Birger Koopmann

Broadening the Genetic Basis of Verticillium longisporum Resistance in Brassica napus by Interspecific Hybridization.

ABSTRACT Verticillium wilt caused by the vascular fungal pathogen Verticillium longisporum is one of the most important pathogens of oilseed rape (Brassica napus sp. oleifera) in northern Europe. Because production of this major oilseed crop is expanding rapidly and no approved fungicides are available for V. longisporum, long-term control of the disease can only be achieved with cultivars carrying effective quantitative resistance. However, very little resistance to V. longisporum is available within the gene pool of oilseed rape, meaning that interspecific gene transfer from related species is the only possibility for broadening levels of resistance in current varieties. The amphidiploid species B. napus can be resynthesized by crossing the two progenitor species Brassica oleracea and Brassica rapa, hence resistant accessions of these two diploid species can be used as resistance donors. In this study a total of 43 potential B. rapa and B. oleracea resistance donors were tested with regard to their reaction to a mixture of two aggressive V. longisporum isolates, and resistances from diverse lines were combined by embryo rescue-assisted interspecific hybridization in resynthesized rapeseed lines. Progenies from crosses of the two B. rapa gene bank accessions 13444 and 56515 to the B. oleracea gene bank accessions BRA1008, CGN14044, 8207, BRA1398, and 7518 showed a broad spectrum of resistance in pathogenicity tests. Of 45 tested resynthesized lines, 41 lines exhibited a significantly higher level of resistance than the moderately V. longisporum-tolerant oilseed rape cultivar Express. These lines represent a promising basis for the combination of different resistance resources in new varieties.

Conversion of cDNA differential display results (DDRT-PCR) into quantitative transcription profiles

BackgroundGene expression studies on non-model organisms require open-end strategies for transcription profiling. Gel-based analysis of cDNA fragments allows to detect alterations in gene expression for genes which have neither been sequenced yet nor are available in cDNA libraries. Commonly used protocols for gel-based transcript profiling are cDNA differential display (DDRT-PCR) and cDNA-AFLP. Both methods have been used merely as qualitative gene discovery tools so far.ResultsWe developed procedures for the conversion of cDNA Differential Display data into quantitative transcription profiles. Amplified cDNA fragments are separated on a DNA sequencer and detector signals are converted into virtual gel images suitable for semi-automatic analysis. Data processing consists of four steps: (i) cDNA bands in lanes corresponding to samples treated with the same primer combination are matched in order to identify fragments originating from the same transcript, (ii) intensity of bands is determined by densitometry, (iii) densitometric values are normalized, and (iv) intensity ratio is calculated for each pair of corresponding bands. Transcription profiles are represented by sets of intensity ratios (control vs. treatment) for cDNA fragments defined by primer combination and DNA mobility. We demonstrated the procedure by analyzing DDRT-PCR data on the effect of secondary metabolites of oilseed rape Brassica napus on the transcriptome of the pathogenic fungus Leptosphaeria maculans.ConclusionWe developed a data processing procedure for the quantitative analysis of amplified cDNA fragments separated by electrophoresis. The system utilizes common software and provides an open-end alternative to DNA microarray analysis of the transcriptome. It is expected to work equally well with DDRT-PCR and cDNA-AFLP data and be useful particularly in reseach on organisms for which microarray analysis is not available or economical.

Identification of Brassica accessions with enhanced resistance to Verticillium longisporum under controlled and field conditions

