Effects of a DNA vaccine in an animal model of Alternaria alternata sensitivity

Sensitivity to the fungus Alternaria is associated with asthma persistence and severity. Current therapeutic options for treating Alternaria-induced airway inflammation are limited. In this study, Brown Norway rats are used to study the effectiveness of a DNA-based vaccine delivered to the airway in attenuating the response to a major Alternaria allergen, rAlt a 2. Compared to untreated sensitized animals, or animals receiving an “out-of-frame” DNA-based vaccine, animals treated with “in-frame” DNA vaccine showed an attenuation in specific IgE antibody titers to rAlt a 2, an increase in IgG^2b (a Th1 response), a reduction in spontaneous IL-13 release by peribronchial lymph node cell suspensions, and an attenuation in the decrease in total lung capacity 72 h post-allergen challenge. Further, histopathologic examination of the lung tissues revealed reduced pulmonary inflammation post-allergen challenge in the DNA-vaccine-treated compared to sensitized, untreated animals. We conclude that a DNA-based vaccine delivered to the airway significantly influences the immunologic, pulmonary physiologic, and histological alterations induced by challenge with a major Alternaria allergen, rAlt a 2, in sensitized animals.     

Volatilization of Selenium by Alternaria alternata

Seleniferous water continues to be a serious problem to wildlife in the central valley of California. Water samples collected from Kesterson Reservoir, Peck Ranch, and Lost Hills evaporation pond facilities contained between 0.005 and 5 mg of Se per liter. The objective of this study was to isolate Se-methylating organisms in evaporation pond water and to assess, through enrichment and manipulation of their optimal growth parameters, the environmental factors which govern microbial Se methylation. Alternaria alternata was isolated as an active Se-methylating organism. The volatile product was identified as dimethylselenide. The effects of pH, temperature, Se substrates, and methyl donors on the ability of A. alternata to methylate Se were investigated in liquid medium containing 100 mg of Se per liter. The optimum pH and temperature for methylation were 6.5 and 30 degrees C, respectively. Selenate and selenite were methylated more rapidly than selenium sulfide and various organic Se compounds (6-selenoguanosine, 6-selenoinosine, seleno-dl-methionine, and 6-selenopurine). l-Methionine and methyl cobalamine (0.1 muM) stimulated dimethylselenide production. This study demonstrates that Se-methylating organisms are present in evaporation pond water and are capable of liberating substantial quantities of Se in the volatile dimethylselenide form. By determining the optimum environmental conditions which stimulate volatilization, it may be possible to design a way to remove Se from seleniferous water in situ.     

Interstrain spheroplast fusion of Alternaria alternata

Summary Auxotrophic and drug resistant colonies of Alternaria alternata were selected following UV mutagenesis of spheroplasts and genetic transformation with pDH25. Intrastrain cell fusion of certain A. alternata parental strains induced by polyethylene glycol occurred at an average rate of 0.35%; interstrain fusions occurred at a rate of 0.08%. Mitotic recombination resulted from UV mutagenesis of spheroplasts from several fusants from 6hy1 × 1ar1. Fusants synthesized different levels of the cyclic tetrapeptide, tentoxin; some colonies produced higher levels than either parent. These results demonstrate that spheroplast fusion may have a potential application for genetic analysis of secondary metabolite production and for strain improvement in A. alternata.Mention of a trademark of proprietary product does not constitute a guarantee or warranty by the U. S. Department of Agriculture and does not imply approval to the exclusion of other products that may also be suitable.     

Two cases of cutaneous phaeohyphomycosis by Alternaria alternata and Alternaria tenuissima

Two cases of cutaneous phaeohyphomycosis, one with a nodular appearance and the other with an erythematous infiltrating patch, are reported in immunocompromised patients. Diagnosis was based on histological examination, which revealed hyphae and round-shaped fungal cells in a granulomatous dermal infiltrate, and on identification of the moulds when biopsy fragments were cultured on Sabouraud-dextrose agar without cycloheximide. The pathogens were Alternaria tenuissima in the first case and A. alternata in the second. The fungi were examined by scanning electron microscopy. The patients were checked for bone and lung involvement and were then treated with surgical excision and itraconazole, and itraconazole only, respectively, with clinical and mycological resolution. This revised version was published online in August 2006 with corrections to the Cover Date.     

