Sclerotiorum Isolate Research Articles

Screening of Diverse Lupinus spp. Highlights New Resistances to Sclerotinia sclerotiorum.

Stem rot caused by Sclerotinia sclerotiorum is a serious and sometimes devastating disease of lupin (Lupinus spp.). A total of 236 lupin accessions from across 12 Lupinus species were screened against the prevalent S. sclerotiorum isolate MBRS-1 (pathotype 76). L. angustifolius accession 21655 and L. albus var. albus accession 20589 showed immune and "near-immune" responses, respectively. Thirteen accessions of L. angustifolius, three accessions each of L. albus and L. albus var. albus, and a single accession each of L. albus var. graecus, L. mutabilis, L. palaestinus, and L. pilosus (totaling ∼4%) showed a highly resistant (HR) response. A further 19 accessions of L. angustifolius, 2 accessions each of L. albus and L. pilosus, and a single accession of L. mutabilis (totaling ∼10%) showed a resistant (R) response. The reactions of 16 (15 L. angustifolius, 1 L. digitatus) of these 236 accessions were also compared with their reactions to a different isolate, Walkaway-3 (WW-3; pathotype 10). Against this isolate, five L. angustifolius accessions showed an HR response and four showed an R response, and the L. digitatus accession showed a moderate resistance response. Overall, isolate WW-3 caused significantly (P < 0.05) smaller lesions than MBRS-1 across tested accessions in common. In addition, 328 plants in a "wild" naturalized field population of L. cosentinii were screened in situ in the field against isolate MBRS-1. Five (∼1.5%) of the 328 plants of wild lupin showed an immune response, 63 (∼19%) showed an HR response, and 146 (∼45%) showed an R response. We believe this is the first examination of diverse Lupinus spp. germplasm responses to a prevalent pathotype of S. sclerotiorum. Lupin genotypes exhibiting high-level resistance to Sclerotinia stem rot identified in this study can be used as parental lines for crosses in lupin breeding programs and/or directly as improved cultivars to reduce the adverse impact of this disease on lupin crops.

Variation in Pathogenicity and Subsequent Production of Sclerotia of Sclerotinia sclerotiorum Isolates in Different Cover Crops, Flower Strips, and Weeds.

Cover crops and flower strips are used in agricultural fields as part of integrated pest management strategies. However, their potential as secondary hosts of soilborne pathogens such as Sclerotinia sclerotiorum in oilseed rape cultivation is not fully comprehended. In the current study, we evaluated the effect of pathogen virulence on the development of Sclerotinia stem/leaf rot and sclerotia production in 33 plant species from 11 botanical families using two S. sclerotiorum isolates. Furthermore, the effect of sclerotial size on carpogenic germination was studied. Results showed that the pathogen's virulence significantly affected the occurrence and development of Sclerotinia stem/leaf rot and the subsequent production of sclerotia. Among all plant species tested, 26 were more susceptible to the highly aggressive S. sclerotiorum isolate, which produced more and bigger sclerotia in 17 species than the less aggressive isolate. Moreover, a stronger positive correlation was found between the relative lesion length of plants inoculated with the highly aggressive isolate and the number of sclerotia produced by this isolate (Spearman's rank correlation coefficient [rs] = 0.572; P = 0.004). Additionally, we found that larger and heavier sclerotia produced stipes and apothecia earlier and at a greater rate than smaller ones. The heavyweight class had the highest carpogenic germination rate (82.4%), followed by the average (67.2%) and lightweight classes (59.5%). Our findings highlight the need for further investigation into the potential risks associated with cover crops, weeds, and flower strips as secondary hosts of soilborne pathogens in agricultural fields.

First report of Sclerotinia sclerotiorum causing stem canker on industrial hemp in South Carolina, USA.

