Strain ofP Research Articles

Adenylate kinase as a virulence factor of Pseudomonas aeruginosa.

Adenylate kinase (AK; ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg. ATP + AMP <--> Mg. ADP + ADP. In this study we show that AK secreted by the pathogenic strains of Pseudomonas aeruginosa appears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain of P. aeruginosa 8821 and hyperproduced it as a fusion protein with glutathione S-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection with P. aeruginosa. Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allows P. aeruginosa to exert its full virulence.

Analysis of the pmsCEAB gene cluster involved in biosynthesis of salicylic acid and the siderophore pseudomonine in the biocontrol strain Pseudomonas fluorescens WCS374.

Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb EcoRI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of P. putida. Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs (pmsC and pmsB) showed homologies with chorismate-utilizing enzymes; a third ORF (pmsE) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also contained a putative histidine decarboxylase gene (pmsA). A putative promoter region and two predicted iron boxes were localized upstream of pmsC. We determined by reverse transcriptase-mediated PCR that the pmsCEAB genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in P. putida and Escherichia coli cells. In E. coli, deletions affecting the first ORF (pmsC) diminished SA production, whereas deletion of pmsB abolished it completely. The pmsB gene induced low levels of SA production in E. coli when expressed under control of the lacZ promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn5 mutant (374-05) that is simultaneously impaired in SA and pseudomonine production.

The green fluorescent protein gene functions as a reporter of gene expression in Phanerochaete chrysosporium.

The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter in Phanerochaete chrysosporium. Four different constructs were prepared. pUGGM3' and pUGiGM3' contain the P. chrysosporium gpd promoter fused upstream of the egfp coding region, and pUMGM3' and pUMiGM3' contain the P. chrysosporium mnp1 promoter fused upstream of the egfp gene. In all constructs, the egfp gene was followed by the mnp1 gene 3' untranslated region. In pUGGM3' and pUMGM3', the promoters were fused directly with egfp, whereas in pUGiGM3' and pUMiGM3', following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the gpd gene were inserted at the 5' end of the egfp gene. All constructs were ligated into a plasmid containing the ura1 gene of Schizophyllum commune as a selectable marker and were used to transform a Ural1 auxotrophic strain of P. chrysosporium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5' of the egfp gene (pUGiGM3' and pUMiGM3') exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5' intron resulted in more egfp RNA than was found in transformants carrying an intronless egfp. These results suggest that the presence of a 5' intron affects the expression of the egfp gene in P. chrysosporium. The expression of GFP in the transformants carrying pUMiGM3' paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studying cis-acting elements in the mnp1 gene promoter.

The gdhB gene of Pseudomonas aeruginosa encodes an arginine-inducible NAD(+)-dependent glutamate dehydrogenase which is subject to allosteric regulation.

The NAD(+)-dependent glutamate dehydrogenase (NAD-GDH) from Pseudomonas aeruginosa PAO1 was purified, and its amino-terminal amino acid sequence was determined. This sequence information was used in identifying and cloning the encoding gdhB gene and its flanking regions. The molecular mass predicted from the derived sequence for the encoded NAD-GDH was 182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa). Cross-linking studies established that the native NAD-GDH is a tetramer of equal subunits. Comparison of the derived amino acid sequence of NAD-GDH from P. aeruginosa with the GenBank database showed the highest homology with hypothetical polypeptides from Pseudomonas putida, Mycobacterium tuberculosis, Rickettsia prowazakii, Legionella pneumophila, Vibrio cholerae, Shewanella putrefaciens, Sinorhizobium meliloti, and Caulobacter crescentus. A moderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) from Saccharomyces cerevisiae or Neurospora crassa. Comparison with the yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and for catalytic function. NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate. Primer extension experiments established that transcription of gdhB is initiated from an arginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins. NAD-GDH was purified to homogeneity from a recombinant strain of P. aeruginosa and characterized. The glutamate saturation curve was sigmoid, indicating positive cooperativity in the binding of glutamate. NAD-GDH activity was subject to allosteric control by arginine and citrate, which function as positive and negative effectors, respectively. Both effectors act by influencing the affinity of the enzyme for glutamate. NAD-GDH from this organism differs from previously characterized enzymes with respect to structure, protomer mass, and allosteric properties indicate that this enzyme represents a novel class of microbial glutamate dehydrogenases.

