Defect in translation of poliovirus RNA at the restrictive temperature.

Translation of the RNA of LSc type 1 poliovirus was examined in vivo at the restrictive temperature (39 °C). During the first two hours of infection at 39 °C the levels of viral polyribosomes were 50% lower than at 35 °C (permissive temperature). During the third hour of infection at 39 °C, only 4 to 10% of the control levels of polyribosomes were observed. Three experiments indicate that the elongation of viral peptides was not occurring properly at 39 °C. First, cultures incubated at 39 °C during the third hour of infection with both [³⁵S]methionine and [³H]uridine exhibit a fourfold increase in the ratio of viral protein/viral RNA in the polyribosome region of sucrose gradients in comparison to controls kept at 35 °C. However, at both temperatures the relative size distribution of polyribosomes was similar. Second, the ratios of released protein/nascent protein after 90-second and 5-minute pulses with [³⁵S]methionine indicate that elongation of peptide chains was inhibited at 39 °C. Third, when initiation of synthesis of viral protein was blocked with 150 mM-NaCl, the polyribosomes disaggregated four to five times more rapidly at 35 °C than at 39 °C. The data indicate that translation of viral RNA is inhibited at the restrictive temperature because of a reduced rate of elongation of viral proteins. The reduced rate of peptide chain elongation at 39 °C was fully reversible when cultures were shifted to 35 °C in the presence of 150 mm-NaCl. The latter finding indicates a conformational change in viral protein at 39 °C.     

The <Emphasis Type="Italic">ecdysoneless</Emphasis><Superscript>1</Superscript> Gene Regulates Metabolism of the Juvenile Hormone and Dopamine in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The dopamine (DA) content and the level of juvenile hormone (JH) degradation were studied in females of the wild-type Canton S strain and the ecdysoneless ¹ (ecd ¹) mutant, which does not produce ecdysone at a restrictive temperature (29°C). Exposure at the restrictive temperature considerably increased the JH-hydrolyzing activity and the DA content in five-day ecd ¹ females compared with flies of both strains growing at 19°C and Canton S females exposed at 29°C. In one-day ecd ¹ females, the level of JH degradation also increased at the restrictive temperature, but the DA content was low. The effect of ecdysone deficiency on the stress reaction in Drosophila melanogaster females was studied using changes in DA content and JH degradation as the reaction indicators. The ecd ¹ mutation did not prevent the initiation of the stress reaction in females exposed at the restrictive temperature, but changed its intensity (stress reactivity). The interaction of 20-hydroxyecdysone with JH and DA in regulating Drosophila reproduction under normal conditions and in stress is discussed.     

Retransformation of ts SV40 Transformants by Murine Sarcoma Virus at Non-permissive Temperature

Temperature sensitive (ts) SV40 transformed mouse fibroblasts (tsSV3T3) express their transformed phenotype in vitro when growing at 32° C but not when growing at 39° C¹. Viral mRNA is, however, apparently transcribed at 39° C, for SV40 specific T-antigen can be demonstrated and viral mRNA can be found by nucleic acid hybridization: Fusion-rescue experiments show that the transforming virus is wild type but tsSV3T3 cells cannot be re-transformed at 39° C with high multiplicity SV40. This suggests that the temperature sensitive behaviour stems from a cellular rather than a viral mutation. The question then arises of the stringency with which these ts transformants control the expression of viral transformation functions at 39° C.     

Optimized expression of prolyl aminopeptidase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its characteristics after glycosylation

