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.

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.

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.

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.

Monitoring of Benzimidazole Resistance in Botrytis cinerea Isolates from Strawberry in Korea and Development of Detection Method for Benzimidazole Resistance.

Botrytis cinerea is a major fungal plant pathogen that causes gray mold disease in strawberries, leading to a decrease in strawberry yield. While benzimidazole is widely used as a fungicide for controlling this disease, the increasing prevalence of resistant populations to this fungicide undermines its effectiveness. To investigate benzimidazole resistant B. cinerea in South Korea, 78 strains were isolated from strawberries grown in 78 different farms in 2022, and their EC50 values for benzimidazole were examined. As a result, 64 strains exhibited resistance to benzimidazole, and experimental tests using detached strawberry leaves and the plants in a greenhouse confirmed the reduced efficacy of benzimidazole to control these strains. The benzimidazole resistant strains identified in this study possessed two types of mutations, E198A or E198V, in the TUB2 gene. To detect these mutations, TaqMan probes were designed, enabling rapid identification of benzimidazole resistant B. cinerea in strawberry and tomato farms. This study utilizes TaqMan real-time polymerase chain reaction analysis to swiftly identify benzimidazole resistant B. cinerea, thereby offering the possibility of effective disease management by identifying optimum locations and time of application.

Efficient disruption of CmHk1 using CRISPR/Cas9 ribonucleoprotein delivery in Cordyceps militaris.

Cordyceps militaris, an entomopathogenic ascomycete, produces edible medicinal mushrooms known to have medicinal and therapeutic functions. To develop the genetic transformation system in C. militaris, green fluorescent protein (GFP) mutants of C. militaris were generated by PEG-mediated protoplast transformation. The CRISPR/Cas9 ribonucleoprotein (RNP) targeting the class III histidine kinase of C. militaris (CmHk1) was then delivered into protoplasts of C. militaris through the transformation system. Mutations induced by the RNP in selected mutants were detected: 1 nt deletion (6 mutants), 3 nt deletion with substitution of 1 nt (1 mutant), insertion of 85 nts (1 mutant), 41 nts (2 mutants), and 35 nts (5 mutants). An in vitro sensitivity assay of the mutants indicated that knockout of CmHk1 reduced sensitivity to two fungicides, iprodione and fludioxonil, but increased sensitivity to osmotic stresses compared to the wild type. Summing up, the CRISPR/Cas9 RNP delivery system was successfully developed, and our results revealed that CmHk1 was involved in the fungicide resistance and osmotic stress in C. militaris.

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.

Population genomic analysis reveals geographic structure and climatic diversification for Macrophomina phaseolina isolated from soybean and dry bean across the United States, Puerto Rico, and Colombia.

Macrophomina phaseolina causes charcoal rot, which can significantly reduce yield and seed quality of soybean and dry bean resulting from primarily environmental stressors. Although charcoal rot has been recognized as a warm climate-driven disease of increasing concern under global climate change, knowledge regarding population genetics and climatic variables contributing to the genetic diversity of M. phaseolina is limited. This study conducted genome sequencing for 95 M. phaseolina isolates from soybean and dry bean across the continental United States, Puerto Rico, and Colombia. Inference on the population structure using 76,981 single nucleotide polymorphisms (SNPs) revealed that the isolates exhibited a discrete genetic clustering at the continental level and a continuous genetic differentiation regionally. A majority of isolates from the United States (96%) grouped in a clade with a predominantly clonal genetic structure, while 88% of Puerto Rican and Colombian isolates from dry bean were assigned to a separate clade with higher genetic diversity. A redundancy analysis (RDA) was used to estimate the contributions of climate and spatial structure to genomic variation (11,421 unlinked SNPs). Climate significantly contributed to genomic variation at a continental level with temperature seasonality explaining the most variation while precipitation of warmest quarter explaining the most when spatial structure was accounted for. The loci significantly associated with multivariate climate were found closely to the genes related to fungal stress responses, including transmembrane transport, glycoside hydrolase activity and a heat-shock protein, which may mediate climatic adaptation for M. phaseolina. On the contrary, limited genome-wide differentiation among populations by hosts was observed. These findings highlight the importance of population genetics and identify candidate genes of M. phaseolina that can be used to elucidate the molecular mechanisms that underly climatic adaptation to the changing climate.

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.

