Functional Divergence of Two Secreted Immune Proteases of Tomato
Ilyas, M. ; Hörger, A.C. ; Bozkurt, T.O. ; Burg, H.A. van den; Kaschani, F. ; Kaiser, M. ; Belhaj, K. ; Smoker, M. ; Joosten, M. ; Kamoun, S. ; Hoorn, R.A.L. van der - \ 2015
Current Biology 25 (2015)17. - ISSN 0960-9822 - p. 2300 - 2306.
cf-2-dependent disease resistance - pathogen effectors - transcription factors - provides insights - genome sequence - plant-pathogens - gene - defense - target - specialization
Rcr3 and Pip1 are paralogous secreted papain-like proteases of tomato. Both proteases are inhibited by Avr2 from the fungal pathogen Cladosporium fulvum, but only Rcr3 acts as a co-receptor for Avr2 recognition by the tomato Cf-2 immune receptor [ 1, 2, 3 and 4]. Here, we show that Pip1-depleted tomato plants are hyper-susceptible to fungal, bacterial, and oomycete plant pathogens, demonstrating that Pip1 is an important broad-range immune protease. By contrast, in the absence of Cf-2, Rcr3 depletion does not affect fungal and bacterial infection levels but causes increased susceptibility only to the oomycete pathogen Phytophthora infestans. Rcr3 and Pip1 reside on a genetic locus that evolved over 36 million years ago. These proteins differ in surface-exposed residues outside the substrate-binding groove, and Pip1 is 5- to 10-fold more abundant than Rcr3. We propose a model in which Rcr3 and Pip1 diverged functionally upon gene duplication, possibly driven by an arms race with pathogen-derived inhibitors or by coevolution with the Cf-2 immune receptor detecting inhibitors of Rcr3, but not of Pip1.
The genome of the saprophytic fungus Verticillium tricorpus reveals a complex effector repertoire resembling that of its pathogenic relatives
Seidl, M.F. ; Faino, L. ; Shi-Kunne, Xiaoqian ; Berg, G.C.M. van den; Bolton, M.D. ; Thomma, B.P.H.J. - \ 2015
Molecular Plant-Microbe Interactions 28 (2015)3. - ISSN 0894-0282 - p. 362 - 373.
chitin-triggered immunity - hidden markov model - rna-seq - eukaryotic genomes - signal peptides - plant-pathogens - protein family - evolution - annotation - wilt
Vascular wilts caused by Verticillium spp. are destructive plant diseases, affecting hundreds of hosts. Only few Verticillium spp. are causal agents of vascular wilt diseases, of which V. dahliae is the most notorious pathogen, and several V. dahliae genomes are available. In contrast, V. tricorpus is mainly known as saprophyte and causal agent of opportunistic infections. Based on a hybrid approach that combines second and third generation sequencing, a near-gapless V. tricorpus genome assembly was obtained. With comparative genomics, we aimed to identify genomic features in V. dahliae that confer the ability to cause vascular wilt disease. Unexpectedly, both species encode similar effector repertoires and share a genomic structure with genes encoding secreted proteins clustered in genomic islands. Intriguingly, V. tricorpus contains significantly less repetitive elements and an extended spectrum of secreted carbohydrate-active enzymes when compared with V. dahliae. In conclusion, we highlight the technical advances of a hybrid sequencing and assembly approach and reveal that the saprophyte V. tricorpus shares many hallmark features with V. dahliae.
Filamentous pathogen effector functions: of pathogens, hosts and microbiomes
Rövenich, H. ; Boshoven, J.C. ; Thomma, B. - \ 2014
Current Opinion in Plant Biology 20 (2014). - ISSN 1369-5266 - p. 96 - 103.
chitin-triggered immunity - secreted fungal effector - potato famine pathogen - cladosporium-fulvum - protease inhibitor - magnaporthe-oryzae - plant-pathogens - genome evolution - tomato - virulence
Microorganisms play essential roles in almost every environment on earth. For instance, microbes decompose organic material, or establish symbiotic relationships that range from pathogenic to mutualistic. Symbiotic relationships have been particularly well studied for microbial plant pathogens and have emphasized the role of effectors; secreted molecules that support host colonization. Most effectors characterized thus far play roles in deregulation of host immunity. Arguably, however, pathogens not only deal with immune responses during host colonization, but also encounter other microbes including competitors, (myco)parasites and even potential co-operators. Thus, part of the effector catalog may target microbiome co-inhabitants rather than host physiology.