Verticillium longisporum (VL) is a soil-borne vascular fungal pathogen with host-specificity to cruciferous plants such as oilseed rape, threatening its production particularly in the Northern European countries. In a comprehensive screening conducted under greenhouse conditions, 1348 accessions of Brassica napus as well as the progenitor species B. oleracea and B. rapa (syn. campestris) were tested for their resistance to VL. While most of the tested B. napus accessions showed a susceptible to moderate resistant phenotype and the majority of the B. rapa genotypes were highly susceptible, an elevated level of resistance was found in the B. oleracea pool. Resynthesized oilseed rape lines produced on the basis of these data by interspecific hybridization of B. oleracea and B. rapa also exhibited enhanced resistance to VL. In order to verify the greenhouse data, a subset of B. napus breeding lines was further tested for resistance in the field at different sites in North and Northeast Germany. Overall, there was no statistically significant relationship, neither between greenhouse and field data nor between results of different field sites. However, genotypes highly resistant in the greenhouse generally performed well also in the field. Thus, the combination of resistance tests conducted under controlled as well as field conditions is a powerful and reliable approach in the practical selection of resistant breeding lines.ZusammenfassungVerticillium longisporum (VL) ist ein bodenbürtiges, pilzliches Gefäßpathogen mit Wirtsspezialisierung auf kruzifere Pflanzenarten wie Raps, dessen Produktion durch diesen Erreger insbesondere in den nordeuropäischen Ländern gefährdet ist. In einem umfangreichen Screening unter Gewächshausbedingungen wurden 1348 B. napus-Akzessionen sowie die Elternspezies B. oleracea und B. rapa (syn. campestris) auf ihre Anfälligkeit gegenüber VL getestet. Während die meisten der getesteten B. napus-Akzessionen einen anfälligen bis mäßig resistenten Phänotyp zeigten und die Mehrheit der B. rapa-Genotypen hoch anfällig war, wurde ein erhöhtes Resistenzniveau im B. oleracea-Pool gefunden. Resynthetisierte Rapslinien, welche auf der Basis dieser Daten durch interspezifische Hybridisierung von B. oleracea und B. rapa erzeugt worden waren, wiesen ebenfalls eine erhöhte Resistenz gegenüber VL auf. Um die Daten aus dem Gewächshaus zu verifizieren, wurden ausgewählte B. napus-Zuchtlinien weiteren Resistenztests im Feld an verschiedenen Standorten im Norden und Nordosten Deutschlands unterzogen. Insgesamt gab es weder zwischen Gewächshaus- und Felddaten noch Feldversuchsstandorte einen statistisch signifikanten Zusammenhang. Allerdings schnitten Genotypen, welche hoch resistent im Gewächshaus waren, im Allgemeinen auch unter Feldbedingungen gut ab. Somit ist die Kombination von Resistenztests sowohl unter kontrollierten als auch unter Feldbedingungen ein leistungsfähiger und zuverlässiger Ansatz in der praxisbezogenen Selektion von resistenten Zuchtlinien.

Wheat Blast and Fusarium Head Blight Display Contrasting Interaction Patterns on Ears of Wheat Genotypes Differing in Resistance.

The interaction of wheat with two ear pathogens, Magnaporthe wheat blast (MWB) and Fusarium graminearum (Fusarium head blight, FHB), was studied on the phenotypic, histological, and gene expression level. Most of the 27 wheat cultivars inoculated with MWB and F. graminearum displayed inverse disease responses to blast and FHB infection. Two cultivars, Milan and Sumai 3, were selected expressing converse disease phenotypes to blast (Milan, R)/(Sumai 3, S) and FHB (Milan, S)/(Sumai 3, R). Confocal laser scanning microscopy revealed early (12 h postinoculation) colonization of the spikelets by MWB similarly on both cultivars, while F. graminearum infected anthers of the susceptible cultivar earlier. Both pathogens grew much faster in the rachilla of susceptible than resistant cultivars, indicating that resistance is mainly expressed in this part connecting the spikelet with the rachis. In general, O2(-) and H2O2 levels were unrelated to disease expression in the four studied interactions. The differential disease phenotypes, fungal spread in the rachis, and colonization patterns in the spikelets were confirmed by distinct gene expression patterns. Among the eight genes analyzed, seven were more strongly induced by FHB than by blast. Genes for chitinase (Chi2), β-1,3-glucanase (PR2), a plant defensin homolog (PRPI), and peroxidase (Pox2) were strongly upregulated in Milan in response to both pathogens, while PR2 and PR5 (thaumatin-like protein) were transiently triggered by MWB on both cultivars. Upregulation of cinnamoyl-CoA reductase (CCR), cytochrome P450 (CYP709C1), and UDP-glycosyl transferase (UGT) were more prominent in ears infected with F. graminearum, while upregulation of UGT was higher in Sumai 3 when infected with either pathogen. Cultivar resistance to FHB was reflected by clearly higher expression levels of UGT and CYP709C1 in Sumai 3. The differential responses of wheat to the two ear pathogens demonstrated in this study makes it unlikely that common resistance genes exist for control of FHB and blast, suggesting the need to stack many genes associated with resistance in breeding programs for multiple resistance.

