Sporophore production ofAgaricus bisporus in aseptic environments

Production of mature sporophores ofAgaricus bisporus was achieved for the first time in amended, autoclaved soil, gamma-sterilized soil, and soil-extract agar medium. The initiation of sporophores was triggered by metabolites of soil-inhabiting bacteria, particularly nodule forming isolates. Whether a single metabolite or several metabolites of these bacteria caused formation of sporophores could not be established; however, biotin alone when added to soil extract medium produced comparable results. The potentiality of different bacteria to induce sporophore formation varied considerably within species and isolates.Amino acids favored vegetative growth ofA. bisporus, but failed to induce formation of sporophores. Organic acids supported luxuriant growth and poor sporophore formation. Among several growth-promoting substances and vitamins, biotin induced abundant formation of mature sporophores.The authors are thankful to Dr. C. Corke, Department of Soil Microbiology, University of Guelph, Ontario, for providing some bacterial cultures used in this study.     

Accumulation of sugar alcohols by filamentous fungi

Five hundred isolates of different xerophilic and non-xerophilic fungi belonging to 10 genera and 74 species were screened for alditol (sugar alcohol) accumulation. Ninety-two of the isolates failed to grow on a salt medium, most of the isolates (408) produced alditols; 348,44 and 16 of them produced low, moderate and high levels of alditols, respectively. The high alditol producers belonged to five species ofAspergillus, six species ofEurotium andFennellia flavipes. Glycerol andd-mannitol were the main constituents of alditol pools of the 16 high alditol producers.d-Arabinitol andmeso-erythritol were also formed but at low concentrations by several of the tested isolates.     

Quantifying the relationship between disease severity and the amount of Aphanomyces euteiches detected in roots of alfalfa and pea with a real-time PCR assay

A real-time PCR assay was used to quantify the relationship in alfalfa and pea between disease severity and the amount of Aphanomyces euteiches detected in roots. The study included isolates of race 1 and race 2 of the alfalfa pathovar of A. euteiches and an isolate obtained from diseased pea. Spearman rank correlations between pathogen DNA content and disease severity index (DSI) ratings were positive ( ? 0.57) and significant (P  0.0007) for individual alfalfa plants, bulked alfalfa plant samples, and individual pea plants. In all experiments, significantly more pathogen was detected in susceptible populations than in resistant populations. The results clearly demonstrate that resistance to A. euteiches in both alfalfa and pea is characterized by a reduction in pathogen colonization relative to levels observed for susceptible reactions. The assay was very specific for A. euteiches, producing very linear assays with DNA extracted from pathogen isolates obtained from alfalfa, pea, and bean. Possible applications of the assay in conjunction with other real-time PCR assays specific to other legume pathogens are discussed in relation to simultaneous disease screening for multiple plant pathogens and the study of microbial population dynamics in mixed plant infections.     

Studies on the mycotic inhabitants ofCulex pipiens collected from fresh water ponds in Egypt

A total of 20 fungal species belonging to 10 genera were found to be associated with all stages ofCulex pipiens. Alternaria alternata, Aspergillus flavus, A. fumigatus, A. niger andPenicillium chrysogenum were the dominant fungi.Beauveria alba andPhoma herbarum. A well known facultative pathogen have been recorded. Most of fungal isolates (63.22%) showed a moderate growth on a synthetic medium containing partially purified chitin. The water extract of bothArtmesia cina andCleome droserifolia showed an inhibitive effect on the protein content and growth of some selected isolates. One ml dose of crude extract ofA. fumigatus killed 90% of the larvae after 192 hr incubation but 36% of the test larvae were killed by the same dose extracted fromP. chrysogenum at the same period of incubation.     

