Comparative study of the pathogenicity of seabed isolates of Fusarium equiseti and the effect of the composition of the mineral salt medium and temperature on mycelial growth

The pathogenicity of seven strains of Fusarium equiseti isolated from seabed soil was evaluated on different host plants showing pre and post emergence damage. Radial growth of 27 strains was measured on culture media previously adjusted to different osmotic potentials with either KCl or NaCl (-1.50 to - 144.54 bars) at 15º, 25º and 35º C. Significant differences and interactive effects were observed in the response of mycelia to osmotic potential and temperature.

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: Fusarium oxysporum, 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 (Cucumis melo L.) cv. Piñonet and tomato (Lycopersicon esculentum 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.

Comparative study of the pathogenicity of seabed isolates of Fusarium equiseti and the effect of the composition of the mineral salt medium and temperature on mycelial growth.

The pathogenicity of seven strains of Fusarium equiseti isolated from seabed soil was evaluated on different host plants showing pre and post emergence damage. Radial growth of 27 strains was measured on culture media previously adjusted to different osmotic potentials with either KCl or NaCl (-1.50 to -144.54 bars) at 15°, 25° and 35° C. Significant differences and interactive effects were observed in the response of mycelia to osmotic potential and temperature.

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 Komada's 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.

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.

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.

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.

Erwinia persicina Causing Chlorosis and Necrotic Spots in Leaves and Tendrils of Pisum sativum in Southeastern Spain.

In 2003, symptoms of generalized chlorosis as well as necrosis in leaves and tendrils were observed in Pisum sativum L. cv Tirabeque grown in green fields in southeastern Spain (Granada Province), and by 2004, the disease affected approximately 12 ha. Bacteria isolated from symptomatic samples were gram negative, rod shaped, motile, oxidase negative, facultatively anaerobic, and fermentative, which coincided with the general characteristics of the family Enterobacteriaceae. The gene encoding the 16S rRNA from two isolates (LPPA 406 and LPPA 408) was sequenced after PCR amplification (1). The two sequences were identical (EMBL Accession No. AM294946 for LPPA 408) and showed 99% similarity with several strains of Erwinia persicina (including the type strain ATCC 35998, LPPA 373, LMG 11254, GS04, and LMG 2691). Additional biochemical tests were performed using E. persicina ATCC 49742 as a control. The three strains were negative for arginine dihydrolase activity, indol production, hydrolysis of casein, and hydrolysis of gelatin. In contrast, they were positive for assimilation of adonitol, l-lactate, mannitol, m-inositol, erythritol, sorbitol, sucrose, nitrate reduction, hydrolysis of aesculin, and growth in 5% NaCl at 36°C. Nevertheless, E. persicina ATTC 49742, but not the isolates from P. sativum, produced a pink pigment. The latter isolates were also tested for pathogenicity. Bacterial suspensions (108 CFU/ml) were spray inoculated on 10 pea seedlings of cv. Tirabeque. Seedlings 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 conducted twice. Symptoms that developed were similar to those originally observed in the field, whereas symptoms did not occur on control seedlings sprayed with sterile distilled water. Bacteria sharing the characteristics of the inoculated isolates were recovered from symptomatic plants, hence fulfilling Koch's postulates. E. persicina has been isolated previously from bean in the United States (3) and southeastern Spain (1) and from tomato, banana, and cucumber in Japan (2). To our knowledge, this is the first report of the bacterium on P. sativum. References: (1) A. J. González et al. Plant Dis. 89:109, 2005. (2) M. V. Hao et al. Int. J. Syst. Bacteriol. 40:379, 1990. (3) M. L. Schuster et al. Fitopatol. Bras. 6:345, 1990.

Bacterial Wilt of Beans (Phaseolus vulgaris) Caused by Curtobacterium flaccumfaciens in Southeastern Spain.

