bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantification of Trichothecene-Producing
Fusarium
Species in Harvested Grain by Competitive PCR To Determine Efficacies of Fungicides against Fusarium Head Blight of Winter Wheat
Authors: Edwards S. G. , Pirgozliev S. R. , Hare M. C. , Jenkinson P. 
Published: July 2002

ABSTRACT

We developed a PCR-based assay to quantify trichothecene-producing
Fusarium
based on primers derived from the trichodiene synthase gene (
Tri
5). The primers were tested against a range of fusarium head blight (FHB) (also known as scab) pathogens and found to amplify specifically a 260-bp product from 25 isolates belonging to six trichothecene-producing
Fusarium
species. Amounts of the trichothecene-producing
Fusarium
and the trichothecene mycotoxin deoxynivalenol (DON) in harvested grain from a field trial designed to test the efficacies of the fungicides metconazole, azoxystrobin, and tebuconazole to control FHB were quantified. No correlation was found between FHB severity and DON in harvested grain, but a good correlation existed between the amount of trichothecene-producing
Fusarium
and DON present within grain. Azoxystrobin did not affect levels of trichothecene-producing
Fusarium
compared with those of untreated controls. Metconazole and tebuconazole significantly reduced the amount of trichothecene-producing
Fusarium
in harvested grain. We hypothesize that the fungicides affected the relationship between FHB severity and the amount of DON in harvested grain by altering the proportion of trichothecene-producing
Fusarium
within the FHB disease complex and not by altering the rate of DON production. The
Tri
5 quantitative PCR assay will aid research directed towards reducing amounts of trichothecene mycotoxins in food and animal feed.
Characterization of Fluorescent and Nonfluorescent Peptide Siderophores Produced by
Pseudomonas syringae
Strains and Their Potential Use in Strain Identification
Authors: Bultreys Alain , Gheysen Isabelle , Maraite Henri , de Hoffmann Edmond 
Published: July 2002

ABSTRACT

Nonfluorescent highly virulent strains of
Pseudomonas syringae
pv. aptata isolated in different European countries and in Uruguay produce a nonfluorescent peptide siderophore, the production of which is iron repressed and specific to these strains. The amino acid composition of this siderophore is identical to that of the dominant fluorescent peptide siderophore produced by fluorescent
P. syringae
strains, and the molecular masses of the respective Fe(III) chelates are 1,177 and 1,175 atomic mass units. The unchelated nonfluorescent siderophore is converted into the fluorescent siderophore at pH 10, and colors and spectral characteristics of the unchelated siderophores and of the Fe(III)-chelates in acidic conditions are similar to those of dihydropyoverdins and pyoverdins, respectively. The nonfluorescent siderophore is used by fluorescent and nonfluorescent
P. syringae
strains. These results and additional mass spectrometry data strongly suggest the presence of a pyoverdin chromophore in the fluorescent siderophore and a dihydropyoverdin chromophore in the nonfluorescent siderophore, which are both ligated to a succinamide residue. When chelated, the siderophores behave differently from typical pyoverdins and dihydropyoverdins in neutral and alkaline conditions, apparently because of the ionization occurring around pH 4.5 of carboxylic acids present in β-hydroxyaspartic acid residues of the peptide chains. These differences can be detected visually by pH-dependent changes of the chelate colors and spectrophotochemically. These characteristics and the electrophoretic behavior of the unchelated and chelated siderophores offer new tools to discriminate between saprophytic fluorescent
Pseudomonas
species and fluorescent
P. syringae
and
P. viridiflava
strains and to distinguish between the two siderovars in
P. syringae
pv. aptata.
Induction of Laccase Activity in
Rhizoctonia solani
by Antagonistic
Pseudomonas fluorescens
Strains and a Range of Chemical Treatments
Authors: Crowe Jonathan D. , Olsson Stefan 
Published: July 2002

