White Mold Disease Research Articles

Influence of irrigation and plant canopy architecture on white mould disease of dry bean

White mould caused by the fungus Sclerotinia sclerotiorum (Lib.) de Bary is a major constraint to irrigated dry bean production in southern Alberta. Irrigation, coupled with dry bean canopy architecture, may influence white mould by creating conducive environmental conditions. Field experiments were conducted from 2015 to 2017 at Lethbridge to determine the effect of three irrigation levels and five dry bean genotypes with different canopy architectures on white mould. Sensors and data loggers were established to monitor micro-climate data including soil moisture within the top 5 cm, leaf wetness, and soil temperature under the canopy. Canopy porosity, lodging, flower infection, and white mould disease severity were also measured. Higher moisture within the top 5 cm of the soil, lower soil temperature, elevated leaf wetness, and higher white mould incidence were observed in high irrigation plots compared with medium and low irrigation plots. Cultivars varied for leaf wetness, porosity, and lodging. Although a significant interaction between irrigation and cultivar was detected, irrigation levels did not affect disease severity significantly. Lower disease severity and incidence were recorded in AAC Burdett and Island. These cultivars have an upright growth habit, high canopy porosity, and lodging resistance, and therefore, exhibited partial field resistance (avoidance) to white mould. Mean yield across all cultivars was not affected by irrigation; however, the highest yield occurred in the medium irrigation plots. A reduced level of irrigation and development of cultivars with both avoidance and partial physiological resistance may reduce white mould severity and incidence in dry bean fields in Alberta.

Assessing the Aerial Interconnectivity of Distant Reservoirs of Sclerotinia sclerotiorum.

Many phytopathogenic fungi are disseminated as spores via the atmosphere from short to long distances. The distance of dissemination determines the extent to which plant diseases can spread and novel genotypes of pathogens can invade new territories. Predictive tools including models that forecast the arrival of spores in areas where susceptible crops are grown can help to more efficiently manage crop health. However, such models are difficult to establish for fungi with broad host ranges because sources of inoculum cannot be readily identified. Sclerotinia sclerotiorum, the pandemic agent of white mold disease, can attack >400 plant species including economically important crops. Monitoring airborne inoculum of S. sclerotiorum in several French cropping areas has shown that viable ascospores are present in the air almost all the time, even when no susceptible crops are nearby. This raises the hypothesis of a distant origin of airborne inoculum. The objective of the present study was to determine the interconnectivity of reservoirs of S. sclerotiorum from distant regions based on networks of air mass movement. Viable airborne inoculum of S. sclerotiorum was collected in four distinct regions of France and 498 strains were genotyped with 16 specific microsatellite markers and compared among the regions. Air mass movements were inferred using the HYSPLIT model and archived meteorological data from the global data assimilation system (GDAS). The results show that up to 700 km could separate collection sites that shared the same haplotypes. There was low or no genetic differentiation between strains collected from the four sites. The rate of aerial connectivity between two sites varied according to the direction considered. The results also show that the aerial connectivity between sites is a better indicator of the probability of the incoming component (PIC) of inoculum at a given site from another one than is geographic distance. We identified the links between specific sites in the trajectories of air masses and we quantified the frequencies at which the directional links occurred as a proof-of-concept for an operational method to assess the arrival of airborne inoculum in a given area from distant origins.

First Report of a Melanotaenium Species Causing White Mold Disease of Tremella fuciformis in China.