Development of fungicide resistance of wheat and barley pathogens against strobilurins: A methodological approach

A methodological approach was conducted to investigate how fast isolates of a “one-race-population” adapt to an ingredient with fungicidal activity. A major objective was to maintain a continuous selection pressure on the fungal populations. Different concentrations of strobilurin azoxystrobin (Amistar®) were applied on wheat and barley cotyledons to ensure selection pressure. The fungi reproduced on fungicide-treated leaves in order to display a “worst-case-scenario”. Wheat powdery mildew (Blumeria graminis f. sp. tritici) adapted rapidly to higher concentrations of the fungicide. Already the fifth generation of the fungus was able to grow and reproduce on leaves containing high concentrations of azoxystrobin (0.5x of the recommended dosage in the field). Two replicates led to the same results. Although rapidity of the adaptation was remarkable, the adapted isolates apparently lost their fitness. In contrast, Blumeria graminis f. sp. hordei was significantly more susceptible to azoxystrobin. The dosage of the fungicide was reduced by the factor of 10 to 40. The lowest concentration of Amistar® efficient against wheat powdery mildew was equal to the highest tolerable concentration applicable for barley powdery mildew. Even when the fungus could adapt to these low concentrations, a change to resistance is not likely, because in the field, 0.1x of the recommended dosage will normally not be used. The third fungus investigated, glume blotch of wheat (Septoria nodorum) was tested ad planta and in vitro. Ad planta, the fourth and fifth generation of the fungus seemed to be less susceptible against the active ingredient azoxystrobin, but in vitro no such effects were observed. The methods used in these studies seem to offer an easy and fast tool to estimate the risk of adaptation and to compare sensitivity levels of different fungal species. The experiments showed significant differences in fungicide susceptibility between the two powdery mildews. We presume that disruptive as well as adaptive resistance mechanisms occur in both pathogens and that the two resistance mechanisms are correlated. Fungi possessing high affinity to both are more assertive than other fungi. A disadvantage is that these artificial circumstances do not exactly reflect the situation in the field because sexual reproduction is not considered. Nevertheless, the risk of a continuous selection pressure caused by a single-site- inhibiting fungicide was obvious in these experiments.ZusammenfassungIn dieser Arbeit wurde in einem modellhaften Ansatz die Fragestellung untersucht, wie schnell sich Individuen einer reinen Rasse an einen fungiziden Wirkstoff adaptieren können. Besondere Bedeutung wurde dabei der Methodik beigemessen, einen permanenten Selektionsdruck auf die Populationen aufrechtzuerhalten. Dieser wurde durch Applikation des Stroh il urins Amistar® auf Weizenbzw. Gerstenkeimblätter in verschiedenen Konzentrationen gewährleistet. Auch die Weitervermehrung der Pilze wurde unter Einfluss des Fungizids vorgenommen und so der „worst case“ dargestellt. Beim Echten Weizenmehltau (Blumeria graminis f. sp. tritici) erfolgte die Adaption an höhere Wirkstoffkonzentrationen sehr schnell. Bereits nach fünf Generationen war der Pilz in der Lage, trotz Applikation hoher Wirkstoffmengen (0,5x der empfohlenen Aufwandmenge) zu wachsen und sich zu reproduzieren. Diese Konzentration wird häufig auch im Feld appliziert. Der Versuch wurde zwei Mal durchgeführt und erzielte dasselbe Ergebnis. Allerdings schienen die adaptierten Individuen unter einem Fitnessverlust zu leiden. Im Gegensatz dazu reagierte der Echte Gerstenmehltau (Blumeria graminis f. sp. hordei) wesentlich sensitiver. Die geringste Wirkstoffkonzentration beim Echten Weizenmehltau stellte die höchste beim Echten Gerstenmehltau dar. Eine Adaption konnte nicht eindeutig festgestellt werden. Zudem waren die Wirkstoffkonzentrationen so gering (bis zu 0,1x der empfohlenen Aufwandmenge), dass eine langsame Adaption an diese Wirkstoffkonzentrationen keine Resistenzgefahr erwarten lässt. Die Versuche zeigten signifikante Unterschiede in der Strobilurinsensitivität der beiden Mehltauarten.Bei Septoria nodorum, dem Erreger der Blatt- und Spelzenbräune des Weizens, wurden die Versuche sowohl ad planta, als auch in vitro durchgeführt. Ad planta zeigte dieser Pilz eine beginnende Adaption an den Wirkstoff Azoxystrobin, die jedoch in vitro nicht bestätigt werden konnte.Mit Hilfe der durchgeführten Versuche lassen sich relativ einfach und schnell Aussagen über Adaptionsrisiken bei bestimmten Wirkstoffen treffen. Auch Sensitivitätsvergleiche zwischen verschiedenen Pilzen können angestellt werden. Es scheint, dass sowohl die disruptive, als auch die adaptative Strobilurin- resistenz bei beiden Mehltauarten auftritt. Entscheidender Faktor dabei ist, dass der Echte Weizenmehltau wesentlich stärker zu Resistenzen neigt als der Echte Gerstenmehltau. Vermutlich besteht ein Zusammenhang zwischen den beiden Resistenzmechanismen, so dass Pilze, die über beide Mechanismen verfügen; ein hohes Potential für schnell entwickelnde Resistenzen besitzen. Allerdings lassen sich diese Modellversuche nur schwer auf die Feldsituation übertragen, da sexuelle Reproduktion hierbei keine Berücksichtigung findet. Die Gefahren, die ein permanenter Selektionsdruck in Form eines „single-site-inhibiting“ Fungizids birgt, werden in diesen Versuchen jedoch deutlich.

