The interplay of suppressive soil bacteria and plant root exudates determines germination of microsclerotia of Verticillium longisporum.

Dormant microsclerotia play a vital role in the survival and spread of Verticillium longisporum, as they can stay viable in the soil and maintain their infectivity for many years. In our previous work, we revealed that soil bacterial volatiles are a key inhibitory factor causing microsclerotia dormancy in the soil. In this study, we further demonstrate that root exudates collected from both host and non-host plants can effectively rescue microsclerotia from bacterial suppression and initiate germination. To identify the specific compounds in root exudates responsible for microsclerotia germination, we fractionated the collected root exudates into polar and non-polar compounds. Subsequently, we conducted comprehensive bioassays with each fraction on germination-suppressed microsclerotia. The result revealed a pivotal role of primary metabolites in root exudates, particularly glutamic acid, in triggering microsclerotia germination and overcoming bacterial inhibition. Moreover, our studies revealed a decrease in inhibitory bacterial volatile fatty acids when bacteria were cultured in the presence of root exudates or glutamic acid. This suggests a potential mechanism, by which root exudates set-off bacterial suppression on microsclerotia. Here, we reveal for the first time that plant root exudates, instead of directly inducing the germination of microsclerotia, enact a set-off effect by counteracting the suppressive impact of soil bacteria on the microsclerotia germination process. This nuanced interaction advances our understanding of the multifaceted dynamics governing microsclerotia dormancy and germination in the soil environment. IMPORTANCE: Our research provides first-time insights into the crucial interaction between plant root exudates and soil bacteria in regulating the germination of Verticillium longisporum microsclerotia, a significant structure in the survival and proliferation of this soil-borne pathogen. We describe so far unknown mechanisms, which are key to understand how root infections on oilseed rape can occur. By pinpointing primary metabolites in root exudates as key factors in overcoming bacteria-induced dormancy and promote microsclerotia germination, our study highlights the potential for exploiting plant - as well as soil microbe-derived - compounds to control V. longisporum. This work underscores the importance of elucidating the nuanced interactions within the soil ecosystem to devise innovative strategies for managing root infective plant diseases, thereby contributing to the resilience and health of cropping systems.

Lignin Composition and Timing of Cell Wall Lignification Are Involved in Brassica napus Resistance to Stem Rot Caused by Sclerotinia sclerotiorum.

Sclerotinia stem rot (SSR) is an economically and globally significant disease in oilseed rape (Brassica napus) caused by the necrotrophic ascomycete Sclerotinia sclerotiorum. This study explored the role of cell wall reinforcement by lignin as a relevant factor for effective plant defense against attack by this pathogen. Expression of key genes in the phenylpropanoid pathway and the induced synthesis of lignin in infected stem tissues were investigated in a study comparing a susceptible ('Loras') and a moderately resistant cultivar ('Zhongyou 821' [ZY821]). Data revealed an earlier and more rapid defense activation in ZY821 through upregulation of transcript levels of genes related to key steps in the phenylpropanoid pathway associated with increased lignin deposition in the resistant B. napus genotype. Expression level of BnCAD5, encoding a cinnamyl alcohol dehydrogenase, responsible for conversion of monolignol to lignin, was more rapidly upregulated in ZY821 than 'Loras'. The similar expression pattern of BnCAD5 and the gene BnF5H, encoding for ferulate-5-hydroxylase, which catalyzes the synthesis of syringyl (S) lignin precursors, suggests that BnCAD5 is involved in S lignin formation. Histological observations confirmed these results, showing an earlier increase of S lignin deposition in the infected resistant genotype. Deposition of guaiacyl lignin was detected in both genotypes and is thus considered a component of basal, cultivar-independent defense response of B. napus to stem rot. The results indicate the importance of cell wall modification for quantitative stem rot resistance by responses in the phenylpropanoid metabolism generating distinct lignin types on different temporal scales.

Hybrids between Brassica napus and B. nigra show frequent pairing between the B and A/C genomes and resistance to blackleg.