Tremella fuciformis is a widely grown edible mushroom in Asia, especially in China for its medicinal and food value (Stamets 2000). In July 2017, white mold infection was observed on T. fuciformis fruiting bodies in a production unit of Youxi, Fujian, China. Approximately 10% of the mushrooms were affected by the mycoparasite and were found not fit for consumption. Initial symptoms appeared as inconspicuous white spots on the surface of T. fuciformis fruiting bodies. The mycoparasite formed a white mass (1 to 3 cm wide and 2 to 4 cm long) within 2 days. Within 5 to 7 days the mycelial mass covered the whole T. fuciformis fruiting body and caused decay. The mycoparasite was collected directly from infected fruiting bodies with sterile needles, inoculated on potato dextrose agar (PDA) and malt extract agar (MEA) plates, and incubated at 23°C. After 5 to 6 days, many cream-white fungal colonies (0.1 to 3.5 mm wide and 1.0 to 6.0 mm long) with limited growth and irregular margins were observed on PDA and MEA plates. Mycelium was aseptate and hyaline. Ballistospores were sessile, hyaline, aseptate, lunate (2.5 to 3.5 µm wide and 8 to 14 µm long), and attached to the side or tip of the hyphae. Teliospores were not present. Morphological characteristics were consistent with those of Melanotaenium endogenum (Ingold 1988). For molecular identification, the internal transcribed spacer (ITS) region and ELF-1α (White et al. 1990; Rehner and Buckley 2005) were amplified and sequenced. A BLAST search in GenBank indicated that amplified ITS sequences of 668 bp (MG806985) had 89 to 88% similarity to Melanotaenium species sequences (DQ789981, DQ875347, and JN367289) and ELF-1α sequences of 497 bp (MH327256) had 92% similarity to Melanotaenium euphorbiae (JN367365). For inoculum production, mycelial discs were aseptically transferred into potato dextrose broth (PDB) and incubated in dark at 23°C with continuous shaking at 150 rpm for 7 to 10 days. Mycelium was separated from PDB by filtration, and the ballistospore suspension (1 × 10⁷ conidia/ml of sterile water) was prepared with filtrate for inoculation. Each fruiting body of T. fuciformis (30 biological replicates in total) was sprayed with the ballistospore suspension, and sterile distilled water served as a control. Inoculated fruiting bodies and controls were maintained at 23°C and 95% relative humidity. White colonies of the mycoparasite developed on T. fuciformis fruiting bodies 2 days after inoculation. The symptoms were similar to those of the original diseased fruiting bodies, and the controls remained asymptomatic. The mycoparasite was reisolated from the infected fruiting bodies and identified as Melanotaenium sp. based on morphological and molecular analysis. To our knowledge, this is the first report of Melanotaenium sp., causing white mold disease on T. fuciformis worldwide. T. fuciformis is one of the most popular fungi in the cuisine and medicine of China, and the presence of Melanotaenium infection is of great concern to producers of T. fuciformis.

Genetic Diversity Studies Based on Morphological Variability, Pathogenicity and Molecular Phylogeny of the Sclerotinia sclerotiorum Population From Indian Mustard (Brassica juncea).

White mold or stem rot disease are ubiquitously distributed throughout the world and the causal organism of this disease Sclerotinia sclerotiorum (Lib.) de Bary, is known to infect over 400 plant species. Sclerotinia stem rot is one of the most devastating fungal diseases and poses a serious threat to the worldwide cultivation of oilseed Brassica including India. S. sclerotiorum pathogen usually infects the stem but in severe cases leaves and pods also affected at different developmental stages that deteriorate not only the oil quality but also causing the seed and oil yield losses up to 90% depending on the severity of the disease infestation. This study investigated the morphological and molecular characterization of pathogenic S. sclerotiorum (Lib) de Bary geographical isolates from oilseed Brassica including Brassica juncea (Indian mustard). The aim of this study was to compare isolates of S. sclerotiorum originated from different agro-climatic conditions and to analyse similarity or differences between them as well as to examine the virulence of this pathogen specifically in Brassica for the first time. The collection of S. sclerotiorum isolates from symptomatic Brassica plants was done and analyzed for morphological features, and molecular characterization. The virulence evaluation test of 65 isolates on four Brassica cultivars has shown 5 of them were highly virulent, 46 were virulent and 14 were moderately virulent. Phylogenetic analysis encompassing all the morphological features, SSR polymorphism, and ITS sequencing has shown the existence of high genetic diversity among the isolates that categorized all the isolates in three evolutionary lineages in the derived dendrogram. Further, genetic variability analysis based on sequences variation in ITS region of all the isolates has shown the existence of either insertions or deletions of the nucleotides in the ITS region has led to the interspecies variability and observed the variation were in a clade-specific manner. Together this analysis observed the existence of higher heterogeneity and genetic variability in S. sclerotiorum isolates collection and indicates the presence of clonal and sexual progenies of the pathogen in the mustard growing regions of India surveyed in this study. With a higher level of genetic variability and diversity among the S. sclerotiorum population needs robust screening approaches to identify the donor parent and utilize them in resistance breeding program for effectively counter the menace of stem rot disease in Brassica.

