The fungal matrices of <Emphasis Type="Italic">Ophiostoma ips</Emphasis> hinder movement of the biocontrol nematode agent, <Emphasis Type="Italic">Deladenus siricidicola</Emphasis>, disrupting management of the woodwasp, <Emphasis Type="Italic">Sirex noctilio</Emphasis>

Deladenus (=?Beddingia) siricidicola (Tylenchida: Neotylenchidae) is the most effective biocontrol agent used against the invasive wood wasp, Sirex noctilio (Fabricius) (Hymenoptera: Siricidae). The nematodes feed and reproduce on the wood-inhabiting fungus, Amylostereum areolatum (Chaillet ex Fr.) Boidin (Russulales: Amylostereaceae) and parasitise larvae of S. noctilio. In the nematode biocontrol program, the nematodes are inoculated into herbicide-weakened ‘trap trees’. Recent declines in nematode parasitism of S. noctilio in Australia have coincided with an increased incidence of an exotic bark beetle, Ips grandicollis (Eichhoff) (Coleoptera: Curculionidae), attacking trap trees and vectoring a wood-inhabiting fungus, Ophiostoma ips (Rumbold) Nannfelt (Ophiostomatales: Ophiostomataceae), which may inhibit migration of the nematode within the tree to the detriment of S. noctilio biocontrol. Several in vitro and in vivo experiments were conducted to investigate the effect of fungal interactions on the ability of D. siricidicola to locate and reproduce on A. areolatum. Deladenus siricidicola showed preference to A. areolatum in the presence and absence of O. ips, but the presence of O. ips negatively affected the choice response and the number of eggs laid by the nematodes. Deladenus siricidicola was unable to survive and reproduce on O. ips. Results give a clearer understanding of the choice response of D. siricidicola in I. grandicollis infested trees, explaining the disruptive impact of bark beetles on biocontrol of S. noctilio, an effect that could extend from Australia to other important pine growing countries.     

Analysis of compound heterozygotes reveals that the mouse floxed <Emphasis Type="Italic">Pax6</Emphasis><Superscript><Emphasis Type="Italic">tm1Ued</Emphasis></Superscript> allele produces abnormal eye phenotypes

Analysis of abnormal phenotypes produced by different types of mutations has been crucial for our understanding of gene function. Some floxed alleles that retain a neomycin-resistance selection cassette (neo cassette) are not equivalent to wild-type alleles and provide useful experimental resources. Pax6 is an important developmental gene and the aim of this study was to determine whether the floxed Pax6 ^tm1Ued (Pax6 ^fl ) allele, which has a retained neo cassette, produced any abnormal eye phenotypes that would imply that it differs from the wild-type allele. Homozygous Pax6 ^fl/fl and heterozygous Pax6 ^fl/+ mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6 ^fl/fl corneas tended be thicker and smaller in diameter. To aid identification of weak effects, we produced compound heterozygotes with the Pax6 ^Sey-Neu (Pax6 ^?) null allele. Pax6 ^fl/? compound heterozygotes had more severe eye abnormalities than Pax6 ^+/? heterozygotes, implying that Pax6 ^fl differs from the wild-type Pax6 ⁺ allele. Immunohistochemistry showed that the Pax6 ^fl/? corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6 ^fl allele provides a useful addition to the existing Pax6 allelic series and this study demonstrates the utility of using compound heterozygotes with null alleles to unmask cryptic effects of floxed alleles.     

Expression of gene for <Emphasis Type="Italic">N</Emphasis>-acyl-homoserine lactonase <Emphasis Type="Italic">AiiA</Emphasis> affects properties of rhizospheric strain <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

The introduction into strain Pseudomonas chlororaphis 449 of plasmid pME6863 that contains the cloned gene for N-acyl-homoserine lactonase, AiiA, leads to the degradation of all three types of N-acylhomoserine lactones produced by this strain (N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, and N-3-oxo-hexanoyl-homoserine lactone). This causes a drastic reduction in the synthesis of phenazine pigment and decreases the ability of cells to migrate on the surface of nutrient medium. However, the antagonistic activity of P. chlororaphis 449 toward phytopathogenic fungi Sclerotinia sclerotiorum and Rhizoctonia solani is not only decreased, but is even slightly increased; no essential changes in the exoprotease activity were observed. It is assumed that one of the QS systems of P. chlororaphis 449 may exert the repression effect on the expression of genes, which determine the two latter cell activities.     

