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.     

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.     

Molecular and biochemical characterization of boscalid resistance in laboratory mutants of Sclerotinia sclerotiorum

The plant‐pathogenic fungus Sclerotinia sclerotiorum has a broad host range and a worldwide distribution. Boscalid, an inhibitor of succinate dehydrogenase in the electron transport chain of fungi, is highly effective in controlling sclerotinia stem rot caused by S. sclerotiorum. The current study characterized the S. sclerotiorum boscalid‐resistant (BR) mutants obtained by fungicide induction. Among the bioactive fungicides against S. sclerotiorum, cross‐resistance was not detected between boscalid and dimethachlon, fluazinam or carbendazim; positive cross‐resistance was detected between boscalid and carboxin; and negative cross‐resistance was detected between boscalid and kresoxim‐methyl. Compared to their parental isolates, BR mutants had slower radial growth, no ability to produce sclerotia, lower virulence and oxalic acid content but higher mycelial respiration and succinate dehydrogenase (SDH) activity. Moreover, BR mutants had decreased sensitivity to salicylhydroxamic acid (SHAM) but not to oxidative stress. All the results indicated that the risk of resistance to boscalid in S. sclerotiorum is low to moderate. DNA sequence analysis showed that all of the BR mutants had the same point mutation A11V (GCA to GTA) in the iron sulphur protein subunit (SDHB). Interestingly, expression of the cytochrome b (cytb) gene was reduced to different degrees in the BR mutants, and this might be correlated with the negative cross‐resistance between boscalid and kresoxim‐methyl. Such information is vital in the design of resistance management strategies.     

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.     

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.     

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.     

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.     

The control of sclerotinia stem rot on oilseed rape (Brassica napus): current practices and future opportunities

Sclerotinia stem rot (SSR) caused by the phytopathogenic fungus Sclerotinia sclerotiorum is a major disease of oilseed rape (Brassica napus). During infection, large, white/grey lesions form on the stems of the host plant, perturbing seed development and decreasing yield. Due to its ability to produce long‐term storage structures called sclerotia, S. sclerotiorum inoculum can persist for long periods in the soil. Current SSR control relies heavily on cultural practices and fungicide treatments. Cultural control practices aim to reduce the number of sclerotia in the soil or create conditions that are unfavourable for disease development. These methods of control are under increased pressure in some regions, as rotations tighten and inoculum levels increase. Despite their ability to efficiently kill S. sclerotiorum, preventative fungicides remain an expensive gamble for SSR control, as their effectiveness is highly dependent on the ability to predict the establishment of microscopic infections in the crop. Failure to correctly time fungicide applications can result in a substantial cost to the grower. This review describes the scientific literature pertaining to current SSR control practices. Furthermore, it details recent advances in alternative SSR control methods including the generation of resistant varieties through genetic modification and traditional breeding, and biocontrol. The review concludes with a future directive for SSR control on oilseed rape.     

Meta‐analytic modelling of the incidence–yield and incidence–sclerotial production relationships in soybean white mould epidemics

White mould (Sclerotinia sclerotiorum) is a destructive disease of soybean worldwide. However, little is known of its impact on soybean production in Brazil. A meta‐analytic approach was used to assess the relationship between disease incidence and soybean yield (35 trials) and between incidence and sclerotia production (29 trials) in experiments conducted in 14 locations across four seasons. Region, site elevation and season included as moderators in random‐effects and random‐coefficients models did not significantly explain the variability in the slopes of the incidence–yield relationship. The Pearson's r, obtained from back‐transforming the Fisher's Z estimated by an overall random‐effects model, showed that incidence of white mould was moderately and negatively correlated with yield (r = ?0.76, P < 0.0001). A random‐coefficients model estimated a slope of ?17.2 kg ha^?1%^?1, for a mean attainable yield of 3455 kg ha^?1, indicating that a 10% increase in white mould incidence would result in a mean yield reduction of 172 kg ha^?1. White mould incidence and production of sclerotia were strongly and positively correlated (r = 0.85, P < 0.0001). For every 10% increase in white mould incidence, 1 kg ha^?1 of sclerotia was produced. The relationship between disease incidence and production of sclerotia was stronger in southern regions and at higher elevation. In the absence of management, economic losses associated with white mould epidemics, assuming 43% incidence in 22% of the soybean area, were estimated at approximately US $1.47 billion annually within Brazil.     

