Use of Poultry Manure Combined with Soil Solarization as a Control Method for Meloidogyne incognita in Carnation.

The effectiveness of a combination of soil solarization and poultry manure (raw or pelletized) amendments for the control of root-knot nematode (Meloidogyne incognita) was tested in carnation (Dianthus caryophyllus) crops grown in in-ground beds under plastic-covered greenhouse conditions in southern Spain. Our trials demonstrated that soil solarization alone did not provide sufficient control of root-knot nematode, because the carnation growing season in this region only partly coincides with the most effective period for solarization, resulting in an insufficient duration of treatment during a key period for effectiveness. Chemical fumigation with 1,3-dichloropropene + chloropicrin prior to planting was effective in reducing nematode population densities in soil. Its effects spanned 9 months after planting, resulting in acceptable crop yields. In comparison, the combination of soil solarization and raw or pelletized poultry manure was slightly less effective than chemical fumigation for control of this pathogen but crop yields after 9 months were similar. However, the higher root gall indices observed after 9 months, in comparison with chemically fumigated plots, indicated the need for a reapplication of the organic manure treatment at the start of each successive growing season.

Cephalosporium maydis, the Cause of Late Wilt in Maize, a Pathogen New to Portugal and Spain.

Scattered maize (Zea mays L.) plants with symptoms of premature wilting were observed in two fields in Toledo (Spain) during the summer of 2003. In 2008, affected fields in Toledo and Ribatejo (Portugal) showed incidences as much as 60% and symptoms affecting 50% of the hybrid varieties. Wilting became visible before tasseling and continued until shortly before maturity. It steadily progressed from the lower to upper leaves; the leaf tissues between the veins changing first to a pale green color then the whole leaf rolling inward lengthwise. Some leaves dried up and became brittle. As leaf wilting advanced, yellowish or reddish brown streaks appeared on the basal internodes of the stalk, which dried up and became shrunken. When the stalk was split, a brown discoloration extended along the internodes. The fungus that was consistently isolated from necrotic roots and basal tissues of the stalks of plants from both locations was identified as Cephalosporium maydis Samra, Sabet & Hingorani (1). The internal transcribed spacer (ITS) region of mycelial DNA was amplified (ITS1 and ITS2 primers) and sequenced. BLAST analysis showed 99% homology with C. maydis (GenBank Accession Nos. CM2A1, CM884, CM3B, and CM1A). Pathogenicity was confirmed in a shadehouse experiment from March to July of 2009. One isolate from Ribatejo and another from Toledo were independently inoculated to 24 4-day-old seedlings of each of two maize varieties. The experimental unit consisted of six seedlings planted in an 8-liter pot filled with sand/silt previously infested with 200 g of wheat grains colonized by the fungi. Noncolonized wheat grains were used for the control treatments. Four replications (pots) were established for each genotype/isolate combination according to a complete randomized 2 × 3 factorial design. After 6 weeks, four plants in each pot were randomly selected for evaluation of root necrosis, plant height, and reisolations of C. maydis. The two remaining plants were grown for nine additional weeks, then their weights were determined, and the percentages of aboveground tissues that were prematurely necrotic and dry were evaluated. Six weeks after inoculation, percentage of root necrosis of inoculated plants ranged between 75 and 100%, which was significantly higher than those of the controls (0%). No significant differences of height were recorded. At the end of the experiment, 28 to 53% of aboveground tissues of inoculated plants were necrotic and dry, significantly higher than in the controls. Regardless of the variety, weights of the inoculated plants were significantly reduced to 45 and 65% of the control plants. When root and stalk tissues from both varieties were sampled and incubated on potato dextrose agar at 25°C, the mycelial growth of C. maydis was confirmed for the inoculated plants but not for the control plants: colonies with "rhizoid" appearance of the margin, first white in color and turning to ash gray with age. To our knowledge, this is the first report of the presence of C. maydis outside Egypt, Hungary, and India. This geographical expansion of the pathogen will eventually affect the maize breeding programs for southern Europe. Reference: (1) A. S. Samra et al. Phytopathology 53:402, 1963.

Selection of potential antagonists against asparagus crown and root rot caused by Fusarium spp.