The dependence of intracellular ATP level on the nutrition mode of the acidophilic bacteria <Emphasis Type="Italic">Sulfobacillus thermotolerans</Emphasis> and <Emphasis Type="Italic">Alicyclobacillus tolerans</Emphasis>

The dynamics of the ATP pool in the aerobic spore-forming acidothermophilic mixotrophic bacteria Sulfobacillus thermotolerans Kr1^T and Alicyclobacillus tolerans K1^T were studied in the course of their chemolithoheterotrophic, chemoorganoheterotrophic, and chemolithoautotrophic growth. It was established that, during mixotrophic growth, the maximum ATP concentrations in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 3.8 and 0.6 nmol/mg protein, respectively. The ATP concentrations in sulfobacilli and alicyclobacilli during organotrophic growth were 2.2 and 3.1 nmol/mg protein, respectively. In the cells of the obligately heterotrophic bacterium Alicyclobacillus cycloheptanicus 4006^T, the maximum ATP concentration was several times higher and reached 12.3 nmol/mg protein. During lithotrophic growth, the maximum values of the ATP concentration in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 0.3 and <0.1 nmol/mg protein, respectively; in the cells of the autotrophic bacterium Acidithiobacillus ferrooxidans TFBk, the ATP content was about 60–300 times higher (17.0 nmol/mg protein). It is concluded that low ATP content is among the possible causes of growth cessation of S. thermotolerans Kr1 and A. tolerans K1 under auto-and heterotrophic conditions after several culture transfers.     

<Emphasis Type="Italic">Metarhizium bibionidarum</Emphasis> and <Emphasis Type="Italic">M. purpureogenum</Emphasis>: new species from Japan

Two new species of Metarhizium, M. bibionidarum and M. purpureogenum are described from Japan. Metarhizium bibionidarum is the phylogenetic sister species of M. pemphigi and a member of the M. flavoviride species complex. It is distinguished morphologically from M. pemphigi by its larger conidia. The species is based on a collection of an infected March fly larva (Diptera: Bibionidae) but is also known to occur on fruit beetle (Coleoptera: Scarabaeidae) encountered in France. Metarhizium purpureogenum was isolated from soil by plating and insect baiting methods and represents a unique phylogenetic lineage placed outside the M. anisopliae and M. flavoviride species complexes. Three isolates of M. purpureogenum excreted a distinctive red-purple pigment into agar medium when co-cultured with M. robertsii or Aspergillus oryzae.     

<Emphasis Type="Italic">Lecanicillium muscarium</Emphasis> and <Emphasis Type="Italic">Adalia bipunctata</Emphasis> combination for the control of black bean aphid<Emphasis Type="Italic">, Aphis fabae</Emphasis>

The predator Adalia bipunctata (Coleoptera: Coccinellidae) and the entomopathogenic fungus Lecanicillium muscarium, have been considered as potential biological control against aphids. While it can be difficult to achieve a high control level of Aphis fabae Scopoli (Hemiptera: Aphididae) using only a single beneficial agent, the research presented here aimed to determine the interaction between L. muscarium and A. bipunctata potential for control against A. fabae. Lecanicillium muscarium was found to cause about 30% mortality in A. bipunctata and with a reduction in feeding by about 15%. However, co-application of A. bipunctata and L. muscarium can cause an addititive effect in reducing aphid populations, resulting in about 90% reduction in aphid populations compared with control treatment. Thus, these two biocontrol agents have the potential to be complementary. This research study demonstrates that it is possible to combine A. bipunctata with L. muscarium to provide a sustainable method for management of A. fabae on broad bean cropping system and that field studies are required.     

Bipolar distribution of the cyst-forming dinoflagellate <Emphasis Type="Italic"> Polarella glacialis</Emphasis>

Morphological investigations of motile cells and cysts of a small dinoflagellate (strain CCMP 2088) isolated from Canadian Arctic waters were carried out under both light and scanning electron microscopy. This species strongly resembled Polarella glacialis (strain CCMP 1383), which up to now was known only from Antarctic sea ice. The photosynthetic pigment composition of strain CCMP 2088 is typical of dinoflagellates, with peridinin as a major accessory pigment. Phylogenetic relationships between the two strains and other dinoflagellate species were inferred from SSU nrDNA using Neighbour Joining and weighted parsimony analyses. Our results showed that strain CCMP 2088 and P. glacialis (strain CCMP 1383) grouped in the same clade (Suessiales clade), showing high similarity values (0.99%). Morphological and molecular data support the assignment of the Arctic strain to P. glacialis. The free-living Gymnodinium simplex and the two P. glacialis strains have a basal position in the Suessiales clade, as compared to Symbiodinium spp.     

