V8 Agar Medium Research Articles

Geographic distribution of mating type alleles of Cryptococcus neoformans in four areas of the United States.

To better understand the epidemiology and population structure of Cryptococcus neoformans, we determined mating types for 358 C. neoformans strains isolated through the active surveillance program from 1992 to 1994 in four geographic areas in the United States: San Francisco, California; Georgia; Texas; and Alabama. Two assays were used to determine mating types: (i) crossing with standard laboratory tester strains JEC20 and JEC21 on V8 agar medium; and (ii) PCR with the mating type alpha allele-specific primer of the STE12 gene and with serotype (A and D)- and mating type (a and alpha)-specific primers of the STE20 gene. Using these two methods, we found that this sample consisted of the following: (i) 324 serotype A, mating type (MAT) alpha (Aalpha) strains; (ii) 12 serotype D, alpha (Dalpha) strains; (iii) 14 serotype AD strains with mating type alleles Aa and Dalpha (AaDalpha); (iv) 2 serotype AD strains with mating type alleles Aalpha and Da (AalphaDa); (v) 3 serotype B, alpha (Balpha) strains; and (vi) 3 serotype AD strains but with only one mating type allele. No strain with MATa was found within serotype A, B, or D in this collection. Interestingly, 14 of the 19 serotype AD strains contained the Aa allele at the STE20 locus; 13 of these 14 were from San Francisco. Our results suggest that the environment in San Francisco might contain Aa strains capable of mating with Dalpha strains. In addition, our result demonstrate that the sample from San Francisco had a significantly higher proportion of self-fertile strains than those from the other three areas.

Indole acetic acid attenuates disease severity in potato- Phytophthora infestans interaction and inhibits the pathogen growth in vitro

Auxins are implicated in a wide variety of developmental and physiological processes in plants. Here, we studied the role of auxins on the interaction between a plant pathogen and its host. Excised potato leaves were pre-treated with different IAA concentrations and inoculated with a compatible race of the late blight agent Phytophthora infestans. A 10-μM IAA-treatment resulted in a 50 % reduction of disease severity, the extent of the protection being dose-dependent. In potato leaves upon inoculation with P. infestans, β-1,3 glucanase, chitinase and extensin mRNA levels were down-modulated by 10 and 100 μM IAA. IAA also showed an antimicrobial activity in vitro. A 45 % growth inhibition of the P. infestans mycelia was observed in V8-agar medium containing 1 μM IAA. Again, the percentage of inhibition was dose-dependent. A 60 % depletion of fungal β-1,3 glucanase activity was found in extracts from P. infestans growing in a medium containing 1 μM IAA. Furthermore, the IAA-mediated inhibitory effect of P. infestans mycelium growth was overcome by supplying the media with 1 ng·mL –1 external β-1,3 glucanase 〚(1,3;1,4)β-D-glucan 3(4)-glucano hydrolase〛. Thus, an IAA dose-dependent correlation can be established between inhibition of fungal growth, and down regulation of plant and fungal β-1,3 glucanase. These results support a putative role of auxins in the potato- P. infestans patho-system as natural defence for pathogen spread and disease development.

First Report of Blight of Field Peppers Caused by Phytophthora capsici in Ontario.

In August 1994, a disease of sweet peppers (Capsicum annuum L.) and butternut squash (Cucurbita pepo L.) was observed in a 2-ha field near Harrow, Essex County, ON, Canada. In 1995, a similar disease was noted on peppers at two locations 30 km apart in Essex County. In 1997, the disease occurred on peppers in a 20-ha field in the vicinity of the 1994 outbreak. Yield loss was estimated at 40 to 60% in pepper fields and 20% in the affected squash field. Brown, necrotic lesions were more prevalent on pepper fruits and upper stems and lateral branches than basal stems. Affected plants occurred in lower areas of fields that had been flooded by rain or irrigation. Gray, floccose masses of sporangia were evident on fruits and stems under humid conditions and on the internal surfaces of infected fruits. Symptoms were similar to those described for Phytophthora blight of pepper (1). Squash infections occurred where fruits contacted soil. Isolations were made from sections of fruit, stems, and leaves of pepper and squash plants with symptoms of disease on lima beans (Difco Laboratories, Detroit) or 20% V8 agar medium and incubated at 22°C. Phytophthora capsici was readily isolated from all plants with disease symptoms. Observations of colony morphology and growth were made on cultures on 20% V8 agar at 25°C under continuous fluorescent light. Sporangia were papillate and averaged 45 ± 5.9 × 27 ± 3.5 μm in size (range 28 to 58 × 21 × 39 μm). Oospores were spherical and 23 ± 2.9 μm in diameter (range 16 to 28 μm) and, when the external wall was included, were 28 ±2.7 μm in diameter (range 23 to 37 μm). Pedicels varied in length, averaging 63 ± 30.9 μm (range 9 to 129 μm). These observations are similar to those described for P. capsici (2). Mating type was determined by coculture with isolates obtained from A. F. Schmitthenner (OARDC, Wooster, OH) designated A1 and A2. Oospore development was determined after 10 days growth at 25°C on 20% V8 agar. Mating types A1 and A2 occurred among Ontario isolates from pepper and squash. In 1995, 13 of 15 isolates tested were A1, and in 1997, 1 of 5 was A1. Both mating types were found in the same field. Pathogenicity of pepper and squash isolates was tested by inoculating greenhouse-grown pepper cvs. Merlin and North Star at the 5-leaf stage by adding 5 ml of a spore suspension (1,000 sporangia per ml) to the crown and adjacent soil or sprayed on the foliage until run off. Plants were covered in plastic bags for 24 h. Wilting and plant death occurred at 4 and 10 days, respectively, with both cultivars. Crown-inoculated plants wilted prior to development of brown lesions on lower stems at the soil line. Symptoms on foliar-inoculated plants were first observed on young tissue at growing points and stem nodes. P. capsici was reisolated from affected tissue. This is the first report of Phytophthora blight of sweet pepper in Ontario.