A Cucumber Disease Caused by Alternaria alternata and its Control

A severe leaf spot disease of cucumber caused by a pathotype of Alternaria alternata (Fr.) Keissler was recorded recently in plastic houses in Crete. Lesions ranged in size of a pin point to over 5 cm in diameter, with necrotic tissue on most of their area and a surrounding yellow zone. The pathogen grew satisfactorily on PDA at temperatures between 5 °C–40 °C and spore germination occurred in the range less than 10 °C to over 37 °C. Optimum temperature in both cases was near 26 °C. Of,13 fungicides tested in vitro, sodium omadine, etem, dichlofluanid, captan and folpet were the most inhibitory on spore germination, and iprodione, sodium omadine and dichlofluanid on mycelial growth. Of 25 fungicides applied on two leaf cucumber plants 24 h before inoculation, maneb, etem, dichlofluanid and chlorothalonil were the most effective. When the last fungicides, plus mancozeb, were applied 24 h after inoculation only maneb was effective. In greenhouse experiments, iprodione, prochloraz-manganese-complex, chlorothalonil, dichlofluanid, guazatine, maneb and etem were the most effective for disease control, while mancozeb was less effective. The local cucumber cv. Knossos and the Dutch F₁ hybrids Evadan, Frella, Herta, Malfa, Mazourka, Pepinex 69 and Renova were all susceptible to infection.     

Tenuazonic acid production by Alternaria alternata and Alternaria tenuissima isolated from cotton

Cultures of Alternaria alternata (three isolates) and Alternaria tenuissima (three isolates) obtained from cottonseeds and bolls were toxigenic when cultured on various laboratory media. A mycotoxin was isolated and identified as tenuazonic acid by using solvent partition, thin-layer chromatography, and instrument analyses. Toxicity was monitored with brine shrimp and chicken embryo bioassays. All cultures except A. alternata 938 produced tenuazonic acid when grown on cottonseed and on yeast extract-sucrose broth. The most toxin (266 mg/kg) was produced by A. tenuissima 843 on cottonseed.     

Mycotoxins of Alternaria alternata produced on ceiling tiles

The production of mycotoxins by Alternaria alternata in cellulosic ceiling tiles was examined with thin-layer chromatography and high-performance liquid chromatography procedures. Alternariol and alternariol monomethyl ether were found in ceiling tile extracts, whereas extracts of control rice cultures of all three isolates produced these mycotoxins plus altenuene and altertoxin I. Extensive fungal growth and mycotoxin production occurred in the ceiling tiles at relative humidities of 84–89% and 97%. Received 28 May 1997/ Accepted in revised form 06 October 1997     

Nitrogen inhibition of mycotoxin production by Alternaria alternata

Alternaria alternata produces the polyketides alternariol (AOH) and alternariol monomethyl ether (AME) during the stationary growth phase. Addition of 12 mM NaNO3 to the cultures before initiation of polyketide production reduced the AOH and AME content to 5 to 10% of that of controls. Glutamate and urea also reduced AOH and AME accumulation, whereas increasing the ionic strength did not affect the polyketide content. Adding NaNO3 after polyketide production had started did not inhibit further AOH accumulation, although over 90% of the added NO3- disappeared from the medium within 24 h. Activity of an AME-synthesizing enzyme, alternariol-O-methyltransferase (AOH-MT), appeared in control mycelia during the early stationary growth phase. No AOH-MT activity appeared in mycelia blocked in polyketide synthesis by addition of NaNO3. Later addition of NaNO3 reduced the AOH-MT specific activity to 50% of that of the control, whereas the total of activity per mycelium was the same. The AOH-MT activity in vitro was not affected by 100 mM NaNO3. The results suggest that nitrogen in some way inhibited the formation of active enzymes in the polyketide-synthesizing pathway in A. alternata when it was added before these enzymes were formed.     

Bicycloalternarenes produced by the phytopathogenic fungus Alternaria alternata

Eleven compounds, ACTG toxins A and B and nine new substances named bicycloalternarenes (BCAs), were isolated and characterized from the culture filtrate of the phytopathogenic fungus Alternaria alternata. Under acidic conditions, these mixed terpenoids convert to pairs of tricycloaltenarenes that can be used for identification of the native compounds. We could not confirm the phytotoxic effects reported with ACTG-toxins in the past. However, BCA 2 at concentrations of 10(-4) M strongly inhibited germination and growth of Spirodela polyrhiza turions.     

