Screening and characterization of resistance to succinate dehydrogenase inhibitors in Alternaria solani

Field isolates of Alternaria solani, which causes early blight of potato in Idaho, USA were evaluated in vitro for their sensitivity towards the succinate dehydrogenase inhibitor (SDHI) fungicides boscalid, fluopyram and penthiopyrad. A total of 20 isolates were collected from foliar‐infected tissue in 2009, 26 in 2010 and 49 in 2011. Fungicide sensitivity was tested using the spiral‐gradient end point dilution method. The frequency of boscalid‐resistant isolates (>50% relative growth when using a spiral dilution gradient starting at 507 mg L^?1) drastically increased over the duration of this study (15% in 2009, 62% in 2010 and 80% in 2011). Increasing resistance to fluopyram and penthiopyrad was observed. However, cross‐resistance was only observed between boscalid and penthiopyrad. The target site of this fungicide class is the succinate dehydrogenase (SDH) enzyme complex, which is vital for fungal respiration. Sequence analysis of the SDH complex revealed mutations in the subunits B and D that were correlated with the emergence of boscalid resistance in potato fields in Idaho. In particular, H277R and H133R were identified in SDH subunits B and D, respectively. The presence of restriction sites in the gene sequences allowed the development of a rapid PCR‐RFLP method to assess boscalid sensitivity in A. solani populations.     

High‐resolution melting analysis for rapid detection and characterization of Botrytis cinerea phenotypes resistant to fenhexamid and boscalid

A novel, high‐resolution melting (HRM) analysis was developed to detect single nucleotide polymorphisms (SNPs) associated with resistance to fenhexamid (hydroxyanilides) and boscalid (succinate dehydrogenase inhibitors) in Botrytis cinerea isolates. Thirty‐six single‐spore isolates arising from 13 phenotypes were selected and tested for fungicide sensitivity. Germ tube elongation assays showed two distinct sensitivity levels for each fungicide. Sequencing revealed that resistance to fenhexamid was due to a nucleotide change in the erg27 gene, resulting in an amino acid replacement of phenylalanine (F) with serine (S) or valine (V) at position 412 of the protein, whereas in isolates resistant to boscalid, a nucleotide change in the sdhB gene resulted in the replacement of histidine (H) with arginine (R) or tyrosine (Y) at position 272 of the respective protein. In each case, melting curve analysis generated three distinct profiles corresponding to the presence of each nucleotide in the targeted areas. HRM analysis successfully detected and differentiated the substitutions associated with resistance to both fungicides. In vitro bioassays, direct sequencing and high‐resolution melting analysis showed a 100% correlation with detection of resistance. The results demonstrate the utility of HRM analysis as a potential molecular tool for routine detection of fungicide resistance using known polymorphic genes of B. cinerea populations.     

Activity of carbendazim,dimethachlon, iprodione,procymidone and boscalid against Sclerotinia stem rot in Jiangsu Province of China

Carbendazim (MBC) was widely used to control Sclerotinia stem rot routinely during the 1980s in China, but development of MBC resistance in the causal agent Sclerotinia sclerotiorum led to control failures of this disease. In this study it was found that the MBC resistance in S. sclerotiorum populations was widespread throughout Jiangsu Province with a resistance frequency of 29.54% in the 1786 collected isolates during the growing seasons of 2006 to 2008. The resistance frequencies differed among sampled cities, ranging from 3.1% to 54.9%. The field MBC-resistant isolates showed comparable mycelial growth, sclerotia production and pathogenicity to the wild-type sensitive isolates, which suggested that the field MBC-resistant isolates might have sufficient parasitic fitness to compete with the field MBC-sensitive isolates in the field. In the in vitro sensitivity test, boscalid showed greater activity against S. sclerotiorum than dicarboximide fungicides (dimethachlon, iprodione and procymidone). The treatment 50% boscalid (WG) 125 g a.i. ha⁻¹ was comparable in efficacy to the treatment 50% iprodione (WP) 600 g a.i. ha⁻¹, and better than other treatments of 6% dimethachlon (WP) 690 g a.i. ha⁻¹ and 50% procymidone (WP) 337.5 g a.i. ha⁻¹, whereas MBC failed to control Sclerotinia stem rot (control efficacy only 16.0%). The most active agent for controlling Sclerotinia stem rot was boscalid in our study.     

