Potato Carrot Agar Research Articles

Yellow Stunt, a Tobacco Disease Caused by Pythium dissotocum, in Southern Parts of Brazil.

Yellow stunt, an emerging disease of tobacco (Nicotiana tabacum), has become increasingly prevalent in tobacco-growing regions in southern Brazil. Major symptoms are moderate to severe stunting, yellowing of leaves, severe wilting, darkened roots, necrosis of stem tissue directly above the soil line, and plant death. Phytophthora glovera was first proposed in 1999 as the primary causal agent of yellow stunt (1), but since then, there has been no data or completion of Koch's postulates to support this. Fifty-six isolates of fungi and fungus-like organisms were obtained from stem and root samples of tobacco plants with typical symptoms of yellow stunt in the Brazilian States of Rio Grande do Sul, Santa Catarina, and Paraná during the growing seasons of 2004/05 to 2006/07. They were identified to species level by analysis of the morphological characteristics (2) and sequence of rDNA internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) (3). The isolates were identified as Pythium dissotocum (29), Fusarium oxysporum (10), P. graminicola (5), Rhizoctonia solani (4), F. solani (3), P. ultimum (3), P. deliense (1), and P. inflatum (1). The ITS sequences of the 29 isolates identified as P. dissotocum were identical. The nucleotide sequence of one isolate, LFM27/2005, has been deposited in GenBank (GQ495982). Analysis of ITS sequences alone was not sufficient to differentiate this isolate from other species in the Pythium subclade B2, such as P. coloratum, P. lutarium, P. marinum, or P. diclinum. However, the combination of morphological and cultural characteristics (2) and sequence data support our identification of LFM27/2005 and similar isolates as P. dissotocum. Colonies of LFM27/2005 on cornmeal agar had filamentous sporangia and formed slightly inflated, dendroid structures. Zoospores formed at 5°C. Daily growth rate on potato carrot agar was 13 mm at 25°C. The oogonia (22 μm in diameter) were nonornamented and either intercalary or terminal. Antheridia, commonly 1 to 2 per oogonium, were sessile, born on unbranched stalks, and either monoclinous or diclinous. Aplerotic or nearly plerotic oospores measured 20 μm in diameter with a smooth wall 2.5 μm thick. Pathogenicity tests for each pathogen were performed in a greenhouse at ~24°C in pots filled with pine bark substrate infested with inoculum at the time Burley tobacco plants showed five expanded leaves. Each test consisted of five plants and was repeated three times. Inoculum for one to three isolates representative of each pathogen was prepared by growing 2-month-old cultures at 28°C in the dark for 7 days on potato dextrose agar medium overlaid with three sterile oat kernels. Noninfested oat kernels were used for control plants. Forty days after inoculation, only plants inoculated with isolates of P. dissotocum exhibited all symptoms associated with yellow stunt. P. inflatum and R. solani did not induce yellow stunt symptoms and the others induced only wilting and root rot. P. dissotocum was recovered from an inoculated, symptomatic plant, fulfilling Koch's postulates. Its morphology was identical to isolates obtained from original field samples. The results demonstrate the association of isolates of P. dissotocum with tobacco yellow stunt in Brazil.

Black grain mycetoma caused byLeptosphaeria tompkinsii

Leptosphaeria tompkinsii is a dematiaceous fungus which is rarely reported as an agent of black-grain mycetoma. We present a case involving a mycetoma of the hand of a former farmer from Mali, West Africa, who has been a resident in France for 27 years. The patient was successfully treated with surgery and the use of oral itraconazole for 6 months. Species identification was based on sexual reproductive structures observed on potato-carrot agar media and the use of internal transcribed spacer sequencing.

Cutaneous fungal granulomas due to Alternaria spp. infection in a horse in New Zealand

Equine cutaneous fungal granulomas have been previously referred to in New Zealand (Fairley 1998), and are described in the veterinary literature from around the world, including North America and Australia (Pascoe and Summers 1981; Genovese et al. 2001; Valentine et al. 2006), but no peer-reviewed reports appear published in the literature in New Zealand. Described here is a case of multiple cutaneous fungal granulomas caused by Alternaria spp. in a horse in New Zealand.

