Arabidopsis thaliana ubiquitin-associated protein 2 (AtUAP2) functions as an E4 ubiquitin factor and negatively modulates dehydration stress response.

E4, a ubiquitin (Ub) chain assembly factor and post-translational modification protein, plays a key role in the regulation of multiple cellular functions in plants during biotic or abiotic stress. We have more recently reported that E4 factor AtUAP1 is a negative regulator of the osmotic stress response and enhances the multi-Ub chain assembly of E3 ligase Arabidopsis thaliana RING Zinc Finger 1 (AtRZF1). To further investigate the function of other E4 Ub factors in osmotic stress, we isolated AtUAP2, an AtUAP1 homolog, which interacted with AtRZF1, using pull-down assay and bimolecular fluorescence complementation analysis. AtUAP2, a Ub-associated motif-containing protein, interacts with oligo-Ub5, -Ub6, and -Ub7 chains. The yeast functional complementation experiment revealed that AtUAP2 functions as an E4 Ub factor. In addition, AtUAP2 is localized in the cytoplasm, different from AtUAP1. The activity of AtUAP2 was relatively strongly induced in the leaf tissue of AtUAP2 promoter-ß-glucuronidase transgenic plants by abscisic acid, dehydration, and oxidative stress. atuap2 RNAi lines were more insensitive to osmotic stress condition than wild-type during the early growth of seedlings, whereas the AtUAP2-overexpressing line exhibited relatively more sensitive responses. Analyses of molecular and physiological experiments showed that AtUAP2 could negatively mediate the osmotic stress-induced signaling. Genetic studies showed that AtRZF1 mutation could suppress the dehydration-induced sensitive phenotype of the AtUAP2-overexpressing line, suggesting that AtRZF1 acts genetically downstream of AtUAP2 during osmotic stress. Taken together, our findings show that the AtRZF1-AtUAP2 complex may play important roles in the ubiquitination pathway, which controls the osmotic stress response in Arabidopsis.

Compensatory Modulation of Seed Storage Protein Synthesis and Alteration of Starch Accumulation by Selective Editing of 13 kDa Prolamin Genes by CRISPR-Cas9 in Rice.

Rice prolamins are categorized into three groups by molecular size (10, 13, or 16 kDa), while the 13 kDa prolamins are assigned to four subgroups (Pro13a-I, Pro13a-II, Pro13b-I, and Pro13b-II) based on cysteine residue content. Since lowering prolamin content in rice is essential to minimize indigestion and allergy risks, we generated four knockout lines using CRISPR-Cas9, which selectively reduced the expression of a specific subgroup of the 13 kDa prolamins. These four mutant rice lines also showed the compensatory expression of glutelins and non-targeted prolamins and were accompanied by low grain weight, altered starch content, and atypically-shaped starch granules and protein bodies. Transcriptome analysis identified 746 differentially expressed genes associated with 13 kDa prolamins during development. Correlation analysis revealed negative associations between genes in Pro13a-I and those in Pro13a-II and Pro13b-I/II subgroups. Furthermore, alterations in the transcription levels of 9 ER stress and 17 transcription factor genes were also observed in mutant rice lines with suppressed expression of 13 kDa prolamin. Our results provide profound insight into the functional role of 13 kDa rice prolamins in the regulatory mechanisms underlying rice seed development, suggesting their promising potential application to improve nutritional and immunological value.

Production of highly pure R,R-2,3-butanediol for biological plant growth promoting agent using carbon feeding control of Paenibacillus polymyxa MDBDO.

BACKGROUND: Chemical fertilizers have greatly contributed to the development of agriculture, but alternative fertilizers are needed for the sustainable development of agriculture. 2,3-butanediol (2,3-BDO) is a promising biological plant growth promoter. RESULTS: In this study, we attempted to develop an effective strategy for the biological production of highly pure R,R-2,3-butanediol (R,R-2,3-BDO) by Paenibacillus polymyxa fermentation. First, gamma-ray mutagenesis was performed to obtain P. polymyxa MDBDO, a strain that grew faster than the parent strain and had high production of R,R-2,3-BDO. The activities of R,R-2,3-butanediol dehydrogenase and diacetyl reductase of the mutant strain were increased by 33% and decreased by 60%, respectively. In addition, it was confirmed that the carbon source depletion of the fermentation broth affects the purity of R,R-2,3-BDO through batch fermentation. Fed-batch fermentation using controlled carbon feeding led to production of 77.3 g/L of R,R-2,3-BDO with high optical purity (> 99% of C4 products) at 48 h. Additionally, fed-batch culture using corn steep liquor as an alternative nitrogen source led to production of 70.3 g/L of R,R-2,3-BDO at 60 h. The fed-batch fermentation broth of P. polymyxa MDBDO, which contained highly pure R,R-2,3-BDO, significantly stimulated the growth of soybean and strawberry seedlings. CONCLUSIONS: This study suggests that P. polymyxa MDBDO has potential for use in biological plant growth promoting agent applications. In addition, our fermentation strategy demonstrated that high-purity R,R-2,3-BDO can be produced at high concentrations using P. polymyxa.

