Sclerotinia Sclerotiorum Research Articles

SsdchA is a novel secretory cellobiohydrolase driving pathogenicity in Sclerotinia sclerotiorum

The necrotrophic fungus, Sclerotinia sclerotiorum, employs an array of cell wall-degrading enzymes (CWDEs), including cellulase, to dismantle host cell walls. However, the molecular mechanisms through which S. sclerotiorum degrades cellulose remain elusive. Here, we unveil a novel secretory cellobiohydrolase, SsdchA, characterized by a signal peptide and a Glyco_hydro_7 (GH7) domain. SsdchA exhibits a robust expression of during early infection stages. Interestingly, colony morphology and growth rates remain unaffected across the wild-type, SsdchA deletion strains and SsdchA overexpression strains on potato dextrose agar (PDA) medium. Nevertheless, the pathogenicity and cellobiohydrolase activity decreased in the SsdchA deletion strains, but enhanced in the SsdchA overexpression strains. Moreover, the heterologous expression of SsdchA in Arabidopsis thaliana leads to reduced cellulose content and heightened susceptibility to S. sclerotiorum. Collectively, our data underscore the pivotal role of the novel cellobiohydrolase SsdchA in the pathogenicity of S. sclerotiorum.

Exploring the Mycovirus Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1 as a Biocontrol Agent of White Mold Caused by Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a necrotrophic fungal pathogen causing white mold on many important economic crops. Recently, some mycoviruses such as S. sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) converted S. sclerotiorum into a beneficial symbiont that helps plants manage pathogens and other stresses. To explore the potential use of SsHADV-1 as a biocontrol agent in the United States and to test the efficacy of SsHADV-1-infected United States isolates in managing white mold and other crop diseases, SsHADV-1 was transferred from the Chinese strain DT-8 to United States isolates of S. sclerotiorum. SsHADV-1 is readily transmitted horizontally among United States isolates of S. sclerotiorum and consistently conferred hypovirulence to its host strains. Biopriming of dry bean seeds with hypovirulent S. sclerotiorum strains enhanced resistance to white mold, gray mold, and Rhizoctonia root rot. To investigate the underlying mechanisms, endophytic growth of hypovirulent S. sclerotiorum in dry beans was confirmed using PCR, and the expression of 12 plant defense-related genes were monitored before and after infection. The results indicated that the endophytic growth of SsHADV-1-infected strains in plants stimulated the expression of plant immunity pathway genes that assisted a rapid response from the plant to fungal infection. Finally, application of the seed biopriming technology with SsHADV-1-infected hypervirulent strain has promise for the biological control of several diseases of wheat, pea, and sunflower.

Highly stable and antifungal properties on the oilseed rape of Cu3(MoO4)2(OH)2 nanoflakes prepared by simple aqueous precipitation

In the last few decades, nanoparticles have been a prominent topic in various fields, particularly in agriculture, due to their unique physicochemical properties. Herein, molybdenum copper lindgrenite Cu3(MoO4)2(OH)2 (CM) nanoflakes (NFs) are synthesized by a one-step reaction involving α-MoO3 and CuCO3⋅Cu(OH)2⋅xH2O solution at low temperature for large scale industrial production and developed as an effective antifungal agent for the oilseed rape. This synthetic method demonstrates great potential for industrial applications. Infrared spectroscopy and X-ray diffraction (XRD) results reveal that CM samples exhibit a pure monoclinic structure. TG and DSC results show the thermal stable properties. It can undergo a phase transition form copper molybdate (Cu3Mo2O9) at about 300 °C. Then Cu3Mo2O9 nanoparticles decompose into at CuO and MoO3 at 791 °C. The morphology of CM powder is mainly composed of uniformly distributed parallelogram-shaped nanoflakes with an average thickness of about 30 nm. Moreover, the binding energy of CM NFs is measured to be 2.8 eV. To assess the antifungal properties of these materials, both laboratory and outdoor experiments are conducted. In the pour plate test, the minimum inhibitory concentration (MIC) of CM NFs against Sclerotinia sclerotiorum (S. sclerotiorum) is determined to be 100 ppm, and the zone of inhibiting S. sclerotiorum is 14 mm. When the concentration is above 100 nm, the change rate of the hyphae circle slows down a little and begins to decrease until to 200 ppm. According to the aforementioned findings, the antifungal effects of a nano CM NFs solution are assessed at different concentrations (0 ppm (clear water), 40 ppm, and 80 ppm) on the growth of oilseed rape in an outdoor setting. The results indicate that the application of CM NFs led to significant inhibition of S. sclerotiorum. Specifically, when the nano CM solution was sprayed once at the initial flowering stage at a concentration of 80 ppm, S. sclerotiorum growth was inhibited by approximately 34%. Similarly, when the solution was sprayed once at the initial flowering stage and once at the rape pod stage, using a concentration of 40 ppm, a similar level of inhibition was achieved. These outcomes show that CM NFs possess the ability to bind with more metal ions due to their larger specific surface area. Additionally, their semiconductor physical properties enable the generation of reactive oxygen species (ROS). Therefore, CM NFs hold great potential for widespread application in antifungal products.

