Allelopathic impact of artificially applied coumarin on <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">niveum</Emphasis>

Watermelon production is threatened by fusarium wilt caused by Fusarium oxysporum f.sp. niveum (FON) in continuous cultivation system. Some elements, mainly allelochemicals, released from living roots or decayed plants might be associated with the disease. The purpose of this work was to evaluate the possible impact of coumarin, one kind of watermelon allelochemical, on FON. Furthermore, possible new mechanisms might be investigated during the ecological interactions of plant-microbe. Results showed that coumarin strongly inhibited growth of FON leading to a decrease in its biomass, dry weight of mycelia of FON in a liquid culture. The dry weight was decreased by 62.9% compared with control. The hyphal growth of FON on plates was stopped at high (>400 mg l⁻¹) concentrations of coumarin. At 320 mg l⁻¹, sporulation and enzyme activities of FON were also severely suppressed by coumarin. The yield of conidia, and the activities of proteinase, cellulase, and amylase were reduced by 98.9%, 79.7%, 29.8% and 15.9% respectively. However, conidial germination and mycotoxin (MT) production of FON were greatly stimulated, being increased by 55.7% and 14.9 fold at 320 mg l⁻¹ respectively. We conclude that coumarin acted as an allelochemical substance to inhibit growth and pathogenic enzyme activities of FON but to stimulate mycotoxin production and conidial germination. It was suggested that coumarin acted as a signal transduction element bridging plant and pathogen in the process of plant-microbe interactions.     

Evaluation of the anti-<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp <Emphasis Type="Italic">cicer</Emphasis> and anti-<Emphasis Type="Italic">Alternaria porri</Emphasis> effects of some essential oils

The anti-Fusarium oxysporum f. sp cicer (FOC) and anti-Alternaria porri (A. porri) effects were evaluated for 75 different essential oils. The most active essential oils found were those of lemongrass, clove, cinnamon bark, cinnamon leaf, cassia, fennel, basil and evening primrose. However, the effectiveness of these essential oils with both the tested fungi showed different responses. The level of inhibition was compared with Hexaconazole. GC–MS analysis for five oils amongst the 75 essential oils tested was performed. The potential of these essential oils as an ecofriendly and economic approach as a fungicide for FOC and A. porri is discussed.     

Aviglycine and propargylglycine inhibit conidial germination and mycelial growth of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>f. sp. <Emphasis Type="Italic">luffae</Emphasis>

Two inhibitors, aviglycine and propargylglycine, were tested for their ability to suppress methionine synthesis thus inhibit conidial germination and mycelial growth of Czapek-Dox liquid medium grown Fusarium oxysporum f. sp. luffae μM. The linear inhibition range for mycelial growth was about 7.6–762.9 μM. Although aviglycine did not completely inhibit both conidial germination and mycelial growth, it showed significant inhibitory effect at 1.5 μM. The inhibition range for propargylglycine against conidial germination and mycelial growth were from 0.08 to 8841 μM and from 0.8 to 884.1 μM, respectively. Propargylglycine inhibited conidial germination and mycelial growth at a concentration of 8841 μM. The EC₅₀ values of aviglycine were 1 μM for conidial growth and 122 μM for mycelial growth, and the EC₅₀ values of propargylglycine were 47.7 μM for conidial growth and 55.6 μM for mycelial growth. Supplement of methionine released inhibition of aviglycine or propargylglycine to conidial germination. In addition, a mixture of aviglycine (1.5 μM) and propargylglycine (8841 μM) showed additive inhibitive effect than applied alone on 10 isolates. From these results, both aviglycine and propargylglycine exhibited inhibitory activity, and suggest that they can provide potential tools to design novel fungicide against fungal pathogens.     

Field assessment of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> based mycoherbicide for control of <Emphasis Type="Italic">Striga hermonthica</Emphasis> in Nigeria

Fusarium oxysporum (isolate PSM 197) based mycoherbicide was evaluated for its efficacy under field conditions in trials conducted during 1999--2001 cropping seasons in the Nigerian savanna. In the 1999 cropping season, spot application of 5--10 g of mycoherbicide was found to give effective control of Striga hermonthica. Results of on-farm trials at Barhim and Dutsen-Ma areas showed the application of the mycoherbicide to significantly (p= 0.05) increase stand count at both 3 weeks and at harvest, reduced Striga shoot count and increased crop yield in both improved and local sorghum varieties, as compared with the same varieties not treated with the mycoherbicide. Results establish the efficacy of F. oxysporum as a mycoherbicide and the need for further development of the mycoherbicide into formulated granules or seed treatment for use in control of S. hermonthica under field conditions.     

