Fusarenon X and nivalenol production by Gibberella zeae in liquid and rice cultures

Toxigenesis of Gibberella zeae strains NHL-F-1372 and NHL-F-1373 was compared in liquid and rice culture. Growth of both strains in glucose-yeast extract-peptone medium (GYEP) for 25 days resulted in peak levels of fusarenon X (FX) ranging from 40–200 mg/L with lower levels of nivalenol (NIV) (10 mg/L). Growth of these strains in modified Fries medium amended with 4% corn steep liquor (CSL) resulted in a much lower total 8-ketotrichothecene yield than in GYEP, with NIV being the primary compound produced. Although FX appeared initially in this latter medium, the toxin disappeared concurrently as the pH exceeded 8.0. Growth rates and total mycelial accumulation were lower in GYEP cultures than in the modified Fries with CSL cultures. Appearance of FX and NIV in modified Fries medium with CSL paralleled the order of appearance of these compounds in rice, but the total trichothecene yield in rice was much higher. In general, growth and toxigenesis by the nivalenol-producing fusaria in liquid and rice cultures was qualitatively similar to that previously found for deoxynivalenol-producing isolates.Michigan State University Agricultural Experiment Station Article No. 11700.     

Fusarium toxin contents of maize and maize products purchased in the years 2000 and 2001 in Germany

Samples (n=106) of maize and maize products were analysed for 13 trichothecene toxins and zearalenone (ZON). All 14 toxins examined were detected, although with varying frequency. Cooccurrence of two or more toxins was observed in 96% of samples. The toxins of the scirpenol group scirpentriol, 15-monoacetoxyscirpenol and diacetoxyscirpenol were detected in 14, 27 and 3% of the samples analysed, the toxins of the T-2 group T-2 toxin, HT-2 toxin, T-2 triol und T-2 tetraol were found in 33, 66, 2 and 7%. Toxin content was higher in feeds than in foods (semolina and flour). In food samples, the German regulatory level for DON (500 μg/kg) was not exceeded, three samples of maize flour contained ZON above the regulatory level (50 μg/kg). Presented at the 26^th Mykotoxin-Workshop in Herrching, Germany, May17–19, 2004     

<Emphasis Type="Italic">Fusarium</Emphasis> toxins of the scirpentriol subgroup: a review

Scirpentriol and its seven acetylated derivatives comprise a family of type-A trichothecene toxins produced by several species of Fusarium fungi. Out of this group 4,15-diacetoxyscirpenol has attracted most attention. It elicits toxic responses in several species and was detected in a variety of substrates. Out of the three possible monoacetylated derivatives 15-monoacetoxyscirpenol and the parent alcohol scirpentriol received some attention, whereas the remaining members of the family were mentioned in few reports. The present review deals with the structure, biosynthesis, analysis and toxicity of scirpentriol toxins. Formation by Fusarium species as well as culture conditions used for toxigenicity studies are reviewed; data about the natural occurrence of scirpentriol toxins in different cereal types, cereal associated products as well as in non-grain matrices including potato and soya bean are reported. Basing on literature reports about the toxicity of scirpentriol toxins an attempt is made to summarise the state of knowledge for risk evaluation for human and animal health.     

A survey of the natural occurrence ofFusarium toxins in wheat grown ina southwestern area of Germany

Wheat for feed use (84 samples) was collected after harvest from 79 farms in a southwestern part of Germany (Baden-Wuerttemberg). The 1987 crop year was characterized by heavy rainfall in the summer months. The internal mycoflora of wheat samples was primarily fusaria, and storage fungi were rarely present. TheFusarium toxins, zearalenone (ZON), - and -zearalenol (,-ZOL), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), nivalenol (NIV), T-2 Toxin (T-2), HT-2 toxin (HT-2) and diacetoxyscirpenol (DAS) were analysed by gas chromatography with mass selective detection (detection limit: 1–3 µg/kg). Each of the samples contained at least one of theFusarium toxins examined except DAS. DON, ZON, 3-AcDON, NIV, T-2, HT-2 and -ZOL were detected in 96%, 80%, 59%, 26%, 26%, 7% and 5% of samples, with an average of 1632, 178, 7, 9, 82, 10 and 23 µg/kg, and a maximum of 20538, 8036, 18, 32, 249, 20 and 71 µg/kg, respectively. -ZOL (12 µg/kg) was found in one sample with -ZOL (71 µg/kg). One, two, three, four, five and sixFusarium toxins were detected in 6%, 27%, 37%, 23%, 4%, and 4% of total samples, respectively. The most frequent combination was that of ZON with DON and 3-AcDON, followed by the combinations ZON/DON and ZON/DON/3-AcDON/NIV in 22, 20, and 11% of total samples, respectively.Abbreviations 3-AcDON 3-Acetyldeoxynivalenol - DAS Diacetoxyscirpenol - DON Deoxynivalenol - HT-2 HT-2 toxin - NIV Nivalenol - T-2 T-2 toxin - -ZOL -Zearalenol - -ZOL -Zearalenol - ZON Zearalenone     