Effects of blue light on pigment biosynthesis of <Emphasis Type="Italic">Monascus</Emphasis>

The influence of different illumination levels of blue light on the growth and intracellular pigment yields of Monascus strain M9 was investigated. Compared with darkness, constant exposure to blue light of 100 lux reduced the yields of six pigments, namely, rubropunctatamine (RUM), monascorubramine (MOM), rubropunctatin (RUN), monascorubrin (MON), monascin (MS), and ankaflavin (AK). However, exposure to varying levels of blue light had different effects on pigment production. Exposure to 100 lux of blue light once for 30 min/day and to 100 lux of blue light once and twice for 15 min/day could enhance RUM, MOM, MS, and AK production and reduce RUN and MON compared with non-exposure. Exposure to 100 lux twice for 30 min/day and to 200 lux once for 45 min/day decreased the RUM, MOM, MS, and AK yields and increased the RUN and MON. Meanwhile, the expression levels of pigment biosynthetic genes were analyzed by real-time quantitative PCR. Results indicated that gene MpPKS5, mppR1, mppA, mppB, mmpC, mppD, MpFasA, MpFasB, and mppF were positively correlated with the yields of RUN and MON, whereas mppE and mppR2 were associated with RUM, MOM, MS, and AK production.     

A new flagellated dispersion stage in <Emphasis Type="Italic">Paraphysoderma sedebokerense</Emphasis>, a pathogen of <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>

The blastocladialean fungus Paraphysoderma sedebokerense Boussiba, Zarka and James is a devastating pathogen of the commercially valuable green microalga Haematococcus pluvialis, a natural source of the carotenoid pigment astaxanthin. First identified in commercial Haematococcus cultivation facilities, P. sedebokerense is hypothesised to have a complex life cycle that switches between a vegetative and a resting phase depending on favourable or unfavourable growth conditions. Rather unusually for blastocladialean fungi, P. sedebokerense was described as lacking flagellated zoospores and only propagating via aplanosporic amoeboid cells. However, during repeated microscopic observation of P. sedebokerense cultivated in optimal conditions, we detected fast-swimming, transiently uniflagellated zoospores which rapidly transform into infectious amoeboid swarmers, the existence of which suggests a closer than previously thought relatedness of P. sedebokerense to its sister genera Physoderma and Urophlyctis. Additionally, we found some morphological and physiological differences between amoeboid swarmers and discuss hypotheses about their significance. These amoeboid and flagellated propagules are key to the dissemination of P. sedebokerense and are probably also the life stages most vulnerable to adverse environmental conditions. They are therefore a prime target for the development of disease management protocols in industrial cultivation facilities, a goal which requires a detailed understanding of their physiology.     

Two new <Emphasis Type="Italic">Entoloma</Emphasis> s.L. species with serrulatum-type lamellar edge from Changbai Mountains,Northeast China

Two species of Entoloma with serrulatum-type lamellar edge, E. cyanostipitum and E. subcaesiocinctum from the Changbai Mountains in northeast China, are described as new. E. cyanostipitum is distinguished by a brownish orange pileus with a central depression, black-blue lamellar edge, deep blue stipe, and cylindrical cheilocystidia with intense blue, intracellular pigment. E. subcaesiocinctum is characterized by a depressed pileus covered with brownish squamules, blackish blue lamellar edge, and cylindrical to clavate cheilocystidia with intense blue intracellular pigment. The two new species are further confirmed based on the ITS and nLSU sequences.     

Morphological and genetic differentiation among four pigment producing Indian species of <Emphasis Type="Italic">Phoma</Emphasis> (Saccardo, 1899)

A PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), was used for assessing genetic relatedness among isolates of the genus Phoma. Randomly Amplified Polymorphic DNA (RAPD) revealed the presence of interspecific genetic variation among the pigment producing isolates of Phoma and has shown distinct phylogenetic cluster. The major objective of the study was to study the genetic variation, if any. Study was aimed to differentiate four pigment producing species of Phoma based on morphological studies and molecular markers in general and RAPD in particular. We found that the test species of Phoma can be very well differentiated using molecular markers. Phoma sorghina was differentiated from P. exigua, P. fimeti and P. herbarum. RAPD profiles of P. herbarum and P. fimeti has shown the maximum similarity, which indicates the genetic relatedness among these two species which were considered earlier as distinct species based on morphological observation.     