SOME FACTORS AFFECTING SEXUAL REPRODUCTION OF APHANOMYCES EUTEICHES

Papavizas , G. C, and C. B. Davey . (USDA, ARS, Crops Res. Div., Beltsville, Maryland.) Some factors affecting sexual reproduction of Aphanomyces euteiches. Amer. Jour. Bot. 47(10) : 884–889. Illus. 1960.—Oospores of Aphanomyces euteiches Drechsler were consistently formed in abundance on a medium (SM–2) consisting of mineral salts, D-glucose, thioglycolic acid, Difco “Special Agar-Noble,” and a mixture of amino acids added in proportions found in powdered yeast extract. The isolates of A. euteiches studied differed in their nutritional requirements for sexual reproduction. Isolates 4 and 7 formed oospores abundantly on SM–1 which was smiliar to SM–2 except that DL-glutamic acid was substituted for the amino acid mixture. Isolate 572 did not form oospores on medium SM–1 except when DL-methionine was substituted for thioglycolic acid. Sexual reproduction of isolates 4 and 7 was observed at a wider concentration range of reduced-sulfur-containing compounds than that of isolate 572. Sexual reproduction of the 3 isolates studied was influenced in a dissimilar way by varying concentrations of D-glucose and DL-glutamic acid, or by varying concentrations of D-glucose and of the amino acid mixture. The optimal sugar carbon/amino nitrogen ratio range for sexual reproduction of isolate 572 was about 5–22, whereas that of isolates 4 and 7 was 15–180. The pH range for sexual reproduction was as wide as that for growth. An acid reaction was more favorable than a neutral or alkaline one. The optimal pH range was 4.9–5.4. Vitamins were not essential.     

Diazotrophic endophytes of native black cottonwood and willow

Poplar and willow are economically-important, fast-growing tree species with the ability to colonize nutrient-poor environments. To initiate a study on the possible contribution of endophytes to this ability, we isolated bacteria from within surface-sterilized stems of native poplar (Populus trichocarpa) and willow (Salix sitchensis) in a riparian system in western Washington state. Several of the isolates grew well in nitrogen-limited medium. The presence ofnifH, a gene encoding one of the subunits of nitrogenase, was confirmed in several of the isolates including species ofBurkholderia, Rahnella, Sphingomonas, andAcinetobacter. Nitrogenase activity (as measured by the acetylene reduction assay) was also confirmed in some of the isolates. The presence of these diazotrophic microorganisms may help explain the ability of these pioneering tree species to grow under nitrogen limitation.     

II. Evidence of the presence in yeast extract of substances which stimulate the growth of Histoplasma capsulatum and blastomyces dermatitidis similarly to that found in starling manure extract

Summary A medium consisting of agar plus yeast extract contained the necessary metabolites for rapid growth and sporulation ofHistoplasma capsulatum andBlastomyces dermatitidis. H. capsulatum when harvested after 10 or 30 days incubation period from this medium was shown to have a similar number of spores as well as total particle viability for each period of growth.The growth characteristics ofH. capsulatum and four different isolates ofB. dermatitidis on yeast extract medium were similar to that obtained previously using starling (Sturnis vulgaris) manure extract medium. These characteristics are rapid growth consisting of many viable spores and a low ratio of vegetative mycelium.Several isolations ofH. capsulatum from naturally contaminated soil specimens were made using yeast extract medium.From the Communicable Disease Center, Public Health Service, U. S. Department of Health, Education, and Welfare.     

Aflatoxin B1 producing potential of isolates of Aspergillus flavus Link ex Fries from cotton,maize and wheat

Twenty-one isolates ofAspergillus flavus Link ex Fries obtained from cotton, maize and wheat were screened for their ability to produce aflatoxins on two liquid media. Of these, sixteen isolates were toxigenic and produced only aflatoxin B₁ as assessed by bioassay on okra seedlings and TLC method. For screening isolates ofA. flavus for aflatoxin formation, 0.7 % YES+ Salt medium was found to be good as also for obtaining higher yields of the toxin. Isolates ofA. flavus produced aflatoxin B₁ ranging from 0.85 to 17.2 mg/50 ml. Maximum yield of aflatoxin was obtained when rice was used as the substrate in case of toxigenic isolates L-27 and C-9, and on maize in isolate M-11.     

Aspergillus terreus and its toxic metabolites as a food contaminant in some Egyptian Bakery products and grains

A. terreus isolates isolated from some bakery products, corn and rice were found to be able to produce territrems. 90% of theA. terreus isolated from bakery products were able to produce territrem A, with a mean of 0.09 ppm, while 80% ofA. terreus isolates produce territrem B with a mean of 0.24 ppm. On the other hand 31.8% of the isolates ofA. terreus from corn were able to produce territrem A with a mean of 0.44 ppm. ConcerningA. terreus isolates from rice, 66.7% were found to produce territrem A, with a mean of 5.28 ppm, and 77.8% of the isolates produced territrem B with a mean of 1.79 ppm.     