Prolyl aminopeptidases are specific exopeptidases that catalyze the hydrolysis of the N-terminus proline residue of peptides and proteins. In the present study, the prolyl aminopeptidase gene (pap) from Aspergillus oryzae JN-412 was optimized through the codon usage of Pichia pastoris. Both the native and optimized pap genes were inserted into the expression vector pPIC9 K and were successfully expressed in P. pastoris. Additionally, the activity of the intracellular enzyme expressed by the recombinant optimized pap gene reached 61.26 U mL^?1, an activity that is 2.1-fold higher than that of the native gene. The recombinant enzyme was purified by one-step elution through Ni-affinity chromatography. The optimal temperature and pH of the purified PAP were 60 °C and 7.5, respectively. Additionally, the recombinant PAP was recovered at a yield greater than 65 % at an extremely broad range of pH values from 6 to 10 after treatment at 50 °C for 6 h. The molecular weight of the recombinant PAP decreased from 50 kDa to 48 kDa after treatment with a deglycosylation enzyme, indicating that the recombinant PAP was completely glycosylated. The glycosylated PAP exhibited high thermo-stability. Half of the activity remained after incubation at 50 °C for 50 h, whereas the remaining activity of PAP expressed in E. coli was only 10 % after incubation at 50 °C for 1 h. PAP could be activated by the appropriate salt concentration and exhibited salt tolerance against NaCl at a concentration up to 5 mol L^?1.     

Verrucarin A and roridin E produced on spinach by <Emphasis Type="Italic">Myrothecium verrucaria</Emphasis> under different temperatures and CO<Subscript>2</Subscript> levels

The behavior of Myrothecium verrucaria, artificially inoculated on spinach, was studied under seven different temperature conditions (from 5 to 35 °C) and under eight different combinations of temperature and CO₂ concentration (14–30 °C and 775–870 or 1550–1650 mg/m³). The isolate used for this study was growing well on spinach, and the mycotoxins verrucarin A and roridin E were produced under all tested temperature and CO₂ conditions. The maximum levels of verrucarin A (18.59 ng/g) and roridin E (49.62 ng/g) were found at a temperature of 26–30 °C and a CO₂ level of 1550–1650 mg/m³. Rises in temperature as well as in temperature and CO₂ concentrations had a significant effect by increasing Myrothecium leaf spots on spinach. The biosynthesis of verrucarin A was significantly increased at the highest temperature (35 °C), while roridin E was influenced by the CO₂ concentration. These results show that a positive correlation between climate condition and macrocyclic trichothecene production is possible. However, because of the ability of M. verrucaria to produce mycotoxins, an increase in temperature could induce the spread of M. verrucaria in temperate regions; this pathogen may gain importance in the future.     

Distribution of bacteriochlorophyll between the pigment-protein complexes of the sulfur photosynthetic bacterium <Emphasis Type="Italic">Allochromatium minutissimum</Emphasis> depending on light intensity at different temperatures

Variation of the distribution of bacteriochlorophyll a (BChl a) between external antenna (LH2) and core complexes (LH1 + RC) of the photosynthetic membrane of the sulfur bacterium Allochromatium minutissimum was studied at light intensities of 5 and 90 Wt/m² in the temperature range of 12–43°C. The increase of light intensity was shown to result in a 1.5-to 2-times increase of a photosynthetic unit (PSU). PSU sizes pass through a maximum depending on growth temperature, and the increase of light intensity (5 and 90 Wt/m²) results in a shift of the maximal PSU size to higher temperatures (15 and 20°C, respectively). In the narrow temperature interval of ～14–17°C, the ratio of light intensity to PSU size is typical of phototrophs: lower light intensity corresponds to larger PSU size. The pattern of PSU size change depending on light intensity was shown to differ at extreme growth temperatures (12°C and over 35°C). The comparison of Alc. minutissimum PSU size with the data on Rhodobacter capsulatus and Rhodopseudomonas palustris by measuring the effective optical absorption cross-section for the reaction of photoinhibition of respiration shows a two to four times greater size of light-harvesting antenna for Alc. minutissimum, which seems to correspond to the maximum possible limit for purple bacteria.     