Identification and Fungicide Control of Bipolaris sorokiniana Causing Leaf Spot and Blight on Common Hop (Humulus lupulus) in Korea.

Hop (Humulus lupulus) is a perennial herbaceous vine belonging to the family Cannabaceae. This crop is commercially grown for the brewing industry for its bitter and aromatic flavor, as well as its antiseptic properties. In June 2021, leaf spot and blight was observed on common hop plants in Buan-gun, Jeollabuk-do, South Korea. The typical symptoms were small to large, dark-brown, necrotic lesions with yellow halos on the leaves. This study aimed to clarify the causal agent of this disease. Two fungal species, Alternaria alternata and Bipolaris sorokiniana, were isolated from the diseased leaf samples and identified by combining morphological observations and phylogenetic analysis using sequence datasets of internal transcribed spacer (ITS), Alt a1, rpb2, endoPG, and OPA10-2; and ITS, gpd, and tef1, respectively. Pathogenicity of the fungal isolates on detached leaves and living plants revealed that B. sorokiniana is the causal pathogen of this disease, while A. alternata is potentially a saprophyte. Fungicide sensitivity of the pathogen B. sorokiniana was further estimated in vitro using three classes of fungicides represented by fluxapyroxad, pyraclostrobin, and hexaconazole. The effective concentrations that inhibited 50% of spore germination (EC50) were 0.72, 1.90, and 0.68 µg ml-1, respectively. Moreover, all of these fungicides were able to control B. sorokiniana on detached common hop leaves at their recommended concentrations. In conclusion, this study reports leaf spot and blight of common hop caused by B. sorokiniana for the first time and proposes potential fungicides for this disease.

First Report of Binucleate Rhizoctonia AG-G Causing Root Rot of Japanese Bay Tree (Machilus thunbergii) in Korea.

Machilus thunbergii Sieb. & Zucc., commonly known as Japanese bay tree, is a large evergreen tree belonging to the Lauraceae family and is widely distributed in Asia, including Korea in subtropical and tropical forest areas (Wu et al., 2006). In April 2021, a root rot disease of 2-year-old Japanese bay trees was observed in a nursery on Wando Island in Korea. Tree roots exhibited brown to black discoloration, root rot, and deterioration, and leaves were severely wilted followed by plant death, with a disease incidence of approximately 30%. Symptomatic roots were surface sterilized with 1% NaOCl for 5 min and washed three times with distilled water. The root tissues were dried and plated on potato dextrose agar (PDA) and vegetable juice agar (V8) media. After 3-4 days of incubation at 25 ˚C, brown Rhizoctonia fungal-like colonies grew on both culture media. Hyphae of two representative isolates (CMML21-35 and CMML21-36) exhibited typical characteristics of Rhizoctonia, including a constriction of branching hyphae (Alvarez et al., 2013). In addition, two nuclei in each mycelial cell were observed after staining of mycelia with 0.1% Safranin O. The two isolates were identified as binucleate Rhizoctonia based on the microscopic observation. To confirm identification of the isolates, the internal transcribed spacer (ITS) and large subunit (LSU) regions were sequenced using two primer sets, ITS1/ITS4 and LROR/LR5 (White et al., 1990; Vilgalys and Hester 1990). BLASTn search analysis revealed that the ITS sequence of isolates had 99.66% (582 base pair matched of 584) sequence similarity with the sequences of binucleate Rhizoctonia (accession numbers JF519837 and AY927327, respectively) and the LSU sequence matched well with the sequence of Rhizoctonia sp. AG-G (accession number MN977413; similarity 99.56% and 910 base pair matched of 914). The sequences were deposited in GenBank under accession numbers OM049427 and OM049428 for ITS, OM679289 and OM679290 for LSU. Phylogenetic analysis of ITS and LSU regions revealed that the isolates grouped with binucleate Rhizoctonia anastomosis group AG-G (Teleomorph: Ceratobasidium sp.) with a high bootstrap value. Accordingly, the morphological and molecular characteristics confirmed the causal pathogen as binucleate Rhizoctonia AG-G (Jiang et al., 2016; Gonzalez et al. 2016). To test pathogenicity, a 2-year-old Japanese bay tree was inoculated by creating a hole in the soil near the root rhizosphere and placing 1.5g of ground mycelia obtained from a 5 day-old broth culture at two time points one week apart (Bartz et al., 2010). The control pot was inoculated with sterilized ddH2O. Inoculated and control plant pots were incubated in plastic boxes with 100% relative humidity at 25 ℃ for five days. After that, the pots were placed in the greenhouse at 23-25 ℃. One month post inoculation, initial disease symptoms were observed, and after two months, severe foliar wilting and eventual plant death occurred. The non-inoculated control remained healthy. The pathogen was re-isolated from infected roots, fulfilling Koch's postulates. The experiment was conducted three times with three replications. This is the first report of root rot of Japanese bay tree caused by binucleate Rhizoctonia AG-G in Korea and in the world. Previously, a pathogenic binucleate Rhizoctonia AG-G was isolated from colonized apple tree roots in orchards in Italy (Kelderer et al., 2012). The present study implies that this pathogen potentially causes a negative impact on the nursery and forest industries, thus further research on the screening for pathogenicity in other tropical and subtropical trees and also apple, which is an important crop in Korea, is needed.