Trichoderma asperelloides antagonism to nine Sclerotinia sclerotiorum strains and biological control of white mold disease in soybean plants

ABSTRACTSclerotinia sclerotiorum is an important plant pathogen with worldwide distribution that causes severe economic losses of various agricultural crops such as soybean. This fungus is normally controlled with synthetic chemical fungicides that pose risks to the environment, and can be harmful to human health, and they can also induce resistance in pests. The aim of this study was to investigate the potential of Trichoderma asperelloides as a biocontrol agent towards white mold disease on soybeans crops. The antagonism of two strains of T. asperelloides (T25 and T42) isolated from soil samples was determined in-vitro by dual-culture confrontation testing on nine S. sclerotiorum strains obtained from sclerotia collected on diseased soybean plants. The mycelial growth and inhibition of carpogenic and ascospore germination by T. asperelloides extracts, as well as the efficacy of these on white mold control in soybeans were evaluated. Both strains of T. asperelloides exhibited high potential of antagonism. Methanolic and ethyl acetate extracts of the two T. asperelloides strains showed excellent growth inhibition (60–100%) on all of the pathogens tested. The ethyl acetate extracts of both T. asperelloides strains exhibited the highest efficacy against carpogenic germination, decreasing by 20–30% the number of ascospores per apothecium. Strains of T. asperelloides tested were more efficient in controlling white mold than two commercial products made from Trichoderma harzianum. The new strains of T. asperelloides have potential for successful biological control of white mold disease of soybean crops in the field.

First Report of Basal Drop and White Mold on Lettuce, Broccoli, and Mustard Caused by Sclerotinia sclerotiorum in Tennessee, U.S.A.

Sclerotinia sclerotiorum causes basal drop and white mold diseases on a wide range of vegetable crops, resulting in significant economic loss (Purdy 1979). Diseased plants of various vegetables with black irregular to cylindrical sclerotia were observed in high tunnels of an organic farm near Knoxville, TN, in February of 2016 and 2017. Leafy mustard (Brassica juncea cv. Purple Osaka) had necrotic leaves and stems, with black sclerotia inside stems. Chinese broccoli (Brassica oleracea var. alboglabra cv. Te You) had water soaked lesions on stems and leaves with white mycelium and black sclerotia. Two kinds of lettuce (Lactuca sativa); butterhead cv. Mirlo and red leaf cv. Cherokee, had symptoms of lettuce drop, i.e., water soaked leaves and crown with white mycelial mats and black sclerotia. Infected plants (1- to 3-cm pieces) and sclerotia, collected in both years, were surface sterilized in 10% commercial bleach for 1 min, followed by 1 min in 10% ethanol. Samples were plated on potato dextrose agar amended with 10 mg/liter rifampicin and 3.45 mg fenpropathrin/liter (aPDA) and incubated at room temperature. After 3 days, white fluffy mycelia grew from infected tissues, and after 7 to 10 days, black sclerotia (1.5 to 10.0 mm × 2.0 to 6.0 mm) were formed. Apothecia from sclerotia were induced on vermiculite according to Pratt (1992). Asci contained eight uniform, binucleate, elliptical ascospores (Kohn 1979). The fungus was identified as S. sclerotiorum (Lib.) de Bary based on morphology. For molecular identification, genomic DNA was extracted (Extract-N-Amp, Sigma-Aldrich, MO) from mycelia. PCR amplicons were produced and sequenced using internal transcribed spacer (ITS) region primers ITS1 and ITS4. Comparison with reference sequences in GenBank (e.g., accession nos. KP340898, KX184720.1, JQ618848) resulted in 100% identity to ITS sequences of S. sclerotiorum. The ITS sequences (520 to 531 bp) were deposited in GenBank (KY859157 [broccoli], KY859158 [lettuce], KY859159 [mustard]). To complete Koch’s postulates, 7-day-old mycelial plugs from aPDA were placed on two 55-day-old lettuce cultivars (PIC 714 and New Red Fire) and 35-day-old broccoli cv. Te You and mustard cv. Purple Osaka grown in soilless growing medium. For each cultivar, the stem at the soil line and newly expanded leaves and petioles of four healthy plants were each inoculated with three to five mycelial plugs (1-cm²-diam.) of the pathogen grown on aPDA, and four more were inoculated with aPDA only as the control. Plants were covered with plastic, and sprayed with water (under the plastic) twice daily for 3 to 5 days to maintain high humidity, and kept in a growth chamber at 25°C. After 7 to 10 days, all pathogen-inoculated plants had symptoms identical to those observed in the field, with or without sclerotia; control plants showed no symptoms. The fungus was reisolated from infected plants, and identity was confirmed with ITS sequence analysis. To our knowledge, this is the first report of S. sclerotiorum on broccoli, mustard, and lettuce in Tennessee. For organic growers where crop rotation is the primary control option, occurrence of this pathogen on multiple crops could cause significant loss.

