M. de Cara

Lethal synergy of solar UV-radiation and H2O2 on wild Fusarium solani spores in distilled and natural well water

Environmentally-friendly disinfection methods are needed in many industrial applications. As a natural metabolite of many organisms, hydrogen peroxide (H(2)O(2))-based disinfection may be such a method as long as H(2)O(2) is used in non-toxic concentrations. Nevertheless, when applied alone as a disinfectant, H(2)O(2) concentrations need to be high enough to achieve significant pathogen reduction, and this may lead to phytotoxicity. This paper shows how H(2)O(2) disinfection concentrations could be significantly reduced by using the synergic lethality of H(2)O(2) and sunlight the first time for fungi and disinfection. Experiments were performed on spores of Fusarium solani, the ubiquitous, pytho- and human pathogenic fungus. Laboratory (250-mL bottles) and pilot plant solar reactors (2 x 14 L compound parabolic collectors, CPCs) were employed with distilled water and real well water under natural sunlight. This opens the way to applications for agricultural water resources, seed disinfection, curing of fungal skin infections, etc.

Species of Fusarium Isolated from River and Sea Water of Southeastern Spain and Pathogenicity on Four Plant Species

Species of Fusarium were isolated from water samples collected from the Andarax River and coastal sea water of the Mediterranean in Granada and Almería provinces of southeastern Spain. In total, 18 water samples were analyzed from the Andarax River, and 10 species of Fusarium were isolated: Fusarium anthophilum, F. acuminatum, F. chlamydosporum, F. culmorum, F. equiseti, F. verticillioides, F. oxysporum, F. proliferatum, F. solani, and F. sambucinum. In addition, five species were isolated from 33 sea water samples from the Mediterranean Sea: F. equiseti, F. verticillioides, F. oxysporum, F. proliferatum, and F. solani. When considering the samples by their origins, 77.8% of the river water samples yielded at least one species of Fusarium, with F. oxysporum comprising 72.2% of the total isolates. In the case of marine water, 45.5% of the samples yielded at least one species of Fusarium, with F. solani comprising 36.3% of the total isolates. The pathogenicity of 41 isolates representing nine of the species collected from river and sea water during the study was evaluated on barley, kohlrabi, melon, and tomato. Inoculation with F. acuminatum, F. chlamydosporum, F. culmorum, F. equiseti, F. verticillioides, F. oxysporum, F. proliferatum F. solani, and F. sambucinum resulted in pre- and post-emergence damping off. Pathogenicity of Fusarium isolates did not seem to be related to the origin of the isolates (sea water or fresh water). However, the presence of pathogenic species of Fusarium in river water flowing to the sea could indicate long-distance dispersal in natural water environments.

Grape Marc Compost Tea Suppressiveness to Plant Pathogenic Fungi: Role of Siderophores

It is important to know about the mechanisms that suppress plant diseases when compost from vegetable residues and/or their liquid extracts (compost tea) are used in order to improve the efficiency of this suppressing effect on pathogens. In this study, we assessed the presence of siderophores in various grape marc aerated compost teas (ACT) and their suppressing effect on nine pathogens: Rhizoctonia solani, Fusarium oxysporum f. sp. radicis-lycopersici, Fusarium oxysporum f. sp. lycopersici race 0, Fusarium oxysporum f. sp. lycopersici race 1, Fusarium oxysporum f. sp. radicis-cucumerinum, Verticillium dahliae, Pythium aphanidermatum, Phytophthora parasitica and the mycopathogen, Verticillium fungicola. Three concentrations (5, 10 and 15%) filtered, microfiltered and sterilized ACT were added to Petri dishes with a PDA medium, and 1 mM of ferric chloride (FeCy. After adding this mixture, a 0.5 cm disc was placed at the center of each dish containing the vegetative and reproductive body of each of the fungi to be tested. All the dishes were incubated at 25°C for seven days, except R. solani y P. aphanidermatum, which developed after 4 days. The addition of 1 mM of FeCl3 deactivated the siderophores present in the ACT, suppressing their inhibition of fungal development. The results obtained with the microfiltered ACT revealed that the microorganisms present in grape marc compost excreted siderophores into the medium which were responsible for inhibiting the growth of the 9 fungi tested. This activity was annulled by the addition of ferric chloride. The same results were achieved with the ACT obtained from filtering. This inhibition was not 100% after adding FeCl3 due to the fact that the microorganisms present in this tea, exhibited other biocontrol mechanisms.