Transcriptome sequencing uncovers the Avr5 avirulence gene of the tomato leaf mould pathogen Cladosporium fulvum
Mesarich, C.H. ; Griffiths, S.A. ; Burgt, A. van der; Okmen, B. ; Beenen, H. ; Etalo, D.W. ; Joosten, M.H.A.J. ; Wit, P.J.G.M. de - \ 2014
Molecular Plant-Microbe Interactions 27 (2014)8. - ISSN 0894-0282 - p. 846 - 857.
fungal effector proteins - cf-2-dependent disease resistance - aspergillus-nidulans - secreted proteins - candidate effectors - neurospora-crassa - allelic variation - virulence factor - signal peptides - plant-pathogens
The Cf-5 gene of tomato confers resistance to strains of the fungal pathogen Cladosporium fulvum carrying the avirulence gene Avr5. Although Cf-5 has been cloned, Avr5 has remained elusive. We report the cloning of Avr5 using a combined bioinformatic and transcriptome sequencing approach. RNA-Seq was performed on the sequenced race 0 strain (0WU; carrying Avr5), as well as a race 5 strain (IPO 1979; lacking a functional Avr5 gene) during infection of susceptible tomato. Forty-four in planta–induced C. fulvum candidate effector (CfCE) genes of 0WU were identified that putatively encode a secreted, small cysteine-rich protein. An expressed transcript sequence comparison between strains revealed two polymorphic CfCE genes in IPO 1979. One of these conferred avirulence to IPO 1979 on Cf-5 tomato following complementation with the corresponding 0WU allele, confirming identification of Avr5. Complementation also led to increased fungal biomass during infection of susceptible tomato, signifying a role for Avr5 in virulence. Seven of eight race 5 strains investigated escape Cf-5-mediated resistance through deletion of the Avr5 gene. Avr5 is heavily flanked by repetitive elements, suggesting that repeat instability, in combination with Cf-5-mediated selection pressure, has led to the emergence of race 5 strains deleted for the Avr5 gene.
Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica
Jiang, R.H.Y. ; Bruijn, I. de; Haas, B.J. ; Belmonte, R. ; Löbach, L. ; Christie, J. ; Ackerveken, G. van den; Bottin, A. ; Bulone, V. ; Díaz-Moreno, S.M. ; Dumas, B. ; Fan, L. ; Gaulin, E. ; Govers, F. ; Grenville-Briggs, L.J. ; Horner, N.R. ; Levin, J.Z. ; Mammella, M. ; Meijer, H.J.G. ; Morris, P. ; Nusbaum, C. ; Oome, S. ; Phillips, A.J. ; Rooyen, D. van; Rzeszutek, E. ; Saraiva, M. ; Secombes, C.J. ; Seidl, M.F. ; Snel, B. ; Stassen, J.H.M. ; Sykes, S. ; Tripathy, S. ; Berg, H. ; Vega-Arreguin, J.C. ; Wawra, S. ; Young, S.K. ; Zeng, Q. ; Dieguez-Uribeondo, J. ; Russ, C. ; Tyler, B.M. ; West, P. van - \ 2013
Plos Genetics 9 (2013)6. - ISSN 1553-7404 - 20 p.
expressed sequence tags - anthocidaris-crassispina eggs - fully automated process - phytophthora-sojae - aphanomyces-euteiches - plant-pathogens - infestans - cells - evolution - reveals
Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.
A Universal Microarray Detection Method for Identification of Multiple Phytophthora spp. Using Padlock Probes
Sikora, K. ; Verstappen, E.C.P. ; Mendes, O. ; Schoen, C.D. ; Ristaino, J. ; Bonants, P.J.M. - \ 2012
Phytopathology 102 (2012)6. - ISSN 0031-949X - p. 635 - 645.
polymerase-chain-reaction - internal transcribed spacer - real-time pcr - ribosomal dna - phylogenetic-relationships - natural ecosystems - plant-pathogens - reaction assay - ramorum - quantification
The genus Phytophthora consists of many species that cause important diseases in ornamental, agronomic, and forest ecosystems worldwide. Molecular methods have been developed for detection and identification of one or several species of Phytophthora in single or multiplex reactions. In this article, we describe a padlock probe (PLP)-based multiplex method of detection and identification for many Phytophthora spp. simultaneously. A generic TaqMan polymerase chain reaction assay, which detects all known Phytophthora spp., is conducted first, followed by a species-specific PLP ligation. A 96-well-based microarray platform with colorimetric readout is used to detect and identify the different Phytophthora spp. PLPs are long oligonucleotides containing target complementary sequence regions at both their 5' and 3' ends which can be ligated on the target into a circular molecule. The ligation is point mutation specific; therefore, closely related sequences can be differentiated. This circular molecule can then be detected on a microarray. We developed 23 PLPs to economically important Phytophthora spp. based upon internal transcribed spacer-1 sequence differences between individual Phytophthora spp. Tests on genomic DNA of many Phytophthora isolates and DNA from environmental samples showed the specificity and utility of PLPs for Phytophthora diagnostics.