Economic Feasibility of Canola Production in the Region of Campos Gerais, Paraná, Brazil

This study aimed to analyze the economic feasibility of canola production (Brassica napus var oleifera) for the region Campos Gerais-PR. We interviewed eight producers, and used information from four of them for economic analysis of agricultural crops in 2012, 2013 and 2014. The variables analyzed were: grain production cost, economical analysis (hurdle rate, net present value, internal rate of return, payback, lucratively index, profitability index and sensitivity analysis) and vegetable oil production costs. We compared the performance of canola, wheat (Triticum aestivum)—as the main crop in the autumn/winter—and soybean (Glycine max)—by liquidity in the world market. It was concluded that although the economic analysis indicates superior results of canola production compared to the wheat crop and vegetable oil production is more attractive financially than soybean, but the culture of canola is still insignificant in the region of Campos Gerais (PR). The producers interviewed pointed to limit the expansion of culture financing the limited farming, production technology in consolidation, the need for machine adjustments and canola be host of the white mold disease (Sclerotinia sclerotiorun).

Parallel evolution of the POQR prolyl oligo peptidase gene conferring plant quantitative disease resistance.

Plant pathogens with a broad host range are able to infect plant lineages that diverged over 100 million years ago. They exert similar and recurring constraints on the evolution of unrelated plant populations. Plants generally respond with quantitative disease resistance (QDR), a form of immunity relying on complex genetic determinants. In most cases, the molecular determinants of QDR and how they evolve is unknown. Here we identify in Arabidopsis thaliana a gene mediating QDR against Sclerotinia sclerotiorum, agent of the white mold disease, and provide evidence of its convergent evolution in multiple plant species. Using genome wide association mapping in A. thaliana, we associated the gene encoding the POQR prolyl-oligopeptidase with QDR against S. sclerotiorum. Loss of this gene compromised QDR against S. sclerotiorum but not against a bacterial pathogen. Natural diversity analysis associated POQR sequence with QDR. Remarkably, the same amino acid changes occurred after independent duplications of POQR in ancestors of multiple plant species, including A. thaliana and tomato. Genome-scale expression analyses revealed that parallel divergence in gene expression upon S. sclerotiorum infection is a frequent pattern in genes, such as POQR, that duplicated both in A. thaliana and tomato. Our study identifies a previously uncharacterized gene mediating QDR against S. sclerotiorum. It shows that some QDR determinants are conserved in distantly related plants and have emerged through the repeated use of similar genetic polymorphisms at different evolutionary time scales.

Large‐seeded Common Bean PRA152, PRA154, and PRA155 with High Levels of Broad‐Spectrum White Mold Resistance

Large‐seeded common bean (Phaseolus vulgaris L.) breeding lines (BL) PRA152 (Reg. No. GP‐304, PI 680637), PRA154 (Reg. No. GP‐302, PI 680635), and PRA155 (Reg. No. GP‐303, PI 680636) were jointly developed at the University of Idaho, Kimberly Research and Extension Center and Colorado State University, Fort Collins. The three BL possess the highest levels of broad‐spectrum white mold resistance (mean disease scores ranging from 3.0 to 3.8) than in previously available large‐seeded common bean BL, germplasm accessions, and cultivars to Sclerotinia sclerotiorum (causal agent of white mold disease) isolates ARS12D, ARS14D, ARS14M, ARS15T, CO467, ND710, and NY133. PRA152 was derived from Cornell 501/G 122//A 195/VCW 55, PRA154 was developed from VA 19/M0162//A 195/G 122, and PRA155 was selected from A 195/4/NY6020‐4/92BG‐7///M0162/I9365‐25//‘ICA Bunsi’/G 122 common bean populations. All BL have a determinate upright growth habit Type I. PRA152 has dark greenish gray–colored seeds with average weight of 45.6 g 100 seed−1. PRA154 has beige mottled seeds with 48.0 g 100 seed−1. Seeds of PRA155 are cream with black spots, with 26.9 g 100 seed−1. PRA154 matures in <100 d, and PRA152 and PRA155 require approximately 105 to 110 d from planting to harvest maturity in southern Idaho. White mold resistance from the three BL should be combined with other complementary sources of resistance to breed for yet higher levels and broader spectrum of resistance and/or introgressed into common bean cultivars of different market classes to combat white mold disease in the United States and elsewhere.