High frequencies of homoeologous and even non-homologous chromosome recombination in Brassica hybrids can transfer useful traits between genomes, but also destabilise synthetic allopolyploids. We produced triploid hybrids (2n = 3x = ABC) from the cross B. napus (rapeseed, 2n = 4x = AACC) × B. nigra (black mustard, 2n = 2x = BB) by embryo rescue and allohexaploid hybrids (2n = 6x = AABBCC = 54) by chromosome doubling of the triploids. These hybrids demonstrated resistance to blackleg disease (causal agent: Leptosphaeria maculans) inherited from their B. nigra parent. In order to assess the possibility of transfer of this resistance between the B genome and the A and C subgenomes of B. napus, as well as to assess the genomic stability of allohexaploids from the cross B. napus × B. nigra, frequencies of non-homologous chromosome pairing in these hybrids were assessed using classical cytogenetics and genomic in-situ hybridization. Meiosis was highly irregular, and non-homologous chromosome pairing between the B genome and the A/C genomes was common in both triploid hybrids (observed in 38% of pollen mother cells) and allohexaploid hybrids (observed in 15% of pollen mother cells). Our results suggest that introgression of blackleg resistance from the B genome into the A or C genomes should be possible, but that allohexaploids from this genome combination are likely unstable.

Potential for Seed Transmission of Verticillium longisporum in Oilseed Rape (Brassica napus).

Verticillium longisporum is a soilborne vascular fungal pathogen that has spread throughout the European oilseed rape cultivation area since the 1980s and was detected in canola fields in Canada in 2014. In a series of greenhouse and field inoculation experiments using V. longisporum-resistant and susceptible cultivars of winter and spring types of oilseed rape, the present study investigated the potential of V. longisporum dissemination by seeds of Brassica napus. Greenhouse inoculation studies with a DsRed-labeled isolate of V. longisporum confirmed the systemic growth of the pathogen from roots to seeds. Further monitoring of plant colonization in the greenhouse with a species-specific real-time polymerase chain reaction assay verified the pathogen growth from roots to stem bases, pods, and seeds in root-inoculated plants. The frequency of recovery of viable colonies of V. longisporum from seeds harvested from greenhouse-grown inoculated plants ranged from 0.08 to 13.3%. The frequency of seed transmission in the greenhouse differed in oilseed rape cultivars varying in susceptibility to V. longisporum. Subsequent studies on transmission of the disease into the offspring revealed that only 1.7 to 2.3% of plants showed disease symptoms as confirmed by the formation of microsclerotia in the stems. Results from field-grown plants differed from the greenhouse studies. The degree of seed transmission in the field was dependent on the crop type. Although only low concentrations of DNA of V. longisporum were detectable in seeds harvested from severely infected winter oilseed rape, significantly greater concentrations of fungal DNA were found in seeds of spring-type oilseed rape, at similar soil conditions and inoculum densities. Correspondingly, plating seeds that were harvested from infected plants on agar yielded viable V. longisporum colonies only from seeds of the spring-type but not of the winter-type plants. Lack of seed infection in the winter-type crop was confirmed in two seasons. Equally, none of the offspring grown from seeds from severely diseased winter oilseed rape plants developed symptoms of Verticillium stem striping. The results suggest that the rate of seed transmission of V. longisporum depends on the degree of plant colonization, which is significantly faster under greenhouse than field conditions and in a spring-sown crop compared with an autumn-sown oilseed rape crop. According to our studies, disease transmission by seeds from European winter oilseed rape production cannot be confirmed.

Contrasting Patterns of Colonization with Verticillium longisporum in Winter- and Spring-Type Oilseed Rape (Brassica napus) in the Field and Greenhouse and the Role of Soil Temperature.

Oilseed rape, an important source of vegetable plant oil, is threatened by Verticillium longisporum, a soil-borne vascular fungal pathogen so far occurring in oilseed rape growing regions in Europe and Canada. Despite intensive research into V. longisporum in the last decades in controlled conditions, basic knowledge is still lacking about the time course of infection, temporal pattern of colonization, and disease development on field-grown plants. In this study, colonization of roots, stem bases, and stems with V. longisporum was followed by real-time PCR from the seedling until mature plant stages in 2-year field experiments with microsclerotia-infested plots and either spring-type or autumn-sown (winter-type) oilseed rape cultivars. The temporal pattern of plant colonization differed between greenhouse and field-grown oilseed rape and between spring- and winter-type plants in the field. Within 28 to 35 days, a continuous systemic colonization with V. longisporum was detected in roots and shoots of young plants in the greenhouse associated with significant stunting. In contrast, real-time PCR analysis of V. longisporum in field-grown winter oilseed rape plants displayed a strongly discontinuous colonization pattern with low fungal growth in roots during juvenile growth stages until flowering, whereas in spring oilseed rape, no root colonization was observed until early flowering stages. Hence, stem colonization with the pathogen required 6 months in winter oilseed rape and 1 month in spring oilseed rape from the time of initial root infection. The different patterns of stem colonization were related to soil temperature. Average soil temperatures in 5-cm depth during 7 days before sampling time points from 2 years of field experiments displayed a significant relationship with fungal colonization in the root. A climate chamber inoculation trial with soil temperature levels that varied from 6 to 18°C revealed a threshold temperature of >12°C in the soil to enable root invasion. This soil condition is reached in winter-type oilseed rape in the field in Germany either until the eight-leaf stage in early autumn or after pod stage in spring, whereas in spring-sown oilseed rape early root infection is delayed owing to the cool conditions during juvenile growth stages. The delay of stem colonization in field-grown oilseed rape may explain the lack of stunting as observed in the greenhouse and the previously reported inconsistent effects of V. longisporum on yield levels and seed quality, which were confirmed in this study.

