Application of RAPD-PCR for Virulence Type Analysis within Verticillium dahliae and V. longisporum

A collection of 24 isolates of Verticillium dahliae, 11 isolates of V. longisporum and one isolate of V. albo‐atrum originating from different host plants and geographical regions was tested for genetic variability by random amplified polymorphic DNA‐polymerase chain reaction (RAPD–PCR). Based on nine primers, the three Verticillium species could be clearly differentiated. Likewise, this analysis provided a distinct separation of vegetative compatibility groups (VCG) 2B, 4A and 4B of V. dahliae by specific DNA banding patterns. Additionally, V. longisporum was found to segregate into two subgroups with only 88% similarity. This molecular‐genetic approach was used for the analysis of randomly selected Verticillium isolates from a field with high intensity of oilseed rape cultivation (33% in crop rotation). RAPD‐PCR analysis revealed that 95 of 100 isolates tested belonged to V. longisporum and 5 to VCG 2B of V. dahliae. This study demonstrates an adaptation of Verticillium soil populations to a specific cropping history.     

Differential resistance of oilseed rape cultivars (Brassica napus ssp. oleifera) to Verticillium longisporum infection is affected by rhizosphere colonisation with antagonistic bacteria, Serratia plymuthica and Pseudomonas chlororaphis

The effect of a seed treatment with the antagonistic bacteria Serratia plymuthica (strain HRO-C48) and/or Pseudomonas chlororaphis (strain MA 342) on the infection of oilseed rape with Verticillium longisporum was assessed with ten different cultivars. Soil was inoculated with microsclerotia and mycelium of a V. longisporum culture. Seeds were treated with rifampicin-resistant antagonistic bacteria at a rate of log₁₀ 6–7 cells per seed. Resistance against V. longisporum infection did not differ between cultivars and was generally low. A significant disease reduction recorded as area under disease progress curve (AUDPC) was obtained with both antagonistic rhizobacteria with no significant difference between the treatments. Percent of healthy plants was approximately 70% in all bacterial treatments. Significant differences were observed between the cultivars ranging from 46.5% (cultivar Titan) to 72.6% (Trabant). The combined use of both bacteria could not provide additional control effects. The bacterial density in the rhizosphere was not related to the control effect, but increased by log₁₀ 2 on infection with V. longisporum. Growth promotion effects were also not related to the control effect. At present, neither the application of chemical fungicides nor breeding for resistance against V. longisporum in oilseed rape can provide a solution for this increasingly problematic plant pathogen. The present results now open perspectives to control V. longisporum in oilseed rape by making use of cultivars, which express resistance against this pathogen on interaction with the antagonistic rhizobacteria S. plymuthica or P. chlororaphis.     

Genetic analysis of phenylpropanoid metabolites associated with resistance against Verticillium longisporum in Brassica napus

Verticillium longisporum is a major threat to production of oilseed rape (Brassica napus) in Europe. The aim of the study was to develop new markers and obtain insights into putative mechanisms and pathways involved in the resistance reaction. A genetic approach was used to identify quantitative trait loci (QTL) for V. longisporum resistance and metabolic traits potentially influencing resistance in a B. napus mapping population. Resistance to V. longisporum was mapped in a doubled haploid (DH) population from a cross between the partially resistant winter oilseed rape variety Express 617 and a resistant resynthesized B. napus line, R53. One major resistance QTL contributed by R53 was identified on chromosome C5, while a further, minor QTL contributed by Express 617 was detected on chromosome C1. Markers flanking the QTL also significantly correlated with V. longisporum resistance in four further DH populations derived from crosses between elite oilseed rape cultivars and other resynthesized B. napus lines originating from genetically and geographically diverse brassica A and C genome donors. The tightly-linked markers developed enable the combination of favorable alleles for novel resistance loci from resynthesized B. napus materials with existing resistance loci from commercial breeding lines. HPLC analysis of hypocotyls from infected DH lines revealed that concentrations of a number of phenylpropanoids were correlated with V. longisporum resistance. QTL for some of these phenylpropanoids were also found to co-localize with the QTL for V. longisporum resistance. Genes from the phenylpropanoid pathway are suggested as candidates for V. longisporum resistance.     