Symptoms of bacterial wilt were observed on common beans (cv. Donna) in southeastern Spain. From samples collected in four different fields (coast of Granada), a bacterium was isolated with the following characteristics: gram positive, aerobic rods with yellow colonies, strictly oxidative, oxidase negative, galactose, sucrose, erythritol, mannitol, sorbitol and m-inositol were not used as a sole carbon source, and hydrolysis of casein was positive. These results coincide with what is expected for Curtobacterium flaccumfaciens pv flaccumfaciens (3). One isolate from each field was selected for pathogenicity tests using two different methods. Bacterial suspensions (approximately 108 CFU/ml) were spray inoculated on bean seedlings of cv. Andecha (10 plants with three true leaves for each isolate). Beans were covered with transparent plastic bags for 2 days and held at 25°C and 80% relative humidity with a 12-h photoperiod. In addition, 10 healthy seeds of cv. Andecha were soaked in bacterial suspensions (approximately 108 CFU/ml) for 1 h and incubated at 25°C (2). Seedlings sprayed with distilled sterile water and seeds soaked in water served as controls. With both methods of inoculation, assays were conducted twice. Results were recorded after 3 weeks. Symptoms that developed on plants after infection with the four isolates were similar to those observed in the field. They included golden yellow necrotic leaf lesions and wilting. Wilting was more pronounced in the field and when inoculation was performed by spraying seedlings rather than by soaking seeds. Control plants did not develop symptoms and grew bigger than the inoculated plants. Two pathogenic isolates were identified through sequencing of the 16S rRNA gene. The genes were amplified by polymerase chain reaction (1) and their nucleotide sequences (1,418 bp) proved to be identical (Accession No. AJ879110). Comparison of these sequences with databases showed that they were also identical to those of C. flaccumfaciens strains LMG 3645 and P 259/26 (Accession Nos. AJ312209 and AJ310414) and Curtobacterium sp. strains 2384 and 3426 (Accession Nos. AY688359 and AY688360). In Spain, the bean pathogen C. flaccumfaciens was first isolated from seeds during 2001 (4). However, to our knowledge, this is the first report of damage caused by this bacterium in the field. Bacterial wilt has been recorded, but often not substantiated, in several countries from North and South America, Africa, Asia, Oceania, and Europe. References: (1) U. Edwards et al. Nucleic Acid Res. 17:7843, 1989. (2) T. F. Hsieh et al. Plant Dis. 86:1275. 2002. (3) K. Komagata and K.-I. Suzuki. Pages 1313-1317 in: Bergey's Manual of Systematic Bacteriology. Vol. 2. Williams and Wilkins, Baltimore, MD, 1986. (4) J. L. Palomo et al. Page 154 in: XI Congreso de la Sociedad Española de Fitopatología, Almería, 2002.

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.

First Report of Pythium tracheiphilum Causing Wilt and Leaf Blight on Lettuce (Lactuca sativa) in Spain.

Since 2002, a disease on lettuce plants (cv. Estibaliz) was observed in the field and in greenhouses in the Principality of Asturias in northern Spain. The disease was estimated to cause losses of 20% in field-grown crops during the summer. Plants with aboveground symptoms of wilt and necrosis and darkening of vessels and necrosis in the tap root were collected. On potato dextrose agar (PDA), a fungus was recovered and identified as Pythium tracheiphilum according to the following characters: terminal or intercalary, globose or subglobose sporangia; zoospores formed in water; chlamydospores present in old cultures; smooth, globose, terminal, and intercalary oogonia; and smooth, globose oospores (2). For pathogenicity tests, two isolates were each inoculated on 9 seedlings (3 true leaves) and 9 plants (6 to 8 true leaves) of lettuce (Batavia type) grown in peat. Inoculum was prepared by suspending mycelium of the isolates collected from two PDA plates in 100 ml of distilled water with 10 ml poured around each plant. Control plants received distilled water only. Plants were maintained at 21°C and 80% relative humidity with a 12-h photoperiod. Symptoms were observed after 4 or 7 days for seedlings and plants, respectively. Within 20 days, 33% of the seedlings and plants died, and all surviving plants showed symptoms. Symptoms did not occur on the control plants. P. tracheiphilum was reisolated from inoculated plants but not from the control plants. This fungus has been described previously as a lettuce pathogen in France and Italy (1) but to our knowledge, this is the first report of P. tracheiphilum infecting lettuce in Spain. Reference: (1) C. M. Messiaen et al. Enfermedades de las hortalizas, Mundi-prensa, Madrid. 1995. (2) A. J. van der Plaats-Niterink. Stud Mycol. 21:1, 1981.

First Report of Phytophthora capsici on Cucumber and Melon in Southeastern Spain.

In autumn 1999, crown and root rot along with wilting of cucumber (Cucumis sativus L.) were observed in greenhouses in southeastern Spain (Granada). Symptoms appeared again during the 2000 to 2001 growing season. In spring 2001, root and crown symptoms were observed also on melon (Cucumis melo L.) in greenhouses in another area of southeastern Spain (Almeria). Isolates from diseased plants from both locations were identified as Phytophthora capsici (Leonian). Isolates produced papillate sporangia of variable shape, some of them with two or three papilla. Sporangia were caducous with pedicels of variable lengths that could be longer than the sporangia. Three isolates were crossed with P. capsici strains of known mating type. All isolates produced amphigynous antheridia and were mating type A1. Isolates grew well at 35°C and did not produce chlamydospores. Pathogenicity was examined for one isolate from cucumber and one from melon. Cucumber and melon plants at the four-leaf stage and pumpkin (Cucurbita maxima × C. moschata) plants at the five-leaf stage were inoculated with a mycelium suspension. Both isolates caused wilting and death of plants on the three host species tested. The pathogen was reisolated from roots and stems of diseased plants. To our knowledge, this is the first time P. capsici has been found on cucumber in Spain. It is also the first time P. capsici has been found on melon in the greenhouses of southeastern Spain, and the first time it has been reported to cause root and crown rot of melon. Previously, P. capsici has been reported to cause disease of field-grown melon (2) and greenhouse-grown pepper (Capsicum annum) (1) in eastern and southeastern Spain, respectively. References: (1) J. C Tello. Comun. INIA 22, 1984. (2) J. J. Tuset Barrachina. An. INIA 7:11, 1977.