Mannitol metabolism in the phytopathogenic fungus Alternaria alternata

Mannitol metabolism in fungi is thought to occur through a mannitol cycle first described in 1978. In this cycle, mannitol 1-phosphate 5-dehydrogenase (EC 1.1.1.17) was proposed to reduce fructose 6-phosphate into mannitol 1-phosphate, followed by dephosphorylation by a mannitol 1-phosphatase (EC 3.1.3.22) resulting in inorganic phosphate and mannitol. Mannitol would be converted back to fructose by the enzyme mannitol dehydrogenase (EC 1.1.1.138). Although mannitol 1-phosphate 5-dehydrogenase was proposed as the major biosynthetic enzyme and mannitol dehydrogenase as a degradative enzyme, both enzymes catalyze their respective reverse reactions. To date the cycle has not been confirmed through genetic analysis. We conducted enzyme assays that confirmed the presence of these enzymes in a tobacco isolate of Alternaria alternata. Using a degenerate primer strategy, we isolated the genes encoding the enzymes and used targeted gene disruption to create mutants deficient in mannitol 1-phosphate 5-dehydrogenase, mannitol dehydrogenase, or both. PCR analysis confirmed gene disruption in the mutants, and enzyme assays demonstrated a lack of enzymatic activity for each enzyme. GC-MS experiments showed that a mutant deficient in both enzymes did not produce mannitol. Mutants deficient in mannitol 1-phosphate 5-dehydrogenase or mannitol dehydrogenase alone produced 11.5 and 65.7 %, respectively, of wild type levels. All mutants grew on mannitol as a sole carbon source, however, the double mutant and mutant deficient in mannitol 1-phosphate 5-dehydrogenase grew poorly. Our data demonstrate that mannitol 1-phosphate 5-dehydrogenase and mannitol dehydrogenase are essential enzymes in mannitol metabolism in A. alternata, but do not support mannitol metabolism operating as a cycle.     

Nitrogen inhibition of mycotoxin production by Alternaria alternata.

Alternaria alternata produces the polyketides alternariol (AOH) and alternariol monomethyl ether (AME) during the stationary growth phase. Addition of 12 mM NaNO3 to the cultures before initiation of polyketide production reduced the AOH and AME content to 5 to 10% of that of controls. Glutamate and urea also reduced AOH and AME accumulation, whereas increasing the ionic strength did not affect the polyketide content. Adding NaNO3 after polyketide production had started did not inhibit further AOH accumulation, although over 90% of the added NO3- disappeared from the medium within 24 h. Activity of an AME-synthesizing enzyme, alternariol-O-methyltransferase (AOH-MT), appeared in control mycelia during the early stationary growth phase. No AOH-MT activity appeared in mycelia blocked in polyketide synthesis by addition of NaNO3. Later addition of NaNO3 reduced the AOH-MT specific activity to 50% of that of the control, whereas the total of activity per mycelium was the same. The AOH-MT activity in vitro was not affected by 100 mM NaNO3. The results suggest that nitrogen in some way inhibited the formation of active enzymes in the polyketide-synthesizing pathway in A. alternata when it was added before these enzymes were formed.     

Interactions of Apple and the Alternaria alternata Apple Pathotype

Apple is one of the most cultivated tree fruits worldwide, and is susceptible to many diseases. Understanding the interactions between the host and pathogen is critical in implementing disease management strategies and developing resistant cultivars. This review provides an update on the interactions of apple with Alternaria alternata apple pathotype, which causes Alternaria blotch, with a brief history about the discovery of the disease and pathogen and its damage and epidemiology. The focus of the review is placed on the physiological and genetic response of the host to pathogen infection, including resistance and susceptibility, and the molecular markers associated with them. Of the response of the pathogen to the host, the emphasis is placed on the role of the selective toxins on pathogenicity and their genetic controls and regulations. The review ends with a perspective on future directions in the research on the apple-A. alternata pathosystem in the era of genomics and post genomics, particularly on how to identify candidate genes from both host and pathogen for potential genetic engineering for disease resistant cultivars.     

Tenuazonic acid production by Alternaria alternata and Alternaria tenuissima isolated from cotton.

Cultures of Alternaria alternata (three isolates) and Alternaria tenuissima (three isolates) obtained from cottonseeds and bolls were toxigenic when cultured on various laboratory media. A mycotoxin was isolated and identified as tenuazonic acid by using solvent partition, thin-layer chromatography, and instrument analyses. Toxicity was monitored with brine shrimp and chicken embryo bioassays. All cultures except A. alternata 938 produced tenuazonic acid when grown on cottonseed and on yeast extract-sucrose broth. The most toxin (266 mg/kg) was produced by A. tenuissima 843 on cottonseed.     