Regulation of the Porphyromonas gingivalis fimA (Fimbrillin) gene.

In common with many bacterial virulence genes, the fimbrillin (fimA) gene of Porphyromonas gingivalis is modulated in response to environmental fluctuation. The trans-acting components that comprise the regulatory system for transcriptional activity of the fimA gene in P. gingivalis were investigated. Three major proteins were found to bind to the upstream region of the fimA promoter. One of these proteins was fimbrillin itself, and the other two were a major arginine protease (Rgp) and lysine protease (Kgp). Production of these proteins was necessary for maximal fimA transcription. An exogenous fimA promoter-lacZ reporter was inactive when introduced into a strain of P. gingivalis carrying a mutation in the indigenous fimA gene. Furthermore, fimA mRNA levels were significantly decreased in rgp and kgp mutant strains. These data indicate that P. gingivalis has evolved multiple levels of control of fimbrial gene expression to enhance its survival in hostile environments.

Karyotype analysis of interspecific fusants of basidiomycetes by pulsed-field gel electrophoresis

The purpose of this research was to analyze the karyotype of the interspecific fusants of twoPleurotus species. Auxotrophic mutants derived from the cultivated strain ofP. ostreatus andP. cornucopiae were used. Protoplasts were fused electrically, and the fusants were selected under auxotrophic complementation. Esterase isozyme analysis showed that several fusants had isozyme bands originating from both parental strains, and others had unilateral isozyme bands. The fusant that had expressed isozyme bands of both parental strains showed chromosomal DNA bands of both of the parental strains in pulsedfield gel electrophoresis analysis. Despite the above results, the chromosomal composition of the fusants obtained by the pulsed-field gel electrophoresis did not exhibit all of the bands of both fusion parents.

Isolation of bacteriophages specific to a fish pathogen, Pseudomonas plecoglossicida, as a candidate for disease control.

Two types of bacteriophage specific to Pseudomonas plecoglossicida, the causative agent of bacterial hemorrhagic ascites disease in cultured ayu fish (Plecoglossus altivelis), were isolated from diseased ayu and the rearing pond water. One type of phage, which formed small plaques, was tentatively classified as a member of the family Myoviridae, and the other type, which formed large plaques, was classified as a member of the family Podoviridae. All 27 strains of P. plecoglossicida examined, which were isolated from diseased ayu from geographically different areas in 1991 to 1999, exhibited quite similar sensitivities to either type of phage. One strain of P. plecoglossicida was highly virulent for ayu, and the 50% lethal dose (LD(50)) when intramuscular injection was used was 10(1.2) CFU fish(-1); in contrast, phage-resistant variants of this organism were less virulent (LD(50), >10(4) CFU fish(-1)). Oral administration of phage-impregnated feed to ayu resulted in protection against experimental infection with P. plecoglossicida. After oral administration of P. plecoglossicida cells of this bacterium were always detected in the kidneys of control fish that did not receive the phage treatment, while the cells quickly disappeared from the phage-treated fish. Bacterial growth in freshwater was lower in the presence of phage, and the number of phage PFU increased rapidly. These results suggest that it may be possible to use phage to control the disease caused by P. plecoglossicida.

Incidence of Prevotella intermedia and Prevotella nigrescens carriage among family members with subclinical periodontal disease.