First report of Diaporthe eres causing leaf spot disease on Machilus thunbergii in Korea.

Machilus thunbergii (Japanese bay tree) is native to warm temperate and subtropical regions in East Asia such as China, Japan, Korea, Taiwan, and Vietnam (Wu et al., 2006). This tree is used for landscape trees, windbreaks, and furniture because the wood is hard and dense (Hong et al., 2016). In May 2020, a leaf spot disease was observed on M. thunbergii in an arboretum on Wando Island, Korea. Among 25 trees surveyed in the arboretum, 7 trees showed 5 to 30% leaf spot disease. Symptoms consisted of gray and dry leaf spots up to approximately one to two centimeters in diameter, surrounded by a deep black margin. Leaf samples containing lesions were collected from the seven diseased trees. Pieces of leaf tissue (5mm × 5mm) were cut from the lesion margins and surface disinfected with 1% sodium hypochlorite (NaOCl) for 1 min and rinsed with sterile distilled water three times, patted dry on sterile paper towel and placed on Potato Dextrose Agar (PDA) in Petri dishes. From the cultures, ten fungal isolates were obtained and two representative isolates (CMML20-5 and CMML20-6) were stored at the Molecular Microbiology Laboratory, Chonnam National University, Gwangju, Korea. Colony morphology of the two isolates on PDA was observed after 7 days at 25°C in the dark. Conidiomata were induced after 7days in a 14h-10h light-dark condition using sufficiently grown mycelium in PDA, and both alpha and beta conidia were observed. Alpha conidia were 7.6 ± 0.9 × 2.8 ± 0.4 µm (n = 30), fusiform, aseptate, and hyaline. Beta conidia were 28.1 ± 3.6 × 2.7 ± 0.4 µm (n = 30), aseptate, hyaline, linear to hooked. Genomic DNA of the two isolates was extracted using the CTAB DNA extraction method (Cubero et al., 1999), followed by PCR using primer sets of the internal transcribed spacer (ITS1/ITS4) (White et al., 1990), elongation factor 1-α (EF1-728F/EF1-986R), calmodulin (CAL228F/CAL737R) (Carbone and Kohn, 1999), and TUB2 (Bt2a/Bt2b) (Glass and Donaldson 1995). PCR products were sequenced and analyzed to confirm species identity. The obtained sequences were deposited in GenBank (accession numbers OM049469, OM049470 for ITS, OM069429, OM069430 for EF1-α, OP130141, OP130142 for CAL, and OP130139, OP130140 for TUB2). BLASTn search analyses for ITS, EF1-α, CAL, and TUB2 sequences of two isolates selected resulted in near identical match (>97% for ITS, 100% for EF1-α, >99% for CAL, and >96% for TUB2) to sequences of Diaporthe eres strain AR4346 (=Phomopsis fukushii) (JQ807429 for ITS, JQ807355 for EF1-α, KJ435003 for CAL, and KJ420823 for TUB2). Phylogenetic analysis using maximum likelihood indicated that the two isolates grouped with reference strains (AR4346, AR4349, and AR4363) of D. eres with 76% bootstrap support. Based on morphological and phylogenetic analyses, the two isolates characterized in this study are members of the Diaporthe eres species complex as described by Udayanga et at. 2014. Pathogenicity tests were conducted using both detached leaf and whole plant assays. Mycelial PDA plugs (5-mm in diameter) or 10µl of 106 conidia suspensions were inoculated on detached leaves of M. thunbergii from 2-year-old trees and placed in 90 mm Petri-dishes containing wet filter papers or water agar medium. Mock inoculated controls used water in place of conidial suspensions. The plates were sealed with Parafilm and incubated at 25°C in the dark. Two year old M. thunbergii trees were inoculated with wet mycelia (1.5g) that was ground with a homogenizer and mixed with 50ml of sterile water and sprayed onto wounded leaves and stems with a needle. Mock inoculated controls were sprayed with water only. The inoculated seedlings were placed in plastic containers at 25 to 30°C to maintain high humidity. The pathogenicity tests were repeated three times with three replications. In detached leaves, symptoms of black spots were observed 6 days after mycelial plug inoculation and 20 days after conidia inoculation. In whole plants, typical symptoms were observed 9 days after inoculation. Symptoms were not observed on the control leaves and plants. Diaporthe eres was re-isolated from the inoculated leaf and whole plants and morphologically identified, fulfilling Koch's postulates. Diaporthe eres has been reported to cause a leaf spot on Photinia × fraseri 'Red Robin' in China (Song et al. 2019). To our knowledge, this is the first report of leaf spot disease caused by Diaporthe eres on Japanese bay tree (Machilus thunbergii) in Korea. It is expected that use of this tree will expand given its utility, however infection with D. eres can cause serious diseases to the leaves and stems. Therefore, further studies on disease management are needed.