Evaluation of the antagonistic activity of black soldier fly frass extracts against plant pathogens using single- and double-layer agar bioassays

Abstract Larval frass from insects which consists of larval excrement, exoskeleton, and undigested diet, is a rich source of organic material and microorganisms. Despite its potential value, research on frass valorisation in agriculture is limited. In this study, single-layer agar (SLA) and double-layer agar (DLA) in vitro bioassays were conducted to evaluate the effect of water-based black soldier fly larvae (BSFL) frass extracts from two different diets on the growth of six plant pathogens: Alternaria solani, Botrytis cinerea, Fusarium oxysporum, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum. The results showed that frass extract from Gainesville house fly diet strongly or completely inhibited the growth of all tested plant pathogens in both SLA and DLA bioassays, while frass extract from fruit/vegetable/bakery/brewery diet strongly inhibited the mycelial growth of A. solani, B. cinerea, and S. sclerotiorum, and moderately inhibited the mycelial growth of P. capsici in both bioassays. For both diets and bioassays, 0.22 μm microfiltered frass extracts which are free of microorganisms showed generally no effect on the growth of the pathogens indicating that growth inhibition is caused by frass-inhabiting microorganisms. Both SLA and DLA bioassays revealed strong antagonistic effect of microorganisms inhabiting BSFL frass against the plant pathogens B. cinerea, A. solani, R. solani, P. capsici, F. oxysporum and S. sclerotiorum. Moreover, the study showed the impact of the BSFL diet on the antagonistic effect of frass extract. In future work, the antagonistic effect of frass extracts against the above-mentioned pathogens will be tested in vivo. BSFL frass could eventually find applications for the control of plant diseases.

Bimetallic nanoparticles and biochar produced by Adansonia Digitata shell and their effect against tomato pathogenic fungi.

Adansonia digitata L. is a royal tree that is highly valued in Africa for its medicinal and nutritional properties. The objective of this study was to use its fruit shell extract to develop new, powerful mono and bimetallic nanoparticles (NPs) and biochar (BC) using an eco-friendly approach. Silver (Ag), iron oxide (FeO), the bimetallic Ag-FeO NPs, as well as (BC) were fabricated by A. digitata fruit shell extract through a reduction process and biomass pyrolysis, respectively, and their activity against tomato pathogenic fungi Alternaria sp., Sclerotinia sclerotiorum, Fusarium equiseti, and Fusarium venenatum were detected by agar dilution method. The Ag, FeO, Ag-FeONPs, and BC were characterized using a range of powerful analytical techniques such as ultraviolet-visible (UV-Vis) spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier Transform-Infra Red (FT-IR), dynamic light scatter (DLS), and zeta potential analysis. The fabricated Ag, FeO and Ag-FeO NPs have demonstrated a remarkable level of effectiveness in combating fungal strains. UV-Vis spectra ofAg, FeO, Ag-FeONPs, and BC show broad exhibits peaks at 338, 352, 418, and 480 nm, respectively. The monometallic, bimetallic NPs, and biochar have indicated the presence in various forms mostly in Spherical-shaped. Their size varied from 102.3 to 183.5 nm and the corresponding FTIR spectra suggested that the specific organic functional groups from the plant extract played a significant role in the bio-reduction process. Ag and Ag-FeO NPs exhibited excellent antifungal activity against pathogenic fungi Alternaria sp., S. sclerotiorum, F. equiseti, and F. venenatum. The current study could be a significant achievement in the field of antifungal agents since has the potential to develop new approaches for treating fungal infections.

First Report of Lecanicillium aphanocladii Causing Rot of Sparassis crispa in China.

