Characterization of Cadophora luteo-olivacea and C. melinii isolates obtained from grapevines and environmental samples from grapevine nurseries in Spain

D. Gramaje et al. S112 Phytopathol. Mediterr. (2011) 50 (Supplement), S112−S126 Corresponding author: D. Gramaje Fax: +34 963879269. E-mail: [email protected] Introduction The genus Cadophora (C.) Lagerberg & Melin was established by Lagerberg et al. (1927), with the type species Cadophora fastigiata Lagerberg & Melin, which causes stains in softwood timber. Melin and Nannfeldt (1934) added five new species to the genus: C. americana Nannf., C. obscura Nannf., C. lagerbergii Melin & Nannf., C. melinii Nannf. and C. richardsiae Nannf. Davidson (1935) described two more species, isolated from stained wood or pulpwood products: C. brunnescens R.W. Davidson and C. repens R.W. Davidson. Subsequently, Conant (1937) determined that Phialophora (P.) Medlar and Cadophora were congeneric and transferred the eight species of Cadophora to Phialophora. This researcher also considered C. americana to be a synonym of P. verrucosa Medlar. Since then a very broad concept of Phialophora has been retained, even in the major revision by Schol-Schwarz (1970). Gams (2000) proposed that the phialophora-like anamorphs with affinities to the Dermateaceae in the Helotiales, such as Mollisia (Fr.) P. Karst. and Pyrenopeziza Fuckel, should be accommodated in the Cadophora, the species of which have pale to hyaline collarettes on top of their phialides with the vegetative hyphae more or less pigmented. This author suggested the following binomials for the genus Cadophora: C. fastigiata, C. melinii, C. repens and C. malorum (Kidd & Beaum.) W. Gams. Harrington and McNew (2003) used morKey words: Cadophora species, grapevine nurseries, Vitis vinifera, young vineyards. Summary. Fifty-eight Cadophora luteo-olivacea and three C. melinii isolates were recovered from grapevines showing black vascular streaking and decline symptoms characteristic of Petri disease, and from different stages of the grapevine nursery process in Spain. The isolates were studied by means of phenotypical characterization, DNA analysis and pathogenicity tests. The morphological characters studied included conidiophore, phialide and conidial morphology. Colony characters and pigment production on MEA, PDA and OA were also examined. Phenotypical data were subjected to cluster analysis, which clearly separated C. luteo-olivacea isolates into four groups. Mating tests were performed on all possible combinations for each Cadophora species but no sexual fruiting bodies were produced. Partial sequences of the nuclear ribosomal internal transcribed spacer (ITS), beta-tubulin (BT) and the elongation factor 1a (EF) were analysed, but no genetic variation occurred within the C. luteo-olivacea isolates or within the C. melinii isolates in any of the regions studied. Pathogenicity tests were conducted on 1-year-old grapevine cuttings of four different rootstocks using four C. luteo-olivacea isolates and one isolate of C. melinii. All Cadophora isolates except the C. melinii isolate caused significantly longer lesions in the xylem of grapevine rootstocks than in the controls. DaviD GRaMaJE1, LizEL MOSTERT2 and JOSEp aRMENGOL1 1 Instituto Agroforestal Mediterráneo, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022, Valencia, Spain 2 Department of Plant Pathology, University of Stellenbosch, Private Bag X1, Stellenbosch 7602, South Africa Characterization of Cadophora luteo-olivacea and C. melinii isolates obtained from grapevines and environmental samples from grapevine nurseries in Spain S113 Vol. 50, Supplement, 2011 D. Gramaje et al. Cadophora luteo-olivacea and C. melinii in Spain phology and rDNA sequences to determine which phialophora-like species might have affinities to the Helotiales, and to confirm the connection of Cadophora to the Dermateaceae. They