Lower Frequency and Diversity of Antibiotic-Producing Fluorescent Pseudomonads in Rhizosphere of Indian Rapeseed–Mustard (Brassica juncea L. Czern.)

Fluorescent pseudomonads are one of the most important microbial communities which play a key role in rhizosphere to enhance plant growth-promotion and protection. The diverse groups of antibiotics viz. 2,4-diacetylphloroglucinol (DAPG), phenazine-1-carboxylic acid (PCA) and pyoluteorin (PLT) are produced by fluorescent pseudomonads inhibiting growth of fungal pathogens which results in health upliftment of plants. The present study, discusses about frequency and diversity of 138 antibiotic-producing fluorescent pseudomonads isolated from eight genotypes of rapeseed mustard rhizosphere (Brassica juncea L. Czern.). The plant growth promoting traits and antibiotics (DAPG, PCA and PLT) production of isolates were examined by using polymerase chain reaction (PCR), thin layer chromatography (TLC) and dot blot-hybridization. Among 138 isolates, 47, 25 and 9 % of isolates were positive in indole production, phosphate solubilization and antagonism potential against Sclerotinia sclerotiorum (causal agent of white mold disease in rapeseed mustard), respectively. PCR amplifications showed that none of the isolates had phlD (DAPG) and phzC (PCA) genes, but four isolates (UKA-2, UKA-8, UKA-11, UKA-66) had pltB (PLT) gene, which was further confirmed by TLC and DNA dot-blot hybridization. BOX profiles of pltB positive isolates were distinct, showing unique genetic diversity in the small population. The four pltB positive fluorescent pseudomonad isolates could be used as promising bio-control and plant growth-promoting inoculants for Indian rapeseed mustard.

English

Common bean (Phaseolus vulgaris) is one of the main Brazilian agricultural crop. It is affected by numerous diseases during its life cycle; one of these includes the white mold caused by the fungus Sclerotinia sclerotiorum. The objective of this study was to verify the control of white mold in bean plants by homeopathic medicines. Two tests were performed: In the first test, the medicine Calcarea carbonica was used, while phosphorus was used in the second test. Both medicines were administered at dynamizations of 6, 12, 24, 36, and 48 CH  (Hahnemanniana Centesimal). In the control treatment, 30% hydroalcoholic solution was used. The study was conducted using a completely randomized design, analyzing the following variables: Area under the disease progress curve and percentage of dead plants. The results were submitted to Tukey’s test and regression analysis (p > 0.05). Calcarea carbonica at 6 CH and Phosphorus at 6 CH, 12 CH, 24 CH, 36 CH, and 48 CH reduced the intensity of white mold in bean plants. With the exception of Calcarea carbonica at 12 CH and 24 CH, no other medicine reduced the percentage of plant death due to white mold disease.   Key words: Homeopathy, alternative control, Sclerotinia sclerotiorum, Phaseolus vulgaris.

Registration of Common Bean Pinto PRP 153 and VCP 13 with High Levels of Broad‐Spectrum White Mold Resistance