Nitric oxide preserves the level of chlorophyll in potato leaves infected by Phytophthora infestans

Nitric oxide (NO) is a bioactive molecule involved in many physiological processes. Among its biological function, NO has been proved to be cytotoxic against microorganisms in cells of the immune response, thus preventing infection. We have specifically studied the effect of a NO donor, sodium nitroprusside (SNP), on the chlorophyll content in potato leaves infected with the pathogenic fungus Phytophthora infestans (Pi). Fifteen days after infection, chlorophyll content strongly decayed in water-treated potato leaf sections. SNP was able to partially revert that loss in a dose-dependent manner, being the effective SNP concentrations between 10 µM and 100 µM. NaNO2 and NaNO3, the SNP-derived residual products, were unable to prevent the chlorophyll loss. Treatments with SNP did not affect the survival of Pi and the fungus was able to grow in a V8-agar medium containing 100 µM SNP. Both the amount and the extent of germination of Pi sporangia resulted similar in the absence and in the presence of SNP. Respiratory inhibitors of the cyanide-sensitive and cyanide-resistant pathways, 2,4-dinitrophenol and salicylhydroxamic acid respectively, did not change the chlorophyll levels in infected potato leaves, suggesting that NO effect should not be on mitochondrial respiration. These results indicate that NO could be a protective molecule, either preserving the chloroplast membrane of infected leaf sections against the toxicity of reactive oxygen species or being directly involved in any step of the chlorophyll metabolic pathway.

In vitro culture of Ascosphaera aggregata (Ascosphaeraceae), a pathogen of the alfalfa leafcutting bee Megachile rotundata (Apidae)

A substrate made of two media, one for initiation of vegetative growth, the other for induction of spore formation, was developed to culture Ascosphaera aggregata, a fungal pathogen of the alfalfa leafcutting bee Megachile rotundata. Plugs from the first medium were placed in voids made in the second medium. Components of both media included low-sodium V8 juice, sucrose, yeast extract, vegetable shortening, agar, thiamine, biotin, magnesium sulfate, and Grace's insect medium without hemolymph. Both media were buffered to pH 6 with potassium phosphate buffer. The composite substrate (referred to as the plug/base medium) supported growth and development of A. aggregata from spore to spore. Koch's postulates were verified. Larvae infected with the in vitro produced spores exhibited the typical characteristics of chalkbrood disease. There was no significant difference ( P = 0.6818) in infection frequency between first instar larvae challenged with either in vitro cultured spores or inoculum from field-collected cadavers.

Characteristics of induced dicarboximide fungicide-resistant strains conferred by filtrates of the resistant strains of Botrytis cinerea.

The characteristics of induced dicarboximide fungicide-resistant strains of Botrytis cinerea developed by post-cultures in the filtrates of the field resistant strains were investigated. Mycelial growth curves of these induced resistant strains were divided into three groups, which were rapid in both radial and non-radial growth, slowed down in growth transiently at about 4 days of inoculation followed by fast growth again, and poor in nonradial growth. Sporulation was scarcely observed on PSA, YGA and V8 agar media except for PSA media containing cucumber leaf (Cu-PSA). But by illuminating with BLB, it was stimulated on PSA media, but not on the other media. Pathogenicity to cucumber plants was examined by cucumber leaf disc method with the mycelia cultured on distilled water agar (WA), WA media containing glucose (GA) and GA containing inosine (IGA), respectively. Any induced resistant strain cultured on WA did not form lesion on cucumber leaf discs. Six strains cultured on GA formed non-spreading lesions (disease incidence rate; DIR=2.5-7.5%), and one strain formed spreading lesion (DIR=37.5%) on the discs. Six strains cultured on IGA formed non-spreading lesions (DIR=0-17.5%), and other ten strains formed spreading lesions (DIR>42.5%), in which four strains showed high pathogenicity (DIR=80-100%). These characteristics of the induced strains had been stable through consecutive subcultures, and were different from those of the original strains of B. cinerea.