Population structure,genetic diversity,and sexual state of the rice brown spot pathogen Bipolaris oryzae from three Asian countries

Bipolaris oryzae causes brown spot in rice (Oryza sativa) inflicting substantial grain yield losses worldwide. Knowledge of the population structure, genetic diversity and sexual recombination of the fungal pathogen can help to implement effective disease management strategies. In this study, B. oryzae isolates sampled from Iran, the Philippines and Japan were analysed with 12 simple‐sequence repeat (SSR) markers, newly developed from the genome sequence of the fungus. Among the 288 B. oryzae isolates genotyped, 278 unique haplotypes were identified. High genotype numbers (richness) with even distribution (evenness) were found within the collection sites. Both mating types, MAT1‐1 and MAT1‐2, were present in each collection area, and the sexual state was induced under controlled conditions with production of viable ascospores. However, the tests of linkage disequilibrium rejected of the hypothesis of random mating. Discriminant analysis of principal components (DAPC) revealed that the B. oryzae collection formed three clusters, each consisting of isolates from different collection sites. Analysis of molecular variance (amova ) showed that genetic variation among clusters was 18.7%, with the rest of the variation distributed within clusters (R_ST = 0.187, P < 0.001). Statistically significant pairwise genetic differentiation was found between the clusters. These results show that Asian B. oryzae isolates are genetically diverse, and, overall, distributed in three groups. These findings will be helpful in managing the disease and guide the use of representative isolates needed for selection of resistant rice varieties.     

Genotypic variation and population structure of Sclerotinia trifoliorum infecting chickpea in California

Sclerotinia trifoliorum, an important pathogen of cool season legumes, displays both homothallism and heterothallism in its life cycle, unique among members of the genus Sclerotinia. Very little is known about its genetic diversity and population structure. A sample of 129 isolates of S. trifoliorum from diseased chickpea in California was investigated for genetic diversity, population differentiation and reproductive mode. Genetic diversity was estimated using mycelial compatibility (MCG) phenotypes, rDNA intron variation, and allelic diversity at seven microsatellite loci. Genetic analysis revealed high levels of genotypic diversity demonstrated by high genotypic richness (0·88). Similarly, high levels of gene diversity (mean expected heterozygosity H_E = 0·68) were observed at the microsatellite loci. Geographic populations of S. trifoliorum were highly admixed as evident from low F_ST values (0–0·11), suggesting high contemporary or historical gene flow. Hierarchical analysis of molecular variance showed that more than 92% of the genetic variation occurred among isolates within populations. Bayesian clustering analysis identified four cryptic genetic populations that were not correlated to geographic location, and index of multilocus association was non‐significant in each of the four genetic populations. However, the presence of identical haplotypes within and among populations indicates clonal reproduction. The high levels of haplotype diversity and population heterogeneity, a lack of correspondence between MCG and microsatellite haplotype, and low levels of population differentiation suggest that populations of S. trifoliorum in chickpea have been undergoing extensive outcrossing and migration events probably shaped by human‐mediated dissemination, the underlying diverse cropping systems, and chickpea disease management practices.     

Hierarchical models for white mould in snap bean

Limitations on the acceptable proportion (incidence) of pods with white mould may lead to the rejection of entire fields of processing snap bean. The low tolerances (no more than 2% to 6% of pods with white mould) are difficult to estimate with sufficient precision in the field when time is limited. These constraints motivated this study of white mould across three spatial hierarchical levels: pods, plants and quadrats consisting of two adjacent plants within rows. Hierarchical relationships are required when designing formal rules for estimating the incidence of pods with white mould from units higher in the spatial hierarchy. Disease assessments were made on all pods from 38 within‐row transects of 40 plants each of the snap bean cultivar Hystyle. Using probability‐based principles, equations were derived and fitted to data on the incidence of white mould on pods (i_pod), plants (i_pad) and quadrats (i_q(2)), which led to a function directly linking i_pod to i_q(2). The variance of i_pod increased with i_q(2), but that may be of little consequence at the lower values of i_q(2) likely to be associated with i_pod values at processor‐set tolerances. For example, at i_q(2) = 0.1 there was a 92% probability that i_pod was less than 0.02. Assessing i_q(2) may be more efficient than directly estimating i_pod because i_q(2) was about an order of magnitude higher than i_pod. Results suggest it may be feasible to design sampling plans for estimating the proportion of pods with white mould from an assessment of the proportion of diseased quadrats.     

