Validation of shikimate dehydrogenase as the herbicidal target of drupacine and screening of target-based compounds with high herbicidal activity.

The discovery of new targets and lead compounds is the key to developing new pesticides. The herbicidal target of drupacine has been identified as shikimate dehydrogenase (SkDH). However, the mechanism of interaction between them remains unclear. This study found that drupacine specifically binds to SkDH with a dissociation equilibrium constant (KD) of 8.88 µM and a Kd value of 2.15 µM, as confirmed by surface plasmon resonance and microscale thermophoresis. Site-directed mutagenesis coupled with fluorescence quenching analysis indicated that residue THR431 was the key amino acid site for drupacine binding to SkDH. Nine compounds with the best binding ability to SkDH were identified by virtual screening from about 120,000 compounds. Among them, compound 8 showed the highest inhibition rate with values of 41.95% against SkDH, also exhibiting the strongest herbicidal activity. This research identifies a novel potential target SkDH and a candidate lead compound with high herbicidal activity for developing new herbicides.

Discovery of Aldehyde Dehydrogenase as a Potential Fungicide Target and Screening of its Natural Inhibitors against Fusarium verticillioides.

Fusarium verticillioides is the primary pathogen causing ear rot and stalk rot in corn (Zea mays). It not only affects yields but also produces mycotoxins endangering both human and animal health. Aldehyde dehydrogenase (ALDH) is essential for the oxidation of aldehydes in living organisms, making it a potential target for human drug design. However, there are limited reports on its function in plant pathogenic fungus. In this study, we analyzed the expression levels and gene knockout mutants, revealing that ALDH genes FvALDH-43 and FvALDH-96 in F. verticillioides played significant roles in pathogenicity and resistance to low-temperature stress by affecting antioxidant capacity. Virtual screening for natural product inhibitors and molecular docking were performed targeting FvALDH-43 and FvALDH-96. Following the biological activity analysis, three natural flavonoid compounds featuring a 2-hydroxyphenol chromene were identified. Among these, Taxifolin exhibited the highest biological activity and low toxicity. Both in vitro and in vivo biological evaluations confirmed that Taxifolin targeted ALDH and inhibited its activity. These findings indicate that aldehyde dehydrogenase may serve as a promising target for the design of novel fungicides.

The genome sequencing and comparative genomics analysis of Rhizoctonia solani reveals a novel effector family owning a uinque domain in Basidiomycetes.

Rhizoctonia solani is a soil-borne pathogen with 14 anastomosis groups (AGs), and different subgroups are genetically diverse. However, the genetic factors contributing to the pathogenicity of the fungus have not been well characterized. In this study, the genome of R. solani AG1-ZJ was sequenced. As the result, a 41.57 Mb draft genome containing 12,197 putative coding genes was obtained. Comparative genomic analysis of 11 different AGs revealed conservation and unique characteristics between the AGs. Furthermore, a novel effector family containing a 68 amino acid conserved domain unique in basidiomycetous fungi was characterized. Two effectors containing the conserved domain in AG4-JY were identified, and named as RsUEB1 and RsUEB2. Furthermore, the spray-induced gene silencing strategy was used to generate a dsRNA capable of silencing the conserved domain sequence of RsUEB1 and RsUEB2. This dsRNA can significantly reduce the expression of RsUEB1 and RsUEB2 and the pathogenicity of AG4-JY on foxtail millet, maize, rice and wheat. In conclusion, this study provides significant insights into the pathogenicity mechanisms of R. solani. The identification of the conserved domain and the successful use of dsRNA silencing of the gene containing the conserved domain will offer a new strategy for controlling sheath blight in cereal crops.

Validamycin A Inhibited FB1 Biosynthesis by the Target FvNth in Fusarium verticillioides.

Validamycin A (VMA) is an antifungal antibiotic derived from Streptomyces hygroscopicus commonly used in plant disease management. Surprisingly, VMA was discovered to impede the production of fumonisin B1 (FB1) in agricultural settings. However, the specific target of VMA in Fusarium verticillioides remained unclear. To unravel the molecular mechanism of VMA, ultrastructural observations unveiled damage to mitochondrial membranes. Trehalase (FvNth) was pinpointed as the target of VMA by utilizing a 3D-printed surface plasmon resonance sensor. Molecular docking identified Trp285, Arg447, Asp452, and Phe665 as the binding sites between VMA and FvNth. A ΔFvnth mutant lacking amino acids 250-670 was engineered through homologous recombination. Transcriptome analysis indicated that samples treated with VMA and ΔFvnth displayed similar expression patterns, particularly in the suppression of the FUM gene cluster. VMA treatment resulted in reduced trehalase and ATPase activity as well as diminished production of glucose, pyruvic acid, and acetyl-CoA. Conversely, these effects were absent in samples treated with ΔFvnth. This research proposes that VMA hinders acetyl-CoA synthesis by trehalase, thereby suppressing the FB1 biosynthesis. These findings present a novel target for the development of mycotoxin control agents.