A Method for the Examination of SDHI Fungicide Resistance Mechanisms in Phytopathogenic Fungi Using a Heterologous Expression System in Sclerotinia sclerotiorum.

Succinate dehydrogenase inhibitors (SDHIs) are a class of broad-spectrum fungicides used for management of diseases caused by phytopathogenic fungi. In many cases, reduced sensitivity to SDHI fungicides has been correlated with point mutations in the SdhB and SdhC target genes that encode components of the succinate dehydrogenase complex. However, the genetic basis of SDHI fungicide resistance mechanisms has been functionally characterized in very few fungi. Sclerotinia sclerotiorum is a fast-growing and SDHI fungicide-sensitive phytopathogenic fungus that can be conveniently transformed. Given the high amino acid sequence similarity and putative structural similarity of SDHI protein target sites between S. sclerotiorum and other common phytopathogenic ascomycete fungi, we developed an in vitro heterologous expression system that used S. sclerotiorum as a reporter strain. With this system, we were able to demonstrate the function of mutant SdhB or SdhC alleles from several ascomycete fungi in conferring resistance to multiple SDHI fungicides. In total, we successfully validated the function of Sdh alleles that had been previously identified in field isolates of Botrytis cinerea, Blumeriella jaapii, and Clarireedia jacksonii (formerly S. homoeocarpa) in conferring resistance to boscalid, fluopyram, or fluxapyroxad and used site-directed mutagenesis to construct and phenotype a mutant allele that is not yet known to exist in Monilinia fructicola populations. We also examined the functions of these alleles in conferring cross-resistance to more recently introduced SDHIs including inpyrfluxam, pydiflumetofen, and pyraziflumid. The approach developed in this study can be widely applied to interrogate SDHI fungicide resistance mechanisms in other phytopathogenic ascomycetes.

Draft Genome Sequence Resource for Blumeriella jaapii, the Cherry Leaf Spot Pathogen.

Blumeriella jaapii is the causal agent of cherry leaf spot (CLS), the most important disease of tart cherry in the Midwestern United States. Infection of leaves by B. jaapii leads to premature defoliation, which places trees at heightened risk of winter injury and death. Current management of CLS relies primarily on the application of three important fungicide classes, quinone outside inhibitors, sterol demethylation inhibitors, and succinate dehydrogenase inhibitors. Here, we present the first high-quality genome of B. jaapii through a hybrid assembly of PacBio long reads and Illumina short reads. The assembled draft genome of B. jaapii is 47.4 Mb and consists of 95 contigs with a N50 value of 1.5 Mb. The genomic information of B. jaapii, representing the most complete sequenced genome of the family Dermateaceae (Ascomycota) to date, provides a valuable resource for identifying fungicide resistance mechanisms of this pathogen and expands our knowledge of the phytopathogenic fungi in this family.

