Impact of carbon sources on growth and oxalate synthesis by the phytopathogenic fungus <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

The impact of various supplemental carbon sources (oxalate, glyoxylate, glycolate, pyruvate, formate, malate, acetate, and succinate) on growth and oxalate formation (i.e., oxalogenesis) by Sclerotinia sclerotiorum was studied. With isolates D-E7, 105, W-B10, and Arg-L of S. sclerotiorum, growth in an undefined broth medium (0.1% soytone; pH 5) with 25 mM glucose and 25 mM supplemental carbon source was increased by the addition of malate and succinate. Oxalate accumulation occurred in the presence of glucose and a supplemental carbon source, with malate, acetate, and succinate supporting the most oxalate synthesis. With S. sclerotiorum Arg-L, oxalate-to-biomass ratios, an indicator of oxalogenic potential, were dissimilar when the organism was grown in the presence of different carbon sources. The highest oxalate-to-biomass ratios were observed with pyruvate, formate, malate, acetate, and succinate. Time-course studies with acetate-supplemented cultures revealed that acetate and glucose consumption by S. sclerotiorum D-E7 coincided with oxalogenesis and culture acidification. By day 5 of incubation, oxalogenesis was halted when cultures reached a pH of 3 and were devoid of acetate. In succinate-supplemented cultures, oxalogenesis essentially paralleled glucose and succinate utilization over the 9-day incubation period; during this time period, culture pH declined but never fell below 4. Overall, these results indicate that carbon sources can regulate the accumulation of oxalate, a key pathogenicity determinant for S. sclerotiorum.     

Ss‐Rhs1, a secretory Rhs repeat‐containing protein,is required for the virulence of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a devastating necrotrophic plant pathogen with a worldwide distribution. Cell wall‐degrading enzymes and oxalic acid are important to the virulence of this pathogen. Here, we report a novel secretory protein, Ss‐Rhs1, which is essential for the virulence of S. sclerotiorum. Ss‐Rhs1 is believed to contain a typical signal peptide at the N‐terminal and eight rearrangement hotspot (Rhs) repeats. Ss‐Rhs1 exhibited a high level of expression at the initial stage of sclerotial development, as well as during the hyphal infection process. Targeted silencing of Ss‐Rhs1 resulted in abnormal colony morphology and reduced virulence on host plants. Microscopic observations indicated that Ss‐Rhs1‐silenced strains exhibited reduced efficiency in compound appressoria formation.     

Aspergillus spp. eliminate Sclerotinia sclerotiorum by imbalancing the ambient oxalic acid concentration and parasitizing its sclerotia

Sclerotinia sclerotiorum, a pathogen of more than 600 host plants, secretes oxalic acid to regulate the ambient acidity and provide conducive environment for pathogenicity and reproduction. Few Aspergillus spp. were previously proposed as potential biocontrol agents for S. sclerotiorum as they deteriorate sclerotia and prevent pathogen's overwintering and initial infections. We studied the nature of physical and biochemical interactions between Aspergillus and Sclerotinia. Aspergillus species inhibited sclerotial germination as they colonized its rind layer. However, Aspergillus-infested sclerotia remain solid and viable for vegetative and carpogenic germination, indicating that Aspergillus infestation is superficial. Aspergillus spp. of section Nigri (Aspergillus japonicus and Aspergillus niger) were also capable of suppressing sclerotial formation by S. sclerotiorum on agar plates. Their culture filtrate contained high levels of oxalic, citric and glutaric acids comparing to the other Aspergillus spp. tested. Exogenous supplementation of oxalic acid altered growth and reproduction of S. sclerotiorum at low concentrations. Inhibitory concentrations of oxalic acid displayed lower pH values comparing to their parallel concentrations of other organic acids. Thus, S. sclerotiorum growth and reproduction are sensitive to the ambient oxalic acid fluctuations and the environmental acidity. Together, Aspergillus species parasitize colonies of Sclerotinia and prevent sclerotial formation through their acidic secretions.     

Oxalic Acid Has an Additional,Detoxifying Function in Sclerotinia sclerotiorum Pathogenesis