Crown and root rot is one of the most important diseases of asparagus crop worldwide. Fusarium oxysporum f.sp. asparagi and F. proliferatum are the two species more frequently associated to this complex and their prevalence depends on the production area. The control of the disease on asparagus crop is difficult to achieve because its perennial condition and the long survival of the pathogen in the soil as chlamydospores or as mycelium in infected plant debris. Furthermore, Fusarium spp. are easily disseminated with asparagus propagation materials. Thus, control measures should aim at obtaining seedlings protection for longer than achieved with conventional pre-planting chemical treatments. The effectiveness of fungal antagonists on the control of diseases caused by soil borne fungi has been reported. The potential of Trichoderma spp. as a biological control agent against diseases caused by Fusarium spp. in tomato and asparagus has been studied . It has been suggested that microorganisms isolated from the root or rhizosphere of a specific crop may be better adapted to that crop and may provide better disease control than organisms originally isolated from other plant species. The objective of this work was the evaluation of the potential of fungal isolates from symptomless asparagus plants as biocontrol agents of Fusarium crown and root rot.

Crown Rot of Zucchini Squash Caused by Fusarium solani f. sp. cucurbitae in Almería Province, Spain.

Cucumber, melon, watermelon, and zucchini are intensively cropped in the southern part of Spain where approximately 20,000 ha of the crops are grown in greenhouses. In the spring of 2007, zucchini plants (Cucurbita pepo) at the fruit-bearing stage in three commercial plastichouses in Almería exhibited necrosis on the basal stem, wilt, and death. The incidence of dead plants was 20 to 30%. Fusarium solani was consistently isolated from the basal stems of symptomatic plants on potato dextrose agar (PDA). Cultures of six single-hyphal transfers were identified on the basis of molecular sequences and morphological characteristics (2). Sequences of ribosomal DNA from ITS1 region, 5.8S rDNA, and ITS2 were identical for all six isolates of F. solani. The rDNA sequence of isolate Fscl-3 of F. solani was deposited as GenBank Accession No. AM940070. The pathogenicity of these six isolates of F. solani was tested in two experiments conducted in one plastichouse in Almería. Pregerminated seeds of zucchini cv. Consul were sown in 1-liter containers filled with vermiculite on 21 May and 22 June, 2007 (experiments 1 and 2, respectively). Plants at the one- to two-true-leaf stage or younger were inoculated with a soil drench of 2.0 to 8.4 × 105 propagules per ml). One colonized PDA petri plate of each isolate was blended and homogenized in 500 ml of distilled water. Inoculum (50 ml per plant) was poured around the stem of zucchini plants growing in vermiculite. The experimental design was a randomized complete block with three replicates with each plot comprising four plants (one plant per container). In both experiments, 12 uninoculated plants of the same cultivar served as controls. Plants were maintained for 1 month following inoculation in a greenhouse with mean temperatures ranging between 20.7 and 24.6°C and 23.3 to 29.8°C for experiments 1 and 2, respectively. Wilting first occurred 9 days after inoculation, and 14 days later, all plants inoculated with the F. solani isolates died. Inoculated plants exhibited lesions on the stem base without rot of secondary roots. At the end of the experiment, the uninoculated plants remained asymptomatic. Results of experiment 2, with higher temperatures, were similar. The pathogen was consistently recovered from symptomatic plants in both experiments, fulfilling Koch's postulates. Although F. solani f. sp. cucurbitae race 1 was reported in field squash (C. maxima) in the province of Valencia of east-central Spain (1), to our knowledge, this is the first report of F. solani as the causal agent of crown rot of zucchini plants in plastichouses in the Almería Province of Spain, one of the world's largest concentrations of greenhouses. References: (1) J. García-Jiménez et al. Plant Dis. 81:1216, 1997. (2) C. M. Messiaen and R. Cassini. Taxonomy of Fusarium. Page 427 in: Fusarium: Diseases, Biology, and Taxonomy. P. E. Nelson et al., eds. Pennsylvania State University, University Park, 1981.

First Report of Root and Crown Necrosis of Bean Caused by Pythium aphanidermatum in Spain.

