Detection and quantification of airborne inoculum of Sclerotinia sclerotiorum using quantitative PCR

This paper reports the development of a new specific diagnostic technique to accurately quantify airborne inoculum of Sclerotinia sclerotiorum and discusses its potential use in disease-forecasting schemes, using examples of three contrasting epidemic seasons: 2007, when there was a severe epidemic of sclerotinia stem rot (SSR) in England and high numbers of airborne ascospores were trapped at Rothamsted, and, in contrast, 2003 and 2004, when the incidence of SSR in England was low and low numbers of airborne ascospores were trapped at Rothamsted. DNA was extracted from wax-coated plastic tapes, such as those used in Burkard (Hirst-type) spore traps and rotating-arm traps. A SYBR-green quantitative PCR (qPCR) method produced a linear relationship between ascospore numbers and S. sclerotiorum DNA (mean 0·35 pg DNA per spore) and was able to detect DNA representing as few as two ascospores. The technique was insensitive to DNA of the host plant, Brassica napus , and other plant pathogens, including Sclerotinia minor , S. trifoliorum and Botrytis cinerea , and common airborne fungal genera such as Cladosporium and Penicillium . There was no relationship between rainfall and numbers of airborne ascospores of S. sclerotiorum present at Rothamsted during the period of infection in the severe SSR season (2007).     

A nested‐PCR method for rapid detection of Sclerotinia sclerotiorum on petals of oilseed rape (Brassica napus)

This study established a quick and accurate method to detect petal infection of oilseed rape (Brassica napus) by Sclerotinia sclerotiorum using a nested‐PCR technique. DNA samples were extracted from each petal using a microwave method, followed by two rounds of PCR amplification. The first‐round PCR amplification was performed using the universal fungal primer pair ITS4/ITS5, and the second‐round amplification with a specific primer pair XJJ21/XJJ222, which was designed using the single‐nucleotide polymorphisms among nuclear rDNA ITS sequences of Sclerotinia spp., Botrytis spp. and other selected fungi. The established technique is rapid and inexpensive, and has a high degree of specificity and sensitivity. This assay can distinguish Sclerotinia spp. from other fungi, including Botrytis cinerea, a closely related and frequent cohabitant on oilseed rape petals, and can detect 50 fg genomic DNA, five ascospores of S. sclerotiorumin vitro or 50 ascospores of S. sclerotiorum on one petal in approximately 6 h, even in the presence of a high background of oilseed rape DNA. This technique was successfully applied in detecting natural petal infections.     

Infection routes for Sclerotinia sclerotiorum in apetalous and fully petalled winter oilseed rape

Near‐isogenic lines (NILs) of apetalous (AP) and fully petalled (FP) winter oilseed rape were used to investigate infection by Sclerotinia sclerotiorum, which occurs mainly via infected petals adhering to leaves in FP oilseed rape. AP1 flowers had an average of 1·4 and 0·8 petals per flower in field and polytunnel experiments, respectively. In field experiments there were no significant differences between counts of FP1 petals, FP1 stamens and AP1 stamens adhered to leaves during flowering. At any one sample time, significantly more stamens tested positive for S. sclerotiorum on AP1 than FP1 NILs, e.g. in 2004, at early flowering 37·5% and 24·2% of stamens tested positive on AP1 and FP1 NILs, respectively. In polytunnel experiments, there were significantly more sclerotinia lesions per plant in the FP1 than in the AP1 NIL. The AP1 NIL did not avoid infection completely, probably because it produced some petals, and lesions were initiated from adhered stamens as well as petals. However, while 8·5% and 16·3% of petals initiated lesions in FP1 and AP1 NILs, respectively, only 2·5% and 1·0% of stamens initiated lesions in FP1 and AP1 NILs, which suggests stamens may be less infective than petals. In field experiments the AP1 NIL had significantly less incidence of sclerotinia stem rot than the FP1 NIL in 2004 (4·9% and 7·0%, respectively). However, there was no significant difference in stem rot incidence between AP and FP lines in 2005 (3·6% and 4·3%, respectively) or 2006 (5·5% and 3·9%, respectively).     

