Sandra Alaniz

Characterization of Cylindrocarpon Species Associated with Black Foot Disease of Grapevine in Spain

In this work, 82 Cylindrocarpon isolates associated with black foot disease of grapevines in Spain were studied by means of phenotypical characterization, DNA analyses, and pathogenicity tests. Partial sequences of the beta-tubulin (BT) gene, BT1, were amplified using primers BT1a and BT1b. A unique and conserved 52-bp insertion in the BT1 sequence, which is a specific marker for C. macrodidymum, was found in 56 of the isolates. The rest of the isolates (26) were identified as C. liriodendri. The BT phylogeny grouped all the isolates of each species into two well-supported clades. Phenotypical data were subjected to multivariate factorial analysis. According to this study, the isolates were clearly separated into two groups which were in agreement with BT species identification. C. macrodidymum isolates were differentiated from C. liriodendri by producing fewer conidia, presenting longer and wider macroconidia, and lower growth rate at 5 and 10°C. Selected isolates of each species inoculated onto rooted cuttings of grapevine rootstock cv. 110 R caused typical black foot disease symptoms. This is the first report of C. liriodendri and C. macrodidymum causing black foot disease of grapevines in Spain.

A Multiplex PCR System for the Specific Detection of Cylindrocarpon liriodendri, C. macrodidymum, and C. pauciseptatum from Grapevine

Cylindrocarpon liriodendri and C. macrodidymum are the causal agents of grapevine black foot disease. Recently, a third species, C. pauciseptatum, has been isolated from roots of grapevine showing decline symptoms. Currently, reliable identification of isolates of these species through phenotypical characteristics has not been possible. The polymerase chain reaction (PCR)-based method developed in this study allows a quick and easy detection of Cylindrocarpon spp. associated with grapevine. Three primer pairs annealing to variable, partly species-specific sites of the internal transcribed spacer regions amplified species-specific PCR fragments of different sizes in C. liriodendri, C. macrodidymum, and C. pauciseptatum in a multiplex assay with DNA obtained with both quick and traditional extraction methods. They did not generate any PCR product in other fungal trunk pathogens or contaminants commonly associated with grapevines. When universal fungal ITS primers were used in a nested multiplex PCR, the three primer pairs also detected C. liriodendri, C. macrodidymum, and C. pauciseptatum in total DNA extracted from roots of inoculated grapevines. The designed methods can be used for the diagnosis of these fungi from pure culture or infected grapevines.

First Report of Phaeoacremonium krajdenii Causing Petri Disease of Grapevine in Spain

In September 2009, symptoms of grapevine (Vitis vinifera L.) decline were observed on 3-year-old grapevines in a vineyard in Roquetas de Mar (Almeria Province, southern Spain). Affected vines were weak with reduced foliage and chlorotic leaves. Black spots and dark streaking of the xylem vessels could be seen in cross- or longitudinal sections of the rootstock trunk. Symptomatic plants were collected and sections (10 cm long) were cut from the basal end of the rootstocks, washed under running tap water, surface disinfested for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally and small pieces of discolored tissues were plated onto malt extract agar (MEA) supplemented with 0.5 g liter-1 of streptomycin sulfate. Dishes were incubated at 25 to 26°C in the dark for 14 to 21 days, and all colonies were transferred to potato dextrose agar (PDA). A Phaeoacremonium sp. was consistently isolated from necrotic tissues. Single conidial isolates were obtained and grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (2). Colonies were grayish brown on PDA and dark brown on MEA. Conidiophores were short and unbranched and 11.5 to 46 (25.5) μm long. Phialides were often polyphialidic. Conidia were hyaline, oblong-ellipsoidal or allantoid, 2.5 to 5 (4.2) μm long, and 1 to 1.7 (1.2) μm wide. On the basis of these characters, the isolates were identified as Phaeoacremonium krajdenii L. Mostert, Summerb. & Crous (1,2). DNA sequencing of a fragment of the beta-tubulin gene of the isolate (Pkr-1) using primers T1 and Bt2b (GenBank Accession No. HM637892) matched P. krajdenii GenBank Accession No. AY579330. Pathogenicity tests were conducted using isolate Pkr-1. Ten 1-year-old callused and rooted cuttings of 110 R rootstock grown in pots with sterile peat were wounded at the uppermost internode with an 8-mm cork borer. A 5-mm mycelium PDA plug from a 2-week-old culture was placed in the wound before being wrapped with Parafilm. Ten control plants were inoculated with 5-mm noncolonized PDA plugs. Plants were maintained in a greenhouse at 25 to 30°C. Within 3 months, shoots on all Phaeoacremonium-inoculated cuttings had weak growth with small leaves and short internodes and there were black streaks in the xylem vessels. The vascular necroses that developed on the inoculated plants were 5.5 ± 1.2 cm long, significantly greater than those on the control plants (P < 0.01). Control plants did not show any symptoms. The fungus was reisolated from discolored tissue of all inoculated cuttings, completing Kochs postulates. P. krajdenii has a worldwide distribution, although these reports are from human infections (1). P. krajdenii was first reported as a pathogen of grapevines in South Africa (1). To our knowledge, this is the first report of P. krajdenii causing young grapevine decline in Spain or any country in Europe. References: (1) L. Mostert et al. J. Clin. Microbiol. 43:1752, 2005. (2) L. Mostert et al. Stud. Mycol. 54:1, 2006.

