Expressing a gene encoding wheat oxalate oxidase enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus)

Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape (Brassica napus). Oxalic acid (OA) secreted by the pathogen is a key pathogenicity factor. Oxalate oxidase (OXO) can oxidize OA into CO₂ and H₂O₂. In this study, we show that transgenic oilseed rape (sixth generation lines) constitutively expressing wheat (Triticum aestivum) OXO displays considerably increased OXO activity and enhanced resistance to S. sclerotiorum (with up to 90.2 and 88.4% disease reductions compared with the untransformed parent line and a resistant control, respectively). Upon application of exogenous OA, the pH values in transgenic plants were maintained at levels slightly lower than 5.58 measured prior to OA treatment, whereas the pH values in untransformed plants decreased rapidly and were markedly lower than 5.63 measured prior to OA treatment. Following pathogen inoculation, H₂O₂ levels were higher in transgenic plants than in untransformed plants. These results indicate that the enhanced resistance of the OXO transgenic oilseed rape to Sclerotinia is probably mediated by OA detoxification. We believe that enhancing the OA metabolism of oilseed rape in this way will be an effective strategy for improving resistance to S. sclerotiorum. Xiangbai Dong and Ruiqin Ji contributed equally to this paper.     

Sensitivity to boscalid in field isolates of Sclerotinia sclerotiorum from rapeseed in Henan Province,China

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is an important disease of oilseed rape in Henan province of China. Boscalid belongs to succinate dehydrogenase inhibitor (SDHI) fungicides, many of which have strong antifungal activity against S. sclerotiorum. In 2015, a total of 175 isolates of S. sclerotiorum were collected from diseased oilseed rape plants in seven different regions of Henan Province. The EC₅₀ values of 175 isolates of S. sclerotiorum to boscalid ranged from 0.0073 to 0.3880 μg ml^?1, and the mean EC₅₀ value was 0.15 ± 0.09 μg ml^?1. The frequency distribution was unimodal. There was no cross‐resistance between boscalid and carbendazim, procymidone, iprodione, dimethachlone, fludioxonil or fluazinam. Field experiments showed that control efficacies of treatments with boscalid (50% WG) at 225, 300 and 375 g ai ha^?1 were 71%, 81% and 90%, respectively. In contrast, the control efficacy of carbendazim (50% WP) at 1,500 g ai ha^?1 was only 52%.     

Effect of Leaf Infection by Kabatiella zeae on Stalk Rot Prevalence and Grain Yield of Maize Hybrids

Eight maize hybrids were tested for resistance to eyespot (Kabatiella zeae) and stalk rot (Fusarium spp.) under field conditions in 1993–1995. The relationship between eyespot and stalk rot and their effect on grain yield of hybrids were studied. Effectiveness of the prochloraz fungicide in controlling both diseases was evaluated. The experiments were performed in three subtrials where plants were naturally infected, inoculated with K. zeae or treated with the fungicide, Sportak 450 EC. The relationship between intensity of leaf damage by eyespot and incidence of stalk rot has been evidently confirmed. High infection of leaf by K. zeae reduced grain yield of hybrids. However, considerable differences in prevalence of disease and yield losses were noted in successive years. The intensity of eyespot depended upon environmental conditions highly variable over the years: it was enhanced by low temperature and high air humidity in July and August. Hybrids showed significant differences in susceptibility to both diseases. Mona – hybrid from the USA and two experimental hybrids from Poland were less affected by diseases than Smolimag and Hidosil (commercial hybrids from Poland). The fungicide Sportak was effective in controlling eyespot and had an indirect influence on decreasing fusarium stalk rot.     

