M. Berbegal

Evidence for Multiple Introductions and Clonality in Spanish Populations of Fusarium circinatum

Fusarium circinatum is thought to have been moved around the world with pine planting stock consisting, most probably, of infected seed. In this effort, we investigate the genetic structure of F. circinatum in Spain and globally. In total, 223 isolates were studied from five regions in northern Spain and eight countries. Eight microsatellite markers revealed 66 multilocus genotypes (MLGs). Minimum spanning network analysis of MLGs by region within Spain as well as globally, discriminant analysis of principal components, and analysis of molecular variance revealed that Spanish populations are significantly differentiated and structured into two distinct groups, each one including one of the dominant genotypes observed. This result suggests that two independent introductions occurred into Spain that subsequently underwent clonal divergence and admixture. This result is further supported by the linkage disequilibrium and clonality observed for F. circinatum populations in northern Spain. The maintenance of differentiation between the clusters could result from the lack of or rare sexual reproduction in Spain. Possible introduction pathways from other countries and subsequent routes of dispersion of F. circinatum in Spain are discussed.

Inoculum Density-Disease Development Relationship in Verticillium Wilt of Artichoke Caused by Verticillium dahliae

The relationship between inoculum density of Verticillium dahliae in soil and disease development was studied in 10 commercial artichoke fields. Inoculum density of V. dahliae varied between 2.2 and 34.2 microsclerotia (ms) g-1 of soil near planting. Artichoke plants were monitored for disease at the beginning and the end of each growing season. There was a significant correlation, which was best described by negative exponential models, between inoculum density and disease incidence, symptom severity, and recovery of the pathogen from the plants. Inoculum densities ranging from 5 to 9 ms g-1 of soil were associated with a mean percentage of infected plants of about 50%. Additionally, three fields were monitored in two consecutive growing seasons to evaluate the population dynamics of V. dahliae microsclerotia in soil and disease development. Numbers of microsclerotia per gram of soil decreased significantly by the end of the first growing season but slightly increased at the end of the second growing season. In these fields, symptom severity was greatest during the second growing season when high percentages of infected plants also were recorded.

Genetic Diversity and Host Range of Verticillium dahliae Isolates from Artichoke and Other Vegetable Crops in Spain

Artichoke is severely affected by Verticillium wilt, caused by Verticillium dahliae, in eastern-central Spain, which is one of the most important vegetable-cropping areas in the country. To determine genetic and virulence variability in local populations of V. dahliae, 18 isolates collected from artichoke and other vegetable species cultivated in eastern-central Spain were selected to represent local vegetative compatibility groups (VCGs). Diversity in the isolates was characterized by molecular markers and virulence in 12 important hosts for that region. Recently developed microsatellite markers (simple-sequence repeats) and polymorphic sequences were used to assess the genetic variation among those isolates to reveal any association occurring among host source, VCG, and virulence. Although all isolates caused severe disease symptoms on artichoke, cardoon, eggplant, and watermelon, those from artichoke had a limited host range and isolates from watermelon, muskmelon, and eggplant were not pathogenic to some of the hosts tested. VCG diversity was related to differential virulence in certain hosts.

Incidence of Verticillium wilt of artichoke in Eastern Spain and role of inoculum sources on crop infection.

Surveys of 94 artichoke fields throughout the artichoke production areas of Comunidad Valenciana (eastern Spain) were conducted from 1999 to 2002 to determine the incidence and distribution of Verticillium wilt.Verticillium dahliae was isolated from 80.9% of the sampled fields, and detected in all artichoke-growing areas, with a mean disease incidence of 53.8% infected plants. The disease was found to cause severe damage to cv. ‘Blanca de Tudela’, which is the most important artichoke cultivar grown in Spain, and was also observed on the seed-propagated cv. ‘Imperial Star’. In field trials to study the role of infected planting material and soil inoculum on infection of artichoke plants during the cropping season,V. dahliae was transmitted from infected stumps to the plants, confirming that the use of infected stumps could have greatly contributed to the dissemination of the pathogen. Inoculum density ofV. dahliae in soil had an effect on crop infection, in that a higher number of microsclerotia per gram of soil resulted in a higher percentage of infected plants. In addition, yield of cv. Blanca de Tudela was significantly affected byV. dahliae infection, showing that a higher percentage of infection corresponded with lower yield.

Verticillium wilt: A threat to artichoke production

Verticillium wilt is becoming an increasing concern in artichoke production because the rapid spread of the disease to new growing areas has led to declining production. Scientists from Italy, Spain, and the United States combine to bring us up to date on diagnosis of the disease, its epidemiology and life cycle, as well as management strategies, current and forthcoming.

