Showing papers on "Sclerotinia sclerotiorum published in 2023"

Activity of Essential Oils and Plant Extracts as Biofungicides for Suppression of Soil-Borne Fungi Associated with Root Rot and Wilt of Marigold (Calendula officinalis L.)

Abstract: Essential oils (EOs) and Plant extracts (PEs) are gaining attention as eco-friendly alternatives to synthetic fungicides for the management of soil-borne fungi related to root rot and the wilt of marigolds. Here, EOs of Cinnamomum zeylanicum (cinnamon), Mentha piperita (peppermint), Syzygium aromaticum (clove), Thymus vulgaris (thyme), PEs of Cymbopogon citratus (lemongrass), Lantana camara (lantana), Ocimum basilicum (basil), and Zingiber officinales (ginger) were first evaluated in vitro for their inhibitory activity against the mycelium growth of the root rot and wilt fungi of marigold: Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, and F. solani, and in vivo for their activity in reducing disease progression. The results show that all EOs had a strong inhibitory activity on the mycelium growth of pathogens. Maximum inhibition of mycelium growth was achieved at a concentration of 1 mL/Lof S. aromaticum, C. zeylanicum, and M. piperita. The inhibition values were 100, 80.9, and 72.4% for F. solani, 100, 81.1, and 70% for S. sclerotiorum, 90.2, 79.4, and 69.1% for F. oxysporum, and 85.4, 78.2, and 68.7% for R. solani, respectively. Regarding plant extracts, the highest inhibition of mycelium growth was attained at a concentration of 20% of Z. officinales, C. citratus, and L. camara. The inhibition values were 77.4, 69.1, and 60.6% for F. solani, 76.5, 67.2, and 58% for S. sclerotiorum, 73.5, 68.2, and 56.3% for F. oxysporum, and 72, 64.8, and 55.2% for R. solani, respectively. In pot experiments, the application of EOs and PEs showed high efficiency in suppressing root rot and wilt of marigold at all concentrations used, especially at 3 mL/L for seed soaking (along with 1.5 mL/L for foliar spraying) for S. aromaticum, C. zeylanicum, and M. piperita EOs, and (40% for seed soaking along with 20% for foliar spraying) for Z. officinales, C. citratus, and L. camara PEs. All the treatments applied in the field greatly reduced the diseases in both seasons, especially S. aromaticum EO, C. zeylanicum EO, Z. officinales PE, and carbendazim treatments. This was accompanied by a significant improvement in morphological, yield, and phytochemical parameters of marigold as well as a significant increase in the activity of defense-related antioxidant enzymes. Overall, essential oils and plant extracts can be used effectively to control root rot and wilt in marigold as sustainable and eco-friendly botanical biofungicides.

Sclerotinia sclerotiorum (Lib.) de Bary: Insights into the Pathogenomic Features of a Global Pathogen

Abstract: Sclerotinia sclerotiorum (Lib.) de Bary is a broad host-range fungus that infects an inclusive array of plant species and afflicts significant yield losses globally. Despite being a notorious pathogen, it has an uncomplicated life cycle consisting of either basal infection from myceliogenically germinated sclerotia or aerial infection from ascospores of carpogenically germinated sclerotia. This fungus is unique among necrotrophic pathogens in that it inevitably colonizes aging tissues to initiate an infection, where a saprophytic stage follows the pathogenic phase. The release of cell wall-degrading enzymes, oxalic acid, and effector proteins are considered critical virulence factors necessary for the effective pathogenesis of S. sclerotiorum. Nevertheless, the molecular basis of S. sclerotiorum pathogenesis is still imprecise and remains a topic of continuing research. Previous comprehensive sequencing of the S. sclerotiorum genome has revealed new insights into its genome organization and provided a deeper comprehension of the sophisticated processes involved in its growth, development, and virulence. This review focuses on the genetic and genomic aspects of fungal biology and molecular pathogenicity to summarize current knowledge of the processes utilized by S. sclerotiorum to parasitize its hosts. Understanding the molecular mechanisms regulating the infection process of S. sclerotiorum will contribute to devising strategies for preventing infections caused by this destructive pathogen.

Green Nanopesticide: pH-Responsive Eco-Friendly Pillar[5]arene-Modified Selenium Nanoparticles for Smart Delivery of Carbendazim to Suppress Sclerotinia Diseases.

Abstract: Controlled-release delivery systems have been widely used to improve the efficacy and bioavailability of pesticides and minimize environmental risks. Herein, a fungicide carbendazim (CBZ)-loaded, a kind of nanovalve including trimethylammoniumpillar[5]arene (AP5), and methyl orange (MO)-functionalized mesoporous selenium (MSe) nanopesticides (CBZ@AP5/MSe⊃MO) were prepared. The nanovalve endowed CBZ@AP5/MSe⊃MO with a pH-responsive property, so the CBZ@AP5/MSe⊃MO can respond to the microenvironment of the pathogen Sclerotinia sclerotiorum (S. sclerotiorum). First, MO was shed due to protonation, and AP5-functionalized MSe gradually dissolved in an acid environment. Finally, CBZ was released rapidly. It is reported that AP5 and MO as the host and guest functionalized mesoporous selenium (MSe) have never been applied to agriculture. In vitro release experiments showed that the cumulative release rate of CBZ at pH 4.5 was 1.74 times higher than that in a neutral environment. In addition, we found that the contact angle of the CBZ@AP5/MSe⊃MO in maize and rape leaves was effectively decreased, which could retain more in the leaves after washout. It can also decrease the dry biomass and the reducing sugar of S.sclerotiorum. The CBZ@AP5/MSe⊃MO holds a good safety profile for plants, animal cells, and the environment owing to the targeted release properties. These results suggest that CBZ@AP5/MSe⊃MO is an environmentally friendly and effective drug-loaded system against S. sclerotiorum. It provides a new strategy for the design and development of nanopesticides and the control of S. sclerotiorum.

