Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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The Genome of the Fungal Pathogen Verticillium dahliae Reveals Extensive Bacterial to Fungal Gene Transfer
Shi-Kunne, Xiaoqian ; Kooten, Mathijs van; Depotter, Jasper R.L. ; Thomma, Bart P.H.J. ; Seidl, Michael F. - \ 2019
Genome Biology and Evolution 11 (2019)3. - ISSN 1759-6653 - p. 855 - 868.
Verticillium - ascomycete - bacteria - fungus - horizontal gene transfer

Horizontal gene transfer (HGT) involves the transmission of genetic material between distinct evolutionary lineages and can be an important source of biological innovation. Reports of interkingdom HGT to eukaryotic microbial pathogens have accumulated over recent years. Verticillium dahliae is a notorious plant pathogen that causes vascular wilt disease on hundreds of plant species, resulting in high economic losses every year. Previously, the effector gene Ave1 and a glucosyltransferase-encoding gene were identified as virulence factor-encoding genes that were proposed to be horizontally acquired from a plant and a bacterial donor, respectively. However, to what extent HGT contributed to the overall genome composition of V. dahliae remained elusive. Here, we systematically searched for evidence of interkingdom HGT events in the genome of V. dahliae and provide evidence for extensive horizontal gene acquisition from bacterial origin.

RNA ‘Information Warfare’ in Pathogenic and Mutualistic Interactions
Chaloner, Thomas ; Kan, Jan A.L. van; Grant-Downton, Robert T. - \ 2016
Trends in Plant Science 21 (2016)9. - ISSN 1360-1385 - p. 738 - 748.
fungus - infection - non-coding RNA - pathogen - resistance

Regulatory non-coding RNAs are emerging as key players in host–pathogen interactions. Small RNAs such as microRNAs are implicated in regulating plant transcripts involved in immunity and defence. Surprisingly, RNAs with silencing properties can be translocated from plant hosts to various invading pathogens and pests. Small RNAs are now confirmed virulence factors, with the first report of fungal RNAs that travel to host cells and hijack post-transcriptional regulatory machinery to suppress host defence. Here, we argue that trans-organism movement of RNAs represents a common mechanism of control in diverse interactions between plants and other eukaryotes. We suggest that extracellular vesicles are the key to such RNA movement events. Plant pathosystems serve as excellent experimental models to dissect RNA ‘information warfare’ and other RNA-mediated interactions.

Genetic diversity in Macrophomina phaseolina, the causal agent of charcoal rot
Sarr, M.P. ; Ndiaye, M. ; Groenewald, J.Z. ; Crous, P.W. - \ 2014
Phytopathologia Mediterranea 53 (2014)2. - ISSN 0031-9465 - p. 250 - 268.
soybean-producing countries - phylogenetic analysis - fungus - botryosphaeriaceae - disease - soil - variability - infection - fusarium - survival
Macrophomina phaseolina (Botryosphaeriaceae) is an important soil- and seed-borne pathogen. This pathogen has a broad geographic distribution, and a large host range. The aim of the present study was to determine the genetic variation among a global set of 189 isolates of M. phaseolina, isolated from 23 hosts and 30 soil samples in 15 countries. To achieve this goal a multi-gene DNA analysis was conducted for the following five loci, ITS, TEF, ACT, CAL and TUB. Based on these results two well-defined clusters could be delineated, one corresponding to M. phaseolina s. str., for which a suitable epitype is designated. The second clade corresponds to M. pseudophaseolina, a novel species occurring on Abelmoschus esculentus, Arachis hypogaea, Hibiscus sabdarifa and Vigna unguiculata in Senegal. No consistent correlation was found among genotype, host and geographic location, and both species could even occur on the same host at the same location. Although M. pseudophaseolina is presently only known from Senegal, further research is required to determine its virulence compared to M. phaseolina, and its geographic distribution.