Optimization of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> (KU923381) for diesel oil degradation using response surface methodology (RSM)

Efficiency of Enterobacter cloacae KU923381 isolated from petroleum hydrocarbon contaminated soil was evaluated in batch culture and bioreactor mode. The isolate were screened for biofilm formation using qualitative and quantitative assays. Response surface methodology (RSM) was used to study the effect of pH, temperature, glucose concentration, and sodium chloride on diesel degradation. The predicted values for diesel oil degradation efficiency by the statistical designs are in a close agreement with experimental data (R ² = 99.66%). Degradation efficiency is increased by 36.78% at pH = 7, temperature = 35°C, glucose = 5%, and sodium chloride concentration = 5%. Under the optimized conditions, the experiments were performed for diesel oil degradation by gas chromatographic mass spectrometric analysis (GC-MS). GC-MS analysis confirmed that E. cloacae had highly degrade hexadecane, heptadecane, tridecane, and docosane by 99.71%, 99.23%, 99.66%, and 98.34% respectively. This study shows that rapid bioremoval of hydrocarbons in diesel oil is acheived by E. cloacae with abet of biofilm formation. The potential use of the biofilms for preparing trickling filters (gravel particles) for the degradation of hydrocarbons from petroleum wastes before their disposal in the open environment is highly suggested. This is the first successful attempt for artificially establishing petroleum hydrocarbon degrading bacterial biofilm on solid substrates in bioreactor.     

Optimization of Nutrients and Culture Conditions for Alkaline Protease Production Using Two Endophytic Micrococci: <Emphasis Type="Italic">Micrococcus aloeverae</Emphasis> and <Emphasis Type="Italic">Micrococcus yunnanensis</Emphasis>

An endophytic species of Micrococcus was isolated from Aloe vera leaf (syn. Aloe barbadensis) and screened for protease production with five other species of Micrococcus. Data indicated that endophytic Micrococcus aloeverae AE-6 MCC 2184^T and Micrococcus yunnanensis DSM 21948^T showed efficient protease production potential and secreted active protease at high salt (10%), temperature (40 °C) and in wide range of pH 8–10. Unlike M. yunnanensis DSM 21948^T, protease production by M. aloeverae AE-6 MCC 2184^T was stringently controlled by pH. Protease induction study using different group of peptides, peptide carbohydrates and peptide macronutrient combinations showed variable response with both the organisms. Result indicated that the amount of protease was not directly related to cell biomass but it depends on nature of inducible peptides. In this study we also developed a modified agar-well assay for semi-quantitative data from large number of replicates.     

Gene cloning and characterization of recombinant L-Asparaginase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain R5

L-asparaginase gene from Bacillus subtilis strain R5 (Asn-R5), comprising 990 nucleotides corresponding to a polypeptide of 329 amino acids, was cloned and expressed in Escherichia coli. Recombinant Asn-R5 was produced in soluble and active form exhibiting a specific activity of 223 μmol min^?1 mg^?1. The optimal temperature and pH for L-asparaginase activity of Asn-R5 were 35 °C and 9.0, respectively. Asn-R5 displayed a 50% activity with D-asparagine and 2% with L-glutamine compared to 100% with L-asparagine. No activity could be detected when D-glutamine was used as substrate. Half-life of the enzyme was 180 min at 35 °C and 40 min at 50 °C. There was no effect of metal ions and EDTA on the activity indicating that Asn-R5 enzyme activity is not metal ion dependent. The K_m and V_max values were 2.4 mM and 265 μmol min^?1 mg^?1, respectively. Activation energy for reaction catalyzed by Asn-R5 was 28 kJ mol^?1. High L-asparaginase activity and thermostability of recombinant Asn-R5 may be beneficial for industrial production and application.     

Deciphering the mode of action,structural and biochemical analysis of heparinase II/III (<Emphasis Type="Italic">Ps</Emphasis>PL12a) a new member of family 12 polysaccharide lyase from <Emphasis Type="Italic">Pseudopedobacter saltans</Emphasis>