First Record of Clavibacter michiganensis subsp. michiganensis Causing Canker of Tomato Plants in Syria

Between March and mid April of 2007, several extensive surveys for Clavibacter michiganensis subsp. michiganensis were carried out among greenhouses in the coastal strip provinces of the Mediterranean Sea in north-west Syria (Latakia and Tartous), where a large proportion of Syrian fresh-market tomatoes are produced. This bacterium causes bacterial canker of tomato and is considered an A2 quarantine pathogen by the European Plant Protection Organization (EPPO). It is currently present in all major tomato-production areas in the EPPO region (4), but has not been previously reported in Syria. The survey revealed typical canker symptoms in 7% of 150 inspected greenhouses that contained cvs. Dima, Huda, and Astona. These symptoms included stunting, dark brown-to-black lesions on the leaf margins, wilting and defoliation of whole plants, and vascular discoloration. The disease incidence in such greenhouses was estimated at 15% at the time of the survey. Diseased plants were surface sterilized and homogenized in sterile water. Serial dilutions were plated on nutrient glucose agar. Suspected colonies were further purified by repeated restreaking on new agar plates. All 10 of the suspected strains obtained from different locations were identified as C. michiganensis subsp. michiganensis on the basis of the following observations: bacterial cells of all strains had a coryneform shape, were nonmotile, gram positive according to Grams reaction test with 3% KOH (2), oxidase-negative, and caused hypersensitive reactions on leaves of Mirabilis jalaba (1) within 24 h. PCR assays were conducted with the C. michiganensis subsp. michiganensis-specific primer set PSA-4/R (3) and template DNA prepared from in-vitro-grown bacteria with the MasterPure Gram Positive DNA Purification Kit (Epicentre Biotechnologies, Madison, WI). The expected 270-bp amplicon was observed for both reference strains as well as the Syrian strains. Pathogenicity of the strains was confirmed by artificial inoculation of 6-week-old tomato plants (Lycopersicon esculentum Mill. cv. Lyconorma). Inoculation was performed by stabbing the stem with a sterile needle through a drop (~35 μl) of bacterial suspension (~108 CFU/ml in 0.01 M MgSO4) placed in the axil of the second or third true leaf. Three tomato seedlings were inoculated with each strain. Control plants were inoculated with sterile 0.01 M MgSO4. Symptoms including lateral wilt of leaflets, stem lesions, and wilting of whole plants were observed within 10 to 15 days after inoculation, except for the negative control. To fulfill Kochs postulates, reisolation and reidentification of the pathogen was conducted as previously described. To our knowledge, this is the first record of the occurrence of bacterial canker of tomato in Syria. References: (1) R. D. Gitaitis. Plant Dis. 74:58, 1990. (2) T. J. Gregersen. Appl. Microbiol. Biotechnol. 5:123, 1978. (3) K. H. Pastrik and F. A. Rainey. J. Phytopathol. 147:687, 1999. (4) I. M. Smith and L. M. F. Charles, eds. Map 253 in: Distribution Maps of Quarantine Pests for Europe. EPPO/CABI, 1998.