Detection and quantification of airborne inoculum of Hymenoscyphus pseudoalbidus using real‐time PCR assays

A method based on real‐time polymerase chain reaction (PCR) and the use of rotating‐arm spore traps was developed for quantifying airborne Hymenoscyphus pseudoalbidus ascospores. The method was sensitive and reproducible, and the collection efficiency was 10% of the spores present in the air. The temporal ascospore dispersal pattern was studied over 3 years by collecting spores every 15 days for a 24 h air‐sampling period during the ash‐growing season. The highest production was detected from the end of June to the beginning of September. The overall ascospore production did not differ significantly among stands within a specific year but there were differences from year to year. There was a positive correlation between air temperature and the number of ascospores trapped, with most of the positive samples being observed at temperatures above 12°C. The vertical profile of ascospore dispersal showed a strong decrease in ascospore density within a height of 3 m, regardless of date of collection. An analysis of the spore traps installed at increasing distances from an infected stand showed that most of the ascospores were deposited downwind within 50 m of the stand. These data are discussed in context of the epidemiology of the disease.     

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.     

Biocontrol of sclerotinia stem rot (Sclerotinia sclerotiorum) of soybean using novel Bacillus subtilis strain SB24 under control conditions

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is a major disease of soybean in Canada. Laboratory and greenhouse experiments were conducted to evaluate potential effectiveness of cell suspensions, cell‐free culture filtrates and broth cultures of Bacillus subtilis strain SB24 for suppression of SSR. The SB24 cell suspensions and cell‐free culture filtrates significantly reduced mycelial growth of S. sclerotiorum by 50 to 75% and suppressed sclerotial formation by > 90%. The severity on soybean was negatively correlated (r < ?0·84, P < 0·01) to the concentrations of cell suspension, cell‐free culture filtrate and broth culture applied. The cell suspension and broth culture preparations significantly (P < 0·01) reduced SSR severity by 45 to 90% at concentrations ranging from 5 × 10⁶ to 10⁹ CFU mL^?1. The most effective concentration was 5 × 10⁸ CFU mL^?1 for all three preparations, reducing the severity by 60 to 90%. The B. subtilis SB24 was most effective in reducing disease severity when applied ≤ 24 h before plant inoculation with S. sclerotiorum and a significant effectiveness was observed up to 15 days after plant inoculation. The population density of B. subtilis on soybean leaves decreased by 1·5 to 2·5 log units over 15 days under field conditions, and by 0·8 log units over 5 weeks under control conditions. The decrease in population density was significantly correlated with rainfall in the field (r < ?0·93, P < 0·01), suggesting that the biocontrol bacteria may be washed away by rain.     

Detection and quantification of Fusarium commune in host tissue and infested soil using real‐time PCR

Fusarium wilt caused by Fusarium commune is a major limiting factor for Chinese water chestnut (Eleocharis dulcis) production in China. A SYBR Green I real‐time quantitative polymerase chain reaction (qPCR) assay was developed based on the mitochondrial small subunit rDNA of F. commune. Assay specificity of the FO1/FO2 primer set was tested on 41 fungal isolates, and only a single PCR band of c. 178 bp from F. commune was amplified. The detection limits of the assay were 1 fg μL^?1 pure F. commune genomic DNA, 1 pg μL^?1 F. commune genomic DNA mixed with host plant genomic DNA (0·5 ng μL^?1), and 1000 conidia/g soil (artificially inoculated). The amount of F. commune DNA in stem tissues detected by qPCR was significantly correlated with the disease severity (DS) ratings; however, the qPCR assay showed no significant positive correlation between spore densities in soil of different fusarium wilt DS groupings and the DS ratings. The qPCR assay was further applied to 76 soil samples collected from commercial fields of E. dulcis during the 2011 and 2012 growing seasons. The spore density of F. commune detected was positively correlated with disease index in the 2012 growing season but not in 2011. The qPCR method can be used for rapid and specific detection of F. commune in plant and soil samples, which will facilitate monitoring of the pathogen and improvement of disease management.     

Ultrastructural and cytochemical studies on the infection process of Sclerotinia sclerotiorum in oilseed rape

Journal of Plant Diseases and Protection - Stem rot, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is one of the major diseases of oilseed rape worldwide. The infection process of S....     

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.     