Growth of Verticillium longisporum in Xylem Sap of Brassica napus is Independent from Cultivar Resistance but Promoted by Plant Aging.

As Verticillium stem striping of oilseed rape (OSR), a vascular disease caused by Verticillium longisporum, is extending into new geographic regions and no control with fungicides exists, the demand for understanding mechanisms of quantitative resistance increases. Because V. longisporum is strictly limited to the xylem and resistance is expressed in the systemic stage post root invasion, we investigated a potential antifungal role of soluble constituents and nutritional conditions in xylem sap as determinants of cultivar resistance of OSR to V. longisporum. Assessment of biometric and molecular genetic parameters applied to describe V. longisporum resistance (net area under disease progress curve, stunting, stem thickness, plant biomass, and V. longisporum DNA content) showed consistent susceptibility of cultivar 'Falcon' in contrast to two resistant genotypes, 'SEM' and 'Aviso'. Spectrophotometric analysis revealed a consistently stronger in vitro growth of V. longisporum in xylem sap extracted from OSR compared with the water control. Further comparisons of fungal growth in xylem sap of different cultivars revealed the absence of constitutive or V. longisporum induced antifungal activity in the xylem sap of resistant versus susceptible genotypes. The similar growth of V. longisporum in xylem sap, irrespective of cultivar, infection with V. longisporum and xylem sap filtration, was correlated with about equal amounts of total soluble proteins in xylem sap from these treatments. Interestingly, compared with younger plants, xylem sap from older plants induced significantly stronger fungal growth. Growth enhancement of V. longisporum in xylem sap of aging plants was reflected by increased contents of carbohydrates, which was consistent in mock or V. longisporum-infected plants and independent from cultivar resistance. The improved nutritional conditions in the xylem of more mature plants may explain the late appearance of disease symptoms, which are observed only in late maturity stages of plants in the field. While falsifying the presence of antifungal activity in xylem sap of resistant cultivars, this study strengthens previous findings that indicated a significant role of physical cell wall bound resistance factors involved in quantitative, cultivar-related resistance of B. napus to V. longisporum.

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.

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), ß-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.

Population Structure, Pathogenicity, and Mating Type Distribution of Magnaporthe oryzae Isolates from East Africa.

Rice blast, caused by Magnaporthe oryzae, is one of the emergent threats to rice production in East Africa (EA), where little is known about the population genetics and pathogenicity of this pathogen. We investigated the genetic diversity and mating type (MAT) distribution of 88 isolates of M. oryzae from EA and representative isolates from West Africa (WA) and the Philippines (Asia) using amplified fragment length polymorphism markers and mating-type-specific primer sets. In addition, the aggressiveness of each isolate was evaluated by inoculating on the susceptible Oryza sativa indica 'Co39', scoring the disease severity and calculating the disease progress. Hierarchical analysis of molecular variance revealed a low level of genetic differentiation at two levels (FST 0.12 and FCT 0.11). No evidence of population structure was found among the 65 isolates from EA, and gene flow among EA populations was high. Moreover, pairwise population differentiation (GST) in EA populations ranged from 0.03 to 0.04, suggesting that >96% of genetic variation is derived from within populations. However, the populations from Asia and WA were moderately differentiated from EA ones. The spatial analysis of principal coordinates and STRUCTURE revealed overlapping between individual M. oryzae isolates from EA, with limited distinctness according to the geographic origin. All the populations were clonal, given the positive and significant index of association (IA) and standardized index of association (rd), which indicates a significant (P<0.001) departure from panmixia (IA and rd=0). Both MAT1-1 and MAT1-2 were detected. However, MAT1-1 was more prevalent than MAT1-2. Pathogenicity analysis revealed variability in aggressiveness, suggesting a potential existence of different races. Our data suggest that either M. oryzae populations from EA could be distributed as a single genetic population or gene flow is exerting a significant influence, effectively swamping the action of selection. This is the first study of genetic differentiation of rice-infecting M. oryzae strains from EA, and may guide further studies on the pathogen as well as resistance breeding efforts.