Adult bean plants (Phaseolus vulgaris cv. Festival) growing in a commercial greenhouse in southeastern Spain developed symptoms of root necrosis, necrotic streaks on the basal stems, and plant wilt. A Pythium sp. was isolated consistently from roots and basal stems on selective agar (P5ARP). Single-hyphal transfers produced intercalary antheridia, oogonia (23 to 26 µm in diameter), oospores (18 to 20 µm in diameter), and zoospores in toruloid sporangia. Cardinal temperatures were a minimum of 10°C, an optimum of 28 to 34°C, and a maximum of 40°C. Daily growth rate on corn meal agar at 25°C was 15 mm. The internal transcribed spacer 1 (ITS 1) rDNA sequence of the isolate matched the sequences of Pythium aphanidermatum in GenBank. The sequence of isolate Py-294 was deposited in GenBank, Accession No. AM396563. This isolate was identified as P. aphanidermatum on the basis of morphological and cultural characteristics (1) and the ITS rDNA sequence. To fulfill Koch's postulates, 50 ml of inoculum of isolate Py-294 was used to inoculate bean plants (cvs. Donna and Emerite) at the five-leaf stage. The inoculum was prepared by homogenizing 2-week-old potato dextrose agar-petri plate cultures in 300 ml of distilled water. The plants were maintained in a greenhouse at temperatures of 18.8 to 30.3°C. Irrigation water had an electric conductivity of 0.5 to 0.6 dS·m-1 while the nutrient solution had 1.9 to 2.1 dS·m-1. Two months after sowing, 35.4 and 100% of cvs. Donna and Emerite, respectively, developed root necrosis, while control plants grown in bags containing noninoculated perlite remained healthy. The pathogen was reisolated from roots and basal stems of symptomatic plants. The test was repeated with similar results. To our knowledge, this is the first report of P. aphanidermatum as the causal agent of root and crown necrosis of adult bean plants in Spain. Reference: (1) A. J. Van der Plaats-Niterink. Stud. Mycol. 21:242, 1981.

Fusarium Wilt of Gerbera in Spain in Soilless Crops.

In 2004, gerbera (Gerbera jamesonii cv. Excellence) plants, grown for cut flowers, were observed in a soilless cultivation system (coconut fiber substrate) in one farm in the Cadiz area (southwestern Spain) exhibiting symptoms of a wilt disease. Gerbera represents a relevant crop for the industry in the region, after rose and carnation. Affected plants were stunted and developed yellow leaves with initially brown and eventually black streaks in the vascular system. The vascular streaks in the yellow leaves were continuous with a brown discoloration in the vascular system of the crown and upper taproot. In some cases, the leaves of affected plants turned red. Fusarium spp. was consistently and readily isolated from symptomatic vascular tissue of infected plants onto a Fusarium-selective medium (3). Colonies were identified as F. oxysporum after subculturing on potato dextrose agar on the basis of morphological observations. Pathogenicity tests were carried out by using two monoconidial isolates, compared with an Italian one, obtained from wilted gerbera plants. Each isolate of F. oxysporum was grown in shake culture (90 rpm) for 10 days on casein hydrolysate at 25°C with 12 h of fluorescent light per day. Healthy rooted 30-day-old plants (cv. Jaska), were inoculated by dipping roots into a conidial suspension (5 × 107 conidia/ml) in one of the three test isolates of F. oxysporum. Plants were transplanted (1 plant per pot) into pots (3.5 liter vol.) containing rockwool-based substrate. Noninoculated plants served as control treatments. Plants (15 per treatment) were grown in a glasshouse at an average day temperature of 30°C and night temperature of 24°C (minimum of 22°C and maximum of 41°C). Wilt symptoms and vascular discoloration in the roots, crown, and veins developed within 30 days on each inoculated plant, while noninoculated plants remained healthy. F. oxysporum was consistently reisolated from infected plants. The pathogenicity test was conducted twice. A wilt of gerbera was described in the Netherlands in 1952 (1) but its presence was not confirmed in further observations (4). Gerbera wilt was recently reported in Italy (2) and identified as F. oxysporum f. sp. chrysanthemi (A. Garibaldi, personal communication). Currently, the wilt of gerbera in Spain is limited to a few farms and a very limited percent (2 to 3%) of plants. References: (1) J. Arx and J. A. von Tijdschr. PlZiekt. 58:5, 1952. (2) A. Garibaldi et al. Plant Dis. 88:311, 2004. (3) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (4) G. Scholten. Neth. J. Plant Pathol. 76:212, 1970.

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.

First Report of Resistance to Metalaxyl in Downy Mildew of Sunflower Caused by Plasmopara halstedii in Spain.