Phylogenetic analysis of <Emphasis Type="Italic">Verticillium</Emphasis> species based on nuclear and mitochondrial sequences

Three different genes were sequenced from isolates of five plant-pathogenic Verticillium species, Verticillium albo-atrum, Verticillium dahliae, Verticillium longisporum, Verticillium nigrescens, and Verticillium tricorpus. The sequences covered parts of the mitochondrial cytochrome b gene (cob), the mitochondrial small subunit rRNA gene (rns) and the nuclear ITS2 region. When the sequences were combined, the five species clustered in five monophyletic groups, with V. nigrescens distantly related to the other species while V. tricorpus displayed a somewhat closer relationship to the three remaining species. V. albo-atrum, V. dahliae and V. longisporum were found to be very similar to each other, with V. albo-atrum and V. longisporum displaying the closest relationship. The species affiliation of V. longisporum is discussed.     

Genetic and environmental control of the <Emphasis Type="Italic">Verticillium</Emphasis> syndrome in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

Verticillium spp. are major pathogens of dicotyledonous plants such as cotton, tomato, olive or oilseed rape. Verticillium symptoms are often ambiguous and influenced by development and environment. The aim of the present study was to define disease and resistance traits of the complex Verticillium longisporum syndrome in Arabidopsis thaliana (L.) Heynh. A genetic approach was used to determine genetic, developmental and environmental factors controlling specific disease and resistance traits and to study their interrelations.     

Endophytic Bacteria Induce Growth Promotion and Wilt Disease Suppression in Oilseed Rape and Tomato

To determine whether bacteria isolated from within plant tissue can have plant growth-promotion potential and provide biological control against soilborne diseases, seeds and young plants of oilseed rape (Brassica napus L. cv. Casino) and tomato (Lycopersicon lycopersicum L. cv. Dansk export) were inoculated with individual bacterial isolates or mixtures of bacteria that originated from symptomless oilseed rape, wild and cultivated. They were isolated after surface sterilization of living roots and stems. The effects of these isolates on plant growth and soilborne diseases for oilseed rape and tomato were evaluated in greenhouse experiments. We found isolates that not only significantly improved seed germination, seedling length, and plant growth of oilseed rape and tomato but also, when used for seed treatment, significantly reduced disease symptoms caused by their vascular wilt pathogens Verticillium dahliae Kleb and Fusarium oxysporum f. sp. lycopersici (Sacc.), respectively.     

Host Specialization among Vegetative Compatibility Groups of Verticillium dahliae in Relation to Verticillium longisporum

A collection of 24 isolates of Verticillium dahliae and 10 isolates of Verticillium longisporum originating from nine different host plants and from several geographic regions was tested for host specificity on 11 economically important crops such as potato, tomato, strawberry, linseed, three legumes and four Brassica species. In order to reveal host specificity the potential of each isolate to induce disease and affect plant yield was recorded for all isolate–host combinations. The collected data were statistically processed by means of a cluster analysis. As a result, the host range of individual isolates was found to be more dependent on the vegetative compatibility group (VCG) of the isolate than on its original host plant provenance. Twenty‐two out of 24 V. dahliae isolates belonged to either VCG 2B or 4B. VCG 2B isolates showed specificity for legumes, strawberry, potato and linseed, whereas VCG 4B was specifically virulent on potato, strawberry and linseed. Subgroups within VCG 2B and 4B almost lacking any host preference were designated 2B* and 4B*. Three isolates from VCG 2B*, however, severely attacked tomato which is a host outside the authentic host range of VCG 2B. The pathogenicity of V. longisporum isolates was restricted to cruciferous hosts. Conversely, cruciferous plants were not affected by isolates from VCGs 2B and 4B of V. dahliae. This lack of cross‐infectivity of certain subpopulations of V. dahliae and of V. longisporum may be useful in the management of this soil‐borne wilt disease.     