In-vitro predatory activity of nematophagous fungi from Costa Rica with potential use for controlling sheep and goat parasitic nematodes

In tropical and subtropical regions of the world, parasitic diseases are a main cause of losses in livestock productivity. The increased acquired resistence to anthelmintics by gastrointestinal nematodes, requires biological control be considered as a potential feasible and effective alternative. The most effective natural soil enemies of nematodes are nematophagous fungi. In order to collect and identify predator nematophagous fungi (PNF), samples were obtained from 51 farms distributed throughout the seven provinces of Costa Rica. The origin samples included: soil from different crops (potatoes, tomatoes, bananas, ornamental plants, squash and coffee); animal feces (cattle, sheep, goat and horse); soil and fallen leaves from forest; and plants with signs of nematode infection. Each sample was processed using three techniques for the extraction of fungi from soil: sprinkling technique, soil dilution and humidity chamber. Twenty four strains of nematophagous fungi were found in 19 farms; 83.3% of the fungi were isolated by sprinkling technique. The following fungi were identified: Arthrobotrys oligospora (n = 13); Candelabrella musiformis (n = 9); and for the first time there was isolation of A. conoides (n = 1) and A. dactyloides (n = 1) in the country. Moreover, 16 strains from Trichoderma (n=13), Beauveria (n = 1), Clonostachys (n = 1) and Lecanicillium (n = 1) were obtained. In addition, pH of each possible fungal isolation source was measured, and it varied from 5.2 to 9.9, however PNF isolates fell within the range of 5.6 to 7.5. The PNF strains were cultivated in four different media for the production of chhlamydospores: potato dextrose agar (PDA); corn meal agar (CMA); malt extract agar (MEA) and potato carrot agar (PCA). Out of these cultures, 95.8% of the strains formed chlamydospores primarily in the PCA. Of these strains, the profilic spore producers were subjected to ruminant artificial gastrointestinal conditions. A total of 14 fungi were tested, out of which 42.9% survived the digestive analysis. Neither A. conoides nor A. dactyloides were viable following the in vitro gastrointestinal test. The PNF isolated in this study demonstrated an action against ovine and caprine gastrointestinal nematodes and are candidates for use in biological control of these organisms. Among these microorganisms, Candelabrella musiformis appears to be the most promising fungi for use as a biological control agent in Costa Rica.

Root Rot of Hydroponically Grown Lettuce Caused by Phytophthora drechsleri in Mexico.

During March of 2008, bibb lettuce (Lactuca sativa L.) plants with severe wilting and root rot were observed in a commercial liquid-hydroponic greenhouse in Guanajuato, Mexico. By July of that year, the disease affected most plants in the facility. A Phytophthora sp. was consistently isolated from diseased roots on potato carrot agar. Several Phytophthora isolates were morphologically characterized. Sporulation was achieved by placing colonized disks of clarified V8 juice agar (V8A) into nonautoclaved soil extract (10 g avocado soil/1,000 ml distilled water, stirred for 3 h, and filtered). Sporangia were persistent, nonpapillate, and 40 to 58 μm long × 30 to 40 μm wide. External and internal proliferation was observed. Hyphal swellings were predominantly rounded. Oospores were not observed. The isolates grew on V8A at 35°C. Pathogenicity tests were conducted twice by utilizing a representative isolate (AC1) on bibb lettuce seedlings (10 replicates per experiment). Seeds were placed on sterile, water-soaked paper in petri dishes. After 10 days, each lettuce seedling was placed into a tube containing approximately 2 ml of sterile distilled water and 2,000 zoospores. Control plants were placed in tubes with water only. Plants were incubated for 7 days in a moist chamber at 25°C. Symptoms of wilting and root necrosis were observed 2 to 3 days after inoculation. All plants were dead 5 to 7 days after inoculation. A Phytophthora sp. was always isolated from the roots of inoculated plants. Control plants remained healthy. The pathogen was identified as Phytophthora drechsleri Tucker according to morphological characteristics. To confirm the identity of the pathogen, sequences of the internal transcribed spacers (ITS) were obtained from three representative isolates. The ITS sequences that were obtained shared 100% homology to several strains of P. dreschleri, including isolates from cucurbits (GenBank Accession No. AF228097). The ITS sequence was deposited in NCBI as Accession No. FJ790770. P. cryptogea and P. dreschleri have been reported as causing root rot on lettuce grown hydroponically in the United States and Korea (1,2). To our knowledge, this is the first report of P. drechsleri causing root rot on lettuce in Mexico.