Invasive alien species under attack: natural enemies of Harmonia axyridis in the Netherlands
Raak-van den Berg, C.L. ; Wielink, P. van; Jong, P.W. de; Gort, G. ; Haelewaters, D. ; Helder, J. ; Lenteren, J.C. van - \ 2014
BioControl 59 (2014)2. - ISSN 1386-6141 - p. 229 - 240.
coleoptera-coccinellidae - ladybird beetle - pallas coleoptera - fungus - laboulbeniales - europe - north - amplification - populations - prevalence
The aphid predator Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) is an invasive alien species in Europe and North America with negative effects on non-target species (including a decline of native ladybird populations), as well as fruit production, and human health. It is, therefore, important to find out which natural enemies could be used to reduce their numbers. Knowledge of H. axyridis’ natural enemies is summarised and data collected from the Netherlands over the past ten years are presented. Beetles were sampled from winter aggregations and from spring through to autumn with illuminated screens at night. Natural enemies were not found in samples of H. axyridis from 2003–2007. From 2008 onward H. axyridis adults were infested by: Hesperomyces virescens Thaxt. fungi (summer and winter), Parasitylenchus bifurcatus Poinar and Steenberg nematodes (winter), Coccipolipus hippodamiae (McDaniel and Morrill) mites (winter), and Dinocampus coccinellae (Schrank) parasitoids (summer and winter). Our results indicate that these natural enemies are starting to use H. axyridis as a host, but are as yet not sufficiently abundant to control the population.
A phylogenetic re-evaluation of Phyllosticta (Botryosphaeriales)
Wikee, S. ; Lombard, L. ; Nakashima, C. ; Motohashi, K. ; Chukeatirote, E. ; Cheewangkoon, R. ; McKenzie, E.H.C. ; Hyde, K.D. ; Crous, P.W. - \ 2013
Studies in Mycology 76 (2013)1. - ISSN 0166-0616 - p. 1 - 29.
citrus black spot - ribosomal dna - south-africa - fungus - banana - identification - eucalyptus - anamorph - vaccinii - nuclear
Phyllosticta is a geographically widespread genus of plant pathogenic fungi with a diverse host range. This study redefines Phyllosticta, and shows that it clusters sister to the Botryosphaeriaceae (Botryosphaeriales, Dothideomycetes), for which the older family name Phyllostictaceae is resurrected. In moving to a unit nomenclature for fungi, the generic name Phyllosticta was chosen over Guignardia in previous studies, an approach that we support here. We use a multigene DNA dataset of the ITS, LSU, ACT, TEF and GPDH gene regions to investigate 129 isolates of Phyllosticta, representing about 170 species names, many of which are shown to be synonyms of the ubiquitous endophyte P. capitalensis. Based on the data generated here, 12 new species are introduced, while epitype and neotype specimens are designated for a further seven species. One species of interest is P. citrimaxima associated with tan spot of Citrus maxima fruit in Thailand, which adds a fifth species to the citrus black spot complex. Previous morphological studies lumped many taxa under single names that represent complexes. In spite of this Phyllosticta is a species-rich genus, and many of these taxa need to be recollected in order to resolve their phylogeny and taxonomy.
Calonectria metrosideri, a highly aggressive pathogen causing leaf blight , root rot, and wilt of Metrosideros spp. in Brazil
Alfenas, R.F. ; Pereira, O.L. ; Ferreira, M.A. ; Jorge, V.L. ; Crous, P.W. ; Alfenas, C.A. - \ 2013
Forest Pathology 43 (2013)4. - ISSN 1437-4781 - p. 257 - 265.
phylogenetic inference - fusarium - fungus - disease
The genus Metrosideros includes several tree, shrub and vine species, native to the Pacific Islands. Seedlings from 25 seed lots of Metrosideros polymorpha and two seed lots of M. tremuloides with symptoms of root rot, stem girdling, wilting and round, purple leaf spots were observed in the Forestry Nursery at the Universidade Federal de Viçosa, Brazil. In the original disease site, seedling mortality reached up to 71% in M. polymorpha and 34% in M. tremuloides. Single conidial cultures obtained from infected leaf, root and stem samples of M. polymorpha were used to identify the fungal species. Morphological characters and DNA sequences of four loci, containing partial sequences of ß-tubulin (TUB2), histone H3 (HIS3), calmodulin (CAL) and the elongation factor (tef-1) genes of three isolates, indicated that they belong to a new species, described here as Calonectria metrosideri sp. nov. Potting medium infestation and inoculation of seedlings of M. polymorpha with an inoculum suspension at 1 × 104 conidia ml-1 induced typical symptoms of the disease (leaf spots, root rot and wilt), similar to those observed under natural conditions. Calonectria metrosideri was re-isolated, which fulfilled Koch's postulates, and confirmed its status as a pathogen.