Overexpression of barley oxalate oxidase gene induces partial leaf resistance to Sclerotinia sclerotiorum in transgenic oilseed rape

Sclerotinia stem rot (SSR) is a severe disease of oilseed rape, which severely impacts the crop productivity worldwide. Sclerotinia sclerotiorum causes SSR, resulting in the secretion of oxalic acid (OA), which can be further degraded to carbon dioxide (CO₂) and hydrogen peroxide (H₂O₂) by oxalate oxidase (OXO). In the present investigation, the barley oxalate oxidase (BOXO, Y14203) gene was introduced into oilseed rape by Agrobacterium‐mediated transformation to investigate the mechanism by which OXO promotes resistance to S. sclerotiorum. Compared to the control 72 h post‐inoculation, there were c. 15–61% fewer lesions on leaves of the transgenic oilseed rape, which thus exhibited a detectable level of partial resistance in leaf tissue to S. sclerotiorum. Transgenic oilseed rape also showed decreased oxalate and increased hydrogen peroxide levels compared to the control, and the expression of defence response genes involved in the hydrogen peroxide signalling pathway was also induced. Therefore, the improved resistance of oilseed rape could be attributed to the enhanced OA metabolism, production of hydrogen peroxide and the hydrogen peroxide‐mediated defence levels during infection.     

Resistance to a highly aggressive isolate of Sclerotinia sclerotiorum in a Brassica napus diversity set

Sclerotinia stem rot (SSR) of oilseed rape (OSR, Brassica napus), caused by Sclerotinia sclerotiorum, is a serious problem in the UK and worldwide. As fungicide‐based control approaches are not always reliable, identifying host resistance is a desirable and sustainable approach to disease management. This research initially examined the aggressiveness of 18 Sclerotinia isolates (17 S. sclerotiorum, one S. subarctica) on cultivated representatives of B. rapa, B. oleracea and B. napus using a young plant test. Significant differences were observed between isolates and susceptibility of the brassica crop types, with B. rapa being the most susceptible. Sclerotinia sclerotiorum isolates from crop hosts were more aggressive than those from wild buttercup (Ranunculus acris). Sclerotinia sclerotiorum isolates P7 (pea) and DG4 (buttercup), identified as ‘aggressive’ and ‘weakly aggressive’, respectively, were used to screen 96 B. napus lines for SSR resistance in a young plant test. A subset of 20 lines was further evaluated using the same test and also in a stem inoculation test on flowering plants. A high level of SSR resistance was observed for five lines and, although there was some variability between tests, one winter OSR (line 3, Czech Republic) and one rape kale (line 83, UK) demonstrated consistent resistance. Additionally, one swede (line 69, Norway) showed an outstanding level of resistance in the stem test. Resistant lines also had fewer sclerotia forming in stems. New pre‐breeding material for the production of SSR resistant OSR cultivars relevant to conditions in the UK and Europe has therefore been identified.     

Selection,characterization and genetic analysis of laboratory mutants of <Emphasis Type="Italic">Botryotinia fuckeliana</Emphasis> (<Emphasis Type="Italic">Botrytis cinerea</Emphasis>) resistant to the fungicide boscalid

Resistance to the fungicide boscalid in laboratory mutants of Botryotinia fuckeliana (Botrytis cinerea) was investigated. The baseline sensitivity to boscalid was evaluated in terms of colony growth (EC₅₀ = 0.3–3 μg ml⁻¹; MIC = 10–30 μg ml⁻¹) and conidial germination (EC₅₀ = 0.03–0.1 μg ml⁻¹; MIC = 1–3 μg ml⁻¹) tests. Mutants were selected in vitro from wild-type strains of the fungus on a fungicide-amended medium containing acetate as a carbon source. Mutants showed two different levels of resistance to boscalid, distinguishable through the conidial germination tests: low (EC₅₀ ∼ 0.3 μg ml⁻¹, ranging from 0.03 to 1 μg ml⁻¹; MIC > 100 μg ml⁻¹) and high (EC₅₀ > 100 μg ml⁻¹) resistance. Analysis of meiotic progeny from crosses between resistant mutants and sensitive reference strains showed that resistant phenotypes were due to mutations in single major gene(s) inherited in a Mendelian fashion, and linked with both the Daf1 and Mbc1 genes, responsible for resistance to dicarboximide and benzimidazole fungicides, respectively. Gene sequence analysis of the four sub-units of the boscalid-target protein, the succinate dehydrogenase enzyme, revealed that single or double point mutations in the highly conserved regions of the iron-sulphur protein (Ip) gene were associated with resistance. Mutations resulted in proline to leucine or phenylalanine replacements at position 225 (P225L or P225F) in high resistant mutants, and in a histidine to tyrosine replacement at position 272 (H272Y) in low resistant mutants. Sequences of the flavoprotein and the two transmembrane sub-units of succinate dehydrogenase were never affected.     