found that the pigmentation of the species studied was often quite variable between species, proposed new combinations in Cadophora and also reviewed the taxonomy of the recognized species. The genus Cadophora then comprised C. fastigiata (the type species), C. malorum, C. melinii, C. lagerbergii, C. finlandia (C.J.K. Wang & H.E. Wilcox) T.C. Harr. & McNew, C. gregata (Allington & D.W. Chamb.) T.C. Harr. & McNew, and C. luteo-olivacea (F.H. Beyma) T.C. Harr. & McNew. The inclusion of P. repens R.W. Davidson in the Cadophora group was not supported by ITS sequence analysis. P. atra F.H. Beyma and C. heteroderae F.H. Beyma were synonymized with C. malorum, and P. goidanichii Delitala with C. luteo-olivacea by Harrington and McNew (2003). The known Cadophora species and their relatives occur in many habitats such as decaying wood (Nilsson, 1973; Morrell and Zabel, 1985; Blanchette et al., 2004; Held et al., 2005; Arenz et al., 2006), soil (Kerry, 1990; Aislabie et al., 2001; Arenz et al., 2006; Hujslová et al., 2010) or plants. Cadophora species on plants are mainly associated with decay symptoms in vines and fruit-rotting in kiwi [Actinidia deliciosa (A. Chev.) C.F. Liang & A.R. Ferguson var. deliciosa] (Di Marco et al., 2004; Johnston et al., 2005; Haegi et al., 2006; Riccioni et al., 2007; Di Marco and Osti, 2008; Prodi et al., 2008). Species of Cadophora have also been recovered from grapevines showing decline symptoms in South Africa, Pennsylvania, California and Uruguay (Halleen et al., 2005; Overton et al., 2005; Rooney-Latham, 2005; Abreo et al., 2008). Halleen et al. (2007) confirmed that C. luteo-olivacea is a vascular pathogen of grapevines in South Africa and can also be recovered from healthy nursery cuttings. Recently, Manning and Mundy (2009) have isolated C. luteo-olivacea and C. melinii from esca-diseased grapevines in New Zealand. Over the last few years, Cadophora spp. have frequently been isolated from nursery grapevines and Petri-diseased grapevines in Spain. The species identity of these isolates was unknown prior to this study. Limited information is available on the phenotypical and molecular variability of Cadophora isolates obtained from grapevine and on their role in the decline of young vines. Therefore, the objective of this research was to study these Cadophora isolates by means of phenotypical characterization, DNA analysis and pathogenicity tests. Materials and methods Fungal isolates In this study we used 58 C. luteo-olivacea and three C. melinii isolates representative of different locations in Spain and recovered in 2007 and 2008 (Table 1). Some of the isolates were recovered from vines in young vineyards, others from planting material showing black vascular streaking and decline symptoms characteristic of Petri disease. Sections were cut from affected areas, washed under running tap water, surface-disinfected for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small pieces of discoloured or decayed tissue were plated on malt extract agar (MEA) (Oxoid Ltd., Basingstoke, Hants, England) supplemented with 0.5 g L-1 streptomycin sulphate (MEAS) (Sigma-Aldrich, St. Louis, MO, USA). Plates were incubated for 10–15 days at 25ºC in the dark. Additionally, some isolates were obtained from nursery samples at three stages of the propagation process: samples from pre-grafting hydration tanks, washings from scissors and washings from grafting tools. Approximately 100 mL of water was sampled from tanks in which cuttings had been soaked for 72 hours. Scissors and grafting tools were washed with a 0.2% sterile solution of Tween-20, and 30 to 60 mL of every washing was placed in a sterile tube. Water samples from the hydration tanks, washings from the scissors and washings from the grafting tools were filtered as described by Eskalen and Gubler (2001). Water samples (10 mL) were forcibly passed through 5 µm and 