DNA barcoding of Mycosphaerella species of quarantine importance to Europe
Quaedvlieg, W. ; Groenewald, J.Z. ; Jesús Yáñez-Morales, M. de; Crous, P.W. - \ 2012
Persoonia 29 (2012). - ISSN 0031-5850 - p. 101 - 115.
eucalyptus leaves - ribosomal dna - leaf-spot - phylogeny - nuclear - fungus - genus - anamorphs - taxonomy - sequence
The EU 7th Framework Program provided funds for Quarantine Barcoding of Life (QBOL) to develop a quick, reliable and accurate DNA barcode-based diagnostic tool for selected species on the European and Mediterranean Plant Protection Organization (EPPO) A1/A2 quarantine lists. Seven nuclear genomic loci were evaluated to determine those best suited for identifying species of Mycosphaerella and/or its associated anamorphs. These genes included ß-tubulin (Btub), internal transcribed spacer regions of the nrDNA operon (ITS), 28S nrDNA (LSU), Actin (Act), Calmodulin (Cal), Translation elongation factor 1-alpha (EF-1a) and RNA polymerase II second largest subunit (RPB2). Loci were tested on their Kimura-2-parameter-based inter- and intraspecific variation, PCR amplification success rate and ability to distinguish between quarantine species and closely related taxa. Results showed that none of these loci was solely suited as a reliable barcoding locus for the tested fungi. A combination of a primary and secondary barcoding locus was found to compensate for individual weaknesses and provide reliable identification. A combination of ITS with either EF-1a or Btub was reliable as barcoding loci for EPPO A1/A2-listed Mycosphaerella species. Furthermore, Lecanosticta acicola was shown to represent a species complex, revealing two novel species described here, namely L. brevispora sp. nov. on Pinus sp. from Mexico and L. guatemalensis sp. nov. on Pinus oocarpa from Guatemala. Epitypes were also designated for L. acicola and L. longispora to resolve the genetic application of these names.
Effector-mediated suppression of chitin-triggered immunity by Magnaporthe oryzae is necessary for rice blast disease
Mentlak, T.A. ; Kombrink, A. ; Shinya, T. ; Ryder, L.S. ; Otomo, I. ; Saitoh, H. ; Terauchi, R. ; Nishizawa, Y. ; Shibuya, N. ; Thomma, B.P.H.J. ; Talbot, N.J. - \ 2012
The Plant Cell 24 (2012)1. - ISSN 1040-4651 - p. 322 - 335.
cladosporium-fulvum - plant infection - defense responses - virulence factor - plasma-membrane - tomato cells - receptor - fungus - elicitor - protein
Plants use pattern recognition receptors to defend themselves from microbial pathogens. These receptors recognize pathogen-associated molecular patterns (PAMPs) and activate signaling pathways that lead to immunity. In rice (Oryza sativa), the chitin elicitor binding protein (CEBiP) recognizes chitin oligosaccharides released from the cell walls of fungal pathogens. Here, we show that the rice blast fungus Magnaporthe oryzae overcomes this first line of plant defense by secreting an effector protein, Secreted LysM Protein1 (Slp1), during invasion of new rice cells. We demonstrate that Slp1 accumulates at the interface between the fungal cell wall and the rice plasma membrane, can bind to chitin, and is able to suppress chitin-induced plant immune responses, including generation of reactive oxygen species and plant defense gene expression. Furthermore, we show that Slp1 competes with CEBiP for binding of chitin oligosaccharides. Slp1 is required by M. oryzae for full virulence and exerts a significant effect on tissue invasion and disease lesion expansion. By contrast, gene silencing of CEBiP in rice allows M. oryzae to cause rice blast disease in the absence of Slp1. We propose that Slp1 sequesters chitin oligosaccharides to prevent PAMP-triggered immunity in rice, thereby facilitating rapid spread of the fungus within host tissue
Durable broad-spectrum powdery mildew resistance in pea er1 plants is conferred by natural loss-of-function mutations in PsMLO1
Humphry, M. ; Reinstädler, A. ; Ivanov, S. ; Bisseling, T. ; Panstruga, R. - \ 2011
Molecular Plant Pathology 12 (2011)9. - ISSN 1464-6722 - p. 866 - 878.