Colletotrichum fructicola is the dominant and one of the most aggressive species causing bitter rot of apple in Uruguay

Forty-one isolates obtained from apple fruit exhibiting the typical symptoms of bitter rot in Uruguay were characterized based on molecular, phenotypic and pathogenicity data. Four species were identified based on the analysis of ITS, GAPDH and BTUB2 genes. The dominant species was Colletotrichum fructicola (33/41 isolates) followed by C. theobromicola (6/41 isolates), both belonging to the C. gloeosporioides species complex. The other species, with one isolate each, were C. melonis and Colletotrichum sp. both belonging to the C. acutatum species complex. Surprisingly, C. gloeosporioides and C. acutatum, commonly reported as the cause of bitter rot, were not found. Phenotypical traits were useful to assign the isolates to a species complex, but not to identify phylogenetic species. Isolates of all four species were able to cause the typical bitter rot disease, but the two species of the C. gloeosporioides species complex were the most aggressive based on lesion size.

Effect of dsRNA on growth rate and reproductive potential of Monosporascus cannonballus

The effect of double stranded RNA (dsRNA) infection on growth rate and the reproductive potential of Monosporascus cannonballus was studied in 21 isolates collected in cucurbit growing areas of Spain and Tunisia. The isolates were incubated on potato dextrose agar (PDA) under different conditions of temperature, pH, and water potential (Ψ(s)). They showed optimal growth temperatures over the range of 27-34°C and perithecia formation was obtained mainly at 25 and 30°C, although some isolates were able to produce perithecia at 35°C. All isolates were able to produce perithecia in a broad range of pHs (4-8). Regarding the effect of Ψ(s,) the isolates were more tolerant to grow on KCl than on NaCl. For each solute, radial growth decreased progressively as Ψ(s) decreased and was severely limited at -5.0 to -6.0MPa. Perithecia formation was highest at -0.5MPa, decreased at -1.0MPa and occurred just in some isolates at -2.0MPa. Nine of the M. cannonballus isolates harboured dsRNA with 2-6 bands each and a size range of 1.9-18.0Kb. Phenotypical data were subjected to multivariate factorial analysis. Most of the isolates clustered in two groups corresponding with the presence/absence of dsRNA elements. Isolates without detectable dsRNA produced more perithecia. However, isolates with dsRNA produced lower number of perithecia depending on the pH, Ψ(s,) or solute used. These results improve our understanding of the behaviour and growth of this pathogen in soil, and can be useful to implement effective disease control.

Genetic Structure of Colletotrichum fructicola Associated to Apple Bitter Rot and Glomerella Leaf Spot in Southern Brazil and Uruguay

Colletotrichum fructicola is the main species causing apple bitter rot (ABR) and Glomerella leaf spot (GLS) in southern Brazil, and ABR in Uruguay where GLS remains unnoticed. Thus, this work aimed to determine the genetic structure of C. fructicola isolates of both the countries. A total of 28 out of 31 Brazilian isolates (90.3%) caused typical symptoms of GLS, while only 6 of 25 Uruguayan isolates (24.0%) originating from fruits were able to infect leaves, but causing atypical symptoms. Both populations showed similar levels of Neis gene diversity (h = 0.088 and 0.079, for Brazilian and Uruguayan populations, respectively), and Bayesian cluster analysis inferred two genetic clusters correlated with the geographical origin of isolates. A principal coordinates analysis scatter plot and an unweighted pair group method with arithmetic mean-based dendrogram also grouped Brazilian and Uruguayan isolates into two groups. By pairwise comparison of nitrate-nonutilizing (nit) mutants with a proposed set of testers, all Uruguayan isolates were grouped into a unique vegetative compatibility group (namely VCG 1), while Brazilian isolates were grouped into four VCGs (VCG 1 to 4). Brazilian and Uruguayan populations of C. fructicola were found to be genetically distinct. Our results suggest that isolates of C. fructicola from Brazil capable of causing GLS and ABR arose independently of those from Uruguay. Possible causes leading to the evolutionary differences between populations are discussed.

