Development of a submerged-liquid sporulation medium for the potential smartweed bioherbicide Septoria polygonorum

Submerged culture experiments were conducted in three phases to determine the optimal medium for rapidly producing conidia of the fungal bioherbicide Septoria polygonorum. In phase I, 47 crude carbon sources were evaluated to determine which would support sporulation. Under the conditions tested, pea brine (5–10% v/v) provided best conidiation. In phase II, a fractional factorial design was utilized to screen 38 different medium adjuncts in combination with pea brine for improved sporulation. MgSO₄ was the only factor that resulted in a significant improvement. In phase III, a central composite design with response surface methodology was used to optimize concentrations of these critical factors. The model predicted optimal sporulation in a medium composed of 8.88% v/v pea brine+0.1 molar MgSO₄ with an expected titer of 1.78×10⁸ conidia/ml. Actual mean titer attained with the model-derived medium was 1.15×10⁸ conidia/ml. No significant difference was observed in virulence of conidia produced on agar vs. the model-derived (liquid) medium.     

Statistical optimization of medium composition for growth of<Emphasis Type="Italic">Leuconostoc citreum</Emphasis>

Leuconostoc citreum is one of the representative strains ofLeuconostoc spp. that show fast growth rates in fermented vegetables. Sequential experimental designs including the Plackett-Burman design, fractional factorial design, steepest ascent analysis, central composite design and response surface methodology were introduced to optimize and improve the medium forL. citreum. Fifteen medium ingredients were examined and glucose (20 g/L), yeast extract (12.5 g/L), sodium acetate trihydrate (6.12 g/L), potassium phosphate (42.55 g/L) and dibasic ammonium citrate (4.12 g/L) were chosen as the best components to give a critical and positive effect for cell-growth. The biomass was increased to 2.79 g/L (169%), compared to the 1.65 g/L in MRS medium.     

Optimization of medium composition for actinomycin X2 production by <Emphasis Type="Italic">Streptomyces </Emphasis><Emphasis Type="Italic">spp</Emphasis> JAU4234 using response surface methodology

The effects of cultivation medium compositions including soybean meal, peptone, soybean oil and cornstarch for actinomycin X2 production by Streptomyces spp JAU4234 were accessed by using response surface methodology. The 2(4) full factorial designs and the paths of steepest ascent were effective in searching for the major factors of actinomycin X2 production. In this study, cornstarch and soybean oil showed negative effect on actinomycin X2 production based on the first-order regression coefficients derived from MINITAB software. Subsequently, a central composite design for optimization was further investigated. Preliminary studies showed that soybean meal and peptone were believed to be the major factors for actinomycin X2 production. Estimated optimum compositions for the production of actionmycin X2 were as follows (g/l): soybean meal 21.65 and peptone 9.41, and result in a maximum actionmycin X2 production of 617.4 mg/l. This value was closed to the 612 mg/l actionmycin X2 production from actual experimental observations. The yield of actionmycin X2 was increased by 36.9% by culturing the strain Streptomyces spp JAU4234 in the nutritionally optimized fermentation medium.     

Evidence of <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> and <Emphasis Type="Italic">Rickettsia helvetica</Emphasis> infection in free-ranging ungulates in central Slovakia

The purpose of this study was to investigate the role of wild animals for Anaplasma phagocytophilum, other ehrlichiae/anaplasmae, Rickettsia helvetica and other rickettsiae and whether different genetic variants of A. phagocytophilum in central Slovakia exist. A total of 109 spleen samples from 49 red deer (Cervus elaphus), 30 roe deer (Capreolus capreolus), 28 wild boar (Sus scrofa) and two mouflon (Ovis musimon) were collected from June 2005 to December 2006. Polymerase chain reaction (PCR) amplification of the16S rRNA gene was used for detection of ehrlichiae/anaplasmae. A nested PCR targeting part (392 bp) of groESL gene was applied for the specific detection of A. phagocytophilum. Fragments of the gltA and ompA genes (381 bp and 632 bp, respectively) were amplified to detect rickettsiae, followed by sequencing. A. phagocytophilum and R. helvetica were detected in wild animals. The prevalence of A. phagocytophilum was 50.0 ± 18.2% in roe deer and 53.1 ± 14.1% in red deer. None of the 28 wild boar was PCR positive for ehrlichiae/anaplasmae. A. phagocytophilum was detected in one mouflon. R. helvetica was found in one roe deer. Our study suggests a role of cervids as a natural reservoir of A. phagocytophilum in Slovakia. However, the role of cervids and wild boars in the circulation of R. helvetica remains unknown. The analysis of sequence variation in the msp4 coding region of A. phagocytophilum showed the presence of different variants previously described in ruminants.     