pisum-sativum-l - barley mlo-gene - erysiphe-pisi - disease resistance - scar markers - identification - protein - fungus - family - defense
Loss-of-function alleles of plant-specific MLO (Mildew Resistance Locus O) genes confer broad-spectrum powdery mildew resistance in monocot (barley) and dicot (Arabidopsis thaliana, tomato) plants. Recessively inherited powdery mildew resistance in pea (Pisum sativum) er1 plants is, in many aspects, reminiscent of mlo-conditioned powdery mildew immunity, yet the underlying gene has remained elusive to date. We used a polymerase chain reaction (PCR)-based approach to amplify a candidate MLO cDNA from wild-type (Er1) pea. Sequence analysis of the PsMLO1 candidate gene in two natural er1 accessions from Asia and two er1-containing pea cultivars with a New World origin revealed, in each case, detrimental nucleotide polymorphisms in PsMLO1, suggesting that PsMLO1 is Er1. We corroborated this hypothesis by restoration of susceptibility on transient expression of PsMLO1 in the leaves of two resistant er1 accessions. Orthologous legume MLO genes from Medicago truncatula and Lotus japonicus likewise complemented the er1 phenotype. All tested er1 genotypes showed unaltered colonization with the arbuscular mycorrhizal fungus, Glomus intraradices, and with nitrogen-fixing rhizobial bacteria. Our data demonstrate that PsMLO1 is Er1 and that the loss of PsMLO1 function conditions durable broad-spectrum powdery mildew resistance in pea.
Medium selection for exopolysaccharide and biomass production in submerged cultures of culinary-medicinal mushrooms from Turkey
Kizilcik, M. ; Yamaç, M. ; Griensven, L.J.L.D. van - \ 2010
International Journal of Medicinal Mushrooms 12 (2010)1. - ISSN 1521-9437 - p. 63 - 71.
induced diabetic-rats - fungus - substances
The present study investigates the exopolysaccharide (EPS) and biomass production of 18 strains of 15 species of culinary-medicinal higher Basidiomycetes in submerged culture under four different media. Gloeophyllum abietinum and Schizophyllum commune produced the highest EPS and biomass amounts—3.81 and 14.68 g L-1, respectively—after 5 days of incubation. The other good EPS producers were Pleurotus eryngii (3.69 g L-1) and Ganoderma carnosum D 21 (3.54 g L-1). On the other hand, Trametes versicolor SV 1, Lentinus strigosus, Clavariadelphus truncatus, and Trametes hirsuta yielded relatively higher biomass values, 11.91, 10.54, 10.50, and 10.18 g L-1, respectively. A considerable increase in EPS and biomass production was observed when the mushrooms were grown in liquid culture (LQ) and potato malt peptone (PMP) medium, respectively. The EPS/biomass ratio of Gloeophyllum abietinum on the 5th day in LQ medium was 3.4, which is 3.6, 1.5, and 15.5 times higher than with PMP, YM, and MCM media, respectively. The results suggest that EPS production is not growth associated for the investigated species and strains. This is the first report on EPS production of native higher Basidiomycetes isolates from Turkey.
RNA-mediated gene silencing of superoxide dismutase (bcsod1) in Botrytis cinerea
Patel, R.M. ; Kan, J.A.L. van; Bailey, A.M. ; Foster, G.D. - \ 2008
Phytopathology 98 (2008)12. - ISSN 0031-949X - p. 1334 - 1339.
fungicide resistance - functional-analysis - neurospora-crassa - expression - plants - transformation - cutinase - disease - fungus - tool
Gene silencing is a powerful tool utilized for identification of gene function and analysis in plants, animals, and fungi. Here, we report the silencing of superoxide dismutase (bcsod1) in Botrytis cinerea through sense and antisense-mediated silencing mechanisms. Because superoxide dismutase (SOD) is a virulence factor, transformants were tested for phenotypic silencing in vitro and reduction in pathogenicity in planta. Plate-based assays with and without paraquat were performed to screen initial silencing efficiency, and a subset of transformants was used for in planta studies of virulence. Transformants exhibiting strongly decreased transcripts levels were recovered with both constructs but none of those exhibited a reduction in virulence in planta. Our investigations may help optimize a high-throughput gene silencing system useful for identifying potential gene targets for future fungal control.
