Genome analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared t

Ascospore release and survival in Sclerotinia sclerotiorum

The release and survival of ascospores of a UK Sclerotinia sclerotiorum isolate were studied. Apothecia placed in a spore clock apparatus with different lighting regimes at 15 °C released ascospores continuously with an increasing rate for the duration of experiments (72–84 h). Spore release was not confined to light or dark periods in alternating regimes and occurred in continuous dark or light. Ascospores were released in both saturated air (90–95% rh) and at 65–75% rh. High temperature and rh were detrimental to ascospore survival but spore viability was maintained for longer periods than previously reported. The significance of these results in relation to disease control is discussed

Fungicide Sensitivity of Sclerotinia sclerotiorum Isolates Selected from Five Different States That Use Different Fungicide Treatments

Sclerotinia sclerotiorum is a plant pathogenic fungus that causes a disease called white mold that can infect more than 450 plant species including soybeans, dry beans, green beans, canola, and sunflower. This pathogen is capable of up to $252M in losses every year (U.S. Canola Association, 2014). Fungicides are widely used in developed agricultural systems to control disease. However, resistance to the most effective fungicides has emerged and spread in pathogen populations and there have been multiple reports of S. sclerotiorum isolates becoming resistant to certain fungicides. Since different fields in different states use different fungicide treatments on plants and different numbers of applications depending on environmental conditions, we hypothesize that isolates with the lowest fungicide sensitivity will be those that come from fields with more intensive fungicide applications. We aim to determine the fungicide sensitivity of S. sclerotiorum isolates from five states to assess risk of resistance. Isolates were selected from dry bean fields from five states from the selection of isolates in the Evertart lab. Isolates were screened against boscalid, tetraconazole, and picoxystrobin fungicides using discriminatory concentrations previously determined by members of the Everhart lab and their EC50(D) was calculated. Interestingly, preliminary results show that the baseline and Washington isolates have a significantly higher EC50(D) than Nebraska isolates when screened against tetraconazole; and that the Washington, Michigan, and Baseline isolates have a significantly higher EC50(D) than Nebraska isolates when screened against picoxystrobin. Differences in EC50(D) in different states hints at S. sclerotiorum developing resistance to fungicides

Use of Coniothyrium minitans transformed with the hygromycin B resistance gene to study survival and infection of Sclerotinia sclerotiorum sclerotia in soil

A Coniothyrium minitans strain (T3) co-transformed with the genes for β-glucuronidase (uidA) and hygromycin phosphotransferase (hph), the latter providing resistance to the antibiotic hygromycin B, was used to investigate the survival and infection of sclerotia of Sclerotinia sclerotiorum by C. minitans over time in four different soils. Infection of sclerotia was rapid in all cases, with the behaviour of transformant T3 and wild type parent A69 being similar. Differences were seen between the soils in the rate of infection of sclerotia by C. minitans and in their indigenous fungal populations. Amendment of agar with hygromycin B enabled the quantification of C. minitans in soil by dilution plating where there was a high background of other microorganisms. In Lincoln soil from New Zealand, which had a natural but low population of C. minitans the hygromycin B resistance marker allowed the umambiguous discrimination of the applied transformed isolate from the indigenous hygromycin B sensitive one. In this soil, although the indigenous C. minitans population was detected from sclerotia, none were recovered on the dilution plates, indicating the increased sensitivity of C. minitans detection from soil using sclerotial baiting. C. minitans was a very efficient parasite, being able to infect a large proportion of sclerotia within a relatively short time from an initially low soil population. The addition of hygromycin B to agar also allowed the detection of C. minitans from decaying sclerotia by inhibiting secondary fungal colonisers. This is the first report to show that fungi colonising sclerotia already infected by C. minitans mask the detection of C. minitans from sclerotia rather than displacing the original parasite

Actividad fungicida/fungistática in vitro del fosfito de manganeso contra hongos patógenos habitantes del suelo con soja