First report ofAlternaria species associated with black point of wheat in Tunisia

Durum wheat (Triticum turgidum) is one of the most important cereal crops in the world and the staple food for the Tunisian population. The main growing areas of durum wheat are located in the Northern part of the country, which is characterised by a sub humid climate favourable for fungal spoilage of grain such asAlternaria species. These species are able to produce plant pathogenic as well as toxic metabolites. For mycological analysis, the agar test was used. After mycelial growth, theAlternaria species were inoculated on potato carrot agar media. A PCR-based assay followed by a sequencing step allowed the identification ofAlternaria alternata; Alternaria tenuissima and Alternaria japonica from grains.

First report of Albugo trianthemae on Delosperma and Lampranthus in the UK

Olive ( Olea europaea ) is of great economic importance for its fruits and oil. In the UK, olive trees are popular ornamentals grown in frost free areas or in glasshouses. Olive trees are benefiting from climate change and are surviving further north in Europe than 30 years ago (Moore & White, 2003). Evidence of this includes the establishment of Britain’s first commercial olive grove of 120 trees, planted in Devon in 2006 (McCarthy, 2006). In May 2005, diseased samples of olive originating from private gardens in Suffolk and Surrey were received at the Royal Horticultural Society, Wisley. The symptoms were diffuse spots on the lower leaf surfaces. Leaves subsequently turned yellow, reddish-brown and then dropped. The fungus was dirty grey in appearance and growing on lower leaf surfaces. Conidia were subcylindrical, guttulate, very pale to medium olivaceous-brown, apex obtuse, straight or often curved, 3–8 septate, 27–55 × 3·5–4·5 μ m. Conidiophores were mostly unbranched, 110–125 × 3–4·5 μ m. Conidiogenous cells were terminal bearing one or more scars. The morphological characteristics fit with Pseudocercospora cladosporioides (Avila et al ., 2005), the causal agent of cercosporiosis or leaf spot disease on olive. Single spore cultures were made on potato carrot agar supplemented with ampicillin and streptomycin. The fungus was slow growing but produced abundant conidia when plated onto potato dextrose agar and incubated for four weeks at 20 ° C in the dark. To fulfil Koch’s postulates, one olive plant was inoculated with a spore suspension (1·5 × 10 5 spores mL − 1 ). Sterile distilled water was used on the control plant. The plants were kept in a glasshouse at 20 ° C, covered with a polythene bag for the first 48 h to increase humidity. After 15 days, the leaves of the inoculated plant became yellow and dropped. Pseudocercospora cladosporioides was reisolated from these infected leaves and confirmed by morphology. The control plant was not infected. The cercosporiosis disease is common and widely distributed in most olive growing regions in the world including Algeria, Argentina, Australia, Brazil, Chile, China, Germany, Greece, India, Italy, Montenegro, Netherlands Antilles, New Zealand, Portugal, Spain, Tanzania, Tunisia and USA (Crous & Braun, 2003; David, 2004). This is, however, the first record of P. cladosporioides in the UK.

Effects of sodium ion and water hyacinth extract in the production of Curvularia pallescens in culture media

An investigation was conducted on the pH, nutritional and cost benefit of the use of different substrates for the production of Curvularia pallescens Boedijn. The growth of the organism was examined at pH range between 5.5 and 8.6 and on different growth media, potato carrot agar (PCA), potato dextrose agar (PDA), sabouraud agar (SA), tap water agar (TWA), water hyacinth agar (WHA) and Czapek-Dox agar (ZA). Also, the growth of the organism was examined on different enrichment media “Czapek-Dox” with its sodium nitrate substituted with equal amount of different nitrogen sources (ammonium chloride, ammonium nitrate, glutamine and sodium glutamate). WHA produced significantly (P < 0.001) highest amount of mycelial growth (84.95 ± 0.05 mm) on day 6 than other media types. The spore concentration was highest, 6.6 E06 ± 3.33 E05 spores/ml with TWA. The colony size was significantly (P < 0.001) highest, 84.9 ± 0.1 mm on day 10 with sodium glutamate compared with other nitrogen sources. Spore count was only observed on ammonium chloride (1.67E06 ± 3.33E05 spores/ml), the optimal growth pH was 5.5. The cost of production of the organism per culture plate on WHA was 61.3 and 82% lesser than on PDA and SA respectively. The formulated water hyacinth agar medium appeared most economically feasible for the mycelial production of C. pallescens Boedijn. Key words: Curvularia pallescence Boedjin, growth measurement, pH measurement.