Resistance of peas to Sclerotinia sclerotiorum in the Pisum core collection

In this study, 497 pea accessions from the Pisum core collection located at the USDA-ARS, Western Regional Plant Introduction Station (WRPIS), Pullman, WA and seven woody-stem pea lines from a private collection in the UK, were screened for resistance to Sclerotinia sclerotiorum , the cause of white mould. All of the Pisum genotypes screened were susceptible to infection, and 237 of the 504 genotypes were highly susceptible since these did not survive 2 weeks post-inoculation. However, 22 pea accessions and one woody-stem line were identified with quantitative partial resistance to white mould. Pea accessions 103709, 166084, 169603, 240515 and 270536 from the core collection demonstrated the greatest quantitative partial resistance to S. sclerotiorum based on nodal resistance and plant survival in replicated greenhouse and laboratory tests. Only five of the 504 genotypes screened had a mean lesion length of between 0 and 1 cm when assessed 3 days post-inoculation. Pea stem diameter was significantly ( P  ≤ 0·03) negatively correlated with stem lesion length in replicated greenhouse and laboratory experiments, and was determined to be the best predictor of quantitative partial resistance to S. sclerotiorum based on lesion length.     

向日葵菌核病接种方法及品种抗病性鉴定

为建立有效的向日葵菌核病田间接种鉴定方法,以核盘菌菌丝体悬浮液和孢子悬浮液作为接种物,分别对抗、感向日葵品种在现蕾期、始花期和盛花期进行人工接种,并对接种后保湿材料和保湿时间进行比较。试验结果表明:两种接种物均可使向日葵抗、感品种产生盘腐症状。用菌丝体悬浮液和孢子悬浮液接种时,浓度分别为10.0~15.0g/L和200~500个/mL,始花期接种,牛皮纸袋保湿2~4d,即可区分出向日葵品种间抗感性差异。用此方法鉴定出13个对盘腐型菌核病表现抗病的向日葵品种。     

Effect of inoculum type and timing of application of Coniothyrium minitans on Sclerotinia sclerotiorum: influence on apothecial production

The effects of different inocula of the mycoparasite Coniothyrium minitans on carpogenic germination of sclerotia of Sclerotinia sclerotiorum at different times of year were assessed. A series of three glasshouse box bioassays was used to compare the effect of five spore-suspension inocula of C. minitans , including three different isolates (Conio, IVT1 and Contans), with a standard maizemeal–perlite inoculum. Apothecial production, as well as viability and C. minitans infection of S. sclerotiorum sclerotia buried in treated soil, were assessed. Maizemeal–perlite inoculum at 10⁷ CFU per cm³ soil reduced sclerotial germination and apothecial production in all three box bioassays, decreasing sclerotial recovery and viability in the second bioassay and increasing C. minitans infection of sclerotia in the first bioassay. Spore-suspension inocula applied at a lower concentration (10⁴ CFU per cm³ soil) were inconsistent in their effects on sclerotial germination in the three box bioassays. Temperature was an important factor influencing apothecial production. Sclerotial germination was delayed or inhibited when bioassays were made in the summer. High temperatures also inhibited infection of sclerotia by C. minitans . Coniothyrium minitans survived these high temperatures, however, and infected the sclerotia once the temperature decreased to a lower level. Inoculum level of C. minitans was an important factor in reducing apothecial production by sclerotia. The effects of temperature on both carpogenic germination of sclerotia and parasitism of sclerotia by C. minitans are discussed.     

Effect of Coniothyrium minitans on sclerotial survival and apothecial production of Sclerotinia sclerotiorum in field-grown oilseed rape

In two field trials with oilseed rape, Coniothyrium minitans was applied to soil as a maizemeal-perlite preparation in order to determine its effect on sclerotial survival and apothecial production of Sclerotinia selerotiorum. The mycoparasite infected sclerotia and decreased sclerotial survival, carpogenic germination and production of apothecia. Effects were greatest when inoculum of C. minitans was applied in autumn, at the time of sowing, rather than when it was applied in spring. C. minitans survived in soil for 2 years and spread to adjacent control plots and infected sclerotia within those plots. However, despite the fact that the inoculum potential of S. selerotiorum was reduced by C. minitans treatment, no disease control was obtained either in trial 1, where disease levels were low (0-20% of plant stems affected), or in trial 2, where disease levels were high (up to 70% of plant stems affected). Possible reasons for this failure of C minitans to control sclerotinia disease in oilseed rape, and strategies to improve its efficacy in the field, are discussed.     

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.     