Las pudriciones de raíz y tallo (PRYT) en el cultivo de soja causadas por patógenos habitantes del suelo son enfermedades comúnmente encontradas en campos de soja y son una de las causas más importantes de pérdidas económicas. La sensibilidad micelial de Fusarium virguliforme, Fusarium tucumaniae, Sclerotinia sclerotiorum y Macrophomina phaseolina fue evaluada en medio de cultivo agar papa glucosa (25 mL) suplementado con diferentes concentraciones (µg mL-1) de fosfito de manganeso (PhiMn) diluido en agua (0; 25; 37.5; 50; 100; 200; 300; 400; 500; 800 y 1000). La sensibilidad del crecimiento micelial fue analizada usando análisis de regresión lineal logarítmico. La concentración de PhiMn necesaria para inhibir el 50% del crecimiento micelial (CI50) fue calculada. Los valores de CI50 fueron desde 105 µg mL-1 (Fusarium spp.) hasta 409 µg mL-1 (M. phaseolina). La formación de esclerocios fue completamente inhibida a 500 µg mL-1. Los resultados del presente estudio representan el primer reporte de la acción fungicida/fungistática in vitro del Phi contra los hongos agentes causales de PRYT en el cultivo de soja.Soybean root and stem rots caused by soil-borne pathogens are diseases commonly found in soybean fields, and one of the most important causes of crop losses. In the present study, the mycelial sensitivity of Fusarium virguliforme, F. tucumaniae, Sclerotinia sclerotiorum and Macrophomina phaseolina was evaluated on potato dextrose agar media (25 mL) supplemented with different concentrations of manganese phosphite (MnPhi) diluted in water (0, 25, 37.5, 50, 100, 200, 300, 400, 500, 800 and 1000 µg/mL). Mycelial growth sensitivity was analyzed using logarithmic linear regression analysis. The MnPhi concentration needed to inhibit 50% of the mycelial growth (IC50) ranged from 105 µg/mL (Fusarium spp.) to 409 µg/mL (M. phaseolina). Sclerotia were completely inhibited at 500 µg/mL. The results of our study represent the first report on the direct in vitro fungicidal/fungistatic action of MnPhi against fungi that are causal agents of soil-borne diseases.Fil: Carmona, Marcelo Anibal. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Vegetal. Cátedra de Fitopatología; ArgentinaFil: Simonetti, Ester. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos. Cátedra de Microbiología Agrícola; ArgentinaFil: Ravotti, M. E.. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Vegetal. Cátedra de Fitopatología; ArgentinaFil: Scandiani, María Mercedes. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Luque, A. G.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Formento, N. A.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Entre Ríos. Estación Experimental Agropecuaria Paraná; ArgentinaFil: Sautua, Francisco. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Vegetal. Cátedra de Fitopatología; Argentin

Molecular studies on intraspecific diversity and phylogenetic position of Coniothyrium minitans

Simple sequence repeat (SSR)±PCR amplification using a microsatellite primer (GACA)% and ribosomal RNA gene sequencing were used to examine the intraspecific diversity in the mycoparasite Coniothyrium minitans based on 48 strains, representing eight colony types, from 17 countries world-wide. Coniothyrium cerealis, C. fuckelii and C. sporulosum were used for interspecific comparison. The SSR±PCR technique revealed a relatively low level of polymorphism within C. minitans but did allow some differentiation between strains. While there was no relationship between SSR±PCR profiles and colony type, there was some limited correlation between these profiles and country of origin. Sequences of the ITS 1 and ITS 2 regions and the 5±8S gene of rRNA genes were identical in all twenty-four strains of C. minitans examined irrespective of colony type and origin. These results indicate that C. minitans is genetically not very variable despite phenotypic differences. ITS and 5±8S rRNA gene sequence analyses showed that C. minitans had similarities of 94% with C. fuckelii and C. sporulosum (which were identical to each other) and only 64% with C. cerealis. Database searches failed to show any similarity with the ITS 1 sequence for C. minitans although the 5±8S rRNA gene and ITS 2 sequences revealed an 87% similarity with Aporospora terricola. The ITS sequence including the 5±8S rRNA gene sequence of Coniothyrium cerealis showed 91% similarity to Phaeosphaeria microscopica. Phylogenetic analyses using database information suggest that C. minitans, C. sporulosum, C. fuckelii and A. terricola cluster in one clade, grouping with Helminthosporium species and 'Leptosphaeria' bicolor. Coniothyrium cerealis grouped with Ampelomyces quisqualis and formed a major cluster with members of the Phaeosphaeriacae and Phaeosphaeria microscopica

Plant pathogens as biocontrol agents of Cirsium arvense : an overestimated approach?

