Homothallism in Sclerotinia minor

Sclerotinia species are sexually reproducing ascomycetes. In the past S. minor and S. sclerotiorum, have been assumed to be homothallic because of the self-fertility of colonies derived from single ascospores. S. trifoliorum has previously been shown to be bipolar heterothallic due to the presence of four self-fertile and four self-sterile ascospores within a single ascus [Uhm, J.Y., Fujii, H., 1983a. Ascospore dimorphism in Sclerotinia trifoliorum and cultural characters of strains from different-sized spores. Phytopathology 73: 565–569]. However, isolates of S. minor and S. sclerotiorum were proven to be homothallic ascomycetes, by self-fertility of all eight ascospores within an ascus. Apothecia were raised from all eight ascospores of a single tetrad from four isolates of S. minor and from an isolate of S. sclerotiorum, indicating that inbreeding may be the predominant breeding mechanism of S. minor. Ascospores from asci of S. minor and S. sclerotiorum were predominantly monomorphic, but rare examples of ascospore dimorphism similar to S. trifoliorum were found.     

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%.     

In vitro inhibitory effects of rosemary and sage extracts on mycelial growth and sclerotial formation and germination of Sclerotinia sclerotiorum

The anti-fungal efficacy for two Labiate plants, rosemary (Rosmarinus officinalis L.) and Greek sage (Salvia fructicosa Mill.), against Sclerotinia sclerotiorum fungus (Lib.) de Bary has been investigated. The inhibitory effect of these plants as crude leaf ethanolic extract on the radial mycelial growth as well as on sclerotial production and germination was measured in vitro at various concentrations (stock?=?0.5?g dry leaf powder/ml ddH₂O) in the growth medium. In general, rosemary extract revealed a remarkable anti-fungal effect against the fungus, being more inhibitory than Greek sage in this respect. This was evident as total inhibition of radial mycelial growth by rosemary occurred at 10% extract concentration, while sage was half as potent producing such an effect at double the concentration (20%). Both rosemary and sage extracts were more inhibitory to sclerotial formation than to mycelial growth as the fungus ceased to produce any sclerotia at the lower concentrations of 5 and 5–10%, respectively. In addition, rosemary was highly effective in inhibiting sclerotia germination as total inhibition of germination occurred at 20% extract concentration at three?days and onward after incubation. Moreover, at this level, the survival of sclerotia was totally lost when examined after 12?days of incubation. For sage, inhibition of sclerotial germination/death was only 20% at 12th day of incubation. The results of this study indicate that the extracts of rosemary and Greek sage leaves could become natural alternatives to synthetic fungicides to manage diseases of S. sclerotiorum.     

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.     

An effector of a necrotrophic fungal pathogen targets the calcium-sensing receptor in chloroplasts to inhibit host resistance

SsITL, a secretory protein of the necrotrophic phytopathogen Sclerotinia sclerotiorum, was previously reported to suppress host immunity at the early stages of infection. However, the molecular mechanism that SsITL uses to inhibit plant defence against S. sclerotiorum has not yet been elucidated. Here, we report that SsITL interacted with a chloroplast-localized calcium-sensing receptor, CAS, in chloroplasts. We found that CAS is a positive regulator of the salicylic acid signalling pathway in plant immunity to S. sclerotiorum and CAS-mediated resistance against S. sclerotiorum depends on Ca²⁺ signalling. Furthermore, we showed that SsITL could interfere with the plant salicylic acid (SA) signalling pathway and SsITL-expressing transgenic plants were more susceptible to S. sclerotiorum. However, truncated SsITLs (SsITL-NT1 or SsITL-CT1) that lost the ability to interact with CAS do not affect plant resistance to S. sclerotiorum. Taken together, our findings reveal that SsITL inhibits SA accumulation during the early stage of infection by interacting with CAS and then facilitating the infection by S. sclerotiorum.     

Does Hyphal‐Tip Ensure the Same Allelic Composition at SSR Loci as Monosporic Isolates of Sclerotinia sclerotiorum?

The proper characterization of individual is a basic stage in population genetic studies. In Sclerotinia sclerotiorum, genetic uniformity of an individual can be obtained by isolation of single ascospore; however, hyphal‐tip isolates are commonly used in genetic studies. The aim of this study was to assess whether hyphal‐tip isolates of S. sclerotiorum can be used as surrogate of monoascosporic (monosporic) isolates. Twenty‐eight isolates of S. sclerotiorum were collected from common bean plants with white mold symptoms and were purified by hyphal‐tip or single ascospore. The correspondence between hyphal‐tip and monosporic isolates was assessed through the allelic composition at 10 microsatellite (SSR) loci of the isolates obtained by both methods. For the SSR loci comprised of dinucleotide repeats in 92% of the cases, the difference (di) between the amplicon size values for hyphal‐tip and monosporic isolates was no more than one base pair. For the loci comprised of tetra or pentanucleotide repeats in 89% of the cases, di was no more than one base pair. The same allelic profile was found for hyphal‐tip or single ascospore isolates of S. sclerotiorum. When monosporic isolates cannot be easily obtained, hyphal‐tip can safeguard the genotypic identity of S. sclerotiorum isolates.     