核盘菌致病性分化研究

为了解核盘菌的致病性分化,本研究用活体定位穿刺接种法,以油菜为供试寄主,对采自四川省10个地区23个县、9种寄主的108个菌株进行了致病性测定,结果表明,所有菌株对供试油菜品种均能致病,但各菌株所致病斑长度差异很大(2.7～82.0 mm),说明核盘菌种群内存在明显的致病性分化,这种分化与地理来源和寄主来源没有明显的关系。     

Population structure and migration of the witches’ broom pathogen Moniliophthora perniciosa from cacao and cultivated and wild solanaceous hosts in southeastern Brazil

Moniliophthora perniciosa, causal agent of witches’ broom disease in cacao plantations in South America and the Caribbean Islands, has co‐evolved with its host cacao, but the pathogen has also emerged in many solanaceous hosts in Brazil, including economically important food crops and wild species. This study was carried out to: (i) determine the existence of host subpopulations of M. perniciosa in Brazil; (ii) estimate gene and genotypic diversity of M. perniciosa host subpopulations infecting solanaceous hosts in southeastern Bahia and Minas Gerais states, Brazil; and (iii) estimate the amount and directionality of historical migration of M. perniciosa subpopulations. Up to 203 M. perniciosa isolates collected from solanaceous hosts with symptoms from Bahia and Minas Gerais states in Brazil and from Theobroma spp. (cacao) and Herrania spp. were characterized with 11 microsatellite markers. Factorial correspondence analyses, minimum‐spanning network and Bayesian clustering revealed genetic clusters associated with their host of origin. Significant subpopulation differentiation was evident (Φ_ST = 0.30, P ≤ 0.05) among M. perniciosa host subpopulations. Most of the multilocus microsatellite genotypes (MLMGs) were host‐specific, with few MLMGs shared among subpopulations. Pairwise comparisons among M. perniciosa host subpopulations were significant, except between jurubeba (Solanum paniculatum) and cultivated solanaceous subpopulations. The combined analyses rejected the null hypothesis that M. perniciosa in Brazil is a single genetic population not structured by host. These findings support a scenario of introduction and subsequent adaptation to solanaceous hosts that should be taken into consideration to improve mitigation and management of M. perniciosa.     

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.     

New experimental hosts of Barley yellow dwarf virus among wild grasses,with implications for grassland habitats

Barley yellow dwarf virus (BYDV), an economically important virus, infects small grain cereal crops and over 150 other Poaceae species. BYDV infection plays an important role in competition among grasses in non‐managed systems, but many grasses remain unexamined as potential BYDV hosts. This study examined grass species that have not been reported as BYDV hosts but are commonly encountered in non‐managed grasslands throughout the United States and Canada. Laboratory inoculations with BYDV‐PAV using the aphid vector Rhopalosiphum padi were performed to examine the ability of 13 grass species and barley to be infected with the virus; eight of the grass species were not documented previously as virus hosts. Serological and molecular assays were used to confirm BYDV‐PAV infection. Plant height, number of leaves, number of tillers and weight were recorded to evaluate susceptibility or sensitivity to BYDV. Infection with BYDV was experimentally achieved for the first time on Achnatherum lettermanii, Achnatherum occidentale, Achnatherum thurberianum, Danthonia intermedia, Poa fendleriana, Sporobolus airoides and Sporobolus cryptandrus, but not on Alopecurus pratensis and Elymus wawawaiensis. Infection was confirmed in Bromus inermis, Elymus elymoides, Poa bulbosa, Poa secunda and Hordeum vulgare, which served as controls. BYDV infection caused reductions in plant height on P. bulbosa and P. fendleriana. BYDV‐infected P. secunda had more leaves per plant compared to healthy plants of the same species. BYDV‐infected A. lettermanii exhibited reduced dry weight in both below‐ground and above‐ground tissue. These findings have implications for the management and conservation of grassland habitats.     

Characterization of the first two viruses described from wild populations of hammer orchids (Drakaea spp.) in Australia

Sequences representing the genomes of two distinct virus isolates infecting wild plants of two members of the genus Drakaea (hammer orchids) in Western Australia are described. The virus isolated from Drakaea livida has a bipartite genome of 4490 nt (RNA1) and 2905 nt (RNA2) that shares closest sequence and structural similarity to members of the genus Pecluvirus, family Virgaviridae, described from legumes in the Indian subcontinent and West Africa. However, it differs from pecluviruses by lacking a P39 protein on RNA2 and having a cysteine‐rich protein gene located 3′ of the triple gene block protein genes. It is the first peclu‐like virus to be described from Australia. The name Drakaea virus A is proposed (DVA; proposed member of the family Virgaviridae, genus unassigned). The second virus isolate was identified from Drakaea elastica, a species classed as endangered under conservation legislation. The genome sequence of this virus shares closest identity with isolates of Donkey orchid symptomless virus (DOSV; proposed member of the order Tymovirales, family and genus unassigned), a species described previously from wild Caladenia and Diuris orchids in the same region. These viruses are the first to be isolated from wild Drakaea populations and are proposed to have an ancient association with their orchid hosts.