Cirsium arvense is one of the worst weeds in agriculture. As herbicides are not very effective and not accepted by organic farming and special habitats, possible biocontrol agents have been investigated since many decades. In particular plant pathogens of C. arvense have received considerable interest and have been promoted as “mycoherbicides” or “bioherbicides”. A total of 10 fungi and one bacterium have been proposed and tested as biocontrol agents against C. arvense. A variety of experiments analysed the noxious influence of spores or other parts of living fungi or bacteria on plants while others used fungal or bacterial products, usually toxins. Also combinations of spores with herbicides and combinations of several pathogens were tested. All approaches turned out to be inappropriate with regard to target plant specificity, effectiveness and application possibilities. As yet, none of the tested species or substances has achieved marketability, despite two patents on the use of Septoria cirsii and Phomopsis cirsii. We conclude that the potential of pathogens for biocontrol of C. arvense has largely been overestimated

Crystal structure of the GalNAc/Gal-specific agglutinin from the phytopathogenic ascomycete Sclerotinia sclerotiorum reveals novel adaptation of a beta-trefoil domain

International audienceA lectin from the phytopathogenic ascomycete Sclerotinia sclerotiorum that shares only weak sequence similarity with characterized fungal lectins has recently been identified. S. sclerotiorum agglutinin (SSA) is a homodimeric protein consisting of two identical subunits of ∼ 17 kDa and displays specificity primarily towards Gal/GalNAc. Glycan array screening indicates that SSA readily interacts with Gal/GalNAc-bearing glycan chains. The crystal structures of SSA in the ligand-free form and in complex with the Gal-β1,3-GalNAc (T-antigen) disaccharide have been determined at 1.6 and 1.97 Å resolution, respectively. SSA adopts a β-trefoil domain as previously identified for other carbohydrate-binding proteins of the ricin B-like lectin superfamily and accommodates terminal non-reducing galactosyl and N-acetylgalactosaminyl glycans. Unlike other structurally related lectins, SSA contains a single carbohydrate-binding site at site α. SSA reveals a novel dimeric assembly markedly dissimilar to those described earlier for ricin-type lectins. The present structure exemplifies the adaptability of the β-trefoil domain in the evolution of fungal lectins

Genomic evidence for genes encoding leucine-rich repeat receptors linked to resistance against the eukaryotic extra- and intracellular Brassica napus pathogens Leptosphaeria maculans and Plasmodiophora brassicae

© 2018 Stotz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Genes coding for nucleotide-binding leucine-rich repeat (LRR) receptors (NLRs) control resistance against intracellular (cell-penetrating) pathogens. However, evidence for a role of genes coding for proteins with LRR domains in resistance against extracellular (apoplastic) fungal pathogens is limited. Here, the distribution of genes coding for proteins with eLRR domains but lacking kinase domains was determined for the Brassica napus genome. Predictions of signal peptide and transmembrane regions divided these genes into 184 coding for receptor-like proteins (RLPs) and 121 coding for secreted proteins (SPs). Together with previously annotated NLRs, a total of 720 LRR genes were found. Leptosphaeria maculans-induced expression during a compatible interaction with cultivar Topas differed between RLP, SP and NLR gene families; NLR genes were induced relatively late, during the necrotrophic phase of pathogen colonization. Seven RLP, one SP and two NLR genes were found in Rlm1 and Rlm3/Rlm4/Rlm7/Rlm9 loci for resistance against L. maculans on chromosome A07 of B. napus. One NLR gene at the Rlm9 locus was positively selected, as was the RLP gene on chromosome A10 with LepR3 and Rlm2 alleles conferring resistance against L. maculans races with corresponding effectors AvrLm1 and AvrLm2, respectively. Known loci for resistance against L. maculans (extracellular hemi-biotrophic fungus), Sclerotinia sclerotiorum (necrotrophic fungus) and Plasmodiophora brassicae (intracellular, obligate biotrophic protist) were examined for presence of RLPs, SPs and NLRs in these regions. Whereas loci for resistance against P. brassicae were enriched for NLRs, no such signature was observed for the other pathogens. These findings demonstrate involvement of (i) NLR genes in resistance against the intracellular pathogen P. brassicae and a putative NLR gene in Rlm9-mediated resistance against the extracellular pathogen L. maculans.Peer reviewe

Industrial Grain Hemp Variety Trial

Hemp is a non-psychoactive variety of cannabis sativa L. The crop is one of historical importance in the U.S. and reemerging in worldwide importance as manufacturers seek hemp as a renewable and sustainable resource for a wide variety of consumer and industrial products. The crop produces a valuable oilseed, rich in Omega-3 and other essential fatty acids that are often absent in western diets. When the oil is extracted from the seed, what remains is a marketable meal co-product, which is used for human and animal consumption. The fiber has high tensile strength and can be used to create cloth, rope, building materials, and even a form of plastic. For twenty years, U.S. entrepreneurs have been importing hemp from China, Eastern Europe and Canada to manufacture travel gear, apparel and accessories, body care and cosmetics, foods like bread, beer, and salad oils, paper products, building materials and animal bedding, textiles, auto parts, housewares, and sporting equipment. Industrial hemp is poised to be a “new” cash crop and market opportunity for Vermont farms that is nutritious, versatile, and suitable for rotation with other small grains and grasses