Effects of experimental warming on fungal disease progress in oilseed rape

Global warming will influence the growth and development of both crops and pathogens. The aims of this study were to investigate potential effects of future warming on oilseed rape growth and the epidemiology of the three economically important pathogens Verticillium longisporum, Sclerotinia sclerotiorum, and Leptosphaeria maculans (anamorph: Phoma lingam). We utilized climate chambers and a soil warming facility, where treatments represented regional warming scenarios for Lower Saxony, Germany, by 2050 and 2100, and compared results of both approaches on a thermal time scale by calculating degree‐days (dd) from day of sowing, December 1st and March 1st until sampling, the latter correlating best with disease progress. Regression analysis showed that plant growth and growth stages in spring responded almost linearly to increasing thermal time until 1000–1500 dd. Colonization of plant tissue by V. longisporum showed an exponential increase when exceeding 1300–1500 dd and reaching plant growth stage BBCH 74/75 (pod development). V. longisporum colonization of plants may be advanced, potentially leading to higher inoculum densities after harvest and increased economic importance of this pathogen under future warming. Sclerotia germination of S. sclerotiorum reached its maximum at 600–900 dd. Advance of these critical degree‐days may lead to earlier apothecia production, potentially advancing the infection window, whereas the future importance of S. sclerotiorum may remain constant. Severity of phoma crown canker increased linearly with increasing thermal time, but showed also large variation in response to the warming scenarios, suggesting that factors such as canopy microclimate in fall or leaf shedding over winter may play a bigger role for L. maculans infection and disease severity than higher soil temperatures. Thermal time was a suitable tool to combine and integrate data on biological responses to soil and air temperature increases from climate chamber and field experiments.     

Biocontrol potential of Microsphaeropsis ochracea on microsclerotia of Verticillium longisporum in environments differing in microbial complexity

The potential of the fungal antagonist Microsphaeropsis ochracea to control the soilborne pathogen Verticillium longisporum was investigated in environments with varying microbial complexity (in vitro vs. in vivo, sterile vs. unsterile, controlled conditions vs. field). A semi-quantitative PCR assay was developed for the detection of M. ochracea on unsterile plant debris. In vitro, M. ochracea caused high levels of mortality to V. longisporum microsclerotia (51–100 %) from 4 to 24 °C, with a broad optimum between 16 and 24 °C. In controlled conditions, M. ochracea significantly reduced the viability of V. longisporum microsclerotia grown on dead rapeseed stems in autoclaved sand, but not in unsterile soil. Likewise, in two experimental years, no significant reduction of V. longisporum inoculum was detectable on rapeseed straw buried in small plots in the field in any of the treatments (soil depths, exposure duration, doses of M. ochracea). Germination of M. ochracea pycnidiospores was inhibited by general soil fungistasis in unsterile soil from a field, botanical garden and grassland. Accordingly, V. longisporum infection of rapeseed plants in the greenhouse was reduced only at artificially high doses of M. ochracea inoculum and no biocontrol efficacy in disease control was recorded in field experiments conducted with winter oilseed rape during two subsequent seasons in an experimental field near Göttingen, with a soil homogenously infested with V. longisporum. The results demonstrate that M. ochracea, although having shown promising potential in controlling pathogens with melanised resting structures on leaf litter, evidently lacks microbial competitiveness to effectively control pathogens in the soil such as V. longisporum, even though the latter is effectively inhibited in vitro.     