On Jun. 20th, 2022, thirty industrial hemp (Cannabis sativa L.) plants (cv. Peach Haze) were vegetatively propagated, grown in a greenhouse for 21 days, and transplanted to a field at The Hemp Mine located in Fair Play, SC. Near the time of harvest (Nov. 17th, 2022), significant mycelial growth was noticed within the floral structure on 30% of plants. Three diseased plants were submitted to the Clemson University Plant and Pest Diagnostic Clinic. Stem cankers were observed on all three plants. Sclerotia typical of Sclerotinia spp. were found inside the stems of two plants. Two pure isolates were obtained by placing a sclerotium from each plant onto an acidified potato dextrose agar (APDA) plate and transferring a hyphal tip to a new APDA plate. After a 7-day-long growth at 25°C under a 24-h photoperiod, both isolates (22-1002-A and B) produced white and sparse mycelia and dark brownish to blackish sclerotia typical of S. sclerotiorum (aver. 36.5 per 90-mm plate). Sclerotia (n=50) were spherical (46%), oval (46%), or irregular (8%) and measured 1.8 to 7.2 × 1.6 to 4.5 mm (aver. 3.6 ± 1.2 × 2.7 ± 0.6 mm). No spores were produced. Sequences of the internal transcribed spacer region including the 5.8S ribosomal RNA gene (GenBank accession no. OQ749889) and the glyceraldehyde 3-phosphate dehydrogenases (G3PDH) gene (OQ790148) of 22-1002-A are 99.8% and 100% identical to those of a S. sclerotiorum isolate LAS01 on industrial hemp (MW079844 and MW082601; Garfinkel 2021). The G3PDH sequence of 22-1002-A is also 100% identical to that of ATCC 18683 (JQ036048), an authenticated S. sclerotiorum strain used for whole genome sequencing (Derbyshire et al. 2017). Ten healthy 'Peach Haze' plants (approx. 10 to 15" tall) grown in 6" pots were used in a pathogenicity test. The epidermis layer of each main stem was slightly wounded (2 × 2 mm2, 1 mm deep) using a sterile dissecting blade. A 5 × 5 mm2 mycelial plug of 22-1002-A was placed onto the wound of each of five plants, while APDA plugs were used for five control plants. Parafilm was used to secure mycelial and sterile agar plugs. All plants were maintained in an indoor controlled environment (25°C, >60% humidity, 24-h photoperiod). Stem cankers were visible on all inoculated plants 5 days after inoculation (DAI). Four of the five inoculated plants had noticeable yellowing and wilting on the foliage 9 DAI, while control plants remained asymptomatic. Elongated and tan-colored cankers (44.3 to 86.2 mm long, aver. 63.1 ± 18.3 mm) were developed at the wounded sites of inoculated plants. Wounded sites of control plants remained green in color and only slightly expanded in length (aver. 3.6 ± 0.8 mm). Tissue was excised from the canker margin of each inoculated plant and the wounded site of each control plant, surface sterilized with 10% bleach for 1 min, rinsed in sterile water, placed onto APDA, and incubated at 25°C. Sclerotia-producing colonies typical of S. sclerotiorum were recovered from all inoculated plants after 6 days, but not from any control plants. Sclerotinia sclerotiorum has a host range of more than 400 plant species (Boland and Hall 1994). This fungus causing stem canker on industrial hemp were reported from MT (Shaw 1973) and OR (Garfinkel 2021) in the USA and Canada (Bains et al. 2000). This is the first report of this disease in SC. Industrial hemp is an emerging crop in SC. The detection of this disease helps SC growers to take actions to monitor and prevent disease outbreak as well as develop an effective management practice when it occurs.

Stem, leaf and cotyledon resistance responses to a prevalent Sclerotinia sclerotiorum pathotype in Australia highlight new opportunities to improve white mould resistance in common bean

Context White mould (Sclerotinia sclerotiorum) inflicts major yield losses on common bean (Phaseolus vulgaris); yet, commercial cultivars known for their high yields and market-adapted grains lack physiological resistance to this disease. Aims This study aimed to test diverse common bean genotypes for resistance in stem, leaf and cotyledon tissues. Methods Thirty-four common bean genotypes with a wide range of agronomic traits and grain types, including genotypes noted previously for susceptible and resistant responses to white mould, were inoculated with the prevalent S. sclerotiorum isolate MBRS-1. Then they were assessed for resistance in stem, leaf and cotyledon tissues under controlled environment conditions, by inoculating plants with a 105 mL−1 hyphal fragment concentration. Key results There was significant (P &lt; 0.001) variation in resistance responses in stem, leaf and cotyledon tissues across the genotypes. Contender, ICA Bunsi, XAN 280 and Taisho-Kintoki showed the highest resistance in stems, whereas Norvell 2558, Pico de Oro, Sanilac, Othelo and Negro Argel exhibited notable resistance in leaves. Metis, Canario 107, Pico de Oro, Pogonion and Jubilejnaja 287 displayed the most resistance in cotyledons. Conclusions This is the first reported attempt to determine the response of common bean germplasm to a prevalent pathotype of S. sclerotiorum in Australia. Bean genotypes exhibiting high-level resistance to white mould identified in this study can be used as parental lines for crosses in common bean breeding programs and/or directly as improved cultivars. Implications The study highlighted both the value of screening under controlled environmental conditions to reliably locate new stem, leaf and/or cotyledon resistances and the possibility of using rapid cotyledon screening to indicate stem resistances because the expression of resistances in cotyledons generally correlated strongly with those in stems.