First Report of Fusarium oxysporum f. sp. radicis-cucumerinum on Cucumber in Spain.

During December 1999, root and stem rot was observed on greenhouse-grown cucumber (cvs. Albatros, Brunex, Acapulco, and Cerrucho) plants in Almería, Spain, using rock wool cultures. The disease caused severe damage, estimated at a loss of up to 75% of the plants, in the first greenhouse affected; afterward, the disease was found in eight additional greenhouses (14 ha) in 1999 and 2000. Stem lesions extended up to 10 to 12 cm above the crown in mature plants, although no fruit damage was observed. In the advanced stages, abundant development of orange sporodochia was evident on crown and stem lesions, without vascular discoloration. Root, crown, and stem pieces that were placed on potato dextrose agar (PDA) after surface-disinfection with 5% sodium hypochlorite, rinsed, and dried resulted in pure fungal colonies. Based on morphological characteristics of conidia, phialides, and chlamydospores from the isolations, the fungus was identified as Fusarium oxysporum Schlechtend.:Fr. Pathogenicity tests were conducted on cucumber (cvs. Marketmore 76 and Cerrucho [F1 hybrid]), melon (cvs. Amarillo oro, Perlita, Piboule, Tania, and Nipper [F1]), watermelon (cvs. Sugar Baby, Sweet Marvel, Jubilee, and Pata Negra and hybrid Crimson sweet), Cucurbita maxima × Cucurbita moschata, zucchini (cv. Senator), and loofah (Luffa aegyptiaca) at several stages: (i) pregermination; (ii) 1 or 2 true leaves; and (iii) more than 10 true leaves. Five fungal isolates were grown on PDA or shaken potato dextrose broth at 25°C for 8 days. Inoculation was performed in pots (10 seeds or plants of each cultivar or hybrid and isolate) by drenching with 100 ml of a fungal suspension (104 to 106 CFU/ml). Sterile water was applied to noninoculated control plants. Tests were repeated in growth chambers at 25°C (night) and 28°C (day) with a 16-h photoperiod. Fifteen to fifty days after inoculation, cucumber and melon plants at all three stages developed symptoms of root and crown rot in 100% of inoculated plants, with no observed vascular discoloration. Fifty days after inoculation, all three stages of C. maxima × C. moschata and zucchini remained symptomless. Loofah and watermelon germinated poorly or not at all when inoculated at the pregermination stage. Fifteen to fifty days after inoculation, 100% of inoculated cucumber and melon plants developed symptoms. Watermelon plants inoculated at the 10 or more true-leaf stage did not develop disease symptoms. No symptoms developed on noninoculated control plants. F. oxysporum was reisolated from infected roots, crowns, and stems of inoculated plants, confirming Koch's postulates. The main symptoms on cucumber infected by F. oxysporum f. sp. cucumerinum are wilt, yellowing, and vascular discoloration. In contrast, based on inoculation of the host differentials and the resulting disease symptoms found in this study, the fungus was identified as F. oxysporum f. sp. radicis-cucumerinum (1). To our knowledge, this is the first report of F. oxysporum f. sp. radicis-cucumerinum causing root and crown rot in cucumber in Spain. Reference: (1) D. J. Vakalounakis. Plant Dis. 80:313, 1996.

In vitro sensitivity of Verticillium fungicola to selected fungicides.

Twenty isolates of Verticillium fungicola var. fungicola collected from diseased fruit-bodies of Agaricus bisporus from prochloraz-treated crops, were exposed to a range of concentrations of six chemicals (benomyl, chlorothalonil, formaldehyde, iprodione, prochloraz-Mn-complex and prochloraz + carbendazim) in vitro. EC(50) values were determined for each fungus-fungicide combination. All isolates were more sensitive to prochloraz-Mn-complex (EC(50) values less than 5 mg 1(-1)) than to the remainder fungicides, and only seven isolates were moderately sensitive (EC(50) values between 5 and 50 mg 1(-1)) to prochloraz + carbendazim. All isolates were moderately sensitive to formaldehyde, whereas the majority of isolates were very resistant to the other three fungicides (benomyl, chlorothalonil and iprodione).