Hydroxamate-siderophore production and utilization by marine eubacteria

Siderophore (iron-binding chelator) production was examined in 30 strains of open ocean bacteria from the generaVibrio, Alteromonas, Alcaligenes, Pseudomonas, andPhotobacterium. The results showed that hydroxamate-type siderophore production was widely distributed in various marine species, except for isolates ofAlteromonas macleodii andV. nereis. In all cases, the ability to produce siderophores was under the control of iron levels in the medium and satisfied the iron requirements of the siderophore bioassay organism. On the basis of chemical assay and bacterial bioassays, none of the examined isolates produced phenolate-type siderophores. Several isolates produces siderophores that were neither hydroxamatenor phenolate-type siderophores. Some strains such asAlteromonas communis produce siderophores that could be used by many other isolates. In contrast, the siderophore produced byAlcaligenes venustus had little cross-strain utilization. These findings suggest that the ability to produce siderophores may be common to open ocean bacteria.     

Nitrogen requirements of certain isolates of alternaria tenuis auct

Three isolates ofA. tenuis isolated from the diseased leaves ofMangifera indica l. Musa paradisiaca l. andPsidium guajava l. were investigated. They were grown on different sources of nitrogen viz., potassium nitrate, sodium nitrate, calcium nitrate, ammonium nitrate, sodium nitrite, ammonium sulphate, ammonium chloride, glycine, DL-valine, L-glutamic acid, urea, thiourea, L-asparagine and peptone. They were also grown on the medium lacking nitrogen. A wide variation was observed in the growth and reproduction of the different isolates. The growth of all of them was good on potassium nitrate, calcium nitrate, glycine, DL-valine, L-glutamic acid, L-asparagine and peptone but the sporulation was satisfactory on calcium nitrate only. Sodium nitrite supported moderate growth of banana leaf isolate whereas there was no growth of the other two isolates. None of the organisms could grow on the medium lacking nitrogen as well as on thiourea. The results obtained with the isolates under study have been compared with those of earlier investigators and it has been clearly established that the different isolates ofA. tenuis could show marked differences in their nitrogen requirements.     

Alternaria on cruciferous plants. 4.Alternaria species on seed of some cruciferous crops and their pathogenicity

The incidence ofAlternaria spp. on seed samples of cruciferous vegetable crops was surveyed between 1990 and 1992. Some commercial seed lots of crucifers which are commonly grown in Japan were infested withAlternaria species. ThreeAlternaria species were encountered on the seed samples ofBrassica campestris, B. orelacea, andRaphanus sativus. The most frequently detected species wereA. japonica andA. alternata onB. campestris, A. brassicicola onB. oleracea, andA. japonica andA. alternata onR. sativus, respectively.Alternaria brassicae was not detected in this study.Alternaria brassicicola isolates from these crops produced necrotic lesions on all of the crucifer seedlings inoculated, whileA. japonica induced different reactions in different plants or plant parts depending on isolates used in inoculation tests. In contrast, most isolates ofA. alternata could not produce necrotic lesions on foliage leaves of crucifers inoculated, although some of them produced clear lesions only on cotyledons.Alternaria alternata associated with these cruciferous crop seeds was considered to be an oppotunistic parasite of these crops.     

Biological species ofArmillaria and their mycoparasitic associations withRhodophyllus abortivus in Hokkaido