Mechanisms regulating grain contamination with trichothecenes translocated from the stem base of wheat (Triticum aestivum) infected with Fusarium culmorum.

Factors limiting trichothecene contamination of mature wheat grains after Fusarium infection are of major interest in crop production. In addition to ear infection, systemic translocation of deoxynivalenol (DON) may contribute to mycotoxin levels in grains after stem base infection with toxigenic Fusarium spp. However, the exact and potential mechanisms regulating DON translocation into wheat grains from the plant base are still unknown. We analyzed two wheat cultivars differing in susceptibility to Fusarium head blight (FHB), which were infected at the stem base with Fusarium culmorum in climate chamber experiments. Fungal DNA was found only in the infected stem base tissue, whereas DON and its derivative, DON-3-glucoside (D3G), were detected in upper plant parts. Although infected stem bases contained more than 10,000 µg kg⁻¹ dry weight (DW) of DON and mean levels of DON after translocation in the ear and husks reached 1,900 µg kg⁻¹ DW, no DON or D3G was detectable in mature grains. D3G quantification revealed that DON detoxification took mainly place in the stem basis, where ≤ 50% of DON was metabolized into D3G. Enhanced expression of a gene putatively encoding a uridine diphosphate-glycosyltransferase (GenBank accession number FG985273) was observed in the stem base after infection with F. culmorum. Resistance to F. culmorum stem base infection, DON glycosylation in the stem base, and mycotoxin translocation were unrelated to cultivar resistance to FHB. Histological studies demonstrated that the vascular transport of DON labeled with fluorescein as a tracer from the peduncle to the grain was interrupted by a barrier zone at the interface between grain and rachilla, formerly described as "xylem discontinuity". This is the first study to demonstrate the effective control of influx of systemically translocated fungal mycotoxins into grains at the rachilla-seed interface by the xylem discontinuity tissue in wheat ears.

Efficacy of Blackleg Major Resistance Genes in B. napus in Germany.

Leptosphaeria maculans is one of the major pathogens of oilseed rape (B. napus). It causes blackleg disease, which accounts for significant yield losses worldwide. Using cultivars that harbor major resistance (R) genes is one of the most effective control methods. However, the efficacy of major R genes is related to the frequency of the corresponding avirulence (Avr) genes in a L. maculans population. In this paper, we report the Avr profiles of L. maculans populations and the ratio of its mating types in Northern and Central regions of Germany. Eleven Avr genes in five-hundred and seventy-four isolates were characterized either by applying cotyledon tests on a B. napus differential set or by amplifying avirulence gene-specific PCR markers. Fifty-two races were determined, among which the most dominant race was Avrlm6, -7, -11, AvrlepR1, -R2. Results showed that the resistance gene Rlm2 is 100% ineffective, some other major R genes such as Rlm1, Rlm3, Rlm4 and LepR3 are partially effective (with corresponding Avr frequencies ≤ 42%), while LepR1, LepR2, Rlm6, Rlm11 and Rlm7 can still provide relatively effective resistance in the German fields investigated (with corresponding Avr frequencies of 63-100%). Sexual reproduction is a factor that enhances the potential of L. maculans to evolve under selection pressure. Mating types of the L. maculans populations did not deviate from the ratio of 1:1 in the examined regions, indicating that sexual reproduction and ascospores play central roles in the L. maculans lifecycle. Overall, this study provides an important dataset for the establishment of a strategic plan to preserve the efficacies of major R genes in Germany by applying cultivar rotations of oilseed rape.

Greenhouse Evaluation of Clubroot Resistant-Brassica napus cv. Mendel and Its Efficacy Concerning Virulence and Soil Inoculum Levels of Plasmodiophora brassicae.