We established a typing system for Prevotella intermedia and Prevotella nigrescens using the combination of PCR ribotyping and arbitrarily primed PCR (AP-PCR) fingerprinting and applied this system to the study of intrafamilial incidence of these species in the oral cavity. PCR ribotyping followed by subtyping by AP-PCR fingerprinting was applied to each type strain of P. intermedia and P. nigrescens and 54 isolates (32 isolates of P. intermedia and 24 isolates of P. nigrescens) from extraoral infections, resulting in an excellent discriminatory power (discrimination index, 0.99) for both species. A total of 18 subjects from six families, with the subjects from each family comprising the mother, the father, and a child who had subclinical early-stage to moderate adult periodontitis or simple gingivitis and who carried P. intermedia or P. nigrescens, or both, were enrolled in the study of intrafamilial carriage. When 20 colonies per specimen of subgingival plaque, if available, were picked from primary culture, 115 P. intermedia and 178 P. nigrescens isolates were recovered from the 18 subjects. Among the subjects studied, family members shared the same subtype strain(s) but non-family members did not. Multiple subtypes were found in 8 (57%) of the 14 P. nigrescens-positive subjects but in only 3 (27%) of the 11 P. intermedia-positive subjects; the difference was, however, not statistically significant (P = 0.14). These results suggest that the combination of PCR ribotyping and AP-PCR fingerprinting is well suited for the epidemiological study of P. intermedia and P. nigrescens and that each family seems to carry a distinct subtype(s) of these species.

Gut Colonization by an Ice Nucleation Active Bacterium,Erwinia(Pantoea)ananasReduces the Cold Hardiness of Mulberry Pyralid Larvae

To evaluate the suitability of using ice nucleation active (INA) bacteria for the biological control of insect pests, the supercooling point (SCP) of larvae of mulberry pyralid,Glyphodes duplicalis,and silkworm,Bombyx mori,ingesting INA strains ofErwinia(Pantoea)ananasandPseudomonas syringaewas determined. Mean SCP of the guts of silkworm larvae ingesting INA strains ofE. ananasranged from −2.5 to −2.8°C, being 5°C higher than that in control treatments. Similarly, mean SCP of mulberry pyralid larvae ingesting INA strain ofE. ananas,which can grow well in the gut, was −4.7°C at 3 days after treatment, being 6.5°C higher than that in control treatments. On the other hand, mean SCP of the larvae-ingesting INA strain ofP. syringae,which cannot grow in the gut, was −9.0°C at 3 days after treatment, rising by only 2.5°C higher than that in the control treatments. In addition, more than 80% of the larvae of mulberry pyralid ingesting the INA strain ofE. ananasfroze and eventually died when exposed to −6°C for 18 h, while only 36% of the larvae ingesting the INA strain ofP. syringae,or approximately 20% of the control larvae, froze and died. Thus, the gut colonization by INA strains ofE. ananasreduced remarkably the cold hardiness of the insects. These findings suggest that INA strains ofE. ananascould be effective as a potential biological control agent of insect pests.

Cloning and characterization of polyphosphate kinase and exopolyphosphatase genes from Pseudomonas aeruginosa 8830.

Pseudomonas aeruginosa accumulates polyphosphates in response to nutrient limitations. To elucidate the function of polyphosphate in this microorganism, we have investigated polyphosphate metabolism by isolating from P. aeruginosa 8830 the genes encoding polyphosphate kinase (PPK) and exopolyphosphatase (PPX), which are involved in polyphosphate synthesis and degradation, respectively. The 690- and 506-amino-acid polypeptides encoded by the two genes have been expressed in Escherichia coli and purified, and their activities have been tested in vitro. Gene replacement was used to construct a PPK-negative strain of P. aeruginosa 8830. Low residual PPK activity in the ppk mutant suggests a possible alternative pathway of polyphosphate synthesis in this microorganism. Primer extension analysis indicated that ppk is transcribed from a sigmaE-dependent promoter, which could be responsive to environmental stresses. However, no coregulation between ppk and ppx promoters has been demonstrated in response to osmotic shock or oxidative stress.

Invasion of human oral epithelial cells by Prevotella intermedia.