Common bean (Phaseolus vulgaris L.) pinto breeding lines PRP 153 (Reg. No. GP‐300, PI 676973) and VCP 13 (Reg. No. GP‐301, PI 676974) possess high levels of broad‐spectrum (i.e., combining two or more sources of resistance) resistance to white mold [caused by Sclerotinia sclerotiorum (Lib.) de Bary]. Both breeding lines were jointly developed at the University of Idaho, Kimberly Research and Extension Center and Colorado State University, Fort Collins. VCP 13 was derived from an interspecific recurrent backcross population between common bean pinto ‘UI 320’ and P. coccineus PI 439534 (UI 320*2/PI 439534). PRP 153 was selected from the double‐cross USPT‐WM‐1/CORN 601//USPT‐CBB‐1/92BG‐7. PRP 153 and VCP 13 have significantly (P ≤ 0.05) higher levels of resistance (mean disease scores ranging from 3.0 to 3.7) than previously reported in common bean to S. sclerotiorum isolates ARS12D, ARS14D, ARS14M, ARS15T, CO467, ND710, and NY133. PRP 153 also has resistance to the less‐aggressive strain ARX8AC of the common blight pathogen, Xanthomonas campestris pv. phaseoli Smith (Dye). Both lines have an indeterminate prostrate growth habit Type III. They are late maturing, requiring 100 d or longer from planting to harvest maturity in southern Idaho. White mold resistance from PRP 153 and VCP 13 should be combined together with other contrasting and complementary sources of resistance to breed for yet higher levels of broad‐spectrum resistance and/or introgressed into common bean cultivars of different market classes to combat white mold disease in the United States and elsewhere.

Biological control of Sclerotinia sclerotiorum (Lib.) de Bary, the causal agent of white mould disease in red cabbage, by some bacteria

Biological control of Sclerotinia sclerotiorum (Lib.) de Bary, the causal agent of white mould disease in red cabbage, by some bacteria

TMT-based quantitative proteomics analyses reveal novel defense mechanisms of Brassica napus against the devastating necrotrophic pathogen Sclerotinia sclerotiorum

The white mould disease, caused by Sclerotinia sclerotiorum, is one of the most important diseases in the vital oil crop Brassica napus. Nevertheless, the defense mechanisms of B. napus against S. sclerotiorum are poorly understood. In this study, we performed comparative quantitative proteomics analyses to reveal B. napus defense mechanisms against S. sclerotiorum. The proteomes of B. napus leaves inoculated with S. sclerotiorum wild-type strain 1980 and nonpathogenic mutant strain Ep-1PB as well as empty agar plug as the control were analyzed using TMT label-based quantitative analysis technique. A total of 79, 299 and 173 proteins consistently differentially expressed between Ep-1PB- and mock-inoculated leaves, 1980- and mock-inoculated leaves, as well as 1980- and Ep-1PB-inoculated leaves, respectively, were identified. The differential expression of 12 selected proteins was confirmed by qRT-PCR analyses. The Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction prediction analyses revealed that redox homeostasis, lipid signaling, calcium signaling, histone and DNA methylation-mediated transcription regulation and defense-related proteins such as defensin and defensin-like proteins and cyanate lyase, contribute to defense against S. sclerotiorum. Our results provide new insights into molecular mechanisms that may be involved in defense responses of B. napus to S. sclerotiorum. SignificanceThe Sclerotinia white mould disease is one of the most important diseases in the significant oil crop Brassica napus. Nevertheless, the defense mechanisms of B. napus against S. sclerotiorum are still largely unknown to date. In this study, we addressed this issue by performing TMT label-based comparative quantitative analyses of the proteomes of B. napus leaves inoculated with S. sclerotiorum wild-type strain 1980 and nonpathogenic mutant strain Ep-1PB as well as empty agar plug as the control. Through comparative analyses on 79, 299, and 173 proteins that are consistently differentially expressed in between Ep-1PB-inoculated and the control leaves, 1980-inoculated and the control leaves, as well as 1980-inoculated and Ep-1PB-inoculated leaves, respectively, we revealed that redox homeostasis, lipid signaling, calcium signaling, histone and DNA methylation-mediated transcription regulation and defense-related proteins such as defensin and defensin-like proteins as well as cyanate lyase, contribute to B. napus defenses against S. sclerotiorum. Notably, the potential role of lipid signaling, calcium signaling, histone and DNA methylation-mediated transcription regulation and cyanate lyase in B. napus defense against S. sclerotiorum are not reported previously but rather unveiled for the first time in this study. The current study represents the most extensive analysis of the protein profile of B. napus in response to S. sclerotiorum inoculation and includes for the first time the results from comparison between plants inoculated with the wild-type strain and a nonpathogenic mutant strain of S. sclerotiorum. Collectively, our results provide new insights into the molecular mechanisms of interactions between B. napus and S. sclerotiorum.