Characterization of mating type (MAT) alleles differentiated by a natural inversion in Sclerotinia minor

A recent study on fungal mating type genes revealed two MAT alleles within homothallic Sclerotinia sclerotiorum differentiated by an inversion, Inv? (inversion negative) and Inv+ (inversion positive). An analysis of mating type in closely related S. minor was conducted to shed light on the evolution of this MAT inversion. Inv? and Inv+ MAT alleles were identified in S. minor and were characterized. Both MAT alleles in S. minor were flanked by APN2 and SLA2, and consisted of two idiomorphs fused as in other homothallic ascomycetes. However, in the Inv+ MAT, the 3·6 kb MAT region was inverted relative to the Inv? MAT. Except for the inversion, both Inv? and Inv+ MAT in S. minor were equal in size and identical in nucleotide sequence. The MAT inversion in Inv+ S. minor was at exactly the same place as in Inv+ S. sclerotiorum and affected three of four MAT genes: MAT1‐1‐1 was truncated and MAT1‐2‐4 and MAT1‐2‐1 were inverted. Unlike S. sclerotiorum, expression of MAT genes did not differ between Inv? and Inv+ S. minor. The 250 bp inverted repeat motif that flanked the inverted MAT region in S. sclerotiorum and believed responsible for the MAT inversion was also found in S. minor, but was 256 bp. Depending on the MAT genes, 93–96% nucleotide identity was observed between Sclerotinia species. Both Inv+ and Inv? MAT S. minor and S. sclerotiorum isolates were commonly found in lettuce fields of Arizona along with MAT heterokaryons.     

Molecular characterization of boscalid- and penthiopyrad-resistant isolates of Didymella bryoniae and assessment of their sensitivity to fluopyram

BACKGROUND: Didymella bryoniae has a history of developing resistance to single‐site fungicides. A recent example is with the succinate‐dehydrogenase‐inhibiting fungicide (SDHI) boscalid. In laboratory assays, out of 103 isolates of this fungus, 82 and seven were found to be very highly resistant (B^VHR) and highly resistant (B^HR) to boscalid respectively. Cross‐resistance studies with the new SDHI penthiopyrad showed that the B^VHR isolates were only highly resistant to penthiopyrad (B^VHR‐P^HR), while the B^HR isolates appeared sensitive to penthiopyrad (B^HR‐P^S). In this study, the molecular mechanism of resistance in these two phenotypes (B^VHR‐P^HR and B^HR‐P^S) was elucidated, and their sensitivity to the new SDHI fluopyram was assessed. RESULTS: A 456 bp cDNA amplified fragment of the succinate dehydrogenase iron sulfur gene (DbSDHB) was initially cloned and sequenced from two sensitive (B^S‐P^S), two B^VHR‐P^HR and one B^HR‐P^S isolate of D. bryoniae. Comparative analysis of the DbSDHB protein revealed that a highly conserved histidine residue involved in the binding of SDHIs and present in wild‐type isolates was replaced by tyrosine (H277Y) or arginine (H277R) in the B^VHR‐P^HR and B^HR‐P^S variants respectively. Further examination of the role and extent of these alterations showed that the H/Y and H/R substitutions were present in the remaining B^VHR‐P^HR and B^HR‐P^S variants respectively. Analysis of the sensitivity to fluopyram of representative isolates showed that both SDHB mutants were sensitive to this fungicide as the wild‐type isolates. CONCLUSION: The genotype‐specific cross‐resistance relationships between the SDHIs boscalid and penthiopyrad and the lack of cross‐resistance between these fungicides and fluopyram should be taken into account when selecting SDHIs for gummy stem blight management. Copyright © 2011 Society of Chemical Industry     

Population structure of Sclerotinia sclerotiorum in crop and wild hosts in the UK