Characterization of a Fungal Virus Representing a Novel Genus in the Family Alphaflexiviridae

Abstract: Sclerotinia sclerotiorum is an ascomycetous fungus and hosts various mycoviruses. In this study, a novel fungal alphaflexivirus with a special genomic structure, named Sclerotinia sclerotiorum alphaflexivirus 1 (SsAFV1), was cloned from a hypovirulent strain, AHS31. Strain AHS31 was also co-infected with two botourmiaviruses and two mitoviruses. The complete genome of SsAFV1 comprised 6939 bases with four open reading frames (ORFs), a conserved 5′-untranslated region (UTR), and a poly(A) tail in the 3′ terminal; the ORF1 and ORF3 encoded a replicase and a coat protein (CP), respectively, while the function of the proteins encoded by ORF2 and ORF4 was unknown. The virion of SsAFV1 was flexuous filamentous 480–510 nm in length and 9–10 nm in diameter. The results of the alignment and the phylogenetic analysis showed that SsAFV1 is related to allexivirus and botrexvirus, such as Garlic virus X of the genus Allexivirus and Botrytis virus X of the genus Botrevirus, both with 44% amino-acid (aa) identity of replicase. Thus, SsAFV1 is a novel virus and a new genus, Sclerotexvirus, is proposed to accommodate this novel alphaflexivirus.

A MAP kinase cascade broadly regulates development and virulence of Sclerotinia sclerotiorum and can be targeted by HIGS for disease control

Abstract: Sclerotinia sclerotiorum causes white mold or stem rot in a broad range of economically important plants, bringing significant yield losses worldwide. Host-induced gene silencing (HIGS) has been showing promising effects in controlling many fungal pathogens, including S. sclerotiorum. However, molecular genetic understanding of signaling pathways involved in its development and pathogenicity is needed to provide effective host-induced gene silencing (HIGS) targets for disease control. Here, by employing a forward genetic screen, we characterized an evolutionarily conserved mitogen-activated protein kinase (MAPK) cascade in S. sclerotiorum, consisting of SsSte50-SsSte11-SsSte7-Smk1, controlling mycelial growth, sclerotia development, compound appressoria formation, virulence, and hyphal fusion. Moreover, disruption of the putative downstream transcription factor SsSte12 led to normal sclerotia but aberrant appressoria formation and host penetration defects, suggestive of diverged regulation downstream of the MAPK cascade. Most importantly, targeting of SsSte50 using host-expressed HIGS double stranded RNA resulted in largely reduced virulence of S. sclerotiorum on Nicotiana benthamiana leaves. Therefore, this MAPK signaling cascade is generally needed for its growth, development, and pathogenesis, and is an ideal HIGS target for mitigating economic damages caused by S. sclerotiorum infection.

First Report of Clonostachys rosea as a Mycoparasite on Sclerotinia sclerotiorum Causing Head Rot of Cabbage in India

Abstract: Clonostachys rosea, an ascomycetous, omnipresent, cellulose-decaying soil fungus, has been reported to be a well-known mycoparasitic biological control agent. In this study, we isolated C. rosea, a mycoparasitic fungus for the first time in India from sclerotia of the notorious plant pathogen Sclerotinia sclerotiorum, causing head rot disease in cabbage. A total of five mycoparasitic fungi were isolated from the sclerotial bodies of S. sclerotiorum (TNAU-CR 01, 02, 03, 04 and 05). All the isolates were tested under morpho-molecular characterization. Among them, TNAU-CR 02 showed the greatest mycelial inhibition of 79.63% over the control. Similarly, the SEM imaging of effective C. rosea isolates indicated the presence of numerous conidia destroying the outer cortex layers of sclerotia. Metabolite fingerprinting of C. rosea TNAU-CR 02 identified 18 chemical compounds using GC-MS analysis. The crude antibiotics of C. rosea TNAU-CR 02 were verified for their antifungal activity against S. sclerotiorum and the results revealed 97.17% mycelial inhibition compared with the control. Similarly, foliar application of TNAU-CR 02 at 5 mL/litre on 30, 45 and 60 days after transplanting showed the lowest disease incidence of 15.1 PDI compared to the control. This discovery expands our understanding of the biology and the dissemination of C. rosea, providing a way for the exploitation of C. rosea against cabbage head rot pathogens.