Fungal microbiota from rain water and pathogenicity of Fusarium species isolated from atmospheric dust and rainfall dust

In order to determine the presence of Fusarium spp. in atmospheric dust and rainfall dust, samples were collected during September 2007, and July, August, and October 2008. The results reveal the prevalence of airborne Fusarium species coming from the atmosphere of the South East coast of Spain. Five different Fusarium species were isolated from the settling dust: Fusariumoxysporum, F.solani, F.equiseti, F.dimerum, and F.proliferatum. Moreover, rainwater samples were obtained during significant rainfall events in January and February 2009. Using the dilution-plate method, 12 fungal genera were identified from these rainwater samples. Specific analyses of the rainwater revealed the presence of three species of Fusarium: F.oxysporum, F.proliferatum and F.equiseti. A total of 57 isolates of Fusarium spp. obtained from both rainwater and atmospheric rainfall dust sampling were inoculated onto melon (Cucumismelo L.) cv. Piñonet and tomato (Lycopersiconesculentum Mill.) cv. San Pedro. These species were chosen because they are the main herbaceous crops in Almeria province. The results presented in this work indicate strongly that spores or propagules of Fusarium are able to cross the continental barrier carried by winds from the Sahara (Africa) to crop or coastal lands in Europe. Results show differences in the pathogenicity of the isolates tested. Both hosts showed root rot when inoculated with different species of Fusarium, although fresh weight measurements did not bring any information about the pathogenicity. The findings presented above are strong indications that long-distance transmission of Fusarium propagules may occur. Diseases caused by species of Fusarium are common in these areas. They were in the past, and are still today, a problem for greenhouses crops in Almería, and many species have been listed as pathogens on agricultural crops in this region. Saharan air masses dominate the Mediterranean regions. The evidence of long distance dispersal of Fusarium spp. by atmospheric dust and rainwater together with their proved pathogenicity must be taken into account in epidemiological studies.

Possibilities of the use of vinasses in the control of fungi phytopathogens

The purpose of this research was to study the biocide effect of three agroindustrial subproducts, concretely sugar beet, sugar cane and wine vinasse. Results from in vitro testing determined that wine vinasse is what shows a 100% capacity to suppress fungal growth with concentrations between 5% and 7% for Fusarium oxysporum f.sp. melonis race 0 and 1, Sclerotinia sclerotiorum, Pythium aphanidermatum and Phytophthora parasitica and 10-15% for F. oxysporum f.sp. radicis-cucumerinum. On the other hand, sugar cane vinasse produced an increase at high concentrations and sugar beet vinasse showed an approximate 100% suppressor effect on fungal growth for only some of the phytopathogens tested: S. sclerotiorum (15%), P. aphanidermatum (7%), P. parasitica (15%) and F. oxysporum f.sp. radicis-cucumerinum (15%). In the soil samples analyzed none of the three vinasse extracts decreased fusaric microbiota, producing an increase in the three samples tested. This would implicitly convey an improvement in soil quality by producing a potential increase in bacterial and fungal microbiota.