Efficient cloning system for construction of gene silencing vectors in Aspergillus niger
Oliveira, J.M. ; Veen, D. van der; Graaff, L.H. de; Qin Ling, - \ 2008
Applied Microbiology and Biotechnology 80 (2008)5. - ISSN 0175-7598 - p. 917 - 924.
transcriptional activator xlnr - double-stranded-rna - neurospora-crassa - caenorhabditis-elegans - functional-analysis - expression - interference - transformation - fungus - inactivation
An approach based on Gateway recombination technology to efficiently construct silencing vectors was developed for use in the biotechnologically important fungus Aspergillus niger. The transcription activator of xylanolytic and cellulolytic genes XlnR of A. niger was chosen as target for gene silencing. Silencing was based on the expression vector pXLNRir that was constructed and used in co-transformation. From all the strains isolated (N = 77), nine showed poor xylan-degrading activities in two semi-quantitative plate assays testing different activities for xylan degradation. Upon induction on D-xylose, transcript levels of xlnR were decreased in the xlnR-silenced strains, compared to a wild-type background. Under these conditions, the transcript levels of xyrA and xynB (two genes regulated by XlnR) were also decreased for these xlnR-silenced strains. These results indicate that the newly developed system for rapid generation of silencing vectors is an effective tool for A. niger, and this can be used to generate strains with a tailored spectrum of enzyme activities or product formation by silencing specific genes encoding, e.g., regulators such as XlnR
Analysis of summer epidemic progress of apple scab at different apple production systems in the Netherlands and Hungary
Holb, I.J. ; Heijne, B. ; Withagen, J.C.M. ; Gáll, J.M. ; Jeger, M.J. - \ 2005
Phytopathology 95 (2005)9. - ISSN 0031-949X - p. 1001 - 1020.
venturia-inaequalis - disease-progress - powdery mildew - orchards - conidia - resistance - infection - spores - fungus - buds
Two, 4-year studies on summer epidemic progress of apple scab were conducted at Randwijk, the Netherlands, from 1998 until 2001 and at Eperjeske, Hungary, from 2000 until 2003. Disease assessments were made on scab-susceptible cv. Jonagold. A range of nonlinear growth functions were fitted to a total of 96 disease progress curves (3 treatment classes x 2 plant parts x 2 disease measures x 4 years x 2 locations) of apple scab incidence and severity. The three-parameter logistic model gave the most consistent fit across three treatment classes in the experiment (integrated, organic-sprayed, and organic-unsprayed). Parameters estimated or calculated from the three-parameter logistic function were used to analyze disease progress. These were disease incidence and severity on the day of the first assessment (Y-s); final disease incidence or upper asymptote for incidence (Y-if) or severity (Y-sf); fruit incidence and severity on day 40, after which no new lesions on fruits appeared (Y-40); leaf incidence and severity on day 75, at which shoot growth stopped (Y-75); relative (beta) and "absolute" (theta) rates of disease progress; inflection point (M); and area under the disease progress curve (AUDPC(5)) standardized by the duration of the total epidemic. Comparisons among disease progress curves were made by Correlation and factor analysis followed by Varimax rotation. There were large differences but high positive correlations among the parameters Y-s, Y-f, theta, and AUDPC(5) across the three treatment classes. In the factor analysis, two factors accounted for more than 85% of the total variance for both incidence and severity. Factor I gave an overall description of epidemic progress of both scab incidence and severity and included the parameters Y-f, Y-40, Y-75, theta, and AUDPCs. Factor 2 identified a relationship between the relative rate parameter (P) and the inflection point (M) for severity and a relationship between disease incidence and severity. For an integrated or an organic orchard, theta, AUDPC(5), and one of Y-f or Y-75 (because of the link with host phenology) can characterize apple scab epidemics during summer. Based on these findings, improved scab management approaches were provided for integrated and organic apple production systems.