Sclerotinia sclerotiorum is an important pathogen of many crop plants which also infects wild hosts. The population structure of this fungus was studied for different crop plants and Ranunculus acris (meadow buttercup) in the UK using eight microsatellite markers and sequenced sections of the intergenic spacer (IGS) region of the rRNA gene and the elongation factor 1‐alpha (EF) gene. A total of 228 microsatellite haplotypes were identified within 384 isolates from 12 S. sclerotiorum populations sampled in England and Wales. One microsatellite haplotype was generally found at high frequency in each population and was distributed widely across different hosts, locations and years. Fourteen IGS and five EF haplotypes were found in the 12 populations, with six IGS haplotypes and one EF haplotype exclusive to buttercup. Analysis of published sequences for S. sclerotiorum populations from the USA, Canada, New Zealand and Norway showed that three of the IGS haplotypes and one EF haplotype were widely distributed, while eight IGS haplotypes were only found in the UK. Although common microsatellite and IGS/EF haplotypes were found on different hosts in the UK, there was evidence of differentiation, particularly for one isolated population on buttercup. However, overall there was no consistent differentiation of S. sclerotiorum populations from buttercup and crop hosts. Sclerotinia sclerotiorum therefore has a multiclonal population structure in the UK and the wide distribution of one microsatellite haplotype suggests spatial mixing at a national scale. The related species S. subarctica was also identified in one buttercup population.     

Mutations in sdh genes in field isolates of Zymoseptoria tritici and impact on the sensitivity to various succinate dehydrogenase inhibitors

Zymoseptoria tritici is the causal agent of septoria tritici blotch (STB), a foliar wheat disease important worldwide. Succinate dehydrogenase inhibitors (SDHIs) have been used in cereals for effective control of STB for several years, but resistance towards SDHIs has been reported in several phytopathogenic fungi. Resistance mechanisms are target‐site mutations in the genes coding for subunits B, C and D of the succinate dehydrogenase (SDH) enzyme. Previous monitoring data in Europe indicated the presence of single isolates of Z. tritici with reduced SDHI sensitivity. These isolates carried mutations leading to amino acid exchanges: C‐T79N, C‐W80S in 2012; C‐N86S in 2013; B‐N225T and C‐T79N in 2014; and C‐V166M, B‐T268I, C‐N86S, C‐T79N and C‐H152R in 2015. The current study provides results from microtitre and greenhouse experiments to give an insight into the impact of different mutations in field isolates on various SDHIs. In microtitre tests, the highest EC₅₀ values for all tested SDHIs were obtained with mutants carrying C‐H152R. Curative greenhouse tests with various SDHIs confirmed the findings of microtitre tests that isolates with C‐H152R are, in general, controlled with lower efficacy than isolates carrying B‐T268I, C‐T79N and C‐N86S. SDHI‐resistant isolates of Z. tritici found in the field were shown to have cross‐resistance towards all SDHIs tested. So far, SDHI‐resistant isolates of Z. tritici have been found in low frequencies in Europe. Therefore, FRAC recommendations for resistance management in cereals, including a limited number of applications, alternation and combination with other MOAs, should be followed to prolong SDHI field efficacy.     

Fungicide resistance in Cercospora species causing cercospora leaf blight and purple seed stain of soybean in Argentina

Cercospora species cause cercospora leaf blight (CLB) and purple seed stain (PSS) on soybean. Because there are few resistant soybean varieties available, CLB/PSS management relies heavily upon fungicide applications. Sensitivity of 62 Argentinian Cercospora isolates to demethylation inhibitor (DMI), methyl benzimidazole carbamate (MBC), quinone outside inhibitor (QoI), succinate dehydrogenase inhibitor (SDHI) fungicides, and mancozeb was determined in this study. All isolates were sensitive to difenoconazole, epoxiconazole, prothioconazole, tebuconazole, and cyproconazole (EC₅₀ values ranged from 0.006 to 2.4 µg/ml). In contrast, 51% of the tested isolates were sensitive (EC₅₀ values ranged from 0.003 to 0.2 µg/ml), and 49% were highly resistant (EC₅₀ > 100 µg/ml) to carbendazim. Interestingly, all isolates were completely resistant to azoxystrobin, trifloxystrobin, and pyraclostrobin, and insensitive to boscalid, fluxapyroxad, and pydiflumetofen (EC₅₀ > 100 µg/ml). The G143A mutation was detected in 82% (53) of the QoI-resistant isolates and the E198A mutation in 97% (31) of the carbendazim-resistant isolates. No apparent resistance mutations were detected in the succinate dehydrogenase genes (subunits sdhB, sdhC, and sdhD). Mancozeb completely inhibited mycelial growth of the isolates evaluated at a concentration of 100 µg/ml. All Argentinian Cercospora isolates were sensitive to the DMI fungicides tested, but we report for the first time resistance to QoI and MBC fungicides. Mechanism(s) other than fungicide target-site modification may be responsible for resistance of Cercospora to QoI and MBC fungicides. Moreover, based on our results and on the recent introduction of SDHI fungicides on soybean in Argentina, Cercospora species causing CLB/PSS are insensitive (naturally resistant) to SDHI fungicides. Insensitivity must be confirmed under field conditions.     