Have biopesticides come of age?
Glare, T. ; Caradus, J. ; Gelernter, W. ; Jackson, T. ; Keyhani, N. ; Köhl, J. ; Marrone, P. ; Morin, L. ; Stewart, A. - \ 2012
Trends in Biotechnology 30 (2012)5. - ISSN 0167-7799 - p. 250 - 258.
biological-control - biocontrol agents - fungal entomopathogens - sclerotinia-minor - theobroma-cacao - plant-pathogens - insect pests - management - efficacy - ipm
Biopesticides based on living microbes and their bioactive compounds have been researched and promoted as replacements for synthetic pesticides for many years. However, lack of efficacy, inconsistent field performance and high cost have generally relegated them to niche products. Recently, technological advances and major changes in the external environment have positively altered the outlook for biopesticides. Significant increases in market penetration have been made, but biopesticides still only make up a small percentage of pest control products. Progress in the areas of activity spectra, delivery options, persistence of effect and implementation have contributed to the increasing use of biopesticides, but technologies that are truly transformational and result in significant uptake are still lacking
Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing
Jonge, R. de; Esse, H.P. van; Maruthachalam, K. ; Bolton, M.D. ; Santhanam, P. ; Keykha Saber, M. ; Zhang, Z. ; Usami, T. ; Lievens, B. ; Subbarao, K.V. ; Thomma, B. - \ 2012
Proceedings of the National Academy of Sciences of the United States of America 109 (2012)13. - ISSN 0027-8424 - p. 5110 - 5115.
horizontal gene-transfer - xanthomonas-axonopodis - plant-pathogens - albo-atrum - resistance - evolution - host - arabidopsis - mechanisms - physiology
Fungal plant pathogens secrete effector molecules to establish disease on their hosts, and plants in turn use immune receptors to try to intercept these effectors. The tomato immune receptor Ve1 governs resistance to race 1 strains of the soil-borne vascular wilt fungi Verticillium dahliae and Verticillium albo-atrum, but the corresponding Verticillium effector remained unknown thus far. By high-throughput population genome sequencing, a single 50-Kb sequence stretch was identified that only occurs in race 1 strains, and subsequent transcriptome sequencing of Verticillium-infected Nicotiana benthamiana plants revealed only a single highly expressed ORF in this region, designated Ave1 (for Avirulence on Ve1 tomato). Functional analyses confirmed that Ave1 activates Ve1-mediated resistance and demonstrated that Ave1 markedly contributes to fungal virulence, not only on tomato but also on Arabidopsis. Interestingly, Ave1 is homologous to a widespread family of plant natriuretic peptides. Besides plants, homologous proteins were only found in the bacterial plant pathogen Xanthomonas axonopodis and the plant pathogenic fungi Colletotrichum higginsianum, Cercospora beticola, and Fusarium oxysporum f. sp. lycopersici. The distribution of Ave1 homologs, coincident with the presence of Ave1 within a flexible genomic region, strongly suggests that Verticillium acquired Ave1 from plants through horizontal gene transfer. Remarkably, by transient expression we show that also the Ave1 homologs from F. oxysporum and C. beticola can activate Ve1-mediated resistance. In line with this observation, Ve1 was found to mediate resistance toward F. oxysporum in tomato, showing that this immune receptor is involved in resistance against multiple fungal pathogens
Reconstruction of Oomycete Genome Evolution Identifies Differences in Evolutionary Trajectories Leading to Present-Day Large Gene Families
Seidl, M.F. ; Ackerveken, G. van den; Govers, F. ; Snel, B. - \ 2012
Genome Biology and Evolution 4 (2012)3. - ISSN 1759-6653 - p. 199 - 211.
