José M. Melero-Vara

Races of Isolates of Plasmopara halstedii from Spain and Studies on Their Virulence

Plants infected with downy mildew were collected from 1994 to 2000 in sunflower fields in Spain. The race of 102 bulk isolates of Plasmopara halstedii obtained from them was determined by inoculation of sunflower lines traditionally used as differentials for characterization of the pathogen. Nine different races of the fungus were determined. Although race 1 was most common and was the only one found in central Spain, races 4, 6, and 7 were widespread in southern Spain. The results allowed the identification of a new race of the pathogen, race 10, and of a race not previously reported in Europe, race 8. According to the proposal of a new system for characterization of the isolates of P. halstedii in the late 1990s, Coded Virulence Formulae (CVF) were assigned to bulk isolates and single-sporangium (ss) isolates obtained from them. The CVF of the bulk isolates (CVFi) did not always fit with the previous designation. Similarly, ss isolates from the same bulk isolate exhibited different CVF, not only among themselves, but also compared with the CVF of the source isolate. Although a revision of the differential lines used to perform the racial characterization of fungal isolates seems to be needed, the occurrence of a diversity of genotypes in field populations of P. halstedii and a high frequency of recombination and/or mutation of the fungal genome is also suggested.

Screening of wild Helianthus species and derived lines for resistance to several populations of Orobanche cernua

Twenty-six different perennial species of Helianthus, 18 wild annual species of the same genus, and 29 lines tracing to wild species were evaluated for resistance to three highly virulent populations of broomrape (Orobanche cernua). Evaluations were carried out in pots containing soil mixture infested with broomrape seeds. Most of the perennial Helianthus species were immune to the populations of broomrape used in the tests. Some wild annual species and wild derived lines were resistant. The resistance found in the wild species, introgressed to cultivated sunflower, could provide unique resistance to the parasite.

Comparison of physical, chemical and biological methods of controlling garlic white rot.

Treatment of garlic cloves with tebuconazole (at 1 ml of Folicur 25% l−1) achieved a significant reduction in the rate of disease progress and the final incidence of plant death by Sclerotium cepivorum: garlic yields were improved. Although soil solarization provided the best control of garlic white rot, bringing soil populations of S. cepivorum to negligible levels, similar levels of disease control and garlic yields were achieved when tebuconazole was sprayed to stem bases of plants grown from cloves also treated with tebuconazole. This double treatment almost doubled the yield compared with untreated plants and significantly increased bulb quality under high disease pressure conditions. Soil solarization was also highly effective in a second consecutive crop of garlic, with significant improvements in yield and garlic quality. In contrast, lower levels of disease control were obtained when selected isolates of Trichoderma harzianum and Bacillus subtilis were applied to the soil and cloves respectively.

Soil solarization in established avocado trees for control of Dematophora necatrix.

Four field experiments on the control of Dematophora necatrix in avocado orchards affected by white root rot were conducted in the Mediterranean coastal area of southern Spain during 1991 to 1994. In the unshaded locations of solarized plots, the maximal temperatures were 35 to 42°C, depending upon the year and soil depth (15 to 60 cm). Temperature increases attributable to soil solarization ranged between 4 and 8°C in unshaded areas, whereas for shaded areas they were approximately 4°C. Inoculum recovery was decreased in root samples buried at 15 to 30 cm in unshaded locations of both solarized and unsolarized plots after 3 to 5 weeks, whereas 4 to 8 weeks of solarization were required for the elimination of the pathogen buried at depths of 45 to 60 cm. In contrast, inoculum recovery ranged from 30 to 60% for samples in shaded locations of unsolarized plots. D. necatrix was not recovered from roots of infected trees in solarized plots sampled 9 months after solarization, whereas recovery from roots in unsolarized plots was similar to levels before solarization. Soil solarization in established orchards was successful in reducing viability of inoculum buried in soil and eliminated inoculum in infected roots of live trees.