Diagnostic qPCR Assay to Detect Fusarium brasiliense, a Causal Agent of Soybean Sudden Death Syndrome and Root Rot of Dry Bean.

Species within clade 2 of the Fusarium solani species complex (FSSC) are significant pathogens of dry bean (Phaseolus vulgaris) and soybean (Glycine max), causing root rot and/or sudden death syndrome (SDS). These species are morphologically difficult to distinguish and often require molecular tools for proper diagnosis to a species level. Here, a TaqMan probe-based quantitative PCR (qPCR) assay was developed to distinguish Fusarium brasiliense from other closely related species within clade 2 of the FSSC. The assay displays high specificity against close relatives and high sensitivity, with a detection limit of 100 fg. This assay was able to detect F. brasiliense from purified mycelia, infected dry bean roots, and soil samples throughout Michigan. When multiplexed with an existing qPCR assay specific to Fusarium virguliforme, accurate quantification of both F. brasiliense and F. virguliforme was obtained, which can facilitate accurate diagnoses and identify coinfections with a single reaction. The assay is compatible with multiple qPCR thermal cycling platforms and will be helpful in providing accurate detection of F. brasiliense. Management of root rot and SDS pathogens in clade 2 of the FSSC is challenging and must be done proactively, because no midseason management strategies currently exist. However, accurate detection can facilitate management decisions for subsequent growing seasons to successfully manage these pathogens.

Convergent Evolution of C239S Mutation in Pythium spp. ß-Tubulin Coincides with Inherent Insensitivity to Ethaboxam and Implications for Other Peronosporalean Oomycetes.

Ethaboxam is a benzamide antioomycete chemical (oomicide) used in corn and soybean seed treatments. Benzamides are hypothesized to bind to ß-tubulin, thus disrupting microtubule assembly. Recently, there have been reports of corn- and soybean-associated oomycetes that are insensitive to ethaboxam despite never having been exposed. Here, we investigate the evolutionary history and molecular mechanism of ethaboxam insensitivity. We tested the sensitivity of 194 isolates representing 83 species across four oomycete genera in the Peronosporalean lineage that were never exposed to ethaboxam. In all, 84% of isolates were sensitive to ethaboxam (effective concentration to reduce optical density at 600 nm by 50% when compared with the nonamended control [EC50] < 5 µg ml-1), whereas 16% were insensitive (EC50 > 11 µg ml-1). Of the insensitive isolates, two different transversion mutations were present in the 239th codon in ß-tubulin within three monophyletic groups of Pythium spp. The transversion mutations lead to the same amino acid change from an ancestral cysteine to serine (C239S), which coincides with ethaboxam insensitivity. In a treated soybean seed virulence assay, disease severity was not reduced on ethaboxam-treated seed for an isolate of Pythium aphanidermatum containing a S239 but was reduced for an isolate of P. irregulare containing a C239. We queried publicly available ß-tubulin sequences from other oomycetes in the Peronosporalean lineage to search for C239S mutations from other species not represented in our collection. This search resulted in other taxa that were either homozygous or heterozygous for C239S, including all available species within the genus Peronospora. Evidence presented herein supports the hypothesis that the convergent evolution of C239S within Peronosporalean oomycetes occurred without selection from ethaboxam yet confers insensitivity. We propose several evolutionary hypotheses for the repeated evolution of the C239S mutation.

Evaluation of a Sclerotinia homoeocarpa Population with Multiple Fungicide Resistance Phenotypes Under Differing Selection Pressures.

Dollar spot, caused by Sclerotinia homoeocarpa, is one of the most significant diseases of cool-season turfgrass on golf courses. Resistance to the benzimidazole, dicarboximide, and succinate dehydrogenase inhibitor (SDHI) classes and reduced sensitivity to the sterol-demethylation inhibitor (DMI) in S. homoeocarpa populations have been widely reported in the United States. Moreover, the occurrence of S. homoeocarpa populations with multiple fungicide resistance (MFR) is a growing problem on golf courses. The present study was undertaken to evaluate the efficacy of DMI, dicarboximide, and SDHI against a S. homoeocarpa population with MFR on a Connecticut golf course fairway from 2014 to 2016. Also, because the S. homoeocarpa population consisted of four different phenotypes with differing resistance profiles to benzimidazole, dicarboximide, and DMI, in vitro sensitivity assays were used to understand the dynamics of the MFR population in the presence and absence of fungicide selection pressures. Results indicated that boscalid fungicide (SDHI) was able to provide an acceptable control of the MFR dollar spot population. Propiconazole or iprodione application selected isolates with both DMI and dicarboximide resistance (DMI-R/Dicar-R). In the absence of fungicide selection pressures, the percent frequency of DMI-R/Dicar-R or DMI and benzimidazole resistance (DMI-R/Ben-R) isolates declined in the population. Out of the four phenotypes, the percent frequency of isolates with DMI, dicarboximide, and benzimidazole resistance (DMI-R/Dicar-R/Ben-R) was the lowest in the population regardless of fungicide selection pressures. Our first report of MFR population dynamics will help develop effective strategies for managing MFR and potentially delay the emergence of future resistant populations in S. homoeocarpa.