nonphotosynthetic protists - phytophthora-infestans - pathogen phytophthora - obligate biotrophy - protein families - plant-pathogens - duplication - reveals - cells - mechanisms
The taxonomic class of oomycetes contains numerous pathogens of plants and animals but is related to nonpathogenic diatoms and brown algae. Oomycetes have flexible genomes comprising large gene families that play roles in pathogenicity. The evolutionary processes that shaped the gene content have not yet been studied by applying systematic tree reconciliation of the phylome of these species. We analyzed evolutionary dynamics of ten Stramenopiles. Gene gains, duplications, and losses were inferred by tree reconciliation of 18,459 gene trees constituting the phylome with a highly supported species phylogeny. We reconstructed a strikingly large last common ancestor of the Stramenopiles that contained ~10,000 genes. Throughout evolution, the genomes of pathogenic oomycetes have constantly gained and lost genes, though gene gains through duplications outnumber the losses. The branch leading to the plant pathogenic Phytophthora genus was identified as a major transition point characterized by increased frequency of duplication events that has likely driven the speciation within this genus. Large gene families encoding different classes of enzymes associated with pathogenicity such as glycoside hydrolases are formed by complex and distinct patterns of duplications and losses leading to their expansion in extant oomycetes. This study unveils the large-scale evolutionary dynamics that shaped the genomes of pathogenic oomycetes. By the application of phylogenetic based analyses methods, it provides additional insights that shed light on the complex history of oomycete genome evolution and the emergence of large gene families characteristic for this important class of pathogens
Dual disease resistance mediated by the immune receptor Cf-2 in tomato requires a common virulence target of a fungus and a nematode
Lozano, J. ; Wilbers, R.H.P. ; Gawronski, P. ; Boshoven, J.C. ; Finkers-Tomczak, A.M. ; Cordewener, J.H.G. ; America, A.H.P. ; Overmars, H.A. ; Klooster, J.W. van t; Baranowski, L. ; Sobczak, M. ; Ilyas, M. ; Hoorn, R.A.L. van der; Schots, A. ; Wit, P.J.G.M. de; Bakker, J. ; Goverse, A. ; Smant, G. - \ 2012
Proceedings of the National Academy of Sciences of the United States of America 109 (2012)25. - ISSN 0027-8424 - p. 10119 - 10124.
potato cyst-nematodes - for-gene specificity - globodera-rostochiensis - phytophthora-infestans - physical interaction - confers resistance - plant-pathogens - protein - defense - effector
Plants lack the seemingly unlimited receptor diversity of a somatic adaptive immune system as found in vertebrates and rely on only a relatively small set of innate immune receptors to resist a myriad of pathogens. Here, we show that disease-resistant tomato plants use an efficient mechanism to leverage the limited nonself recognition capacity of their innate immune system. We found that the extracellular plant immune receptor protein Cf-2 of the red currant tomato (Solanum pimpinellifolium) has acquired dual resistance specificity by sensing perturbations in a common virulence target of two independently evolved effectors of a fungus and a nematode. The Cf-2 protein, originally identified as a monospecific immune receptor for the leaf mold fungus Cladosporium fulvum, also mediates disease resistance to the root parasitic nematode Globodera rostochiensis pathotype Ro1-Mierenbos. The Cf-2-mediated dual resistance is triggered by effector-induced perturbations of the apoplastic Rcr3(pim) protein of S. pimpinellifolium. Binding of the venom allergen-like effector protein Gr-VAP1 of G. rostochiensis to Rcr3(pim) perturbs the active site of this papain-like cysteine protease. In the absence of the Cf-2 receptor, Rcr3(pim) increases the susceptibility of tomato plants to G. rostochiensis, thus showing its role as a virulence target of these nematodes. Furthermore, both nematode infection and transient expression of Gr-VAP1 in tomato plants harboring Cf-2 and Rcr3(pim) trigger a defense-related programmed cell death in plant cells. Our data demonstrate that monitoring host proteins targeted by multiple pathogens broadens the spectrum of disease resistances mediated by single plant immune receptors.
Studies on the interaction between the biocontrol agent, Serratia plymuthica A30, and blackleg-causing Dickeya sp. (biovar 3) in potato (Solanum tuberosum)
Czajkowski, R.L. ; Boer, W.J. de; Veen, J.A. van; Wolf, J.M. van der - \ 2012
Plant Pathology 61 (2012)4. - ISSN 0032-0862 - p. 677 - 688.
protein-tagged strain - biological-control - erwinia-carotovora - pseudomonas-fluorescens - endophytic bacteria - plant-pathogens - rhizosphere - seed - inoculation - colonization
Interactions between Serratia plymuthica A30 and a blackleg-causing biovar 3 Dickeya sp. were examined. In a potato slice assay, S. plymuthica A30 inhibited tissue maceration caused by Dickeya sp. IPO2222 when co-inoculated at a density at least 10 times greater than that of the pathogen. In glasshouse experiments, population dynamics of the antagonist and of the pathogen in planta were studied by dilution plating and confocal laser scanning microscopy (CLSM) using fluorescent protein-tagged strains. Pathogen-free minitubers were vacuum-infiltrated with DsRed-tagged Dickeya sp. IPO2222 and superficially treated during planting with a water suspension containing GFP-tagged S. plymuthica A30. A30 reduced the blackleg incidence from 55% to 0%. Both the pathogen and the antagonist colonized the seed potato tubers internally within 1 day post-inoculation (dpi). Between 1 and 7 dpi, the population of A30 in tubers increased from 101 to c. 103 CFU g)1 and subsequently remained stable until the end of the experiment (28 dpi). Populations of A30 in stems and roots increased from c. 102 to c. 104 CFU g)1 between 7 and 28 dpi. Dilution plating and CLSM studies showed that A30 decreased the density of Dickeya sp. populations in plants. Dilution plating combined with microscopy allowed the enumeration of strain A30 and its visualization in the vascular tissues of stem and roots and in the pith of roots, as well as its adherence to and colonization of the root surface. The implications of these finding for the use of S. plymuthica A30 as a biocontrol agent are discussed
Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer
Abd-Elsalam, K. ; Bahkali, A.H. ; Moslem, M. ; Wit, P.J.G.M. de; Verreet, J.A. - \ 2011
International Journal of Molecular Sciences 12 (2011)1. - ISSN 1661-6596 - p. 682 - 693.