Sparassis crispa, also known as cauliflower mushroom, is a new popularly edible mushroom in China, also a medicinal mushroom, which possesses various biological activities, such as immunopotentiation, anti-diabetes, anti-cancer, and anti-inflammatory effects. (Han et al., 2018). In recent years, the artificial cultivation of S. crispa has gained considerable public attention in China. In 2023, approximately 20% of S. crispa (about 0.05 ha of the planting area) showed obvious rot with white molds symptoms in mushroom hothouse, located in Shuangliu county, Sichuan province, China (GPS, 104°7'51"N, 30°25'2"E). Infected fruiting bodies were covered by white mycelia that later turned red or fuchsia. In the final stages of infection, the S. crispa fruiting bodies turned dark red or brown before rotting. The pathogen was isolated from the margin of the lesions by plating onto potato dextrose agar (PDA), and incubated at 25℃ in the dark for a week. Five pure culture fungal isolates were obtained. Collected isolates with similar morphology were described as Lecanicillium spp. (Zare et al., 2001). The colonies were raised, covered with white, the reverse side were violet brown, produced diffusing reddish-purple pigment. Conidiogenous cells produced singly, in pairs, verticillate or in dense irregular clusters on prostrate hyphae, at first flask-shaped, tapering into threadlike neck, with a size of 3.0-6.2×0.8-2.2 μm. Conidia were solitary, oval to subglobose, and 2.3-4.0×1.1-2.1 μm in size, similar to L. aphanocladii (Higo et al., 2021). For pathogenicity testing, ten fruiting bodies of S. crispa (planted in the bottles) were selected. Fungal cake of the isolate Bx-Ljb of L. aphanocladii were applied to the fruiting body of S. crispa, whereas pieces of sterile PDA medium were used as controls. All the bottles were incubated at 19±1℃, 85-100% relative humidity, and 18 h of light in the mushroom hothouse. A week later, the inoculated fruiting bodies developed brown spots and gradually expanding, with symptoms similar to the original diseased fruiting bodies. The controls remained healthy. The same fungus was reisolated from the infected fruiting bodies and subsequently identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results. For molecular identification, the DNA of the isolates was extracted using a Fungi Genomic DNA Extraction kit (Solarbio, Beijing). The SSU, LSU, and TEF1-α genes were amplified with the primer as previously described (Zhou et al., 2018). The generated sequences were deposited in GenBank with accession numbers OR206377, OR206378, and OR204702, respectively. BLASTn analyses showed >99.2% identity with previously deposited sequences of L. aphanocladii. Based on the maximum likelihood method, phylogenetic analysis revealed 99% bootstrap support values with L. aphanocladii. The fungus was identified as L. aphanocladii based on morphological and multilocus phylogenetic analyses. To our knowledge, there are two reports of L. aphanocladii on fruiting bodies of Tremella fuciformis and Morchella sextelata in China, and this is the first report of this fungus causing rot of S. crispa in China. It may be a reminder that the risk of L. aphanocladii in mushroom production in China is gradually increasing. These results will contribute to developing managemental strategies for this disease in S. crispa.

Attenuation of phytofungal pathogenicity of Ascomycota by autophagy modulators

Autophagy in eukaryotes functions to maintain homeostasis by degradation and recycling of long-lived and unwanted cellular materials. Autophagy plays important roles in pathogenicity of various fungal pathogens, suggesting that autophagy is a novel target for development of antifungal compounds. Here, we describe bioluminescence resonance energy transfer (BRET)-based high-throughput screening (HTS) strategy to identify compounds that inhibit fungal ATG4 cysteine protease-mediated cleavage of ATG8 that is critical for autophagosome formation. We identified ebselen (EB) and its analogs ebselen oxide (EO) and 2-(4-methylphenyl)−1,2-benzisothiazol-3(2H)-one (PT) as inhibitors of fungal pathogens Botrytis cinerea and Magnaporthe oryzae ATG4-mediated ATG8 processing. The EB and its analogs inhibit spore germination, hyphal development, and appressorium formation in Ascomycota pathogens, B. cinerea, M. oryzae, Sclerotinia sclerotiorum and Monilinia fructicola. Treatment with EB and its analogs significantly reduced fungal pathogenicity. Our findings provide molecular insights to develop the next generation of antifungal compounds by targeting autophagy in important fungal pathogens.

Discovery of Potential Rosin-Based Triazole Antifungal Candidates to Control Valsa mali for Sustainable Crop Protection.