Soybean Sudden Death Syndrome Causal Agent Fusarium brasiliense Present in Michigan.

Sudden death syndrome (SDS), caused by members of Fusarium solani species complex (FSSC) clade 2, is a major and economically important disease in soybean worldwide. The primary causal agent of SDS isolated to date in North America has been F. virguliforme. In 2014 and 2016, SDS symptoms were found in two soybean fields located on the same farm in Michigan. Seventy Fusarium strains were isolated from roots of the SDS-symptomatic soybeans in two fields. Phylogenetic analysis of partial sequences of elongation factor-1α, the nuclear ribosomal DNA intergenic spacer region, and the RNA polymerase II beta subunit revealed that the primary FSSC species isolated was F. brasiliense (58 and 36% in each field) and the remaining Fusarium strains were identified as F. cuneirostrum, F. phaseoli, an undescribed Fusarium sp. from FSSC clade 2, and strains in FSSC clade 5 and FSSC clade 11. Molecular identification was supported with morphological analysis and a pathogenicity assay. The soybean seedling pathogenicity assay indicated that F. brasiliense was capable of causing typical foliar SDS symptoms. Both root rot and foliar disease severity were variable by strain, just as they are in F. virguliforme. Both FSSC 5 and FSSC 11 strains were also capable of causing root rot, but SDS foliar symptoms were not detected. To our knowledge, this is the first report of F. brasiliense causing SDS in soybean in the United States and the first report of F. cuneirostrum, F. phaseoli, an as-yet-unnamed Fusarium sp., and strains in FSSC clade 5 and FSSC clade 11 associated with or causing root rot of soybean in Michigan.

Annotation resource of tandem repeat-containing secretory proteins in sixty fungi.

Fungal secretory proteins that interact with host plants are regarded as effectors. Because fungal effectors rarely contain conserved sequence features, identification and annotation of fungal effectors from predicted secretory proteins are difficult using outward comparison methods such as BLAST or hidden Markov model. In desire of more sequence features to prioritize research interests of fungal secretory proteins, this study developed a pipeline to identify tandem repeat (TR) domain within putative secretory proteins and tested a hypothesis that at least one type of TR domain in non-orthologous secretory proteins has emerged from convergent evolution for plant pathogenicity. There were 2804 types of TR domains and a total of 2925 TR-containing secretory proteins found from 60 fungi. There was no conserved type of TR domain shared only by plant pathogens, indicating functional divergence for different types of TR domain and TR-containing secretory proteins. The annotation resource of putative fungal TR-containing secretory proteins provides new sequence features that will be useful for the community interested in fungal effector biology.

Nucleic adaptability of heterokaryons to fungicides in a multinucleate fungus, Sclerotinia homoeocarpa.

Sclerotinia homoeocarpa is the causal organism of dollar spot in turfgrasses and is a multinucleate fungus with a history of resistance to multiple fungicide classes. Heterokaryosis gives rise to the coexistence of genetically distinct nuclei within a cell, which contributes to genotypic and phenotypic plasticity in multinucleate fungi. We demonstrate that field isolates, resistant to either a demethylation inhibitor or methyl benzimidazole carbamate fungicide, can form heterokaryons with resistance to each fungicide and adaptability to serial combinations of different fungicide concentrations. Field isolates and putative heterokaryons were assayed on fungicide-amended media for in vitro sensitivity. Shifts in fungicide sensitivity and microsatellite genotypes indicated that heterokaryons could adapt to changes in fungicide pressure. Presence of both nuclei in heterokaryons was confirmed by detection of a single nucleotide polymorphism in the ß-tubulin gene, the presence of microsatellite alleles of both field isolates, and the live-cell imaging of two different fluorescently tagged nuclei using laser scanning confocal microscopy. Nucleic adaptability of heterokaryons to fungicides was strongly supported by the visualization of changes in fluorescently labeled nuclei to fungicide pressure. Results from this study suggest that heterokaryosis is a mechanism by which the pathogen adapts to multiple fungicide pressures in the field.