Invasion of oral epithelial cells by pathogenic oral bacteria may represent an important virulence factor in the progression of periodontal disease. Here we report that a clinical isolate of Prevotella intermedia, strain 17, was found to invade a human oral epithelial cell line (KB), whereas P. intermedia 27, another clinical isolate, and P. intermedia 25611, the type strain, were not found to invade the cell line. Invasion was quantified by the recovery of viable bacteria following a standard antibiotic protection assay and observed by electron microscopy. Cytochalasin D, cycloheximide, monodansylcadaverine, and low temperature (4 degreesC) inhibited the internalization of P. intermedia 17. Antibodies raised against P. intermedia type C fimbriae and against whole cells inhibited invasion, but the anti-type-C-fimbria antibody inhibited invasion to a greater extent than the anti-whole-cell antibody. This work provides evidence that at least one strain of P. intermedia can invade an oral epithelial cell line and that the type C fimbriae and a cytoskeletal rearrangement are required for this invasion.

Protection against exotoxin A (ETA) and Pseudomonas aeruginosa infection in mice with ETA-specific antipeptide antibodies.

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious and sometimes fatal infections in the compromised host, especially in patients with major trauma or thermal injuries. Exotoxin A (ETA) is the major and most lethal virulence factor produced by this ubiquitous microorganism. In a recent study (H. S. Elzaim, A. K. Chopra, J. W. Peterson, R. Goodheart, and J. P. Heggers, Infect. Immun. 66:2170-2179, 1998), we identified two major epitopes, one within the translocation domain (amino acid [aa] residues 289 to 333) of ETA and another within the enzymatic domain (aa 610 to 638), by using a panel of antipeptide antibodies. Synthetic peptides representing these two epitopes induced ETA-specific antibodies which were able to abrogate the cytotoxic activity of ETA, as measured by incorporation of [3H]leucine into 3T3 fibroblasts. In the present study, these antibodies were tested for the ability to provide protection against ETA and infection with a toxin-producing strain of P. aeruginosa in a mouse model. Antibodies to either of the synthetic peptides conferred protection against ETA. Also, when used for immunization, both peptides induced active immunity to ETA in mice. Antibodies to the peptide representing a region within the enzymatic domain of ETA, in combination with the antibiotic amikacin, enhanced the survival of mice infected with a toxin-producing strain of P. aeruginosa. Thus, antipeptide antibodies specific for ETA might be paired with antibiotic treatment for passive immunization of patients suffering from P. aeruginosa infection.

An Ocular Strain ofPseudomonas aeruginosais Inflammatory but not Virulent in the Scarified Mouse Model

Pseudomonas aeruginosais the most common pathogen among contact lens-associated infections. This study investigated the response of the murine cornea to infection with an ocular strain ofP. aeruginosaisolated from a subject with an inflammatory adverse response to contact lens wear termed CLARE. Although this bacterium was isolated in confluency (greater than 2000 cfu lens-1) from the lens at the time of the inflammatory episode, no infection of the cornea subsequently developed. Male C57BL/6J mice (20 per strain) had their corneas scratched with a 26 gauge needle (3 parallel 1.0 mm wounds in the left eye only). The incisions were centered over the pupillary axis and penetrated the epithelial cell basal lamina and into the superficial stroma. The CLARE strain was found to persist (viable bacteria could be cultured from corneal homogenates) up to 8 hr, as did the virulent control strain ATCC 19660. At 24 hr, only ATCC 19660 could be cultured, indicating an inability of the strain isolated from CLARE, Paer1, to persist in the eye consistent with the human inflammatory episode. Histological examination of the mouse tissue showed further differences between infection by the two strains. Infection with ATCC 19660 resulted in tissue necrosis and a large population of polymorphonuclear leukocytes (PMNs) recruited to the wound site. In contrast, during infection with the CLARE strain, PMN recruitment was reduced and temporally delayed. The CLARE strain grew as well as ATCC 19660in vitrobut produced less protease activity, in particular less elastase. The decreased PMN response and decreased protease production by the CLARE strain may have been responsible for the lack of ocular damage and apparent healing of the wound.P. aeruginosastrains are considered to be invasive or cytotoxic to corneal tissue, however this strain may represent a third inflammatory type consistent with its differing pathology.