Interspecific hybridization between sunflower and wild perennial Helianthus species via embryo rescue

Interspecific crosses were made between the cultivated sunflower inbred line HA89 and accessions of five wild perennial Helianthus species (H. giganteus L., H. laevigatus T. & G., H. resinosus Small, H. pauciflorus Nutt. and H. decapetalus L.) resistant to broomrape ( Orobanche cernua Loefl.). Using the genetic male-sterile isogenic version of that line as female, successful reciprocal crosses were also obtained with all these wild species except with H. decapetalus. Five-day-old hybrid embryos were excised and cultured in vitro. In all cases, few mature plants were obtained from embryos in early developmental stages (early heart and globular) but up to 28% mature plants were obtained from embryos in later stages of development. Very immature embryos were difficult to excise without damage. Hybrid embryos and mature plants were obtained from all five wild species. Interspecific hybrid embryos from different wild species showed distinct developmental potentials, the proportion of hybrid embryos in different developmental stages varying among species. Differences in the proportion of hybrid embryos at the several developmental stages were also obtained for the reciprocal crosses (cultivated × wild), which showed higher proportion of fully developed embryos. Hybrids involving H. giganteus and cultivated sunflower were difficult to obtain without the use of embryo culture. Embryo culture proved to be an useful tool to overcome post-zygotic hybrid incompatibility in different Helianthus spp., and facilitated interspecific transfer of resistance to O. cernua.

Loss of viability of Dematophora necatrix in solarized soils

To study the relationship between temperature regimes and loss of viability of Dematophora necatrix in soil, two field experiments were conducted to determine the effectiveness of soil solarization on reducing the population of D. necatrix colonizing avocado root segments buried at a depth of 15–60 cm. Increase of maximum hourly temperatures attributable to soil solarization reached, depending on depth, 6.7–4.6°C in unshaded areas and 3.9–1.5°C for shaded areas in the first experiment (starting in early June, 1995). The better environmental conditions in the second experiment (starting by mid-July, 1995) led to higher temperature increases (8.6–5.6°C, depending on depth) when solarization was conducted in unshaded areas. One, 4, 5 and 6 weeks of solarization were required to eliminate the viability of D. necatrix at 15, 30, 45 and 60 cm depths in the first experiment, whereas only 8, 10, 15 and 22 days of solarization were needed for the loss of viability of D. necatrix at the same depths in the second experiment. In shaded areas, however, soil solarization attained significant effectiveness at 15 cm depth.Regression analyses of fungal viability (ln-transformed data) over accumulated temperature–time showed best fits when the minimum threshold temperature was 30°C. Although eradication of D. necatrix in soil can be achieved down to 60 cm depth in solarized plots, and at 15 cm depth in unsolarized unshaded plots, the accumulation of temperature–time appeared less effective in reducing inoculum viability in the latter.

Relationships between Weather Variables, Airborne Spore Concentrations and Severity of Leaf Blight of Garlic Caused by Stemphylium vesicarium in Spain

Ascospores and conidia released into the air were recorded around plots on which garlic debris infected by Stemphylium vesicarium were fixed onto the soil surface. Symptoms in garlic trap plots located in the vicinity of infected debris, started in March and developed during April–May to reach disease incidence close to 100%, final disease severity values being lower in 1993 and 1995 than in 1994 and 1996. Whereas daily concentrations of ascospores were rather erratic, with 30% of captures between 0 and 6 h, conidia showed a daily periodicity with highest concentrations between 12 and 18 h, with a pronounced peak between 14 and 16 h, and lowest values at night. Ascospore release occurred mainly in February and March. It coincided with rainfall periods, 14 h with vapour pressure deficit ≤5 mb and solar radiation <145 W m−2 on the current day of the capture. In contrast, greatest captures of conidia started in late April and were prevalent in May, and were associated with rainfall in days previous to the capture in which rather high temperature occurred and solar radiation was 109–345 W m−2. Among the weather variables considered, rainfall appeared directly related to the aerial concentration of ascospores and conidia. The role of relative humidity seemed essential when rainfall did not occur. There was a relationship between conidia concentration in the air and number of hours with temperature in the range 12–21 °C. Ascospore production was not essential for infections to take place, since primary infection from conidia may occur and disease can develop from them readily.