Draft genome sequencing and secretome profiling of Sclerotinia sclerotiorum revealed effector repertoire diversity and allied broad-host range necrotrophy

White mold commonly known as Sclerotinia sclerotiorum causes stem rot disease and has emerged as one of the major fungal pathogens of oilseed Brassica across the world. In the present study, consistently virulent S. sclerotiorum isolate “ESR-01” was sequenced and an assembly size of ~ 41 Mb with 328 scaffolds having N50 of 447,128 was obtained. Additionally, 27,450 single nucleotide polymorphisms (SNPs) were identified from 155 scaffolds against S. sclerotiorum 1980 isolate, with an average SNP density of ~ 1.5 per kb genome. 667 repetitive elements were identified and approximately comprised 7% of the total annotated genes. The DDE_1 with 454 in numbers was found to be the most abundant and accounts for 68% of the total predicted repetitive elements. In total, 3844 simple sequence repeats are identified in the 328 scaffolds. A total of 9469 protein-coding genes were predicted from the whole genome assembly with an average gene length of 1587 bp and their distribution as 230.95 genes per Mb in the genome. Out of 9469 predicted protein-coding genes, 529 genes were observed encoding the CAZymes (Carbohydrate-Active enzymes) capable of degradation of the complex polysaccharides. Glycosyltransferase (GT) families were most abundant (49.71%) among the predicted CAZymes and GT2 (23%), GT4 (20%), and glycoside hydrolase (GH) 23% with GH18 (11%) were the prominent cell wall degrading enzyme families in the ESR-01 secretome. Besides this, 156 genes essential for the pathogen-host interactions were also identified. The effector analysis in the whole genome proteomics dataset revealed a total of 57 effector candidates (ECs) and 27 of them were having their analogs whereas the remaining 30 were novel ones. Eleven selected ECs were validated experimentally by analyzing the expression profile of the ESR-01 isolate of S. sclerotiorum. Together, the present investigation offers a better understanding of the S. sclerotiorum genome, secretome, and its effector repertoire which will help in refining the present knowledge on S. sclerotiorum-Brassica interactions and necrotrophic lifestyle of the phytopathogen in general.

A detailed in silico analysis of secondary metabolite biosynthesis clusters in the genome of the broad host range plant pathogenic fungus Sclerotinia sclerotiorum

BackgroundThe broad host range pathogen Sclerotinia sclerotiorum infects over 400 plant species and causes substantial yield losses in crops worldwide. Secondary metabolites are known to play important roles in the virulence of plant pathogens, but little is known about the secondary metabolite repertoire of S. sclerotiorum. In this study, we predicted secondary metabolite biosynthetic gene clusters in the genome of S. sclerotiorum and analysed their expression during infection of Brassica napus using an existing transcriptome data set. We also investigated their sequence diversity among a panel of 25 previously published S. sclerotiorum isolate genomes.ResultsWe identified 80 putative secondary metabolite clusters. Over half of the clusters contained at least three transcriptionally coregulated genes. Comparative genomics revealed clusters homologous to clusters in the closely related plant pathogen Botrytis cinerea for production of carotenoids, hydroxamate siderophores, DHN melanin and botcinic acid. We also identified putative phytotoxin clusters that can potentially produce the polyketide sclerin and an epipolythiodioxopiperazine. Secondary metabolite clusters were enriched in subtelomeric genomic regions, and those containing paralogues showed a particularly strong association with repeats. The positional bias we identified was borne out by intraspecific comparisons that revealed putative secondary metabolite genes suffered more presence / absence polymorphisms and exhibited a significantly higher sequence diversity than other genes.ConclusionsThese data suggest that S. sclerotiorum produces numerous secondary metabolites during plant infection and that their gene clusters undergo enhanced rates of mutation, duplication and recombination in subtelomeric regions. The microevolutionary regimes leading to S. sclerotiorum secondary metabolite diversity have yet to be elucidated. Several potential phytotoxins documented in this study provide the basis for future functional analyses.