Histopathology of Sclerotinia sclerotiorum infection and oxalic acid function in susceptible and resistant soybean

The success of the necrotrophic fungus Sclerotinia sclerotiorum is largely dependent on its major virulence factor, oxalic acid (OA). Virulence is lost in transgenic plants that express OA degrading enzymes, e.g. oxalate oxidase (OxO). The histopathology of S. sclerotiorum infection and OA accumulation was examined in a transgenic soybean line over‐expressing OxO (OxO‐OE) and its isogenic parent (WT). In situ flower inoculation showed that the OxO‐OE plants were highly resistant to the pathogen while the WT parents were susceptible. This difference in resistance was not apparent in the floral tissues, as aggressive hyphal activity was similar on both hosts, showing that high OxO activity and low OA accumulation in OxO‐OE was not a deterrent. However, the process of fungal infection on excised leaf tissue differed on the two hosts. Primary lesions developed and showed similar severe ultrastructural damage on both hosts but rapid lesion expansion (colonization) proceeded only on the WT, concomitant with OA accumulation. Oxalic acid rose in OxO‐OE 1 day post‐inoculation and did not change over the following 3 days, showing that colonization can be blocked by maintaining low levels of OA. However, OxO degradation of OA did not deter initial host penetration and primary lesion formation. This shows that OA, the major virulence factor of S. sclerotiorum, is critical for host colonization but may not be required during primary lesion formation, suggesting that other factors are contributing to the establishment of the primary lesion.     

Resistance to Sclerotinia sclerotiorum in wild Brassica species and the importance of Sclerotinia subarctica as a Brassica pathogen

Brassica crops are of global importance, with oilseed rape (Brassica napus) accounting for 13% of edible oil production. All Brassica species are susceptible to sclerotinia stem rot caused by Sclerotinia sclerotiorum, a generalist fungal pathogen causing disease in over 400 plant species. Generally, sources of plant resistance result in partial control of the pathogen although some studies have identified wild Brassica species that are highly resistant. The related pathogen S. subarctica has also been reported on Brassica but its aggressiveness in relation to S. sclerotiorum is unknown. In this study, detached leaf and petiole assays were used to identify new sources of resistance to S. sclerotiorum within a wild Brassica ‘C genome’ diversity set. High‐level resistance was observed in B. incana and B. cretica in petiole assays, whilst wild B. oleracea and B. incana lines were the most resistant in leaf assays. A B. bourgeai line showed both partial petiole and leaf resistance. Although there was no correlation between the two assays, resistance in the detached petiole assay was correlated with stem resistance in mature plants. When tested on commercial cultivars of B. napus, B. oleracea and B. rapa, selected isolates of S. subarctica exhibited aggressiveness comparable to S. sclerotiorum indicating it can be a significant pathogen of Brassica. This is the first study to identify B. cretica as a source of resistance to S. sclerotiorum and to report resistance in other wild Brassica species to a UK isolate, hence providing resources for breeding of resistant cultivars suitable for Europe.     

Distribution and molecular characterization of Corynespora cassiicola isolates resistant to boscalid