FvOshC Is a Key Global Regulatory Target in Fusarium verticillioides for Fumonisin Biosynthesis and Disease Control.

Fusarium verticillioides has a substantial impact on maize production, commonly leading to maize ear rot and the production of fumonisin, a mycotoxin that poses health risks to both humans and animals. Currently, there is a lack of molecular targets for preventing the disease and controlling the toxin. The biological functions of oxysterol-binding proteins (OSBP) in filamentous fungi remain unclear. In this research, 7 oxysterol-binding protein-related proteins were identified in F. verticillioides, and these proteins were obtained through prokaryotic expression and purification. FvOshC was identified as the specific protein that binds to ergosterol through fluorescence titration. Gene knockout complementation techniques confirmed that FvOSHC plays a positive role, establishing it as a novel global regulatory protein involved in the pathogenicity and FB1 biosynthesis in F. verticillioides. Additionally, the interaction between FvOshC and FvSec14 was identified using yeast two-hybrid techniques. Moreover, computer-aided drug design technology was utilized to identify the receptor molecule Xanthatin based on FvOshC. The inhibitory effect of Xanthatin on the growth of F. verticillioides and the synthesis of FB1 was significantly demonstrated. These findings provide valuable insights that can aid in the management of mycotoxin pollution.

The catalytic subunit of type 2A protein phosphatase negatively regulates conidiation and melanin biosynthesis in Setosphaeria turcica.

Northern corn leaf blight caused by Setosphaeria turcica is a major fungal disease responsible for significant reductions in maize yield worldwide. Eukaryotic type 2A protein phosphatase (PP2A) influences growth and virulence in a number of pathogenic fungi, but little is known about its roles in S. turcica. Here, we functionally characterized S. turcica StPP2A-C, which encodes the catalytic C subunit of StPP2A. StPP2A-C deletion slowed colony growth, conidial germination, and appressorium formation but increased conidiation, melanin biosynthesis, glycerol content, and disease lesion size on maize. These effects were associated with expression changes in genes related to calcium signaling, conidiation, laccase activity, and melanin and glycerol biosynthesis, as well as changes in intra- and extracellular laccase activity. A pull-down screen for candidate StPP2A-c interactors revealed an interaction between StPP2A-c and StLac1. Theoretical modeling and yeast two-hybrid experiments confirmed that StPP2A-c interacted specifically with the copper ion binding domain of StLac1 and that Cys267 of StPP2A-c was required for this interaction. StPP2A-C expression thus appears to promote hyphal growth and reduce pathogenicity in S. turcica, at least in part by altering melanin synthesis and laccase activity; these insights may ultimately support the development of novel strategies for biological management of S. turcica.

FGCD: a database of fungal gene clusters related to secondary metabolism.

Fungal secondary metabolites are not necessary for growth, but they are important for fungal metabolism and ecology because they provide selective advantages for competition, survival and interactions with the environment. These various metabolites are widely used as medicinal precursors and insecticides. Secondary metabolism genes are commonly arranged in clusters along chromosomes, which allow for the coordinate control of complete pathways. In this study, we created the Fungal Gene Cluster Database to store, retrieve, and visualize secondary metabolite gene cluster information across fungal species. The database was created by merging data from RNA sequencing, Basic Local Alignment Search Tool, genome browser, enrichment analysis and the R Shiny web framework to visualize and query putative gene clusters. This database facilitated the rapid and thorough examination of significant gene clusters across fungal species by detecting, defining and graphically displaying the architecture, organization and expression patterns of secondary metabolite gene clusters. In general, this genomic resource makes use of the tremendous chemical variety of the products of these ecologically and biotechnologically significant gene clusters to our further understanding of fungal secondary metabolism. Database URL: https://www.hebaubioinformatics.cn/FungalGeneCluster/.