Colony Age of Trichoderma azevedoi Alters the Profile of Volatile Organic Compounds and Ability to Suppress Sclerotinia sclerotiorum in Bean Plants

Abstract: Common bean (Phaseolus vulgaris L.) is one of the most important crops in human food production. The occurrence of diseases, such as white mold, caused by Sclerotinia sclerotiorum can limit the production of this legume. The use of Trichoderma has become an important strategy in the suppression of this disease. The aim of the present study was to evaluate the effect of volatile organic compounds (VOCs) emitted by Trichoderma azevedoi CEN1241 in five different growth periods on the severity of white mold in common bean. The in vitro assays were carried out in double-plate and split-plate, and the in vivo assays, through the exposure of the mycelia of S. sclerotiorum to the VOCs of T. azevedoi CEN1241 and subsequent inoculation in bean plants. Chemical analysis by gas chromatography coupled to mass spectrometry detected 37 VOCs produced by T. azevedoi CEN1241, covering six major chemical classes. The profile of VOCs produced by T. azevedoi CEN1241 varied according to colony age and was shown to be related to the ability of the biocontrol agent to suppress S. sclerotiorum. T. azevedoi CEN1241 VOCs reduced the size of S. sclerotiorum lesions on bean fragments in vitro and reduced disease severity in a greenhouse. This study demonstrated in a more applied way that the mechanism of antibiosis through the production of volatile compounds exerted by Trichoderma can complement other mechanisms, such as parasitism and competition, thus contributing to a better efficiency in the control of white mold in bean plants.

Jasmonic Acid as a Mediator in Plant Response to Necrotrophic Fungi

Abstract: Jasmonic acid (JA) and its derivatives, all named jasmonates, are the simplest phytohormones which regulate multifarious plant physiological processes including development, growth and defense responses to various abiotic and biotic stress factors. Moreover, jasmonate plays an important mediator’s role during plant interactions with necrotrophic oomycetes and fungi. Over the last 20 years of research on physiology and genetics of plant JA-dependent responses to pathogens and herbivorous insects, beginning from the discovery of the JA co-receptor CORONATINE INSENSITIVE1 (COI1), research has speeded up in gathering new knowledge on the complexity of plant innate immunity signaling. It has been observed that biosynthesis and accumulation of jasmonates are induced specifically in plants resistant to necrotrophic fungi (and also hemibiotrophs) such as mostly investigated model ones, i.e., Botrytis cinerea, Alternaria brassicicola or Sclerotinia sclerotiorum. However, it has to be emphasized that the activation of JA-dependent responses takes place also during susceptible interactions of plants with necrotrophic fungi. Nevertheless, many steps of JA function and signaling in plant resistance and susceptibility to necrotrophs still remain obscure. The purpose of this review is to highlight and summarize the main findings on selected steps of JA biosynthesis, perception and regulation in the context of plant defense responses to necrotrophic fungal pathogens.

Drug repurposing strategy II: from approved drugs to agri-fungicide leads

Abstract: Epidemic diseases of crops caused by fungi deeply affected the course of human history and processed a major restriction on social and economic development. However, with the enormous misuse of existing antimicrobial drugs, an increasing number of fungi have developed serious resistance to them, making the diseases caused by pathogenic fungi even more challenging to control. Drug repurposing is an attractive alternative, it requires less time and investment in the drug development process than traditional R&D strategies. In this work, we screened 600 existing commercially available drugs, some of which had previously unknown activity against pathogenic fungi. From the primary screen at a fixed concentration of 100 μg/mL, 120, 162, 167, 85, 102, and 82 drugs were found to be effective against Rhizoctonia solani, Sclerotinia sclerotiorum, Botrytis cinerea, Phytophthora capsici, Fusarium graminearum and Fusarium oxysporum, respectively. They were divided into nine groups lead compounds, including quinoline alkaloids, benzimidazoles/carbamate esters, azoles, isothiazoles, pyrimidines, pyridines, piperidines/piperazines, ionic liquids and miscellaneous group, and simple structure-activity relationship analysis was carried out. Comparison with fungicides to identify the most promising drugs or lead structures for the development of new antifungal agents in agriculture.

QTL mapping and transcriptome analysis identify novel QTLs and candidate genes in Brassica villosa for quantitative resistance against Sclerotinia sclerotiorum

Abstract: Abstract Key message Novel QTLs and candidate genes for Sclerotinia-resistance were identified in B. villosa , a wild Brassica species, which represents a new genetic source for improving oilseed rape resistance to SSR. Abstract Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is one of the most destructive diseases in oilseed rape growing regions. To date, there is no effective genetic resistance against S. sclerotiorum in the B. napus germplasm and knowledge of the molecular plant–fungal interaction is also limited. To identify new resistance resources, we screened a set of wild Brassica species and identified B. villosa (BRA1896) with a high level of Sclerotinia-resistance. Two segregating F 2 populations for Sclerotinia-resistance, generated by interspecific crosses between the resistant B. villosa (BRA1896) and the wild susceptible B. oleracea (BRA1909) were assessed for Sclerotinia-resistance. Genetic mapping using a 15-k Illumina Infinium SNP-array resulted in a high-density genetic map containing 1,118 SNP markers and spanning a total genetic length of 792.2 cM. QTL analysis revealed seven QTLs explaining 3.8% to 16.5% of phenotypic variance. Intriguingly, RNAseq-based transcriptome analysis identified genes and pathways specific to B. villosa, of which a cluster of five genes encoding putative receptor-like kinases (RLKs) and two pathogenesis-related (PR) proteins are co-localized within a QTL on chromosome C07. Furthermore, transcriptomic analysis revealed enhanced ethylene (ET)-activated signaling in the resistant B. villosa, which is associated with a stronger plant immune response, depressed cell death, and enhanced phytoalexin biosynthesis compared to the susceptible B. oleracea. Our data demonstrates that B. villosa represents a novel and unique genetic source for improving oilseed rape resistance against SSR.