First report of Colletotrichum karstii causing glomerella leaf spot on apple in Santa Catarina State, Brazil.

Glomerella leaf spot (GLS) is an emerging disease of apple (Malus domestica Borkh.) that has been reported in regions with a humid subtropical climate, such as southern Brazil, the southeastern United States, and more recently eastern China. GLS is favored by high humidity and temperatures between 23 and 28°C and can result in extensive defoliation when the severity is high. The disease was first reported 1988 in Brazil on cvs. Gala and Golden Delicious in orchards in Paraná State (3), but now is widespread in the countrys producing areas. Two Colletotrichum species of different complexes have been associated with GLS, C. gloeosporioides (Penz.) Penz. & Sacc. and its sexual stage Glomerella cingulata (Stoneman) Spaulding & Scherenk, and C. acutatum J. H. Simmonds, although GLS is more commonly associated with the former. In the summer of 2012, necrotic spots were observed on apple leaves (cv. Gala) in Santa Catarina state, Brazil. The first symptoms were reddish-brown spots, evolving to small necrotic lesions 1 to 10 mm long at 7 to 10 days after symptoms were first noted. Pure cultures were obtained by monosporic isolation and grown on PDA at 25°C and with a 12-h photoperiod under fluorescent light. The color of the upper surface of the colony varied from white to gray and the reverse was pink. The conidia length and width ranged from 9.1 to 17.1 μm ( = 12.8) and from 2.9 to 6.8 μm ( = 4.9), respectively, and were cylindrical, hyaline, and straight. After germination, conidia formed oval or circular appressoria measuring between 4.0 and 10.0 ( = 6.3) × 3.0 and 9.0 ( = 5.7). To confirm pathogenicity, susceptible apple seedlings (cv. Gala) were inoculated with a suspension of 1 × 106 conidia.mL-1. Seedlings sprayed with sterile distilled water served as controls. Seedlings were incubated in a moist chamber at 25°C and 100% RH for 48 h. First symptoms appeared 4 days after inoculation and were similar to those observed in the field. The control treatment remained symptomless. The pathogen was reisolated from lesions, confirming Kochs postulates. Fungus was molecularly characterized by sequencing the internal transcribed spacer (ITS) rDNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the nucleotide sequence was deposited in the GenBank database (KC876638 and KC875408). C. karstii, considered as part of the C. boninense species complex (1), was identified with 100% sequence homology. This species was previously reported in China (4), Thailand, and the United States, affecting Orchidaceae plants (2), and in Brazil it has been reported affecting Carica papaya, Eugenia uniflora, and Bombax aquaticum (1). To our knowledge, this is the first report of C. karstii causing GLS on apple in Brazil. The development of pre-harvest management practices may be warranted to manage this disease. References: (1) U. Damm et al. Stud. Mycol. 73:1, 2012. (2) I. Jadrane. Plant Dis. 96:1227, 2012. (3) T. B. Sutton. Plant Dis. 82:267, 1998. (4) Y. Yang. Cryptogamie Mycologie 32:229, 2011.

First Report of Colletotrichum acutatum and C. fragariae Causing Bitter Rot of Apple in Uruguay