Specimens of basidiomes and/or rhizomorphs ofArmillaria mellea complex and basidiomes ofRhodophyllus abortivus, developing on the same decaying stumps or stems of forest trees, were collected in three forests in Hokkaido. Normal basidiomes ofR. abortivus were found near to, but free from, the rhizomorphs and/or basidiomes ofArmillaria, while abnormal basidiomes, as carpophoroid forms, were developed on the rhizomorphs ofArmillaria. Of three mycoparasiticArmillaria isolates found withR. abortivus, one was identified asA. gallica and two asA. jezoensis. The isolates ofR. abortivus showed excellent mycelial growth and rhizomorph formation on PDA. However, on MDA, RMDA and BMDA, they showed poor aerial mycelia growth and no rhizomorphs. In the contrapositional cultures, the growth ofA. gallica was completely inhibited byR. abortivus on PDA but only slightly inhibited on MDA and RMDA. On the other hand, mutual inhibition at a distance was observed on BMDA. The mycelial growth and rhizomorph formation inA. jezoensis were severely inhibited by the colony ofR. abortivus on PDA, but only slightly inhibited on MDA. On RMDA and BMDA, the colonies of twoArmillaria species andR. abortivus showed mutual inhibition at a distance and apparent rhizomorph formation by bothArmillaria species.     

Identification of Cryptococcus neoformans in a routine clinical laboratory

Summary Eight taxonomic tests were compared for their ability to distinguishCryptococcus neoformans from the non-pathogenic species ofCryptococcus. Eight isolates ofCryptococcus were obtained from the American Type Culture Collection and 43 isolates were obtained directly from human and natural sources. The tests which appeared to be most valuable to the routine diagnostic laboratory were growth at 37° C, characteristic growth on Guizotia seed agar and virulence for mice.     

Synthesis of sterigmatocystin on a chemically defined medium by species ofAspergillus andChaetomium

Sterigmatocystin (ST) is a secondary metabolite and a principal mycotoxin known to be produced by over 30 species of filamentous fungi. It is also one of the late intermediates in aflatoxin biosynthesis. We have tested the ability of 7 species ofAspergillus, including 4 strains ofA. versicolor, one species ofBipolaris, and two species ofChaetomium, to produce ST on a sucrose-salts-phenylalanine defined medium as well as on three complex substrates. Highest ST production in our survey was by a strain ofA. versicolor grown on wheat, whereas, the highest ST production on defined medium was byC. cellulolyticum. To our knowledge, this is the first report of ST production byC. cellulolyticum on any substrate. In precursor feeding studies, resting cultures of wild typeA. nidulans andA. versicolor were unable to biotransform O-methylsterigmatocystin (OMST), the last known intermediate in aflatoxin biosynthesis. These results suggest that ST is the end product of polyketide metabolism in the strains tested.     

Based on biochemical and physiological behavior,where isAspergillus egyptiacus better placed?

Physiological and biochemical properties were tested in 45 isolates ofAspergillus egyptiacus (16 isolates),Emericella nidulans (16) andAspergillus versicolor (13). The three fungal species exhibited common and similar features. The big similarity betweenA. egyptiacus andE. nidulans was greater than betweenA. egyptiacus andA. versicolor. It included the inability to produce base either from sodium citrate or lactic acid media, growth at 45 °C (thermophilicity), and production of very similar pigmentations onAspergillus flavus andparasiticus agar.A. egyptiacus is therefore better placed in theAspergillus nidulans-Emericella assemblage.     

Nitrogen modulation of Medicago truncatula resistance to Aphanomyces euteiches depends on plant genotype

Nitrogen (N) availability can impact plant resistance to pathogens by the regulation of plant immunity. To better understand the links between N nutrition and plant defence, we analysed the impact of N availability on Medicago truncatula resistance to the root pathogen Aphanomyces euteiches. This oomycete is considered to be the most limiting factor for legume production. Ten plant genotypes were tested in vitro for their resistance to A. euteiches in either complete or nitrate‐deficient medium. N deficiency led to enhanced or reduced susceptibility depending on the plant genotype. Focusing on four genotypes displaying contrasting responses, we determined the impact of N deficiency on plant growth and shoot N concentration, and performed expression analyses on N‐ and defence‐related genes, as well as the quantification of soluble phenolics and different amino acids in roots. Our analyses suggest that N modulation of plant resistance is not linked to plant response to N deprivation or to mechanisms previously identified to be involved in plant resistance. Furthermore, our studies highlight a role of glutamine in mediating the susceptibility to A. euteiches in M. truncatula.     