real-time pcr - septoria-tritici - stagonospora-nodorum - plant-pathogens - rapid detection - markers - assay - diagnostics - resistance - diseases
Early detection of infection is very important for efficient management of Mycosphaerella graminicola leaf blotch. To monitor and quantify the occurrence of this fungus during the growing season, a diagnostic method based on real-time PCR was developed. Standard and real-time PCR assays were developed using SYBR Green chemistry to quantify M. graminicola in vitro or in wheat samples. Microsatellite dinucleotide specific-primers were designed based on microsatellite repeats of sequences present in the genome of M. graminicola. Specificity was checked by analyzing DNA of 55 M. graminicola isolates obtained from different geographical origins. The method appears to be highly specific for detecting M. graminicola; no fluorescent signals were observed from 14 other closely related taxa. Primer (CT) 7 G amplified a specific amplicon of 570 bp from all M. graminicola isolates. The primers did not amplify DNA extracted from 14 other fungal species. The approximate melting temperature (Tm) of the (CT) 7 G primer was 84.2 °C. The detection limit of the real-time PCR assay with the primer sets (CT) 7 G is 10 fg/25 µL, as compared to 10 pg/25 µL using conventional PCR technology. From symptomless leaves, a PCR fragment could be generated two days after inoculation. Both conventional and real-time PCR could successfully detect the fungus from artificially inoculated wheat leaves. However, real-time PCR appeared much more sensitive than conventional PCR. The developed quantitative real-time PCR method proved to be rapid, sensitive, speci¿c, cost-effective and reliable for the identi¿cation and quanti¿cation of M. graminicola in wheat
Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism
Spanu, P.D. ; Abbott, J.C. ; Amselem, J. ; Burgis, T.A. ; Soanes, D.M. ; Stüber, K. ; Loren van Themaat, E. Ver; Brown, J.K.M. ; Butcher, S.A. ; Gurr, S.J. ; Lebrun, M.H. ; Ridout, C.J. ; Schulze-Lefert, P. ; Talbot, N.J. ; Ahmadinejad, N. ; Ametz, C. ; Barton, G.R. ; Benjdia, M. ; Bidzinski, P. ; Bindschedler, L.V. ; Both, M. ; Brewer, M.T. ; Cadle-Davidson, L. ; Cadle-Davidson, M.M. ; Collemare, J. ; Cramer, R. ; Frenkel, O. ; Godfrey, D. ; Harriman, J. ; Hoede, C. ; King, B.C. ; Klages, S. ; Kleemann, J. ; Knoll, D. ; Koti, P.S. ; Kreplak, J. ; López-Ruiz, F.J. ; Lu, X. ; Maekawa, T. ; Mahanil, S. ; Micali, C. ; Milgroom, M.G. ; Montana, G. ; Noir, S. ; O'Connell, R.J. ; Oberhaensli, S. ; Parlange, F. ; Pedersen, C. ; Quesneville, H. ; Reinhardt, R. ; Rott, M. ; Sacristán, S. ; Schmidt, S.M. ; Schön, M. ; Skamnioti, P. ; Sommer, H. ; Stephens, A. ; Takahara, H. ; Thordal-Christensen, H. ; Vigouroux, M. ; Weßling, R. ; Wicker, T. ; Panstruga, R. - \ 2010
Science 330 (2010)6010. - ISSN 0036-8075 - p. 1543 - 1546.
plant-pathogens - virulence - proteins
Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.