0.45 µm pore-size syringe filters (Sartorius Stedium Biotech, Göttingen, Germany) attached in tandem to sterile 20 mL syringes (BD Biosciences, Madrid, Spain). The 5 µm filters trapped debris and large fungal spores. The 0.45 µm filters trapped small particulates and smaller spores, including spores of Cadophora species. The filters were rinsed with 4 mL of sterile water to backwash any trapped spores and collected in microfuge tubes. Aliquots of 1 mL were then spread Phytopathologia Mediterranea D. Gramaje et al. S114 Table 1. Isolation details and GenBank accession numbers of the Cadophora isolates studied. Species/ Isolate Geographical origin GenBank accessionsb Year Town Province Source of collectionaITS β-tubulin EF C. luteo-olivacea Clo-1 2007 Ayelo Valencia Tempranillo/110 R HQ661084 HQ661054 HQ661069 Clo-2, CBS 128571 2007 Ciudad Real Ciudad Real Tempranillo/110 R HQ661085 HQ661055 HQ661070 Clo-3 2007 Ciudad Real Ciudad Real Tempranillo/110 R Clo-4 2007 Ayelo Valencia Grafting machine HQ661086 HQ661056 HQ661071 Clo-5, CBS 128572 2007 Ayelo Valencia Hydration tank Clo-6 2007 Ciudad Real Ciudad Real Tempranillo/110 R Clo-7, CBS 128573 2007 Ciudad Real Ciudad Real Tempranillo/110 R HQ661087 HQ661057 HQ661072 Clo-8 2007 Ayelo Valencia Hydration tank Clo-9 2007 Ayelo Valencia Grafting machine Clo-10 2007 Ayelo Valencia Pruning scissors Clo-11 2007 Ayelo Valencia Grafting machine Clo-12 2007 Ayelo Valencia Grafting machine Clo-13 2007 Ayelo Valencia Grafting machine Clo-14 2007 Ayelo Valencia Hydration tank Clo-15, CBS 128574 2007 Ayelo Valencia Hydration tank HQ661088 HQ661058 HQ661073 Clo-16 2007 Ayelo Valencia Grafting machine Clo-17 2007 Ayelo Valencia Grafting machine Clo-18, CBS 128575 2007 Ciudad Real Ciudad Real Tempranillo/110 R HQ661089 HQ661059 HQ661074 Clo-19 2007 Olivenza Badajoz Syrah Clo-20 2007 Requena Valencia Tempranillo/110 R Clo-21 2007 Ayelo Valencia 1103 P Clo-22 2007 Ayelo Valencia 1103 P HQ661090 HQ661060 HQ661075 Clo-23 2007 Ayelo Valencia 1103 P Clo-24 2007 Ayelo Valencia 1103 P Clo-25 2007 Ayelo Valencia 1103 P Clo-26 2007 Ayelo Valencia 1103 P Clo-27 2007 Ayelo Valencia 1103 P Clo-28 2007 Ayelo Valencia 1103 P HQ661091 HQ661061 HQ661076 Clo-29 2007 Ayelo Valencia 1103 P Clo-30 2007 Ayelo Valencia 1103 P Clo-31 2007 Ayelo Valencia 1103 P Clo-32 2007 Ayelo Valencia 110 R Clo-33, CBS 128576 2007 Ayelo Valencia 110 R HQ661092 HQ661062 HQ661077 Clo-34, CBS 128577 2007 Ayelo Valencia 110 R Clo-35 2007 Ayelo Valencia 110 R Clo-36 2007 Ayelo Valencia 110 R Clo-37 2007 Ayelo Valencia 110 R Clo-38 2007 Ayelo Valencia 110 R Clo-39 2007 Ayelo Valencia 110 R Clo-40 2007 Socuéllamos Ciudad Real Garnacha/110 R HQ661093 HQ661063 HQ661078 Clo-41 2007 Socuéllamos Ciudad Real Airen/1103 P Clo-42 2008 Ayelo Valencia 1103 P Clo-43 2008 Ayelo Valencia 110 R Clo-44 2008 Ayelo Valencia 110 R Clo-45 2008 Ayelo Valencia 110 R Clo-46, CBS 128578 2008 Ayelo Valencia 110 R HQ661094 HQ661064 HQ661079 Clo-47 2008 Ayelo Valencia 110 R continues S115 Vol. 50, Supplement, 2011 D. Gramaje et al. Cadophora luteo-olivacea and C. melinii in Spain on a semi-selective culture medium adapted from Tello et al. (2009) [2% MEA amended with 1.5 g L-1 streptomycin sulphate; 0.02 g L-1 Folpet (Aragonesas Agro, Madrid, Spain); and 0.015 g L-1 ampicillin (Laboratorio Reig Jofré S.A., Sant Joan Despí, Spain)]. Plates were air dried under a laminar flow hood with the lids partially lifted for 30 min. Plates were then sealed with parafilm and incubated at 25ºC in darkness for one month. Petri dishes were inspected daily for mycelial growth and reisolation. All isolates were transferred to potato dextrose agar (2% PDA; Biokar-Diagnostics, Beauvais, France). The isolates were single-spored by serial dilution prior to use (Dhingra and Sinclair, 1995) and were stored in a 15% glycerol solution at -80ºC in 1.5 mL cryovials. Reference isolates of C. luteo-olivacea (CBS 141.41) and C. melinii (CBS 268.33) were obtained from the Centraalbureau voor Schimmelcultures in the Netherlands (CBS) (Table 1). Morphological and cultural characterization Morphological characters used in this study included conidiophore, phialide and conidial morphology. Colony characters and pigment production were determined on MEA, PDA (2% PDA; Biolab, Midrand, South Africa) and oatmeal agar (OA; 60 g oatmeal; 12.5 g agar; Difco, Osi, Maurepas, France) (Gams et al. 2007) incubated at 25ºC for 8 and 16 d. Colony colours were determined using the colour charts of Rayner (1970). Cardinal temperatures for growth were determined by incubating three MEA plates per isolate in the dark at temperatures ranging from 5 to 40ºC at 5ºC intervals. Radial growth was measured after 8 d at 25ºC on MEA. Aerial mycelium of colonies cultivated on MEA were inspected under the microscope or by a slide culture technique (Arzanlou et al., 2007). Slide cultures were set up in Petri dishes containing 2 mL of sterile water, into which a U-shaped glass rod was placed, extending above the water surface. A block of an actively growing fungal colony, approx. 1 cm square, was placed on a sterile microscope slide, covered with a somewhat larger, sterile glass covering slip, and incubated in a moist chamber. Fungal sporulation was monitored, and when it was optimal, images Clo-48 2008 Ayelo Valencia 110 R Clo-49 2008 Ayelo Valencia 110 R Clo-50 2008 Ayelo Valencia 110 R Clo-51 2008 Ayelo Valencia 110 R Clo-52 2008 Ayelo Valencia 110 R Clo-53 2008 Ayelo Valencia 110 R Clo-54 2008 Cariñena Zaragoza Syrah/110 R Clo-55 2008 Sa Punta Mallorca Merlot/110 R HQ661095 HQ661065 HQ661080 Clo-56 2008 Sa Punta Mallorca Merlot/110 R Clo-57 2008 Petra Mallorca Manto Negro Clo-58 2008 Petra Mallorca Manto Negro CBS 141.41 1939 Munksund Sweden Waste water of Schleiferei Byske C. melinii Cme-1 2007 Olivenza Badajoz Syrah HQ661096 HQ661066 HQ661081 Cme-2 2007 Olivenza Badajoz Syrah HQ661097 HQ661067 HQ661082 Cme-3 2007 Olivenza Badajoz Syrah HQ661098 HQ661068 HQ661083 CBS 268.33 1933 - Sweden - a Isolates were collected from Vitis vinífera plants (indicated as rootstock or scion/rootstock combinations) or from different stages in the grapevine nursery process (hydration tanks, grafting machines or pruning scissors). b The sequences of a selection of isolates have been deposited in GenBank. Species Geographical origin GenBank accessionsb Isolate Year Town Province Source of collectionaITS β-tubulin EF Table 1. Continued Phytopathologia Mediterranea D. Gramaje et al. S116 of the sporulating fungus were photographed with a Nikon camera system (Digital Sight DXM 1200, Nikon Corporation, Japan). Slides were mounted in lactic acid, and 30 measurements (×1000 magnification) of the relevant structures were made. The 5th and 95th percentiles were defined for all measurements with the extremes given in parentheses. Mating tests with all C. luteo-olivacea and C. melinii isolates were performed as described by Mostert et al. (2003). Cadophora spp. isolates were grown on MEA plates for 2 weeks, using 5 plates per isolate. Conidia were dislodged from the agar surface with a glass rod, and suspensions were prepared in 5 mL sterile distilled water. Two aliquots of 100 mL each, representing two different isolates, were pipetted onto twice-autoclaved pieces of grapevine canes placed on 2% water agar plates (GWA) (Difco, France). Each Cadophora species was mated in all possible combinations. Controls consisted of a 200 mL aliquot of one isolate only. Plates were incubated at 20ºC and 25ºC under continuous white light (Philips TLD18W/33) and microscopically examined at weekly intervals for fruiting bodies. Each pair of isolates was replicated three times. The experiment was repeated. To group C. luteo-olivacea isolates by their phenotypical characters, cluster analysis was conducted on the Euclidean