Heparinases are widely used for production of clinically and therapeutically important bioactive oligosaccharides and in analyzing the polydisperse, heterogeneous, and complex structures of heparin/heparan sulfate. In the present study, the gene (1911 bp) encoding heparinase II/III of family 12 polysaccharide lyase (PsPL12a) from Pseudopedobacter saltans was cloned, expressed, and biochemically and functionally characterized. The purified enzyme PsPL12a of molecular size approximately 76 kDa exhibited maximum activity in the temperature range 45–50 °C and at pH 6.0. PsPL12a gave maximum activity at 1% (w/v) heparin under optimum conditions. The kinetic parameters, K _m and V_max, for PsPL12a were 4.6?±?0.5 mg/ml and 70?±?2 U/mg, respectively. Ten millimolars of each Mg²⁺ and Mn²⁺ ions enhanced PsPL12a activity by 80%, whereas Ni²⁺ inhibited by 75% and Co²⁺ by 10%, and EDTA completely inactivated the enzyme. Protein melting curve of PsPL12a gave a single peak at 55 °C and 10 mM Mg²⁺ ions and shifted the peak to 60 °C. The secondary structure analysis of PsPL12a by CD showed 65.12% α-helix, 11.84% β-strand, and 23.04% random coil. The degradation products of heparin by PsPL12a analyzed by ESI-MS spectra displayed peaks corresponding to heparin di-, tetra-, penta-, and hexa-saccharides revealing the endolytic mode of enzyme action. Heparinase II/III (PsPL12a) from P. saltans can be used for production of low molecular weight heparin oligosaccharides for their utilization as anticoagulants. This is the first report on heparinase cloned from P. saltans.     

Trichloroethylene removal and bacterial variations in the up-flow anaerobic sludge blanket reactor in response to temperature shifts

Trichloroethylene (TCE) degradation and the variations of bacteria composition and structure in the up-flow anaerobic sludge blanket (UASB) reactor were investigated by increasing the operating temperature from 20 to 40 °C. The influent was supplemented with 36.5 mg/L of TCE. There was a rise in the chemical oxygen demand (COD) removal efficiency from 20 to 35 °C and a decline when temperature enhanced to 40 °C. It reached maximum at 35 °C. In addition, TCE removal efficiency increased with temperature varying from 20 to 35 °C, and it dropped dramatically to 78.38 % at 40 °C, which presumably because the genus of Dehalobacter, a kind of bacteria with the ability to dechlorinate TCE to the corresponding chlorinated products, was not detected at 40 °C according to sequencing results. The Illumina MiSeq platform was adopted to explore the bacteria composition and structure in response to temperature shifts. The results indicated that temperature impacted greatly on the dominance and presence of specific populations at different taxonomic levels. Importantly, the class Dehalococcoidia was detected from 25 to 40 °C, in which there were many well-known Dehalococcoides sp. strains that were capable of complete dechlorination of TCE to ethene. It also suggested the potential function of the dominant genera (non-dechlorinating bacteria and dechlorinating bacteria) in the reactor.     

Defective DNA Synthesis in Permeabilized Yeast Mutants

THE simple eukaryote, Saccharomyces cerevisiae, is suitable for combined genetic and biochemical analysis of the cell division cycle. More than forty temperature-sensitive mutants of S. cerevisiae defective in fifteen genes that control various steps of the yeast cell cycle have been detected by screening a collection of mutants with time-lapse photomicroscopy¹. Mutations in two genes, cdc4 and cdc8, result in defective DNA synthesis at the restrictive temperature². The product of cdc8 is apparently required throughout the period of DNA synthesis, because if a strain defective in this gene is shifted to 36° C within the S period, DNA replication ceases. In contrast, the product of cdc4 is apparently required only at the initiation of DNA synthesis because when a strain carrying a defect in this gene is shifted to 36° C, DNA replication already in progress is not impaired. Cells defective in cdc4, however, fail to initiate new rounds of DNA synthesis at the restrictive temperature. Based on these observations the DNA mutants have been tentatively classified as defective in DNA replication (cdc8) and in the initiation of DNA synthesis (cdc4).     

