In vitro growth of wheat and flax rust fungi on complex and chemically defined media

Growth from uredospores seeded in axenic culture is described for several races of Puccinia graminis Pers. f. sp. tritici (Erikss. and Henn.) and race 3 of Melampsora lini (Ehrenb.) Lév. on complex media containing peptone, yeast extract, and bovine serum albumin (BSA); and for an Australian isolate of Puccinia graminis, race 126-ANZ 6,7, and Melampsora lini, race 3, on chemically defined, liquid media.Of six North American isolates of Puccinia graminis only race 38 formed colonies approaching those of race 126-ANZ 6,7 in final size and general morphology on complex media. 5′AMP had no effect on the growth of 126-ANZ 6,7, but cyclic AMP inhibited growth after uredospore germination. Good growth and sporulation were obtained with 126-ANZ 6,7, but not with the other isolates tested, using a new, chemically defined liquid medium, sterilized by millipore filtration, and containing glucose, Czapek's minerals plus micronutrients, Ca2+, glucose and aspartic acid, glutathione, and cysteine. Uredospores produced in culture reinfected exposed mesophyll tissue, but not intact seedling leaves of wheat.Highly reproducible growth and sporulation of Melampsora lini, race 3, were obtained routinely on a solid medium containing Difco-Bacto agar, sucrose, Knop's minerals, micronutrients, yeast extract, peptone, and BSA. Vegetative cultures, capable of reinfecting the cut ends of surface-sterilized flax cotyledons, could be maintained indefinitely by subdivision before sporulation and transfer to the same medium minus BSA. Evidence is presented that BSA stimulated the development of colonies and the formation of uredospores. The mode of action of BSA is unknown, but it could not be replaced by putrescine.A new chemically defined, liquid medium containing sucrose, Knop's mineral salts, micronutrients, aspartic (or glutamic) acid, and cysteine supported the growth of colonies of Melampsora lini in a highly reproducible manner. The formation of uredospores and teliospores by these colonies was controlled by (a) the level of Ca2+ (as Ca(NO3)2∙4H2O), (b) the concentration of aspartic acid, and (c) the number of colonies per flask. At inoculum levels giving 40 to 60 colonies per flask, in media containing 8.5 mM Ca+ and 45 mM aspartic acid, uredospore formation occurred in 60 to 70% of the colonies. A decrease in the Ca2+ level to 4.25 mM, or a decrease in aspartic acid to 22.5 mM, or adjustment of the inoculum level to give about 10 colonies per flask each resulted in only infrequent sporulation. The uredospores produced in vitro infected intact, 1-week-old flax cotyledons in a normal manner.

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Sporulation and pathogenicity of an Australian isolate of wheat rust grown in vitro

Canadian Journal of Botany ◽

10.1139/b71-274 ◽

1971 ◽

Vol 49 (11) ◽

pp. 1961-1964 ◽

Cited By ~ 9

Author(s):

A. Bose ◽

Michael Shaw

Keyword(s):

Surface Coating ◽

Germ Tube ◽

Australian Isolate ◽

Puccinia Graminis ◽

Lower Epidermis ◽

Mineral Salts ◽

Wheat Leaves ◽

Wheat Rust ◽

Serum Albumen

Colonies of Puccinia graminis Pers. f. sp. tritici Erikss. & Henn., race ANZ 126-6,7 were grown from uredospores on Czapek's mineral salts, 3% glucose, 0.1% Evans' peptone, plus defatted bovine serum albumen. Dikaryotic vegetative hyphae apparently developed from centers of germ tube anastomosis, without the formation of typical infection structures. Typical pigmented uredospores and teliospores were formed after 6 to 8 weeks growth. Both spore forms were coated with a layer of material which was visible under the scanning electron microscope and was not observed on uredospores grown on intact wheat leaves. The uredospores were capable of infecting the mesophyll of wheat leaves exposed by stripping back the lower epidermis. The possibility is considered that the surface coating of uredospores grown in vitro is related to their inability to infect intact leaves via the stomata.

