaceous due to diffuse pigment; on OA flat µm; type II phialides most predominant, either subcylindrical to low convex, entire to slightly undulate margin, white to pale or navicular, some elongate-ampulliform and attenuated at the rosy vinaceous, surface covert almost entirely with white aerial base, (9–)10–16.5(–17) × 1.5–2.5 (av. = 13.5 × 2) µm; type III mycelium, reverse salmon to flesh, white towards the margin; phialides cylindrical to subcylindrical, 17–27 × 1.5–2.5(–3) (av. on MEA flat to low convex, undulate margin, dark vinaceous, = 21 × 2) µm. Conidia hyaline, oblong ellipsoidal or obovoid, surface covered partly or entirely with rosy to vinaceous aerial (3–)4–5 × 1.5–3 (av. = 4.5 × 2) µm, L/W ratio = 2.1. mycelium; reverse blood colour, medium around culture red On surface or submerged in the agar — Phialides hyaline, due to diffusing pigment. mostly cylindrical, 4–12 × 1–2 (av. = 7.5 × 1.5) µm. Conidia Specimens examined. Spain, Mallorca, Sant Llorenç del Cardassar, hyaline, mostly allantoid, few reniform, (5–)5.5–7(–10) × 1–2 isolated from branches of Prunus dulcis trees, June 2010, J. Armengol, (av. = 6.5 × 1.5) µm, L/W ratio = 4.4. 10 Persoonia – Volume 28, 2012 Culture characteristics — Colonies reaching a radius of known species. A distinguishing morphological feature is the 9.5–10 mm after 8 d at 25 °C. Minimum temperature for growth frequent occurrence of polyphialides. Other Phaeoacremonium 15 °C, optimum 25 °C, maximum 30–35 °C. Colonies on MEA species that also form polyphialides include Pm. australiense, flat, with entire margin; after 8 d and 16 d, white to brownish Pm. fuscum, Pm. krajdenii, Pm. occidentale, Pm. pallidum, Pm. drab above, buff-yellow to yellowish olive in reverse; on PDA flat, prunicolum, Pm. scolyti, the anamorph of Togninia africana felt-like with few woolly tufts near the centre, with entire margin; and the anamorph of T. griseo-olivacea. Phaeoacremonium after 8 d and 16 d, white to olive-brown or olive-green above, amygdalinum can be distinguished from these species based pale brownish drab towards the edge to dark greyish brown on brown colonies, the production of pale brown pigment on in reverse; on OA flat, with entire margin; after 8 d and 16 d PDA, and by the predominance of the type II phialides. This white to pale olive-grey above. Colonies producing pale brown species often produced microcyclic conidia under slide culture pigment on PDA. conditions. Specimens examined. Spain, Mallorca, Sant Llorenç del Cardassar, isolated from Prunus dulcis trees, June 2009, D. Gramaje, CBS H-20509 DISCUSSION holotype, culture ex-type CBS 128570 = Psp-3; Mallorca, Sant Llorenç del Cardassar, isolated from Prunus dulcis trees, June 2009, D. Gramaje, Psp-1 This study shows the high incidence and diversity of fungal herb, CBS H-20507, culture Psp-1; Mallorca, Sant Llorenç del Cardassar, trunk pathogens associated with wood decay symptoms on isolated from Prunus dulcis trees, June 2009, D. Gramaje, Psp-2 herb, CBS almond trees on the island of Mallorca. These include species H-20508, culture Psp-2; Mallorca, Sant Llorenç del Cardassar, isolated from of Botryosphaeriaceae, Eutypa lata, Phaeoacremonium irani- Prunus dulcis trees, June 2009, D. Gramaje, Psp-4 herb, CBS H-20510, anum, Phomopsis amygdali and the new species described culture Psp-4. here, namely Collophora hispanica and Phaeoacremonium Notes — The phylogeny of the combined sequence dataset amygdalinum. These species could be distinguished based showed that Pm. amygdalinum does not group with any of the on DNA sequence data and unique morphological characters. a b c e f g h i d n l m j k u v r o p q s t w Fig. 5 Phaeoacremonium amygdalinum. a–c. Sixteen-day-old colonies incubated at 25 °C on MEA (a), PDA (b) and OA (c); d–j. aerial structures on MEA; d–f. conidiophores with polyphialides (indicated by arrows); g, h. type III phialides; i. type III and type II phialides (indicated by arrow); j. type I phialides; k–t. aerial structures by using slide culture technique; k–m. branched conidiophores and type I phialides (indicated by arrows); n–p. type II phialides; q. type I phialides; r–s. Microcyclic conidiation; t. conidia; u–w. structures on the surface of and in MEA; u–v. adelophialides with conidia; w. conidia. — Scale bars: d = 10 µm; scale bar for d applies to d–w.