Effect of pH and Temperature on Enzyme Activity of Chitosanase Produced Under Solid Stated Fermentation by <Emphasis Type="Italic">Trichoderma</Emphasis> spp<Emphasis Type="Italic">.</Emphasis>

Trichoderma strains were extensively studied as biocontrol agents due to their ability of producing hydrolytic enzymes, which are considered key enzymes because they attack the insect exoskeleton allowing the fungi infection. The present work aimed to evaluate the ability of chitosanase production by four Trichoderma strains (T. harzianum, T. koningii, T. viride and T. polysporum) under solid stated fermentation and to evaluate the effect of pH and temperature on enzyme activity. pH strongly affected the enzyme activity from all tested strains. Chitosanase from T. harzianum and T. viride presented optimum activity at pH 5.0 and chitosanase from T. koningii and T. polysporum presented optimum activity at pH 5.5. Temperature in the range of 40–50°C did not affect enzyme activity. T. polysporum was found as the most promising strain to produce chitosanase with maximal enzyme activity of about 1.4 IU/gds, followed by T. viride (~1.2 IU/gds) and T. harzianum (1.06 IU/gds).     

Relationship between <Emphasis Type="Italic">Psoroptes cuniculi</Emphasis> and the Internal Bacterium <Emphasis Type="Italic">Serratia marcescens</Emphasis>

The bacterium Serratia marcescens isolated from surface-sterilised Psoroptes cuniculi was found sensitive to the antibiotic Amikacin. Mites placed in this antibiotic for 48–72 h and then washed by centrifugation were found to be alive and S. marcescens-free. Two experimental infestations were undertaken in order to verify the ability of the S. marcescens-free mites to infect and to give ear skin lesions in healthy rabbits and to evaluate the differential ability of the S. marcescens-free and S. marcescens-infected mites to give ear skin lesions. All rabbits were found to be infested, but only rabbits infested with S. marcescens-free mites presented crusts in their ears, whereas mites and/or eggs were only detected in the ear cerumen of all rabbits infested with S. marcescens-infected mites. S. marcescens was isolated only from P. cuniculi mites taken from these latter rabbits. Results indicate that P. cuniculi mites do not need S. marcescens to live and to be able to infest a healthy rabbit. In addition, S. marcescens was not isolated from eggs and newly born larvae of S. marcescens-infected P. cuniculi, demonstrating that in a population of P. cuniculi this bacterium is not transmitted transovarially.     

Response surface optimization of fermentation conditions for producing xylanase by <Emphasis Type="Italic">Aspergillus</Emphasis><Emphasis Type="Italic">niger</Emphasis> SL-05

Fermentation conditions were statistically optimized for producing extracellular xylanase by Aspergillus niger SL-05 using apple pomace and cotton seed meal. The primary study shows that culture medium with a 1:1 ratio of apple pomace and cotton seed meal (carbon and nitrogen sources) yielded maximal xylanase activity. Three significant factors influencing xylanase production were identified as urea, KH(2)PO(4), and initial moisture content using Plackett-Burman design study. The effects of these three factors were further investigated using a design of rotation-regression-orthogonal combination. The optimized conditions by response surface analysis were 2.5% Urea, 0.09% KH(2)PO(4), and 62% initial moisture content. The analysis of variance indicated that the established model was significant (P < 0.05), "while" or "and" the lack of fit was not significant. Under the optimized conditions, the model predicted 4,998 IU/g dry content, whereas validation experiments produced an enzymatic activity of xylanase at 5,662 IU/g dry content after 60 h fermentation. This study innovatively developed a fermentation medium and process to utilize inexpensive agro-industrial wastes to produce a high yield of xylanase.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Feeding strategy of the sacoglossan opisthobranch <Emphasis Type="Italic">Oxynoe olivacea</Emphasis> on the tropical green alga <Emphasis Type="Italic">Caulerpa taxifolia</Emphasis>