The occurrence of HT-2 toxin and other trichothecenes in Norwegian cereals

A total of 449 grain samples, 102 barley, 169 wheat and 178 oat samples were collected from different regions of Norway from 1996–1998 crops, mainly from grain loads and silos. The samples were analysed for type A and B trichothecenes, the largest groups of mycotoxins produced by the Fusarium species, by gas chromatography with mass spectrometric detection (GC-MS). Factors affecting the presence of the different trichothecenes are discussed. Deoxynivalenol (DON) and HT-2 toxin were the trichothecenes most frequently detected, followed by T-2 toxin, nivalenol, and scirpentriol, scirpentriol being detected only in seven samples (>20 g/kg).Oats were the grain species most heavily contaminated with an incidence(% >20 g/kg) and mean concentration of positive samples of 70%(115 g/kg) for HT-2 toxin, 30% (60 g/kg) for T-2 toxin, 57%(104 g/kg) for DON, and 10% (56 g/kg) for nivalenol. The corresponding values for barley were 22% (73 g/kg), 5% (85 g/kg),17% (155 g/kg) and 6% (30 g/kg), and for wheat 1.2% (20 g/kg),0.6% (20 g/kg), 14% (53 g/kg) and 0% for HT-2, T-2, DON and nivalenol, respectively. Norwegian oats were found to contain HT-2 and T-2 toxin in concentrations that might be at threat to human health for high consumers of oats. The amount of DON was significantly lower than in the crop from previous years.This revised version was published online in October 2005 with corrections to the Cover Date.     

A case of sporotrichosis caused by two genetically differentSporothrix schenckii strains

A survey for the natural occurrence of Fusarium mycotoxins, deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN), in Dutch cereals (totaling 29 samples) harvested in 1984/1985, showed that 90%, 79% and 62% of samples were contaminated with DON, NIV and ZEN, respectively. Average contents (ng/g) in the total of positive samples were 221 (DON), 123 (NIV) and 61 (ZEN). Among the cereals examined, the highest concentrations (ng/g) was 3198 (DON), 1875 (NIV) and 677 (ZEN) in a yellow corn sample for animal feed. The results of this survey show that Dutch cereals were relatively significantly contaminated with Fusarium mycotoxins.     

Patterns of mycotoxin production by Fusarium gmminearum isolated from Argentine wheat

Fusarium graminearum was isolated from several wheat samples of the 1985/86 Argentine crop, taken from lots that had suffered extensive invasion by this fungus. Previous chemical analysis of the cereal, had revealed contamination with deoxynivalenol (DON), but the presence of the other mycotoxins could not be excluded with certainty, due to the low sensitivity of the analytical methodology employed.Twenty four F. graminearum isolates were grown on white corn with 50% water, for 21 days at 28 °C, or in liquid medium (Sucrose 3%, Peptone 0,1% and Yeast Extract 0,1%) for 7 days at 28 °C without shaking, and tested for the production of mycotoxins. Eight isolates (33% of the total) were found to produce toxins in one or both media. Toxins detected were: DON (6 isolates), 15-AcetylDON (5), 3-AcetylDON (2) and Zearalenone (3). No traces of Nivalenol, Fusarenon-X or other trichothecenes were found.These results suggest that strains of F. graminearum, prevailing in Argentine wheat-growing regions, might belong to the DON/AcetylDON chemotype, since no organisms of the Nivalenol/Fusarenon-X chemotype were detected in this study.     