Toxicity of hydrolysis volatile products of Brassica plants to Sclerotinia sclerotiorum,in vitro

Oilseed rape stem rot disease caused by Sclerotinia sclerotiorum causes serious yield losses worldwide. Glucosinolates as specific secondary metabolites of Brassicaceae are produced in various parts of the host plants. Their enzymatic hydrolysis releases chemical components, particularly isothiocyanates, with fungitoxic activity and volatile characteristics. To investigate the effect of volatiles derived from Brassica tissues, the pathogen was exposed to hydrolysis products of Brassica shoot parts as sources of glucosinolates including oilseed rape varieties and two species, black and white mustard. The results showed significant differences in inhibition of S. sclerotiorum growth between varieties and species. All tissues of black mustard inhibited completely the exposed colonies of the pathogen and oilseed rape varieties Dunkeld, Oscar and Rainbow had significant inhibitory effect on the fungus. The genotypes demonstrated significant differences for the production of toxic volatiles, indicating that GSL contents in Brassica species and even cultivars have different potentials for toxic products.     

Effects of experimental warming on fungal disease progress in oilseed rape

Global warming will influence the growth and development of both crops and pathogens. The aims of this study were to investigate potential effects of future warming on oilseed rape growth and the epidemiology of the three economically important pathogens Verticillium longisporum, Sclerotinia sclerotiorum, and Leptosphaeria maculans (anamorph: Phoma lingam). We utilized climate chambers and a soil warming facility, where treatments represented regional warming scenarios for Lower Saxony, Germany, by 2050 and 2100, and compared results of both approaches on a thermal time scale by calculating degree‐days (dd) from day of sowing, December 1st and March 1st until sampling, the latter correlating best with disease progress. Regression analysis showed that plant growth and growth stages in spring responded almost linearly to increasing thermal time until 1000–1500 dd. Colonization of plant tissue by V. longisporum showed an exponential increase when exceeding 1300–1500 dd and reaching plant growth stage BBCH 74/75 (pod development). V. longisporum colonization of plants may be advanced, potentially leading to higher inoculum densities after harvest and increased economic importance of this pathogen under future warming. Sclerotia germination of S. sclerotiorum reached its maximum at 600–900 dd. Advance of these critical degree‐days may lead to earlier apothecia production, potentially advancing the infection window, whereas the future importance of S. sclerotiorum may remain constant. Severity of phoma crown canker increased linearly with increasing thermal time, but showed also large variation in response to the warming scenarios, suggesting that factors such as canopy microclimate in fall or leaf shedding over winter may play a bigger role for L. maculans infection and disease severity than higher soil temperatures. Thermal time was a suitable tool to combine and integrate data on biological responses to soil and air temperature increases from climate chamber and field experiments.     

Carbendazim resistance and dimethachlone sensitivity of field isolates of Sclerotinia sclerotiorum from oilseed rape in Henan Province,China

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most important diseases in oilseed rape‐growing areas of China. To determine the frequency of resistance of field isolates of S. sclerotiorum to carbendazim and dimethachlone, a total of 556 isolates from 10 different regions of Henan Province were obtained between 2015 and 2016. The frequency of isolates with a high‐resistance phenotype and a moderate‐resistance phenotype to carbendazim was 69.2% and 10.8%, respectively. However, S. sclerotiorum isolates resistant to dimethachlone were not detected. The baseline sensitivity of S. sclerotiorum to dimethachlone was distributed as a unimodal curve with a mean EC₅₀ value of 0.39 ± 0.09 μg ml^?1 for the inhibition of mycelial growth. Four dimethachlone‐resistant mutants were obtained from 20 wild‐type isolates induced by exposure to increasing concentrations of the fungicide in vitro. The mutants showed high levels of resistance to dimethachlone, with resistance factors that ranged from 179 to 323. Positive cross‐resistance occurred between dimethachlone and procymidone, iprodione, and fludioxonil; however, no cross‐resistance was observed for carbendazim and boscalid. The fitness of the dimethachlone‐resistant mutants was significantly lower than that of the wild‐type isolates, as measured by mycelial growth, hyphal dry weight, sclerotium number and dry weight, and pathogenicity. Additionally, based on osmotic tests, the inhibition of mycelial growth caused by NaCl applied at different concentrations was significantly higher for the dimethachlone‐resistant mutants than for their wild‐type parents.     