ABSTRACT

Fungi often produce the phenoloxidase enzyme laccase during interactions with other organisms, an observation relevant to the development of biocontrols. By incorporating the laccase substrate 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) into agar, we analyzed laccase induction in the plant-pathogenic fungus
Rhizoctonia solani
when paired against isolates of the soil bacterium
Pseudomonas fluorescens
. Substantial induction of
R. solani
laccase was seen only in pairings with strains of
P. fluorescens
known to produce antifungal metabolites. To study laccase induction further, a range of chemical treatments was applied to
R. solani
liquid cultures.
p
-Anisidine, copper(II), manganese(II), calcium ionophore
A23187
, lithium chloride, calcium chloride, cyclic AMP (cAMP), caffeine, amphotericin B, paraquat, ethanol, and isopropanol were all found to induce laccase; however, the
P. fluorescens
metabolite viscosinamide did not do so at the concentrations tested. The stress caused by these treatments was assessed by measuring changes in lipid peroxidation levels and dry weight. The results indicated that the laccase induction seen in pairing plate experiments was most likely due to calcium or heat shock signaling in response to the effects of bacterial metabolites, but that heavy metal and cAMP-driven laccase induction was involved in sclerotization.
Characterization of the Properties of Human- and Dairy-Derived Probiotics for Prevention of Infectious Diseases in Fish
Authors: Nikoskelainen Sami , Salminen Seppo , Bylund Göran , Ouwehand Arthur C. 
Published: July 2002

ABSTRACT

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use,
Lactobacillus rhamnosus
ATCC 53103,
Lactobacillus casei
Shirota,
Lactobacillus bulgaricus, L. rhamnosus
LC 705,
Bifidobacterium lactis
Bb12, and
Lactobacillus johnsonii
La1, and one for animal use,
Enterococcus faecium
Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens,
Vibrio anguillarum, Aeromonas salmonicida
, and
Flavobacterium psychrophilum
, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of
A. salmonicida
. Coculture experiments showed significant inhibition of growth of
A. salmonicida
, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except
L. casei
Shirota showed tolerance against fish bile.
L. rhamnosus
ATCC 53103 and
L. bulgaricus
were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth,
L. rhamnosus
ATCC 53103 and
L. bulgaricus
can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.
Kinetics of Perchlorate- and Chlorate-Respiring Bacteria
Authors: Logan Bruce E. , Zhang Husen , Mulvaney Peter , Milner Michael G. , Head Ian M. , Unz Richard F. 
Published: July 2002

ABSTRACT

Ten chlorate-respiring bacteria were isolated from wastewater and a perchlorate-degrading bioreactor. Eight of the isolates were able to degrade perchlorate, and all isolates used oxygen and chlorate as terminal electron acceptors. The growth kinetics of two perchlorate-degrading isolates, designated “
Dechlorosoma
” sp. strains KJ and PDX, were examined with acetate as the electron donor in batch tests. The maximum observed aerobic growth rates of KJ and PDX (0.27 and 0.28 h
−1
, respectively) were only slightly higher than the anoxic growth rates obtained by these isolates during growth with chlorate (0.26 and 0.21 h
−1
, respectively). The maximum observed growth rates of the two non-perchlorate-utilizing isolates (PDA and PDB) were much higher under aerobic conditions (0.64 and 0.41 h
−1
, respectively) than under anoxic (chlorate-reducing) conditions (0.18 and 0.21 h
−1
, respectively). The maximum growth rates of PDX on perchlorate and chlorate were identical (0.21 h
−1
) and exceeded that of strain KJ on perchlorate (0.14 h
−1
). Growth of one isolate (PDX) was more rapid on acetate than on lactate. There were substantial differences in the half-saturation constants measured for anoxic growth of isolates on acetate with excess perchlorate (470 mg/liter for KJ and 45 mg/liter for PDX). Biomass yields (grams of cells per gram of acetate) for strain KJ were not statistically different in the presence of the electron acceptors oxygen (0.46 ± 0.07 [
n
= 7]), chlorate (0.44 ± 0.05 [
n
= 7]), and perchlorate (0.50 ± 0.08 [
n
= 7]). These studies provide evidence that facultative microorganisms with the capability for perchlorate and chlorate respiration exist, that not all chlorate-respiring microorganisms are capable of anoxic growth on perchlorate, and that isolates have dissimilar growth kinetics using different electron donors and acceptors.
Purification, Characterization, and Functional Role of a Novel Extracellular Protease from
Pleurotus ostreatus
Authors: Palmieri Gianna , Bianco Carmen , Cennamo Giovanna , Giardina Paola , Marino Gennaro , Monti Maria , Sannia Giovanni 
Published: July 2002