Clubroot resistance of oilseed rape (OSR) cultivars frequently relies on a major resistance gene originating from cv. Mendel. The efficacy of this resistance was studied in greenhouse experiments using two Plasmodiophora brassicae isolates, which were either virulent (P1(+)) or avirulent (P1) on Mendel. Seeds of clubroot-susceptible cultivar Visby and clubroot-resistant cultivar Mendel were sown in soil mixtures inoculated with different concentrations of resting spores (101, 103, 105, and 107 resting spores/g soil). Clubroot severity, plant height, shoot and root weight as well as resting spore propagation were assessed for each isolate and cultivar separately at four dates after sowing. The OSR cultivars behaved significantly different in the measured parameters. The threshold of inoculum density to cause disease depended strongly on the virulence of the pathogen and susceptibility of the host plant. In Visby grown in soil infested with P1, clubroot symptoms and increases in root weight and the number of propagated resting spores occurred at inoculum levels of 101 resting spores and higher, whereas Mendel was not affected in soils under the three lowest inoculum densities. In contrast, the P1(+) isolate led to earlier and more severe symptoms, heavier galls, and a significantly higher number of new resting spores in both cultivars.

Methods for Assessment of Viability and Germination of Plasmodiophora brassicae Resting Spores.

Clubroot caused by the obligate biotrophic parasite Plasmodiophora brassicae is a destructive soil borne disease of cruciferous crops. Resting spores of P. brassicae can survive in the soil for a long period without hosts or external stimulants. The viability and germination rate of resting spores are crucial factors of the inoculum potential in the field. The accurate assessment of viability and germination rate is the foundation to evaluate the effect of control methods. In this study, we evaluated several methods for the assessment of viability and germination rate of P. brassicae resting spores. Dual staining with calcofluor white-propidium iodide (CFW-PI) or single stain with Evans blue showed reliable accuracy in estimating viability. CFW-PI was capable of reliably determining the viability within 10 min, while Evans blue required overnight incubation to obtain accurate results. Due to DNA degradation of heat treatments, acetone was selected to evaluate the efficiency of propidium monoazide (PMA)-quantitative PCR (qPCR) used for the quantification of DNA from viable cells. The staining with 4,6-Diamidine-2-phenylindole dihydrochloride (DAPI) and the use of differential interference contrast microscopy were suitable for the determination of resting spore germination rates. The latter method also allowed recording individual germination states of spores. Alternatively, dual staining with CFW-Nile red was successfully used to assess the germination rate of resting spores with a lethal pre-treatment. This study evaluates and confirms the suitability of various microscopic and molecular genetic methods for the determination of viability and germination of P. brassicae resting spores. Such methods are required to study factors in the soil regulating survival, dormancy and germination of P. brassicae resting spores causing clubroot disease in Brassicaceae hosts and therefore are fundamental to develop novel strategies of control.

First Characterisation of the Phoma Species Complex on Maize Leaves in Central Europe.

In the last decade, the cultivated area of maize has increased in Central Europe due to its high yield potential and diverse uses for feed and bio-energy. This has led to more intense maize cultivation, with narrowed crop rotations resulting in the increase in maize leaf diseases. During 2012 and 2013, an inventory of maize leaf spot diseases was carried out in various regions in Central Europe. In addition to the major leaf pathogens, isolates of Phoma-like species were obtained from oval to elliptical spots on leaves or found in lesions produced by other leaf pathogens. A total of 16 representative Phoma-like strains were characterised for their pathogenicity on maize leaves, for their morphological characteristics and with a phylogenetic analysis based on multilocus sequence analysis using part of the actin (ACT), calmodulin (CAL), ß-tubulin (TUB), internal transcribed spacer (ITS) region of ribosomal DNA and large subunit ribosomal RNA (LSU) genes. The strains were grouped into four clades, and morphological studies supported this classification for most of them. Strains were compared with six reference Phoma-like species strains from the Westerndijk Fungal Biodiversity Institute collection reported to colonise maize. The pathogenic group of strains from our collection (after completion of Koch's postulates) did not cluster with any of these species, indicating a different and novel Phoma-like species infecting maize leaves. To our knowledge, this is the first study dissecting the Phoma species complex on maize leaves in Central Europe.

Dissection of Quantitative Blackleg Resistance Reveals Novel Variants of Resistance Gene Rlm9 in Elite Brassica napus.

