A. Vicent

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.

First Report of Alternaria Brown Spot of Citrus in Spain

In 1998, a new disease of Fortune mandarin trees was detected in orchards in the eastern province of Valencia. This is one of the most important late-maturing cultivars grown in Spain. Symptoms were typical of Alternaria brown spot of citrus (2). Young leaves showed brown necrotic and irregular blighted areas with characteristic yellow halos. The necrosis had a tendency to follow the veins. On fruits, symptoms included light brown, slightly depressed spots to circular and dark brown areas on the external surface. Infected young fruits and leaves often fell and the mature fruits were unmarketable due to lesions, resulting in important economic losses. Isolations on potato dextrose agar supplemented with 0.5 mg/ml of streptomycin sulfate (PDAS) from affected leaves and fruits consistently yielded Alternaria alternata (Fr.:Fr.) Keissl., which was identified based on conidial morphological characteristics. Pathogenicity tests were conducted using 15 isolates from fruit and leaves by inoculating detached immature Fortune leaves with a sterile water suspension of 5 × 105 conidia per ml. Drops of this suspension (40 μl each) were placed on the lower surfaces of each leaflet using four leaves per isolate. Leaves were incubated in a moist chamber in the dark at 27°C (1). After 48 h, most of these isolates caused necrotic lesions on the leaves similar to those observed in the field, and the fungus was reisolated, confirming Kochs postulates. In 1999, the fungus spread to other citrus-growing areas, and to date the disease has been detected affecting Fortune and Nova mandarins and Minneola tangelo. This is the first report of Alternaria brown spot of citrus in Spain. References: (1) K. Kohmoto et al. Phytopathology 81:719, 1991. (2) J. O. Whiteside. Plant Dis. Rep. 60:326, 1976.

Identification, incidence and characterization of Fusarium proliferatum on ornamental palms in Spain.

During a survey conducted from 1998 to 2002, Fusarium proliferatum was found associated with young and adult palms belonging to the genera Chamaerops, Phoenix, Trachycarpus and Washingtonia showing symptoms of wilt and dieback. The fertility and toxicological profile of 36 strains representing different locations and hosts were studied. All of them except two, which were infertile, belonged to mating population D. Both mating types (MATD-1 and MATD-2) were isolated from the same host species, showing a high potential of genetic recombination in the field. Additionally, eight strains were fertile once crossed as female. Toxin analysis showed differences in the ability of strains to produce fumonisin B1, moniliformin, beauvericin, fusaric acid and fusaproliferin. Only 17 of them produced all the toxins analyzed. Pathogenicity tests were conducted on Phoenix dactylifera and P. canariensis using nine F. proliferatum Spanish strains and two reference strains from Saudi Arabia. Eight months after inoculation all strains caused disease, with palms showing lesions on the bases of leaves and development of wilt symptoms similar to those originally observed in affected plants. This is the first report on the occurrence of F. proliferatum on P. dactylifera in Spain and also the first report of this pathogen on C. humilis, P. canariensis, P. reclinata, T. fortunei, W. filifera and W. robusta.

Laboratory Evaluation of Citrus Cultivars Susceptibility and Influence of Fruit Size on Fortune Mandarin to Infection by Alternaria alternata pv. citri

Young leaves of 62 citrus cultivars were inoculated with conidia of three Spanish isolates of Alternaria alternata pv. citri, the causal agent of brown spot of citrus. Hybrids with Dancy mandarin, King mandarin or their derivates as a parent, grapefruit cultivars and the mandarin cultivars Guillermina, Emperor, Clemenpons and Esbal were highly susceptible to the pathogen. Satsuma cultivar Clausellina and orange cultivars, with the exception of Sanguinelli, were slightly susceptible. Lemon and lime cultivars were not susceptible, with the exception of Mexican lime (Citrus aurantifolia), which was slightly susceptible. Although this study shows a range of potential hosts for this pathogen, to date the only affected cultivars in Spain are Fortune and Nova mandarins, and Minneola tangelo. The susceptibility of Fortune fruits decreased as diameter increased, being susceptible through the whole season. This was confirmed with field observations in autumn where fruit infections have been detected when the diameter reaches 6–7 cm.