First Report of Neofusicoccum parvum Associated with Dieback of Mango Trees in Peru.

Mango (Mangifera indica L) is one of the most important cash crops of northern Peru. Since 2003, adult mango trees (cvs. Criollo and Kent) located in Piura Province developed symptoms of dieback characterized by the death of twigs and branches in the tree canopy. Additional disease symptoms involved darkened, elongated lesions on the peduncle, causing an early maturation of the fruit, and in advanced symptoms, stem-end rot of fruits. Symptoms were frequent in the spring months (September to November) when the lesions expand rapidly. Diseased tissues from branches and fruits were collected and small pieces of necrotic tissues were surface disinfected and plated onto potato dextrose agar (PDA) with 0.5 g L-1 streptomycin sulfate. Plates were incubated at 25°C in the dark. All affected tissues consistently developed colonies with a white mycelium, moderately dense, and becoming olivaceous gray after 5 to 6 days. Pycnidia were produced on sterile mango twigs placed on the surface of potato carrot agar (PCA) after 10 days. Conidia were hyaline, guttulate, aseptate, measuring (15-) 18.5 (-22.5) × (4-) 5.2 (-7.5) μm. Conidia became olivaceous and developed one or two septa before germination. Isolates were identified as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, & A.J.L. Phillips (1). DNA sequences of the rDNA internal transcribed spacer region (ITS) and part of the translation elongation factor 1-alpha (EF1-α) genes were used to confirm the identification through BLAST searches in GenBank (ITS: 99% identity to Accession No. EU080928; EF1-α: 98% identity to Accession No. AY343367). Representative sequences of the studied DNA regions were deposited at GenBank (ITS: Accession No. FJ528596; EF1-α: Accession No. FJ528597). Pathogenicity tests were conducted on 18-month-old potted mango plants cv. Kent with two N. parvum strains (A4 and A5). A mycelial plug (3 cm in diameter) taken from the margin of an actively growing colony of each isolate was put in a wound made with a cork borer of the same diameter on the stem of each plant. Inoculation wounds were wrapped with Parafilm. Controls were inoculated with sterile PDA plugs. Ten replicates for each isolate were used with an equal number of control plants. Plants were maintained in a greenhouse with a temperature range of 22 to 28°C. After 4 weeks, mango plants showed necrotic stem lesions originating from the inoculation point affecting also the branches of the inoculated plants. No differences in lesion area between strains were obtained. No lesions developed in the control plants. Reisolations from necrotic tissues were successful and both isolates were morphologically identical to those used for inoculations. N. parvum was isolated from all symptomatic trees in all surveyed areas. This pathogen has already been reported on mango (2) and currently represents a serious problem in the mango-producing areas of Peru. To our knowledge, this is the first report of N. parvum affecting mango in Peru.

Potato carrot agar with manganese as an isolation medium for Alternaria, Epicoccum and Phoma

A semi-selective medium for isolation of Alternaria spp., Epicoccum sp. and Phoma spp. from soil and plant samples was developed. The basal medium was a modified potato carrot agar (PCA), containing 10 g/L of potato and carrot. It is known that the target genera sporulate well on standard PCA when grown at 25 °C with an alternating light/dark cycle consisting of 8 h of cool-white daylight followed by 16 h darkness. Addition of 1.5% MnCl 2 4 H 2O (w/v) inhibited most other fungi than Alternaria, Epicoccum and Phoma species when tested on pure cultures. The mycobiota of two soil samples and eight grain samples were examined using PCA-Mn and three commonly used isolation media, DRYES, DG18 and V8. On the three conventional media growth of several genera was observed with the predominant being Aspergillus, Eurotium, Fusarium, Mucor, Penicillium and Rhizopus. Of these only F. oxysporum and F. verticillioides were able to grow on PCA-Mn. Alternaria infectoria and Epicoccum nigrum were present in three cereal grain samples, but emerged to a far lower degree on the three conventional media compared to PCA-Mn. Three black spored fungi, identified as Phoma eupyrena, Paraconiothyrium minitan and one unknown species, were isolated from the two soil samples when incubated on PCA-Mn but were absent on the three conventional media.