Daily changes of infections by Pythium ultimum after a nutrient impulse in organic versus conventional soils
He, M. ; Ma, W. ; Tian, G. ; Blok, W.J. ; Khodzaeva, A. ; Zelenev, V.V. ; Semenov, A.M. ; Bruggen, A.H.C. van - \ 2010
Phytopathology 100 (2010)6. - ISSN 0031-949X - p. 593 - 600.
damping-off - bacterial-populations - root-rot - pseudomonas-fluorescens - biological indicators - microbial communities - disease suppression - plant-pathogens - wheat roots - compost
Bacterial populations (CFU) have been shown to oscillate in wavelike patterns after nutrient impulses in previous studies. The amplitudes and periods of oscillations could possibly be used as indicators of soil health analogous to the stability and resilience of biological populations widely accepted as indicators for ecosystem health. Limited plant and animal disease outbreaks can also be viewed as a manifestation of a healthy soil ecosystem. Two pot experiments were carried out to verify whether damping-off of beet seedlings by Pythium ultimum, measured as area under the disease progress curve (AUDPC), fluctuated over time after incorporation of organic materials into organic versus conventional soils, and to investigate whether daily dynamics of AUDPCs were linked to the dynamics of microbial populations and chemical parameters. AUDPCs oscillated significantly over time when Pythium bioassays were initiated daily after addition of ground grass and clover shoots (GC) into unplanted soils. Similar oscillations with significant harmonics of AUDPC were also observed in composted manure (CM)-amended soils but with smaller amplitudes than in GC-amended soils. The AUDPC harmonics in amended soils had periods similar to those of CFU of copiotrophic bacteria. Cross-correlation analysis demonstrated that periodic fluctuations of P. ultimum infections (AUDPCs) did not coincide with those of copiotrophic CFU but were shifted in phase. It appears that competition or antagonism from some fast-growing bacteria influenced pathogen infections, because these bacterial populations were growing and dying. Soil chemical variables, including pH, dissolved organic carbon, and NO(3)(-)-N, and NH(4)(+)-N contents, changed significantly in the initial 7 days after a nutrient impulse into soils. These changes were cross-correlated with copiotrophic CFU with time lags of approximately 1 to 2 days but were seldom associated with daily changes in AUDPCs. Organically managed soils always had lower AUDPC ratios of amended to nonamended treatments, indicating that organic materials showed stronger suppressive abilities to P. ultimum in organic than in conventional soils. The oscillations in AUDPCs and copiotrophic CFU in amended organic soil also had smaller amplitudes than in amended conventional soil. These results suggested that organically managed soils had a greater resistance and resilience to the disturbance of the amendments and, therefore, could be considered healthier than conventionally managed soils
Robust Detection and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay
Doorn, R. van; Slawiak, M. ; Szemes, M. ; Dullemans, A.M. ; Bonants, P.J.M. ; Kowalchuk, G.A. ; Schoen, C.D. - \ 2009
Applied and Environmental Microbiology 75 (2009)12. - ISSN 0099-2240 - p. 4185 - 4193.
real-time pcr - polymerase-chain-reaction - dna microarray - oligonucleotide microarrays - microbial communities - phytophthora-ramorum - pathogen detection - plant-pathogens - universal - quantification
Simultaneous detection and identification of multiple pathogenic microorganisms in complex environmental samples are required in numerous diagnostic fields. Here, we describe the development of a novel, background-free ligation detection (LD) system using a single compound detector probe per target. The detector probes used, referred to as padlock probes (PLPs), are long oligonucleotides containing asymmetric target complementary regions at both their 5' and 3' ends which confer extremely specific target detection. Probes also incorporate a desthiobiotin moiety and an internal endonuclease IV cleavage site. DNA samples are PCR amplified, and the resulting products serve as potential targets for PLP ligation. Upon perfect target hybridization, the PLPs are circularized via enzymatic ligation, captured, and cleaved, allowing only the originally ligated PLPs to be visualized on a universal microarray. Unlike previous procedures, the probes themselves are not amplified, thereby allowing a simple PLP cleavage to yield a background-free assay. We designed and tested nine PLPs targeting several oomycetes and fungi. All of the probes specifically detected their corresponding targets and provided perfect discrimination against closely related nontarget organisms, yielding an assay sensitivity of 1 pg genomic DNA and a dynamic detection range of 10(4). A practical demonstration with samples collected from horticultural water circulation systems was performed to test the robustness of the newly developed multiplex assay. This novel LD system enables highly specific detection and identification of multiple pathogens over a wide range of target concentrations and should be easily adaptable to a variety of applications in environmental microbiology.