Controle biológico de Monosporascus cannonballus com Chaetomium

Melon collapse caused by Monosporascus cannonballus is one of the main diseases that affect this cucurbit. The objective of this research was to study the biological control of M. cannonballus by Chaetomium. Infested substrates with 0.5, 1, 2, 4 and 8 x 105 CFU g-1 were introduced in plates where seeds of melon type Piel de Sapo cv. PS 1430 were sowed in seeding trays after two weeks of incubation. The seedlings were transplanted to pots with a similar substrate, infested additionally with 2.5 x 104 CFU of Chaetomium per gram and 20 CFU of M. cannonballus per gram of substrate. Three controls were considered: one infested with Chaetomium alone, another with M. cannonballus and one without any treatment. The experimental design was completely randomized with five replicates. General disease index and control percentage were evaluated forty-five days after transplant. The General disease index varied according to the increased Chaetomium concentration. The results demonstrated that 4 and 8 x 105 CFU of Chaetomium g-1 were equivalent to 0.9 and 0.6, respectively. The statistical analysis showed that 4 and 8 x 105 CFU g-1 of Chaetomium concentration were the most efficient treatments, with control percentage higher than 55 %, indicating the potential of Chaetomium to control M. cannonballus.

Comparative Epidemiology of Monosporascus Root Rot and Vine Decline in Muskmelon, Watermelon, and Grafted Watermelon Crops

Disease progression and soil population dynamics of Monosporascus cannonballus ascospores were studied and compared through the entire growing season in muskmelon, watermelon, and watermelon grafted onto Cucurbita rootstock crops. In muskmelon and watermelon, there was a decrease of ascospore counts in soil from transplanting until first symptoms of vine decline were observed. This decrease probably was related to ascospore germination and subsequent root infection. Later, there was a remarkable increase of ascospore counts in a parallel way to the quick development of vine decline symptoms, which lead to plant death. In these crops, root colonization adjusted to first-order linear equations and vine decline progression best correlated with exponential and logistic models. In contrast, there were no symptoms of vine decline on grafted watermelon. Furthermore, the isolation of M. cannonballus from Cucurbita roots was considerably lower, perithecia were never observed, and ascospore soil populations remained stable. These results suggest that disease control by grafting onto genus Cucurbita seems to be related primarily by the increased resistance of its root system to infection by M. canonballus, and provide further evidence of the use of grafting as a disease management measure for this disease.

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.

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.

Comparison of Application Methods of Systemic Fungicides to Suppress Branch Cankers in Clementine Trees Caused by Phytophthora citrophthora

Since 2002, considerable losses of Clementine trees (Citrus clementina) have been observed in Spain due to Phytophthora branch canker of citrus caused by Phytophthora citrophthora. Due to the low efficiency of the available cultural and genetic control measures, application of fungicides is required for economic management of the disease. Fosetyl-Al, metalaxyl, and its enantiomer mefenoxam are the only systemic fungicides registered for control of Phytophthora diseases in Spain. However, their efficacy has not been tested against Phytophthora branch canker. Greenhouse and field experiments were conducted for 3 years in Spain to evaluate the ability of these fungicides and application methods to reduce lesion expansion. Nevertheless, with the inoculation technique used, it was not possible to evaluate the protective activity of fungicides, which can play an important role in their performance under field conditions. None of the fungicide treatments inhibited lesion expansion when applications were made on a curative basis. The residual effect was better on young than on mature trees. Paint treatments were generally more effective in reducing lesion expansion that drip chemigation or foliar sprays. However, this application method is laborious and becomes uneconomical in Spain. Trunk and branch sprays as well as long-term programs of foliar sprays or drip chemigation for control of spring and autumn infections are proposed as targets for future research.

Risk of establishment of non-indigenous diseases of citrus fruit and foliage in Spain: An approach using meteorological databases and tree canopy climate data

Based on macroclimate comparisons of monthly means of temperature and rainfall, the Mediterranean-type climate might be considered unfavorable for the establishment of the quarantine pathogens of fruit and foliage of citrus regulated by the EC Council Directive 2000/29. The presence of free water on the canopy during periods with temperatures favorable for disease development seems to be limited by the characteristic rainless summer. However, our field study showed that due to the formation of dew, rainfall and rain days were not positively correlated with canopy wetness. Dew periods were quite frequent during summer nights with temperatures over 15°C and even 20°C. Nevertheless, wetness periods were seldom continuous and they were usually interrupted by dry periods approximately 10–14 h long. In contrast to some endemic foliar pathogens such asAlternaria alternata, no data are currently available on the performance of these non-indigenous pathogens under interrupted wetness conditions. Due to the lack of rain during the summer in semi-arid areas, the natural spread of rain-disseminated citrus pathogens, such asElsinoë spp. andXanthomonas axonopodis pv.citri, might be rather limited. However, windborne pathogens, such asGuignardia citricarpa andPseudocercospora angolensis, would have considerable potential for dissemination under the Mediterranean climate. We consider that more information about the effect of microclimate on the epidemiology of these diseases is needed to estimate accurately their risk of establishment in Spain and in other citrus-growing countries of the Mediterranean Basin.