To investigate the potential application value of dehydroabietic acid, 27 novel dehydroabietyl-1,2,4-triazole-5-thioether-based derivatives were designed and characterized by IR, 1H NMR, 13C NMR, and LC-MS. Their antifungal activities were evaluated against five plant fungi, namely, Valsa mali, Colletotrichum orbiculare, Fusarium graminearum, Sclerotinia sclerotiorum, and Gaeumannomyces graminis; the results showed that compound 5h-1 (Co. 5h-1) exhibited a considerable inhibitory effect against V. mali. Moreover, in vivo experiments indicated that Co. 5h-1 had a certain protective effect on apple branches. The preliminary structure-activity relationship analysis suggested that the electron-withdrawing group on the benzyl group was significantly better than that of other substituent derivatives. Through electron microscopy analysis, it was found that Co. 5h-1 hindered the growth of mycelia, damaged their cell structure, and caused the large accumulation of reactive oxygen species (ROS). Preliminary research on the mode of action indicated that Co. 5h-1 could affect the activity of CAT by increasing the α-helix (0.790%), decreasing the β-sheet (0.170%), which led to the accumulation of ROS. In addition, Co. 5h-1 also affected the activity of CYP51, hindered the biosynthesis of ergosterol, and increased cell membrane permeability. Overall, this above research proposed that Co. 5h-1 can be a novel leading structure for development of a fungicide agent.

Antifungal Activity and Mechanism of Xenocoumacin 1, a Natural Product from Xenorhabdus nematophila against Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum (Lib.) de Bary, a polyphagous necrotrophic fungal pathogen, has brought about significant losses in agriculture and floriculture. Until now, the most common method for controlling S. sclerotiorum has been the application of fungicides. Xenocoumacin 1 (Xcn1) is a potential biopesticide having versatile antimicrobial activities, generated by Xenorhabdus nematophila. This study was intended to isolate Xcn1 from X. nematophila YL001 and clarify its efficacies for S. sclerotiorum control. Xcn1 demonstrated a wider antifungal spectrum against 10 plant-pathogenic fungi. It also exhibited a strong inhibitory effect on the mycelial growth of S. sclerotiorum with an EC50 value of 3.00 μg/mL. Pot experiments indicated that Xcn1 effectively inhibited disease extension on oilseed rape and broad bean plants caused by S. sclerotiorum. Morphological and ultrastructural observations revealed that the hyphae of S. sclerotiorum became twisted, shriveled, and deformed at the growing points after treatment with Xcn1 at 3.00 μg/mL and that the subcellular fractions also became abnormal concurrently, especially the mitochondrial structure. Moreover, Xcn1 also increased cell membrane permeability and decreased the content of exopolysaccharide as well as suppressing the activities of polygalacturonase and cellulase of S. sclerotiorum, but exerted no effects on oxalic acid production. This study demonstrated that Xcn1 has great potential to be developed as a new biopesticide for the control of S. sclerotiorum.

Antagonistic potential of forestry compost bacteria on Sclerotinia sclerotiorum (Lib.) de Bary, causal agent of carrot white mould

Composts are known to contain beneficial bacteria, which may be antagonistic to plant pathogens. This work evaluated whether carrot white mould, causal agent Sclerotinia sclerotiorum, can be reduced using antagonistic bacteria isolated from forestry compost. In vitro and in vivo experiments demonstrated that bacteria from the genera Pseudomonas and Bacillus can inhibit mycelial growth and reduce white mould. Bacillus subtilis strains F9–2 and F9–12 and Pseudomonas arsenicoxydans strain F9–7 showed the highest inhibitory properties. Three cyclic dipeptides (diketopiperazines) were characterized from the antifungal culture filtrates of P. arsenicoxydans F9–7. When assayed against S. sclerotiorum, the diketopiperazines showed the following inhibitory activity, in increasing order: cyclo-(l-Pro-l-Val), cyclo-(l-Pro-l-Phe) and cyclo-(l-Pro-l-Leu). The combination of these diketopiperazines indicated additive and, occasionally, synergistic antifungal effects. These results indicated a potential for some bacteria to inhibit the growth of S. sclerotiorum and reduce its associated disease on carrots postharvest.

First Report of Sclerotinia Rot Caused by Sclerotinia sclerotiorum on Artemisia capillaris in China.