The feeding behaviour of the shelled sacoglossan Oxynoe olivacea was investigated to better understand the role and importance of this species in influencing encroachments of the alien alga Caulerpa taxifolia in the Mediterranean sea. We tested whether this slug preferred, as preliminary field observations suggested, an aggregative feeding behaviour and which part of the algal thallus, phylloid vs rhizoid, it preferred. Results showed that O. olivacea fed in groups and actively selected phylloid. This outcome poses important questions regarding the possibility that this species, fragmenting the alga thallus, could enhance dispersion and regeneration of C. taxifolia.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Four novel <Emphasis Type="Italic">Candida</Emphasis> species in the <Emphasis Type="Italic">Candida albicans</Emphasis>/<Emphasis Type="Italic">Lodderomyces elongisporus</Emphasis> clade isolated from the gut of flower beetles

Flower-visiting beetles belonging to three species of Cetoniidae were collected on three mountains near Beijing, China, and yeasts were isolated from the gut of the insects collected. Based on the 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region sequence analysis and phenotypic characterization, four novel anamorphic yeast species located in the Candida albicans/Lodderomyces elongisporus clade were identified from 18 of the strains isolated. The new species and type strains are designated as Candida blackwellae AS 2.3639^T (=CBS 10843^T), Candida jiufengensis AS 2.3688^T (=CBS 10846^T), Candida oxycetoniae AS 2.3656^T (=CBS 10844^T), and Candida pseudojiufengensis AS 2.3693^T (=CBS 10847^T). C. blackwellae sp. nov. was basal to the branch formed by C. albicans and C. dubliniensis with moderately strong bootstrap support. The closest relative of C. oxycetoniae was L. elongisporus. C. jiufengensis sp. nov. and C. pseudojiufengensis sp. nov. were closely related with each other and formed a branch in a subclade represented by C. parapsilosis and L. elongisporus.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Submerged culture of a mycelial formulation of a bioherbicidal strain of <Emphasis Type="Italic">Myrothecium </Emphasis><Emphasis Type="Italic">verrucaria</Emphasis> with mitigated mycotoxin production

A mycelial formulation of the fungus Myrothecium verrucaria (IMI 361690) containing 0.20% Silwet L-77 surfactant was found to be highly efficacious in controlling the exotic invasive weed kudzu. The mycelium can be rapidly (48–72 h) produced in several media, including an inexpensive soy flour–corn meal medium. Mycelial yields were 2, 10, and 25 g dry weight l⁻¹ in Czapek-Dox, Richard’s V-8, and soy flour–corn meal media, respectively. Scale-up production in soy flour–corn meal medium using laboratory fermenters (10–25 l), resulted in a mycelial formulation that caused 90% mortality of naturally-occurring mature (0.9–1.0 m in height) kudzu within 48 h after application in field experiments. HPLC analyses revealed that the mycelium produced in this liquid culture contained no detectable amounts of the trichothecene mycotoxins roridin A and verrucarin A (limit of detection 2 μg ml⁻¹). This has resulted in a safer, yet effective bioherbicidal product. We anticipate that these findings should improve the probability of EPA registration and subsequent commercial development of this bioherbicide.     

Alginate production by <Emphasis Type="Italic">Pseudomonas mendocina</Emphasis> in a stirred draft fermenter

In this study alginate production by Pseudomonas mendocina in a laboratory-scale fermenter was investigated. In the experiments the effect of temperature (25–31°C) and agitation (500–620 rev min⁻¹) at a constant air flow of 10 v/v/h were evaluated in relation to the rate of glucose bioconversion to alginate using response surface methodology (RSM). The fermenter configuration was also adapted to a system with a screw mixer and draft tube, due to the change in rheological characteristics of the fermentation broth. The adjusted model indicates a temperature of 29.1°C and agitation of 553 rev min⁻¹ for optimum alginate synthesis. In this fermentation system a Y _p/s of 44.8% was achieved. The alginate synthesized by P. mendocina showed a partially acetylated pattern as previously reported for alginates obtained from other Pseudomonas spp and Azotobacter vinelandii.     

Mutational analysis of the <Emphasis Type="Italic">gum</Emphasis> gene cluster required for xanthan biosynthesis in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv <Emphasis Type="Italic">oryzae</Emphasis>

Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sang-Yoon Kim and Jeong-Gu Kim contributed equally to this work.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type