First Report of Choke, Caused by Epichloe typhina, on Orchardgrass in Oregon.

During July 1997, Epichloe typhina (Pers.:Fr.) Tul. in Tul. & C. Tul., the cause of choke disease, was found in four fields of an unnamed, experimental cultivar of orchardgrass (Dactylis glomerata L.) grown for seed near Halsey, OR. Disease occurrence in each of three fields was estimated by counting choked tillers in about 50 quadrats, 1 × 0.3 m, taken at 30-m intervals along three or four diagonal transects. In two fields, the disease was present in most quadrats (3% tillers infected). In the third field, choke was clustered in two areas, each with 1 to 8% infected tillers. A collection of E. typhina was deposited at the Oregon State University Mycological Herbarium (accession number 56,395). The disease had not been previously observed in commercial cultivars grown for seed in Oregon, with the exception of an infected tiller collected from an orchard-grass seed field during 1996. This is the first report of choke in Oregon on orchardgrass. Choke is an important disease in France, where it reduces seed yields of orchardgrass. Ten Oregon cultivars of orchardgrass were evaluated under field conditions in France in 1993 and 1994 for susceptibility to E. typhina. All cultivars were found susceptible to the disease; incidence of infected tillers ranged from 4 to 11%, with a mean of 7% (G. Sicard and R. E. Welty, unpublished). During 1996, several fragments of stroma of E. typhina were found among seed from a seed lot submitted to the Oregon State University Seed Lab for purity testing. This indicates that stroma may occur as a contaminant with seed, although it is not known if E. typhina would survive with the seed. E. typhina has not been reported to be seed-borne in orchardgrass.

Improved extraction and HPLC methods for ergovaline from plant material and rumen fluid.

Ergovaline, the main ergopeptine alkaloid produced in tall fescue infected with Acremonium coenophialum, is known to cause tall fescue toxicosis. Current methods in use for quantifying ergovaline in plant material have several disadvantages, including large solvent volumes and long analysis time. We report here improvements in extraction and cleanup and the high-pressure liquid chromatographic methods. Our improvements include a 24-hour extraction time, the use of smaller solvent volumes during sample preparation, and fast analysis on the polymeric reverse-phase column. In addition to allowing the analysis of large batches to assist practitioners in the accurate diagnosis of fescue toxicosis, our method is also easily modified for other matrices, such as rumen fluid.

Fungi isolated from flue-cured tobacco sold in Southeast United States, 1968-1970.

Flue-cured tobacco leaves, from low- and middle-stalk positions, offered for sale in each of two markets, within each of five tobacco types, were evaluated for moisture content (MC) and filamentous fungi during August through October in 1968, 1969, and 1970. Alternaria alternata, Penicillium cyclopium, Aspergillus niger, Aspergillus repens, and Aspergillus flavus were most frequently isolated from cultured tissue. Other filamentous fungi that grew from the tissue included species from four genera of field fungi and seven species of storage fungi. Although the MC ranged from 11.0 to 22.5%, it averaged 16.4, 16.8, and 15.9% for samples taken in 1968, 1969, and 1970, respectively. Average populations of fungi per sample over the three years ranged from 0 to 1,528,500 colonies/g of tobacco.

Antifungal spectra of actinomycetes isolated from tobacco.

Five species (28 strains) of actinomycetes isolated from tobacco were tested for antagonism against 12 species of storage and field fungi associated with tobacco. Two strains of Streptomyces albus were antagonistic against all test fungi. The actinomycetes grew more rapidly, produced more pigment, and had more pronounced antibiotic activity when grown at 36 C than at 28 C. Krasilnikov's synthetic medium, SMK-1, supported the greatest antifungal activity. More of the actinomycetes were antagonistic against more test fungi when grown for 20 days rather than 10 days.

Growth of Aspergillus repens in Flue-Cured Tobacco.

In laboratory tests, flue-cured tobacco inoculated with Aspergillus repens was stored at 75, 80, 85, 87, and 95% relative humidity at 20 and 30 C. Samples were taken weekly for 4 weeks and evaluated for mold growth (colony count) and moisture content (MC). The weekly rate of fungus increase was slower at 20 C than at 30 C. Tobacco at 20 C with MC between 25 to 30% supported a slight to moderate increase in A. repens after 3 weeks of storage. However, tobacco at the same MC stored at 30 C was subject to rapid invasion by the fungus in as few as 1 to 2 weeks. Tobacco with MC above 30% stored at either 20 or 30 C became moldy in about 1 week. A mold index is proposed for evaluating populations of A. repens in tobacco.

Fungi isolated from flue-cured tobacco inoculated in the field with storage fungi.