Soil suppressiveness and functional diversity of the soil microflora in organic farming systems
Postma, J. ; Schilder, M.T. ; Bloem, J. ; Leeuwen, W.K. van - \ 2008
Soil Biology and Biochemistry 40 (2008)9. - ISSN 0038-0717 - p. 2394 - 2406.
rhizoctonia-solani - microbial communities - disease suppression - plant-pathogens - root-rot - pythium-aphanidermatum - bacterial - streptomyces - biocontrol - management
Arable fields of 10 organic farms from different locations in The Netherlands were sampled in three subsequent years. The soil samples were analysed for disease suppressiveness against Rhizoctonia solani AG2.2IIIB in sugar beet, Streptomyces scabies in radish and Verticillium longisporum in oilseed rape. In addition, a variety of microbial, chemical and physical soil characteristics were assessed. All data were correlated by multiple regression and multivariate analyses with the objective to find correlations between soil suppressiveness and biotic or abiotic soil characteristics. Significant differences in soil suppressiveness were found between the fields for all three diseases. Multiple regression indicated a significant correlation between suppressiveness against Rhizoctonia and the number of antagonistic Lysobacter spp., as well as with % active fungi and bacterial diversity. Grass-clover stimulated Rhizoctonia suppression as well as the presence of antagonistic Lysobacter spp. (mainly L. antibioticus and L. gummosus) in clay soils. Streptomyces suppression correlated with the number of antagonistic Streptomyces spp., % of active fungi and bacterial population size. The presence of antagonistic Streptomyces spp. correlated with a high fungal/bacterial biomass ratio. Verticillium suppression was only measured in 2004 and 2005, due to the inconsistent suppressiveness along the years. Nevertheless, a significant correlation with pH, potential nitrogen mineralization and bacterial biomass was found. Bacterial and fungal PCR-denaturing gel electrophoresis fingerprinting of bacterial and fungal communities, in general, did not significantly correlate with disease suppression. Highly significant explanatory factors of the composition of the dominating bacterial and fungal populations were % lutum, pH, C/N quotient, biomass and growth rate of bacteria. Additionally, the % of organic matter and years of organic farming were explaining significantly the composition of the bacterial population
Recent developments in the molecular discrimination of formae speciales of Fusarium oxysporum
Lievens, B. ; Rep, M. ; Thomma, B.P.H.J. - \ 2008
Pest Management Science 64 (2008)11. - ISSN 1526-498X - p. 781 - 788.
vegetative compatibility groups - polymerase-chain-reaction - f-sp basilici - sp radicis-cucumerinum - transposable elements - genetic diversity - plant-pathogens - sp ciceris - i-3-mediated resistance - nectria-haematococca
Rapid and reliable detection and identification of potential plant pathogens is required for taking appropriate and timely disease management measures. For many microbial species of which all strains generally are plant pathogens on a known host range, this has become quite straightforward. However, for some fungal species this is quite a challenge. One of these is Fusarium oxysporum Schlechtend:Fr., which, as a species, has a very broad host range, while individual strains are usually highly host-specific. Moreover, many strains of this fungus are non-pathogenic soil inhabitants. Thus, with regard to effective disease management, identification below the species level is highly desirable. So far, the genetic basis of host specificity in F. oxysporum is poorly understood. Furthermore, strains that infect a particular plant species are not necessarily more closely related to each other than to strains that infect other hosts. Despite these difficulties, recently an increasing number of studies have reported the successful development of molecular markers to discriminate F. oxysporum strains below the species level.
Screening of bacterial isolates from various European soils for in vitro antagonistic activity towards Rhizoctonia solani and Fusarium oxysporum: Site-dependent composition and diversity revealed
Adesina, M.F. ; Lembke, A. ; Costa, R. ; Speksnijder, A.G.C.L. ; Smalla, K. - \ 2007
Soil Biology and Biochemistry 39 (2007)11. - ISSN 0038-0717 - p. 2818 - 2828.
microbial community structure - biological-control - suppressive soils - plant-pathogens - pseudomonas - wilt - microorganisms - rhizosphere - mechanisms - diseases
A cultivation-based approach was used to determine the in vitro antagonistic potential of soil bacteria towards Rhizoctonia solani AG3 and Fusarium oxysporum f. sp. lini (Foln3). Four composite soil samples were collected from four agricultural sites with previous documentation of disease suppression, located in France (FR), the Netherlands (NL), Sweden (SE) and the United Kingdom (UK). Similarly, two sites from Germany (Berlin, G-BR; and Braunschweig, G-BS) without documentation of disease suppression were sampled. Total bacterial counts were determined by plating serial dilutions from the composite soil samples onto R2A, AGS and King's B media. A total of 1,788 isolates (approximately 100 isolates per medium and site) was screened for antifungal activity, and in vitro antagonists (327 isolates) were found amongst the dominant culturable bacteria isolated from all six soils. The overall proportion of antagonists and the number of isolates with inhibitory activity against F. oxysporum were highest in three of the suppressive soils (FR, NL and SE). Characterization of antagonistic bacteria revealed a high phenotypic and genotypic diversity. Siderophore and protease activity were the most prominent phenotypic traits amongst the antagonists. The composition and diversity of antagonists in each soil was site-specific. Nevertheless, none of the antimicrobial traits of bacteria potentially contributing to soil suppressiveness analyzed in this study could be regarded as specific to a given site.