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Nutritional Factors Affecting Growth and Production of Antimicrobial Substances by Streptococcus lactis subsp. diacetylactis S1-67/C

Journal of Food Protection ◽

10.4315/0362-028x-46.6.514 ◽

1983 ◽

Vol 46 (6) ◽

pp. 514-517 ◽

Cited By ~ 8

Author(s):

N. S. REDDY ◽

B. RANGANATHAN

Keyword(s):

Amino Acids ◽

Yeast Extract ◽

Casein Hydrolysate ◽

Basal Medium ◽

Good Growth ◽

Mineral Salts ◽

Nutritional Factors ◽

Maximum Production ◽

Antimicrobial Substances ◽

Streptococcus Lactis

The present study pertains to the effect of nutritional factors on the growth and production of antimicrobial substances (AS) by Streptococcus lactis subsp. diacetylactis S1-67/C. Among nine media tested, yeast extract dextrose broth supported good growth and maximum production of AS. Addition of beef extract and yeast extract at 1.0 and 0.6% levels, respectively, increased growth as well as production of AS. Of ten carbohydrates examined, maximum production of AS was achieved with 1% glucose followed by fructose, 4% molasses, lactose, sucrose, galactose, mannitol, maltose and 2% molasses. Xylose inhibited production of AS, although it stimulated growth of the organism. Peptone, tryptone and tryptose (each at the 1.5% level) significantly stimulated production of AS. Other nitrogen sources, including soytone, casein hydrolysate and proteose peptone, retarded production of inhibitory substances. Among the amino acids, L-leucine, DL-methionine and L-glutamic acid were most essential for growth and production of AS, whereas L-lysine, L-proline, DL-serine, DL-asparatic acid, L-arginine-HCl and DL-tryptophan were stimulatory. Other amino acids such as DL-ornithine, L-cysteine-HCl and DL-citrulline slightly stimulated AS production. In the presence of cynocobalmin, niacin, folic acid, calcium pantothenate and riboflavin, S. lactis subsp. diacetylactis S1-67/C produced maximum amounts of inhibitory substances. Omission of individual mineral salts from the basal medium did not affect production of AS by the organism.

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In vitro growth of gelatin suspensions of uredospores of Puccinia graminis f. sp. tritici

Canadian Journal of Botany ◽

10.1139/b69-181 ◽

1969 ◽

Vol 47 (8) ◽

pp. 1291-1293 ◽

Cited By ~ 13

Author(s):

M. D. Coffey ◽

A. Bose ◽

Michael Shaw

Keyword(s):

Basic Medium ◽

Australian Isolate ◽

Brown Pigment ◽

Puccinia Graminis ◽

Partial Recovery ◽

It Diffusion ◽

The Media ◽

Agar Surface ◽

Bacto Agar

A comparison was made of the growth of the Australian isolate of Puccinia graminis Pers. f. sp. tritici Eriks. & E. Henn. race ANZ 126-6,7 on 1.5% Difco Bacto-Agar using water and 15% gelatin as suspension media for seeding uredospores on the media. A range of media was tested, the basic constituents being 3% glucose, Czapek's minerals, peptone, and 1% bovine serum albumin. Spores embedded in gelatin gave much better growth than those applied to the agar surface in water suspension in all media. Sodium citrate (0.2%) inhibited growth; pectin (0.2%) did also, but partial recovery occurred later. Best growth was on the basic medium seeded with uredospores in gelatin. The mycelium was white and fluffy in appearance at first, but collapsed later as a dense brown stroma developed beneath it. Diffusion of brown pigment into the agar medium took place with the gelatin, but not the water series. Sporulation was observed after 60 days in a few colonies and was located in the brown stroma. Spores were non-pigmented, oval in shape, and similar in size to typical uredospores. It is suggested that the physical environment around the spores affects growth.

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In vitro culture of the flax rust, Melampsora lini

Canadian Journal of Botany ◽

10.1139/b70-105 ◽

1970 ◽

Vol 48 (4) ◽

pp. 773-776 ◽

Cited By ~ 18

Author(s):

M. D. Coffey ◽

A. Bose ◽

Michael Shaw

Keyword(s):

Ambient Temperature ◽

In Vitro Culture ◽

Yeast Extract ◽

Coconut Milk ◽

Melampsora Lini ◽

Different Strains ◽

Transfer Procedures ◽

Inorganic Constituents

Two different strains of Melampsora lini (Pers.) Lév. were grown on media similar to those described by Turel (Can. J. Bot. 47: 821–823). Alterations in organic and inorganic constituents did not, in general, prevent growth. Peptone and yeast extract, singly or in combination, supported growth, but coconut milk did not. Maintenance of the ambient temperature below 17 °C during transfer procedures increased the number of colonies which grew. The mycelium was generally binucleate. Spore-like cells of varying shapes were found, some of which resembled uredospores and teliospores.