The Vascular Pathogen Verticillium longisporum Does Not Affect Water Relations and Plant Responses to Drought Stress of Its Host, Brassica napus

Verticillium longisporum is a host-specific vascular pathogen of oilseed rape (Brassica napus L.) that causes economic crop losses by impairing plant growth and inducing premature senescence. This study investigates whether plant damage through Verticillium stem striping is due to impaired plant water relations, whether V. longisporum affects responses of a susceptible B. napus variety to drought stress, and whether drought stress, in turn, affects plant responses to V. longisporum. Two-factorial experiments on a susceptible cultivar of B. napus infected or noninfected with V. longisporum and exposed to three watering levels (30, 60, and 100% field capacity) revealed that drought stress and V. longisporum impaired plant growth by entirely different mechanisms. Although both stresses similarly affected plant growth parameters (plant height, hypocotyl diameter, and shoot and root dry matter), infection of B. napus with V. longisporum did not affect any drought-related physiological or molecular genetic plant parameters, including transpiration rate, stomatal conductance, photosynthesis rate, water use efficiency, relative leaf water content, leaf proline content, or the expression of drought-responsive genes. Thus, this study provides comprehensive physiological and molecular genetic evidence explaining the lack of wilt symptoms in B. napus infected with V. longisporum. Likewise, drought tolerance of B. napus was unaffected by V. longisporum, as was the level of disease by drought conditions, thus excluding a concerted action of both stresses in the field. Although it is evident that drought and vascular infection with V. longisporum impair plant growth by different mechanisms, it remains to be determined by which other factors V. longisporum causes crop loss.

Elevated temperature increases in planta expression levels of virulence related genes in Magnaporthe oryzae and compromises resistance in Oryza sativa cv. Nipponbare

Temperature changes have the potential to alter the incidence and severity of plant disease epidemics and pressures, as well as to reshape the co-evolutionary relationships between plants and pathogens. However, the molecular basis of temperature modulation of pathogenicity of plant pathogens is still unclear. Here, we studied the effect of temperature on biomass of Magnaporthe oryzae in planta using qPCR. Additionally, the transcriptomes of M. oryzae and rice were analysed using RNA-seq. Rice seedlings were exposed to 35°C and 28°C for 7 days before pathogen inoculation. Inoculated plants were kept in the dark at 28°C for 24h and later re-exposed to 35°C and 28°C for an additional 24h before sample collection. Plants grown and predisposed to 35°C prior to inoculation exhibited accelerated tissue necrosis compared with plants grown and inoculated at 28°C. In accordance with the disease severity observed on infected leaves, in planta fungal biomass was significantly higher at 35°C than 28°C. Moreover, M. oryzae exhibited increased expression levels of putative fungal effector genes in plants exposed to 35°C compared with plants exposed to 28°C. Collectively, this study revealed that temperature elevation could favour M. oryzae infection by compromising plant resistance and accelerating plant tissue colonisation with the pathogen.

Detection of Bacterial Diseases of Tomatoes by the Polymerase Chain Reaction (PCR) and Serology

Tomatoes contribute a large portion of vegetables grown in Turkey. Production is still increasing and bacterial diseases are causing high yield losses [1]. Because effective bactericides are not available and tomatoes are mainly grown on small farms with limited budgets, disease control is limited [2,3]. The present strategy to reduce yield losses by bacterial diseases is to use healthy seeds and resistant cultivars in combination with chemical, biological or cultural measures [4,5]. Therefore, highly sensitive detection methods are required for improved indexing of seed by the plant protection service in Turkey.

The Use of PCR to Detect Pseudomonas syringae pv. tomato in Planta

The PCR technique was used to detect Pseudomonas syringae pv. tomato, the cause of bacieriai speck of tomato.