Eradication of Polymyxa betae by thermal and anaerobic conditions and in the presence of compost leachate
Rijn, E. van; Termorshuizen, A.J. - \ 2007
Journal of Phytopathology 155 (2007)9. - ISSN 0931-1785 - p. 544 - 548.
plantenziekten - compost - anaërobe omstandigheden - overleving - temperatuur - polymyxa betae - bietenrhizomanievirus - suikerbieten - plant diseases - composts - anaerobic conditions - survival - temperature - polymyxa betae - beet necrotic yellow vein virus - sugarbeet - yellow-vein-virus - plant-pathogens - sugar-beet - manure
The abiotic conditions required for eradication of Polymyxa betae, the vector of Beet necrotic yellow vein virus in sugar beet, were investigated. Survival of resting spores of P. betae was determined under aerobic (30 min, 4 days and 21 days) and anaerobic (4 days) conditions under several temperature regimes in a water suspension and in leachate extracted from an aerobic compost heap. In water under aerobic conditions the lethal temperature was 60, 55 and 40°C for exposure times of 30 min, 4 days and 21 days, respectively. The effect of compost leachate and/or anaerobic conditions on survival of P. betae depended on temperature. After incubation for 4 days at 20°C, no significant effects of anaerobic conditions or leachate on the survival of P. betae were found. However, at 40°C for 4 days under anaerobic conditions, survival of P. betae was significantly lower than survival under aerobic conditions in water as well as in leachate. In leachate taken from an aerobic compost heap, aerobically incubated at 40°C for 4 days, survival of P. betae was significantly lower than survival in water at the same temperature. As anaerobic spots are prevalent in aerobic compost heaps, especially during the thermophilic phase, actual inactivation temperatures under composting conditions are likely to be lower than the temperatures we found for eradication in water under aerobic conditions.
Biological soil disinfestation (BSD), a new control method for potato brown rot, caused by Ralstonia solanacearum race 3 biovar 2
Messiha, N.A.S. ; Diepeningen, A.D. van; Wenneker, M. ; Beuningen, A.R. van; Janse, J.D. ; Coenen, G.C.M. ; Termorshuizen, A.J. ; Bruggen, A.H.C. van; Blok, W.J. - \ 2007
European Journal of Plant Pathology 117 (2007)4. - ISSN 0929-1873 - p. 403 - 415.
gradient gel-electrophoresis - pseudomonas-solanacearum - organic amendments - plant-pathogens - fatty-acid - survival - resistance - strains - tubers - crops
The potential of Biological Soil Disinfestation (BSD) to control potato brown rot, caused by Ralstonia solanacearum race 3 biovar 2, was investigated. BSD involves the induction of anaerobic soil conditions by increasing microbial respiration through incorporation of fresh organic amendments (here: grass or potato haulms) and by reducing re-supply of oxygen by covering with airtight plastic sheets. Control treatments were left without cover and amendment, or amended without covering or covered only without amendment. The effect of BSD on survival of R. solanacearum was tested at three different scales: in 1-l glass mesocosms under laboratory conditions, in 1.2-m-diam microplots positioned in an outdoor quarantine field, and in a naturally infested commercial field. Within a few days, anaerobic conditions developed in the BSD-treated soils. In the mesocosm and microplot experiment, anaerobic conditions persisted till the end of the 4-week experimental period. In the field experiment, the period of anaerobiosis was shorter due to birds damaging the plastic cover. In all three experiments, BSD reduced soil populations of R. solanacearum significantly by 92.5% to >99.9% compared to the non-amended and uncovered control treatments. In the field experiment, BSD also resulted in a significant reduction of R. solanacearum survival in potato tubers buried at 15 or 35 cm and in the rapid decomposition of superficially buried potatoes remaining after harvesting, thus destroying an important inoculum reservoir of R. solanacearum. The treatments with grass amendment only or covering with only plastic did not result in anaerobic conditions and did not decrease R. solanacearum populations during the experimental period. PCR-DGGE analyses of 16S-rDNA from soil samples of the various treatments in the mesocosm and microplot experiments revealed that BSD hardly affected bacterial diversity but did result in clear shifts in the composition of the bacterial community. The possible implications of these shifts are discussed. It is concluded that BSD has the potential to strongly decrease soil infestation levels of R. solanacearum and to become an important element in a sustainable and effective management strategy for potato brown rot, especially in areas where the disease is endemic.