Synergistic mechanism and environmental behavior of tank-mix adjuvants to topramezone and atrazine.

An effective way to reduce herbicide quantity is to use adjuvants in order to optimize the amount of herbicide and improve its control efficiency. In order to screen for efficient herbicide tank-mix adjuvants, improve the control of weeds in maize fields, reduce the amount of effective ingredients, and improve the adsorption and digestion behavior of herbicides in soil, this study evaluated the synergistic effects and soil behavior of four types of tank-mix adjuvants combined with herbicides. Different types of adjuvants can enhance herbicide production. Surface tension was significantly reduced by 13% after the pesticide solution was applied with AgroSpred™ Prime. The contact angle with the foliar surface was significantly reduced and solution wettability improved using Atp Lus 245-LQ-(TH). The permeability of topramezone and atrazine in leaves of Amaranthus retroflexus L. and Digitaria sanguinalis (L.) Scop. was increased by 22-96% after adding either tank-mix adjuvant. The solution drying time and maximum retention on leaves were not affected by the tank-mix adjuvants. Ethyl and methylated vegetable oils can reduce the adsorption of topramezone in the soil, thus reducing its half-life in soil. The tank-mix adjuvants had no significant effect on soil dissipation or adsorption of atrazine. AgroSpred™ Prime and Atp Lus 245-LQ-(TH) have the best synergistic effect on topramezone and atrazine in the control of A. retroflexus L. and D. sanguinalis (L.) Scop. in maize fields.

Purine metabolism in plant pathogenic fungi.

In eukaryotic cells, purine metabolism is the way to the production of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and plays key roles in various biological processes. Purine metabolism mainly consists of de novo, salvage, and catabolic pathways, and some components of these pathways have been characterized in some plant pathogenic fungi, such as the rice blast fungus Magnaporthe oryzae and wheat head blight fungus Fusarium graminearum. The enzymatic steps of the de novo pathway are well-conserved in plant pathogenic fungi and play crucial roles in fungal growth and development. Blocking this pathway inhibits the formation of penetration structures and invasive growth, making it essential for plant infection by pathogenic fungi. The salvage pathway is likely indispensable but requires exogenous purines, implying that purine transporters are functional in these fungi. The catabolic pathway balances purine nucleotides and may have a conserved stage-specific role in pathogenic fungi. The significant difference of the catabolic pathway in planta and in vitro lead us to further explore and identify the key genes specifically regulating pathogenicity in purine metabolic pathway. In this review, we summarized recent advances in the studies of purine metabolism, focusing on the regulation of pathogenesis and growth in plant pathogenic fungi.

2,4-Di-tert-butylphenol and 7-hydroxy-3-(2-methylpropyl)-2,3,6,7,8,8a-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione: two natural products from Serratia marcescens Ha1 and their herbicidal activities.

BACKGROUND: Weeds are one of the critical factors that negatively affect crop yield and quality. Microbial herbicides are a research hotspot for novel herbicides owing to their environmental safety and lack of weed resistance. In the current study, the active ingredients of Serratia marcescens Ha1, a new microbial herbicide, were investigated for their effectiveness against agricultural weeds using bioassay-guided fractionation. RESULTS: The results revealed that petroleum ether and ethyl acetate extracts of S. marcescens Ha1 had high herbicidal activity. Forty-nine compounds were identified from the petroleum ether extract, including 2,4-di-tert-butylphenol (DB; C14 H22 O, 38.82%), ethyl 14-methyl-hexadecanoate, 1-nonadecene, and [1,1'-biphenyl]-2,3'-diol, 3,4',5,6'-tetrakis. Of these, DB showed significant inhibitory effects on root and shoot growth in Amaranthus retroflexus, with half-maximal inhibitory concentration (IC50 ) values of 389.17 and 832.44 mg L-1 , respectively. In addition, 7-hydroxy-3-(2-methylpropyl)-2,3,6,7,8,8a-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (HPD) was identified as the major active ingredient in the ethyl acetate extract of S. marcescens Ha1 using bioassay-guided fractionation, with IC50 values of 439.86 and 476.95 mg L-1 against A. retroflexus shoot and root growth, respectively. Scanning electron microscopy indicated that DB and HPD exert destructive effects on A. retroflexus root, and the damage is gradually aggravated with increasing treatment time and concentration. CONCLUSION: The S. marcescens Ha1 extract and its active compounds DB and HPD exhibit significant herbicidal activity, which could be utilized further for the development of microbial herbicides. © 2023 Society of Chemical Industry.