Electrofusion of protoplasts from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>, <Emphasis Type="Italic">S. bulbocastanum</Emphasis> and <Emphasis Type="Italic">S. pinnatisectum</Emphasis>

This paper discusses a number of experiments performed, involving the fusion by an electric field of mesophyll protoplasts from Solanum tuberosum cv. Bintje, S. tuberosum dihaploid clones 243, 299 and the wild tuberous disease-resistant species S. bulbocastanum and S. pinnatisectum. Three fusion experiments (S. bulbocastanum + S. tuberosum dihaploid 243, S. pinnatisectum + S. tuberosum cv. Bintje and S. pinnatisectum + S. tuberosum dihaploid 299) yielded 542 calli, the 52 ones of which produced shoots. Obtained regenerants were estimated by the flow-cytometry (FC) and RAPD analysis to determine hybrid plants.The utilisation of the FC as a useful method for detecting somatic hybrids is also discussed in this paper. The combination S. bulbocastanum + S. tuberosum dihaploid 243 led to the creation of eight somatic hybrids, the combination S. pinnatisectum + S. tuberosum cv. Bintje yielded four somatic hybrids and the combination S. pinnatisectum + S. tuberosum dihaploid 299 resulted in no hybrid regenerants. Morphology in vitro, growth vigour and production of tuber-like structures were evaluated in hybrid plants. Plants were transferred in vivo for further estimation (acclimatization, habitus evaluation and tuberization ability).     

Influence of Temperature on Virulence of Fungal Isolates of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and <Emphasis Type="Italic">Beauveria bassiana</Emphasis> to the Two-Spotted Spider Mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Twenty-three isolates of Metarhizium anisopliae (Metschnikoff) Sokorin and three isolates of Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales: Clavicipitaceae) were assessed for their virulence against the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). Based on the screening results, nine isolates of M. anisopliae and two isolates of B. bassiana were tested for their virulence against young adult (1- to 2-day-old) female T. urticae at constant temperatures of 20, 25, 30 and 35°C. At all temperatures tested, all the fungal isolates were pathogenic to T. urticae but mortality varied with isolates and temperatures. Fungal isolates were more virulent at 25, 30 and 35°C than at 20°C. The lethal time to 50% mortality (LT₅₀) and lethal time to 90% mortality (LT₉₀) values decreased with increased temperature. There were no significant differences in virulence between fungal isolates at 30 and 35°C; however, significant differences were observed at 20 and 25°C.     

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.     

In vitro efficacy of <Emphasis Type=Italic>Hyptis suaveolens</Emphasis> L. (Poit.) essential oil on growth and morphogenesis of <Emphasis Type=Italic>Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type=Italic>gladioli</Emphasis> (Massey) Snyder &; Hansen

Hyptis suaveolens L. (Poit.) essential oil was tested in vitro on the growth and morphogenesis of Fusarium oxysporum f.sp. gladioli (Massey) Snyder & Hansen, which causes Fusarium corm rot and yellows in various susceptible cultivars of gladiolus. The fungitoxicity of the oil was measured by percentage radial growth inhibition using the poisoned food technique (PF) and volatile activity assay (VA). The mycelial growth of the test fungus was completely inhibited at 0.998 and 0.748 μg ml⁻¹ concentration of oil in PF and VA, respectively. Essential oil was found to be fungicidal in nature at 1.247 and 0.998 μg ml⁻¹ concentration of oil in PF and VA, respectively. Determination of conidial germination in the presence of oil was also carried out and it was found that the oil exhibited 100% inhibition of conidial germination at 0.450 μg ml⁻¹ concentration. The effect of essential oil on the yield of mycelial weight was observed and it was found that at 0.873 μg ml⁻¹ concentration no mycelium was recorded and 100% inhibition was observed. The fungitoxicity of oil did not change even on exposure to 100°C temperature or to autoclaving, and the oil also retained its fungicidal nature even after storage of 24 months. The main changes observed under light microscopy after oil treatment were a decrease and loss of conidiation and anomalies in the hyphae such as a decrease in the diameter of hyphae and granulation of cytoplasm. The treatment of the oil also showed highly reduced cytoplasm in the hyphae, showing clear retraction of the cytoplasm from the hyphae and ultimately in some areas hyphae without cytoplasm were also found. GC-MS studies of the essential oil revealed that the oil consisted of 24 compounds with 1,8-cineole as major component accounting for 44.4% of the total constituents.     

Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

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.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

Cutinase production by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> in liquid medium using central composite design

The objective of the present study was to measure the production of cutinase by Fusarium oxysporum in the presence of several carbon and nitrogen sources (glycides, fatty acids and oils, and several organic and inorganic nitrogen sources), trying to find a cost-effective substitute for cutin in the culture medium as an inducer of cutinase production. The results were evaluated by the Tukey test, and flaxseed oil was found to give the best results as a cutinase inducer. The authors optimized the composition of the growth medium employing response surface methodology. The experimental results were fitted to a second-order polynomial model at a 95% level of significance (p < 0.05). The greatest cutinolytic activity was obtained in a liquid mineral medium supplemented with flaxseed oil, showing an increase in enzymatic activity from 11 to 22.68 U/mL after 48 h of fermentation. A CCD study of the fermentation conditions was carried out, and the best production of cutinase was registered with the use of 30 °C and 100 rpm. These results support the use of flaxseed oil as a substitute for cutin, which is difficult and expensive to obtain, for the production of cutinase in a larger scale.     