Melanization of <Emphasis Type="Italic">Fusarium keratoplasticum</Emphasis> (<Emphasis Type="Italic">F. solani</Emphasis> Species Complex) During Disseminated Fusariosis in a Patient with Acute Leukemia

Fusarium spp. are recognized as the second most frequently filamentous fungi causing opportunistic infections and particularly important due to the increasing number of immunocompromised patients. F. keratoplasticum (a member of F. solani species complex) is one of the Fusarium species commonly associated with human infection, and therefore, studies on the virulence of this fungus are needed. This study aimed to confirm the presence of melanin in F. keratoplasticum from a patient with systemic fusariosis. Immunofluorescence labeling with anti-melanin monoclonal antibody (MAb) was used to examine an expression of melanin in F. keratoplasticum in vitro and during infection. Electron spin resonance identified the particles extracted from F. keratoplasticum as stable free radical consistent with melanin. Lesional skin from the sites with fusariosis contained hyphal structures that could be labeled by melanin-binding MAb, while digestion of the tissue yielded dark particles that were reactive. These findings suggest that F. keratoplasticum hyphae and chlamydospores can produce melanin in vitro and that hyphae can synthesize pigment in vivo. Given the potential role of melanin in virulence of other fungi, this pigment in F. keratoplasticum may play a role in the pathogenesis of fusariosis.     

Mutation <Emphasis Type="Italic">clp</Emphasis>A::<Emphasis Type="Italic">kan</Emphasis> of the gene for an Hsp100 family chaperone impairs the DnaK-dependent refolding of proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The rate and level of DnaK-dependent refolding of heat-inactivated Vibrio fischeri luciferase in the clp A mutant (clp A:: kan) were considerably lower then in wild-type cells. The decline in refolding level progressed with increasing heat inactivation time. A mutation of clp P had no influence on the kinetics and level of luciferase refolding. Approximately equal amounts of the DnaKJE chaperone were synthesized upon heat shock induction in E. coli clp A + and E. coli clpA::kan cells. It was assumed that, like homologous chaperone ClpB, ClpA is involved in disaggregation of denatured proteins, increasing the refolding efficiency. This in vivo phenomenon occurred only upon a prolonged incubation of cells at a higher temperature, which led to the formation of large protein aggregates that were poorly refoldable by the DnaKJE system.     

Filamentous Aggregation of Sequestosome-1/p62 in Brain Neurons and Neuroepithelial Cells upon <Emphasis Type="Italic">Tyr-Cre</Emphasis>-Mediated Deletion of the Autophagy Gene <Emphasis Type="Italic">Atg7</Emphasis>

Defects in autophagy and the resulting deposition of protein aggregates have been implicated in aging and neurodegenerative diseases. While gene targeting in the mouse has facilitated the characterization of these processes in different types of neurons, potential roles of autophagy and accumulation of protein substrates in neuroepithelial cells have remained elusive. Here we report that Atg7^f/f Tyr-Cre mice, in which autophagy-related 7 (Atg7) is conditionally deleted under the control of the tyrosinase promoter, are a model for accumulations of the autophagy adapter and substrate sequestosome-1/p62 in both neuronal and neuroepithelial cells. In the brain of Atg7^f/f Tyr-Cre but not of fully autophagy competent control mice, p62 aggregates were present in sporadic neurons in the cortex and other brain regions as well in epithelial cells of the choroid plexus and the ependyma. Western blot analysis confirmed a dramatic increase of p62 abundance and formation of high-molecular weight species of p62 in the brain of Atg7^f/f Tyr-Cre mice relative to Atg7^f/f controls. Immuno-electron microscopy showed that p62 formed filamentous aggregates in neurons and ependymal cells. p62 aggregates were also highly abundant in the ciliary body in the eye. Atg7^f/f Tyr-Cre mice reached an age of more than 2 years although neurological defects manifesting in abnormal hindlimb clasping reflexes were evident in old mice. These results show that p62 filaments form in response to impaired autophagy in vivo and suggest that Atg7^f/f Tyr-Cre mice are a model useful to study the long-term effects of autophagy deficiency on the homeostasis of different neuroectoderm-derived cells.