Surveillance of Landraces’ Seed Health in South Italy and New Evidence on Crop Diseases

Abstract: During the last three years, more than 300 landraces belonging to different plant species have been the main focus of an Italian valorization research project (AgroBiodiversità Campana, ABC) aiming at analyzing, recovering, preserving, and collecting local biodiversity. In this context, phytosanitary investigation plays a key role in identifying potential threats to the preservation of healthy seeds in gene banks and the successful cultivation of landraces. The surveillance carried out in this study, in addition to highlighting the expected presence of common species-specific pathogens such as Ascochyta pisi in peas, Ascochyta fabae in broad beans, and Macrophomina phaseolina, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans in beans, pointed to the presence of novel microorganisms never detected before in the seeds of some hosts (Apiospora arundinis in common beans or Sclerotinia sclerotiorum and Stemphylium vesicarium in broad beans). These novel seedborne pathogens were fully characterized by (i) studying their morphology, (ii) identifying them by molecular methods, and (iii) studying their impact on adult crop plants. For the first time, this study provides key information about three novel seedborne pathogens that can be used to correctly diagnose their presence in seed lots, helping prevent the outbreaks of new diseases in the field.

Association mapping combined with whole genome sequencing data reveals candidate causal variants for sclerotinia stem rot resistance in <i>Brassica napus</i>

Abstract: Canola ( Brassica napus) yield can be significantly reduced by the disease sclerotinia stem rot (SSR), which is caused by Sclerotinia sclerotiorum, a necrotrophic fungal pathogen with an unusually large host range. Breeding cultivars that are physiologically resistant to SSR is desirable to enhance crop productivity. However, the development of resistant varieties has proved challenging due to the highly polygenic nature of S. sclerotiorum resistance. Here, we identified regions of the B. napus genome associated with SSR resistance using data from a previous study by association mapping. We then validated their contribution to resistance in a follow-up screen. This follow-up screen also confirmed high levels of SSR resistance in several genotypes from the previous study. Using publicly available whole genome sequencing data for a panel of 83 B. napus genotypes, we identified nonsynonymous polymorphisms linked to the SSR resistance loci. A qPCR analysis showed that two of the genes containing these polymorphisms were transcriptionally responsive to S. sclerotiorum infection. In addition, we provide evidence that homologues of three of the candidate genes contribute to resistance in the model Brassicaceae species Arabidopsis thaliana. The identification of resistant germplasm and candidate genomic loci associated with resistance are important findings that can be exploited by breeders to improve the genetic resistance of canola varieties.

Design, synthesis and antifungal evaluation of novel nopol derivatives as potent laccase inhibitors.

Abstract: BACKGROUND To explore more potential natural product-based antifungal agents, a series of novel nopol-based carboxamide and hydrazide derivatives containing natural pinene structure was designed, synthesized, and evaluated for their inhibitory activities against seven phytopathogenic fungi and oomycetes. RESULTS The bioassay results indicated that some compounds exhibited good inhibitory activities against Gibberella zeae, Sclerotinia sclerotiorum, and Phytophthora capsici. Among them, compound 3h displayed excellent in vitro activities against G. zeae, with EC50 values of 1.09 mg/L, which was comparable to commercial fungicides bixafen and carbendazim (EC50 = 1.21 and 0.89 mg/L, respectively). Notably, in vivo bioassay results suggested that compound 3h also showed prominent protective and curative effects (95.6% and 94.2%) at 200 mg/L against G. zeae. The SEM study indicated that compound 3h could destroy the morphological integrity of G. zeae hyphae. The in vitro enzyme inhibitory bioassay revealed that compound 3h exhibited potent inhibitory activity against laccase with IC50 values of 4.93 μM, superior to positive control cysteine (IC50 = 35.50 μM), and its binding modes with laccase were elucidated by molecular docking study. In addition, the fluorescent imaging of the dansylamide-labeled derivatives 8 on wheat leaf epidermal cells and the hyphae of G. zeae revealed that this class of hydrazide derivatives could readily permeate into wheat leaves and reached the laccase target in fungal cells. CONCLUSION Some nopol-based hydrazide derivatives exhibited excellent anti-G. zeae activity and laccase inhibitory activity, which merits further development as a new fungicide candidate for controlling Fusarium head blight. This article is protected by copyright. All rights reserved.