Partial stem resistance in Brassica napus to highly aggressive and genetically diverse Sclerotinia sclerotiorum isolates from Australia

Sclerotinia sclerotiorum is a fungal pathogen that causes stem rot in oilseed rape (Brassica napus). Previously, B. napus accessions with partial stem resistance to a Canadian S. sclerotiorum isolate (#321) were identified using a stem test in which flowering plants were inoculated with mycelium plugs. The present study examined the partial stem resistance of four of these accessions, PAK54, PAK93, DC21 and K22, following inoculation with Australian isolates. Mycelial compatibility groups and intergenic spacer (IGS) region haplotypes were identified among 71 isolates from Australian oilseed rape and lupin fields. Eleven genetically diverse isolates showed differences in aggressiveness when inoculated onto nine oilseed rape varieties and one Chinese accession. Isolates CU8.24, CU10.17 and CU11.19 were selected based on genetic diversity, growth rate in vitro and high aggressiveness in the initial screen and subsequently inoculated onto the four B. napus accessions. These accessions developed significantly smaller lesions compared with the susceptible control varieties (‘AV Garnet’ and ‘Westar’), with the average frequency of soft and collapsed lesions being less than 20% in PAK54, DC21 and K22, 29% in PAK93 and greater than 88% in the susceptible controls. Microscopic examination revealed that hyphae were typically confined to the stem cortex in the smallest lesions, but could be found in the stem pith in larger lesions. These results show that B. napus accessions PAK54, PAK93, DC21 and K22 can be used in Australia for development of varieties with partial stem resistance to S. sclerotiorum.

Host response of Arabidopsis thaliana ecotypes is determined by Sclerotinia sclerotiorum isolate type

Studies were undertaken to characterise the interactions of eight Sclerotinia sclerotiorum isolates/pathotypes on leaves of intact plants of 17 Arabidopsis thaliana ecotypes. For lesion diameter and lesion incidence, there were significant (P ≤ 0.001) effects of pathogen isolates, A. thaliana ecotypes, and a significant interaction between isolates and ecotypes in all three experiments. Resistance to S. sclerotiorum infection across the ecotypes ranged from highly susceptible through to resistant and the bioassay was sensitive enough to allow assessment of small differences in partial relative resistances across the ecotypes. While some A. thaliana ecotypes, such as Sha, Bay-0, Ws-1 and Ws-2, were found to be highly susceptible to all S. sclerotiorum isolates tested, others, such as Er-0, Jea and Cvi-0, showed a consistent level of resistance, and of these latter, Er-0 showed the highest and most consistent expression of resistance. In contrast, Col-0, Nd-0 and Oy-0 responses ranged from relative resistant to highly susceptible, depending upon the isolate being tested. Some isolates, such as MBRS1, elicited a wide range of resistance responses across the ecotypes, making these isolates most useful for screening the full range of A. thaliana ecotypes for their responses to S. sclerotiorum. In contrast, other isolates such as ‘Cabbage’ only generated lesions over a very narrow size range of host resistance response and hence could limit the ability of such isolates to differentiate resistances across diverse A. thaliana populations. Increasing the number of ecotypes tested increased the capacity to differentiate both the levels of relative resistance amongst test ecotypes and the varying levels of aggressiveness between different isolates. This is the first known study to demonstrate how Arabidopsis resistance responses can be expressed or compromised by variation in aggressiveness amongst different S. sclerotiorum isolates and/or pathotypes.

Inoculum potential of Sclerotinia sclerotiorum sclerotia depends on isolate and host plant

The soilborne fungus Sclerotinia sclerotiorum infects many important crop plants. Central to the success of this pathogen is the production of sclerotia, which enables survival in soil and constitutes the primary inoculum. This study aimed to determine how crop plant type and S. sclerotiorum isolate impact sclerotial production and germination and hence inoculum potential. Three S. sclerotiorum isolates (L6, L17, L44) were used to inoculate plants of bean, carrot, lettuce, oilseed rape (OSR) and potato, and the number and weight of sclerotia per plant quantified. Carpogenic germination of sclerotia collected from different hosts was also assessed for L6. Production of sclerotia was dependent on both crop plant type and S. sclerotiorum isolate, with OSR and lettuce supporting the greatest number (42–122) and weight (1.6–3.0 g) of sclerotia per plant. The largest sclerotia were produced on OSR (33–66 mg). The three S. sclerotiorum isolates exhibited a consistent pattern of sclerotial production irrespective of crop type; L6 produced large numbers of small sclerotia while L44 produced smaller numbers of large sclerotia, with L17 intermediate between the two. Germination rate and percentage was greatest for larger sclerotia (4.0–6.7 mm) and also varied between host plants. Combining sclerotial production data and typical field crop densities suggested that infected carrot and OSR could produce the greatest number (3944 m−2) and weight (73 g m−2) of S. sclerotiorum sclerotia, respectively, suggesting these crops potentially contribute a greater increase in inoculum. This information, once further validated in field trials, could be used to inform future crop rotation decisions.

