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.     

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.     

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.     

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.     

小核盘菌菌丝生长和乙二酸合成影响因素研究

小核盘菌菌丝在V8溶液培养基和酵母浸膏溶液培养基中生长显著优于其他供试培养基,菌丝干重分别为6.666 mg/mL和6.632 mg/mL。在葡萄糖土豆浸汁培养基中产生的乙二酸量显著高于其他培养基,为1.981 mg/mL。在蔗糖液培养基中,培养7 d菌丝干重达到最大,为4.455 mg/mL。培养6 d乙二酸产生量达到最大,为0.966 mg/mL。培养基初始pH4.5最适合菌丝生长,干重为4.678 mg/mL。乙二酸含量随初始pH增加而增加,pH7.0的培养基中乙二酸含量为1.835 mg/mL。氮源含量增加可以促进乙二酸的合成,当蔗糖与大豆水解蛋白之比为25 g/10 g时,乙二酸含量为1.897 mg/mL。添加琥珀酸钠乙二酸含量显著增加,为3.741 mg/mL。     

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.     

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.     

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.     

内蒙古和黑龙江的核盘菌菌丝融合群分化及致病性研究

为明确核盘菌的遗传多样性,对采自内蒙古和黑龙江不同地区的44株核盘菌进行了菌丝融合群确定,并比较了不同菌丝融合群间菌丝生长速度、致病力、草酸和总酸产量的差异。结果表明:供试44个菌株分为25个融合群,其中有14个融合群仅由单一菌株组成,所占比例为56.0%。菌丝融合群内和菌丝融合群间菌丝生长速度、致病力、草酸和总酸产量都表现出显著差异(P0.001),并与菌株的地理来源无关。相关分析表明核盘菌菌株的致病力与菌株草酸产量呈正相关(r=0.484,P≤0.01),与pH呈负相关(r=-0.580,P≤0.01),与菌株的生长速度无关;草酸产量与pH高低(表示总酸的分泌量)负相关(r=-0.392,P≤0.01),进一步表明核盘菌菌株产生的总酸中草酸量占了很大的比例。     

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.     

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.     

Expression of an oxalate oxidase gene in tomato and severity of disease caused by Botrytis cinerea and Sclerotinia sclerotiorum

A cDNA clone of wheat oxalate oxidase (OxO) under the control of the constitutive CAMV 35S promotor was expressed in tomato plants by Agrobacterium -mediated transformation. Twenty-six transgenic tomato lines were obtained and analysed. PCR experiments confirmed the incorporation of the OxO gene in all tested tomato lines. The transgenic tomato plants expressed a 124-kDa protein showing OxO activity, and were able to convert different oxalic acid (OA) concentrations in vitro . In a detached leaf assay, most of the transgenic lines showed reduced disease symptoms compared with controls, following inoculation with Botrytis cinerea . In addition, leaves of the line T15 showed a marked reduction in symptoms compared with the control following inoculation with Sclerotinia sclerotiorum .     

草酸对重寄生真菌盾壳霉分生孢子萌发和菌丝生长的影响

 本文研究了草酸对核盘菌的重寄生真菌盾壳霉(Coniothyrium minitans)分生孢子萌发和菌丝生长的影响。结果表明:草酸对盾壳霉分生孢子萌发没有明显促进作用,在酸碱性非缓冲基质(水琼脂)和酸碱性缓冲基质中,抑制盾壳霉分生孢子萌发的最低浓度分别为150和700μg/mL。在马铃薯葡萄糖琼脂培养基中,当草酸浓度为100~2000μg/mL时,盾壳霉菌丝能够生长,且浓度为300~500μg/mL的草酸对盾壳霉的菌丝生长具有明显的促进作用。在以草酸为唯一碳源的合成培养基中,在酸碱性非缓冲的条件下,当草酸浓度为100~2000μg/mL时,盾壳霉菌丝能够生长,且草酸浓度为500μg/mL时对盾壳霉菌丝生长具有促进作用,而当草酸浓度为2500μg/mL时,盾壳霉菌丝则停止生长。在酸碱性缓冲的合成基质中,草酸浓度为100~4000μg/mL时,盾壳霉菌丝能够生长,且草酸浓度为1500~2500μg/mL时对盾壳霉菌丝生长具有促进作用。在含草酸钙的混浊培养基(以草酸为唯一碳源)上,盾壳霉菌落区域形成了透明圈。上述结果说明盾壳霉能忍耐一定浓度的草酸而进行分生孢子萌发及菌丝生长,且这种真菌可能对草酸分子具有分解作用。     