First Report of Fusarium proliferatum Causing Rot of Garlic Bulbs in Spain

In October of 2008, decayed garlic bulbs (Allium sativum L. cv. Blancomor de Vallelado) were received from a producer in Segovia, Spain. In November of 2009, similar symptoms were observed on stored bulbs (cvs. Blancomor de Vallelado and Garcua) from each of 30 municipalities in northwest Segovia and Valladolid. A minimum of one sample was collected from 12 localities. Pieces of symptomatic bulbs were surface disinfested for 2 to 3 min in 0.5% NaOCl and transferred to potato dextrose agar (PDA) and Komadas media. Colonies had catenate microconidia and curved macroconidia that were usually three to five septate. Microconidia were club shaped with a flattened base, aseptate, and were produced on both mono- and polyphialides. On the basis of morphological features, the fungus was identified as Fusarium proliferatum (T. Matsushima) Nirenberg (2,3). Pathogenicity tests were conducted with 12 isolates of the fungi following the method of Dugan et al. (1). Each assay with an isolate consisted of six cloves (cv. Blancomor de Vallelado) disinfested in 0.5% NaOCl for 45 s, rinsed with sterile water, and injured to a depth of 4.5 mm with a probe 1 mm in diameter. The wound was filled with PDA colonized by the appropriate isolate. Six cloves for each tested isolate received sterile agar as a control. The cloves were incubated at 25°C for 5 weeks. The test was repeated once with cv. Garcua. All isolates produced water-soaked, tan lesions. An isolate of the fungus was deposited in the collection of the Plant Production Department of the University of Almeria. No fungi were recovered from the control cloves. F. proliferatum has been reported on garlic in the northwestern United States (1) and Serbia (4). To our knowledge, this is the first report of a Fusarium sp. in the section Liseola attacking garlic in Spain. The fungus seems to be well established on this host in Spain. References: (1) F. M. Dugan et al. Phytopathology 155:437, 2007. (2) P. E. Nelson et al. Fusarium Species: An Illustrated Manual for Identification. Pennsylvania State University Press, University Park, 1983. (3) H. Nirenberg et al. Mycologia 90:434, 1998. (4) S. Stankovic et al. Eur. J. Plant Pathol. 118:165, 2007.

Association of Olpidium bornovanus and Melon necrotic spot virus with Vine Decline of Melon in Guatemala

Thirty-one soil samples from 14 different fields of Guatemala melon with vine decline symptoms were analyzed for the presence of organisms associated with the disease. With a soil-dilution plating method, only Macrophomina phaseolina was detected in five samples. With a melon bait plant technique, Olpidium bornovanus, often together with Melon necrotic spot virus (MNSV), was found in nearly all the samples, corresponding with all the fields studied. Other pathogens that were detected less frequently included Pythium aphanidermatum, Monosporascus cannonballus, and Rhizoctonia solani. Consequently, O. bornovanus and MNSV were uniquely associated with disease occurrence and thus are the most probable cause of melon vine decline in the fields studied.

First Report of Erwinia persicina from Phaseolus vulgaris in Spain

A previously unreported leaf spot disease of common bean, which caused losses as much as 50% of the crops, was observed in southeastern Spain (Almería, Granada, and Málaga provinces) in November 2003. In 2004, samples of cv. Donna with chlorotic and necrotic leaf spots were collected from Granada and processed for microbiological analysis. Bacteria isolated from the symptomatic leaves were determined to be fermentative on the basis of the ability to metabolize glucose in aerobic and anaerobic conditions. Three isolates were selected for pathogenicity tests. Bacterial suspensions (108 CFU/ml) were spray inoculated on bean seedlings (3 true leaves) of cv. Andecha. Beans were covered with transparent plastic bags for 2 days and held in an incubation chamber at 22°C and 80% relative humidity with a 12-h photoperiod. Assays were repeated at least twice. Symptoms that developed on plants inoculated with the three isolates were similar to those originally observed, while symptoms did not occur on control plants (inoculated with distilled water). The pathogenic isolates were identified by sequencing of the 16S rDNA after amplification (2). The amplified sequences were compared to available DNA sequences in databases by using BLAST (1); 99% homology with 16S rDNA of Erwinia persicina was shown. Microbiological characteristics (gram staining, motility, morphology, and results of biochemical tests) were in agreement with the molecular identification of the isolates. E. persicina has been isolated from bean in the United States (4) and described on tomato, banana, and cucumber in Japan (3). To our knowledge, this is the first report of E. persicina from common bean in Spain and in Europe. References: (1) S. F. Altschul et al. J. Mol. Biol. 215:403, 1990. (2) U. Edwards et al. Nucleic Acids Res. 17:7843, 1989. (3) M. V. Hao et al. Int. J. Syst. Bacteriol. 40:379, 1990. (4) M. L. Schuster et al. Fitopatol. Bras. 6:345, 1981.