Design, Synthesis and Antifungal Activity of Novel 1,4-Pentadiene-3-one Containing Quinazolinone

Abstract: Twenty 1,4-pentadiene-3-one derivatives containing quinazolinone (W1–W20) were designed and synthesized. The bioactivity test results showed that some compounds had antifungal activities in vitro. W12 showed excellent bioactivity against Sclerotinia sclerotiorum (S. sclerotiorum) and Phomopsis sp., with EC50 values of 0.70 and 3.84 μg/mL, which are higher than those of the control drug azoxystrobin at 8.15 and 17.25 μg/mL. In vivo activity tests were carried out on oilseed rape and kiwifruit. The protective effect of W12 on oilseed rape infected with S. sclerotiorum (91.7 and 87.3%) was better than that of azoxystrobin (90.2 and 79.8%) at 100 and 50 μg/mL, respectively, and the protective effect on kiwifruit infected with Phomopsis sp. (96.2%) was better than that of azoxystrobin (94.6%) at 200 μg/mL. Scanning electron microscopy results showed the hyphae of S. sclerotiorum treated with compound W12 abnormally collapsed and shriveled, inhibiting the growth of mycelium and, thus, laying the inhibiting effect on S. sclerotiorum. The results of the mechanism research showed that the action of W12 changed the mycelial morphology of S. sclerotiorum, affected the permeability of cells, increased the leakage of cytoplasm and allowed the cell membrane to break down. This study shows that 1,4-pentadiene-3-one derivatives containing quinazolinone have good effects on plant fungi and the potential for becoming new fungicides.

Isolation and evaluation of Bacillus subtilis RSS-1 as a potential biocontrol agent against Sclerotinia sclerotiorum on oilseed rape

Abstract: Abstract Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most important diseases of oilseed rape in the world. Because of the absence of resistant varieties and the disadvantages of chemical control, application of antifungal microbes has become an eco-friendly and effective measure to control this disease. In this study, Bacillus subtilis strain RSS-1, isolated from soil samples, was identified based on morphological, physiological and biochemical tests, and DNA gyrase subunit A ( gyrA ), gyrB , DNA-directed RNA polymerase subunit beta ( rpoB ) and rpoC gene sequence analysis. It significantly inhibited mycelial growth and sclerotial production of S. sclerotiorum in vitro . In greenhouse experiments, all three tested concentrations (10 6 , 10 7 , 10 8 cfu mL −1 ) of cell fermentation broth and culture filtrate significantly reduced the severity of sclerotinia stem rot on oilseed rape ( P < 0.05). RSS-1 was more effective at reducing disease severity when applied 24 h before inoculation with S. sclerotiorum than at 24 h post inoculation, suggesting that RSS-1 should be applied as a prophylactic rather than a curative biological agent. Colonization tests indicated that the population density of RSS-1 on rapeseed leaves significantly decreased ( P < 0.05) over 6 days. However, RSS-1 could stably colonize in rhizospheric soil of rapeseed over 30 days. Challenge inoculation tests showed that RSS-1 significantly inhibited the activities of polygalacturonase and cellulase and accumulation of oxalic acid during the S. sclerotiorum infection. These results suggest that RSS-1 was a potential biological agent for controlling sclerotinia stem rot caused by S. sclerotiorum on oilseed rape.

Design, synthesis and antifungal evaluation of phenylthiazole-1,3,4-oxadiazole thione (ketone) derivatives inspired by natural thiasporine A.

Abstract: BACKGROUND Plant pathogenic fungal infections have become a severe threat to the yield and quality of agricultural products, and new green antifungal agents with high-efficiency and low-toxicity are needed. In this study, a series of thiasporine A derivatives containing phenylthiazole-1,3,4-oxadiazole thione (ketone) structures were designed and synthesized, and their antifungal activities against six invasive and highly destructive phytopathogenic fungi were evaluated. RESULTS The results showed that all compounds showed moderate to potent antifungal activity against six phytopathogenic fungi, and most of E the series compounds showed remarkable antifungal activity against S. sclerotiorum and C. camelliaet. In particular, compounds E1 - E5, E7, E8, E13, E14, E17, and E22 showed more significant antifungal activity against S. sclerotiorum, with EC50 values of 0.22，0.48, 0.56, 0.65, 0.51, 0.39, 0.60, 0.56, 0.60, 0.63, and 0.45 μg/mL, respectively, which were superior to that of carbendazim (0.70 μg/mL). Further activity studies showed that compound E1 showed superior curative activities against S. sclerotiorum in vivo and better inhibitory effects on sclerotia germination and formation of S. sclerotiorum compared to those of carbendazim. CONCLUSIONS Therefore, this study indicates that these thiasporine A derivatives containing phenylthiazole-1,3,4-oxadiazole thione structures might be applied as antifungal agents against S. sclerotiorum. This article is protected by copyright. All rights reserved.

