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.     

Screening resistance against Sclerotinia sclerotiorum in Brassica crops with use of detached stem assay under controlled environment

Sclerotinia sclerotiorum is a major pathogen that infects stem tissue, causing yield loss and poor seed quality in rapeseed (Brassica napus). Here we report a reliable detached stem inoculation method for evaluating resistance under controlled environment. Two sets of Brassica materials were employed, including 17 genotypes from five Brassica crops in experiment 1, and 71 F₂ lines derived from a cross between susceptible and resistant lines of B. oleracea in experiment 2. High correlations were detected between stem and branch for lesion length in both experiments and between stem and sections of stem in experiment 1. Although the lesion length of detached stem inoculation under controlled environment was positively correlated with that of toothpick inoculation in the field, the variation of lesion length in the detached stem inoculation was lower than that in toothpick inoculation in experiment 2. Moreover, no significant association was detected between lesion length and the diameter of stem or branch. These findings suggest that the detached stem inoculation under controlled conditions is a large-scale, flexible and reliable method of screening for resistance against S. sclerotiorum in Brassica crops. The application of detached stem inoculation is discussed in resistance breeding programs against S. sclerotiorum in Brassica crops.     

A Glasshouse Cropping Method for Screening Lettuce Lines for Resistance to Sclerotinia Sclerotiorum

A soil-based glasshouse crop procedure was developed to screen lettuce lines for resistance to Sclerotinia sclerotiorum. Six sequential crops of 19 different lettuce lines with a range of cultural morphologies, reported previously to exhibit some form of resistance to S. sclerotiorum, were planted in a glasshouse infested with S. sclerotiorum and natural disease development compared with a standard susceptible commercial butterhead cultivar, Rachel. Concomitantly, the same lettuce lines were planted in pots in a nearby glasshouse, were artificially inoculated with ascospores of S. sclerotiorum, assessed for infection and scored for disease severity. Most of lines exhibited resistance in at least one of the crop or direct inoculation assessments with wild form, PI 251246, and stem lettuce, Taiwan, exhibiting resistance in three of the assessments and wild form, PI 271938 (Lactuca serriola), and Iceberg (crisp) line, 74-1076, exhibiting resistance in all four assessments. Cos line, PI 250427, was less resistant than the standard control in all assessments. The crop based screen with predictable, natural disease development was the most discriminating overall assessment and enabled growth habit to be taken into account during the screening process which was not possible through the direct inoculation procedures. Nevertheless, the novel ascospore inoculation screening process provided information on the type of resistance expressed that could not be identified from the cropping procedure.     

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.     

甘蓝型油菜对核盘菌及其毒素的抗性遗传分析

采用Griffing双列杂交第四类遗传试验设计,运用朱军发展的加性-显性遗传模型,直接估算了甘蓝型油菜抗核盘菌及其毒素草酸的遗传方差、遗传力和基因效应.抗病性鉴定采用温室病圃和草酸浸根鉴定法,它们分别鉴定了对核盘菌和草酸的抗性.结果表明,油菜对核盘菌及草酸的抗性加性方差和显性方差均极显著(P＜0.01),抗病性主要由加性和显性基因控制,且对核盘菌抗性的加性方差大于显性方差,而对草酸抗性则是显性方差大于加性方差.油菜对核盘菌和草酸的广义遗传力分别为0.750和0.576,狭义遗传力分别为0.403和0.236.高遗传力表明可通过适当的抗病性鉴定方法有效地培育抗病品种(系).基因效应评价结果表明,抗、感亲本的基因效应是不同的,其中抗病亲本783-3具有较理想的加性效应值,同时在多数组合中有较低的显性效应值,是抗病育种的优良亲本,而感病亲本相反.抗×感病的后代既可能为抗病,也可能为感病.     