Molecular cloning and characterization of calmodulin-like protein CaLP from the Scleractinian coral <Emphasis Type="Italic">Galaxea astreata</Emphasis>

Optimal temperature and light are both necessary conditions for coral survival. Light enhances calcification, and thermal stress disrupts Ca²⁺ homeostasis. As calcium is involved in many important metabolic activities, in this study, we cloned the calmodulin-like protein (CaLP) gene of one of the scleractinian corals, Galaxea astreata. We also detected the relative mRNA expression levels of gaCaLP using the calcium channel blocker verapamil and CaCl₂ treatment under conditions of light and dark, and compared expression levels under controlled temperature conditions. Full-length gaCaLP cDNA comprised 1290 nucleotides and contained 498 bp open reading frame that encoded a protein with 165 amino acids. With CaCl₂, expression levels of gaCaLP only increased in the presence of light, suggesting that light may be a restrictive factor in CaLP expression when sufficient calcium is available in the environment. In addition, after verapami treatment, we noted that a down regulation of gaCaLP, suggesting that the expression of CaLP is closely related to extracellular Ca²⁺ influx. Under temperature stress at both high (30 °C) and low (20 °C) temperatures, expression levels of gaCaLP showed an initial increase, followed by a decreasing trend as treatment progressed. Expression levels reached their maximum value at 24 h. This result showed that CaLP participated in a temperature stress response, and Ca²⁺ homeostasis was disrupted during stress. The findings of the present study will help determine the function and regulatory mechanisms of gaCaLP.     

Effect of Temperature on Development and Reproduction of <Emphasis Type="Italic">Epilachna dodecastigma</Emphasis> (Wied.) (Coleoptera: Coccinellidae)

The effect of five constant temperatures of 21, 24, 27, 30 and 33 °C on adult life span, reproduction, oviposition behavior and larval developmental time of a bitter gourd inhabited coleopteran insect Epilachna dodecastigma (Wied.) (Coccinellidae) was determined in laboratory conditions under 70 ± 5 % relative humidity and a photoperiod of 12 L : 12 D. Larval developmental time of E. dodecastigma decreased as temperature increased from 21 to 33 °C. Life table data revealed that overall mortality was lowest at 27 °C and highest at 21 °C. Females lived longer than males at all temperatures; but longevity decreased with increase in temperature. Pre-oviposition period decreased significantly with increase in temperature up to 27 °C and thereafter increased at a slower rate; whereas oviposition period decreased significantly with increase in temperature. Fecundity and egg viability increased significantly with an increase in temperature up to 27 °C and thereafter decreased at a slower rate. The intrinsic rate of increase (r _m ) was 0.1703, 0.1984, 0.2235, 0.2227 and 0.2181 day^?1 at 21, 24, 27, 30 and 33 °C, respectively. The net reproductive rate and finite rate of increase was highest at 27 °C (R _o  = 112.05; λ = 1.4233) and lowest at 21 °C (R _o  = 51.23; λ = 1.2581).     

Thermal Stability Investigation and the Kinetic Study of Folnak<Superscript>®</Superscript> Degradation Process Under Nonisothermal Conditions

The nonisothermal degradation process of Folnak^® drug samples was investigated by simultaneous thermogravimetric and differential thermal analysis in the temperature range from an ambient one up to 810°C. It was established that the degradation proceeds through the five degradation stages (designated as I, II, III, IV, and V), which include: the dehydration (I), the melting process of excipients (II), as well as the decomposition of folic acid (III), corn starch (IV), and saccharose (V), respectively. It was established that the presented excipients show a different behavior from that of the pure materials. During degradation, all excipients increase their thermal stability, and some kind of solid–solid and/or solid–gas interaction occurs. The kinetic parameters and reaction mechanism for the folic acid decomposition were established using different calculation procedures. It was concluded that the folic acid decomposition mechanism cannot be explained by the simple reaction order (ROn) model (n?=?1) but with the complex reaction mechanism which includes the higher reaction orders (RO, n?>?1), with average value of <n?>?=?1.91. The isothermal predictions of the third (III) degradation stage of Folnak^® sample, at four different temperatures (T _iso?=?180°C, 200°C, 220°C, and 260°C), were established. It was concluded that the shapes of the isothermal conversion curves at lower temperatures (180–200°C) were similar, whereas became more complex with further temperature increase due to the pterin and p-amino benzoic acid decomposition behavior, which brings the additional complexity in the overall folic acid decomposition process.     