Identification of Triazolo-quinazolinone Derivatives as Novel and Potent Chitinase OfChi-h Inhibitors Based on Structure-Based Virtual Screening.

Insect chitinase, OfChi-h, from Ostrinia furnacalis, is considered as a promising target for the development of green pesticides. On the basis of the crystal structure of OfChi-h, we successfully obtained a triazolo-quinazolinone scaffold as the novel class of OfChi-h inhibitor via a structure-based virtual screening approach. Rational compound screening enabled us to acquire a potent OfChi-h inhibitor TQ19 with a Ki value of 0.33 µM. Furthermore, the in vivo biological activity of target compounds was assayed. The results showed that compounds TQ8 and TQ19 could dramatically inhibit the growth and development of Ostrinia nubilalis larvae, and most of the compounds showed higher insecticidal activity than hexaflumuron. This present work reveals that triazolo-quinazolinone derivatives can serve as novel candidates for insect growth regulators.

The Septin Gene StSep4 Contributes to the Pathogenicity of Setosphaeria turcica by Regulating the Morphology, Cell Wall Integrity, and Pathogenic Factor Biosynthesis.

Septins are a conserved group of GTP-binding proteins found in all eukaryotes and are the fourth-most abundant cytoskeletal proteins. Septins of some pathogenic fungi are involved in morphological changes related to infection. Our previous studies have identified four core septins (StSep1-4) in Setosphaeria turcica, the causal agent of northern corn leaf blight, while only StSep4 is significantly upregulated during the invasive process. We therefore used forchlorfenuron (FCF), the specific inhibitor of septin, and ΔStSep4 knockout mutants to further clarify the role of septins in S. turcica pathogenicity. FCF treatment caused a dose-dependent reduction in S. turcica colony growth, delayed the formation of infection structures, and reduced the penetration ability. ΔStSep4 knockout mutants displayed abnormal mycelium morphology, slow mycelial growth, conidiation deficiency, delayed appressorium development, and weakened pathogenicity. StSep4 deletion also broke cell wall integrity, altered chitin distribution, decreased the melanin content, and disrupted normal nuclear localization. A transcriptomic comparison revealed that genes differentially expressed between ΔStSep4 and WT were enriched in terms of ribosomes, protein translation, membrane components, and transmembrane transport activities. Our results demonstrate that StSep4 is required for morphology and pathogenicity in S. turcica, making it a promising target for the development of novel fungicides.

Natural product drupacine acting on a novel herbicidal target shikimate dehydrogenase.

Natural products are one of the important sources for the creation of new pesticides. Drupacine ((1R,11S,12S,13R,15S)-13-methoxy-5,7,21-trioxa-19-azahexacyclo[11.7.1.02,10.04,8.011,15.015,19]henicosa-2,4(8),9-trien-12-ol), isolated from Cephalotaxus sinensis (Chinese plum-yew), is a potent herbicidal compound containing an oxo-bridged oxygen bond structure. However, its molecular target still remains unknown. In this study, the targets of drupacine in Amaranthus retroflexus were identified by combining drug affinity responsive target stability (DARTS), cellular thermal shift assay coupled with mass spectrometry (CETSA MS), RNA-seq transcriptomic, and TMT proteomic analyses. Fifty-one and sixty-eight main binding proteins were identified by DARTS and CETSA MS, respectively, including nine co-existing binding proteins. In drupacine-treated A. retroflexus seedlings we identified 1389 up-regulated genes and 442 down-regulated genes, 34 up-regulated proteins, and 194 down-regulated proteins, respectively. Combining the symptoms and the biochemical profiles, Profilin, Shikimate dehydrogenase (SkDH), and Zeta-carotene desaturase were predicted to be the drupacine potential target proteins. At the same time, drupacine was found to bind SkDH stronger by molecular docking, and its inhibition on ArSkDH increased with the treatment concentration increase. Our results suggest that the molecular target of drupacine is SkDH, a new herbicide target, which lay a foundation for the rational design of herbicides based on new targets from natural products and enrich the target resources for developing green herbicides.

Uptake and Biotransformation of Spirotetramat and Pymetrozine in Lettuce (Lactuca sativa L. var. ramosa Hort.).