Occurrence and distribution of <Emphasis Type="Italic">Microdochium nivale</Emphasis> and <Emphasis Type="Italic">Fusarium</Emphasis> species isolated from barley,durum and soft wheat grains in France from 2000 to 2002

Fusarium Head Blight of small grain cereal is a disease of growing concern in Europe. Along with Microdochium nivale, several species of Fusarium may be associated with the disease, including species that are potentially toxigenic. This paper describes the results of a large scale survey of the variety and frequency of different Fusarium species and M. nivale in France. A total of 749 soft wheat, durum wheat and barley samples were collected and analyzed from 2000 to 2002. The most frequent species isolated were F.graminearum, F. avenaceum and F. poae. The frequency of F. poae seems to have increased while M.nivale and F. culmorum appear less frequent than previously described in France. Other Fusarium species detected in decreasing prevalence were F. tricinctum, F. equiseti, F. acuminatum, F. sambucinum, F.sporotrichioides, F. moniliforme, F. heterosporum, F. subglutinans and F. oxysporum. All the most frequent pathogenic species and also the less pathogenic ones were frequently associated with individual fields. The implications of these associations for the protection of cereals crops and for contamination by mycotoxins are discussed.     

Genetic and mutational tools for investigating the genetics and molecular biology of trichothecene production inGibberella pulicaris (Fusarium sambucinum)

Gibberella pulicaris (Fusarium sambucinum) is a promising organism for studying the genetics and regulation of trichothecene biosynthesis; conditions for obtaining fertile crosses have been defined (Desjardins & Beremand, 1987) and crosses between natural variants have provided some information about the number, location, arrangement, and role of genes which determine trichothecene production (Desjardins & Beremand, 1987; Beremand & Desjardins, 1988). The development of some additional experimental tools and methodologies required for the further genetic analysis of trichothecene production inG. pulicaris are described in the present study. A highly fertile, isogenic line was constructed forG. pulicaris strain R-6380. The ability to readily generate mutants in this strain was also demonstrated. Both biochemical and morphological mutants were obtained following UV-mutagenesis. The inheritance of some of these mutations through meiosis indicated that they will be useful genetic markers for crosses and mapping studies. Since strain R-6380 is also transformable (Salch & Beremand, 1988), it is an excellent choice for transmission and molecular genetic studies involving trichothecene production.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Gene-enzyme relationships of the purine biosynthetic pathway in Bacillus subtilis

Summary The gene-enzyme relationship has been established for most of the steps of the purine de novo biosynthetic pathway in Bacillus subtilis. The synthesis of inosine monophosphate (IMP) involves ten steps, and the branching from IMP to AMP and to guanosine monophoshate (GMP) synthesis both require two steps. To avoid confusion in the nomenclature of the pur genes we have adopted the Escherichia coli system for B. subtilis. The two genes specifying the enzymes catalysing the conversion of IMP to succinyl-AMP (purA), and the conversion of IMP to xanthosine monophosphate (guaB), occur as single units whilst the other purine genes are clustered at 55 degrees on the B. subtilis linkage map. Based on transformation and transduction studies, and on complementation studies using B. subtilis pur genes cloned in plasmids, the arrangement of some of the clustered genes has been determined relative to outside markers. The following gene order has been established: pbuG-purB-purF-purM-purH-purD-tre. Three other genes were also found to be located in the cluster, guaA, purL and purE/C. However, we were not able to find their exact location. When the purF, purM, purD and purB genes of B. subtilis are present in plasmids they are capable of directing the synthesis in E. coli of phosphoribosylpyrophosphate amidotransferase (purF), aminoimidazole ribonucleotide synthetase (purM), glycinamide ribonucleotide synthetase (purD) and adenylosuccinate lyase (purB), respectively.     

First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 g/kg (DON) and 17 g/kg (ZEA), maximum concentrations were 1800 g kg^–1 and 120 g kg^–1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1 % and 0.7% of the samples, at at maximum level of about 100 g kg^–1. Only one sample was positive for T-2 (55 g/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.Abbreviations 3-AcDON 3-acetyldeoxynivalenol - 15-AcDON 15-acetyldeoxynivalenol - DAS diacetoxyscirpenol - DON deoxynivalenol - EIA enzyme immunoassay - T-2 T-2 toxin - ZEA zearalenone     

The effect of increasing concentrations ofFusarium toxins in the diets for piglets on histological parameters of the uterus

The effects of feeding diets containing 0.01, 0.06, 0.15, 0.22 and 0.42 mg zearalenone and 0.2, 0.8, 1.0, 1.9 and 3.9 mg deoxynivalenol per kg, coming fromFusarium toxin contaminated maize, on the uterus of 50 prepubertal piglets were investigated. The mean weight of the uteri of the animals which received the most contaminated diet was significantly increased at the time of slaughtering. The histological investigation showed no marked differences between the feeding groups. Histometrical parameters of the surface epithelium of the uterus and of the uterine glands were not altered by the treatment. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Cloning and partial characterization of the putative rifamycin biosynthetic gene cluster from the actinomycete Amycolatopsis mediterranei DSM 46095