Blackleg is one of the major fungal diseases in oilseed rape/canola worldwide. Most commercial cultivars carry R gene-mediated qualitative resistances that confer a high level of race-specific protection against Leptosphaeria maculans, the causal fungus of blackleg disease. However, monogenic resistances of this kind can potentially be rapidly overcome by mutations in the pathogen's avirulence genes. To counteract pathogen adaptation in this evolutionary arms race, there is a tremendous demand for quantitative background resistance to enhance durability and efficacy of blackleg resistance in oilseed rape. In this study, we characterized genomic regions contributing to quantitative L. maculans resistance by genome-wide association studies in a multiparental mapping population derived from six parental elite varieties exhibiting quantitative resistance, which were all crossed to one common susceptible parental elite variety. Resistance was screened using a fungal isolate with no corresponding avirulence (AvrLm) to major R genes present in the parents of the mapping population. Genome-wide association studies revealed eight significantly associated quantitative trait loci (QTL) on chromosomes A07 and A09, with small effects explaining 3-6% of the phenotypic variance. Unexpectedly, the qualitative blackleg resistance gene Rlm9 was found to be located within a resistance-associated haploblock on chromosome A07. Furthermore, long-range sequence data spanning this haploblock revealed high levels of single-nucleotide and structural variants within the Rlm9 coding sequence among the parents of the mapping population. The results suggest that novel variants of Rlm9 could play a previously unknown role in expression of quantitative disease resistance in oilseed rape.

Diversity of Expression Types of Ht Genes Conferring Resistance in Maize to Exserohilum turcicum.

Northern corn leaf blight (NCLB) is an important leaf disease in maize (Zea mays) worldwide and is spreading into new areas with expanding maize cultivation, like Germany. Exserohilum turcicum, causal agent of NCLB, infects and colonizes leaf tissue and induces elongated necrotic lesions. Disease control is based on fungicide application and resistant cultivars displaying monogenic resistance. Symptom expression and resistance mechanisms differ in plants carrying different resistance genes. Therefore, histological studies and DNA quantification were performed to compare the pathogenesis of E. turcicum races in maize lines exhibiting compatible or incompatible interactions. Maize plants from the differential line B37 with and without resistance genes Ht1, Ht2, Ht3, and Htn1 were inoculated with either incompatible or compatible races (race 0, race 1 and race 23N) of E. turcicum. Leaf segments from healthy and inoculated plants were collected at five different stages of infection and disease development from penetration (0-1 days post inoculation - dpi), until full symptom expression (14-18 dpi). Symptoms of resistance responses conveyed by the different Ht genes considerably differed between Ht1 (necrotic lesions with chlorosis), Ht2 (chlorosis and small lesions), Ht3 (chlorotic spots) and Htn1 (no lesions or wilt-type lesions). In incompatible interactions, fungal DNA was only detected in very low amounts. At 10 dpi, DNA content was elevated in all compatible interactions. Histological studies with Chlorazol Black E staining indicated that E. turcicum formed appressoria and penetrated the leaf surface directly in both types of interaction. In contrast to incompatible interactions, however, the pathogen was able to penetrate into xylem vessels at 6 dpi in compatible interactions and strongly colonized the mesophyll at 12 dpi, which is considered the crucial process differentiating susceptible from resistant interactions. Following the distinct symptom expressions, resistance mechanisms conferred by Ht1, Ht2, Ht3, and Htn1 genes apparently are different. Lower disease levels and a delayed progress of infection in compatible interactions with resistant lines imply that maize R genes to E. turcicum are associated with or confer additional quantitative resistance.

Role of Salicylic Acid and Components of the Phenylpropanoid Pathway in Basal and Cultivar-Related Resistance of Oilseed Rape (Brassica napus) to Verticillium longisporum.