Suppressive effects of Bacillus spp. on mycelia, apothecia and sclerotia formation of Sclerotinia sclerotiorum and potential as biological control of white mold on mustard

Biological control, especially with Bacillus-based biocontrol agents, offers an attractive alternative to synthetic pesticides for sustainable management of white mold disease caused by Sclerotinia sclerotiorum. In this study, eight effective Bacillus isolates were isolated from rhizospheric soil samples as potential bacterial biocontrol agents. Cultural, biochemical, and molecular analyses of 16S rDNA and gyrase subunit A (gyrA) confirmed that all isolates were identified as Bacillus amyloliquefaciens subsp. plantarum. The production of hydrolytic enzymes and the plant growth-promotional attributes of these isolates confirmed their multifaceted potential. Molecular analysis of the eight biosynthetic genes, which are related to antibiotic properties of bacilli, revealed that all of the isolates possess five genes: bacA for bacilysin, dfnM for difficidin, fenA for fengycin, ituA for iturin, and sfp for surfactin. The Bacillus isolates inhibited mycelial growth and suppressed formation of sclerotia during an in vitro test against S. sclerotiorum. Deformities and cell-wall lysis of mycelia, abnormalities of apothecia, and germination failure of ascospores through interaction with the Bacillus isolates were observed with light and scanning electron microscopes, suggesting that they have high antagonistic effects against S. sclerotiorum. Seed bacterization with the Bacillus isolates protected mustard seedlings in vitro up to 98 % against S. sclerotiorum. In a pot experiment, damages of mustard plants against the pathogen decreased up to 90 % after foliar spray of the Bacillus isolates. In addition, the isolates increased seed germination and accelerated seedling vigor of mustard, suggesting that they have plant growth-promoting functions.

Use of petal test in early-flowering varieties of oilseed rape (Brassica napus L.) for predicting the infection pressure of Sclerotinia sclerotiorum (Lib.) de Bary

Sclerotinia sclerotiorum is one of the most important pathogens of winter oilseed rape plants. It causes the white mould disease of rape, thus significantly reducing the yield. The aim of our research was to use a spring oilseed rape variety sown in autumn or an early-flowering variety of winter oilseed rape to predict the infection pressure of S. sclerotiorum in a given year. Field experiments were conducted in 2008–2012 at the experimental station of the Czech University of Life Sciences Prague at Červený Újezd, 20 km west of Prague. In the experiment, we used one variety of spring oilseed rape (Lužnice, LU) and one early-flowering variety of winter oilseed rape (Californium, CA). The methodology of the petal test involved isolating pathogenic fungi from fallen petals on a nutrient medium (potato dextrose agar) in Petri dishes. The first term of collection was the beginning of petal fall (BBCH 61) and the last term was the end of flowering (BBCH 69). The dishes with petals were visually analysed after 1 week. The results were compared with the actual occurrence of white mould of rape in the stand. The occurrence of white mould of rape was strongly influenced by the progress of weather conditions over the given year. Infected petals and stems with symptoms of white mould of rape were found to be moderately correlated (r = 0.80). Spread of white mould spores was mostly observed in two terms (BBCH 62 and BBCH 65). Statistically significant differences were not observed in the infection of petals of spring (LU) and winter (CA) oilseed rape. Spring oilseed rape (LU) and early-flowering varieties of winter oilseed rape (CA) can be used to determine the strength of infection pressure of S. sclerotiorum in the stand in a given year, thereby improving protection against white mould of rape.

Adaptation and Resistance to Diseases in Brazil of Putative Sources of Common Bean Resistance to White Mold.

Common bean breeding programs for white mold (WM) resistance are in their initial stages in Brazil. Sources of partial resistance to WM are available abroad but their performance in Brazil is unknown. In two greenhouse (straw test) and three field experiments conducted in three districts in the state of Minas Gerais, Brazil, we evaluated a total of 23 lines with putative WM resistance with the objective to select lines with resistance to WM and other diseases associated with high yield potential. Two field-resistant local lines, two susceptible local cultivars, and two susceptible international lines were also included in the study. In the greenhouse, Cornell 605, A 195, and G122 were among the lines with the highest partial resistance to WM. In the field, these three lines were highly resistant to WM and had intermediate resistance or were resistant to anthracnose, angular leaf spot, rust, and Fusarium wilt. Cornell 605 and A 195 had high-yield potential but G122 yielded 47% less than the local lines under WM pressure. Our results suggest that Cornell 605 and A 195 are the most useful sources of resistance to WM for use in common bean breeding programs in Brazil.