distance matrix with the unweighted pair group method based on arithmetic averages (UPGMA). The morphological characters used in the analysis were: colony colour on MEA and PDA as nominal variables; the maximum number of guttules in conidia and the optimum growth temperatures as symmetric-binary variables; conidophores, phialides, collarettes and conidia measurements and radial growth as interval variables; and yellow pigmentation on PDA and OA as asymmetric-binary variables. The clustering method and similarity coefficient were tested using the NCSS 2007 procedure (Statistical Solutions Ltd, Cork, Ireland). Molecular analysis Fungal mycelium and conidia were scraped from pure cultures grown on PDA for 2 weeks at 25ºC in the dark and ground to a fine powder with liquid nitrogen using a mortar and pestle. Total DNA was extracted using the E.Z.N.A. Plant Miniprep Kit (Omega Bio-tek, Norcross, GA, USA) following manufacturer’s instructions. DNA was visualized on 0.7% agarose gels (agarose D-1 Low EEO, Conda, Madrid, Spain) stained with ethidium bromide. The DNA was stored at –20ºC. The internal transcribed spacers 1 and 2 as well as the 5.8S ribosomal RNA gene of approximately 595 bp for C. luteo-olivacea isolates and 555 bp for C. melinii isolates was amplified with the primers ITS1 and ITS4 (White et al., 1990). Oligonucleotide primers BT1a and BT1b were used to amplify the partial β-tubulin (BT) gene of approximately 480 bp for the C. luteo-olivacea isolates and 300 bp for the C. melinii isolates (Petit and Gubler, 2005). Amplification of the partial translation elongation-factor 1a (EF) gene of approximately 555 bp for C. luteo-olivacea isolates and 525 bp for C. melinii isolates was done with the primers EF1688F and EF1-1251R (Alves et al., 2008). Each PCR reaction contained 1× PCR buffer, 2.5 mM MgCl2, 200 µM each dNTP, 0.4 µM of each primer, 1 U of DNA Taq polymerase (Dominion MBL, Córdoba, Spain), and 1 µL of template DNA. The PCR reaction mix was adjusted to a final volume of 25 µL with water (Chromasolv Plus, Sigma-Aldrich, Steinheim, Germany). PCR amplifications were performed on a Peltier Thermal Cycler-200 (MJ Research, Waltham, USA). The program consisted of an initial step of 4 min at 94°C, followed by 30 cycles (35 cycles for BT) of denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and an elongation at 72°C for 1.5 min. A final extension was performed at 72°C for 10 min. The PCR products were visualized in 1.5% agarose gels. A 100 bp ladder was used as a molecular weight marker (Dominion MBL). PCR products were purified with the High Pure PCR product purification kit (Roche Diagnostics, Germany) and sequenced in both directions by the DNA Sequencing Service of the Universidad Politécnica de Valencia-CSIC. The sequences were aligned using the MAFFT sequence alignment program version 6 (Katoh and Toh, 2008) followed by manual adjustments of the alignments in the Sequence Alignment Editor v. 2.0a11 (Rambaut, 2002). Reference sequences of the ex-type cultures of the relevant Cadophora species and reference sequences used by Harrington and McNew (2003) were obtained from GenBank. Phylogenetic analysis of the aligned S117 Vol. 50, Supplement, 2011 D. Gramaje et al. Cadophora luteo-olivacea and C. melinii in Spain sequence data was performed with Phylogenetic analysis using parsimony (PAUP) v. 4.0b10 (Swofford, 2003). Alignment gaps were treated as missing data and all characters were unordered and of equal weight. Any ties were broken randomly when encountered. For parsimony analysis, alignment gaps were treated as missing data and all characters were unordered and of equal weight. Maximum parsimony analysis was performed using the heuristic search option with 10 random simple taxon additions and tree bisection and reconstruction (TBR) as the branch-swapping algorithm with the option of saving no more than 10 trees with a score greater than or equal to 5 (Harrison and Langdale, 2006). Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. The robustness of the trees obtained was evaluated by 1000 bootstrap replications. Tree length (TL), the consistency index (CI), the retention index (RI) and the rescaled consistency index (RC) were calculated. Sequences derived in this study were lodged at GenBank (Table 1) and the alignment and phylogenetic tree in TreeBASE (number S10849). Pathogenicity tests One isolate of C. luteo-olivacea of each of the four morphological groups defined by cluster analysis and one isolate of C. melinii were used. Pathogenicity tests were conducted on 1-year-old grapevine cuttings of four rootstocks (140 Ruggeri, 161-49 Couderc, 1103 Paulsen and 110 Richter). In total 96 dormant cuttings of each rootstock were cut into equal lengths containing four to five buds, and then hot-water treated at 53ºC for 30 min to eliminate any fungal trunk pathogens (Gramaje et al., 2009). In order to enhance callusing and rooting, dormant cuttings were buried in sterilized peat moss in plastic boxes, and placed in a callusing room at 25ºC and 100% humidity for 4 weeks. After callusing and rooting, the cuttings were wounded between the two upper internodes with a 5 mm cork borer. A 5 mm mycelium agar plug from a 2-weekold culture was placed in the wound. Wounds were wrapped with parafilm. Eight cuttings per fungal isolate were used for each rootstock. Eight cuttings of each rootstock were inoculated with 5 mm noncolonized PDA agar plugs from two different plates for negative controls. Inoculated cuttings were planted immediately in individual pots, placed in a greenhouse at 25ºC and watered every 3 days or as needed. Plants were arranged in a completely randomized design. The experiment was repeated. Cuttings were collected after 14 weeks and inspected for lesion development. The extent of vascular discolouration was measured upwards and downwards from the inoculation point. Additionally, shoot dry weight was evaluated for the shoots above the inoculation point. Small pieces (0.5 to 1 cm) of necrotic tissue from the edge of each lesion were cut and placed on MEAS in an attempt to recover the inoculated fungi and fulfill Koch’s postulates. Fungi were identified as previously described. Lesion length and shoot dry weight data were subjected to analysis of variance using SAS v. 8.1 (SAS Institute, Cary, North Carolina, USA) and Student’s t-test for Least Significant Difference was calculated at the 5% significance level to compare the treatment means for the different fungal species and grapevine rootstocks. Results Morphological characterization The isolates were identified as C. luteo-olivacea or C. melinii based on morphological characters (Cole and Kendrick, 1969; Schol-Schwarz, 1970; Gams, 2000; Harrington and McNew, 2003). Conidiophore, phialide and conidial morphology were similar in all C. luteo-olivacea isolates (Figure 1). Aerial mycelium on MEA consisted of branched, septate hyphae occurring singly or in bundles of up to 5; hyphae tuberculate with warts up to 3 µm diam, verruculose to smooth, medium brown and 2.5-4 µm wide. Conidiophores were mostly short, usually unbranched, arising from aerial or submerged hyphae, erect to flexuous, up to 7-septate, pale brown, (9–)11.5–65(–69) (av.=26) µm long and 2–2.5 (av.=2) µm wide. Phialides were terminal or lateral, mostly monophialidic, smooth to verruculose, hyaline, with collarettes 2.5–3 µm long and 2–2.5 µm wide, mostly cylindrical, some elongateampulliform, attenuated at the base or navicular, (4–)8.5–26(–31) × 1.5–3 (av.=5×2) µm. Conidia were hyaline, with up to 3 guttules, ovoid or oblong ellipsoidal, (3–)3.5–7.5×2–3 (av.