Artemisia capillaris (Asteraceae) is an annual herb found in ˃10 provinces in China. It is cultivated on ˃670 ha, with annual production around 2,500 tons. Its shoot is used in traditional Chinese medicine (Liu et al. 2021). From April to May 2023, Sclerotinia rot symptoms were seen at the Institute of Medicinal Plant Development (40.04°N, 116.28°E), Beijing, China. Disease incidence was up to 10% in the field through investigation of 300 plants. Initial symptoms were irregular tan-brown lesions (0.5 to 5.0 mm) that expended to circumferential necrosis on the roots and basal stem, aerial mycelia and sclerotia were developed on them. The leaves and stem tips were withered and droopy in severe cases. Twelve symptomatic primary roots of 12 plants from two sites were cut into 5 × 5 mm pieces, surface sterilized with 75% ethanol for 30 s and 5% NaClO for 60 s, rinsed with distilled water for three times, dried with sterile filter paper, put on potato dextrose agar (PDA), and incubated at 25°C in the dark for 2 days. Two Sclerotinia-like isolates were obtained using the hyphaltip method. White aerial mycelia were sparse and appressed for isolate YC1-3 and dense for isolate YC1-7. After incubated at 25°C in the dark for 15 days, 10 to 25 sclerotia were developed near the colony margin. Sclerotia of isolate YC1-3 were 1.0 to 3.9 × 1.2 to 4.5 (mean 1.8 × 2.2) mm (n = 60), ovoid or arc-shaped. Sclerotia of isolate YC1-7 were 1.5 to 3.4 × 2.7 to 9.2 (mean 2.3 × 4.3) mm (n = 60), ovoid, dumbbell shaped or curved. The isolates were identified as Sclerotinia sclerotiorum based on morphology (Maas 1998). To further identify the pathogens, molecular identification was performed with isolates YC1-3 and YC1-7. DNA of the two isolates were extracted by the cetyltrimethylammonium bromide (CTAB) method. Polymerase chain reaction was performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) region (Choi et al. 2020; White et al. 1990) and primers G3PDHfor/G3PDHrev for the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene (Garfinkel. 2021). BLAST search analysis revealed that the ITS sequence (GenBank OR229758 and OR229762) was ≥99% similar to S. sclerotiorum (MN099281, MZ379265, KX781301, etc.), and the G3PDH sequence (OR778388 and OR761975) was too (MZ493894, JQ036048, OQ790148, etc.). Phylogenetic trees were computed with ITS and G3PDH sequences using the Maximum Likelihood in MEGA 11. Nine two-month-old seedlings of A. capillaris were used to test pathogenicity. The epidermis layer of each primary root was slightly wounded (2 × 2 mm, 1 mm deep) using a sterile dissecting blade. Three plants were inoculated with mycelial plugs (5 mm in diameter) of YC1-3 and YC1-7 that cultured on PDA for 7 days. Control plants were inoculated with sterile PDA plugs. All seedlings were then incubated at 25oC and 90% relative humidity. After isolate YC1-7 inoculation 3 days and isolate YC1-3 inoculation 5 days, inoculated roots had symptoms like those in the field, controls had no symptoms. S. sclerotiorum was consistently re-isolated from diseased roots, fulfilling Koch's postulates. Diseases caused by S. sclerotiorum have been reported threatens several important economical crops (Marin and Peres 2020; Guan et al. 2022). To our knowledge, this is the first report of S. sclerotiorum causes Sclerotinia rot on A. capillaris. To avoid of significant economic losses, it is urgent to establish an effective disease-management strategy.

Design, Synthesis, and Biological Activities of Novel Coumarin Derivatives as Pesticide Candidates.

Food security is an important issue in the 21st century; preventing and controlling crop diseases and pests are the key to solve this problem. The creation of new pesticides based on natural products is an important and effective method. Herein, coumarins were selected as parent structures, and a series of their derivatives were designed, synthesized, and evaluated for their antiviral activities, fungicidal activities, and insecticidal activities. We found that coumarin derivatives exhibited good to excellent antiviral activities against tobacco mosaic virus (TMV). The antiviral activities of I-1, I-2a, I-4b, II-2c, II-2g, II-3, and II-3b are better than that of ribavirin at 500 μg/mL. Molecular docking research showed that these compounds had a strong interaction with TMV CP. These compounds also showed broad-spectrum fungicidal activities against 14 plant pathogenic fungi. The EC50 values of I-1, I-2a, I-3c, and II-2d are in the range of 1.56-8.65 μg/mL against Rhizoctonia cerealis, Physalospora piricola, Sclerotinia sclerotiorum, and Pyricularia grisea. Most of the compounds also displayed good insecticidal activities against Mythimna separata. Pesticide-likeness analysis showed that these compounds are following pesticide-likeness and have the potential to be developed as pesticide candidates. The present work lays a foundation for the discovery of novel pesticide lead compounds based on coumarin derivatives.