Synthesis of Pogostone‐Inspired Dehydroacetic Acid Derivatives and Their Antifungal Activity against Sclerotinia sclerotiorum (Lib.) de Bary

Abstract: In an effort to exploit the natural antifungal pogostone, its simplified scaffold dehydroacetic acid (DHA) was used as a lead compound to semi‐synthesize 56 DHA derivatives (I1–48, II, III, and IV1–6). Among them, compound IV4 exhibited the most potent antifungal activity with 11.0 μM EC50 against mycelial growth of Sclerotinia sclerotiorum (Lib.) de Bary whose sclerotia production was also completely suppressed at this concentration. Furthermore, IV4 could completely inhibit infection cushion formation of S. sclerotiorum on rape leaves and achieved a preventive efficacy of 90.2 % at 500 μM, which was on the same level as that of commercial boscalid at 30 μM (88.7 %). The results of physiological and ultrastructural studies indicated that IV4 might disrupt the cell membrane permeability or induce the imbalance of mitochondrial membrane potential homeostasis to exert the antifungal mode of action. Besides, the robust and predicative three‐dimensional quantitative structure‐activity relationship (3D‐QSAR) models were developed and discussed herein.

Fungicide sensitivity of <i>Sclerotinia sclerotiorum</i> from U.S. soybean and dry bean, compared to different regions and climates

Abstract: Fungicide use is integral to reduce yield loss from Sclerotinia sclerotiorum on dry bean and soybean. Increasing fungicide use against this fungus may lead to resistance to the most common fungicides. Resistance has been reported in Brazil (Glycine max) and China (Brassica napus subsp. napus), however, few studies have investigated fungicide sensitivity of S. sclerotiorum in the U.S. The work presented here was conducted to determine if there was a difference in fungicide sensitivity of S. sclerotiorum isolates in the U.S. from: (i) dry bean versus soybean and (ii) fields with different frequencies of fungicide application. We further hypothesized that isolates with fungicide applications of a single active ingredient from tropical Brazil and sub-tropical Mexico were less sensitive compared to temperate U.S. isolates due to different management practices and climates. The EC50(D) fungicide sensitivity of 512 S. sclerotiorum isolates from the U.S. (443), Brazil (36), and Mexico (33) was determined using a discriminatory concentration (DC) previously identified for tetraconazole (2.0 ppm; EC50(D) ranged of 0.197 to 2.27 ppm), boscalid (0.2; 0.042 to 0.222), picoxystrobin (0.01; 0.006 to 0.027), and thiophanate-methyl, which had a qualitative DC of 10 ppm. Among the 10 least sensitive isolates to boscalid and picoxystrobin, two presented mutations known to confer resistance in the SdhB (qualitative) and SdhC (quantitative) genes; however no strong resistance was found. This study established novel DCs that can be used for further resistance monitoring and baseline sensitivity of S. sclerotiorum to tetraconazole throughout the world plus baseline sensitivity to boscalid in the U.S.

Bacillus amyloliquefaciens PKM16 acts as an antagonist of white mold and an inducer of defense enzymes in tomato plants

Abstract: This study aimed to investigate the potential of rhizobacteria isolated from tomato plants to control Sclerotinia sclerotiorum and induce the activity of pathogenesis-related enzymes in Micro-Tom tomato plants. Three rhizobacterial isolates were evaluated to determine the most efficient antagonist agent, which was later identified by gene sequencing as Bacillus amyloliquefaciens PKM16. The antagonistic effects of B. amyloliquefaciens against S. sclerotiorum were assessed in vivo and in vitro using live and autoclaved cultures at concentrations of 0% (control), 20%, 30%, and 40% (v/v). The residual effects of four treatments (20% live culture, 20% autoclaved culture, a Bacillus subtilis-based commercial product, and autoclaved distilled water) on tomato plants inoculated with S. sclerotiorum were determined. The same treatments were also used to assess the myceliogenic germination of sclerotia and induction of plant defense enzymes (peroxidase, catalase, polyphenol oxidase, phenylalanine ammonia-lyase, and β-1,3-glucanase) in tomato plants. The live culture had a residual effect for 4 days and inhibited sclerotial germination by approximately 30%. Furthermore, live and autoclaved bacterial growth cultures stimulated enzyme activity. Therefore, B. amyloliquefaciens PKM16 was antagonistic to S. sclerotiorum, effectively inhibiting mycelial growth and activating defense mechanisms in Micro-Tom tomato plants.

Airborne versus soilborne inoculum: white mould, where do you come from?

Abstract: Identifying the sources of fungal inoculum that induces epidemics is instrumental to managing crop health in a more efficient way by implementing prophylactic methods and by using better targeted biocontrol and fungicide applications. For some phytopathogenic fungi, this identification is hampered by the multiplicity of inoculum types. This is the case for Sclerotinia sclerotiorum, the agent of white mould, which can produce ascospores disseminated via the atmosphere, sclerotia stored in the soil and mycelium in plant debris of a large variety of hosts. The present study aims to assess the relative importance of airborne and soilborne inocula in white mould disease development and its persistence across two crop seasons. S. sclerotiorum isolates were collected from air, soil and carrot plants during two crop seasons in south-western France and genotyped with 16 microsatellite markers. Among the 490 isolates, 162 clonal lineages were identified. The genetic characteristics did not differ significantly among isolates collected from air, soil and carrots. The epidemics of white mould on carrots were initiated either by airborne inoculum or by soilborne inoculum. It also appears that some isolates from airborne inoculum persisted from year to year.