Biological control of Sclerotinia disease by Aspergillus sp. on oilseed rape in the field

Sclerotinia sclerotiorum causes serious yield losses to many crops worldwide. Aspergillus sp. Asp-4, previously shown to inhibit germination of sclerotia of S. sclerotiorum in vitro and in the field, was evaluated in field trials for suppression of this pathogen on oilseed rape. Spray application of Asp-4 to the soil prior to sowing rice in a rice–oilseed rape rotation resulted in a significant reduction in incidence of Sclerotinia stem rot on oilseed rape compared with the non-treated control in two field trials. This application of Asp-4 also resulted in a significant reduction in germination of sclerotia relative to the non-treated control in these field trials, suggesting that this reduction in sclerotial germination led to disease control. Microscopic examination demonstrated that Asp-4 could effectively colonise external and internal portions of sclerotia of S. sclerotiorum in vitro. Incubation of Asp-4 with sterile sclerotial material induced production of β-glucanase and chitinase activities by this isolate; β-glucanase and chitinase being potentially capable of degrading the glucan and chitin polymeric components of sclerotia. Incubation of Asp-4 with sterile sclerotial material also resulted in a significant reduction in dry weight of this sclerotial material relative to the non-treated control in 96?h in vitro experiments. Experiments reported here indicate that Aspergillus sp. Asp-4 has promise as a biological control agent for S. sclerotiorum on oilseed rape. Experiments reported here suggest that disease control results from inhibition of germination of sclerotial resting structures due to mycoparasitic colonisation by Asp-4.     

Pre-germinated conidia of Coniothyrium minitans enhances the foliar biological control of Sclerotinia sclerotiorum

The relatively slow germination rate of Coniothyrium minitans limits its control efficiency against Sclerotinia sclerotiorum. Pre-germinated conidia of C. minitans enhanced its efficiency significantly: in foliar experiments with oilseed rape, hyphal extension of S. sclerotiorum was inhibited by 68%, while formation of sclerotia was completely inhibited when pre-germinated conidia were applied.Revisions requested 27 July 2004; Revisions received 7 September     

The use of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> P13 to control sclerotinia stem rot (<Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>) of oilseed rape

Sclerotinia stem rot (SSR) caused by the fungus Sclerotinia sclerotiorum has been an increasing threat to oilseed rape (Brassica napus L.) cultivation. Efficient and environment—friendly treatments are much needed. Here we focus on microbial control. The Pseudomonas fluorescens P13 that was isolated from oilseed rape cultivation soil, proved to be a useful biocontrol strain for application. Morphology, physiological and biochemical tests and 16S rDNA analysis demonstrated that it was P. fluorescens P13 and that it had a broad antagonistic spectrum, significantly lessening the mycelial growth of S. sclerotiorum by 84.4% and suppressing sclerotial formation by 95–100%. Scanning electron microscopy studies attested that P13 deformed S. sclerotiorum mycelia when they were cultured together. P13 did not produce chitinase but did produce hydrogen cyanide (HCN) which was likely one of the antagonistic mechanisms. The density of P13 remained at a high level (≥10⁶ CFU/ml) during 5 weeks in the rhizosphere soil and roots. P13 reduced SSR severity at least by 59% in field studies and also promoted seedling growth (p<0.05) at the seedling stage. From these data, our work provided evidence that P13 could be a good alternative biological resource for biocontrol of S. sclerotiorum.     

Decreased incidence of disease caused by Sclerotinia sclerotiorum and improved plant vigor of oilseed rape with Bacillus subtilis Tu-100