Specific Labelling of Virus Proteins by Quantitative Reaction of Cysteine Residues with <Superscript>35</Superscript>S-Sulphite

POLYPEPTIDES from different sources can be compared conveniently by digesting them with proteolytic enzymes and fingerprinting the resulting smaller peptides. If peptides with identical electrophoretic and chromatographic properties are obtained, the implication is very strong that the sequences of the original polypeptides were, at least in part, the same. The need for such comparisons arises in studies of in vitro polypeptides synthesized in coupled systems directed by viral DNAs. The material synthesized in vitro must be compared with authentic virus-coded material to verify that the system is transcribing DNA to RNA and translating RNA to protein with fidelity. For viruses such as SV40 and polyoma, which can be grown in tissue culture, the virus particles grown in the presence of ³⁵S-methionine are a convenient source of virus-coded proteins. Proteolytic digests of these particles can be compared with digests of ³⁵S-methionine labelled material synthesized in vitro. Preliminary results have shown that, in the case of polyoma virus, matching peptides are obtained from virus particles and polypeptides synthesized in vitro¹.     

<Emphasis Type="Italic">Pedobacter panacis</Emphasis> sp. nov., isolated from <Emphasis Type="Italic">Panax ginseng</Emphasis> soil

A novel strain, DCY108^T was isolated from soil of a Panax ginseng field, Yeoncheon province (38°04′N 126°57′E), Republic of Korea. Strain DCY108^T is Gram-negative, non-motile, non-flagellate, rod-shaped, and aerobic. The bacterium grows optimally at 25–30 °C, pH 6.5–7.0 and 1 % NaCl. Phylogenetically, strain DCY108^T is closely related to Pedobacter jejuensis JCM 18824^T, Pedobacter aquatilis JCM 13454^T, Pedobacter kyungheensis LMG 26577^T and the type strain of the genus Pedobacter heparinus DSM 2366^T. The DNA–DNA relatedness values between strain DCY108^T and its close phylogenetic neighbors were below 30.0 %. The DNA G+C content of strain DCY108^T was determined to be 45.1 mol%. The predominant quinone was menaquinone 7 (MK-7). The major polar lipids were identified as phosphatidylethanolamine and three unidentified aminolipids AL1, AL13 and AL17. Iso-C_15:00, iso-C_17:03OH and summed feature 3 (C_16:1 ω7c/C_16:1 ω6c) were identified as the major fatty acids present in strain DCY108^T. The results of physiological and biochemical tests allowed strain DCY108^T to be differentiated phenotypically from other recognized species belonging to the genus Pedobacter. Therefore, it is suggested that the newly isolated organism represents a novel species, for which the name Pedobacter panacis sp. nov is proposed with the type strain designated as DCY108^T (=CCTCCAB 2015196^T = KCTC 42748^T).     

Molecular Cloning,Expression and Characterization of Pectin Methylesterase (<Emphasis Type="Italic">Ct</Emphasis>PME) from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0–9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca²⁺ or Mg²⁺ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca²⁺ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.     

<Emphasis Type="Italic">Plectosphaerella cucumerina</Emphasis> as a bioherbicide for <Emphasis Type="Italic">Cirsium arvense</Emphasis>: proof of concept

Plectosphaerella cucumerina (Lindf.) W. Gams was evaluated as a bioherbicide for Cirsium arvense L. (Scop.) using a Canadian and a New Zealand isolate. Both isolates defoliated C. arvense when applied at 10¹³ conidia ha^?1 in water volumes ranging from 250 to 6400 l ha^?1 with a rapid decline in effect with declining conidial dose. Repeat application and the addition of the adjuvant Pulse^® penetrant to the conidial suspension increased the disease severity in C. arvense. Maximum disease occurred at 20 °C with a 48 h post-application dew period. The experiments demonstrate that P. cucumerina can defoliate C. arvense under the environmental conditions of temperate pastures where the weed is problematic. The results also show that modifications to formulation and strategic application may reduce the 48 h dew period requirement and risk to non-target species respectively, supporting the conclusion that the fungus has potential as a bioherbicide for C. arvense.