The actinomycete Amycolatopsis mediterranei produces the commercially and medically important polyketide antibiotic rifamycin, which is widely used against mycobacterial infections. The rifamycin biosynthetic (rif) gene cluster has been isolated, cloned and characterized from A. mediterranei S699 and A. mediterranei LBGA 3136. However, there are several other strains of A. mediterranei which also produce rifamycins. In order to detect the variability in the rif gene cluster among these strains, several strains were screened by PCR amplification using oligonucleotide primers based on the published DNA sequence of the rif gene cluster and by using dEBS II (second component of deoxy-erythronolide biosynthase gene) as a gene probe. Out of eight strains of A. mediterranei selected for the study, seven of them showed the expected amplification of the DNA fragments whereas the amplified DNA pattern was different in strain A. mediterranei DSM 46095. This strain also showed striking differences in the banding pattern obtained after hybridization of its genomic DNA against the dEBS II probe. Initial cloning and characterization of the 4-kb DNA fragment from the strain DSM 46095, representing a part of the putative rifamycin biosynthetic cluster, revealed nearly 10% and 8% differences in the DNA and amino acid sequence, respectively, as compared to that of A. mediterranei S699 and A. mediterranei LBGA 3136. The entire rif gene cluster was later cloned on two cosmids from A. mediterranei DSM 46095. Based on the partial sequence analysis of the cluster and sequence comparison with the published sequence, it was deduced that among eight strains of A. mediterranei, only A. mediterranei DSM 46095 carries a novel rifamycin biosynthetic gene cluster.     

Differential toxinogenesis in the genusPichia detected by an anti-yeast killer toxin monoclonal antibody

The differential toxinogenesis of 25 isolates belonging to species of the potential yeast killer genusPichia that were previously classified in the genusHansenula was comparatively demonstrated by two serologic techniques (indirect immunofluorescence and double immunodiffusion) by using a monoclonal antibody against a yeast killer toxin produced by a selected strain ofPichia anomala (UCSC 25F). The killer phenotypes of thePichia isolates were evaluated by their ability to kill each other. The results, although of insufficient taxonomic value for a reliable separation of either species or genera, attest to the genomic heterogeneity for the killer character in the genusPichia as well as the presumptive dual killer/sensitive identity for each single isolate.     

Molecular characterization of Alg8, a putative glycosyltransferase, involved in alginate polymerisation

The topology of Alg8, the proposed catalytic subunit of the alginate polymerase, was assessed using PhoA and LacZ fusion protein analysis. This analysis suggested that the periplasmic loop comprises only three amino acid residues with the adjacent transmembrane helices at positions 361–387 and 393–416. Accordingly, the extended cytosolic loop could be located at positions 71–361 and was proposed to contain important catalytic residues. Further experimental evidence for this cytosolic domain was obtained by independently demonstrating this protein region as purified soluble protein domain. The soluble protein domain was identified by MALDI-TOF/MS and presumably represents the cytosolic catalytic domain of Alg8. Site-directed mutagenesis of 11 conserved residues in the cytosolic loop showed that D-188/D-190 (DXD motif), D-295/D-296 (acid–base catalysts) and K-297 were each essential for in vivo polymerase activity, whereas D-179/D-181 (DXD motif), C-244, R-263, D-279, and E-282 were not directly involved in the polymerisation reaction. The role of these amino acid residues with respect to the catalysed alginate polymerisation reaction was discussed with the aid of the recently developed structural model of Alg8.     

Control of the pyrimidine biosynthetic pathway in Pseudomonas pseudoalcaligenes

The five de novo enzyme activities unique to the pyrimidine biosynthetic pathway were found to be present in Pseudomonas pseudoalcaligenes ATCC 17440. A mutant strain with 31-fold reduced orotate phosphoribosyltransferase (encoded by pyrE) activity was isolated that exhibited a pyrimidine requirement for uracil or cytosine. Uptake of the nucleosides uridine or cytidine by wild-type or mutant cells was not detectable; explaining the inability of the mutant strain to utilize either nucleoside to satisfy its pyrimidine requirement. When the wildtype strain was grown in the presence of uracil, the activities of the five de novo enzymes were depressed. Pyrimidine limitation of the mutant strain led to the increase in aspartate transcarbamoylase and dihydroorotate dehydrogenase activities by more than 3-fold, and dihydroorotase and orotidine 5-monophosphate decarboxylase activities about 1.5-fold, as compared to growth with excess uracil. It appeared that the syntheses of the de novo enzymes were regulated by pyrimidines. In vitro regulation of aspartate transcarbamoylase activity in P. pseudoalcaligenes ATCC 17440 was investigated using saturating substrate concentrations; transcarbamoylase activity was inhibited by P_i, PP_i, uridine ribonucleotides, ADP, ATP, GDP, GTP, CDP, and CTP.     