Sclerotinia sclerotiorum causes serious yield losses in oilseed crops worldwide. Bacillus subtilis Tu-100 significantly reduced (P≤0.05) the incidence of disease caused by S. sclerotiorum on oilseed rape at harvest in two trials conducted in fields artificially infested with this pathogen. Mean plant dry weight was significantly greater (P≤0.05) and mean plant length was significantly greater (P≤0.07) at the seven-true-leaf stage with the Tu-100 treatment than with the control. Mean seed yield per 120 plants at harvest was significantly greater (P≤0.05) in the second field trial with treatments containing isolate Tu-100. B. subtilis Tu-100 also promoted the growth of hydroponically grown oilseed rape. Plants were approximately 15% greater in dry weight (P≤0.0001) and 6% greater in length (P≤0.0025) when grown in the presence of isolate Tu-100 in Hoagland’s solution, compared with the noninoculated control. In gnotobiotic studies, the lacZ-tagged strain B. subtilis Tu-100(pUC18) was detected within all roots of oilseed rape. Isolate Tu-100 did not persist in the ectorhizosphere of oilseed rape. Populations of this isolate decreased from 8.5×10⁸ colony-forming units (CFU) per seed to approximately 10² CFU in the plant ectorhizosphere within 30 days of sowing in autoclaved soil.     

Baseline sensitivity and control efficacy of fluazinam against Sclerotinia sclerotiorum in Henan Province,China

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is a devastating disease in Henan Province, of the main rapeseed production areas in China. Fluazinam belongs to the broad‐spectrum phenylpyridinamine fungicides, which have high activity in inhibiting the mycelial growth of S. sclerotiorum. In this study, 191 field isolates were obtained from different oilseed rape fields in Henan Province, before being exposed to fluazinam in 2015. The baseline sensitivity of S. sclerotiorum to fluazinam was established. The effective concentration for 50% inhibition of mycelial growth (EC₅₀) ranged from 0.0019 to 0.0337 μg/ml, and the mean EC₅₀ value was 0.0084 ± 0.0055 μg/ml. The range of the frequency distribution was narrow. The results of a cross‐resistance assay revealed no cross‐resistance between fluazinam and carbendazim, dimethachlone, boscalid or fludioxonil. Field efficacy tests showed that the control efficacies of fluazinam (50% WG) applied at 150, 225 and 300 g ai ha^?1 were 67%, 73% and 88%, respectively. In contrast, the control efficacies of boscalid (50% WG) and carbendazim (50% WP) applied at 225 and 1,500 g ai ha^?1 were 71% and 52%, respectively.     

Antifungal substances produced by Penicillium oxalicum strain PY-1—potential antibiotics against plant pathogenic fungi

This paper reports the isolation from soil of Penicillium strain PY-1 with strong antagonistic activity against plant pathogenic fungi. On the basis of its morphological characteristics and the sequence of the ITS region, strain PY-1 was identified as P. oxalicum. Strain PY-1 produces antifungal substances that suppress the mycelial growth of Sclerotinia sclerotiorum and many other plant pathogenic fungi tested; the highest antagonistic activity was detected at 72 h when cultured in a 250-ml flask containing 80 ml potato dextrose broth. Compared with carbendazim, the relative activity of the antifungal substances produced by strain PY-1 was approximately 4 μg active ingredient (a.i.) per milliliter. The antifungal substances were extracted with ethyl acetate and further separated by high-performance liquid chromatography (HPLC); at least two active components were discovered. The ability to control plant disease with strain PY-1 was confirmed with S. sclerotiorum, a widespread pathogenic fungus that attacks rapeseed (Brassica napus) and other plants. Spores (10⁶ or 10⁷ ml⁻¹) and filtrate (tenfold diluted or undiluted) of strain PY-1 could significantly suppress infection and/or the extent of infection by S. sclerotiorum of plants at seven-true-leaves stage. The potential of strain PY-1 for identifying new antibiotics to control fungal disease and for biological control of plant disease, for example oilseed rape stem rot, is discussed.     

Candidate gene association mapping of Sclerotinia stalk rot resistance in sunflower (Helianthus annuus L.) uncovers the importance of COI1 homologs

Key message

Functional markers for Sclerotinia basal stalk rot resistance in sunflower were obtained using gene-level information from the model species Arabidopsis thaliana.