Detection of airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae in oilseed rape crops by polymerase chain reaction (PCR) assays

The potential use of DNA-based methods for detecting airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae , both damaging pathogens of oilseed rape, was investigated. A method for purifying DNA from spores collected using Hirst-type spore samplers and detecting it using polymerase chain reaction (PCR) assays is described. For both pathogens, the sensitivities of the DNA assays were similar for spore-trap samples and pure spore suspensions. As few as 10 spores of L. maculans or P. brassicae could be detected by PCR and spores of both species could be detected against a background of spores of six other species. The method successfully detected spores of P. brassicae collected using spore traps in oilseed rape crops that were infected with P. brassicae. Leptosphaeria maculans spores were detected using spore traps on open ground close to L. maculans -infected oilseed rape stems. The potential use of PCR detection of airborne inoculum in forecasting the diseases caused by these pathogens is discussed.     

Reducing the build‐up of Plasmodiophora brassicae inoculum by early management of oilseed rape volunteers

Clubroot of oilseed rape (OSR), caused by Plasmodiophora brassicae, is a disease of increasing economic importance worldwide. Previous studies indicated that OSR volunteers, Brassica crops and weeds play a critical role in the predisposition of the disease. To determine the effect of timing of foliar application of the herbicide glyphosate or mechanical destruction of OSR volunteers in reduction of clubroot severity and resting spore production, a series of studies was conducted under controlled conditions with a susceptible OSR cultivar and an isolate of P. brassicae. Plants were inoculated by injecting a spore suspension beside the root hairs at growth stage 11–12 (BBCH scale) and were terminated at 7 (early) or 21 (late) days post‐inoculation (dpi). Under controlled conditions, the first symptoms on roots were observed as early as 7 dpi. The early application of glyphosate as well as early mechanical destruction resulted in significant (P ≤ 0.05) reduction in the development of clubroot symptoms, root fresh weight and the number of resting spores?g root. Furthermore, the effect of volunteer management on clubroot severity in the succeeding OSR was studied by inoculating plants with the resting spores obtained from treated clubbed roots. Inoculated OSR exhibited root clubs similar to the initial symptoms after 35 dpi. Plants that were inoculated with spore suspension from early treated roots resulted in significant reductions in clubroot incidence and severity. Conversely, plants inoculated with the spore suspension from the late treated roots displayed levels of clubroot similar to the plants inoculated with the spore solutions of positive controls.     

油菜内生生防菌BY-2在油菜体内的定殖与对油菜菌核病的防治作用

 从油菜植株体内分离出的内生细菌BY-2,经过生物学鉴定为枯草芽孢杆菌(Bacillus subtilis)。用BY-2回接油菜,重新分离到具有BY-2相同形态特征和抑制病原真菌能力的内生菌株。油菜接种后的第10 d,体内的BY-2菌数达(2.24~9.02)×10³ cfu/g鲜植株,25 d仍然保持在(3.13~8.59)×10³ cfu/g鲜植株。BY-2与油菜核盘菌[Sclerotinia sclerotiorum(Lib.) de Bary]对峙培养可以形成直径为3.1 cm的抑菌圈;可使油菜核盘菌菌丝细胞浓缩变短,细胞壁破裂,原生质外溢,从而抑制真菌生长发育;同时还能抑制菌核的萌发,抑制率达60%~70%;在油菜离体叶片试验中,BY-2对菌核病的防治效果达100%。     

Identification of Greenhouse Characteristics which Affect the Incidence of Sclerotinia sclerotiorum (Lib.) de Bary in Pepper Crops in a Mediterranean Climate

The effect of the main construction factors and design features of greenhouses on the incidence of white rot disease, caused by Sclerotinia sclerotiorum (Lib.) de Bary, was studied in peppers grown under plastic-roofed greenhouses typical of Almería (SE Spain). At the height of the pepper-growing season in the province, fifty greenhouses were repeatedly sampled. Incidence of the disease was measured and the different construction features of the greenhouses were evaluated: types of roof cover, colour and age of the plastic cover, dimensions (ground plan, surface area, average height), estimated ventilation capacity and the presence of netting on the sides of the greenhouses (laterals). Statistical analyses were undertaken to see if the choice of various design options for each factor had any significant influence on the incidence of the disease. To achieve this single factor ANOVA was undertaken. The incidence of this disease was significantly affected (p<0.05) by the following greenhouse characteristics: ground plan, surface area, mean height, colour of the plastic cover, estimated ventilation and use of lateral netting.     

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.