Characterization of SsHog1 and Shk1 Using Efficient Gene Knockout Systems through Repeated Protoplasting and CRISPR/Cas9 Ribonucleoprotein Approaches in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is the causal agent of sclerotinia stem rot in over 400 plant species. In a previous study, the group III histidine kinase gene of S. sclerotiorum (Shk1) revealed its involvement in iprodione and fludioxonil sensitivity and osmotic stress. To further investigate the fungicide sensitivity associated with the high-osmolarity glycerol (HOG) pathway, we functionally characterized SsHog1, which is the downstream kinase of Shk1. To generate knockout mutants, split marker transformation combined with a newly developed repeated protoplasting method and CRISPR/Cas9 ribonucleoprotein (RNP) delivery approach were used. The pure SsHog1 and Shk1 knockout mutants showed reduced sensitivity to fungicides and increased sensitivity to osmotic stress. In addition, the SsHog1 knockout mutants demonstrated reduced virulence compared to Shk1 knockout mutants and wild-type. Our results indicate that the repeated protoplasting method and RNP approach can generate genetically pure homokaryotic mutants and SsHog1 is involved in osmotic adaptation, fungicide sensitivity, and virulence in S. sclerotiorum.

Chelation of the Optimal Antifungal Pogostone Analogue with Copper(II) to Explore the Dual Antifungal and Antibacterial Agent.

In an ongoing effort to explore more potent antifungal pogostone (Po) analogues, we maintained the previously identified 3-acetyl-4-hydroxy-2-pyrone core motif while synthesizing a series of Po analogues with variations in the alkyl side chain. The in vitro bioassay results revealed that compound 21 was the most potent antifungal analogue with an EC50 value of 1.1 μg/mL against Sclerotinia sclerotiorum (Lib.) de Bary. Meanwhile, its Cu(II) complex 34 manifested significantly enhanced antibacterial activity against Xanthomonas campestris pv campestris (Xcc) with a minimum inhibitory concentration (MIC) value of 300 μg/mL compared with 21 (MIC = 700 μg/mL). Complex 34 exhibited a striking preventive effect against S. sclerotiorum and Xcc in rape leaves, with control efficacies of 98.8% (50 μg/mL) and 80.7% (1000 μg/mL), respectively. The 3D-QSAR models generated using Topomer comparative molecular field analysis indicated that a shorter alkyl chain (carbon atom number <8), terminal rings, or electron-deficient groups on the alkyl side chain are beneficial for antifungal potency. Further, bioassay results revealed that the component of 21 in complex 34 dominated the antifungal activity, but the introduction of Cu(II) significantly enhanced its antibacterial activity. The toxicological observations demonstrated that 21 could induce abnormal mitochondrial morphology, loss of mitochondrial membrane potential, and reactive oxygen species (ROS) accumulation in S. sclerotiorum. The enzyme assay results showed that 21 is a moderate promiscuous inhibitor of mitochondrial complexes II and III. Besides, the introduction of Cu(II) to 34 could promote the disruption of the cell membrane and intracellular proteins and the ROS level in Xcc compared with 21. In summary, these results highlight the potential of 34 as a dual antifungal and antibacterial biocide for controlling rape diseases or as a promising candidate for further optimization.

The Transcription Factor SsZNC1 Mediates Virulence, Sclerotial Development, and Osmotic Stress Response in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a fungal pathogen with a broad range of hosts, which can cause diseases and pose a great threat to many crops. Fungal-specific Zn2Cys6 transcription factors (TFs) constitute a large family prevalent among plant pathogens. However, the function of Zn2Cys6 TFs remains largely unknown. In this study, we identified and characterized SsZNC1, a Zn2Cys6 TF in S. sclerotiorum, which is involved in virulence, sclerotial development, and osmotic stress response. The expression of SsZNC1 was significantly up-regulated in the early stages of S. sclerotiorum infection on Arabidopsis leaves. The target deletion of SsZNC1 resulted in reduced virulence on Arabidopsis and oilseed rape. In addition, sclerotial development ability and growth ability under hyperosmotic conditions of SsZNC1 knockout transformants were reduced. A transcriptomic analysis unveiled its regulatory role in key cellular functions, including cellulose catabolic process, methyltransferase activity, and virulence, etc. Together, our results indicated that SsZNC1, a core regulatory gene involved in virulence, sclerotial development and stress response, provides new insight into the transcription regulation and pathogenesis of S. sclerotiorum.