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Bioprocess development for enhanced endoglucanase production by newly isolated bacteria, purification, characterization and in-vitro efficacy as anti-biofilm of Pseudomonas aeruginosa

Scientific Reports ◽

10.1038/s41598-021-87901-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Atef M. Ibrahim ◽

Ragaa A. Hamouda ◽

Noura El-Ahmady El-Naggar ◽

Fatma M. Al-Shakankery

Keyword(s):

Pseudomonas Aeruginosa ◽

Yeast Extract ◽

Statistical Design ◽

Life Time ◽

Maximum Activity ◽

Bioprocess Development ◽

Ammonium Sulfate Precipitation ◽

Face Centered ◽

Central Composite

AbstractEndoglucanase producing bacteria were isolated from Egyptian soils and the most active bacterial strain was identified as Bacillus subtilis strain Fatma/1. Plackett–Burman statistical design was carried out to assess the effect of seven process variables on endoglucanase production. Carboxymethyl cellulose (CMC), yeast extract and peptone were the most significant variables that enhanced the endoglucanase production and thus were selected for further optimization using face-centered central composite design. The highest yield of endoglucanase (32.37 U/mL) was obtained in run no. 9, using 18 g/L CMC, 8 g/L peptone, 7 g/L yeast extract and 0.1 g/L FeSO4.7H2O. The optimized medium showed about eightfold increase in endoglucanase production compared to the unoptimized medium. The produced crude enzyme was further purified by ammonium sulfate precipitation, then DEAE-Sepharose CL6B column. The purified enzyme was shown to have a molecular weight of 37 kDa. The enzyme showed maximum activity at pH 8.0, temperature of 50 °C, incubation time of 60 min. The half-life time (T1/2) was 139.53 min at 50 °C, while being 82.67 min at 60 °C. Endoglucanase at concentration of 12 U/mL effectively removed 84.61% of biofilm matrix of Pseudomonas aeruginosa with marked reduction in carbohydrate content of the biofilm from 63.4 to 7.9 μg.

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Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase

Biochemical Journal ◽

10.1042/bj3010577 ◽

1994 ◽

Vol 301 (2) ◽

pp. 577-583 ◽

Cited By ~ 8

Author(s):

K Oda ◽

J Cheng ◽

T Saku ◽

N Takami ◽

M Sohda ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Endoplasmic Reticulum ◽

Aspartic Acid ◽

Chimeric Protein ◽

Attachment Site ◽

In Vitro Translation ◽

Placental Alkaline Phosphatase ◽

Wild Type ◽

Aspartic Acid Residue

Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.

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Amended structure of side chains in a cell wall mannan from Candida albicans serotype A strain grown in yeast extract-Sabouraud liquid medium under acidic conditions: detection of the branched side chains corresponding to antigenic factor 4

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.1997.tb10433.x ◽

2006 ◽

Vol 152 (2) ◽

pp. 235-242 ◽

Cited By ~ 15

Author(s):

Hidemitsu Kobayashi ◽

Shizu Tanaka ◽

Junko Suzuki ◽

Yoko Kiuchi ◽

Nobuyuki Shibata ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Liquid Medium ◽

Yeast Extract ◽

Side Chains ◽

Serotype A ◽

A Cell ◽

Antigenic Factor ◽

Acidic Conditions ◽

Cell Wall Mannan

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Characterization of Phospholipase Activity of the Pseudomonas aeruginosa Type III Cytotoxin, ExoU

Journal of Bacteriology ◽

10.1128/jb.187.3.1192-1195.2005 ◽

2005 ◽

Vol 187 (3) ◽

pp. 1192-1195 ◽

Cited By ~ 42

Author(s):

Hiromi Sato ◽

Jimmy B. Feix ◽

Cecilia J. Hillard ◽

Dara W. Frank

Keyword(s):

Pseudomonas Aeruginosa ◽

Substrate Specificity ◽

Yeast Extract ◽

Enzyme Activation ◽

Phospholipase Activity ◽

Eukaryotic Protein ◽

Type Iii

ABSTRACT Recombinant ExoU (rExoU) and yeast extract were used to optimize an in vitro phospholipase assay as a basis for identifying the mechanism for enzyme activation and substrate specificity. Our results support a model in which a eukaryotic protein cofactor or complex facilitates the interaction of rExoU with phospholipid substrates.

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