An accurate in vitro assay for high-throughput disease testing of Phytophthora infestans in potato
Huang, S. ; Vleeshouwers, V.G.A.A. ; Visser, R.G.F. ; Jacobsen, E. - \ 2005
Plant Disease 89 (2005)12. - ISSN 0191-2917 - p. 1263 - 1267.
broad-spectrum resistance - major gene resistance - late blight - solanum-bulbocastanum - complementary races - bary - fungus - mont
Research and breeding for resistance to Phytophthora infestans, the causal agent of the devastating late blight disease in potato (Solanum tuberosum), requires extensive disease assessments. Especially for large-scale experiments such as recombinant screenings for genetic mapping of novel resistance genes, a quick and space-effective method is required. Potato breeding clones and collections of wild Solanum germ plasm are often maintained in vitro, and this environment is extremely favorable for late blight disease development. In this paper, we describe the development of a high-throughput in vitro disease testing assay. We compared the new method with the well-established detached-leaf assay and proved that the in vitro assay is accurate and reliable, and can routinely be used for investigating the qualitative interaction between potato and P. infestans
An ancestral oomycete locus contains late blight avirulence gene Avr3a, encoding a protein that is recognized in the host cytoplasm
Armstrong, R. ; Whisson, S.C. ; Pritchard, L. ; Bos, J.I.B. ; Venter, E. ; Avrova, A.O. ; Rehmany, A.P. ; Bohme, U. ; Brooks, K. ; Cherevach, I. ; Hamlin, N. ; White, B. ; Fraser, A. ; Lord, A. ; Quail, M.A. ; Churcher, C. ; Hall, N. ; Berriman, M. ; Huang, S. ; Kamoun, S. ; Beynon, J.L. ; Birch, P.R.J. - \ 2005
Proceedings of the National Academy of Sciences of the United States of America 102 (2005)21. - ISSN 0027-8424 - p. 7766 - 7771.
phytophthora-infestans - comparative genomics - disease resistance - potato famine - plant-cells - arabidopsis - evolution - products - elicitor - fungus
Plants sense phosphate (Pi) deficiency and initiate signaling that controls adaptive responses necessary for Pi acquisition. Herein, evidence establishes that AtSIZ1 is a plant small ubiquitin-like modifier (SUMO) E3 ligase and is a focal controller of Pi starvation-dependent responses. T-DNA insertional mutated alleles of AtSIZ1 (At5g60410) cause Arabidopsis to exhibit exaggerated prototypical Pi starvation responses, including cessation of primary root growth, extensive lateral root and root hair development, increase in root/shoot mass ratio, and greater anthocyanin accumulation, even though intracellular Pi levels in siz1 plants were similar to wild type. AtSIZ1 has SUMO E3 ligase activity in vitro, and immunoblot analysis revealed that the protein sumoylation profile is impaired in siz1 plants. AtSIZ1-GFP was localized to nuclear foci. Steadystate transcript abundances of Pi starvation-responsive genes AtPT2, AtPS2, and AtPS3 were moderate but clearly greater in siz1 seedlings than in wild type, where Pi is sufficient. Pi starvation induced the expression of these genes to the same extent in siz1 and wild-type seedlings. However, two other Pi starvation-responsive genes, AtIPS1 and AtRNS1, are induced more slowly in siz1 seedlings by Pi limitation. PHR1, a MYB transcriptional activator of AtIPS1 and AtRNS1, is an AtSIZ1 sumoylation target. These results indicate that AtSIZ1 is a SUMO E3 ligase and that sumoylation is a control mechanism that acts both negatively and positively on different Pi deficiency responses
Novel species of Cylindrocarpon (Neonectria) and Campylocarpon gen. nov. associated with black foot disease of grapevines (Vitis spp.).
Halleen, F. ; Schroers, H.J. ; Groenewald, J.Z. ; Crous, P.W. - \ 2004
Studies in Mycology 50 (2004)2. - ISSN 0166-0616 - p. 431 - 455.