Soybean plants expressing an active oligomeric oxalate oxidase from the wheat gf-2.8 (germin) gene are resistant to the oxalate-secreting pathogen Sclerotina sclerotiorum

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum (Lib.) De Bary is a serious fungal disease of soybean. Senescing petals provide a starting nutrient source for the invasion of healthy tissue by the advancing oxalic acid secreting fungal hyphae. Since oxalic acid is a major pathogenicity factor of SSR, transgenic soybean capable of degrading oxalic acid may be resistant to the pathogen. Transgenic soybean plants were produced byAgrobacterium -mediated transformation with the wheat germin gene (gf-2.8) encoding an oligomeric protein, oxalate oxidase (OxO), which oxidizes oxalic acid to carbon dioxide and hydrogen peroxide (H₂O₂). Transgenic soybean homozygous for 35S- gf-2.8 produced an approx. 130 kDa protein indistinguishable from wheat germin, and with OxO activity. OxO activity was prominent in cell walls proximal to the site of pathogen attack. The transgenics had greatly reduced disease progression and lesion length following cotyledon and stem inoculation with S. sclerotiorum indicating that the germin gene product conferred resistance to SSR. This is the first report of plant resistance to the fungal pathogen S. sclerotiorum in transgenic plants expressing OxO.     

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.     

Ss-Bi1 encodes a putative BAX inhibitor-1 protein that is required for full virulence of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a destructive necrotrophic plant pathogen with global distribution. Although S. sclerotiorum has been studied extensively, substantial research on aspects of the pathogen's ability to cause disease is still needed. Bax inhibitor-1 protein functions as a suppressor of programmed cell death and is involved in the response to biotic and abiotic stress in animals, plants and yeast. In this study, we functionally characterized a putative Bax inhibitor-1 protein, Ss-Bi1, from S. sclerotiorum. Ss-Bi1 is predicted to contain a BAX inhibitor-1-like super family domain and shows significant homology with many BAX inhibitor-1 proteins. High expression levels of Ss-Bi1 were observed in hyphae under various stresses. Targeted silencing of Ss-Bi1 resulted in reduced virulence in host plants. Ss-Bi1 gene-silenced strains were more sensitive to heat stress and ER stress than the wild-type strain. The results suggest that Ss-Bi1 encodes a putative BAX inhibitor-1 protein that is required for full virulence of S. sclerotiorum.     

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.     

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.     

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.     

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.     

Comparative resistance to foliar fungal pathogens in transgenic carrot plants expressing genes encoding for chitinase, β-1,3-glucanase and peroxidise

Genes encoding an acidic wheat class IV chitinase (383), an acidic wheat β 1,3-glucanase (638) and a rice cationic peroxidase (POC1) were introduced into ‘Nantes Coreless’ carrot (Daucus carota) by Agrobacterium-mediated transformation. The genes were introduced singly or in various combinations followed by selection imposed by the herbicide phosphinothricin. Regenerated plantlets were screened for presence and expression of the three transgenes using PCR, Southern and Northern hybridisations. Eighteen transgenic lines expressing a single transgene and 2 lines each co-expressing 638/383 and 383/POC1 were assessed for resistance to the necrotrophic fungal pathogens Botrytis cinerea and Sclerotinia sclerotiorum. Percentage leaf area diseased was measured 4 and 7 days after inoculation (dai) and compared to non-transformed control plants. Six lines expressing β-1,3-glucanase 638 alone had no enhanced resistance to B. cinerea at 4 dai and only slight resistance to S. sclerotiorum; there was no effect at 7 dai. Two out of the six lines expressing 383 alone had enhanced tolerance to both pathogens with a 20–50% reduction in disease development at 7 dai. Two lines co-expressing 638/383 had slight reductions in disease by (10–20%) similar to that of the lines expressing chitinase 383 alone. Highest levels of disease resistance were seen in transgenic lines expressing POC1, alone or in combination with chitinase 383. Disease symptoms were slower to develop and symptoms were reduced by up to 90% for B. cinerea and 70% for S. sclerotiorum. The 383/POC1 co-expressing plants developed disease at levels similar to that of POC1 alone. Petioles of plants over-expressing POC1 had higher levels of lignin accumulation constitutively compared to control plants, which was greatly enhanced following inoculation with S. sclerotiorum. These results indicate that peroxidase over-expression can lead to significant disease reduction against necrotrophic pathogens in transgenic carrot plants.     