ABSTRACT

A new extracellular protease (PoSl;
Pleurotus ostreatus
subtilisin-like protease) from
P. ostreatus
culture broth has been purified and characterized. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, a pI of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl activity is positively affected by calcium. A 10-fold decrease in the
K

m

value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca
2+
binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of
P. ostreatus
laccase activity by degrading and/or activating different isoenzymes.
Burkholderia
, a Genus Rich in Plant-Associated Nitrogen Fixers with Wide Environmental and Geographic Distribution
Authors: Estrada-De Los Santos Paulina , Bustillos-Cristales Rocı́o , Caballero-Mellado Jesús 
Published: July 2002

ABSTRACT

The genus
Burkholderia
comprises 19 species, including
Burkholderia vietnamiensis
which is the only known N
2
-fixing species of this bacterial genus. The first isolates of
B. vietnamiensis
were recovered from the rhizosphere of rice plants grown in a phytotron, but its existence in natural environments and its geographic distribution were not reported. In the present study, most N
2
-fixing isolates recovered from the environment of field-grown maize and coffee plants cultivated in widely separated regions of Mexico were phenotypically identified as
B. cepacia
using the API 20NE system. Nevertheless, a number of these isolates recovered from inside of maize roots, as well as from the rhizosphere and rhizoplane of maize and coffee plants, showed similar or identical features to those of
B. vietnamiensis
TVV75
T
. These features include nitrogenase activity with 10 different carbon sources, identical or very similar
nifHDK
hybridization patterns, very similar protein electrophoregrams, identical amplified 16S rDNA restriction (ARDRA) profiles, and levels of DNA-DNA reassociation higher than 70% with total DNA from strain TVV75
T
. Although the ability to fix N
2
is not reported to be a common feature among the known species of the genus
Burkholderia
, the results obtained show that many diazotrophic
Burkholderia
isolates analyzed showed phenotypic and genotypic features different from those of the known N
2
-fixing species
B. vietnamiensis
as well as from those of
B. kururiensis
, a bacterium identified in the present study as a diazotrophic species. DNA-DNA reassociation assays confirmed the existence of N
2
-fixing
Burkholderia
species different from
B. vietnamiensis
. In addition, this study shows the wide geographic distribution and substantial capability of N
2
-fixing
Burkholderia
spp. for colonizing diverse host plants in distantly separated environments.
Quantitative PCR Method for Diagnosis of Citrus Bacterial Canker
Authors: Cubero J. , Graham J. H. , Gottwald T. R. 
Published: July 2002

ABSTRACT
For diagnosis of citrus bacterial canker by PCR, an internal standard is employed to ensure the quality of the DNA extraction and that proper requisites exist for the amplification reaction. The ratio of PCR products from the internal standard and bacterial target is used to estimate the initial bacterial concentration in citrus tissues with lesions.
Effect of High-Rate Algal Ponds on Viability of
Cryptosporidium parvum
Oocysts
Authors: Araki S. , Martı́n-Gomez S. , Bécares E. , De Luis-Calabuig E. , Rojo-Vazquez F. 
Published: July 2002

ABSTRACT

The physicochemical conditions of high-rate algal ponds were responsible for a more than 97% reduction in the infectivity of
Cryptosporidium parvum
oocysts in neonatal mice. The use of semipermeable bags of cellulose showed that pH, ammonia, and/or light seems to be a major factor for the inactivation of oocysts in wastewater, supporting the importance of alga-based systems for safer reuse of treated wastewater.
Contamination of River Water by
Cryptosporidium parvum
Oocysts in Western Japan
Authors: Ono Kazuo , Tsuji Hidetaka , Rai Shiba Kumar , Yamamoto Akio , Masuda Kuniyoshi , Endo Takuro , Hotta Hak , Kawamura Takashi , Uga Shoji 
Published: July 2002