=4.5×2) µm, L/W=2.1. Some differences were found in colony characters and pigment production of the C. luteo- Phytopathologia Mediterranea D. Gramaje et al. S118 olivacea isolates studied (Figure 2 and Table 2). Colonies on MEA were flat, felty, with even edge and varying in colour from white to greenish-olivaceous (23’’’b). Colonies on PDA were flat, felty and cottony in the middle, with an even edge and varying in colour from white to grey-olivaceous (21’’’’i). Colonies on OA were flat, felty and cottony in the middle, with an even edge and varying in colour from white to olivaceous-buff (21’’’d). For all isolates, colonies reached a radius of 15.5–17.2 mm after 8 d at 25ºC. The minimum temperature for growth was 5ºC, optimum 20–25ºC and maximum 30ºC. Some C. luteo-olivacea isolates produced yellow pigmentation on PDA and/or MEA plates. The C. luteo-olivacea isolates were different from the type description since they had predominantly monophialidic phialides, which were thinner. The conidial shape and the diversity of the colony colours on the media studied also differed. Van Beyma (1940) described colonies of C. luteoolivacea as having thick phialides often in bunches, conidia ellipsoid or elongate, and colonies oliveyellow with white mycelium in the middle. A comparison based on the morphological and colony characters of the 58 C. luteo-olivacea isolates studied are shown in a dendrogram (Figure 3). Analysis revealed that isolates were distributed in two clusters at a Euclidean distance with a cut off value of 1.26. Four distinct groups were formed. Cluster A comprised group I. Cluster B was divided into three groups, II, III and IV. The cophenetic correlation value was 0.9419. Group I contained 6 isolates. This group was the most diverse, with a dissimilarity ranging from 0 to 0.92. Group II contained 21 isolates and had a dissimilarity ranging from 0 to 0.88. Isolate CBS 141.41 was included in this group and joined the other isolates of the same group at a dissimilarity of 0.88. Group III contained 23 isolates with a dissimilarity ranging from 0 to 0.81. Group IV contained 9 isolates and was the most homogeneous (dissimilarity ranging from 0 to 0.51). Colony characters and pigment production were the most useful parameters distinguishing these groups (Figure 2 and Table 2). The majority of C. luteo-olivacea isolates were from grapevines. Seven isolates were from the grafting machines, four from the hydration tank and one from pruning scissors. The morphological and colony characters of C. melinii isolates are shown in Figure 4. Aerial mycelium on MEA consisted of branched, septate hyphae occurring singly or in bundles of up to 13; hyphae tuberculate with warts up to 2.5 µm diam, verruculose to smooth, medium brown and 3–3.5 µm wide. Conidiophores mostly short, usually branched, arising from aerial or submerged Figure 1. Cadophora luteo-olivacea. a–t, Aerial structures on MEA; a-d, conidiophores; e-f, hyphal swellings; g-s, conidiophores and phialides; t, conidia. Scale bar: a–t=10 µm. Scale bar for a also applies to b–t. S119 Vol. 50, Supplement, 2011 D. Gramaje et al. Cadophora luteo-olivacea and C. melinii in Spain hyphae, erect to flexuous, up to 4-septate, pale brown, (11–)11.5–50.5(–65) (av.=24.5) µm long and 2–3 (av.=2.5) µm wide. Phialides terminal or lateral, mostly monophialidic, smooth to verruculose, hyaline, with 1.5–3.5 µm long, 2–3 µm wide, subcylindrical or navicular collarettes, (4– )5–13.5(–15)×1.5–3(–4) (av.= 8×2.5) µm. Conidia hyaline, with up to 2 guttules, mostly ovoid, (3–)4–5.5×1.5–2.5 (av.