Viruslike particles in tentoxin-producing strains of Alternaria alternata.

Double-stranded (ds) RNAs associated with viruslike particles have been found in six isolates of Alternaria alternata which produce tentoxin. Isolates had from one to three dsRNAs ranging in size from 1.0 to 5.1 kilobase pairs. In two isolates the dsRNAs were associated with 30-nm particles. No dsRNA was detected in any of six other tentoxin-producing isolates or nine isolates which did not produce tentoxin.     

Alternaria alternata, a new pathotype pathogenic to aphids

A fungus, identified as Alternaria alternata, was isolated from dying or dead aphids and proved to be pathogenic. It was isolated from different parts of Greece from aphid specimens on cultivated plants, ornamentals and weeds. In the laboratory, disease development started with the germination of spores on the insect integument and the subsequent growth of mycelium. The fungus formed apical and intercalary, globose or lobate appressoria which were firmly attached onto the host exoskeleton and facilitated entrance of the mycelium into the insect body. Under favorable conditions of temperature (15–35 °C) and relative humidity (100%), infected aphids died in 2–4 days. A characteristic brown discoloration accompanied the death of the insects. Both mycelial growth and sporulation were profuse on dead specimens. The pathogen infected all 26 aphid species tested but was unable to infect other insects (Drosophila melanogaster and Ceratitis capitata) or aphid host plants. There were significant differences in mortality rate among aphid species only during the first two days after inoculation. It is suggested that A. alternata may be a good candidate to be exploited for the biological control of aphids.     

Biosynthesis of the bicycloalternarenes, mixed terpenoids of Alternaria alternata

By incorporation of [2-13C]-mevalonate, [1-13C]-acetate and [1-13C]-glucose we could reveal that the phytopathogenic fungus Alternaria alternata biosynthesized the mixed terpenoids bicycloalternarenes via the classic mevalonate pathway. The polyketid pathway does not participate in the biosynthesis of bicycloalternarenes, because there is no incorporation of [13C]-acetate into the C-ring of these compounds. The labelling pattern in this nonterpenoid part of bicycloalternarenes after feeding with [1-13C]-glucose and [U-13C6]-glucose, respectively, allows the assumption that metabolites of the shikimate pathway are involved.     

Effect of substrate on metabolite production of Alternaria alternata.

Alternariol and alternariol monomethyl ether are commonly associated with weathered grain sorghum. Production of these metabolites and altenuene by isolates of Alternaria alternata was evaluated on various sterile grain substrates. At 35% moisture content and 25 C, metabolite yields were highest on rice, intermediate on sorghums, and lowest on wheat and yellow corn. Fourteen-to 21-day cultures on milled rice were best in terms of ease of metabolite recovery, even though yields were higher on 28-day cultures of rough and brown rice. Metabolite production was reduced when rice was supplemented with yeast extract or yeast extract plus Czapek-Dox broth.     

Mutagenicity of stemphyltoxin III, a metabolite of Alternaria alternata

Some common decay organisms of vegetables and ripened fruits are Alternaria species. Even fruits and vegetables kept under refrigeration can be spoiled by Alternaria species because the mold grows at low temperatures. Alternaria alternata is commonly found in grain in areas with a high incidence of esophageal cancer. Three metabolites, altertoxins I, II, and III, have been isolated from A. alternata and have hydroxyperylenequinone structures. Although other perylenequinone metabolites such as stemphyperylenol and stemphyltoxins I, II, III, and IV, have been isolated from Stemphylium botryosum var. lactucum, a plant pathogen and mold, we isolated and identified stemphyltoxin III from A. alternata. This metabolite was tested for mutagenicity in the Ames Salmonella typhimurium plate incorporation assay with and without Aroclor 1254-induced rat S-9 metabolic activation. A positive response was noted with and without metabolic activation in S. typhimurium TA98 and TA1537, and there was a marginal response in strain TA100.     

Tenuazonic Acid, a Toxin Produced by Alternaria alternata

Fifty-seven of 87 isolates of Alternaria alternata (Fr) Keissler grown on autoclaved, moist corn-rice substrate and fed to rats were lethal. The major toxin produced was isolated and characterized as tenuazonic acid. Twenty of 23 toxigenic Alternaria isolates examined produced tenuazonic acid. No tenuazonic acid could be detected in either of the field samples of sorghum or blackeyed peas, which were heavily invaded by Alternaria.