Here, we investigated the uptake, transport, and subcellular distribution of the pesticides pymetrozine and spirotetramat, and spirotetramat metabolites B-enol, B-glu, B-mono, and B-keto, under hydroponic conditions. Spirotetramat and pymetrozine exhibited high bioconcentrations in lettuce roots, with both having root concentration factor (RCF) values >1 after exposure for 24 h. The translocation of pymetrozine from roots to shoots was higher than that of spirotetramat. Pymetrozine is absorbed in roots mainly via the symplastic pathway and is primarily stored in the soluble fraction of lettuce root and shoot cells. The cell wall and soluble fractions were the major enrichment sites of spirotetramat and its metabolites in root cells. Spirotetramat and B-enol were mainly enriched in the soluble fractions of lettuce shoot cells, whereas B-keto and B-glu accumulated in cell walls and organelles, respectively. Both symplastic and apoplastic pathways were involved in spirotetramat absorption. Pymetrozine and spirotetramat uptake by lettuce roots was passive, with no aquaporin-mediated dissimilation or diffusion. The findings of this study enhance our understanding of the transfer of pymetrozine, spirotetramat, and spirotetramat metabolites from the environment to lettuce, and their subsequent bioaccumulation. This study describes a novel approach for the efficient management of lettuce pest control using spirotetramat and pymetrozine. At the same time, it is of great significance to evaluate the food safety and environmental risks of spirotetramat and its metabolites.

Identification and analysis of the secretome of plant pathogenic fungi reveals lifestyle adaptation.

The secretory proteome plays an important role in the pathogenesis of phytopathogenic fungi. However, the relationship between the large-scale secretome of phytopathogenic fungi and their lifestyle is not fully understood. In the present study, the secretomes of 150 plant pathogenic fungi were predicted and the characteristics associated with different lifestyles were investigated. In total, 94,974 secreted proteins (SPs) were predicted from these fungi. The number of the SPs ranged from 64 to 1,662. Among these fungi, hemibiotrophic fungi had the highest number (average of 970) and proportion (7.1%) of SPs. Functional annotation showed that hemibiotrophic and necrotroph fungi, differ from biotrophic and symbiotic fungi, contained much more carbohydrate enzymes, especially polysaccharide lyases and carbohydrate esterases. Furthermore, the core and lifestyle-specific SPs orthogroups were identified. Twenty-seven core orthogroups contained 16% of the total SPs and their motif function annotation was represented by serine carboxypeptidase, carboxylesterase and asparaginase. In contrast, 97 lifestyle-specific orthogroups contained only 1% of the total SPs, with diverse functions such as PAN_AP in hemibiotroph-specific and flavin monooxygenases in necrotroph-specific. Moreover, obligate biotrophic fungi had the largest number of effectors (average of 150), followed by hemibiotrophic fungi (average of 120). Among these effectors, 4,155 had known functional annotation and pectin lyase had the highest proportion in the functionally annotated effectors. In addition, 32 sets of RNA-Seq data on pathogen-host interactions were collected and the expression levels of SPs were higher than that of non-SPs, and the expression level of effector genes was higher in biotrophic and hemibiotrophic fungi than in necrotrophic fungi, while secretase genes were highly expressed in necrotrophic fungi. Finally, the secretory activity of five predicted SPs from Setosphearia turcica was experimentally verified. In conclusion, our results provide a foundation for the study of pathogen-host interaction and help us to understand the fungal lifestyle adaptation.

MicroRNAs involved in the trans-kingdom gene regulation in the interaction of maize kernels and Fusarium verticillioides.

Maize ear rot is a widespread disease and the main pathogen is Fusarium verticillioides. Plant microRNAs (miRNAs) have great effects on disease resistance and it has been reported that maize miRNA participates in defense responses in maize ear rot. However, the trans-kingdom regulation of miRNAs between maize and F. verticillioides remains uncharacterized. In this study, the relationship between miRNA-like RNAs (milRNAs) of F. verticillioides and pathogenicity was investigated, followed by sRNA analysis and degradome sequencing of miRNA profiles and the target genes of maize and F. verticillioides after inoculation. It was found that the milRNA biogenesis positively regulated the pathogenicity of F. verticillioides by knocking out the gene FvDicer2-encoded Dicer-like protein in F. verticillioides. Following inoculation with F. verticillioides, 284 known and 6571 novel miRNAs were obtained in maize, including 28 miRNAs differentially expressed at multiple time points. The target genes of maize differentially expressed miRNAs in F. verticillioides mediated multiple pathways, including autophagy and MAPK signaling pathway. Fifty-one novel F. verticillioides milRNAs were predicted to target 333 genes in maize involved in MAPK signaling pathways, plant hormone signaling transduction and plant-pathogen interaction pathways. Additionally, the miR528b-5p in maize targeted the mRNA of FvTTP which encoded a twice transmembrane protein in F. verticillioides. The FvTTP-knockout mutants displayed decreased pathogenicity and reduced synthesis of fumonisins. Thus, by interfering with the translation of FvTTP, the miR528b-5p inhibited F. verticillioides infection. These findings suggested a novel function of miR528 in resisting F. verticillioides infection. The miRNAs identified in this research and their putative target genes can be used to further elucidate the trans-kingdom functions of microRNAs in plant pathogen interaction.