Almost 50% of deciduous fruit produced in Uruguay are apples and bitter rot is ubiquitous in the apple-production regions in Uruguay. In rainy and hot seasons (25 to 32°C by day), severe outbreaks of bitter rot occur. In summer 2010, when apple rot incidence reached 70% in some orchards, fruit with typical symptoms of bitter rot were collected from orchards in the south-central region, the main apple-production area. Symptoms included 0.5 to 6.0 cm in diameter, circular, sunken, light brown-to-brown lesions on the fruit surface that contained black, pinhead-sized fruiting structures that produced orange-to-brown conidial masses under high relative humidity. Each lesion progressed to the core of the fruit in a V-shaped pattern. Single-conidial isolates from lesions were examined morphologically (3), and based on sequences of the internal transcribed spacer (ITS) rDNA determined using ITS1/ITS4 primers (4), three species were identified: Colletotrichum acutatum with white-to-pale orange colonies and one-celled, hyaline, fusiform to cylindrical conidia that averaged 14.5 (9.3 to 17.8) × 5.0 (6.9 to 4.0) μm (isolates C11 and C18, GenBank Nos. JN413081 and JN413082, respectively); C. fragariae with white-to-pale gray and/or dark gray colonies and one-celled, hyaline, cylindrical to fusiform conidia that averaged 20.5 (14.3 to 22.9) × 6.0 (4.6 to 7.6) μm (isolate C15 and C37, GenBank Nos. JN413083 and JN413084, respectively); and C. gloeosporioides with white-to-pale gray or gray colonies and one-celled, hyaline, cylindrical to fusiform conidia that averaged 16.5 (13.1 to 20.3) × 6.5 (3.7 to 7.6) μm (isolates C5 and C29, GenBank Nos. JN413079 and JN413080) when grown on potato dextrose agar (PDA) at 25°C. To confirm pathogenicity, two isolates of each Colletotrichum spp. were inoculated onto mature, asymptomatic fruit of cv. Pink Lady (eight fruit per isolate). Each fruit was surface disinfested with 70% ethanol, wounded with a sterile needle, and inoculated with 10 μl of a spore suspension (5 × 105 conidia/ml) of the appropriate isolate. Eight control fruit were each inoculated with 10 μl of sterile water. Inoculated fruit and the control fruit were placed in plastic bags (eight fruit per bag) and incubated at 25°C. Symptoms (sunken, brown lesions each with a V-shaped pattern extending to the core) developed on all inoculated fruit 2 to 4 days after inoculation. No lesions were observed on control fruit. When fungi were reisolated from lesions of inoculated fruit onto PDA and incubated at 25°C, colony and conidial morphology were identical to those of the original isolates, confirming Kochs postulates. This study confirms a previous report of C. gloeosporioides causing bitter rot on apple in Uruguay (1). C. acutaum and C. gloeosporioides are known to cause bitter rot on apple. C. fragariae has traditionally been associated with strawberry and recently with other host plants (2), but not with bitter rot of apple. To our knowledge, this is the first report of bitter rot of apple caused by C. fragariae, and the first report of this disease caused by C. acutatum and C. fragariae in Uruguay. References: (1) S. García. Page 49 in: Guía Para el Manejo Integrado de Plagas y Enfermedades en Frutales. INIA Las Brujas, Canelones, 1998. (2) S. J. MacKenzie et al. Plant Dis. 92:1432, 2008. (3) B. C. Sutton. The Coelomycetes. CAB International Publishing, New York, 1980. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First report of Campylocarpon fasciculare causing black foot disease of grapevine in Spain.

In May 2008, symptoms of black foot disease were observed on 8-year-old grapevines (Vitis vinifera L.) cv. Garnacha in Albuñol (Granada Province, southern Spain). Affected plants showed delayed budding with low vigor. Roots showed black discoloration and necrosis of wood tissues. Root fragments were cut, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small pieces of discolored or necrotic tissues were plated onto potato dextrose agar (PDA) supplemented with 0.5 g liter-1 of streptomycin sulfate. Plates were incubated at 25°C in the dark for 10 days and all colonies were transferred to PDA. A Cylindrocarpon-like fungus was consistently isolated from necrotic root tissues. Single conidial isolates were obtained and grown on PDA and Spezieller Nährstoffarmer Agar (SNA) and incubated at 25°C for 10 days in darkness. On PDA, the isolates developed white, thick, and cottony to felty abundant mycelium. On SNA, all isolates produced slightly to moderately curved one-septate (22.5-) 25.6 (-27.5) × (5-) 5.63 (-6.25) μm, two-septate (30-) 36.1 (-45) × (6.25-) 7.08 (-7.5) μm, three-septate (37.5-) 47.9 (-52.5) × (6.25-) 7.5 (-8.75) μm, four-septate (47.5-) 53.3 (-62.5) × (7.5-) 7.89 (-8.75) μm, and five-septate (52.5-) 61.8 (-67.5) × (7.5-) 8 (-8.75) μm macroconidia. Microconidia were not observed. DNA sequence of the rDNA internal transcribed spacer region (ITS) was obtained for isolate Cf-270 and deposited in GenBank (Accession No. HQ441249). This sequence showed high similarity (99%) to the sequence of Campylocarpon fasciculare Schroers, Halleen & Crous (GenBank Accession No. AY677303), in agreement with morphological features (1). Pathogenicity tests were conducted with inoculum produced on wheat (Triticum aestivum L.) seeds that were soaked for 12 h in flasks filled with distilled water. Each flask contained 300 ml of seeds that were subsequently autoclaved three times after excess water was drained. Two fungal disks of a 2-week-old culture of C. fasciculare (isolate Cf-270) grown on PDA were placed aseptically in each flask. The flasks were incubated at 25°C for 4 weeks and shaken once a week to avoid clustering of inoculum. Plastic pots (220 cm3) were filled with a mixture of sterilized peat moss and 10 g of inoculum per pot. One-month-old grapevine seedlings were planted individually in each pot and placed in a greenhouse at 25 to 30°C in a completely randomized design. Control plants were inoculated with sterile uninoculated seeds. Six replicates (each one in individual pots) were used, with an equal number of control plants. The experiment was repeated. Symptoms developed on all plants 20 days after inoculation and consisted in reduced vigor, interveinal chlorosis and necrosis of the leaves, necrotic root lesions with a reduction in root biomass, and plant death. The fungus was reisolated from the roots of affected seedlings and identified as C. fasciculare, completing Kochs postulates. No symptoms were observed on the control plants. Black foot disease of grapevines can be caused by different species of Cylindrocarpon and Campylocarpon. C. fasciculare was first reported in South Africa in 2004 (1). To our knowledge, this is the first report of C. fasciculare causing black foot disease of grapevine in Spain as well as other countries in Europe. Reference: (1) F. Halleen et al. Stud. Mycol. 50:431, 2004.