H2O2 is involved in cAMP-induced inhibition of sclerotia initiation and maturation in the sunflower pathogen Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a destructive pathogen and has a wide range of hosts, including sunflower. The fungus produces sclerotia, which is a dormant structure and plays a vital role in the infection cycle of S. sclerotiorum . The initiation and maturation of sclerotia can be influenced by many factors. In this study, S. sclerotiorum isolate X-8, was collected from a diseased sunflower plant, and used to determine the role of H 2 O 2 and cyclic adenosine 3', 5'-monophosphate (cAMP) in the sclerotia initiation and maturation. Results showed that the transition of S. sclerotiorum from vegetative growth to the initial stage of sclerotia is accompanied by a decrease of H2O2 and exogenously supplying H2O2 or diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase, in the culture medium promoted or inhibited the initiation of sclerotia, respectively. In addition, the cAMP-induced inhibition of sclerotia initiation and maturation were also accompanied by significantly decrease of H2O2. This was achieved by down- regulating the transcripts of Nox1 and Nox2 genes, the isoform of NADPH oxidase. Additionally, a decreased enzymatic activity of both superoxide dismutase (SOD) and catalase (CAT) were also detected in S. sclerotiorum after cAMP treatment. We concluded that H2O2 not only regulates the formation of sclerotia, but also cross-talks with cAMP in regulating the inhibition of sclerotial initiation and maturation.

Inheritance of White Mold Resistance in an Andean Common Bean A 195 and its Relationship with Andean G 122

ABSTRACTWhite mold caused by Sclerotinia sclerotiorum (Lib.) de Bary is a devastating disease of common bean (Phaseolus vulgaris L.) in cool and wet production regions of the Americas and elsewhere. Partial resistance is found in common bean, wild bean, and Phaseolus species of the secondary gene pool such as P. coccineus L. The objectives were to determine (i) the inheritance of white mold resistance in a large‐seeded Andean common bean breeding line A 195 in response to contrasting S. sclerotiorum isolates and (ii) the genetic relationship between A 195 and an Andean germplasm accession G 122. White mold resistant (scores ≤ 4) A 195 was crossed with susceptible (scores &gt; 7) pinto ‘Othello’ and resistant G 122. The F1 and the three parents were inoculated with an aggressive S. sclerotiorum isolate ND710 in 2011. Parents and F2 from resistant F1 plants were inoculated with the less aggressive isolate ARS12D at the fifth internode on the main stem and reinoculated (primary branch) with ND710 and evaluated at 35 d in 2012. Two independent complementary dominant genes controlled resistance in the F2 of A 195/Othello in response to either isolate. There was no segregation in A 195/G 122 F2 in response to ARS12D. But a single dominant gene controlled the difference in resistance between A 195 and G 122 to ND710. The F3 results corroborated the F2 segregation ratios in both populations. This information should help introgress and pyramid higher levels of white mold resistance into common bean cultivars of different market classes.

Molecular characterisation of an endornavirus infecting the phytopathogen Sclerotinia sclerotiorum

The complete sequence and genome organisation of an endornavirus from the phytopathogenic fungus Sclerotinia sclerotiorum isolate 11691 was described and the name Sclerotinia sclerotiorum endornavirus 1 (SsEV1/11691) proposed. The genome is 10,513 nucleotides (nts) long with a single open reading frame (ORF) that codes for a single polyprotein of 3459 amino acid (aa) residues. The polyprotein contains cysteine-rich region (CRR), viral methyltransferase (MTR), putative DEXDc, viral helicase (Hel), phytoreo_S7 (S7) and RNA-dependent RNA polymerase (RdRp) domains. The polyprotein and the conserved domains are phylogenetically related to endornaviruses. However, the coding strand of SsEV1/11691 does not contain a site-specific nick characteristic of most previously described endornaviruses. The elimination of SsEV1/11691 did not result in any significant changes in the host phenotype and virulence.

Delineation of Sclerotinia sclerotiorum pathotypes using differential resistance responses on Brassica napus and B. juncea genotypes enables identification of resistance to prevailing pathotypes