Ammonia oxidation by the methane oxidising bacterium Methylococcus capsulatus strain bath

Soluble extracts of Methylococcus capsulatus (Bath) that readily oxidise methane to methanol will also oxidise ammonia to nitrite via hydroxylamine. The ammonia oxidising activity requires O₂, NADH and is readily inhibited by methane and specific inhibitors of methane mono-oxygenase activity. Hydroxylamine is oxidised to nitrite via an enzyme system that uses phenazine methosulphate (PMS) as an electron acceptor. The estimated K _mvalue for the ammonia hydroxylase activity was 87 mM but the kinetics of the oxidation were complex and may involve negative cooperativity.Abbreviations PMS Phenazine methosulphate - NADH nicotinamide adenine dinucleotide, reduced form - K _m Michaelis constant - NO ₂ ^- nitrite - NH₂OH hydroxylamine     

Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin,fusicoccin

The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60–70% of the complexes can be solubilized with 50–60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml^-1 soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9-nor-fusicoccin-8-alcohol-[³H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4–10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (M_r) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an M_r of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9-nor-8[(3,5-[³H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([³H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an M_r of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [³H]ABE-FC in the presence of 0.1–1 M fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.Abbreviations ABE-FC [(4-azidobenzoyl)-ethylenediamine]-fusicoccin - ABE-NHS (4-azidobenzoyl)-N-hydroxysuccinimide ester - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-norfusicoccin-8-alcohol - MAB monoclonal antibody - Mega-9(10) nonanoyl(decanoyl)-N-methylglucamide - M_r apparent molecular mass - PMSF phenylmethyl-sulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

A new method for isolating physiologically active Mg-protoporphyrin monomethyl ester, the substrate of the cyclase enzyme of the chlorophyll biosynthetic pathway

Mg-protoporphyrin monomethyl ester (MPE) is a biosynthetic intermediate of chlorophyll and converted by MPE cyclase to protochlorophyllide. Limited availability of MPE has so far hampered cyclase research. In a new, simplified, method MPE was prepared from freeze dried bchE mutant Rhodobacter capsulatus DB575 cells by extraction with acetone/H(2)O/25% NH(3). Isolated MPE was identified by absorption and fluorescence spectroscopy, and its purity was analyzed by HPLC. The extracted MPE was dried and redissolved in buffered DMSO and its substrate activity is shown by enzymatic cyclase assays. A linear time course was observed for MPE conversion to protochlorophyllide by enzymes from barley etioplasts. Our innovation of freeze drying the R. capsulatus cells before extraction provides a high yield method for MPE, which is significantly faster and more reproducible than previous extraction methods.     

Kinetic studies on ammonia and methane oxidation by Nitrosococcus oceanus

The kinetics of ammonia oxidation and the ability of a marine ammonia-oxidizing bacterium, Nitrosococcus oceanus, to metabolize methane were investigated in semicontinuous batch culture. The effects of inhibitors (acetylene and nitrapyrin) and coreactants were determined in order to elucidate the behavior of the ammonia oxygenase enzyme in N. oceanus. Acetylene and nitrapyrin were potent inhibitors and their effects were not mitigated by increased ammonia concentrations. Oxygen concentration had the effect of a mixed-type inhibitor; reduced oxygen inhibited the rate or ammonia oxidation at high substrate concentration but may enhance the rate at low substrate concentrations. Substrate affinity in terms of NH ₄ ⁺ increased (K _m decreased) with increasing pH. Optimal pH was about 8. Methane inhibited ammonia oxidation; the interaction was not simple competitive inhibition and the presence of multiple active sites on the enzyme was indicated by the behaviour of the inhibited treatments. Half-saturation constants for methane (K _i=6.6 M) and ammonia (K _m=8.1 M) were similar. N. oceanus oxidized methanol and methane linearly over time, with CO₂ and cell material being produced at approximately equal rates.