Overexpression of an nsLTPs-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus)

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most important diseases of oilseed rape worldwide and leads to considerable yield losses. In this study, a non-specific lipid transfer protein-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) was introduced into oilseed rape (Zhongyou 821) by Agrobacterium-mediated transformation. In vitro experiments revealed that the mycelial growth of S. sclerotiorum was significantly inhibited when supplied with crude leaf extracts from transgenic oilseed rape plants overexpressing LJAMP2. Furthermore, in vivo studies showed that transgenic LJAMP2 plants had enhanced resistance to S. sclerotiorum. Semi-quantitative RT-PCR analysis showed that the LJAMP2 gene was transcribed in all transformed plants. In addition, we also found that overexpression of LJAMP2 in transgenic plants caused constitutive activation of the defense-related gene PR-1 and an increase of H₂O₂ production, but did not enhance PDF1.2 expression. Our results suggest that constitutive expression of the LJAMP2 gene from motherwort seeds might be exploited to improve the resistance of oilseed rape against S. sclerotiorum.     

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.     

Pathogenicity of morphologically different isolates of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> with <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">B. juncea</Emphasis> genotypes

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is a serious threat to oilseed production in Australia. Eight isolates of S. sclerotiorum were collected from Mount Barker and Walkway regions of Western Australia in 2004. Comparisons of colony characteristics on potato dextrose agar (PDA) as well as pathogenicity studies of these isolates were conducted on selected genotypes of Brassica napus and B. juncea. Three darkly-pigmented isolates (WW-1, WW-2 and WW-4) were identified and this is the first report of the occurrence of such isolates in Australia. There was, however, no correlation between pigmentation or colony diameter on PDA with the pathogenicity of different isolates of this pathogen as measured by diameter of cotyledon lesion on the host genotypes. Significant differences were observed between different isolates (P ≤ 0.001) in two separate experiments in relation to pathogenicity. Differences were also observed between the different Brassica genotypes (P ≤ 0.001) in their responses to different isolates of S. sclerotiorum and there was also a significant host × pathogen interaction (P ≤ 0.001) in both experiments. Responses between the two experiments were significantly correlated in relation to diameter of cotyledon lesions caused by selected isolates (r = 0.79; P < 0.001, n = 48). Responses of some genotypes (e.g., cv. Charlton) were relatively consistent irrespective of the isolates of the pathogen tested, whereas highly variable responses were observed in some other genotypes (e.g., Zhongyou-ang No. 4, Purler) against the same isolates. Results indicate that, ideally, more than one S. sclerotiorum isolate should be included in any screening programme to identify host resistance. Unique genotypes which show relatively consistent resistant reactions (e.g., cv. Charlton) across different isolates are the best for commercial exploitation of this resistance in oilseed Brassica breeding programmes.     

In vitro inhibition of Sclerotinia sclerotiorum by mixtures of azoxystrobin, SHAM, and thiram

The necrotrophic fungal phytopathogen Sclerotinia sclerotiorum (Lib.) de Bary has a broad host range and frequently causes destructive diseases. The extensive use of common fungicides to control these diseases has selected for resistance in populations of S. sclerotiorum. In this study, 105 isolates of S. sclerotiorum from different geographical regions in Jiangsu Province of China were characterized for baseline sensitivity to azoxystrobin, and the average EC₅₀ value was 0.2932 μg/mL for mycelial growth. Of the mixtures of the fungicides thiram and azoxystrobin that were tested using an in vitro mycelial growth assay, the 1:4 ratio provided the greatest inhibition of S. sclerotiorum. When tested against nine isolates, the 1:4 mixture resulted in a mean synergy ratio of 2.31, indicating synergistic inhibition. Mycelial respiration was inhibited for about 2 h by azoxystrobin alone but for 48 h by the mixture of thiram and azoxystrobin. Salicylhydroxamic acid (SHAM, a known inhibitor of alternative respiration) also increased the inhibition of mycelial growth and respiration caused by azoxystrobin. These results suggest the need for further study of effects of combinations of azoxystrobin with thiram or SHAM in planta to evaluate their potential for management of diseases caused by S. sclerotiorum.