Effect of Cauliflower Residue Amendments and Soil Solarization on Verticillium Wilt Control in Artichoke

The effect of fresh cauliflower residue amendment alone and with a low dose of metham sodium (MS) combined with soil solarization was investigated for the control of Verticillium wilt of artichoke in two commercial fields under artichoke-cauliflower rotation. Treatments were a factorial combination of three main plots (an unamended control, soil amended with cauliflower residue, and a combination of cauliflower residue and a low dose of MS) and two subplots (application of a plastic cover or uncovered). Inoculum densities of Verticillium dahliae were measured before and after soil treatments as well as disease incidence, symptom severity, and yield. Although soil solarization reduced inoculum of V. dahliae and the incidence of Verticillium wilt of artichoke, no added benefit was obtained when solarization was used with cauliflower residue amendments. In addition to toxic volatile compounds, other mechanisms could be involved in disease suppression because the effects of incorporating cauliflower residue were not enhanced by the plastic covering. The effect of cauliflower residues on populations of V. dahliae microsclerotia in soil was inconsistent, possibly due to varying pretreatment inoculum levels. Treatments with cauliflower residue amendments and low doses of MS maintained low inoculum densities in the fields until the end of the growing season and significantly reduced the percentage of infected plants.

Complex molecular relationship between vegetative compatibility groups (VCGs) in Verticillium dahliae: VCGs do not always align with clonal lineages.

Verticillium wilts caused by the soilborne fungus Verticillium dahliae are among the most challenging diseases to control. Populations of this pathogen have been traditionally studied by means of vegetative compatibility groups (VCGs) under the assumption that VCGs comprise genetically related isolates that correlate with clonal lineages. We aimed to resolve the phylogenetic relationships among VCGs and their subgroups based on sequences of the intergenic spacer region (IGS) of the ribosomal DNA and six anonymous polymorphic sequences containing single-nucleotide polymorphisms (VdSNPs). A collection of 68 V. dahliae isolates representing the main VCGs and subgroups (VCGs 1A, 1B, 2A, 2B, 3, 4A, 4B, and 6) from different geographic origins and hosts was analyzed using the seven DNA regions. Maximum parsimony (MP) phylogenies inferred from IGS and VdSNP sequences showed five and six distinct clades, respectively. Phylogenetic analyses of individual and combined data sets indicated that certain VCG subgroups (e.g., VCGs 1A and 1B) are closely related and share a common ancestor; however, other subgroups (e.g., VCG 4B) are more closely related to members of a different VCG (e.g., VCG 2A) than to subgroups of the same VCG (VCG 4B). Furthermore, MP analyses indicated that VCG 2B is polyphyletic, with isolates placed in at least three distinct phylogenetic lineages based on IGS sequences and two lineages based on VdSNP sequences. Results from our study suggest the existence of main VCG lineages that contain VCGs 1A and 1B; VCGs 2A and 4B; and VCG 4A, for which both phylogenies agree; and the existence of other VCGs or VCG subgroups that seem to be genetically heterogeneous or show discrepancies in their phylogenetic placement: VCG 2B, VCG 3, and VCG 6. These results raise important caveats regarding the interpretation of VCG analyses: genetic homogeneity and close evolutionary relationship between members of a VCG should not be assumed.

First Report of Alternaria Black Spot of Pomegranate Caused by Alternaria alternata in Spain

Since 2010, a new foliar and fruit disease was observed in pomegranate (Punica granatum L.) orchards in Alicante Province (eastern Spain). Symptoms included black spots on leaves and fruits, as well as chlorosis and premature abscission of leaves. Fungal isolates were obtained by surface-disinfecting small fragments of symptomatic leaf and fruit tissues in 0.5% NaOCl, double-rinsing in sterile water, and plating them onto potato dextrose agar (PDA) amended with 0.5 g/liter of streptomycin sulfate. Gray-to-black colonies were obtained, which were identified as Alternaria sp. based on the dark, brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa tapering to a prominent beak attached in chains on a simple and short conidiophore (4). Conidia (n = 100) measured (12.2-) 20.2 (-27.6) × (5.7-) 9.2 (-12.0) μm, and had 3 to 6 transverse and 0 to 5 longitudinal septa. Single spore cultures were obtained and their genomic DNA was extracted. The internal transcribed spacer (ITS) region of rDNA and partial sequences of the beta tubulin gene were amplified and sequenced with primers ITS1-ITS4 and Bt1a-Bt1b, respectively (3). BLAST analysis of the sequences showed that they were 100% identical to a pathogenic A. alternata (Fr.) Keissl. isolate obtained from black spot disease of pomegranate in Israel (Accession No. JN247826.1, ITS and Accession No. JN247836.1, beta tubulin) (2). As all the sequences obtained showed 100% homology, ITS and beta tubulin sequences of a representative isolate (1516B) were submitted to GenBank (KF199871 and KF199872, respectively). In addition, a PCR reaction with specific primers (C_for/C_rev) designed to recognize highly virulent isolates of A. alternata causing black spot of pomegranate was used with all isolates (2). A characteristic fragment of ~950 bp was amplified in two isolates: 1552B and 1707B. Pathogenicity was assessed on plants and detached fruit of pomegranate cv. Mollar (1). Two-year-old pomegranate trees were inoculated with isolates 1552B and 1707B by spraying a conidial suspension (106 conidia/ml) onto the upper and lower leaf surfaces. Five plants per fungal isolate were used and five control plants were sprayed with sterile water. Plants were covered with transparent plastic bags and incubated in a growth chamber for 1 month at 25°C, with a 12-h photoperiod. One-month-old fruits were surface sterilized in 1.5% sodium hypochlorite solution for 1 min and rinsed twice in water. Two filter paper squares (5 × 5 mm) were dipped in the conidial suspensions and placed on the fruit surface. Inoculated fruit were incubated in a humid chamber in the dark at 25°C. Ten fruit per fungal isolate were used and 10 control fruit were inoculated with sterile water. Black spots were visible on inoculated leaves and fruit, 10 and 3 days after inoculation, respectively. Symptoms were not observed on controls. The fungus was re-isolated from leaf and fruit lesions, confirming Kochs postulates. Leaf black spot of pomegranate caused by A. alternata was first described in India in 1988, and later in Israel in 2010 affecting both fruit and leaves (1). To our knowledge, this is the first report of the disease in Spain, where it could represent a threat for pomegranate cultivation due to the increasing amount of area dedicated to this crop. References: (1) D. Ezra et al. Australas. Plant Dis. Notes 5:1, 2010. (2) T. Gat et al. Plant Dis. 96:1513, 2012. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (4) E. G. Simmons. Alternaria: An identification manual. CBS Fungal Biodiversity Center, Utrecht, Netherlands, 2007.