Sclerotinia stem rot caused by the fungus Sclerotinia sclerotiorum is one of the most damaging and difficult-to-manage diseases of oilseed rape (Brassica napus) and mustard (B. juncea). Identifying oilseed Brassica genotypes with effective resistance offers the best long-term prospect for improved management of the disease. Despite some significant interactions between oilseed Brassica genotypes and S. sclerotiorum isolates reported in earlier studies, mostly a single pathogen isolate has been used to identify resistant genotypes. This paper reports the results of studies involving 53 isolates of S. sclerotiorum from the northern and southern agricultural regions of Western Australia where Sclerotinia stem rot is a serious disease of oilseed rape. Colony characteristics of isolates on potato dextrose agar were determined, and two field virulence (i.e., levels of disease severity) studies conducted. The first field study included 14 Brassica genotypes against three S. sclerotinia isolates and the second had eight Brassica genotypes against 50 S. sclerotinia isolates. Only colony diameter of isolates growing on potato dextrose agar was correlated with stem lesion length in the field. In both field experiments, there were significant effects of isolates and host genotypes, as well as a significant interaction between isolates and genotypes in relation to stem lesion length. In the first field experiment, mean stem lesion length ranged from 1cm in the highly resistant B. napus ZY006 to at least 9cm for susceptible genotypes Brassica juncea #2 and B. juncea Montara. The latter genotype was the most susceptible with a mean stem lesion length of 11.1. Expression of high-level resistance in B. napus ZY006 was largely independent of S. sclerotiorum isolate. In contrast, responses in B. napus genotypes, Zhongyou 821, 06-6-3792, RT108 and Charlton were much more isolate-dependent. In the first field experiment, with a greater number of host genotypes than S. sclerotiorum isolates, a higher variance ratio occurred for isolates (VR=158.4) than for host genotypes (VR=10.7). In the second field experiment, with a larger number of S. sclerotinia isolates than hosts, host genotypes had a higher variance ratio (VR=458.9) compared to the isolates (VR=71.2). Increasing the number of isolates greatly improved the capacity to differentiate levels of resistance among test genotypes. In this experiment, some genotypes showed more consistent resistant reactions (e.g., B. napus Mystic and B. juncea Xinyou 9) across different isolates and these are ideal targets for commercial exploitation of this resistance in oilseed Brassica breeding programs. From this experiment, a standardized set of three B. napus and three B. juncea genotypes were shown to be suitable for use as universal differentials to characterize pathotypes of S. sclerotiorum using octal nomenclature. Eight distinct pathotypes of S. sclerotiorum were delineated and characterized and these six host differential genotypes can now be utilized to identify and monitor the incidence and distribution of current and future pathotypes of S. sclerotiorum. Further, by providing a reliable means to characterize pathotypes of S. sclerotiorum, for the first time not only allows identification of resistance(s) against the predominant pathotype(s) of S. sclerotiorum prevailing in a particular region, but also now allows oilseed rape breeding programs to combine host resistances against several specific pathotypes of S. sclerotiorum into future cultivars.

In vitro and in vivo antifungal activity of synthetic pure isothiocyanates against Sclerotinia sclerotiorum

Isothiocyanates (ITCs) released by the enzymatic hydrolysis of glucosinolates in the Brassicaceae are potentially useful for controlling fungal pathogens. In vitro activity of pure ITCs against Sclerotinia sclerotiorum (Lib.) de Bary was studied by adding them to glass filters in petri dishes and dissolving them in the growing media. Methyl, allyl and benzyl ITCs were the most fungitoxic of the compounds in bioassays with S. sclerotiorum isolate Ss31. In the volatile phase, mycelial growth was completely inhibited by these three compounds. Aromatic ITCs were less toxic in the petri dishes but were more toxic than aliphatic ITCs when dissolved in the agar. Benzyl ITC exhibited the highest inhibitory effect on sclerotial germination, with an EC(50) value of 75.1 µmol L(-1) . Butyl and benzyl ITCs reduced apothecial production of S. sclerotiorum by 92.5% at the highest concentration. In in vivo assay, only allyl and 2-phenylethyl ITCs reduced disease incidence (by 76.7 and 70% respectively) at low concentrations. Sclerotinia sclerotiorum in the soil might be suppressed by the higher concentrations of allyl and benzyl ITCs released from decomposition of Brassica juncea, B. carinata, B. nigra and Sinapis spp.

Pathogenicity of morphologically different isolates of Sclerotinia sclerotiorum with Brassica napus and B. juncea 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.

First Report of Sclerotinia sclerotiorum Infecting Peanut in Texas.