Abstract

Sclerotinia stalk rot, caused by Sclerotinia sclerotiorum, is one of the most destructive diseases of sunflower (Helianthus annuus L.) worldwide. Markers for genes controlling resistance to S. sclerotiorum will enable efficient marker-assisted selection (MAS). We sequenced eight candidate genes homologous to Arabidopsis thaliana defense genes known to be associated with Sclerotinia disease resistance in a sunflower association mapping population evaluated for Sclerotinia stalk rot resistance. The total candidate gene sequence regions covered a concatenated length of 3,791 bp per individual. A total of 187 polymorphic sites were detected for all candidate gene sequences, 149 of which were single nucleotide polymorphisms (SNPs) and 38 were insertions/deletions. Eight SNPs in the coding regions led to changes in amino acid codons. Linkage disequilibrium decay throughout the candidate gene regions declined on average to an r ² = 0.2 for genetic intervals of 120 bp, but extended up to 350 bp with r ² = 0.1. A general linear model with modification to account for population structure was found the best fitting model for this population and was used for association mapping. Both HaCOI1-1 and HaCOI1-2 were found to be strongly associated with Sclerotinia stalk rot resistance and explained 7.4 % of phenotypic variation in this population. These SNP markers associated with Sclerotinia stalk rot resistance can potentially be applied to the selection of favorable genotypes, which will significantly improve the efficiency of MAS during the development of stalk rot resistant cultivars.     

Transgenic <Emphasis Type="Italic">Brassica napus</Emphasis> L. lines carrying a two gene construct demonstrate enhanced resistance against <Emphasis Type="Italic">Plutella xylostella</Emphasis> and <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Plant diseases and insect pests are serious threat to the growth and yield of oilseed rape. In this study, a binary vector carrying sporamin and chitinase PjChi-1 genes in tandem was introduced into Brassica napus cv. ZS 758 via Agrobacterium tumefaciens for dual resistance against disease and insect attack. Thirty-two regenerated plantlets exhibiting hygromycin resistance were selected following Agrobacterium-mediated transformation of 600 leaf petiole explants. Of these, 27 transformants were confirmed to carry the two transgenes as detected by polymerase chain reaction (PCR) with 4.5% transformation efficiency. Eight plantlets were randomly selected for further confirmation by Southern and northern blot hybridization analyses. Four plants carried single copy of the transgenes, while the remaining four plants carried either two or three copies of the transgenes. Moreover, expression of the sporamin transgene was detected by northern blot hybridization in transgenic lines, but not in wild-type plants. These eight T₀ plants were grown in vitro, and inoculated with the Lepidoptera larvae of Plutella xylostella and with spores of the fungal pathogen of Sclerotinia sclerotiorum. Transgenic plants exhibited high levels of resistance to P. xylostella and S. sclerotiorum when compared to untransformed wild-type plants. Genetic analysis of T₁ progeny confirmed Mendelian segregation of the introduced genes. Therefore, these transgenic lines demonstrate a promising potential for variety development of oilseed rape lines with enhanced resistance against both P. xylostella and S. sclerotiorum.     

一株高效拮抗向日葵菌核菌的细菌菌株YC16的筛选及其作用效果研究

[目的]筛选高效拮抗向日葵菌核菌的细菌菌株,为开发防治菌核菌病害、提高向日葵产量的生物菌剂提供菌种资源。[方法]以羧甲基纤维素钠(CMC)、小麦秸秆纤维素为唯一碳源的无机盐培养基,分离高效降解纤维素的细菌菌株;采用纤维素降解菌与菌核菌的平板对峙方法,进一步筛选拮抗菌核菌的菌株;利用16S rDNA序列鉴定菌株、PDYA平板对峙实验检验上述所选拮抗菌株的抑菌谱;采用离体向日葵新鲜叶片、草炭土基质盆栽实验,观察拮抗菌菌株抑制菌核菌生长的能力;温室盆栽和田间试验条件下,研究其防治向日葵菌核菌病害、促进生长和提高产量的效果。[结果]筛选了一株高效抑制菌核菌的细菌YC16,经过16S rDNA序列分析,鉴定为解淀粉芽孢杆菌。YC16菌株能够抑制8种病原真菌生长,包括齐整小核菌、腐皮镰孢菌、尖孢镰刀菌、稻梨孢、辣椒疫霉、镰刀菌、尖镰孢黄瓜专化型和向日葵菌核菌;抑制菌核菌感染叶片,抑制率达到了80.42%;抑制盆栽基质中菌核菌的菌丝生长,基质表面菌丝密度比对照减少了50%以上。盆栽接种YC16的向日葵生物量比对照提高54.9%,田间向日葵接种YC16菌剂对菌核菌引发的盘腐病防治效果达39%-100%,产量提高24.4%-30.2%。[结论]YC16生物菌剂施用于土壤,能够有效防治向日葵的茎腐病和盘腐病,展现了防治向日葵菌核病和提高产量的双重效果,是一株具有良好应用前景的高效菌种资源。     