ribosomal dna-sequences - phylogenetic-relationships - anamorphs - fusarium - hypocreales - nectria - fungus - bionectriaceae - identification - destructans
Four Cylindrocarpon or Cylindrocarpon-like taxa isolated from asymptomatic or diseased Vitis vinifera plants in nurseries and vineyards of South Africa, New Zealand, Australia, and France were morphologically and phylogenetically compared with other Neonectria/Cylindrocarpon taxa. Sequences of the partial nuclear large subunit ribosomal DNA (LSU rDNA), internal transcribed spacers 1 and 2 of the rDNA including the 5.8S rDNA gene (ITS), and partial -tubulin gene introns and exons were used for phylogenetic inference. Neonectria/ Cylindrocarpon species clustered in mainly three groups. One monophyletic group consisted of three subclades comprising (i) members of the Neonectria radicicola/Cylindrocarpon destructans complex, which contained strains isolated from grapevines in South Africa, New Zealand, and France; (ii) a Neonectria/Cylindrocarpon species isolated from grapevines in South Africa, Canada (Ontario), Australia (Tasmania), and New Zealand, described here as Cylindrocarpon macrodidymum; and (iii) an assemblage of species closely related to strains identified as Cylindrocarpon cylindroides, the type species of Cylindrocarpon. This monophyletic group excluded two other groups, which comprised (i) members of the Neonectria mammoidea complex, with anamorphs characterised by curved macroconidia, violet or purple pigments in cultures of most of its members, and lack of microconidia and chlamydospores; and (ii) two undescribed Cylindrocarpon-like species, both from grapevines in South Africa. The latter two clades formed a paraphyletic group in LSU rDNA analysis but were supported as a monophyletic group in ITS and -tubulin gene analysis. Strains of the Neonectria radicicola/Cylindrocarpon destructans complex isolated from grapevines matched C. destructans in morphology and DNA sequences. Cylindrocarpon macrodidymum formed micro- and macroconidia, but rarely formed chlamydospores. Its mostly 3-septate macroconidia were more or less straight, minutely widening towards the tip, and had an apical cell slightly bent to one side. Its teleomorph, Neonectria macrodidyma, was obtained in mating experiments, and was characterised by smooth to finely warted ascospores, smooth to finely warted perithecia, and moderately sized angular to subglobose cells in the outer region of the perithecial wall. The other two undescribed Cylindrocarpon-like species mentioned above were characterised by mostly 3-5-septate, curved macroconidia, and by the lack of microconidia. Both species differed from members of the Neonectria mammoidea group by brownish colonies and by brownish hyphal strands formed in the aerial mycelium. For these species a new genus, Campylocarpon gen. nov., is proposed. It comprises the new species Campylocarpon fasciculare and Campylocarpon pseudofasciculare, respectively. Inoculation of 6-mo-old potted grapevine rootstocks (cv. Ramsey) with selected isolates of Cylindrocarpon destructans, Neonectria macrodidyma, Campylocarpon fasciculare, and Campylocarpon pseudofasciculare resulted in a reduced root and shoot mass of inoculated plants and appearance of symptoms typical of black foot disease.
Isolation of a fluffy mutant of Aspergillus niger from chemostat culture and its potential use as a morphologically stable host for protein production
Vondervoort, P.J.I. van de; Poulsen, B.R. ; Ruijter, G.J.G. ; Schuleit, T. ; Visser, J. ; Iversen, J.J.L. - \ 2004
Biotechnology and Bioengineering 86 (2004)3. - ISSN 0006-3592 - p. 301 - 307.
recombinant glucoamylase production - fusarium-graminearum a3/5 - penicillium-chrysogenum - genetic-analysis - argb gene - nidulans - transformation - growth - ph - fungus
Chemostat cultivation of Aspergillus niger and other filamentous fungi is often hindered by the spontaneous appearance of morphologic mutants. Using the Variomixing bioreactor and applying different chemostat conditions we tried to optimize morphologic stability in both ammonium- and glucose-limited cultures. In most cultivations mutants with fluffy (aconidial) morphology became dominant. From an ammonium-limited culture, a fluffy mutant was isolated and genetically characterized using the parasexual cycle. The mutant contained a single morphological mutation, causing an increased colony radial growth rate. The fluffy mutant was subjected to transformation and finally conidiospores from a forced heterokaryon were shown to be a proper inoculum for fluffy strain cultivation. (C) 2004 Wiley Periodicals, Inc.
Frequency, diversity and activity of 2,4-diacetylphloroglucinol-producing Pseudomonas spp. in Dutch take-all decline soils
Souza, J.T. ; Weller, D.M. ; Raaijmakers, J.M. - \ 2003
Phytopathology 93 (2003)1. - ISSN 0031-949X - p. 54 - 63.