Isolation and Functional Characterization of Soybean BES1/BZR1 Homolog 3-Like 1 (GmBEH3L1) Associated with Dehydration Sensitivity and Brassinosteroid Signaling in Arabidopsis thaliana

Brassinosteroid (BR) is an important steroid hormone that regulates plant development, abscisic acid (ABA) signaling, and responses to abiotic stress. We previously demonstrated that BEH3 (BES1/BZR1 Homolog 3) of Arabidopsis thaliana regulates dehydration and ABA responses by mediating proline metabolism. Furthermore, BEH3 negatively regulates BR-mediated hypocotyl elongation in dark-grown seedlings. However, the roles of BEH3 ortholog genes in the osmotic stress response of plants have remained largely unknown. Here, GmBEH3L1 (Glycine max BEH3-Like 1), a soybean (G. max) ortholog of the BEH3 gene of A. thaliana, was isolated and functionally characterized. GmBEH3L1 is induced by ABA, dehydration, and drought conditions. The GmBEH3L1-overexpressing transgenic lines (GmBEH3L1-OE/beh3) with the beh3 mutant background have ABA- and dehydration-sensitive phenotypes during early seedling growth, implying that GmBEH3L1 is involved in both osmotic stress and ABA sensitivity as a negative regulator in A. thaliana. Consistent with these results, GmBEH3L1-OE/beh3 complemental lines exhibit decreased expression levels of ABA- or dehydration-inducible genes. Under darkness, GmBEH3L1-OE/beh3 complemental lines display a short hypocotyl length compared to the beh3 mutant, indicating that GmBEH3L1 is linked to BR signaling. Together, our data suggest that GmBEH3L1 participates negatively in ABA and dehydration responses through BR signaling.

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.

Genome-wide transcriptional response of the causal soybean sudden death syndrome pathogen Fusarium virguliforme to a succinate dehydrogenase inhibitor fluopyram.

BACKGROUND: Succinate dehydrogenase inhibitors (SDHIs) have been widely used to manage plant diseases caused by phytopathogenic fungi. Although attention to and use of SDHI fungicides has recently increased, molecular responses of fungal pathogens to SDHIs have often not been investigated. A SDHI fungicide, fluopyram, has been used as a soybean seed treatment and has displayed effective control of Fusarium virguliforme, one of the causal agents of soybean sudden death syndrome. To examine genome-wide gene expression of F. virguliforme to fluopyram, RNA-seq analysis was conducted on two field strains of F. virguliforme with differing SDHI fungicide sensitivity in the absence and presence of fluopyram. RESULTS: The analysis indicated that several xenobiotic detoxification-related genes, such as those of deoxygenase, transferases and transporters, were highly induced by fluopyram. Among the genes, four ATP-binding cassette (ABC) transporters were characterized by the yeast expression system. The results revealed that expression of three ABCG transporters was associated with reduced sensitivity to multiple fungicides including fluopyram. In addition, heterologous expression of a major facilitator superfamily (MFS) transporter that was highly expressed in the fluopyram-insensitive F. virguliforme strain in the yeast system conferred decreased sensitivity to fluopyram. CONCLUSION: This study demonstrated that xenobiotic detoxification-related genes were highly upregulated in response to fluopyram, and expression of ABC or MFS transporter genes was associated with reduced sensitivity to the SDHI fungicide. This is the first transcriptomic analysis of the fungal species response to fluopyram and the finding will help elucidate the molecular mechanisms of SDHI resistance. © 2021 Society of Chemical Industry.

Biocontrol of the causal brown patch pathogen Rhizoctonia solani by Bacillus velezensis GH1-13 and development of a bacterial strain specific detection method.

Brown patch caused by the basidiomycete fungus Rhizoctonia solani is an economically important disease of cool-season turfgrasses. In order to manage the disease, different types of fungicides have been applied, but the negative impact of fungicides on the environment continues to rise. In this study, the beneficial bacteria Bacillus velezensis GH1-13 was characterized as a potential biocontrol agent to manage brown patch disease. The strain GH1-13 strongly inhibited the mycelial growth of turf pathogens including different anastomosis groups of R. solani causing brown patch and large patch. R. solani AG2-2(IIIB) hyphae were morphologically changed, and fungal cell death resulted from exposure to the strain GH1-13. In addition, the compatibility of fungicides with the bacterial strain, and the combined application of fungicide azoxystrobin and the strain in brown patch control on creeping bentgrass indicated that the strain could serve as a biocontrol agent. To develop strain-specific detection method, two unique genes from chromosome and plasmid of GH1-13 were found using pan-genome analysis of 364 Bacillus strains. The unique gene from chromosome was successfully detected using both SYBR Green and TaqMan qPCR methods in bacterial DNA or soil DNA samples. This study suggests that application of GH1-13 offers an environmentally friendly approach via reducing fungicide application rates. Furthermore, the developed pipeline of strain-specific detection method could be a useful tool for detecting and studying the dynamics of specific biocontrol agents.

Fungi Associated with Postharvest Diseases of Sweet Potato Storage Roots and In Vitro Antagonistic Assay of Trichoderma harzianum against the Diseases.

