Screening of antifungal activity of 12 essential oils against eight pathogenic fungi of vegetables and mushroom.

The antifungal properties of 12 Eos, that is, Syzygium aromaticum, Pelargonium graveolens, Lavandula angustifolia, Cupresus sempervirens, Mentha piperita, Santolina chamaecyparissus, Citrus sinensis, Pogostemon patchouli, Thymus mastichina, Thymus vulgaris, Eucalyptus globulus and Rosmarinus officinalis, were screened. The influence of five doses of each EOs was tested against Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium oxysporum, Phytophthora parasitica, Pythium aphanidermatum, Alternaria brassicae, Cladobotryum mycophilum and Trichoderma aggressivum f.sp. europaeum using disc-diffusion method. The mycelial growth inhibition and ED50 were calculated. The chemical analysis of the EOs was analysed using gas chromatography-mass spectroscopy. A total of 58 compounds were identified in the 12 EOs. All essential oils (EOs) analysed showed antifungal activity against the test pathogens in the range of 5·32-100%. The inhibitory effect of oils showed dose-dependent activity on the tested fungus. Based on the ED50 values, clove, rose geranium, peppermint and patchouli were the most effective. This study warrants further research into the practical use of EOs for the control of important myco- and phytopathogens in intensive horticulture. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the serious damage caused by fungal pathogens of vegetables and mushrooms, it is necessary to search for integrated strategies of disease control. This study provides relevant information about the effects of 12 essential oils (EOs) against eight pathogens of agricultural interest, included mycopathogens with emphasis on the possible future application of the EOs as alternative antifungal agents.

First Report of Cladobotryum mycophilum Causing Cobweb on Cultivated King Oyster Mushroom in Spain.

In 2010, symptoms of cobweb were observed on cultivated king oyster mushroom (Pleurotus eryngii) in Castilla-La Mancha (Spain) affecting 16% of the blocks of substrate cultivated. Cobweb appeared at the end of the crop cycle, first as small, white patches on the casing soil, subsequently spreading to the nearest king oyster mushroom by means of a fine gray-white mycelium, and eventually sporulating to produce masses of dry spores. The mycelium can quickly cover pinheads, stalks, pileus, and gills, eventually resulting in decomposition of the entire fruit body. Infected tissues of P. eryngii were plated onto potato dextrose agar (PDA) and the parasitic fungus was isolated. Fungal colonies consisted of abundant and cottony aerial mycelium spreading rapidly on PDA and red pigment spreading in the agar. Conidiogenous cells were 24 to 35 µm long, 3.5 to 5 µm wide basally, and tapered slightly to the tip. Conidia were cylindrical to narrowly ellipsoidal, 17 to 25 (-28) × 8 to 10 µm, and zero to three septate. Total DNA was extracted and the internal transcribed spacer (ITS) region of rDNA was amplified for one isolate using ITS1F/ITS4 primers (1,3). The amplicon was sequenced (GenBank Accession No. JF505112). BLAST analysis showed 100% similarity of the obtained ITS sequence with two sequences of Cladobotryum mycophilum (teleomorph Hypomyces odoratus) (GenBank Accession Nos. Y17096 and Y17095) (2). Pathogenicity tests were performed using 24 blocks containing sterilized, spawned, and incubated P. eryngii substrate (3.6 kg, 352 cm2 in area). The blocks were placed in a mushroom-growing room and cased with a 40-mm layer of a casing soil (0.7 liter block-1) made with mineral soil + Sphagnum peat 4:1 (vol/vol). Five days after casing, a conidial suspension (7 × 103 conidia ml-1) of one isolate of C. mycophilum was sprayed (5 ml per block) onto the surface of the casing layer at a rate of 106 conidia m-2. Twenty-two blocks were sprayed with sterile distilled water as a control. A temperature of 17 to 18°C and 85 to 90% relative humidity were maintained throughout cropping. The first cobweb symptoms developed 23 days after inoculation and C. mycophilum was consistently reisolated from nine (37.5%) of the inoculated blocks. Noninoculated blocks remained healthy. In a second test, conidial suspensions (3.4 × 105 conidia ml-1) of one isolate of C. mycophilum were inoculated onto 20 P. eryngii fruit bodies. Ten fruit bodies were inoculated externally while the other 10 fruit bodies were cut in half and inoculated internally with 50 µl of conidial suspension per fruit body. Sterilized distilled water was used as a control. All fruit bodies were then incubated at 22°C in a moist chamber. Assays were conducted twice and the results were recorded after 7 days. C. mycophilum grew on 85% of the internally inoculated fruit bodies and on 40% of those inoculated superficially, while the control mushrooms remained symptomless. To our knowledge, this is the first report of C. mycophilum causing cobweb in king oyster mushroom in Spain. This finding will have a potentially significant impact on button mushroom farms where cobweb is one of the most common diseases. References: (1) M. Gardes and T. D. Bruns. Mol. Ecol. 2:113, 1993. (2) G. J. McKay et al. Appl. Environ. Microbiol. 65:606, 1999. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Brown Blotch Caused by Pseudomonas tolaasii on Cultivated Pleurotus eryngii in Spain.