Construction of an intraspecific linkage map of lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> ssp. <Emphasis Type="Italic">culinaris</Emphasis>)

The first intraspecific linkage map of the lentil genome was constructed with 114 molecular markers (100 RAPD, 11 ISSR and three RGA) using an F₂ population developed from a cross between lentil cultivars ILL5588 and ILL7537 which differed in resistance for ascochyta blight. Linkage analysis at a LOD score of 4.0 and a maximum recombination fraction of 0.25 revealed nine linkage groups comprising between 6 and 18 markers each. The intraspecific map spanned a total length of 784.1 cM. The markers were distributed throughout the genome, however markers were clustered in the middle or near the middle of the linkage groups, suggesting the location of centromeres. Of 114 mapped markers, 16 (14.0%) were distorted, usually at the end or middle of the linkage groups. The utility of ISSR and RGA markers for mapping in lentil was explored, and the primer with an (AC) repeat motif was found to be useful.Communicated by H.C. Becker     

Evolutionary Relationships Among <Emphasis Type="Italic">Aspergillus terreus</Emphasis> Isolates and their Relatives

Aspergillus terreus is a ubiquitous fungus in our environment. It is an opportunistic human pathogen and economically important as the main producer of lovastatin, a cholesterol lowering drug. Our aim was to examine the genetic variability of A. terreus and closely related species using molecular and analytical techniques. Lovastatin production was examined by HPLC. Lovastatin was produced by seven isolates belonging to the species A. terreus. RAPD analyses were carried out using 25 different random primers. Neighbor-joining analysis of RAPD data (120 characters) resulted in clustering of the A. terreus isolates into distinct groups. Some correlation was observed between lovastatin producing abilities of the isolates and their position on the dendrogram based on RAPD profiles. The internal transcribed spacer region and the 5.8S rRNA gene of A. terreus and related isolates was also sequenced. Phylogenetic analysis of sequence data let us classify the isolates into different clades which mostly correspond to the species Aspergillus terreus, Aspergillus flavipes, Aspergillus niveus, Aspergillus carneus and Aspergillus janus/A. janus var. brevis. Aspergillus allahabadii, A. terreus var. aureus and A. niveus var. indicus belonged to the A. niveus clade, while an Aspergillus isolate previously classified as A. niveus was most closely related to A. flavipes isolates. Aspergillus anthodesmis formed a distinct branch on the tree. Although it was previously suggested based on 28S rDNA sequence data that Aspergillus section Terrei should include A. carneus and A. niveus isolates, phylogenetic analysis of ITS sequences indicate that A. flavipes isolates are more closely related to A. terreus than A. carneus isolates. Our data suggest that sections Terrei and Flavipedes should be merged. However, further loci should be analysed to draw more definite conclusions.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

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.     

Life cycle of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> var. <Emphasis Type="Italic">azukicola</Emphasis> on <Emphasis Type="Italic">Vigna angularis</Emphasis>

Field observations and inoculation experiments revealed that Uromyces appendiculatus var. azukicola has an autoecious and macrocyclic life cycle and produces spermogonia, aecia, uredinia, and telia on Vigna angularis var. angularis and V. angularis var. nipponensis. From inoculation experiments, it was suggested that this rust fungus has different host relationships from other varieties. Morphological examinations revealed that the characteristics of urediniospores and teliospores are different among varieties, although aeciospores are morphologically similar to each other.Contribution no. 182, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

New species in the genus <Emphasis Type="Italic">Francisella</Emphasis> (Gammaproteobacteria; Francisellaceae); <Emphasis Type="Italic">Francisella piscicida</Emphasis> sp. nov. isolated from cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella strain, GM2212, previously isolated from moribund farmed Atlantic cod (Gadus morhua) in Norway, is closely related to Francisella philomiragia among Francisella spp. according to its complete 16S rDNA, 16S-23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and hypothetical lipoprotein (LpnB) sequences. A comparison between GM2212 and the type strain of Francisella philomiragia were performed by DNA–DNA hybridization and fatty acid analysis. The DNA–DNA hybridization showed a 70% similarity. The fatty acid analysis showed only minor differences between the Francisella isolates. Due to the inconclusive result from the DNA–DNA hybridisation, major emphasis concerning the status of this isolate is made on previously published molecular, phenotypic and biochemical characters. All characteristics taken together support the establishment of GM2212 as a novel species, for which the name Francisella piscicida sp. nov. is proposed (=CNCM I-3511^T = DSM 18777^T = LMG registration number not yet available).     

Mineralization of chlorpyrifos by co-culture of <Emphasis Type="Italic">Serratia</Emphasis> and <Emphasis Type="Italic">Trichosporon</Emphasis> spp.

A bacterial strain (Serratia sp.) that could transform chlorpyrifos to 3,5,6-trichloro-2-pyridinol (TCP) and a TCP-mineralizing fungal strain (Trichosporon sp.) were isolated from activated sludge by enrichment culture technique. The fungus could also degrade 50 mg chlorpyrifos l(-1) within 7 days. Co-cultures completely mineralized 50 mg chlorpyrifos l(-1) within 18 h at 30 degrees C and pH 8 using a total inocula of 0.15 g biomass l(-1).