Histopathology of infection and colonization of Quercus ilex fine roots by Phytophthora cinnamomi

Quercus ilex is one of the European forest species most susceptible to root rot caused by the oomycete Phytophthora cinnamomi. This disease contributes to holm oak decline, a particularly serious problem in the ‘dehesas’ ecosystem of the southwestern Iberian Peninsula. This work describes the host–pathogen interaction of Q. ilex and P. cinnamomi, using new infection indices at the tissue level. Fine roots of 6‐month‐old saplings inoculated with P. cinnamomi were examined by light microscopy and a random pool of images was analysed in order to calculate different indices based on the measured area of pathogen structures. In the early stages of invasion, P. cinnamomi colonizes the apoplast and penetrates cortical cells with somatic structures. On reaching the parenchymatous tissues of the central cylinder, the pathogen develops different reproductive and survival structures inside the cells and then expands through the vascular system of the root. Some host responses were identified, such as cell wall thickening, accumulation of phenolic compounds in the middle lamella of sclerenchyma tissues, and mucilage secretion blocking vascular cells. New insights into the behaviour of P. cinnamomi inside fine roots are described. Host responses fail due to rapid expansion of the pathogen and a change in its behaviour from biotrophic to necrotrophic.     

Induction of systemic resistance to Sclerotiniasclerotiorum by oxalic acid in oilseed rape

Treatment of the lowermost leaf of flowering plants of oilseed rape cv. Linetta with 20 m m oxalic acid induced systemic disease resistance in the three next youngest leaves. Resistance was not caused by the translocation of fungitoxic concentrations of oxalic acid. Resistance was also expressed in the stem; in oxalic acid-treated plants the vertical spread of stem lesions was halted and the signal travelled both acropetally and basipetally. Expression of resistance in the leaves was detected 6 h after treatment and continued for at least 5 weeks. Twenty-four h after treatment, resistance was detectable in the leaves following the use of as little as 0.5 m m oxalic acid. Local resistance in the oxalic acid-sprayed leaf was detectable only at certain times (12 and 48 but not 24 h after treatment) and at very low (0.25 m m ) or high (≥40 m m ) oxalic acid concentrations. When oxalic acid was applied to a discrete area of the leaf, significant local resistance was expressed in the surrounding leaf tissue, maximum resistance being exhibited by the tissue closest to the site of petiole attachment.     

Environmental conditions influencing Sclerotinia sclerotiorum infection and disease development in lettuce

The environmental factors that influence infection of lettuce by ascospores of Sclerotinia sclerotiorum , and subsequent disease development, were investigated in controlled environment and field conditions. When lettuce plants were inoculated with a suspension of ascospores in water or with dry ascospores and exposed to a range of wetness durations or relative humidities at different temperatures, all plants developed disease but there was no relationship between leaf wetness duration or humidity and percentage of diseased plants. Ascospores started to germinate on lettuce leaves after 2–4 h of continuous leaf wetness at optimum temperatures of 15–25°C. The rate of development of sclerotinia disease and the final percentage of plants affected after 50 days were greatest at 16–27°C, with disease symptoms first observed 7–9 days after inoculation, and maximum final disease levels of 96%. At lower temperatures, 8–11°C, disease was first observed 20–26 days after inoculation, with maximum final disease levels of 10%. Disease symptoms were always observed first at the stem base. In field-grown lettuce in Norfolk, 2000 and 2001, inoculated with ascospore suspensions, disease occurred only in lettuce planted in May and June, with a range of 20–49% of plants with disease by 8 weeks after inoculation. In naturally infected field-grown lettuce in Cheshire, 2000, disease occurred mainly in lettuce planted throughout May, with a maximum of 31% lettuce diseased within one planting, but subsequent plantings had little (≤ 4%) or no disease. Lack of disease in the later plantings in both Norfolk and Cheshire could not be attributed to differences in weather factors.