First Report of Circular Leaf Spot of Persimmon Caused by Mycosphaerella nawae in Spain

Production of persimmon (Diospyros kaki L. f.) has increased significantly during the last decade in Spain as a profitable alternative for fruit growers. In August 2008, after a mild and rainy spring, symptoms of a new disease were observed in commercial persimmon fields located in Valencia Province (eastern-central Spain). Symptoms included circular necrotic spots on the leaves and defoliation. Early fruit maturation and premature abscission were associated with early symptom development in the trees. A fungus was consistently isolated from the margins of leaf lesions. All isolates obtained were hyphal-tipped twice and transferred to potato dextrose agar (PDA). The cultures grew slowly and reached a diameter of 21 to 29 (mean 26) mm within 4 weeks on PDA at 25°C in the dark. Mycelium was initially dark green and ultimately became dark gray to black. Several media and incubation conditions were tested to induce sporulation, but conidia formation was not observed. In April 2009, mature spherical pseudothecia were observed in lesions on fallen leaves that had remained in affected fields during the winter. Ascospores were uniseptate and mostly spindle shaped, 10 to 11.5 (mean 10.3) μm long, and 3 to 3.9 (mean 3.4) μm wide. Fungal colonies obtained from the ascospores were identical to those isolated from the leaf lesions. Morphological characters observed matched those described for the pathogen Mycosphaerella nawae Hiura & Ikata (1). In Korea, the circular leaf spot of persimmon caused by M. nawae was considered an economically important disease in the 1990s, especially in the southern regions (2). Sequences of the internal transcribed spacer (ITS) region of the rDNA were obtained for isolates MY2 and MY3 and deposited in GenBank (Accession Nos. GQ465767 and GQ465768). These sequences were identical to each other and to the sequence obtained from a Korean isolate of M. nawae. Symptoms of the disease were reproduced after inoculation of 2-year-old persimmon trees growing in individual pots. A ground mycelial suspension (5 × 105 CFU ml-1) of strain MY2 was sprayed onto 20 potted trees (200 ml per individual tree) in late May of 2009. Ten trees were sprayed with sterile distilled water as a control. Trees were incubated at 20°C in a growth chamber with a 12-h photoperiod and covered with a semitransparent plastic hood for the first 10 days after inoculation, after which the plastic was punctured for ventilation and trees were incubated at 22°C. The first symptoms (small circular spots on the leaves) appeared on inoculated trees 15 days after inoculation. One month after inoculation, all inoculated trees showed circular leaf spots and severe defoliation, whereas noninoculated trees remained healthy. M. nawae was successfully reisolated from the lesions. To our knowledge, this is the first report of M. nawae causing circular leaf spot of persimmon in Spain. References: (1) J. H. Kwon et al. Plant Dis. Agric. 1:18, 1995. (2) J. H. Kwon et al. Korean J. Plant Pathol. 14:397, 1998.

Identification of Pythium tracheiphilum as the causal agent of vascular necrosis of endive (Cichorium endivia) in Spain

Pythium isolates were recovered from endive plants (Cichorium endivia) showing vascular necrosis collected from commercial fields located in Castellón province (eastern Spain). They were identified as Pythium tracheiphilum on the basis of their phenotypical and molecular profile. Pathogenicity tests conducted with two P. tracheiphilum isolates, obtained from endive and lettuce (Lactuca sativa), respectively, in this region, confirmed that both isolates were pathogenic to endive, with no significant differences in virulence between them. This is the first report of vascular necrosis caused by P. tracheiphilum on endive in Spain.