Enhanced resistance is a key strategy of controlling 'Verticillium stem striping' in Brassica napus caused by the soil-borne vascular pathogen Verticillium longisporum. The present study analyses the role of a broad range of components in the phenylpropanoid and salicylic acid (SA) pathways in basal and cultivar-related resistance of B. napus towards V. longisporum. A remarkable increase of susceptibility to V. longisporum in SA-deficient transgenic NahG plants indicated an essential role of SA in basal resistance of B. napus to V. longisporum. Accordingly, elevated SA levels were also found in a resistant and not in a susceptible cultivar during early asymptomatic stages of infection (7 dpi), which was associated with increased expression of PR1 and PR2. In later symptomatic stages (14 or 21 dpi), SA responses did not differ anymore between cultivars varying in resistance. In parallel, starting at 7 dpi, an overall increase in phenylpropanoid syntheses developed in the resistant cultivar, including the activity of some key enzymes, phenylalanine ammonium lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POX) and the expression of key genes, PAL4, CCoAMT, CCR, POX. As a consequence, a remarkable increase in the levels of phenolic acids (t-cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid) occurred associated with cultivar resistance. A principal component analysis including all 27 traits studied indicated that component 1 related to SA synthesis (PR1, PR2, POX, level of free SA) and component 2 related to lignin synthesis (level of free ferulic acid, free p-coumaric acid, conjugated t-cinnamic acid) were the strongest factors to determine cultivar-related resistance. This study provides evidence that both SA and phenolic acid synthesis are important in cultivar-related resistance, however, with differential roles during asymptomatic and symptomatic stages of infection.

Starch Hydrolysis and Vessel Occlusion Related to Wilt Symptoms in Olive Stems of Susceptible Cultivars Infected by Verticillium dahliae.

This study investigated starch content, amount of pathogen DNA and density of occluded vessels in healthy and Verticillium dahliae infected olive shoots and stems. Starch hydrolysis is considered a mechanism to refill xylem vessels that suffered cavitation by either, drought conditions or pathogen infections. The main objective of this work was to evaluate this mechanism in olive plants subjected to V. dahliae infection or to drought conditions, in order to know the importance of cavitation in the development of wilting symptoms. In initial experiments starch content in the shoots was studied in trees of cultivars differing in the level of resistance growing in fields naturally infested with V. dahliae. The starch content, esteemed by microscopic observation of stem transversal sections stained with lugol, decreased with the level of symptom severity. Results were confirmed in a new experiment developed with young plants of cultivars 'Picual' (highly susceptible), 'Arbequina' (moderately susceptible) and 'Frantoio' (resistant), growing in pots under greenhouse conditions, either inoculated or not with V. dahliae. In this experiment, the pathogen DNA content, quantified by real-time PCR, and the density of occluded vessels, recorded by microscopic observations of transversal sections stained with toluidine blue, were related to the symptoms severity caused by the pathogen. Finally, a drought experiment was established with young plants of the cultivar 'Picual' grown in pots under greenhouse conditions in order to compare the effects caused by water deficit with those caused by the pathogen infection. In both cases, results show that starch hydrolysis occurred, what indirectly evidence the importance of xylem cavitation in the development of the symptoms caused by V. dahliae but in the water stressed plants no vessel occlusion was detected.

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.

High temperature effects on Pi54 conferred resistance to Magnaporthe oryzae in two genetic backgrounds of Oryza sativa.

The global temperatures are predicted to rise due to climate change. However, knowledge on the mechanisms underlying the effect of high temperature (HT) on plant pathogen interaction is limited. We investigated the effect of elevated temperature on host phenotypic, biochemical and gene expression patterns in the rice-Magnaporthe oryzae (Mo) pathosystem using two genetic backgrounds, Co39 (Oryzae sativa-indica) and LTH (O. sativa-japonica) with (CO and LT) and without (Co39 and LTH) R gene (Pi54). After exposure to 28°C and 35°C the two genetic backgrounds showed contrasting responses to Mo. At 28°C, CO, Co39 and LTH displayed a more severe disease phenotype than LT. Surprisingly, CO became resistant to Mo after exposure to 35°C. CO and LT were used for further analysis to determine the defence related biochemical and transcriptome changes associated with HT induced resistance. Pre-exposure to 35°C triggered intense callose deposits and cell wall fluorescence of the attacked epidermal cells, as well as, increased hydrogen peroxide (H2O2) and salicylic acid (SA) levels. Transcriptional changes due to combined stress (35°C+Mo) were largely overridden by pathogen infection in both backgrounds, suggesting that the plants tended to shift their response to the pathogen. However, significant differences in global gene expression patterns occurred between CO and LT in response to both single (35°C and Mo) and double stress (35°C+Mo). Collectively, our results suggest that rice lines carrying Pi54 respond to Mo by rapid induction of callose and H2O2, and that these resistance mechanisms are amplified at HT. The relative difference in disease severity between CO and LT at 28°C suggests that the genetic background of japonica rice facilitates the function of Pi54 more than the background of indica rice. The phenotypic plasticity and gene expression differences between CO and LT reveal the presence of intricate background specific molecular signatures that may potentially influence adaptation to plant stresses.