graminis var tritici - gaeumannomyces-graminis - metabolite 2,4-diacetylphloroglucinol - antibiotic biosynthesis - ecological fitness - biological-control - suppressive soils - wheat roots - rhizosphere - fungus
Natural suppressiveness of soils to take-all disease of wheat, referred to as take-all decline (TAD), occurs worldwide, It has been postulated that different microbial genera and mechanisms are responsible for TAD in soils from different geographical regions. In growth chamber experiments,,we demonstrated that fluorescent Pseudomonas spp. that produce the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) play a key role in the natural suppressiveness of two Dutch TAD soils. First, 2,4-DAPG-producing fluorescent Pseudomonas spp. were present on roots of wheat grown in both of the TAD soils at densities at or above the threshold density required to control take-all of wheat; in a complementary take-all conducive soil, population densities of 2,4-DAPG-producing Pseudomonas spp. were below this threshold level. Second, introduction of 2,4-DAPG-producing strain SSB17, a representative of the dominant geriotypic group found in the Dutch TAD soils, into the take-all conducive soil at population densities similar to the densities, of indigenous 2,4-DAPG producers found in TAD soils provided control of take-all similar to that observed in the TAD soil, Third, a mutant of strain SSB17 deficient in 2,4-DAPG production was not able to control take-all of wheat, indicating that 2,4-DAPG is a key determinant in take-all suppression, These results show that in addition to the physicochemically different TAD soils from Washington State, 2,4-DAPG-producing fluorescent Pseudomonas spp. are also a key component of the natural suppressiveness found in Dutch TAD soils. Furthermore, it is the first time since the initial studies of Gerlagh (1968) that at least part of the mechanisms and microorganisms that operate in Dutch TAD soils are identified. Although quantitatively similar, the genotypic composition of 2,4-DAPG-producing Pseudomonas spp. varied between the Dutch TAD soils and the TAD soils from Washington State.
The role of the haematopoietic tissue in haemocyte production and maturation of the black tiger shrimp (Penaeus monodon)
Braak, C.B.T. van de; Botterblom, M.H.A. ; Liu, W. ; Taverne, N. ; Knaap, W.P.W. van der; Rombout, J.H.W.M. - \ 2002
Fish and Shellfish Immunology 12 (2002)3. - ISSN 1050-4648 - p. 253 - 272.
crustacean hemocytes - sicyonia-ingentis - hematopoiesis - morphology - origin - fungus - cells - prawn
The haematopoietic tissue (HPT) of the black tiger shrimp (Penaeus monodon) is located in different areas in the cephalothorax, mainly at the dorsal side of the stomach and in the onset of the maxillipeds and, to a lesser extent, towards the antennal gland. In young and in experimentally stimulated animals, the HPT is expanded in relatively larger and more numerous lobules throughout the cephalothorax. Four cell types could be identified in the HPT by electron microscopy. The type 1 cells are the presumed precursor cells that give rise to a large- and a small-granular young haemocyte, denominated as the type 2 and type 3 cells, respectively. A gradient of maturation from the type 1 towards the type 2 or 3 cells could frequently be observed. The presumed precursor cells are located towards the exterior of the lobules and maturing young haemocytes towards the inner part, where they can be released into the haemal lacunae. The type 4 cells show typical features of interstitial cells. Different stimulation experiments were carried out and various techniques were used to study the HPT in relation to the (circulating) haemocytes. The majority of the cells in the HPT are able to proliferate and proliferation can be increased significantly after the injection of saline and, to a much higher extent, after LPS injection. The circulating haemocytes of crustaceans are generally divided into hyaline (H), semigranular (SG) or granular (G) cells, of which large- and small-granular variants of each of these were suggested in the present study. Even after stimulation in this study, the circulating haemocytes scarcely divide. The high variations that were found in the total haemocyte count in the stimulation experiments were not accompanied by significant differences in differential haemocyte count and, therefore, appeared to be a less useful indicator of stress or health in P. monodon. Light and electron microscopical observations support the regulation of the populations of the different haemocyte types in the circulation by (stored) haemocytes from the connective tissue. In conclusion, according to morphological and immuno-chemical criteria, it is proposed in the present study to divide the haemocytes into a large and a small-granular developmental series. After extensive morphologicalobservations, it is suggested that the hyaline cells are the young and immature haemocytes of both the large- and small-granular cell line that are produced in the HPT, and can be released into the haemolymph. Indications were found that the granular cells, of at least the large-granular cell line, mature and accumulate in the connective tissue and are easily released into the haemolymph. Combining the results of the present study with literature, this proposed model for haemocyte proliferation, maturation and reaction will be discussed
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