Progress in plant tolerance to the fungal disease Sclerotinia in breeding experiments on winter oilseed rape

Abstract: Summary Sclerotinia sclerotiorum is a pathogen which causes a disease of oilseed rape. Severe plant infection contributes to a decrease in the quality of the crop. It is therefore important to pay attention to whether hybrids are highly tolerant to this fungal disease at the early stages of breeding. In this study, the question of interest is whether there has been progress in increasing the tolerance of new hybrids to this pathogen. Three years of breeding experiments (2014, 2015, 2017) are included in the analysis. Each year, three to five experiments were carried out, with several dozen varieties and three standards. Each series of experiments was repeated in several locations. Because the degree of infection was assessed on a scale (from 1 – the highest infection – to 9 – the least infection), the analysis is carried out using an ordinal logistic model. It is noted that in earlier years the standard varieties’ probability of infection with this disease had a smaller range (empirical probability 0.5–0.83) than in the last analyzed year (empirical probability 0.33–1.00). The results of the analysis show that in 2014 and 2015 several hybrids exhibited a significantly higher tolerance to Sclerotinia, but in 2017 none of the hybrids were significantly better than the standard. Perhaps breeding selection of hybrids has eliminated the less tolerant varieties. However, to be able to draw more general conclusions, it would be necessary to repeat the study in controlled conditions (a greenhouse), where the level of fungal spores and their effect on plants could be controlled. Obtaining tolerant hybrids will enable a reduction in production costs, since there will be no need to monitor whether disease infestation occurs and no need to use corresponding plant protection products.

CARACTERIZAÇÃO DE ISOLADOS DE Trichoderma spp. DO CERRADO TOCANTINENSE E APLICAÇÃO COMO AGENTE DE CONTROLE BIOLÓGICO CONTRA O FITOPATÓGENO Sclerotinia sclerotiorum

Abstract: O fitopatógeno Sclerotinia sclerotiorum, causador da doença conhecida como mofo-branco acomete diversas culturas agrícolas em todo o mundo, seu alto grau de patogenicidade está associado à formação de escleródios que atuam como mecanismo de resistência e sobrevivência. Considerando a necessidade de métodos que contribuam no desenvolvimento agrícola sem promover danos ao ambiente e a população, os agentes de controle biológico são hoje vistos como um excelente artifício. Fungos do gênero Trichoderma são considerados agentes de biocontrole sagazes por apresentarem inúmeros mecanismos com alto potencial antagônico que prejudicam a germinação de esporos, o crescimento das hifas e o desenvolvimento de escleródios e clamidósporo de diversos fitopatógenos. Buscando avaliar a eficiência de isolados de Trichoderma spp. provenientes de áreas do Cerrado do Tocantins no biocontrole do fitopatógeno S. sclerotiorum foram realizados testes de pareamento, enzimáticos e de produção de metabólitos secundários. Através dos resultados obtidos foi possível concluir que o isolado Trichoderma spp. 2, oriundo de áreas do Cerrado tocantinense, possui potencial para ser considerado como um biocontrolador ao fitopatógeno S. Sclerotiorum, sendo seu antagonismo mais evidente por meio da produção dos metabólitos secundários voláteis.

mRNA Turnover Protein 4 Is Vital for Fungal Pathogenicity and Response to Oxidative Stress in Sclerotinia sclerotiorum

Abstract: Ribosome assembly factors have been extensively studied in yeast, and their abnormalities may affect the assembly process of ribosomes and cause severe damage to cells. However, it is not clear whether mRNA turnover protein 4 (MRT4) functions in the fungal growth and pathogenicity in Sclerotinia sclerotiorum. Here, we identified the nucleus-located gene SsMRT4 using reverse genetics, and found that knockdown of SsMRT4 resulted in retard mycelia growth and complete loss of pathogenicity. Furthermore, mrt4 knockdown mutants showed almost no appressorium formation and oxalic acid production comparing to the wild-type and complementary strains. In addition, the abilities to ROS elimination and resistance to oxidative and osmotic stresses were also seriously compromised in mrt4 mutants. Overall, our study clarified the role of SsMRT4 in S. sclerotiorum, providing new insights into ribosome assembly in regulating pathogenicity and resistance to environmental stresses of fungi.

Metodologias de avaliação e compatibilidade de produtos químicos e biológicos, visando o controle de Sclerotinia sclerotiorum em soja