Sensitivity of the Alternaria infectoria species-group 1 to cycloheximide

A possibility of using cycloheximide tolerance and/or sensitivity as an additional diagnostic tool for distinguishing morphologically related species within common small-spored Alternaria has been tested during this study. A total of 33 strains from four Alternaria species-groups, namely Alternaria alternata, Alternaria arborescens, Alternaria infectoria and Alternaria tenuissima were tested for their growth response to 100 microg m(-1) cycloheximide in potato carrot agar. All A. infectoria strains were completely inhibited, showing no growth at all even after prolonged incubation. In contrast, all other strains representing the remnant three species exhibited a high resistance to this antibiotic. Cycloheximide sensitivity represents a further important physiological character for distinguishing A. infectoria from the three similar species. The relevance of these findings corresponds with the potential ability of the Alternaria species produce mycotoxins. Cycloheximide may be in future used in the design of selective media for the isolation of some potentially toxigenic food-borne Alternaria species such as A. alternata, A. tenuissima and/or A. arborescens, for example in screening cereals for toxigenic Alternaria spp. and for their direct separation from nontoxigenic representatives of A. infectoria species-group.

Alternaria infectoria species‐group associated with black point of wheat in Argentina

Regional surveys are being conducted in Argentina to assess the presence of wheat (Triticum aestivum) pathogens on grains across the main cropping area. During 2001 and 2002, grain samples with a dark brown or blackish discoloration around the embryo end, known as black point, were observed on several cultivars across the wheat region of Buenos Aires Province. Seed analysis by blotter and agar tests (Neergaard, 1979) showed up to 55% of prevalence (number of samples infected over the total) of Alternaria spp. and a mean natural infection of 37%. Morpho-cultural studies were carried out on potato carrot agar. The isolates were typified as belonging to the A. infectoria species-complex according to their sporulation pattern and the production of long secondary conidiophores (Andersen et al., 2002). Pathogenicity tests were carried out in Petri dishes following two standard methods: blotter test and agar test using 2% potato dextrose agar, inoculating wheat seeds with a conidial suspension (2 × 105 conidia mL−1) of 20 selected isolates. Seeds were incubated for seven days at 20 ± 2°C in a growth chamber under 12 h fluorescent plus near ultraviolet photoperiod. No disease symptoms were observed in the controls. Infected seeds became totally or partially black and seedlings from them appeared weak with reduced vigour. Black point is known to affect grain quality adversely, impairing flour, semolina and their products (Mathur & Cunfer, 1993). Simmons (1992) organized the genus Alternaria into 14 species-groups based on characteristics of conidia and chain formation. The A. infectoria species-group comprises nine known species and an unknown number of distinct taxa yet to be described. In Europe, North America and Australia studies have shown that grain samples are infected with A. alternata and A. infectoria species-groups ranging from low levels to 100%. In Argentina, previous records of Alternaria spp. refer to A. alternata associated with black point in wheat. However, in this study the vast majority of Alternaria strains conformed to the A. infectoria complex. The incidence levels of this group are gaining importance and have increased in recent years probably due to changes in cropping systems in most of the different agroclimatic zones of Argentina. This is the first published report of Alternaria infectoria species-complex causing black point in wheat grains in Argentina. This highlights the necessity to better understand the relationship of this group with the deterioration of wheat sub-products and the risk of harmful mycotoxins production.

Association of Pythium aphanidermatum with Root and Crown Rot of Melons in Honduras.