Investigations into the taxonomy of the mushroom pathogen <Emphasis Type="Italic">Verticillium fungicola</Emphasis> and its relatives based on sequence analysis of nitrate reductase and ITS regions

The full sequence of the nitrate reductase gene was obtained from a type isolate of Verticillium fungicola var. fungicola and used for phylogenetic analysis against other ascomycete fungi. Sequencing obtained 2749 bp of coding region, 668 bp of 5′ flanking sequence and 731 bp of 3′ flanking sequence. In silico analysis indicated that the coding region contains a single intron and translates into an 893 amino acid protein, with BLAST analysis identifying five conserved nitrate reductase domains within the protein. The 5′ flanking sequence contains numerous conserved sites putatively involved in binding nitrogen regulatory proteins, indicating that the regulation of the gene is likely to be subject to the same regulation as that of model fungi such as Aspergillus nidulans. The central portion of this gene was amplified and sequenced from a number of V. fungicola isolates and related fungi and the resulting phylogenies compared to those obtained from analysis of the rDNA internal transcribed spacer regions for these fungi. Both nitrate reductase and ITS analyses provide additional evidence that reinforces previous findings that suggest the mushroom pathogenic Verticillium species are more related to other chitinolytic fungi such as the insect pathogens Verticillium lecanii and Beauveria bassiana than to the plant pathogenic Verticillia.     

Protection of Oilseed Rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) Toward Fungal Pathogens by Strains of Plant-associated <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis>

In this report, four Bacillus strains were tested for effects on plant fitness and disease protection of oilseed rape (Brassica napus). The strains belonged to newly discovered plant-associated Bacillus amyloliquefaciens and a recently proposed species, Bacillus endophyticus. The fungal pathogens tested represented different infection strategies and included Alternaria brassicae, Botrytis cinerea, Leptosphaeria maculans, and Verticillium longisporum. The B. amyloliquefaciens strains showed no or a weak plant growth promoting activity, whereas the B. endophyticus strain had negative effects on the plant as revealed by phenological analysis. On the other hand, two of the B. amyloliquefaciens strains conferred protection of oilseed rape toward all pathogens tested. In vitro experiments studying the effects of Bacillus exudates on fungal growth showed clear growth inhibition in several but not all cases. The protective effects of Bacillus can therefore, at least in part, be explained by production of antibiotic substances, but other mechanisms must also be involved probably as a result of intricate plant–bacteria interaction. The protective effects observed for certain Bacillus strains make them highly interesting for further studies as biocontrol agents in Brassica cultivation.     

Potential effects of global warming on oilseed rape pathogens in Northern Germany

This review analyses potential effects of global warming, i.e. higher mean temperatures, on the life cycle of five economically important oilseed rape pathogens in Northern Germany using a meta-analytical approach. First, the currently available but strongly fragmented knowledge about temperature influences on individual life cycle stages of the referred pathogens, such as survival, sporulation, infection and disease progress is summarised. These data are compared with current regional climate projections (REMO model) based on emission scenario A1B for the periods 2001–2030 and 2071–2100 in three different oilseed rape growing regions in Lower Saxony, Germany, using a baseline historical series of meteorological data collected from 1971 to 2000. Our analysis suggests that warming might lead to shifts in the future prevalence of these pathogens. Verticillium longisporum, Sclerotinia sclerotiorum and Alternaria brassicae could be particularly favoured, but beneficial effects may also occur for certain life cycle stages of Phoma lingam, such as infection and stem canker development. Pyrenopeziza brassicae might even lose importance under future warming. These changes may be particularly explicit in the long-term view (2071–2100). However, predictions on potential disease shifts are restricted by a lack of reliable, high-resolution future climate scenarios and complicated by the prospective adaptations of farming techniques and crop genotypes to climate change.     