Abstract: A doença mofo-branco, causada por Sclerotinia sclerotiorum, pode reduzir a produtividade da soja de 30% até 70%. O manejo dessa doença consiste de controles químico e biológico. Objetivou-se no presente estudo avaliar a compatilibidade de produtos químicos e biológicos no controle in vitro, in vivo e na germinação carpogênica de isolados de S. sclerotiorum de soja e aprimorar metodologias de avaliação. Foram testados três isolados do fungo. Os fungicidas testados foram: Tiofanato metílico; Procimidona; Boscalida + Dimoxistrobina; Fluazinam e Fluazinam + Tiofanato metílico, em cinco concentrações (0; 0,1; 1,0; 10, 100 e 1000 ppm do i.a.). Os produtos de controle biológico foram: Trichordemil® (Trichoderma harzianum); Tricho-turbo® (T. Asperellum); Serenade® (Bacillus subtilis) e Bio-Imune® (B. subtilis), na concentração de 1000 ppm do p.c., bem como as misturas dos fungicidas + produtos de controle biológico. O teste de patogenicidade dos isolados foi feito em folhas destacadas de soja, seguido de inoculação de disco de BDA com os isolados. Avaliou-se a severidade da doença com escala diagramática e contou-se o número de escleródios produzidos. O pH das caldas dos fungicidas, dos produtos de controle biológico e das misturas foi medido bem como avaliada a compatibilidade. O efeito in vitro dos fungicidas sobre os produtos de controle biológico foi avaliado por cultura pareada. No ensaio in vitro, incorporou-se os fungicidas, os produtos de controle biológico e as misturas em BDA, seguido de inoculação dos isolados do fungo. Mediu-se o diâmetro da colônia e contaram-se os escleródios produzidos. Calculou-se a equação da perfomance de cada produto e misturas por isolado, o R2 e as concentrações letais CL50 e CL95. No ensaio in vivo, os fungicidas, os produtos de controle biológico e suas misturas foram aplicados nas folhas destacadas de soja, seguido de inoculação dos isolados do fungo. A avaliação foi idêntica ao do teste de patogenicidade. As equações, R2 e CL50 e CL95 por produto e por isolado também foram calculadas. O teste de germinação carpogênica dos isolados foi feita em caixas gerbox com solo autoclado, infestado com escleródios de cada isolado e regado com fungicidas, produtos de controle biológico e suas misutras. Avaliou-se a germinação dos viii escleródios. Calculou-se a equação da perfomance, o R2 e as concentrações letais CL50 e CL95. Todos os isolados de S. sclerotiorum foram patogênicos em folhas de soja destacadas, com diferenças de agressividade entre eles. Os valores de pH dos fungicidas e, principalmente, da mistura fungicidas + produtos de controle biológico ficaram dentro da normalidade para aplicação no campo. Não há incompatibilidade física na calda dos fungicidas com produtos de controle biológico, quando a mesma ficou em água em constante agitação. Os fungicidas interferiram mais efetiva e negativamente sobre o crescimento do antagonista Bacillus do que do Trichoderma. Os fungicidas associados aos antagonistas Trichoderma e Bacillus proporcionaram maiores porcentagens de inibição de S. sclerotiorum, quando comparados ao uso isolado dos fungicidas, bem como inibiram nova produção de escleródios e impediram a germinação carpogênica das estruturas. Dentre as metodologias testadas, o método de inoculação com disco de BDA em folhas destacadas de soja reproduziu de forma eficiente os sintomas da doença e pode demonstrar melhor o que pode acontecer no campo com a aplicação, principalmente, das misturas nas plantas de soja visando o controle do mofo branco. Todos os isolados de S. sclerotiorum mostraram-se altamente sensíveis aos fungicidas quanto ao crescimento micelial, com exceção dos isolados de Jaciara – MT e Muitos Capões – RS que enquadraram-se como moderadamente sensíveis especificamente ao tiofanato-metilico. Os isolados mostraram-se moderadamente sensíveis aos fungicidas quando testados para o controle da severidade da doença, com exceção dos isolados de Pilar do Sul – SP e de Muitos Capões – RS que foram pouco sensíveis ao fluazinam e tiofanato-metilico. Na germinação carpogênica, os isolados comportaram-se desde altamente sensíveis a pouco sensíveis aos fungicidas, em especial o de Muitos Capões-RS que foi pouco sensível aos fungicidas fluazinam, procimidona e tiofanato-metílico.

Crescimento micelial e produção de escleródios de isolados de Sclerotinia sclerotiorum sob diferentes regimes de luz e temperatura

Abstract: O mofo-branco causado pelo fungo Sclerotinia slerotiorum está entre as principais doenças do feijoeiro. A avaliação de genótipos resistentes é de grande importância para identificação de fontes de resistência a esse fungo. Para as avaliações fenotípicas, é necessário a inoculação em plantas diretamente com o fungo e para isso, o seu cultivo em meio artificial faz-se necessário. O objetivo deste estudo, foi estabelecer um protocolo para crescimento micelial e formação de escleródios do fungo S. sclerotiorum testando diferentes regimes de luz e temperatura. Foram utilizados dois isolados diferentes (UFLA154 e UFLA22). Estes foram cultivados em meio de cultura BDA em três regimes de luz/temperatura: 01: Escuro em geladeira (temperatura de aproximadamente 4 ºC); 02: Fotoperíodo e temperatura ambiente (fotoperíodo de aproximadamente 12 h); 03: escuro e temperatura ambiente). Foi realizada a medição radial do crescimento micelial e contagem do número de escleródios. Dos isolados testados, o UFLA154 apresentou crescimento micelial e produção de esclerodios maior. Na indisponibilidade de equipamentos apropriados de controle de luz e temperatura, como uma BOD por exemplo, o crescimento dos isolados testados do fungo S. Sclerotiorum em fotoperíodo e temperatura ambientes é satisfatório. A temperatura apresentou maior influência que o fotoperíodo no crescimento micelial e produção de escleródio.