Resistance of Sclerotinia homoeocarpa Field Isolates to Succinate Dehydrogenase Inhibitor Fungicides.

Sclerotinia homoeocarpa isolates were collected from golf courses in Japan and the United States (2016-2017). Japan isolates were collected during a monitoring study and the U.S. isolates were collected due to field failure. Five succinate dehydrogenase inhibitor (SDHI) active ingredients (boscalid, fluopyram, fluxapyroxad, isofetamid, and penthiopyrad) were examined using in vitro sensitivity assays to determine cross-resistance. Sequence analysis revealed a point mutation leading to an amino acid substitution (H267Y) and a silent mutation (CTT to CTC) at codon 181 in the SdhB subunit gene. Isolates with the B-H267Y (n = 10) mutation were resistant to boscalid and penthiopyrad and had increased sensitivity to fluopyram. SdhB silent mutation 181C>T isolates (n = 2) were resistant to boscalid, isofetamid, and penthiopyrad. Sequence analysis revealed 3 mutations leading to an amino acid substitution (G91R, n = 5; G150R, n = 1; G159W, n = 1) in the SdhC subunit gene. Isolates harboring the SdhC (G91R or G150R) mutations were resistant to boscalid, fluxapyroxad, isofetamid, and penthiopyrad. A genetic transformation system was used to generate mutants from a SDHI sensitive isolate to confirm the B-H267Y and C-G91R mutations were direct determinants of SDHI resistance and associated with in vitro sensitivity assay results.

Antifungal mechanisms of ZnO and Ag nanoparticles to Sclerotinia homoeocarpa.

Fungicides have extensively been used to effectively combat fungal diseases on a range of plant species, but resistance to multiple active ingredients has developed in pathogens such as Sclerotinia homoeocarpa, the causal agent of dollar spot on cool-season turfgrasses. Recently, ZnO and Ag nanoparticles (NPs) have received increased attention due to their antimicrobial activities. In this study, the NPs' toxicity and mechanisms of action were investigated as alternative antifungal agents against S. homoeocarpa isolates that varied in their resistance to demethylation inhibitor (DMI) fungicides. S. homoeocarpa isolates were treated with ZnO NPs and ZnCl2 (25-400 µg ml-1) and Ag NPs and AgNO3 (5-100 µg ml-1) to test antifungal activity of the NPs and ions. The mycelial growth of S. homoeocarpa isolates regardless of their DMI sensitivity was significantly inhibited on ZnO NPs (≥200 µg ml-1), Ag NPs (≥25 µg ml-1), Zn2+ ions (≥200 µg ml-1), and Ag+ ions (≥10 µg ml-1) amended media. Expression of stress response genes, glutathione S-transferase (Shgst1) and superoxide dismutase 2 (ShSOD2), was significantly induced in the isolates by exposure to the NPs and ions. In addition, a significant increase in the nucleic acid contents of fungal hyphae, which may be due to stress response, was observed upon treatment with Ag NPs using Raman spectroscopy. We further observed that a zinc transporter (Shzrt1) might play an important role in accumulating ZnO and Ag NPs into the cells of S. homoeocarpa due to overexpression of Shzrt1 significantly induced by ZnO or Ag NPs within 3 h of exposure. Yeast mutants complemented with Shzrt1 became more sensitive to ZnO and Ag NPs as well as Zn2+ and Ag+ ions than the control strain and resulted in increased Zn or Ag content after exposure. This is the first report of involvement of the zinc transporter in the accumulation of Zn and Ag from NP exposure in filamentous plant pathogenic fungi. Understanding the molecular mechanisms of NPs' antifungal activities will be useful in developing effective management strategies to control important pathogenic fungal diseases.

Molecular Mechanisms Involved in Qualitative and Quantitative Resistance to the Dicarboximide Fungicide Iprodione in Sclerotinia homoeocarpa Field Isolates.