Peanut (Arachis hypogaea L.) is grown extensively in several counties in West Texas. Sclerotinia blight, caused by the soilborne fungus Sclerotinia minor Jagger, is an increasingly important disease throughout this region. In September of 2007, diseased peanut plants (cv. Tamrun OL02) exhibiting symptoms of Sclerotinia blight (2,4) were collected from a commercial farm in Gaines County during a regional survey. Infected stem sections were surface disinfested in 0.5% sodium hypochlorite for 1 min, air dried, and placed in petri dishes containing water agar. Hyphae were subsequently transferred to petri dishes containing potato dextrose agar (PDA) after 3 days of incubation at room temperature. Pure cultures formed abundant, aerial, white mycelia, which later became darkly pigmented. Black, spherical to elongated sclerotia, 3.8 ± 0.8 mm, formed on the outer edge of petri dishes after 1 week. These characteristics are consistent with S. sclerotiorum (Lib.) de Bary (1,2). Pathogenicity tests were conducted by inoculating stems of greenhouse-grown peanut plants (cvs. Tamrun OL02, n = 4 and Flavorrunner 458, n = 4) with PDA plugs containing S. sclerotiorum. Mounting pins were used to create a shallow wound and affix inoculum plugs to stems. Control plants (n = 4) were inoculated with noncolonized PDA plugs. Plants were placed in a dew chamber at 20°C and 95% relative humidity in a randomized complete block design. The experiment was conducted two times. Symptoms identical to those observed on the original plant specimens were observed after 3 days on all inoculated plants; the controls remained healthy. Darkly pigmented cultures of S. sclerotiorum were consistently reisolated from all symptomatic stem sections. Fungal DNA was extracted from mycelia and sclerotia with a Qiagen DNeasy Plant Mini kit (Valencia, CA) and amplified by PCR using three S. sclerotiorum-specific primer pairs (3). PCR products for three replicates (two from mycelia and one from sclerotia) were sequenced and subjected to a NCBI BLAST search of highly similar sequences (megablast). The BLAST search revealed that our sequences are highly similar only with reported sequences of S. sclerotiorum. Sequences generated from three primer pairs in this study were 99, 95, and 95% homologous to S. sclerotiorum Accessions Nos. AF377925.1, AF377919.1, and AF377904.1 over 373, 376, and 300 bp of aligned sequence, respectively. Results from the pathogenicity tests and molecular study confirm that the S. sclerotiorum isolate obtained in this study is capable of inciting Sclerotinia blight of peanut. While S. minor is the primary causal agent of the disease, recent reports indicate that S. sclerotiorum is becoming more prevalent throughout the peanut-producing regions of the United States (2,4), and must therefore be considered in disease diagnosis.

Characterization of Sclerotinia sclerotiorum Isolated from Paprika

A fungal isolate collected from infected paprika (Capsicum annuum var. grossum) was characterized as Sclerotinia sclerotiorum based on its ability of selerotium formation, physiological and molecular properties. When the isolate was grown on potato dextrose agar, oatmeal agar, and malt extract agar, it grew most well on PDA. Optimal temperature and pH for its growth were 25°C and pH 7, respectively. The fungal isolate produced sclerotia on PDA within 10 days, and the color and shape of the sclerotia were similar to those of S. sclerotiorum. The ITS rDNA regions including ITS1 and ITS2 and 5.8S sequences were amplified using ITS1F and ITS4 primers from the genomic DNAs of the paprika isolate and other known pathogenic S. sclerotiorum isolated from different crops in Korea, and their nucleotide sequences were determined. Sequence comparison analysis showed the ITS rDNA of the paprika isolate shares 100% sequence identity with those of S. sclerotiorum isolated from red pepper, lettuce and a S. sclerotiorum isolate registered in Gen Bank DNA database. Neighbor joining analysis based on the ITS rDNA sequence revealed the paprika isolate has very close phylogenetic relationships with known Sclerotinia sclerotiorum isolates. This is the first report that S. sclerotiorum has been found associated with paprika rot in paprika growing countries.

First Report of Soft Rot Caused by Sclerotinia sclerotiorum on Borage in North Dakota.