A total of 618 isolates of corynespora leaf spot fungus (Corynespora cassiicola) collected from 24 commercial cucumber greenhouses in 12 cities in Ibaraki Prefecture, Japan, were tested for their sensitivity to boscalid. Boscalid‐resistant isolates were detected in 17 out of 19 greenhouses with a history of use of this fungicide and detection frequencies of the resistant isolates exceeded 47% in nine greenhouses. Frequencies of very highly resistant (VHR) isolates with 50% effective concentration (EC₅₀) values of boscalid exceeding 30 μg mL^?1 were higher than those of moderately resistant (MR) isolates with EC₅₀ ranging from 2·0 to 5·9 μg mL^?1 in 11 greenhouses. Additionally, highly resistant (HR) isolates with EC₅₀ from 8·9 to 10·7 μg mL^?1 were first detected. Furthermore, molecular characterization of genes encoding succinate dehydrogenase (SDH) subunits (SdhA, SdhB, SdhC and SdhD) was carried out to elucidate the amino acid substitution responsible for the resistance to boscalid. All 23 VHR isolates had the same mutation from CAC to TAC in the SdhB gene leading to the substitution of histidine with tyrosine at amino acid position 278 (B‐H278Y). At the same position, the substitution to arginine conferred by a mutation to CGC (B‐H278R) was detected in all four HR isolates. Some MR isolates showed a substitution from serine to proline at position 73 in SdhC (C‐S73P), from serine to proline or from glycine to valine at position 89 (D‐S89P) and 109 (D‐G109V), respectively, in SdhD. There was no common mutation in SDH genes of all MR isolates.     

Inoculum potential of Sclerotinia sclerotiorum sclerotia depends on isolate and host plant

The soilborne fungus Sclerotinia sclerotiorum infects many important crop plants. Central to the success of this pathogen is the production of sclerotia, which enables survival in soil and constitutes the primary inoculum. This study aimed to determine how crop plant type and S. sclerotiorum isolate impact sclerotial production and germination and hence inoculum potential. Three S. sclerotiorum isolates (L6, L17, L44) were used to inoculate plants of bean, carrot, lettuce, oilseed rape (OSR) and potato, and the number and weight of sclerotia per plant quantified. Carpogenic germination of sclerotia collected from different hosts was also assessed for L6. Production of sclerotia was dependent on both crop plant type and S. sclerotiorum isolate, with OSR and lettuce supporting the greatest number (42–122) and weight (1.6–3.0 g) of sclerotia per plant. The largest sclerotia were produced on OSR (33–66 mg). The three S. sclerotiorum isolates exhibited a consistent pattern of sclerotial production irrespective of crop type; L6 produced large numbers of small sclerotia while L44 produced smaller numbers of large sclerotia, with L17 intermediate between the two. Germination rate and percentage was greatest for larger sclerotia (4.0–6.7 mm) and also varied between host plants. Combining sclerotial production data and typical field crop densities suggested that infected carrot and OSR could produce the greatest number (3944 m^?2) and weight (73 g m^?2) of S. sclerotiorum sclerotia, respectively, suggesting these crops potentially contribute a greater increase in inoculum. This information, once further validated in field trials, could be used to inform future crop rotation decisions.     

Biological control of Pythium,Rhizoctonia and Sclerotinia in lettuce: association of the plant protective activity of the bacterium Paenibacillus alvei K165 with the induction of systemic resistance

The soilborne fungi Sclerotinia sclerotiorum, Rhizoctonia solani and the oomycete Pythium ultimum are among the most destructive pathogens for lettuce production. The application of the biocontrol agent Paenibacillus alvei K165 to the transplant soil plug of lettuce resulted in reduced S. sclerotiorum, R. solani and P. ultimum foliar symptoms and incidence compared to untreated controls, despite the suppressive effect of the pathogens on the rhizosphere population of K165. In vitro, K165 inhibited the growth of S. sclerotiorum and R. solani but not P. ultimum. Furthermore, the expression of the pathogenesis‐related (PR) gene PR1, a marker gene of salicylic acid (SA)‐dependent plant defence, and of the Lipoxygenase (LOX) and Ethylene response factor 1 (ERF1) genes, markers of ethylene/jasmonate (ET/JA)‐dependent plant defence was recorded. K165‐treated plants challenged with P. ultimum showed up‐regulation of PR1, whereas challenge with R. solani resulted in up‐regulation of LOX and ERF1, and challenge with S. sclerotiorum resulted in up‐regulation of PR1, LOX and ERF1. This suggests that K165 triggers the SA‐ and the ET/JA‐mediated induced systemic resistance against P. ultimum and R. solani, respectively, while the simultaneous activation of the SA and ET/JA signalling pathways is proposed for S. sclerotiorum.     