Approximately 10,000 ha of melon (Cucumis melo L.), primarily cantaloupe and honeydew types, are grown in Honduras for export to U.S. markets. In 2004 and 2005, several soil surveys were conducted in areas with a history of vine decline. Twenty-nine soil samples from six farms were collected from the rhizosphere of wilted plants. Thirty-six melon plants were planted in a mixture of each rhizosphere sample and vermiculite (1:6 v/v). The plants were maintained in a growth chamber at 23 to 25°C with a 16-h photoperiod. The first symptoms, which appeared at the one- or two-true-leaf stage, were girdling of the lower stem, leaf chlorosis, and wilting. Affected plants exhibited necrotic crowns and roots and half of all plants died less than 3 days after wilting. Isolations from washed and dried crown and roots pieces from affected plants were placed on malt extract agar. Colonies were transferred to potato carrot agar and into dishes of sterile water and immature carnation petals to aid in the identification of recovered fungi. Nearly 500 isolates of Pythium species were cultured, and approximately 60% were identified as P. aphanidermatum (Edson) Fitzp. on the basis of their toruloid sporangia, aplerotic oospores, terminal and smooth oogonia, monoclinous sac-shaped antheridia (one to two per oogonium), and abundant appressoria. The pathogenicity of nine isolates was confirmed in a growth chamber. Ten plants of melon cv. Amarillo Canario, grown in sterilized vermiculite, were inoculated at the two- or three-true-leaf stage by drenching pots with 100 ml of a suspension of each isolate (103 CFU ml-1). Noninoculated plants served as controls. There were three replicates per isolate. Plants began to die 7 days after inoculation and the incidence of the affected plants reached an average of 70%. P. aphanidermatum causing decline of melon plants has been previously reported in hot and semi-arid areas in Israel and Spain (1,2). To our knowledge, this is the first report of P. aphanidermatum pathogenic to melon plants in Honduras.

Influence of culture media and environmental factors on mycelial growth, sporulation and spore germination behaviour of Curvularia eragrostidis (P. Hennings) Mayer

Curvularia eragrostidis is a foliar fungal pathogen of young tea plants. It causes leaf spot disease of tea. Mycelial growth, sporulation and spore germination behaviour of the pathogen were studied. Six different media were tested for mycelial growth. Among these, potato carrot agar (PCA) was found best for the mycelial growth and sporulation. Maximum mycelial growth was attained after 15 days of incubation. Mycelial growth was also studied in different temperatures and pH. Optimum temperature of growth was 25 °C and best growth was obtained at pH 6.0. Glucose and peptone were best carbon and nitrogen sources respectively for growth and sporulation of the fungus. The optimum conditions of spore germination were found to be at pH 7.25 and at incubation temperature of 25 °C. Keywords: Sporulation; Curvularia eragrostidis; Mycelial growth, fungus

First Report of Seedling Damping-Off of Fennel Caused by Alternaria petroselini in the Netherlands.