The broad host range pathogen Sclerotinia sclerotiorum produces multiple effector proteins that induce host cell death intracellularly

Sclerotinia sclerotiorum is a broad host range necrotrophic fungal pathogen, which causes disease on many economically important crop species. S. sclerotiorum has been shown to secrete small effector proteins to kill host cells and acquire nutrients. We set out to discover novel necrosis-inducing effectors and characterize their activity using transient expression in Nicotiana benthamiana leaves. Five intracellular necrosis-inducing effectors were identified with differing host subcellular localization patterns, which were named intracellular necrosis-inducing effector 1–5 (SsINE1–5). We show for the first time a broad host range pathogen effector, SsINE1, that uses an RxLR-like motif to enter host cells. Furthermore, we provide preliminary evidence that SsINE5 induces necrosis via an NLR protein. All five of the identified effectors are highly conserved in globally sourced S. sclerotiorum isolates. Taken together, these results advance our understanding of the virulence mechanisms employed by S. sclerotiorum and reveal potential avenues for enhancing genetic resistance to this damaging fungal pathogen.     

Some environmental factors affect growth and antibiotic production by the mycoparasite Coniothyrium minitans

The effects of temperature and pH on growth and antibiotic production by three isolates of Coniothyrium minitans (Conio, Contans and IVT1), known to produce the macrolide antibiotic macrosphelide A, were examined in modified Czapek Dox broth (MCD). Antibiotic production was determined by incorporating heated (60°C for 5 min) C. minitans spent culture filtrates of MCD (10%, v/v) into potato dextrose broth and assessing the ability of the filtrates to inhibit growth of S. sclerotiorum. All isolates grew over the temperature range of 10–30°C, with the optimum at approximately 15–20°C. Antibiotics were produced by all isolates at 10–30°C. Culture filtrates of MCD from all isolates incorporated into PDB inhibited growth of S. sclerotiorum by >50%, whereas there was a reduction in inhibition at 30°C for Conio and IVT1 but not Contans. All three isolates grew over the pH range of 3–7, with greater biomass production in buffered pH 3–5 than the unbuffered control (pH 4.8) media. Antibiotics were produced by all isolates at pH 3–5. Culture filtrates of MCD from all three isolates grown at pH 3–5 inhibited growth of S. sclerotiorum, with the greatest effect on inhibition observed at pH 3. There were no differences in growth inhibition between isolates at pH 3 and 4, but culture filtrates from Conio grown at pH 5 inhibited S. sclerotiorum more than those of IVT1 grown at the same pH. The significance of these results for biocontrol and optimizing antibiotic production by C. minitans is discussed.     

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。     

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.     

Metabolic processes and carbon nutrient exchanges between host and pathogen sustain the disease development during sunflower infection by <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Interactions between the necrotrophic fungus Sclerotinia sclerotiorum and one of its hosts, Helianthus annuus L., were analyzed during fungal colonization of plant tissues. Metabolomic analysis, based on ¹³C- and ³¹P-NMR spectroscopy, was used to draw up the profiles of soluble metabolites of the two partners before interaction, and to trace the fate of metabolites specific of each partner during colonization. In sunflower cotyledons, the main soluble carbohydrates were glucose, fructose, sucrose and glutamate. In S. sclerotiorum extracts, glucose, trehalose and mannitol were the predominant soluble carbon stores. During infection, a decline in sugars and amino acids was observed in the plant and fungus total content. Sucrose and fructose, initially present almost exclusively in plant, were reduced by 85%. We used a biochemical approach to correlate the disappearance of sucrose with the expression and the activity of fungal invertase. The expression of two hexose transporters, Sshxt1 and Sshxt2, was enhanced during infection. A database search for hexose transporters homologues in the S. sclerotiorum genome revealed a multigenic sugar transport system. Furthermore, the composition of the pool of reserve sugars and polyols during infection was investigated. Whereas mannitol was produced in vitro and accumulated in planta, glycerol was exclusively produced in infected tissues and increased during colonization. The hypothesis that the induction of glycerol synthesis in S. sclerotiorum exerts a positive effect on osmotic protection of fungal cells and favors fungal growth in plant tissues is discussed. Taken together, our data revealed the importance of carbon–nutrient exchanges during the necrotrophic pathogenesis of S. sclerotiorum.     