Proteomic Profiling Unravels Insights into the Molecular Background Underlying Increased <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis>-Tolerance of <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To investigate the molecular mechanisms underlying susceptibility of legumes to the root pathogen Aphanomyces euteiches (oomycota), comparative proteomic studies have been carried out. In a first approach, we have analysed two Medicago truncatula lines of the French CORE collection (F83.005-5 (R2002) and F83.005-9 (R2002)), which showed either increased or decreased susceptibility to A. euteiches as compared to the widely adopted line A17. Several proteins were identified to be differentially induced after pathogen challenge in the two M. truncatula accessions with altered disease susceptibility, whereof proteins with increased abundances in the more resistant line F83.005-9 could be involved in mechanisms that lead to an improved disease resistance. Among these proteins, we identified two proteasome alpha subunits, which might be involved in defense response. To broaden our studies on A. euteiches-tolerance of M. truncatula, we investigated two other phenomena that lead to an either increased A. euteiches-resistance or to an enhanced susceptibility. The topic of an enhanced plant resistance to A. euteiches was studied in plants showing a bioprotective effect of a pre-established arbuscular mycorrhiza (AM) symbiosis. Evaluation of root fresh weights and pathogen spreading in the root system clearly indicate that mycorrhizal plants show increased A. euteiches-resistance as compared to non-mycorrhizal plants. Proteome analyses revealed the induction of similar protein patterns as in the M. truncatula accessions with comparatively high resistance level to A. euteiches. In a third approach, increased A. euteiches susceptibility was effected by exogenous abscisic acid (ABA) application prior to root infection. Evaluation of the abundance levels of a group of pathogenesis related class 10 (PR10)-like proteins, which were previously identified to be regulated after A. euteiches infection, revealed a correlation between the abundance levels of these proteins and the A. euteiches infection level or severity. Requests concerning seeds from the Medicago truncatula lines F83.005-5 and F83.005-9 should be addressed to Jean-Marie Prospéri, INRA-SGAP Laboratory, Laboratoire de Ressources Génétiques et d’Amélioration des Luzernes méditerranéennes, Mauguio, France, jean-marie.prosperi@ensam.inra.fr.     

Studies in the differentiation between Microsporum ferrugineum Ota and Trichophyton soudanense Joyeux

A study, conducted with 20 isolates of Microsporum ferrugineum and 12 isolates of Trichophyton soudanense, revealed that some of the discrepancies in the literature regarding their characteristics and differentiation were due to methodology, strain variation and the use of an insufficient number of isolates. We found all isolates of T. soudanense to be urease negative and gelatinase positive (usually by the first week); to produce brown to black colonies on Lowenstein-Jensen medium; to rapidly decompose casein and more slowly tyrosine; to grow well or better at 37°C as compared to room temperature; to produce reflexive branching on cornmeal Tween agar and abundant microconidia on casero medium and to exhibit no sexual reaction with either mating type of Arthroderma simii. All but one isolate demonstrated restricted growth on lactose agar and only three isolates perforated hair.In contrast, we found 18 of 20 isolates of M. ferrugineum to be urease positive in urea broth (most isolates were negative on urea agar); all produced light-colored colonies on Lowenstein-Jensen medium; spreading colonies on lactose agar and failed to perforate hair in vitro or to produce reflexive branching. Most isolates manifested poorer to no growth at 37°C compared to room temperature and all but one failed to decompose casein and tyrosine. A few strains produced macroconidia and/ or microconidia on casero medium and some reacted sexually with A. simii (a) or (–) mating type. Gelatin hydrolysis was variable.We suggest the following key tests to differentiate M. ferrugineum from T. soudanense: urease activity in urea broth; colony color on Lowenstein-Jensen medium; growth on lactose agar; growth at 37° C compared to room temperature; presence of reflexive branching on cornmeal Tween agar.     

Armillaria jezoensis,a new symbiont ofGaleola septentrionalis (Orchidaceae) in Hokkaido

NineArmillaria isolates obtained from the roots ofGaleola septentrionalis in Hokkaido were identified asA. jezoensis by means of mating tests. Cultures of these isolates were similar in colony morphology, mycelial growth and rhizomorph formation on each of malt extract-dextrose agar (MDA), potato-dextrose agar (PDA), andG. septentrionalis root extractdextrose agar (GDA) media, showing better mycelial growth and rhizomorph formation on GDA medium.