The mechanism of the diseases caused by the necrotroph plant pathogen Sclerotinia sclerotiorum is not well understood. To investigate the role of oxalic acid during infection high resolution, light-, scanning-, transmission electron microscopy and various histochemical staining methods were used. Our inoculation method allowed us to follow degradation of host plant tissue around single hyphae and to observe the reaction of host cells in direct contact with single invading hyphae. After penetration the outer epidermal cell wall matrix appeared degraded around subcuticular hyphae (12-24 hpi). Calcium oxalate crystals were detected in advanced (36-48 hpi) and late (72 hpi) infection stages, but not in early stages. In early infection stages, surprisingly, no toxic effect of oxalic acid eventually secreted by S. sclerotiorum was observed. As oxalic acid is a common metabolite in plants, we propose that attacked host cells are able to metabolize oxalic acid in the early infection stage and translocate it to their vacuoles where it is stored as calcium oxalate. The effects, observed on healthy tissue upon external application of oxalic acid to non-infected, living tissue and cell wall degradation of dead host cells starting at the inner side of the walls support this idea. The results indicate that oxalic acid concentrations in the early stage of infection stay below the toxic level. In plant and fungi oxalic acid/calcium oxalate plays an important role in calcium regulation. Oxalic acid likely could quench calcium ions released during cell wall breakdown to protect growing hyphae from toxic calcium concentrations in the infection area. As calcium antimonate-precipitates were found in vesicles of young hyphae, we propose that calcium is translocated to the older parts of hyphae and detoxified by building non-toxic, stable oxalate crystals. We propose an infection model where oxalic acid plays a detoxifying role in late infection stages.     

Transcriptome analyses suggest a disturbance of iron homeostasis in soybean leaves during white mould disease establishment

Sclerotinia sclerotiorum is a serious pathogen of numerous crops around the world. The major virulence factor of this pathogen is oxalic acid (OA). Mutants that cannot produce OA do not cause disease, and plants that express enzymes that degrade OA, such as oxalate oxidase (OxO), are very resistant to S. sclerotiorum. To examine the effect of OA on plants, we infiltrated soybean leaves with 5 mm OA and examined the gene expression changes at 2 h post‐infiltration. By comparing the gene expression levels between leaves of a transgenic soybean carrying an OxO gene (OxO) and its parent AC Colibri (AC) infiltrated with OA (pH 2.4) or water (pH 2.4 or 5.5), we were able to compare the effects of OA dependent or independent of its pH. Gene expression by microarray analysis identified 2390 genes that showed changes in expression, as determined using an overall F‐test P‐value cut‐off of 0.001. The additional requirement that at least one pairwise t‐test false discovery rate (FDR)‐corrected P value should be less than 0.001 reduced the list of the most highly significant differentially expressed genes to 1054. Independent of pH, OA altered the expression levels of 78 genes, with ferritin showing the strongest induction by OA. The combination of OA plus its low pH caused 1045 genes (99% of all significant genes) to be differentially expressed, with many of the up‐regulated genes being related to basal defence, such as genes of the phenylpropanoid pathway and various cytochrome P450s. RNA‐seq was also conducted on four samples: OxO and AC genotypes infiltrated with either OA pH 2.4 or water pH 2.4. The RNA‐seq analysis also identified ferritin paralogues as being strongly induced by OA. As the expression of ferritin, a gene that encodes for an iron storage protein, is induced by free iron, these results suggest that S. sclerotiorum benefits from the ability of OA to free iron from plant proteins, as this induces host cell death, and also allows the uptake and assimilation of the iron for its own metabolic needs.     

Calcium Oxalate Crystals: An Integral Component of the Sclerotinia sclerotiorum/Brassica carinata Pathosystem

Oxalic acid is an important virulence factor for disease caused by the fungal necrotrophic pathogen Sclerotinia sclerotiorum, yet calcium oxalate (CaOx) crystals have not been widely reported. B. carinata stems were infected with S. sclerotiorum and observed using light microscopy. Six hours post inoculation (hpi), CaOx crystals were evident on 46% of stem sections and by 72 hpi on 100%, demonstrating that the secretion of oxalic acid by S. sclerotiorum commences before hyphal penetration. This is the first time CaOx crystals have been reported on B. carinata infected with S. sclerotiorum. The shape of crystals varied as infection progressed. Long tetragonal rods were dominant 12 hpi (68% of crystal-containing samples), but by 72 hpi, 50% of stems displayed bipyramidal crystals, and only 23% had long rods. Scanning electron microscopy from 24 hpi revealed CaOx crystals in all samples, ranging from tiny irregular crystals (< 0.5 μm) to large (up to 40 μm) highly organized arrangements. Crystal morphology encompassed various forms, including tetragonal prisms, oval plates, crystal sand, and druses. Large conglomerates of CaOx crystals were observed in the hyphal mass 72 hpi and these are proposed as a strategy of the fungus to hold and detoxify Ca²⁺ions. The range of crystal morphologies suggests that S. sclerotiorum growth and infection controls the form taken by CaOx crystals.     