ABSTRACT

In Japan, only a few rivers have been inspected for
Cryptosporidium parvum
contamination, and the methods used had low sensitivity. In 1998 and 1999, we used a method with higher sensitivity to examine all large rivers used as sources of water supply in one prefecture (which we divided into four areas) in western Japan for
Cryptosporidium
oocysts. One sample was collected at each of 156 sites along 18 rivers, and samples were tested for
Cryptosporidium
oocysts by immunomagnetic separation. Samples were classified as being obtained on an island with livestock and fishing industries, a densely populated urban area, a western region including farming villages, or a still more rural northern area with agriculture and fishing. Restriction fragment length polymorphism analysis was used for identification of the
C. parvum
found as the bovine or human type.
C. parvum
was detected in at least one sample from 13 of the 18 rivers and in 47% (74 of 156) of the samples. One-third to all of the samples from each area contained
C. parvum
oocysts. The number of
C. parvum
oocysts per 20 liters of river water varied in the same pattern as the number of cattle kept in the four kinds of areas (as determined by the Mantel extension test). Oocysts isolated were of the bovine type; the
C. parvum
detected in rivers probably came from cattle kept in that valley. As we had expected, when tested with a more sensitive method, river water in western Japan was found to be greatly contaminated with
C. parvum
oocysts, as reported in other countries.
Low-Temperature Lipase from Psychrotrophic
Pseudomonas
sp. Strain KB700A
Authors: Rashid Naeem , Shimada Yuji , Ezaki Satoshi , Atomi Haruyuki , Imanaka Tadayuki 
Published: July 2002

ABSTRACT

We have previously reported that a psychrotrophic bacterium,
Pseudomonas
sp. strain KB700A, which displays sigmoidal growth even at −5°C, produced a lipase. A genomic DNA library of strain KB700A was introduced into
Escherichia coli
TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open reading frame (
KB-lip
) consisting of 1,422 nucleotides that encoded a protein (KB-Lip) of 474 amino acids with a molecular mass of 49,924 Da. KB-Lip showed 90% identity with the lipase from
Pseudomonas fluorescens
and was found to be a member of Subfamily I.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activity in the presence of Ca
2+
. Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca
2+
-dependent lipase. Addition of Mn
2+
and Sr
2+
also led to enhancement of lipase activity but to a much lower extent than that produced by Ca
2+
. The optimal pH of KB-Lip was 8 to 8.5. The addition of detergents enhanced the enzyme activity. When
p
-nitrophenyl esters and triglyceride substrates of various chain-lengths were examined, the lipase displayed highest activity towards C
10
acyl groups. We also determined the positional specificity and found that the activity was 20-fold higher toward the 1(3) position than toward the 2 position. The optimal temperature for KB-Lip was 35°C, lower than that for any previously reported Subfamily I.3 lipase. The enzyme was also thermolabile compared to these lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily I.3, indicating that KB-Lip has evolved to function in cold environments, in accordance with the temperature range for growth of its psychrotrophic host, strain KB700A.
Effect of Challenge Temperature and Solute Type on Heat Tolerance of
Salmonella
Serovars at Low Water Activity
Authors: Mattick K. L. , Jørgensen F. , Wang P. , Pound J. , Vandeven M. H. , Ward L. R. , Legan J. D. , Lappin-Scott H. M. , Humphrey T. J. 
Published: July 2002

ABSTRACT

Salmonella
spp. are reported to have an increased heat tolerance at low water activity (a
w
; measured by relative vapor pressure [rvp]), achieved either by drying or by incorporating solutes. Much of the published data, however, cover only a narrow treatment range and have been analyzed by assuming first-order death kinetics. In this study, the death of
Salmonella enterica
serovar Typhimurium DT104 when exposed to 54 combinations of temperature (55 to 80°C) and a
w
(rvp 0.65 to 0.90, reduced using glucose-fructose) was investigated. The Weibull model (LogS = −