Design and Synthesis of Novel Diphenyl Ether Carboxamide Derivatives To Control the Phytopathogenic Fungus Sclerotinia sclerotiorum.

Sclerotinia stem rot (SSR) caused by the phytopathogenic fungus Sclerotinia sclerotiorum has led to serious losses in the yields of oilseed rape and other crops every year. Here, we designed and synthesized a series of carboxamide derivatives containing a diphenyl ether skeleton by adopting the scaffold splicing strategy. From the results of the mycelium growth inhibition experiment, inhibition rates of compounds 4j and 4i showed more than 80% to control S. sclerotiorum at a dose of 50 μg/mL, which is close to that of the positive control (flubeneteram, 95%). Then, the results of a structure-activity relationship study showed that the benzyl scaffold was very important for antifungal activity and that introducing a halogen atom on the benzyl ring would improve antifungal activity. Furthermore, the results of an in vitro activity test suggested that these novel compounds can inhibit the activity of succinate dehydrogenase (SDH), and the binding mode of 4j with SDH was basically similar to that of the flutolanil derivative. Morphological observation of mycelium revealed that compound 4j could cause a damage on the mycelial morphology and cell structure of S. sclerotiorum, resulting in inhibition of the growth of mycelia. Furthermore, in vivo antifungal activity assessment of 4j displayed a good control of S. sclerotiorum (>97%) with a result similar to that of the positive control at a concentration of 200 mg/L. Thus, the diphenyl ether carboxamide skeleton is a new starting point for the discovery of new SDH inhibitors and is worthy of further development.

Simulations of multiple breeding strategy scenarios in common bean for assessing genomic selection accuracy and model updating.

The aim of this study was to evaluate the accuracy of the ridge regression best linear unbiased prediction model across different traits, parent population sizes, and breeding strategies when estimating breeding values in common bean (Phaseolus vulgaris). Genomic selection was implemented to make selections within a breeding cycle and compared across five different breeding strategies (single seed descent, mass selection, pedigree method, modified pedigree method, and bulk breeding) following 10 breeding cycles. The model was trained on a simulated population of recombinant inbreds genotyped for 1010 single nucleotide polymorphism markers including 38 known quantitative trait loci identified in the literature. These QTL included 11 for seed yield, eight for white mold disease incidence, and 19 for days to flowering. Simulation results revealed that realized accuracies fluctuate depending on the factors investigated: trait genetic architecture, breeding strategy, and the number of initial parents used to begin the first breeding cycle. Trait architecture and breeding strategy appeared to have a larger impact on accuracy than the initial number of parents. Generally, maximum accuracies (in terms of the correlation between true and estimated breeding value) were consistently achieved under a mass selection strategy, pedigree method, and single seed descent method depending on the simulation parameters being tested. This study also investigated model updating, which involves retraining the prediction model with a new set of genotypes and phenotypes that have a closer relation to the population being tested. While it has been repeatedly shown that model updating generally improves prediction accuracy, it benefited some breeding strategies more than others. For low heritability traits (e.g., yield), conventional phenotype-based selection methods showed consistent rates of genetic gain, but genetic gain under genomic selection reached a plateau after fewer cycles. This plateauing is likely a cause of faster fixation of alleles and a diminishing of genetic variance when selections are made based on estimated breeding value as opposed to phenotype.

First Report of White Mold Caused by Sclerotinia sclerotiorum on Cabbage in Mexico.