Isolation of Oxalic acid tolerating fungi and decipherization of its potential to control Sclerotinia sclerotiorum through oxalate oxidase like protein

Oxalic acid plays major role in the pathogenesis by Sclerotinia sclerotiorum; it lowers the pH of nearby environment and creates the favorable condition for the infection. In this study we examined the degradation of oxalic acid through oxalate oxidase and biocontrol of Sclerotinia sclerotiorum. A survey was conducted to collect the rhizospheric soil samples from Indo-Gangetic Plains of India to isolate the efficient fungal strains able to tolerate oxalic acid. A total of 120 fungal strains were isolated from root adhering soils of different vegetable crops. Out of 120 strains a total of 80 isolates were able to grow at 10?mM of oxalic acid whereas only 15 isolates were grow at 50?mM of oxalic acid concentration. Then we examined the antagonistic activity of the 15 isolates against Sclerotinia sclerotiorum. These strains potentially inhibit the growth of the test pathogen. A total of three potential strains and two standard cultures of fungi were tested for the oxalate oxidase activity. Strains S7 showed the maximum degradation of oxalic acid (23?%) after 60?min of incubation with fungal extract having oxalate oxidase activity. Microscopic observation and ITS (internally transcribed spacers) sequencing categorized the potential fungal strains into the Aspergillus, Fusarium and Trichoderma. Trichoderma sp. are well studied biocontrol agent and interestingly we also found the oxalate oxidase type activity in these strains which further strengthens the potentiality of these biocontrol agents.     

An atypical forkhead‐containing transcription factor SsFKH1 is involved in sclerotial formation and is essential for pathogenicity in Sclerotinia sclerotiorum

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic plant pathogen with a worldwide distribution. The sclerotia of S. sclerotiorum are pigmented multicellular structures formed from the aggregation of vegetative hyphae. These survival structures play a central role in the life and infection cycles of this pathogen. Here, we characterized an atypical forkhead (FKH)‐box‐containing protein, SsFKH1, involved in sclerotial development and virulence. To investigate the role of SsFkh1 in S. sclerotiorum, the partial sequence of SsFkh1 was cloned and RNA interference (RNAi)‐based gene silencing was employed to alter the expression of SsFkh1. RNA‐silenced mutants with significantly reduced SsFkh1 RNA levels exhibited slow hyphal growth and sclerotial developmental defects. In addition, the expression levels of a set of putative melanin biosynthesis‐related laccase genes and a polyketide synthase‐encoding gene were significantly down‐regulated in silenced strains. Disease assays demonstrated that pathogenicity in RNAi‐silenced strains was significantly compromised with the development of a smaller infection lesion on tomato leaves. Collectively, the results suggest that SsFkh1 is involved in hyphal growth, virulence and sclerotial formation in S. sclerotiorum.     

Pathogenicity Determinants of Sclerotinia sclerotiorum and Their Association to Its Aggressiveness on Brassica juncea

White rot or stem rot caused by Sclerotinia sclerotiorum is one of the most destructive fungal diseases that have become a serious threat to the successful cultivation of oilseed Brassicas. The study was designed with an aim to investigate the association between the pathogenic aggressiveness and pathogenicity determinants of this pathogen specifically in Brassica for the first time. For this, a total of 58 isolates of S. sclerotiorum from different geographical regions were collected and purified. These isolates were inoculated on a Brassica juncea cv. RL-1359 and they exhibited high level of variation in their disease progression. The isolates were grouped and then 24 isolates were selected for the biochemical analysis of pathogenicity determinants. The isolates varied significantly with respect to their total organic acids, oxalic acid production and pectin methyl esterase and polygalacturonase activity. The oxalic acid production corresponded to the disease progression of the isolates; the isolates with higher oxalic acid production were the more aggressive ones and vice-versa. This is, in our knowledge, the first study to establish a correlation between oxalic acid production and pathogenic aggressiveness of S. sclerotiorum on B. juncea. However, the pectinases’ enzyme activity did not follow the trend as of disease progression. These suggest an indispensable role of oxalic acid in pathogenicity of the fungus and the potential to be used as biochemical marker for preliminary assessment of pathogenic aggressiveness of various isolates before incorporating them in a breeding program.     

AtWRKY63基因过表达拟南芥对核盘菌抗性的研究

为研究草酸在核盘菌致病过程中可能的作用,以模式植物拟南芥为材料,采用30mmol/L草酸喷施3周龄拟南芥,发现草酸显著诱导拟南芥AtWRKY63的表达。通过构建AtWRKY63过表达载体转化拟南芥,获得过表达AtWRKY63的纯系转基因植株,再用核盘菌活体接种拟南芥,结果表明过表达AtWRKY63植株对核盘菌的抗性显著增强。组织化学染色结果表明,AtWRKY63是通过诱导植物的氧爆发,抑制核盘菌菌丝的生长来抵御核盘菌的侵染;qRT-PCR对拟南芥转录水平分析表明,AtWRKY63可能激活了过表达植株的水杨酸与茉莉酸依赖的抗病信号途径,从而增强对核盘菌的抗性。     