Impact of formulation procedures on the effect of the biocontrol agent Serratia plymuthica HRO-C48 on Verticillium wilt in oilseed rape

Verticillium wilt is an important disease in oilseed rape with an increasing importance worldwide. Currently, there are no methods available to suppress the pathogen. A biological protection strategy on the basis of the plant-beneficial bacterium Serratia plymuthica HRO-C48 to control Verticillium dahliae in oilseed rape was developed. Three different techniques to apply the biocontrol agent to seeds, namely pelleting, film coating and bio-priming, were evaluated considering the influence on the control activity, cell stability during storage and practical feasibility. Neither the treatment nor the inoculum density was found to influence the abundances of HRO-C48 in the rhizosphere after 30 days. Serratia treatment using bio-priming and pelleting resulted in a statistically significant biocontrol in comparison to the non-bacterized controls. Additionally, survival of HRO-C48 differed between treatments, and was the highest using bio-priming at 20°C, and pelleting at 4°C. In conclusion, the procedure of bio-priming, which was developed in line with this study, resulted in a stable and efficient formulation of S. plymuthica on rape seed. This technology opens a possibility to develop a commercial Serratia formulation to protect oilseed against V. dahliae.     

The rhizosphere effect on bacteria antagonistic towards the pathogenic fungus Verticillium differs depending on plant species and site

Rhizobacteria with antagonistic activity towards plant pathogens play an essential role in root growth and plant health and are influenced by plant species in their abundance and composition. To determine the extent of the effect of the plant species and of the site on the abundance and composition of bacteria with antagonistic activity towards Verticillium dahliae, bacteria isolated from the rhizosphere of two Verticillium host plants, oilseed rape and strawberry, and from bulk soil were analysed at three different locations in Germany over two growing seasons. A total of 6732 bacterial isolates screened for in vitro antagonism towards Verticillium resulted in 560 active isolates, among which Pseudomonas (77%) and Serratia (6%) were the most dominant genera. The rhizosphere effect on the antagonistic bacterial community was shown by an enhanced proportion of antagonistic isolates, by enrichment of specific amplified ribosomal DNA restriction analysis types, species and genotypes, and by a reduced diversity in the rhizosphere in comparison to bulk soil. Such an effect was influenced by the plant species and by the site of its cultivation. Altogether, 16S rRNA gene sequencing of 66 isolates resulted in the identification of 22 different species. Antagonists of the genus Serratia were preferentially isolated from oilseed rape rhizosphere, with the exception of one site. For isolates of Pseudomonas and Serratia, plant-specific and site-specific genotypes were found.     

Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays

Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers.     

Pseudomonas community structure and antagonistic potential in the rhizosphere: insights gained by combining phylogenetic and functional gene-based analyses

The Pseudomonas community structure and antagonistic potential in the rhizospheres of strawberry and oilseed rape (host plants of the fungal phytopathogen Verticillium dahliae) were assessed. The use of a new PCR-DGGE system, designed to target Pseudomonas-specific gacA gene fragments in environmental DNA, circumvented common biases of 16S rRNA gene-based DGGE analyses and proved to be a reliable tool to unravel the diversity of uncultured Pseudomonas in bulk and rhizosphere soils. Pseudomonas-specific gacA fingerprints of total-community (TC) rhizosphere DNA were surprisingly diverse, plant-specific and differed markedly from those of the corresponding bulk soils. By combining multiple culture-dependent and independent surveys, a group of Pseudomonas isolates antagonistic towards V. dahliae was shown to be genotypically conserved, to carry the phlD biosynthetic locus (involved in the biosynthesis of 2,4-diacetylphloroglucinol - 2,4-DAPG), and to correspond to a dominant and highly frequent Pseudomonas population in the rhizosphere of field-grown strawberries planted at three sites in Germany which have different land use histories. This population belongs to the Pseudomonas fluorescens phylogenetic lineage and showed closest relatedness to P. fluorescens strain F113 (97% gacA gene sequence identity in 492-bp sequences), a biocontrol agent and 2,4-DAPG producer. Partial gacA gene sequences derived from isolates, clones of the strawberry rhizosphere and DGGE bands retrieved in this study represent previously undescribed Pseudomonas gacA gene clusters as revealed by phylogenetic analysis.