The dicarboximide fungicide class is commonly used to control Sclerotinia homoeocarpa, the causal agent of dollar spot on turfgrass. Despite frequent occurrences of S. homoeocarpa field resistance to iprodione (dicarboximide active ingredient), the genetic mechanisms of iprodione resistance have not been elucidated. In this study, 15 field isolates (seven suspected dicarboximide resistant, three multidrug resistance (MDR)-like, and five dicarboximide sensitive) were used for sequence comparison of a histidine kinase gene, Shos1, of S. homoeocarpa. The suspected dicarboximide-resistant isolates displayed nonsynonymous polymorphisms in codon 366 (isoleucine to asparagine) in Shos1, while the MDR-like and sensitive isolates did not. Further elucidation of the Shos1 function, using polyethylene glycol-mediated protoplast transformation indicated that S. homoeocarpa mutants (Shos1I366N) from a sensitive isolate gained resistance to dicarboximides but not phenylpyrrole and polyols. The deletion of Shos1 resulted in higher resistance to dicarboximide and phenylpyrrole and higher sensitivity to polyols than Shos1I366N. Levels of dicarboximide sensitivity in the sensitive isolate, Shos1I366N, and Shos1 deletion mutants were negatively correlated to values of iprodione-induced expression of ShHog1, the last kinase in the high-osmolarity glycerol pathway. Increased constitutive and induced expression of the ATP-binding cassette multidrug efflux transporter ShPDR1 was observed in six of seven dicarboximide-resistant isolates. In conclusion, S. homoeocarpa field isolates gained dicarboximide resistance through the polymorphism in Shos1 and the overexpression of ShPDR1.

Exploring SDHI fungicide resistance in Botrytis cinerea through genetic transformation system and AlphaFold model-based molecular docking.

BACKGROUND: Gray mold caused by Botrytis cinerea is one of the most serious diseases affecting strawberry. Succinate dehydrogenase inhibitor (SDHI) fungicides have been used for more than a decade to control the disease. Monitoring resistance and improving in-depth understanding of resistance mechanisms are essential for the control of B. cinerea. RESULTS: In this study, resistance monitoring of a SDHI fungicide boscalid was conducted on B. cinerea isolated from strawberries in Korea during 2020 and 2021, with resistance rates of 76.92% and 72.25%, respectively. In resistant strains, mutations P225F/H and H272R were found in SdhB, with P225F representing the dominant mutation type. Simultaneous mutations G85A, I93V, M158V, and V168I in SdhC were detected in 54.84% of sensitive strains. Sensitivity profiles of different Sdh genotypes of B. cinerea strains to six SDHIs were determined in vitro and in vivo. In addition, the mutation(s) were genetically validated through in situ SdhB (SdhC) expression. Docking assays between SDHIs and AlphaFold model-based Sdh complexes revealed generally consistent patterns with their in vitro phenotypes. CONCLUSION: Resistance of B. cinerea to SDHI fungicide on strawberry was systematically investigated in this study. Deciphering of SDHI resistance through the genetic transformation system and AlphaFold model-based molecular docking will provide valuable insights into other target site-based fungicide resistance in fungal pathogens. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Characterization of soybean chitinase genes induced by rhizobacteria involved in the defense against Fusarium oxysporum.

Rhizobacteria are capable of inducing defense responses via the expression of pathogenesis-related proteins (PR-proteins) such as chitinases, and many studies have validated the functions of plant chitinases in defense responses. Soybean (Glycine max) is an economically important crop worldwide, but the functional validation of soybean chitinase in defense responses remains limited. In this study, genome-wide characterization of soybean chitinases was conducted, and the defense contribution of three chitinases (GmChi01, GmChi02, or GmChi16) was validated in Arabidopsis transgenic lines against the soil-borne pathogen Fusarium oxysporum. Compared to the Arabidopsis Col-0 and empty vector controls, the transgenic lines with GmChi02 or GmChi16 exhibited fewer chlorosis symptoms and wilting. While GmChi02 and GmChi16 enhanced defense to F. oxysporum, GmChi02 was the only one significantly induced by Burkholderia ambifaria. The observation indicated that plant chitinases may be induced by different rhizobacteria for defense responses. The survey of 37 soybean chitinase gene expressions in response to six rhizobacteria observed diverse inducibility, where only 10 genes were significantly upregulated by at least one rhizobacterium and 9 genes did not respond to any of the rhizobacteria. Motif analysis on soybean promoters further identified not only consensus but also rhizobacterium-specific transcription factor-binding sites for the inducible chitinase genes. Collectively, these results confirmed the involvement of GmChi02 and GmChi16 in defense enhancement and highlighted the diverse inducibility of 37 soybean chitinases encountering F. oxysporum and six rhizobacteria.