Improving nutritional quality and fungal tolerance in soya bean and grass pea by expressing an oxalate decarboxylase

Soya bean (Glycine max) and grass pea (Lathyrus sativus) seeds are important sources of dietary proteins; however, they also contain antinutritional metabolite oxalic acid (OA). Excess dietary intake of OA leads to nephrolithiasis due to the formation of calcium oxalate crystals in kidneys. Besides, OA is also a known precursor of β‐N‐oxalyl‐L ‐α,β‐diaminopropionic acid (β‐ODAP), a neurotoxin found in grass pea. Here, we report the reduction in OA level in soya bean (up to 73%) and grass pea (up to 75%) seeds by constitutive and/or seed‐specific expression of an oxalate‐degrading enzyme, oxalate decarboxylase (FvOXDC) of Flammulina velutipes. In addition, β‐ODAP level of grass pea seeds was also reduced up to 73%. Reduced OA content was interrelated with the associated increase in seeds micronutrients such as calcium, iron and zinc. Moreover, constitutive expression of FvOXDC led to improved tolerance to the fungal pathogen Sclerotinia sclerotiorum that requires OA during host colonization. Importantly, FvOXDC‐expressing soya bean and grass pea plants were similar to the wild type with respect to the morphology and photosynthetic rates, and seed protein pool remained unaltered as revealed by the comparative proteomic analysis. Taken together, these results demonstrated improved seed quality and tolerance to the fungal pathogen in two important legume crops, by the expression of an oxalate‐degrading enzyme.     

The eukaryotic protein kinase superfamily of the necrotrophic fungal plant pathogen,Sclerotinia sclerotiorum

Protein kinases have been implicated in the regulation of many processes that guide pathogen development throughout the course of infection. A survey of the Sclerotinia sclerotiorum genome for genes encoding proteins containing the highly conserved eukaryotic protein kinase (ePK) domain, the largest protein kinase superfamily, revealed 92 S. sclerotiorum ePKs. This review examines the composition of the S. sclerotiorum ePKs based on conserved motifs within the ePK domain family, and relates this to orthologues found in other filamentous fungi and yeasts. The ePKs are also discussed in terms of their proposed role(s) in aspects of host pathogenesis, including the coordination of mycelial growth/development and deployment of pathogenicity determinants in response to environmental stimuli, nutrients and stress.     

Degradation of oxalic acid by the mycoparasite Coniothyrium minitans plays an important role in interacting with Sclerotinia sclerotiorum

Coniothyrium minitans (Cm) is a mycoparasite of the phytopathogenic fungus Sclerotinia sclerotiorum (Ss). Ss produces a virulence factor oxalic acid (OA) which is toxic to plants and also to Cm, and Cm detoxifies OA by degradation. In this study, two oxalate decarboxylase genes, Cmoxdc1 and Cmoxdc2, were cloned from Cm strain Chy‐1. OA and low pH induced expression of Cmoxdc1, but not Cmoxdc2. Cmoxdc1 was partially responsible for OA degradation, whereas Cmoxdc2 had no effect on OA degradation. Disruption of Cmoxdc1 in Cm reduced its ability to infect Ss in dual cultures where OA accumulated. Compared with Chy‐1, the Cmoxdc1‐disrupted mutants had reduced expression levels of two mycoparasitism‐related genes chitinase (Cmch1) and β‐1,3‐glucanase (Cmg1), and had no detectable activity of extracellular proteases in the presence of OA. On the other hand, the cultural filtrates of the Cmoxdc1‐disrupted mutants in OA‐amended media showed enhanced antifungal activity, possibly because of increased production of antifungal substances under acidic pH condition resulted from reduced Cmoxdc1‐mediated OA degradation. This study provides direct genetic evidence of OA degradation regulating mycoparasitism and antibiosis of Cm against Ss, and sheds light on the sophisticated strategies of Cm in interacting with metabolically active mycelia and dormant sclerotia of Ss.     

Molecular genotyping of Sclerotinia sclerotiorum isolates from different regions and host plants in Iran

Genetic variation among Sclerotinia sclerotiorum isolates from different regions and host plants were investigated using pathogenicity test, mycelial compatibility groups (MCGs) and molecular markers. Six MCGs were identified and significant differences of virulence variability were observed within and among MCGs. Cluster analysis of combined repetitive sequence-based polymerase chain reaction and randomly amplified polymorphic DNA data discriminated 12 isolates into 11 genotypes, indicating high level of genetic polymorphism among tested isolates. Twelve isolates clustered into four major groups corresponding to their hosts andgeographical region. The variability found within closely related isolates of S.sclerotiorum indicated that such morphological and molecular markers are useful in population studies of this pathogen.