Development of Pleospora allii on garlic debris infected by Stemphylium vesicarium

Pseudothecia of Pleospora allii developed best on garlic leaf debris infected by Stemphylium vesicarium incubated at low temperature (5–10°C) and relative humidity (RH) close to saturation. RH of less than 96% prevented the formation of pseudothecia, while an incubation temperature of 15–20°C led to the early degeneration of pseudothecia. Under natural conditions, colonization by pseudothecia of unburied garlic leaf debris varied between seasons from 6.0 to 15.5 pseudothecia/mm2, whereas lower colonization levels were recorded when samples were buried. Pseudothecial maturity was reached 1–4 mo after the deposition of garlic debris on the soil surface and 15 days after the burial of residues. In the later case, pseudothecia degenerated with degradation of the plant debris. Ascospore release, which required rainfall or dew periods, occurred between late January and late April depending upon the year. A high correlation was found between pseudothecia maturation and four meteorological variables. Two of which, i.e. the number of hours with RH≥98% and with a mean temperature of 4.5–10.5°C, and the accumulated rainfall, explained most variability (adjusted R2=0.82−0.98 depending upon the year). A multiple regression equation relating the pseudothecia maturity index with these two variables could be used to forecast the epidemic onset of Stemphylium leaf spots in Southern Spain. Temporal progress of pseudothecia maturation was best fitted by a monomolecular model.

Characterization of Isolates of Fusarium spp. Obtained from Asparagus in Spain

Microbial analysis of asparagus plants (Asparagus officinalis) obtained from four nurseries in Spain in 2002 to 2003 indicated high frequencies of Fusarium proliferatum, F. oxysporum, and F. moniliforme in the rhizomes and storage roots. Out of 201 isolates of Fusarium obtained from nursery crowns and from plants sampled in nine established asparagus fields, the highest frequency of highly pathogenic isolates was observed from samples collected from fields, and included some extremely virulent isolates of F. solani. For isolates of low to moderate virulence, the percentage of those significantly (P = 0.01) associated with root dry weight loss was larger for F. proliferatum (53.8%) than for the other Fusarium species (10.3 to 23.1%). Random amplified polymorphic DNA (RAPD) analysis of 19 isolates of Fusarium spp. grouped all F. proliferatum and F. moniliforme isolates together and, in a second cluster, five of the eight isolates of F. oxysporum. Asparagus cultivars Verde-Morado and Dariana were the least susceptible of 11 cultivars commonly grown in Spain; isolates of F. solani and F. moniliforme proved highly virulent; and a significant interaction was observed among pathogen isolates and asparagus cultivars when representative pathogenic isolates of F. proliferatum, F. oxysporum, F. moniliforme, and F. solani were tested on those cultivars. Larger reductions in root dry weight were associated with F. proliferatum and F. solani than with F. oxysporum and F. moniliforme, and differences in root and stem dry weights among cultivars were significant.

Aflatoxin (B1, B2, G1, and G2) contamination in rice of mexico and spain, from local sources or imported

Rice is an important cereal but it is often contaminated with aflatoxins (AFs). The purpose of this study was to identify and quantify AF (B1 , B2 , G1 , and G2 ) in 67 rice samples cultivated in Mexico and Spain, and from imported crops collected in 2008 and 2009. The methodology was validated, the rice samples were concentrated and purified with immunoaffinity columns and were quantified by high-pressure liquid chromatography (HPLC). The average total AF (AFt) in the Spanish rice was 37.3 μg/kg, the range was from 1.6 to 1383 μg/kg, the most contaminated samples being from San Juan de Aznalfarache, Sevilla (AFt = 138.6 μg/kg), from Tortosa, Tarragona (AFt = 104.6 μg/kg), and Calasparra, Murcia (AFt = 103.9 μg/kg). The rice imported from France to Spain had AFt of 26.6 μg/kg and from Pakistan AFt of 18.4 μg/kg, showing less AF contamination than the local one. The rice which originated from Mexico contained (AFt = 16.9 μg/kg), and those imported from the United States (AFt = 14.4 μg/kg) and Uruguay (AFt = 15.6 μg/kg). The imported rice had better quality in terms of the presence of AFs.