Flue-cured tobacco inoculated in the field with A. amstelodami, A. flavus, A. ochraceus, A. repens, A. ruber, and a species of Penicillium was rarely invaded by these fungi. Regardless of inoculum, the predominant fungi reisolated from green tissue were species of Alternaria and Cladosporium. After curing, A. repens, A. niger, and species of Alternaria and a species of Penicillium were the most commonly isolated fungi. The fungus used as inoculum was not the predominant fungus reisolated from green or cured tissue. Conditions during handling and storage prior to marketing probably determine when storage fungi become associated with the leaf and which species becomes predominant.

Mouse toxicty of fungi of tobacco.

A bioassay for fungal toxins based on the intraperitoneal injection of test materials into mice was used to screen 976 cultures isolated from tobacco and grown in a high-protein baby cereal and also to determine whether samples of tobacco damaged by fungi are more toxic than samples of apparently sound tobacco. Of 236 fungal isolates from noncured tobacco, 79% were lethal when homogenized cultures of these isolates were tested. Forty-nine per cent of 740 fungi isolated from cured tobacco were lethal. Of the genera from which 30 or more isolates were tested, Epicoccum, Alternaria, and Penicillium had the highest percentage of toxic isolates from non-cured tobacco, whereas Epicoccum, Aspergillus, and Alternaria had the highest percentage from cured tobacco. Samples of tobacco naturally infected with brown spot, caused by Alternaria tenuis, did not have a significantly different LD(50) value after 48 hr than comparable disease-free samples. However, animals which died from doses near the LD(50) dose of tobacco infected with Alternaria generally died in 24 to 48 hr with signs associated with a depressant rather than a stimulant, such as nicotine, which caused death in 15 to 30 min. These signs were duplicated by injecting homogenized pure cultures of Alternaria. These studies, although inconclusive with regard to the effects of fungal contaminants on the quality or usability of tobacco, have developed evidence that suggests the advisability of a study on smoke or smoke condensates from moldy and nonmoldy tobacco.

Fungi Isolated from Flue-cured Tobacco at Time of Sale and After Storage.

The fungi isolated from 100 samples of flue-cured tobacco from 12 markets in 2 tobacco belts comprised 11 genera, including 10 species of Aspergillus. The mean percentage per sample isolated from 62 samples of tobacco from Middle Belt markets was Alternaria, 40.6%; Aspergillus niger, 47.8%; Aspergillus repens, 38.0%; and Penicillium, 25.8%. The mean percentage per sample isolated from 38 samples of tobacco from Old Belt markets was Alternaria, 74.0%; Penicillium, 52.5%; Aspergillus repens, 38.0%; and Aspergillus ruber, 36.2%. Damaged (74 samples) and nondamaged (26 samples) stored tobacco yielded species of six genera of fungi, including eight species of Aspergillus. Species of Aspergillus and Penicillium were commonly isolated from both damaged and nondamaged tobacco, whereas species of Alternaria, Cladosporium, Fusarium, and Rhizopus were isoalted more frequently from nondamaged tobacco. The fungi that occurred in the highest population in damaged tobacco were Aspergillus repens, A. niger, A. ruber, and Penicillium species.

Fungi isolated from tobacco leaves and brown-spot lesions before and after flue-curing.

Discs of tissue excised from ripe nonflue-cured and flue-cured tobacco leaves, harvested on six dates and cultured on three selective media, yielded 21 and 24 genera of fungi, respectively. Of 5,094 fungi isolated from 3,240 pieces of nonfluecured leaf tissue, 89.5% comprised five genera, Alternaria, Cladosporium, Epicoccum, Trichoderma, and Nigrospora. Of 2,494 fungi isolated from 3,240 pieces of flue-cured leaf tissue, 70.9% were Alternaria, Cladosporium Epicoccum, Aspergillus, and Nigrospora. Flue-cured and nonflue-cured brown-spot lesions harvested at two locations yielded 12 and 14 genera of fungi, respectively. Alternaria, Penicillium, Phoma, and Stemphyllium comprised 91.5% of the 2,245 fungi isolated from noncured and 87.1% of the 1,118 fungi isolated from the cured lesions. The number and kinds of fungi obtained from diseased and healthy tissue were reduced but not eliminated by flue-curing.

Fungi Isolated from Damaged Flue-cured Tobacco.

Species of Aspergillus were the most prevalent fungi isolated from 51 samples of damaged flue-cured tobacco of the 1966 U.S. crop, comprising 57% of the total isolates. Other prevalent fungi were Penicillium (16%), Alternaria (8%), Cladosporium (4%), and Chaetomium (4%). Members of the Aspergillus glaucus group were isolated most frequently from samples with moisture contents ranging from 18 to 28%, whereas Alternaria, Cladosporium, and Penicillium were isolated consistently from samples containing 24 to 32% moisture. Aspergillus niger was prevalent in tobacco ranging in moisture content from 18 to 30%.