bt
n

) was used to describe microbial inactivation, and surface response models were developed to predict death rates for serovar Typhimurium at all points within the design surface. The models were evaluated with data generated by using six different
Salmonella
strains in place of serovar Typhimurium DT104 strain 30, two different solutes in place of glucose-fructose to reduce a
w
, or six low-a
w
foods artificially contaminated with
Salmonella
in place of the sugar broths. The data demonstrate that, at temperatures of ≥70°C,
Salmonella
cells at low a
w
were more heat tolerant than those at a higher a
w
but below 65°C the reverse was true. The same patterns were generated when sucrose (rvp 0.80 compared with 0.90) or NaCl (0.75 compared with 0.90) was used to reduce a
w
, but the extent of the protection afforded varied with solute type. The predictions of thermal death rates in the low-a
w
foods were usually fail-safe, but the few exceptions highlight the importance of validating models with specific foods that may have additional factors affecting survival.
Mutagenic Analysis of a Conserved Region of Domain III in the Cry1Ac Toxin of
Bacillus thuringiensis
Authors: Masson Luke , Tabashnik Bruce E. , Mazza Alberto , Préfontaine Gabrielle , Potvin Léna , Brousseau Roland , Schwartz Jean-Louis 
Published: July 2002

ABSTRACT

We used site-directed mutagenesis to probe the function of four alternating arginines located at amino acid positions 525, 527, 529, and 531 in a highly conserved region of domain III in the Cry1Ac toxin of
Bacillus thuringiensis
. We created 10 mutants: eight single mutants, with each arginine replaced by either glycine (G) or aspartic acid (D), and two double mutants (R525G/R527G and R529G/R531G). In lawn assays of the 10 mutants with a cultured
Choristoneura fumiferana
insect cell line (Cf1), replacement of a single arginine by either glycine or aspartic acid at position 525 or 529 decreased toxicity 4- to 12-fold relative to native Cry1Ac toxin, whereas replacement at position 527 or 531 decreased toxicity only 3-fold. The reduction in toxicity seen with double mutants was 8-fold for R525G/R527G and 25-fold for R529G/R531G. Five of the mutants (R525G, R525D, R527G, R529D, and R525G/R527G) were tested in bioassays with
Plutella xylostella
larvae and ion channel formation in planar lipid bilayers. In the bioassays, R525D, R529D, and R525G/R527G showed reduced toxicity. In planar lipid bilayers, the conductance and the selectivity of the mutants were similar to those of native Cry1Ac. Toxins with alteration at position 527 or 529 tended to remain in their subconducting states rather than the maximally conducting state. Our results suggest that the primary role of this conserved region is to maintain both the structural integrity of the native toxin and the full functionality of the formed membrane pore.
Optimization of the Cell Wall Microenvironment Allows Increased Production of Recombinant
Bacillus anthracis
Protective Antigen from
B. subtilis
Authors: Thwaite Joanne E. , Baillie Les W. J. , Carter Noel M. , Stephenson Keith , Rees Mark , Harwood Colin R. , Emmerson Peter T. 
Published: July 2002

ABSTRACT

The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the
trans
side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases. In
Bacillus subtilis,
degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached. The former process generally involves membrane- and/or cell wall-bound proteases, while the latter involves proteases that are released into the culture medium. The identification and manipulation of factors that influence the folding of heterologous proteins has the potential to improve the yield of secreted heterologous proteins. Recombinant anthrax protective antigen (rPA) has been used as a model secreted heterologous protein because it is sensitive to proteolytic degradation both before and after folding into its native conformation. This paper describes the influence of the microenvironment on the
trans
side of the cytoplasmic membrane on the stability of rPA. Specifically, we have determined the influence of net cell wall charge and its modulation by the extent to which the anionic polymer teichoic acid is
d
-alanylated on the secretion and stability of rPA. The potential role of the
dlt
operon, responsible for
d
-alanylation, was investigated using a
Bacillus subtilis
strain encoding an inducible
dlt
operon. We show that, in the absence of
d
-alanylation, the yield of secreted rPA is increased 2.5-fold. The function of
d
-alanylation and the use of rPA as a model protein are evaluated with respect to the optimization of
B. subtilis
for the secretion of heterologous proteins.