The Interactive Effects of Temperature and Osmotic Potential on the Growth of Aquatic Isolates of Fusarium culmorum

The mycelial growth of 10 Fusarium culmorum strains isolated from water of the Andarax riverbed in the provinces of Granada and Almeria in southeastern Spain was tested on potato-dextrose-agar adjusted to different osmotic potentials with either KCl or NaCl (−1.50 to −144.54 bars) at 10°C intervals ranging from 15° to 35°C. Fungal growth was determined by measuring colony diameter after 4 d of incubation. Mycelial growth was maximal at 25°C. The quantity and capacity of mycelial growth of F. culmorum were similar at 15 and 25°C, with maximal growth occurring at −13.79 bars water potential and a lack of growth at 35°C. The effect of water potential was independent of salt composition. The general growth pattern of Fusarium culmorum growth declined at potentials below −13.79 bars. Fungal growth at 25°C was always greater than growth at 15°C, at all of the water potentials tested. Significant differences were observed in the response of mycelia to water potential and temperature as main and interactive effects. The number of isolates that showed growth was increasingly inhibited as the water potential dropped, but some growth was still observable at −99.56 bars. These findings could indicate that F. culmorum strains isolated from water have a physiological mechanism that permits survival in environments with low water potential. Propagules of Fusarium culmorum are transported long distances by river water, which could explain the severity of diseases caused by F. culmorum on cereal plants irrigated with river water and its interaction under hydric stress or moderate soil salinity. The observed differences in growth magnitude and capacity could indicate that the biological factors governing potential and actual growth are affected by osmotic potential in different ways.

First Report of Fusarium verticillioides Causing Stalk and Root Rot of Sorghum in Spain

Sweet sorghum (Sorghum bicolor L.) is considered one of the most promising crops for bioethanol production in many countries and is a focus of bioenergy research worldwide. In July 2011, plants of the sweet sorghum cv. Suchro 506 in Oropesa (Toledo, Spain, 40.048577°N, 5.360298°W) (European Datum 1950 UTM zone 30 N) were observed with severe wilting. Upon examination, the lower internodes were found to be straw colored. When the plant was split, the internal pith was reddish, soft, and disintegrating. Small pieces of symptomatic stems and roots were surface disinfected in sodium hypochlorite (0.5% wt/vol) for 2 min and air dried. The sections were then placed on either PDA (potato dextrose agar) medium or Komada agar and incubated for 5 days at 25°C. Isolations from diseased stem and root tissue consistently yielded Fusarium verticillioides (Sacc.) Nirenberg (3). The small, hyaline, mostly single-celled, oval to club-shaped microconidia of F. verticillioides were produced in long catenate chains arising from monophialides. PCR amplification of the ITS1-5.8S-ITS2 was performed using the primers and protocols described elsewhere (4) and the fragments obtained were subsequently sequenced in both directions. Sequences were deposited in the EMBL Sequence Database (Accession Nos. HE652878, HE652879, HE652880, and HE652881). Four of the recovered F. verticilliodes isolates were tested in pathogenicity assays. One-week-old cultures of each isolate were homogenized in 400 ml of sterile water and 200 ml were used to inoculate water-growth-chamber-grown plants in 500-ml pots. Two pots each with three plants of cv. Suchro 506 were inoculated for each isolate. Water with sterile PDA was used as a control. All plants were kept at 20 to 25°C under a photoperiod of 14 h at 12,000 lux. After 21 days, above- and belowground parts were dried for 24 h at 60°C. Total length and dry weight of both sections were obtained. Inoculated plants produced root rot symptoms characteristic of F. verticillioides with dark red discolorations of the cortex of seedling roots (1), whereas the plants watered with water containing only PDA did not produce symptoms. Inoculated plants also had a decrease in dry weight for above- and belowground sections (P = 0.05) compared with the control with 43 and 47% reductions, respectively. The length of aerial parts was approximately 5% less in inoculated plants compared with control plants. F. verticillioides was reisolated from all inoculated plants. Sorghum stalk and root rot caused by F. verticillioides has been reported in different countries including India (2) and the United States (3). To our knowledge, this is the first report of F. verticillioides causing stalk and root rot of sorghum in Spain. An increase of production of this crop is expected to meet targets of the renewable energy share in Spain and any disease compromising yield may be a threat to this endeavour. References: (1) R. A. Frederiksen and G. N. Odvody. Compendium of Sorghum Diseases. The American Phytopathological Society. St. Paul, MN, 2000. (2) N. N. Khune et al. Indian Phytopathol. 37:316, 1984. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, New York, 1990.