In 2006, symptoms of brown blotch were observed on cultivated Pleurotus eryngii (king oyster mushroom) in Castilla-La Mancha (Spain). Subsequently, between January and May of 2008, brown blotch affected 39.75% of the blocks of substrate cultivated, resulting in a considerable loss of production. Symptoms observed were principally characterized by a yellowish brown-to-orangish color, first of the cap and then of the stalk. Some samples also showed a slightly concave cap. From samples collected from four different king oyster mushroom farms, a fluorescent gram-negative bacterium was recovered on King's B medium and identified as Pseudomonas tolaasii by the LOPAT scheme and other tests (2). The bacterium isolated had the following characteristics: oxidative, positive for oxidase and arginine dihydrolase, negative for levan production, pectinolitic activity on potato slices, and tobacco hypersensitivity. Results from other tests were as follows: negative for esculin hydrolysis and positive for gelatine, casein, and Tween 80 hydrolysis; mannitol, erythritol, sorbitol, m-inositol, and adonitol were used as a sole carbon source, but not sucrose, d-tartrate, or trigonelline. The white line test was performed (4) using P. reactans LPPA 540 and the presumptive isolates of P. tolaasii were positive. The gene encoding the 16S rRNA from two isolates (LPPA532 and LPPA533) was sequenced after PCR amplification (2) and their nucleotide sequences (1,400 bp; EMBL Accession No. FM864215 for LPPA 532) proved to be identical. The amplified sequences were compared with DNA sequences available in databases (GenBank, EMBL, DDBJ, and PDB) by using BLAST. An identity of 99% was obtained with 16S rDNA of three P. tolaasii strains (GenBank Accession Nos. AF320990, AF094750, and AF255336). Four isolates were selected for pathogenicity tests. Bacterial suspensions were grown for 16 h in yeast peptone glucose broth (approximately 108 CFU/ml) and were inoculated by puncture into 10 mushroom caps using sterilized wooden toothpicks (4). Sterilized distilled water was used as a control. These were then incubated at room temperature in glass dishes. Assays were conducted twice and the results were recorded after 10 days. The symptoms that developed after infection were similar to those observed in the crop, while the control mushrooms remained symptomless. Bacteria sharing the characteristics of the inoculated isolates were recovered from symptomatic caps. P. tolaasii has been described as causing brown blotch on Pleurotus eryngii (1,3), but to our knowledge, this is the first report of P. tolaasii causing brown blotch on Pleurotus eryngii in Spain. References: (1) J. F. Bradbury. No. 891 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1987. (2) A. J. González et al. Appl. Environ. Microbiol. 69:2936, 2003. (3) A. Russo et al. Microbiol. Res. 158:265, 2003. (4) J. M. Wells et al. Phytopathology 86:1098, 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).