Transfection of Sclerotinia sclerotiorum with in vitro transcripts of a naturally occurring interspecific recombinant of Sclerotinia sclerotiorum hypovirus 2 significantly reduces virulence of the fungus.

A recombinant strain of Sclerotinia sclerotiorum hypovirus 2 (SsHV2) was identified from a North American Sclerotinia sclerotiorum isolate (328) from lettuce (Lactuca sativa L.) by high-throughput sequencing of total RNA. The 5'- and 3'-terminal regions of the genome were determined by rapid amplification of cDNA ends. The assembled nucleotide sequence was up to 92% identical to two recently reported SsHV2 strains but contained a deletion near its 5' terminus of more than 1.2 kb relative to the other SsHV2 strains and an insertion of 524 nucleotides (nt) that was distantly related to Valsa ceratosperma hypovirus 1. This suggests that the new isolate is a heterologous recombinant of SsHV2 with a yet-uncharacterized hypovirus. We named the new strain Sclerotinia sclerotiorum hypovirus 2 Lactuca (SsHV2L) and deposited the sequence in GenBank with accession number KF898354. Sclerotinia sclerotiorum isolate 328 was coinfected with a strain of Sclerotinia sclerotiorum endornavirus 1 and was debilitated compared to cultures of the same isolate that had been cured of virus infection by cycloheximide treatment and hyphal tipping. To determine whether SsHV2L alone could induce hypovirulence in S. sclerotiorum, a full-length cDNA of the 14,538-nt viral genome was cloned. Transcripts corresponding to the viral RNA were synthesized in vitro and transfected into a virus-free isolate of S. sclerotiorum, DK3. Isolate DK3 transfected with SsHV2L was hypovirulent on soybean and lettuce and exhibited delayed maturation of sclerotia relative to virus-free DK3, completing Koch's postulates for the association of hypovirulence with SsHV2L. A cosmopolitan fungus, Sclerotinia sclerotiorum infects more than 400 plant species and causes a plant disease known as white mold that produces significant yield losses in major crops annually. Mycoviruses have been used successfully to reduce losses caused by fungal plant pathogens, but definitive relationships between hypovirus infections and hypovirulence in S. sclerotiorum were lacking. By establishing a cause-and-effect relationship between Sclerotinia sclerotiorum hypovirus Lactuca (SsHV2L) infection and the reduction in host virulence, we showed direct evidence that hypoviruses have the potential to reduce the severity of white mold disease. In addition to intraspecific recombination, this study showed that recent interspecific recombination is an important factor shaping viral genomes. The construction of an infectious clone of SsHV2L allows future exploration of the interactions between SsHV2L and S. sclerotiorum, a widespread fungal pathogen of plants.

Identification of QTLs of resistance to white mold in common bean from multiple markers by using Bayesian analysis.

In this study, we identified simple sequence repeat, ampli-fied fragment length polymorphism, and sequence-related amplified poly-morphism markers linked to quantitative trait loci (QTLs) for resistance to white mold disease in common bean progenies derived from a cross between lines CNFC 9506 and RP-2, evaluated using the oxalic acid test and using Bayesian analysis. DNA was extracted from 186 F₂ plants and their parental lines for molecular analysis. Fifteen experiments were car-ried out for phenotypic analysis, which included 186 F₂:₄ progenies, the F₁ generation, the F₂ generation, and the lines CNFC 9506, RP-2, and G122 as common treatments. A completely randomized experimental design with 3 replications was used in controlled environments. The adjusted means for the F₂:₄ generation were to identify QTLs by Bayesian shrink-age analysis. Significant differences were observed among the progenies for the reaction to white mold. The moving away method under the Bayes-ian approach was effective for identifying QTLs when it was not possible to obtain a genetic map because of low marker density. Using the Wald test, 25 markers identified QTLs for resistance to white mold, as well as 16 simple sequence repeats, 7 amplified fragment length polymorphisms, and 2 sequence-related amplified polymorphisms. The markers BM184, BM211, and PV-gaat001 showed low distances from QTLs related white mold resistance. In addition, these markers showed, signal effects with increasing resistance to white mold and high heritability in the analysis with oxalic acid, and thus, are promising for marker-assisted selection.

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.