Herbicidal Active Compound Ferulic Acid Ethyl Ester Affects Fatty Acid Synthesis by Targeting the 3-Ketoacyl-Acyl Carrier Protein Synthase I (KAS I).

Exploring new herbicide targets based on natural product derivatives is an important research aspect for the generation of innovative pesticides. Ferulic acid ethyl ester (FAEE), a natural product derivative from ferulic acid, has significant herbicidal activity mainly by inhibiting the normal growth of weed seedling roots. However, the FAEE target protein underlying its herbicidal activity has not been identified. In this study, we synthesized an FAEE probe to locate its site of action. We discovered that FAEE entry point was via the root tips. Fourteen major binding proteins were identified using Drug affinity responsive target stability (DARTS) combined with LC-MS/MS, which included 3-ketoacyl-acyl carrier protein synthase I (KAS I) and phenylalanine ammonia-lyase I (PAL I). The KAS I and PAL I proteins/genes expression was changed significantly after exposure to FAEE, as evidenced by combined transcriptomic and proteomic analysis. A molecular docking assay indicated that KAS I and FAEE had a strong binding ability. Combined with previous studies on FAEE mechanism of action, and based on our results, we conclude that FAEE targeting KAS I lead to the blockage of the fatty acid synthesis pathway and result in plant death.

Potent insecticidal activity of Eleocharis dulcis (Burm. f.) Trin peel extract and its main components against aphids.

BACKGROUND: Aphids are significant pests of cash crops and food farm crops. Botanical insecticides are safe for aphid control, especially for organic farming. In this study, Eleocharis dulcis (Burm. f.) Trin. peel extract (EDPE), a new botanical insecticide, was investigated for its active compositions against several agricultural aphids. RESULTS: The results showed that the EDPE had high insecticidal activity against Sitobion avenae Fabricius, Aphis gossypii Glover, Megoura crassicauda Mordvilko, and Acyrthosiphon pisum Harris, with half-lethal concentration (LC50 ) values of 95.92, 81.04, 140.31, and 255.73 mg/L after 48 h of treatment. In the pot culture assay, the aphicidal effects of 25% EDPE soluble liquid (SL) at a concentration of 0.016% were 68.98 ± 5.61%, 79.33 ± 8.27%, and 88.82 ± 3.91% after the first, third, and seventh days of treatment, respectively. Nine compounds were identified by bioactivity-directed fractionation: 4',5'-dimethoxy-6,6-dimethylpyranoisoflavone (1), 3-methoxy-4-hydroxylonchocarpin (2), 4-hydroxylonchocarpin (3), 4-methoxylonchocarpin (4), barbigerone (5), lonchocarpusone (6), 6a,12a-dehydrodeguelin (7), 13-homo-13-oxa-6a, 12a-dehydrodeguelin (8) and deguelin (9). Among them, 4-hydroxylonchocarpin (3) showed the highest aphidicidal activity against M. crassicauda, S. avenae, and A. pisum, with LC50 values of 97.24, 140.63, and 112.31 mg/L, respectively. CONCLUSION: These data contribute to a better understanding of the aphicidal activity of EDPE and its main component, 4-hydroxylonchocarpin. This will help to develop new botanical insecticides to contro aphids. © 2022 Society of Chemical Industry.

StRAB4 gene is required for filamentous growth, conidial development, and pathogenicity in Setosphaeria turcica.