Characterization of the Cryptic Plasmids of thePseudomonas alcaligenesType Strain

The species type strain ofPseudomonas alcaligenescontains three small cryptic plasmids (designated pECB1, 2, and 3) of 7740, 4480, and 2700 bp, respectively. Partial restriction enzyme maps have been constructed for pECB1 and 2 which on this basis do not appear to be related. pECB3 proved refractile to cutting with commonly used restriction enzymes, though it was completely rendered by those enzymes which recognize 4-bp sequences containing only G + C. This suggested that pECB3 is especially rich in these bases. Hybridization studies using labeled pECB2 as probe revealed homology with pECB3 and with regions of the bacterial chromosome, but not with pECB1. A 1214-bp region of pECB2 showed great sequence similarity to the basic replicon of pPS10, a 10-kbPseudomonas-specific plasmid isolated fromPseudomonas syringaepv.savastonoi.The putative replicon (including the gene for a replication protein) was subcloned and both DNA strands were sequenced. Introduction of the putative replicon into theEscherichia coliplasmid pUC19 created a recombinant vector able to replicate in bothE. coliandPseudomonasspp. Minicell analyses did not reveal any peptides which could be attributed to the remaining region of pECB2 or to pECB1—a finding supported by sequencing studies. Attempts at plasmid curing were unsuccessful. A phenotypic comparison with a non-plasmid-harboring strain ofP. alcaligenes,based on nutritional versatility and antibiotic susceptibility, revealed a single difference of note: the type strain alone was able to utilize benzoate for growth. Transformation of the non-harboring strain with pECB1–3, followed by selection on a minimal medium containing benzoate, gave no colonies. The advantages gained by possession of pECB1–3, if any, are at present unknown.

Occurrence of Pseudomonas tolaasii on fruiting bodies of Lentinula edodes formed on Quercus logs

A bacterial disease occurred on fruiting bodies ofLentinula edodes that formed outdoors onQuercus bedlogs during winter. The pathogen was identified asPseudomonas tolaasii based on morphological and bacteriological characteristics. Symptoms exhibited by infected fruiting bodies ranged from mild browning to severe necrotic cavities that characteristically developed in the cap tissue along the periphery of the attachment area to the stalk. The mode of symptom development was greatly influenced by the internal tissue structure of fruiting bodies. Multiplication of bacterial cells within the fruiting bodies was strictly intercellular and thus differed from previously reported bacterial disease ofL. edodes incited by an unidentified rod-shaped bacterium. The present strain ofP. tolaasii was capable of attacking theL. edodes mycelium in the inner bark and outer sapwood regions and caused lysis of heavily infected hyphae.

Resistance to cadmium salts and metal absorption by different microbial species

The present study evaluates the inhibitory activity and the absorption of cadmium (Cd) salts by different microbial species, including Gram-positive (Staphylococcus aureus andS. epidermidis), Gram-negative (Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, andProteus mirabilis) bacteria and one yeast (Candida albicans). The metal absorption by growing cells was considered both in liquid and in solid medium. For one strain ofP. aeruginosa the presence of Cd deposits inside the cell was investigated by transmission electron microscopy. Generally, the Gram-negative species tested proved to be highly resistant to Cd ions and accumulated great amounts of Cd during growth. Two strains ofP. aeruginosa showed a high degree of resistance to Cd and were particularly efficient in removing the metal from solutions. The Gram-positive bacteria showed a heterogeneous behavior: anS. aureus strain susceptible to Cd absorbed, at low metal concentrations, higher amounts of metal than a Cd-resistant one. The metal absorption for Gram-negative species was dose dependent, while for the Cd-resistant staphylococci it reached a plateau. Our results suggest that microorganisms can represent a good model to study the interactions between heavy metals and living organisms.