Fifty-two isolates of Plasmopara halstedii Farl. Berl. & de Toni (causal agent of sunflower downy mildew) collected from sunflower (Helianthus annuus L.) in Spain from 1994 to 2000 were evaluated for metalaxyl resistance. The pathogen was identified on the basis of the morphology of the sporangiophores and zoosporangia recovered on the underside of the leaves (2). Metalaxyl (Apron 20% LS) at 2.0 g a.i./kg of seed (labeled European rate) was applied as seed dressing to the susceptible sunflower 'Peredovik'. There were two replications of 40 plants, and the test was repeated three times. Inoculum (sporangia bearing zoospores) was produced on artificially inoculated plants. Seed were germinated in a humidity chamber at 28°C for 24 to 48 h. When the radicle was 0.5 to 1.0 cm long, untreated and treated seedlings were inoculated by dipping the entire plant in an aqueous suspension of 6.0 × 104 sporangia per ml for 4 h, planted in a sand/perlite mixture (2:3 vol/vol), and grown at 16 to 21°C with a 12-h photoperiod. Plants were incubated for 24 to 48 h at 100% relative humidity and 15°C in the dark to enhance sporulation. After 12 days, disease incidence (DI) of inoculated plants was determined as a percentage of plants displaying sporulation of the fungus on the cotyledons and/or true leaves (3). DI was 95 to 100% for the untreated seedlings, but mildew did not develop on seedlings treated with metalaxyl for 51 of the isolates. The remaining isolate caused symptoms on 67% of the treated plants. This isolate was tested in another experiment in which 'Peredovik' seed was treated with metalaxyl at 0, 0.5, 2.0, 3.5, and 5 g a.i./kg of seed. There were four replications of 12 seedlings per treatment, and seedlings were inoculated as described previously. DI in the untreated control was 77%, which was not significantly different from the DI for seed treated with metalaxyl at 0.5, 2.0, and 3.5 g a.i./kg of seed (97, 73, and 96%, respectively). DI for seed treated with metalaxyl at 5.0 g a.i./kg of seed was 37%, which was significantly lower than the other treatments. Although resistance of P. halstedii to metalaxyl has been reported in France (1), to our knowledge, this is the first report of resistance of sunflower downy mildew to metalaxyl in Spain. References: (1) J. M. Albourie et al. Eur. J. Plant Pathol. 104:235, 1998. (2) G. Hall, Mycopathologia 106:205, 1989. (3) M.L. Molinero-Ruiz et al. Plant Disease 86:736, 2002.

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.

First Report of Stem Rot and Wilt of Sunflower Caused by Sclerotinia minor in Spain.

In 2001, sunflower (Helianthus annuus L.) plants with symptoms of stem and root rot and wilt were observed in Soria, Spain. Light brown, water-soaked lesions developed on the collar of infected plants and extended along the stem, affecting the pith and causing early and sudden wilt. White mycelium and sclerotia (0.5 to 2 mm long) formed in the pith of stems. The sclerotia were disinfested in NaClO (10% vol/vol) for 1 min, transferred to potato dextrose agar (PDA), and incubated at 20°C. The fungus consistently obtained was identified as Sclerotinia minor Jagger (1). Pathogenicity was confirmed in a greenhouse experiment (15 to 25°C, 13 h light). Seven-week-old plants of six genotypes of sunflower ('Peredovik', HA89, HA821, HA61, RHA274, and HA337) were inoculated by placing one PDA disk with active mycelial growth adjacent to each basal stem just below the soil line and covering it with peat/sand/silt (2:2:1, vol/vol). Six plants of each genotype were inoculated without wounding, and another six were inoculated immediately after stem base wounding with a scalpel; six wounded and uninoculated plants were used as controls. First symptoms (wilting) appeared 4 days after inoculation in all genotypes. Two weeks after inoculation, the percentage of dead plants ranged from 33 to 92% (depending on cultivar), white mycelium was observed at the base of affected plants, and sclerotia were present in the pith of diseased plants. There was no effect of plant wounding on disease incidence or severity, and the fungus was reisolated from inoculated plants. To our knowledge, this is the first report of S. minor in Spain. Reference: (1) L. M. Kohn. Mycotaxon IX 2:365, 1979.

Temperature Effects on the Disease Reactions of Sunflower to Infection by Orobanche cumana.

Three virulent populations (CU194, SE193, and SE194) of the parasitic plant Orobanche cu-mana were inoculated onto four lines (KA-41, J-8281, HA-89, and RHA-273) of sunflower (Helianthus annuus L.). Pots were transferred to growth chambers set at 15, 19, 23, and 27°C. Emergence of broomrape plants and infection incidence were determinants of disease reaction. All broomrape populations were pathogenic to the sunflower lines KA-41, HA-89, and RHA-273, although differences in virulence were found. At 15 to 23°C, the populations of broomrape infected these three sunflower lines, but a delay in emergence of broomrape was found at 15°C; whereas, at 27°C, the level of infection was restricted. Only population CU194 infected the resistant line J-8281, with infection occurring mainly at 23 and 27°C, but few broomrape plants emerged. Our results suggest that the effect of temperature on the host-parasite relationship is complex.

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.