Role of camalexin, indole glucosinolates, and side chain modification of glucosinolate-derived isothiocyanates in defense of Arabidopsis against Sclerotinia sclerotiorum

Plant secondary metabolites are known to facilitate interactions with a variety of beneficial and detrimental organisms, yet the contribution of specific metabolites to interactions with fungal pathogens is poorly understood. Here we show that, with respect to aliphatic glucosinolate‐derived isothiocyanates, toxicity against the pathogenic ascomycete Sclerotinia sclerotiorum depends on side chain structure. Genes associated with the formation of the secondary metabolites camalexin and glucosinolate were induced in Arabidopsis thaliana leaves challenged with the necrotrophic pathogen S. sclerotiorum. Unlike S. sclerotiorum, the closely related ascomycete Botrytis cinerea was not identified to induce genes associated with aliphatic glucosinolate biosynthesis in pathogen‐challenged leaves. Mutant plant lines deficient in camalexin, indole, or aliphatic glucosinolate biosynthesis were hypersusceptible to S. sclerotiorum, among them the myb28 mutant, which has a regulatory defect resulting in decreased production of long‐chained aliphatic glucosinolates. The antimicrobial activity of aliphatic glucosinolate‐derived isothiocyanates was dependent on side chain elongation and modification, with 8‐methylsulfinyloctyl isothiocyanate being most toxic to S. sclerotiorum. This information is important for microbial associations with cruciferous host plants and for metabolic engineering of pathogen defenses in cruciferous plants that produce short‐chained aliphatic glucosinolates.     

Prevalence of multidrug-resistant Staphylococcus aureus in diabetics clinical samples

Antibiotic resistance in 40 Staphylococcus aureus clinical isolates from 110 diabetic patients (36%) was evaluated. Of these, 32 (80%) of the isolates showed multidrug-resistance to more than eight antibiotics and 35% isolates were found to be methicillin resistant S. aureus (MRSA). All 40 S. aureus strains (100%) screened from diabetic clinical specimens were resistant to penicillin, 63% to ampicillin, 55% to streptomycin, 50% to tetracycline and 50% to gentamicin. Where as low resistance rate was observed to ciprofloxacin (20%) and rifampicin (8%). In contrast, all (100%) S. aureus strains recorded susceptibility to teicoplanin, which was followed by vancomycin (95%). Genotypical examination revealed that 80% of the aminoglycoside resistant S. aureus (ARSA) have aminoglycoside modifying enzyme (AME) coding genes; however, 20% of ARSA which showed non-AME mediated (adaptive) aminoglycoside resistance lacked these genes in their genome. In contrast all MRSA isolates possessed mecA, femA genetic determinants in their genome.

     

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.     

Endophytic Fungi Isolated from Oil-Seed Crop Jatropha curcas Produces Oil and Exhibit Antifungal Activity

Jatropha curcas L., a perennial plant grown in tropics and subtropics is popularly known for its potential as biofuel. The plant is reported to survive under varying environmental conditions having tolerance to stress and an ability to manage pest and diseases. The plant was explored for its endophytic fungi for use in crop protection. Endophytic fungi were isolated from leaf of Jatropha curcas, collected from New Delhi, India. Four isolates were identified as Colletotrichum truncatum, and other isolates were identified as Nigrospora oryzae, Fusarium proliferatum, Guignardia cammillae, Alternaria destruens, and Chaetomium sp. Dual plate culture bioassays and bioactivity assays of solvent extracts of fungal mycelia showed that isolates of Colletotrichum truncatum were effective against plant pathogenic fungi Fusarium oxysporum and Sclerotinia sclerotiorum. Isolate EF13 had highest activity against S. sclerotiorum. Extracts of active endophytic fungi were prepared and tested against S. sclerotiorum. Ethyl acetate and methanol extract of C. truncatum EF10 showed 71.7% and 70% growth inhibition, respectively. Hexane extracts of C. truncatum isolates EF9, EF10, and EF13 yielded oil and the oil from EF10 was similar to oil of the host plant, i.e., J. curcas.     