In April 2005, serious seedling damping-off was noted on fennel (Foeniculum vulgare Mill. cv. Rondo) in a transplant greenhouse facility in Maasdijk, the Netherlands. Symptoms appeared 3 to 4 weeks after sowing and included black, sunken lesions aboveground on the stem and belowground on the hypocotyls. Mortality of seedlings was 6 to 10% (10 to 15 seedlings per 150-plant tray). Following removal of diseased seedlings, further transplant mortality in the field was not evident. Samples of diseased tissue were collected, surface disinfested, and placed in petri dishes containing water agar. After 7 to 10 days of incubation at 25°C under fluorescent lights, an Alternaria sp. was growing from each sample. Single conidium cultures were obtained from representative colonies and cultured on potato dextrose agar (PDA) and potato carrot agar (PCA) for morphological examination. On PDA, colonies were dark olive brown, cottony, subsurface microsclerotia production abundant, and no production of pigments in the medium. On PCA, conidia were darkly pigmented, broadly ellipsoidal to subsphaerical, and produced singly. Mature conidia were 28 to 45 × 20 to 25 μm with two to four transepta and one to three longisepta. Characteristics were consistent with those of Alternaria petroselini (2,3). In subsequent freezer-blotter assays (ISTA blotter method; www.seedtest.org ) of seed lot used in the original planting, the same fungus was recovered at an infestation level of 30%, confirming that it was seedborne. To confirm pathogenicity on fennel, pathogenicity tests were conducted on a common fennel cultivar (Floro F1) in the greenhouse and on fennel stalks in the laboratory. Fennel seeds were soaked in a conidia suspension (106/ml in sterile H2O) for 10 min. Control seeds were soaked in sterile H2O. Seeds were dried on paper, sown in soil plugs, and grown in the greenhouse at 16 to 20°C. After 4 weeks, black lesions were observed on the fennel stems and symptoms were similar to those observed on the original infected material. Control plants remained healthy. A fungus was reisolated from the lesions of symptomatic plants and was identical to the fungus isolated from the original infected material. For the fennel stalk assay, two surface-sterilized fennel stalks were sliced longitudinally and three 4-mm plugs from a 10-day-old culture of each isolate were placed along the fennel stalks. Sterile agar plugs were used as negative controls. After 7 to 10 days of incubation at 25°C in plastic boxes, test isolates grew extensively from agar plugs and resulted in extensive black necrosis of the fennel stalks. No necrosis resulted from control plugs. DNA was extracted from field isolates, and the nuclear internal transcribed spacer region was sequenced using protocol previously described (1). A representative sequence was deposited in GenBank (Accession No. EF636901). A BLAST search of the NCBI database revealed A. petroselini Accession No. AY154685 as the closest match (total score = 1,014, 100% coverage, 99% sequence identity). The next closest match was A. radicina Accession No. DQ394074 (total score 987, 100% coverage, 98% sequence identity). To our knowledge, this is the first report of A. petroselini causing disease of fennel and the fungus being seedborne on fennel seed. An isolate has been deposited at the Centraalbureau voor Schimmelcultures (Accession No. 118228).

Phoma tracheiphila

<i>Phoma tracheiphila</i>

Avaliação da produção de melanina por espécies de Cryptococcus em quatro diferentes meios de cultura

The capacity of Cryptococcus spp to produce melanin in media containing phenol compounds is widely used for identifying these species in the laboratory. The aim of the present study was to compare the production of this pigment by Cryptococcus spp. in four culture media. Sixteen strains of Cryptococcus neoformans, 17 of Cryptococcus albidus, 13 of Cryptococcus laurentii and two of Cryptococcus uniguttulatus were tested in the following media: potato-carrot agar, Niger seed agar, sunflower seed agar and L-dopa agar. The melanin production was evaluated on the basis of colony pigmentation. Its production after five days of incubation was demonstrated by 93.8% of the strains of Cryptococcus neoformans in the media of potato-carrot agar, sunflower seed agar and L-dopa agar. From the isolates of Cryptococcus albidus, 29.4% produced the pigment in potato-carrot agar and L-dopa agar, 11.8% in Niger seed agar and 36% in sunflower seed agar. From Cryptococcus laurentii, 53.8% produced the pigment in potato-carrot agar and sunflower seed agar, 61.5% in L-dopa agar and 84.6% in Niger seed agar. Only one strain of Cryptococcus uniguttulatus presented slight production of the pigment, in potato-carrot agar.

First Report of Pythium intermedium Causing Root Rot on Rosa canina Rootstock in France.