The state of Puebla is the main producer of cabbage (Brassica oleracea var. capitata) in Mexico, with an area of approximately 1,858 ha (SIAP 2023). In April 2023, a field sampling was conducted in the San Luis Ajajalpan, Tecali de Herrera (18°55.57'N, 97°55.607'W), Puebla, Mexico. The average temperature was 24°C and the relative humidity was 95% for five consecutive days. Cabbage plants cv. 'American Taki San Juan' close to harvest, with head rot symptoms were found in a commercial area of approximately 3 ha, at an estimated incidence of 35 to 45%. More than 70% of the leaves were symptomatic on severely affected plants. Typical symptoms included chlorosis of older foliage, soft rot with abundant white to gray mycelium, and abundant production of large and irregularly-shaped sclerotia. The fungus was isolated from 30 symptomatic plants. Sclerotia were collected from symptomatic heads, surface sterilized in 3% NaOCl, rinsed twice with sterile distilled water, and plated on Potato Dextrose Agar (PDA) with sterile forceps. Subsequently, a dissecting needle was used to place fragments of mycelium directly on PDA. Plates were placed in an incubator at 25°C in the dark. A total of 30 representative isolates were obtained by the hyphal-tip method, one from each diseased plant (15 isolates from sclerotia and 15 from mycelial fragments). After 8 days, colonies had fast-growing, dense, cottony-white aerial mycelium forming irregular sclerotia of 3.75 ± 0.8 mm (mean ± standard deviation, n=100). Each Petri dish produced 14-25 sclerotia (mean = 18, n = 50), after 10 days. The sclerotia were initially white and gradually turned black. The isolates were identified as Sclerotinia sclerotiorum based on morphological characteristics (Saharan and Mehta 2008). Two representative isolates were chosen for molecular identification, and genomic DNA was extracted by a CTAB protocol. The ITS region and the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene were sequenced for two isolates (White et al. 1990; Staats et al. 2005). The ITS and G3PDH sequences of a representative isolate (SsC.1) were deposited in the GenBank (ITS- OR286628; G3PDH- OR333495). BLAST analysis of the partial sequences ITS (509 bp) and G3PDH (915 bp) showed 100% similarity to S. sclerotiorum isolates (GenBank: MT436756.1 and OQ790148). Pathogenicity was confirmed by inoculating 10 detached cabbage heads of 'American Taki San Juan', using the SsC.1 isolate, according to Sanogo et al. (2015). Heads were placed on the rim of a plastic container and inserted in a moisture box with 2 cm of water on its bottom. The box was covered with a plastic sheet to maintain humidity. The control plants were inoculated with a plug of noncolonized PDA. The inoculated cabbages were covered with white to gray mycelia and abundant sclerotia within 10 days, whereas no symptoms were observed on non-inoculated controls. The fungus was re-isolated from the inoculated cabbages as described above, fulfilling Koch's postulates. The pathogenicity tests were repeated three times. White mold caused by S. sclerotiorum on Brussels sprouts was recently reported in Mexico (Ayvar-Serna et al. 2023). In 2015, S. sclerotiorum was reported on cabbage in New Mexico, causing head rot (Sanogo et al. 2015). To our knowledge, this is the first report of S. sclerotiorum causing white mold on cabbage in Mexico. This research is essential for designing management strategies and preventing spread to other production areas.

Botrytis cinerea type II inhibitor of apoptosis BcBIR1 enhances the biocontrol capacity of Coniothyrium minitans.

Apoptosis-like programmed cell death is associated with fungal development, ageing, pathogenicity and stress responses. Here, to explore the potential of Botrytis cinerea type II inhibitor of apoptosis (IAP) BcBIR1 in elevating the biocontrol efficacy of Coniothyrium minitans, the BcBIR1 gene was heterologously expressed in C. minitans. Results indicated that the strains expressing BcBIR1 had higher rates of conidiation, mycelial growth and biomass growth than the wild-type strain. Moreover, BcBIR1 was found to inhibit apoptosis, indicating its role as an IAP in C. minitans. Under various abiotic stresses, the growth rates of BcBIR1-expressing strains were significantly higher than that of the wild-type strain. Moreover, the conidial survival rate of the BcBIR1-expressing strains treated with ultraviolet irradiation was enhanced. In antifungal activity assay, the culture filtrates of BcBIR1-expressing strains displayed a stronger inhibitory effect on B. cinerea and Sclerotinia sclerotiorum than the wild-type strain. The study also found that BcBIR1 expression increased the mycoparasitism against the sclerotia, but not the hyphae of S. sclerotiorum. Taken together, these results suggest that BcBIR1 enhances vegetative growth, conidiation, anti-apoptosis activity, abiotic stress resistance, antifungal activity and mycoparasitism in C. minitans. As an IAP, BcBIR1 may improve the control capacity of C. minitans against S. sclerotiorum.

The schizotrophic lifestyle of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a cosmopolitan and typical necrotrophic phytopathogenic fungus that infects hundreds of plant species. Because no cultivars highly resistant to S. sclerotiorum are available, managing Sclerotinia disease caused by S. sclerotiorum is still challenging. However, recent studies have demonstrated that S. sclerotiorum has a beneficial effect and can live mutualistically as an endophyte in graminaceous plants, protecting the plants against major fungal diseases. An in-depth understanding of the schizotrophic lifestyle of S. sclerotiorum during interactions with plants under different environmental conditions will provide new strategies for controlling fungal disease. In this review, we summarize the pathogenesis mechanisms of S. sclerotiorum during its attack of host plants as a destructive pathogen and discuss its lifestyle as a beneficial endophytic fungus.