=4.5×2) µm, L/W=2.1. Regarding cultural characteristics, colonies reached a radius of 12–14.5 mm after 8 d at 25ºC. The minimum temperature for growth was 5–10ºC, the optimum 20–25ºC and the maximum 30ºC. Colonies on MEA were flat, felty, with an even edge; after 16 d, olivaceous-black (27’’’’k) to greyolivaceous (23’’’’i) from the top, olivaceous-black (27’’’’k) from the bottom. Colonies on PDA were flat, felty, with an even edge; after 16 d, white to olivaceous-buff (21’’’f) from the top and the bottom. Colonies on OA were raised with striating furrows, Figure 2. Cadophora luteo-olivacea. Group 1: a–c. Sixteen-day-old colonies on MEA (a), PDA (b) and OA (c). Group 2: d-f, Sixteen-day-old colonies on MEA (d), PDA (e) and OA (f). Group 3: g-i. Sixteen-day-old colonies on MEA (g), PDA (h) and OA (i). Group 4: j-l. Sixteen-day-old colonies on MEA (j), PDA (k) and OA (l). Phytopathologia Mediterranea D. Gramaje et al. S120 Table 2. Summary of colony colour useful for the identification of each group of Cadophora luteo-olivacea. C. luteo-olivacea group Colony colour on MEA Colony colour on PDA Colony colour on OA Yellow pigmentation on PDA Yellow pigmentation on OA Group 1 Greenish olivaceous (23’’’b) to olivaceous buff (21’’’b) above and in reverse Olivaceous black (27’’’’k) to grey olivaceous (21’’’’i) above and in reverse Olivaceous (21’’k) to olivaceous buff (21’’’d) above No No Group 2 Olivaceous black (25’’’’k) to white towards the margin above and in reverse White to greenish olivaceous (21’’’i) close to the centre White to olivaceous buff (21’’’d) close to the centre above No No Group 3 White to grey olivaceous (21’’’’i) close to the centre above and in reverse White to straw (19’f) above, straw (19’f) topure yellow (21’b) in reverse Olivaceous buff (21’’’d) to straw (21’d) above Yes Yes Group 4 White to oliveaceous black (25’’’’k) close to the centre above and in reverse White to olivaceous buff (23’’’b) close to the centre above and in reverse Greenish olivaceous (23’’’b) to straw (21’f) above No Yes woolly when close to the centre, with an even edge; after 16 d, they were pale olivaceous-grey (21’’’’’d) to olivaceous-black (27’’’’k) from the top. The C. melinii isolates differed from the type description in hyphal colour and in phialide shape. Cole and Kendrick (1969) described colonies of C. melinii as having hyphae light-brown to hyaline, with the phialides subcylindrical or flexuous. A useful feature of C. melinii not observed in that work but mentioned by Cole and Kendrick (1973) and by Domsch et al., (1980) was that the collarettes were inwardly curved. The isolates did not correspond to the type description of C. fastigiata in that very few of the multiple branched or verticillate conidiophores characteristic of C. fastigiata were seen. C. fastigiata also had globose and subglobose conidia. Such conidia were very rarely found in the C. luteo-olivacea and C. melinii isolates. After 2 months of evaluation, no sexual fruiting bodies were produced by crossing C. luteo-olivacea with each other, or C. melinii isolates with each other. Molecular analysis The ITS phylogeny (Figure 5) clearly showed that the isolates were either grouped with the reference sequences of C. luteo-olivacea, with a bootstrap support of 85%, or with the reference sequences of C. melinii, with a bootstrap support of 72%. No BT or EF sequences of any Cadophora species were available on GenBank, therefore, no analyses were made with these sequences. Minor differences were found among the C. luteo-olivacea sequences with 3 nucleotides varying in the ITS region, 3 nucleotides in the BT region, and 2 nucleotides in the EF region. The C. melinii isolates from grapevines were very similar, with 1 nucleotide varying in the ITS region, 2 nucleotides in the BT region, and 2 nucleotides in the EF region. Sequences in the C. melinii clade exhibited more variation when the isolates came from European silver fir or kiwi. Pathogenicity tests The assays on lesion length and on shoot dry weight gave similar results (P=0.4224 and P=0.5180 respectively) so that these results were combined in a single analysis. Analysis of variance on the lesion length of the rootstocks detected a significant treatment effect (P<0.001; ANOVA not shown). All Cadophora isolates except C. melinii caused lesions in the rootstock xylem that were significantly longer than the lesions in the con-