Temperature adaptation in isolates of Sclerotinia sclerotiorum affects their ability to infect Brassica carinata

Sclerotinia stem rot (Sclerotinia sclerotiorum) is a serious disease in oilseed Brassica crops worldwide. In this study, temperature adaptation in isolates of S. sclerotiorum collected from differing climatic zones is reported for the first time on any crop. Sclerotinia sclerotiorum isolates from oilseed rape (Brassica napus) crops in warmer northern agricultural regions of Western Australia (WW3, UWA 7S3) differed in their reaction to temperature from those from cooler southern regions (MBRS‐1, UWA 10S2) in virulence on Brassica carinata, growth on agar, and oxalic acid production. Increasing temperature from 22/18°C (day/night) to 28/24°C increased lesion diameter on cotyledons of B. carinataBC054113 more than tenfold for warmer region isolates, but did not affect lesion size for cooler region isolates. Mean lesion length averaged across two B. carinata genotypes (resistant and susceptible) fell from 4·6 to 2·4 mm for MBRS‐1 when temperature increased from 25/21°C to 28/24°C but rose for WW3 (2·35 and 3·21 mm, respectively). WW3, usually designated as low in virulence, caused as much disease on stems at 28/24°C as MBRS‐1, historically designated as highly virulent. Isolates collected from cooler areas grew better at low temperatures on agar. While all grew on potato dextrose agar between 5 and 30°C, with maximum growth at 20–25°C, growth was severely restricted above 32°C, and only UWA 7S3 grew at 35°C. Oxalate production increased as temperature increased from 10 to 25°C for isolates MBRS‐1, WW3 and UWA 7S3, but declined from a maximum level of 101 mg g^?1 mycelium at 20°C to 24 mg g^?1 mycelium at 25°C for UWA 10S2.     

Monitoring of plant and airborne inoculum of Sclerotinia sclerotiorum in spring oilseed rape using real‐time PCR

Sclerotinia stem rot of spring oilseed rape (Brassica napus) is caused by Sclerotinia sclerotiorum. In Sweden, the disease leads to severe crop damage that varies from year to year. A real‐time PCR assay was developed and used to determine the incidence of S. sclerotiorum DNA on petals and leaves of spring oilseed rape as well as in air samples, with the aim of finding tools to improve precision in disease risk assessment. Five field experiments were conducted from 2008 to 2010 to detect and study pathogen development. Assessments of stem rot showed significant differences between experimental sites. The real‐time PCR assay proved fast and sensitive and the relationship between percentage of infected petals determined using a conventional agar test and the PCR assay was linear (R² > 0·76). There were significant differences in S. sclerotiorum incidence at different stages of flowering. The incidence of S. sclerotiorum DNA on the leaves varied (0–100%), with significantly higher incidence on leaves at lower levels. In one field experiment, S. sclerotiorum DNA was not detected on petals during flowering, whereas the pathogen was detected on leaves, with a corresponding stem rot incidence of 7%. The amount of S. sclerotiorum DNA in sampled air revealed that spore release did not coincide with flowering on that experimental site. Thus, using a real‐time PCR assay to determine the incidence of S. sclerotiorum on oilseed rape leaves, rather than on petals, could potentially improve disease risk assessment.     

Host‐induced gene silencing in the necrotrophic fungal pathogen Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic fungus that causes a devastating disease called white mould, infecting more than 450 plant species worldwide. Control of this disease with fungicides is limited, so host plant resistance is the preferred alternative for disease management. However, due to the nature of the disease, breeding programmes have had limited success. A potential alternative to developing necrotrophic fungal resistance is the use of host‐induced gene silencing (HIGS) methods, which involves host expression of dsRNA‐generating constructs directed against genes in the pathogen. In this study, the target gene chosen was chitin synthase (chs), which commands the synthesis of chitin, the polysaccharide that is a crucial structural component of the cell walls of many fungi. Tobacco plants were transformed with an interfering intron‐containing hairpin RNA construct for silencing the fungal chs gene. Seventy‐two hours after inoculation, five transgenic lines showed a reduction in disease severity ranging from 55·5 to 86·7% compared with the non‐transgenic lines. The lesion area did not show extensive progress over this time (up to 120 h). Disease resistance and silencing of the fungal chs gene was positively correlated with the presence of detectable siRNA in the transgenic lines. It was demonstrated that expression of endogenous genes from the very aggressive necrotrophic fungus S. sclerotiorum could be prevented by host induced silencing. HIGS of the fungal chitin synthase gene can generate white mould‐tolerant plants. From a biotechnological perspective, these results open new prospects for the development of transgenic plants resistant to necrotrophic fungal pathogens.