Setosphaeria turcica, the fungal pathogen responsible for northern corn leaf blight in maize, forms specialized infectious structures called appressoria that are critical for fungal penetration of maize epidermal cells. The Rab family of proteins play a crucial role in the growth, development, and pathogenesis of many eukaryotic species. Rab4, in particular, is a key regulator of endocytosis and vesicle trafficking, essential for filamentous growth and successful infection by other fungal pathogens. In this study, we silenced StRAB4 in S. turcica to gain a better understanding the function of Rab4 in this plant pathogen. Phenotypically, the mutants exhibited a reduced growth rate, a significant decline in conidia production, and an abnormal conidial morphology. These phenotypes indicate that StRab4 plays an instrumental role in regulating mycelial growth and conidial development in S. turcica. Further investigations revealed that StRab4 is a positive regulator of cell wall integrity and melanin secretion. Functional enrichment analysis of differentially expressed genes highlighted primary enrichments in peroxisome pathways, oxidoreductase and catalytic activities, membrane components, and cell wall organization processes. Collectively, our findings emphasize the significant role of StRab4 in S. turcica infection and pathogenicity in maize and provide valuable insights into fungal behavior and disease mechanisms.

First Report of Rhizoctonia solani AG-4 HG-III Causing Sheath Blight on Foxtail Millet in China.

Foxtail millet (Setaria italica) is an important grain and forage crop. This crop is widely grown in Northern China (Yang et al.2020). In Aug 2021, foxtail millet variety of Jigu42 showing lodging were found in Baoding China with the incidence of 30% and irregular brown lesions were found in sheaths and leaves of infected plants. The center of the lesions was kraurotic and pale, and the edges were gray-brown or dark brown. Twelve samples with typical lesions were collected from the surveyed field to isolate the pathogen. The infected samples were cut into square pieces of about 3 to 5 mm and were immersed into NaOCl (1%) for 1 min followed by washing with sterile water for three times. Then all sterilized tissues were inoculated on potato dextrose agar (PDA) plates and incubated at 25℃. After 3 days, fresh mycelial tips grown from the tissues were transferred to new plates for purification and incubated in the dark at 25°C for 4-5 days until the hyphae covered the whole plates. The colonies of 15 isolates on PDA medium showed similar colonial characteristics, which were fluffy and white initially, gradually turned light brown, and no sclerotia was observed even at 20 days later. Micro-examination revealed that all isolates showed the identical morphological features as Rhizoctonia sp. (Sneh et al. 1991), which contained the septate and right-angled branching hyphae with slight constriction at the base of mycelial branches, and three to seven nuclei per cell (Yang et al. 2013). Total genomic DNA was extracted from 5-day-old cultures, and the internal transcribed spacer (ITS) region of rDNA was amplified with ITS1 and ITS4 as the primers (Garibaldi et al. 2019). The sequencing results showed that the nucleotide sequences of 15 amplicons were identical and shared 100% identity with the corresponding fragments of R. solani AG-4 HG-III from sugar beet (GenBank accession No. MH172666 and MH172663) in Blastn search. The sequencing size of ITS in this study was 3 bp shorter than that of sugar beet, with a length of 722, because the base 'T' in the beginning and 'GA' in the end of the sequences did not detected in our study. Phylogenetic tree of 16 isolates of different AG4 subgroups was created by the software MEGA 7.0 through the NJ method, and the showed that the isolates were clustered to the clade of AG-4 HG-III group. The sequences of three isolates were deposited in GenBank under the accession No. ON810364, ON810365 and ON810366. For pathogenicity test, 5 mm diameters plate of the 5-day-old fungus which cultured on PDA were inoculated to the sheath of 10 foxtail millet plants grown in pots at 5- or 6-leaf stage. Then, the inoculated plants were placed into a growth chamber, and the inoculated sheaths were covered with wet cotton ball for 2 days to keep humidity, while sterile water was inoculated as the control. All plants were cultivated at 26°C with 14 h light and 10 h dark for 14 days. The experiment was repeated for three times. As the result, the same lesions observed in the field appeared on the inoculated plants at 10-14 days post inoculation, whereas the mock was healthy. The pathogen was re-isolated from the infected samples. The morphological characteristics and the nucleotide sequences of ITSs were same as that of the original isolates. All in above, the pathogen cusing sheath blight on foxtail millet was identified as R. solani AG-4 HG-III. To our knowledge, this is the first report of R. solani AG-4 HG-III causing sheath blight on S. italica in China. This finding expands the host range known for R. solani AG-4 HG-III and will be helpful for developing effective control strategies of foxtail millet sheath blight.