First report of Cylindrocladiella parva and C. peruviana associated with black-foot disease of grapevine in Spain

From 2007 to 2009, Cylindrocladiella-like isolates were recovered from grapevine (Vitis vinifera L.) roots with symptoms of black-foot disease in Spain, where the causal agents of this disease have been previously reported as Campylocarpon and Cylindrocarpon species (1,2). Three representative isolates were selected to confirm their identity: CPa1 and CPa2 from Asturias (northern Spain), and CPe523 from Cuenca (central Spain). Isolates were incubated on malt extract agar (MEA) and Spezieller Nährstoffarmer Agar (SNA) with carnation leaves (4) at 25°C for 10 days in darkness. On MEA, colonies developed light brown, cottony mycelium. On SNA, all three isolates produced chlamydospores in chains, and conidia were zero-to one-septate, but CPa1 and CPa2 produced longer conidia (10.4 to 18.9 [15.3] × 1.7 to 3.1 [2.4] μm) than CPe523 (6.4 to 12.3 [9.7] × 1.6 to 3.3 [2.4] μm). A fragment of the beta-tubulin gene from all isolates was sequenced with primers T1 and Bt2b (1) and deposited in GenBank (Accession Nos. JQ693133, JQ693134, and JQ693135). CPa1 and CPa2 showed high similarity (99%) to Cylindrocladiella parva (AY793486) and CPe523 showed high similarity (99%) to C. peruviana (AY793500), which is in agreement with the corresponding morphological features of these species (4). Pathogenicity tests were conducted with inoculum produced on wheat (Triticum aestivum L.) seed soaked for 12 h in 300 ml of distilled water and autoclaved three times. Inoculum was prepared by inoculating two fungal disks (8 mm in diameter) of a 2-week-old culture of each isolate grown on potato dextrose agar to wheat seed and incubation at 25°C for 4 weeks. One-month-old grapevine seedlings were planted individually in 220-cc pots filled with a potting medium of sterilized peat moss and 10 g of inoculum, and grown in the greenhouse at 25°C in a completely randomized design. Controls were inoculated with sterile, noninoculated wheat seed. There were six replicate plants per isolate, with an equal number of controls, and the experiment was repeated once. Symptoms developed in all plants by 20 days post-inoculation and consisted of reduced vigor, necrotic root lesions, and occasionally mortality, all of which resembled the symptoms from grapevines in the field from which the isolates were originally recovered. Mean shoot dry weights of inoculated plants (0.25, 0.16, and 0.28 g for CPa1, Cpa2, and CPa523, respectively) were significantly lower (P < 0.05) than that of the controls (0.74 g). Mean root dry weights of inoculated plants (0.28, 0.16, and 0.29 g for CPa1, Cpa2, and CPa523, respectively) were also significantly lower (P < 0.05) than that of the controls (0.68 g). Isolates recovered from the roots of inoculated plants were identical morphologically and molecularly to C. parva and C. peruviana, thereby satisfying Kochs postulates. No symptoms were observed on the control plants. These Cylindrocladiella spp. have been reported from nurseries or vineyards in South Africa and New Zealand (3). To our knowledge, this is the first report of C. parva and C. peruviana associated with black-foot disease of grapevine in Spain, and in Europe. References: (1) S. Alaniz et al. Plant Dis. 91:1187, 2007. (2) S. Alaniz et al. Plant Dis. 95:1028, 2011. (3) E. E. Jones et al. Plant Dis. 96:144, 2012. (4) L. Lombard et al. Mycol. Progress DOI 10.1007/s11557-011-0799-1, 2012.