Microsatellite and Morphological Markers Reveal Genetic Variation within a Population of Sclerotinia sclerotiorum from Oilseed Rape in the Çanakkale Province of Turkey

The genetic variation among a population of Sclerotinia sclerotiorum collected from oilseed rape fields in the Çanakkale Province of Turkey was assessed using molecular and morphological markers. Seven microsatellite primer pairs (out of eight) revealed 32 clear polymorphic alleles among the 36 fungal isolates examined. An unweighted pair‐group mean analysis dendrogram was generated using the genetic distance matrix with the 32 microsatellite alleles. The level of similarity was as low as 15% between some isolates indicating a high level of genetic diversity within the fungal population; 23 distinct isolates were found (at a genotypic diversity level of 63%). Among the collection of 36 isolates, 19 mycelial compatibility groups (MCGs) were identified; 10 MCGs included at least two isolates. Molecular and morphological data suggest that most of the isolates within a single MCG were identical; however, the isolates belonging to the MCG2 and MCG4 had variable microsatellite haplotypes and were morphologically dissimilar. The data suggest that there is possibly a high rate of outcrossing as well as evolutionary potential within the population of the pathogen in oilseed rape fields. This is the first report demonstrating the genetic and morphological variation within a population of S. sclerotiorum in Turkey.     

Control of Pratylenchus brachyurus in soybean with Trichoderma spp. and resistance inducers

Trichoderma spp. is a fungus with nematode control potential; however, its potential to control the root lesion nematode Pratylenchus brachyurus remains poorly studied. Thus, the aim of this study was to select Trichoderma spp. isolates and assess their ability to control P. brachyurus in soybean crops. Different experiments were conducted aiming at selecting isolates, assessing whether they were able to reduce nematode penetration in plants or cause mortality in vitro, and whether they were able to induce resistance in soybean, as well as at studying the possibility of using the selected isolates associated with resistance inducers (acibenzolar‐S‐methyl, Ecolife^? and AgroMos^?). The selection experiment found three isolates showing satisfactory results, namely GF422, GF425 and GF427; the GF362 isolate was assessed in the subsequent experiments. These four isolates reduced P. brachyurus penetration in soybean roots and promoted nematode mortality in vitro. Increased total protein and catalase activity were recorded, mainly in the 72‐hr assessments. Overall, the protein production was different between isolates. The best results were found in the combination between the GF362 isolate and the three resistance inducers, between GF427 and Ecolife^?, between GF427 and AgroMos^? and between GF422 and Ecolife^?.     

Mitochondrial haplotypes in populations of the plant-infecting fungus Sclerotinia sclerotiorum: wide distribution in agriculture, local distribution in the wild

Analysis of mitochondrial DNA (mtDNA) haplotypes of Sclerotinia sclerotiorum points to a common origin of some genotypes from agricultural populations, especially when compared with two wild populations that are sharply distinguished from the agricultural sample and from each other. Five agricultural population samples from canola (Alberta, Canada and Norway), cabbage (North Carolina, USA), sunflower (Manitoba, Canada and Queensland, Australia) and two Norwegian populations from a wild plant, Ranunculus ficaria were compared. Haplotypes were determined by Southern hybridization of purified organelle DNA from S. sclerotiorum and Neurospora crassa to total genomic DNA of S. sclerotiorum. Each isolate had one haplotype. Haplotypes of S. sclerotiorum from R. ficaria were different between the two wild populations and also from all haplotypes observed in the agricultural populations. Among the wild isolates, DNA fingerprint, mtDNA haplotype and location in the sampling transect were all associated. Among the agricultural isolates, four haplotypes were observed in at least two agricultural populations and one haplotype was observed in all agricultural populations. In the Canadian canola sample some clones had one mtDNA haplotype, indicating association with DNA fingerprint, some clones had more than one haplotype, and some groups of clones shared haplotypes. Some of the haplotype diversity may be due to the presence of extra-chromosomal elements associated with the mitochondria of S. sclerotiorum.     

Effect of Eucalyptus Essential Oil on Respiratory Bacteria and Viruses

The activity of Eucalyptus globulus essential oil was determined for 120 isolates of Streptococcus pyogenes, 20 isolates of S. pneumoniae, 40 isolates of S. agalactiae, 20 isolates of Staphylococcus aureus, 40 isolates of Haemophilus influenzae, 30 isolates of H. parainfluenzae, 10 isolates of Klebsiella pneumoniae, 10 isolates of Stenotrophomonas maltophilia and two viruses, a strain of adenovirus and a strain of mumps virus, all obtained from clinical specimens of patients with respiratory tract infections. The cytotoxicity was evaluated on VERO cells by the MTT test. The antibacterial activity was evaluated by the Kirby Bauer paper method, minimum inhibitory concentration, and minimum bactericidal concentration. H. influenzae, parainfluenzae, and S. maltophilia were the most susceptible, followed by S. pneumoniae. The antiviral activity, assessed by means of virus yield experiments titered by the end-point dilution method for adenovirus, and by plaque reduction assay for mumps virus, disclosed only a mild activity on mumps virus.