Sweet potato is the 11th most important food crop in the world and an excellent source of nutrition. Postharvest diseases were monitored in sweet potato storage roots collected from the local markets in Korea during 2021. Several diseases including Fusarium surface and root rot, charcoal rot, dry rot, and soft rot were observed in the postharvest sweet potatoes. A total of 68 fungal isolates were obtained from the diseased samples, and the isolates were grouped into 8 different fungal colony types. Based on multilocus phylogeny and morphological analysis of 17 representative isolates, the isolates were identified as Fusarium oxysporum, F. ipomoeae, F. solani, Penicillium citrinum, P. rotoruae, Aspergillus wentii, Mucor variicolumellatus (Mu. circinelloides species complex), and Macrophomina phaseolina. F. oxysporum was the predominant pathogen as this is the most common pathogen of sweet potato storage roots causing the surface rot disease, and M. phaseolina caused the most severe disease among the pathogens. Dual culture antagonistic assays were evaluated using Trichoderma harzianum strains CMML20-26 and CMML20-27. The results revealed that the two strains showed strong antifungal activity in different ranges against all tested pathogens. This study provides an understanding of diverse postharvest diseases in sweet potatoes and suggests potential biocontrol agents to manage the diseases. In addition, this is the first report of sweet potato storage root rot diseases caused by A. wentii, and P. rotoruae worldwide.

Photochemically derived 1-aminonorbornanes provide structurally unique succinate dehydrogenase inhibitors with in vitro and in planta activity.

Agrochemical fungicidal leads have been prepared from photochemically derived 1-aminonorbornane building blocks. The unique 1-aminonorbornane core is generated via direct excitation of a Schiff base precursor, leveraging the N-centered radical character of the excited state species to facilitate a series of radical reactions that construct the norbornane core. This process requires no exogenous reagents, only solvent and photons; thus, it represents an exceptionally simple and efficient means of generating the key building blocks. These (hetero) arene-fused 1-aminonorbornanes are unprecedented in both the agrochemical and pharmaceutical discovery literature; therefore, photochemical advances have provided the unique opportunity to explore the functional utility of novel chemical space. Toward this end, the 1-aminonorbornanes were used to generate next-generation succinate dehydrogenase inhibitors. In vitro fungicidal activity is demonstrated against three fungal plant pathogens affecting field crops, specifically: Fusarium graminearum, Sclerotinia sclerotiorum, and Macrophomina phaseolina. The in vitro performance against F. graminearum was shown to translate into a greenhouse setting. The discovery of in planta fungicidal activity illustrates the interdisciplinary value available via photochemical innovation.

Plant and Fungal Genome Editing to Enhance Plant Disease Resistance Using the CRISPR/Cas9 System.

Crop production has been substantially reduced by devastating fungal and oomycete pathogens, and these pathogens continue to threaten global food security. Although chemical and cultural controls have been used for crop protection, these involve continuous costs and time and fungicide resistance among plant pathogens has been increasingly reported. The most efficient way to protect crops from plant pathogens is cultivation of disease-resistant cultivars. However, traditional breeding approaches are laborious and time intensive. Recently, the CRISPR/Cas9 system has been utilized to enhance disease resistance among different crops such as rice, cacao, wheat, tomato, and grape. This system allows for precise genome editing of various organisms via RNA-guided DNA endonuclease activity. Beyond genome editing in crops, editing the genomes of fungal and oomycete pathogens can also provide new strategies for plant disease management. This review focuses on the recent studies of plant disease resistance against fungal and oomycete pathogens using the CRISPR/Cas9 system. For long-term plant disease management, the targeting of multiple plant disease resistance mechanisms with CRISPR/Cas9 and insights gained by probing fungal and oomycete genomes with this system will be powerful approaches.

Aphid estrogen-related receptor controls glycolytic gene expression and fecundity.

Aphids, the major insect pests of agricultural crops, reproduce sexually and asexually depending upon environmental factors such as the photoperiod and temperature. Nuclear receptors, a unique family of ligand-dependent transcription factors, control insect development and growth including morphogenesis, molting, and metamorphosis. However, the structural features and biological functions of the aphid estrogen-related receptor (ERR) are largely unknown. Here, we cloned full-length cDNA encoding the ERR in the green peach aphid, Myzus persicae, (Sulzer) (Hemiptera: Aphididae) (MpERR) and demonstrated that the MpERR modulated glycolytic gene expression and aphid fecundity. The phylogenetic analysis revealed that the MpERR originated in a unique evolutionary lineage distinct from those of hemipteran insects. Moreover, the AF-2 domain of the MpERR conferred nuclear localization and transcriptional activity. The overexpression of the MpERR significantly upregulated the gene expression of rate-limiting enzymes involved in glycolysis such as phosphofructokinase and pyruvate kinase by directly binding to ERR-response elements in their promoters. Moreover, ERR-deficient viviparous female aphids showed decreased glycolytic gene expression and produced fewer offspring. These results suggest that the aphid ERR plays a pivotal role in glycolytic transcriptional control and fecundity.

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.