Borage (Borago officinalis) is an oilseed crop that is being evaluated as an alternative crop in North Dakota. During September 2004, borage plants in a field in Cass County, North Dakota were dying from a watery soft rot. The main stems and lateral branches were affected, and affected plants were usually completely lodged and prostrate. Dead plants had bleached and shredded stems with black sclerotia (9.1 ± 3.0 × 2.6 ± 0.5 mm) inside the pith and on the epidermis. At the time of observation, borage plants were flowering and forming pods and seed. Approximately 60% of the plants were visually affected by the watery soft rot. Sclerotia were collected from diseased plants, soaked in a 0.5% NaOCL solution for 30 s, air dried, and placed in petri dishes containing potato dextrose agar (PDA). A fungus grew from the plated sclerotia that subsequently produced white mycelium and black sclerotia (4.8 ± 1.2 × 2.5 ± 1.0 mm), which is characteristic of Sclerotinia sclerotiorum (Lib.) de Bary (3). To confirm pathogenicity, borage plants were inoculated in the greenhouse with a S. sclerotiorum isolate from field-infected borage. Thirteen borage plants were grown from seed in the greenhouse under natural sunlight at a temperature range of 24 ± 3°C. When plants were at the four-leaf stage (approximately 16 cm high), the second leaf was excised from each plant with the petiole remaining on the plant. The leafless petioles were inoculated using a method previously described (2). Petioles of 10 plants were inoculated with PDA containing mycelium of the S. sclerotiorum borage isolate, while petioles of five plants were inoculated with PDA to serve as a control. Three days after inoculation, plants inoculated with the S. sclerotiorum borage isolate were beginning to wilt and 5 days after inoculation, these plants were completely wilted and prostrate, similar to observations made on field-infected plants. Sclerotia collected from the diseased, inoculated plants were placed on PDA, and S. sclerotiorum was successfully recovered. Control plants inoculated with PDA did not show any disease symptoms. Other plant genera in the Boraginaceae are known hosts of S. sclerotiorum (1); however, to our knowledge, this is the first report of borage as a host.

Interaction of Sclerotinia sclerotiorum with Brassica napus: cloning and characterization of endo- and exo-polygalacturonases expressed during saprophytic and parasitic modes

Five major and several minor PG isoenzymes were identified in a Sclerotinia sclerotiorum isolate from Brassica napus by isoelectric focusing and pectin gel overlays. Using a combination of degenerate PCR and expressed sequence tags (ESTs) four endo-polygalacturonase (PG) genes, designated as sspg1d, sspg3, sspg5, and sspg6, and two exo-PG genes, ssxpg1 and ssxpg2, were identified. SSPG1d is a member of the PG gene family previously described by Fraissinet-Tachet et al. [Curr. Genet. 29 (1995) 96]. The mature SSPG1d is a neutral PG, whereas fully processed SSPG3, SSPG5, and SSPG6 are acidic enzymes. Under saprophytic growth conditions, sspg1d, sspg3, sspg5, and ssxpg1 expression was induced by pectin and galacturonic acid and subject to catabolite repression by glucose. Conditions could not be identified under which sspg6 or ssxpg2 were expressed well. Transfer of mycelia from liquid media to solid substrates induced expression of sspg1d suggesting that it may also be regulated by thigmotrophic interactions. Under pathogenic conditions, sspg1d was highly expressed during infection. sspg3 was also expressed during infection, albeit at lower levels than sspg1d, whereas sspg5, sspg6, and ssxpg1 were expressed only weakly. Index Descriptors: Sclerotinia sclerotiorum; Endo-polygalacturonase; Exo-polygalacturonase; Brassica napus; Pathogenesis; Regulation

First Report of Sclerotinia sclerotiorum on Niger (Guizotia abyssinica).

Niger is a new crop being grown in North Dakota and Minnesota for the commercial birdseed market. In 2002, approximately 60 and 150 ha of niger were grown in North Dakota and Minnesota, respectively. In September 2002, niger plants in a field located near Prosper, ND showed the following symptoms and signs: bleached, shredded, and broken stems at the basal area, and presence of white mycelium and black sclerotia (2.2 ± 0.8 mm diameter) inside the pith cavity. Approximately 40% of plants in that field showed signs or symptoms of infection. Sclerotia were collected from the pith cavity, soaked in a 0.53% NaOCL solution for 30 s, air dried, and placed in petri dishes containing potato dextrose agar (PDA). A fungal colony grew out from the plated sclerotia that subsequently produced aerial white mycelium and black sclerotia characteristic of Sclerotinia sclerotiorum (Lib.) de Bary. To confirm pathogenicity, 2 groups of 10 30-day-old niger seedlings each were inoculated using the straw test method (2) or petiole inoculation test method (1) with mycelium from a S. sclerotiorum isolate obtained from an infected niger plant; 20 seedlings served as a noninoculated control. At the time of inoculation, seedlings were healthy and approximately 14 cm high. Three days after inoculation using either method, tissue at the inoculated area turned gray. The gray lesions progressed across the majority of the seedling tissue and 1 week after inoculation, all inoculated plants were wilted and dead. S. sclerotiorum was reisolated from infected tissue that was placed on PDA. To our knowledge, this is the first report of S. sclerotiorum causing a stem rot disease of niger or any species in the Guizotia genus. Several of the major crops grown in North Dakota and Minnesota, such as canola, dry edible bean, soybean, and sunflower are susceptible to S. sclerotiorum. Growing niger in rotation with these or other susceptible crops could increase S. sclerotiorum inoculum levels and lead to severe disease outbreaks if conditions are favorable.