Assessing in vitro efficacy of certain fungicides to control Sclerotinia sclerotiorum in peanut

The fungal pathogen Sclerotinia sclerotiorum is responsible for Sclerotinia blight in several crops around the world, including peanut. This study was conducted under laboratory conditions to determine the effects of four registered fungicides, Propulse?, Fontelis^®, Omega^® and Endura^® on mycelial growth and pigmentation, as well as sclerotia and oxalic acid production on a growth medium modified with a fungicide and on the pathogenicity of S. sclerotiorum on leaflets detached from Valencia peanut. Propulse, Omega and Fontelis inhibited mycelial growth of S. sclerotiorum, while, mycelial growth on a modified support with Endura was similar to the control treatment. All fungicides, except Endura, inhibited the production of oxalic acid. Pigmentation of the mycelium was observed in both the control and endura treatments. Sclerotia production was observed only in the control treatment. With the exception of Endura, all fungicides were effective in controlling the development of lesions on Valencia peanut leaflets.     

Characterization of Defense Signaling Pathways of <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Brassica carinata</Emphasis> in Response to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> Challenge

Canola (Brassica napus L.) is an agriculturally and economically important crop in Canada, and its growth and yield are frequently influenced by fungal pathogens. Sclerotinia sclerotiorum is among those fungal pathogens and causes stem rot disease in B. napus whereas it has been reported that Brassica carinata is moderately tolerant to S. sclerotiorum. Jasmonic acid/ethylene (JA/ET) and salicylic acid (SA) are phytohormones that are known to be involved in plant disease responses. To investigate the defense signaling cascades involved in the interaction of B. napus and B. carinata with S. sclerotiorum, we examined the expression of five orthologs of B. napus genes involved in JA/ET or SA signaling pathways using quantitative RT-PCR. Our results indicated that there are differences in the timing of JA/ET and SA signaling pathways between B. napus and B. carinata. Our results in these two Brassica species also support previous observations that necrotrophic pathogens trigger JA/ET signaling in response to infection. Finally, we observed that transgenic canola expressing 1-aminocyclopropane-1-carboxylate-deaminase producing low levels of ET was relatively more susceptible to S. sclerotiorum than its wild-type counterpart, suggesting that ET inhibits S. sclerotiorum-induced symptom development.     

Synthesis of Pogostone-Inspired Dehydroacetic Acid Derivatives and Their Antifungal Activity against Sclerotinia sclerotiorum (Lib.) de Bary

In an effort to exploit the natural antifungal pogostone, its simplified scaffold dehydroacetic acid (DHA) was used as a lead compound to semi-synthesize 56 DHA derivatives ( I1 – 48 , II , III , and IV1 – 6 ). Among them, compound IV4 exhibited the most potent antifungal activity with 11.0 μM EC₅₀ against mycelial growth of Sclerotinia sclerotiorum (Lib.) de Bary whose sclerotia production was also completely suppressed at this concentration. Furthermore, IV4 could completely inhibit infection cushion formation of S. sclerotiorum on rape leaves and achieved a preventive efficacy of 90.2 % at 500 μM, which was on the same level as that of commercial boscalid at 30 μM (88.7 %). The results of physiological and ultrastructural studies indicated that IV4 might disrupt the cell membrane permeability or induce the imbalance of mitochondrial membrane potential homeostasis to exert the antifungal mode of action. Besides, the robust and predicative three-dimensional quantitative structure-activity relationship (3D-QSAR) models were developed and discussed herein.