Old cultivars of roses (Rosa spp.) established on Rosa canina as rootstock are grown in field collections in the repository at Gaillard in eastern France, close to the Swiss border. During June 2005 and July 2006, several rose plants in full vegetation were severely wilted, leading ultimately to plant death. Necrotic lesions were present on the roots, and marginal tissue was excised and incubated in the dark at 20°C on PARP V8 agar plates (1). Similar isolates were obtained in both years, which were inoculated within 4 days on V8 agar medium and incubated at 25°C. The pathogen colonies grew approximately 30 mm daily on V8 agar. Colonies on corn meal agar (CMA) (1) were submerged, with or without a vague radiate pattern on potato carrot agar. Main hyphae were as much as 7 μm wide. Sporangia and zoospores were not produced. Hyphal swellings as much as 25 μm in diameter were abundant, terminal, and sometimes intercalary, often forming regular, dense chains in basipetal succession at hyphal tips and readily liberated. The morphological characters matched those described for Pythium intermedium (2). DNA sequencing of the ITS rDNA (ITS1, ITS2, and 5.8S rDNA) was generated using primers ITS5 and ITS4 in primary PCR (35 cycles: 96°C for 1 min, 55°C for 1 min, and 72°C for 2 min) and sequencing reactions (1). All isolates yielded identical ITS sequences, which were identical to the ex-type strain of P. intermedium, CBS 266.38 (GenBank Accession No. AY598647). The ITS sequence was deposited as GenBank Accession No. EF078693 (National Center for Biotechnology Information, NCBI, Bethesda, MD). Pathogenicity testing was performed on 3-year-old Rosa canina rootstocks from clonal propagation in 25 cm in diameter and 40 cm deep pots of compost 297 (Ricoter, Aarberg, Switzerland). Five-day-old mycelial plugs of our isolates grown on CMA (1) were inoculated in two pots, and roots were mechanically wounded. Sterile plugs were used on two control plants set aside. All plants were kept for 2 weeks at 20°C in a glasshouse with sufficient watering. Extensive necrotic lesions developed on inoculated roots, and severe wilting and leaves drying out were also observed. The controls showed no symptoms. P. intermedium was reisolated from margins of root lesions, thus fulfilling the postulates of Koch. P. intermedium was originally isolated from dead plant material (2) but is known as a typical soil inhabitant. It also has been isolated from water and living material from numerous plant species, but never from the genus Rosa until now. To our knowledge, this is the first record of P. intermedium on Rosa spp. grafted on Rosa canina in France.

A new species, Embellisia astragali sp. nov., causing standing milk-vetch disease in China

A new species, Embellisia astragali sp. nov., is described from necrotic leaves, petioles and stems of Astragalus adsurgens in China. The morphology of E. astragali is compared and contrasted to that of four similar species, E. abundans, E. oxytropis, E. phragmospora and E. telluster. The fungus grew intercellularly in stems and leaf blades and intracellularly in leaves. It was isolated from most sources of seeds at frequencies of 0.1–44.6%. Growth rates of colonies on potato-carrot agar, potato-dextrose agar, wheat hay decoction (WHDA) and V8 at 25 C were 0.48, 0.32, 0.68 and 0.27 mm d−1, respectively. The optimal temperature for colony growth on WHDA was 20–25 C, and no growth was measured above 30 C. Five week old standing milk-vetch seedlings were inoculated with E. astragali by dipping whole roots and pruned roots in a conidial suspension and pouring the suspension onto the soil surface in which two seedlings had been planted. After 20 wk 66.5%, 62.1% and 85.0% plants were diseased and 24.1% , 20.7% and 17.5% plants were dead, respectively. Symptoms included the development of more side shoots with small, curved, necrotic and yellowed young leaves, plant stunting, reddish brown lesions, stem browning, dieback, shoot blight, crown rot, root black rot and plant death. This is first report of a pathogenic Embellisia on legumes.

A new species, Embellisia astragali sp. nov., causing standing milk-vetch disease in China

A new species, Embellisia astragali sp. nov., is described from necrotic leaves, petioles and stems of Astragalus adsurgens in China. The morphology of E. astragali is compared and contrasted to that of four similar species, E. abundans, E. oxytropis, E. phragmospora and E. telluster. The fungus grew intercellularly in stems and leaf blades and intracellularly in leaves. It was isolated from most sources of seeds at frequencies of 0.1-44.6%. Growth rates of colonies on potato-carrot agar, potato-dextrose agar, wheat hay decoction (WHDA) and V8 at 25 C were 0.48, 0.32, 0.68 and 0.27 mm d(-1), respectively. The optimal temperature for colony growth on WHDA was 20-25 C, and no growth was measured above 30 C. Five week old standing milk-vetch seedlings were inoculated with E. astragali by dipping whole roots and pruned roots in a conidial suspension and pouring the suspension onto the soil surface in which two seedlings had been planted. After 20 wk 66.5%, 62.1% and 85.0% plants were diseased and 24.1%, 20.7% and 17.5% plants were dead, respectively. Symptoms included the development of more side shoots with small, curved, necrotic and yellowed young leaves, plant stunting, reddish brown lesions, stem browning, dieback, shoot blight, crown rot, root black rot and plant death. This is first report of a pathogenic Embellisia on legumes.