Metabolism of 2,5-dichlorobenzoic acid inPseudomonas stutzeri

4-Chroropyrocatechol is formed as a results of the oxidation of 2,5-dichlorobenzoate byPseudomonas stutzeri. 3-Chloro-cis,cis-muconic acid is the product of the oxidation of 4-chloropyrocatechol. Pyrocatechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, but not pyrocatechol 2,3-dioxygenase or protocatechuate 3,4-dioxygenase activities were found in cell-free extracts. Theortho cleavage activity for catechols appeared to involve induction of isoenzymes with different stereospecificity towards chlorocatechols. A catablic pathway for the degradation of 2,5-dichlorobenzoate by a newly isolated strain ofP. stutzeri was proposed.

Pathogenicity ofPythium species on cucumber in peat-sand, rockwool and hydroponics

Pythium spp. that cause damping-off of seedlings also can cause root rot of older plants and lead to yield reductions. This can be especially severe in soilless cultures where the fungus can spread easily with the nutrient solution. 39Pythium isolates obtained from discolored roots were assayed for their ability to cause damping-off on cucumber seedlings in sand-peat and for their pathogenicity in soilless culture of cucumber in rockwool or hydroponic solution. Isolates ofPythium aphanidermatum, P. irregulare, P. sylvaticum andP. ultimum were highly pathogenic in sand-peat, but onlyP. aphanidermatum strains were pathogenic in soilless conditions and led to root decay, plant death in rockwool culture and growth reduction in hydroponic culture. One strain ofP. aphanidermatum significantly reduced the yield of cucumber grown in rockwool under conditions similar to those of commercial cultures.

Secretion of Ligninolytic Enzymes and Mineralization of 14 C-Ring-Labelled Synthetic Lignin by Three Phlebia tremellosa Strains

Production of ligninolytic enzymes by three strains of the white rot fungus Phlebia tremellosa (syn. Merulius tremellosus) was studied in bioreactor cultivation under nitrogen-limiting conditions. The Mn(II) concentration of the growth medium strongly affected the secretion patterns of lignin peroxidase and laccase. Two major lignin peroxidase isoenzymes were expressed in all strains. In addition, laccase and glyoxal oxidase were purified and characterized in one strain of P. tremellosa. In contrast, manganese peroxidase was not found in fast protein liquid chromatography profiles of extracellular proteins under either low (2.4 muM) or elevated (24 and 120 muM) Mn(II) concentrations. However, H(2)O(2)- and Mn-dependent phenol red-oxidizing activity was detected in cultures supplemented with higher Mn(II) levels. Mineralization rates of C-ring-labelled synthetic lignin (i.e., dehydrogenation polymerizate) by all strains under a low basal Mn(II) level were similar to those obtained for Phanerochaete chrysosporium and Phlebia radiata. A high manganese concentration repressed the evolution of CO(2) even when a chelating agent, sodium malonate, was included in the medium.

Comparison of dimethoate and methidathion tolerance in four strains ofPhytoseiulus persimilis (Athias-Henriot) (Acarina: Phytoseiidae) in Australia

The toxicity of dimethoate 30EC and methidathion 40EC was tested against four strains ofPhytoseiulus persimilis obtained from commercial greenhouse rose crops. A single strain ofP. persimilis demonstrated tolerance to the field rate of 1.0 ml/l dimethoate used to controlHeliothrips haemorrhoidalis as indicated by the LC50 of 1.5 ml/l ai and a significantly different response at the LC90 level (P<0.05) to the three other strains tested. None of the strains was different in level of response to methidathion (P>0.05) and were unable to tolerate the recommended field rate of methidathion. Selection to dimethoate by routine field spraying has produced improved tolerance inP. persimilis. The dimethoate-tolerant strain was not different in developmental period and mean number of eggs produced per female when compared with the Sydney strain (P>0.05).