Mining Biosynthetic Gene Clusters of Pseudomonas vancouverensis Utilizing Whole Genome Sequencing.

Natural product (NP)-based pesticides have emerged as a compelling alternative to traditional chemical fungicides, attracting substantial attention within the agrochemical industry as the world is pushing toward sustainable and environmentally friendly approaches to safeguard crops. Microbes, both bacteria and fungi, are a huge source of diverse secondary metabolites with versatile applications across pharmaceuticals, agriculture, and the food industry. Microbial genome mining has been accelerated for pesticide/drug discovery and development in recent years, driven by advancements in genome sequencing, bioinformatics, metabolomics/metabologenomics, and synthetic biology. Here, we isolated and identified Pseudomonas vancouverensis that had shown antifungal activities against crop fungal pathogens Colletotrichum fragariae, Botrytis cinerea, and Phomopsis obscurans in a dual-plate culture and bioautography assay. Further, we sequenced the whole bacterial genome and mined the genome of this bacterium to identify secondary metabolite biosynthetic gene clusters (BGCs) using antiSMASH 7.0, PRISM 4, and BAGEL 4. An in-silico analysis suggests that P. vancouverensis possesses a rich repertoire of BGCs with the potential to produce diverse and novel NPs, including non-ribosomal peptides (NRPs), polyketides (PKs), acyl homoserine lactone, cyclodipeptide, bacteriocins, and ribosomally synthesized and post-transcriptionally modified peptides (RiPPs). Bovienimide-A, an NRP, and putidacin L1, a lectin-like bacteriocin, were among the previously known predicted metabolites produced by this bacterium, suggesting that the NPs produced by this bacterium could have biological activities and be novel as well. Future studies on the antifungal activity of these compounds will elucidate the full biotechnological potential of P. vancouverensis.

Antifungal and Phytotoxic Activities of Isolated Compounds from Helietta parvifolia Stems.

The identification of natural and environmentally friendly pesticides is a key area of interest for the agrochemical industry, with many potentially active compounds being sourced from numerous plant species. In this study, we report the bioassay-guided isolation and identification of phytotoxic and antifungal compounds from the ethyl acetate extract of Helietta parvifolia stems. We identified eight compounds, consisting of two coumarins and six alkaloids. Among these, a new alkaloid, 2-hydroxy-3,6,7-trimethoxyquinoline-4-carbaldehyde (6), was elucidated, along with seven known compounds. The phytotoxicity of purified compounds was evaluated, and chalepin (4) was active against Agrostis stolonifera at 1 mM with 50% inhibition of seed germination and it reduced Lemna pausicotata (duckweed) growth by 50% (IC50) at 168 µM. Additionally, we evaluated the antifungal activity against the fungal plant pathogen Colletotrichum fragariae using a thin-layer chromatography bioautography assay, which revealed that three isolated furoquinoline alkaloids (flindersiamine (3), kokusagenine (7), and maculine (8)) among the isolated compounds had the strongest inhibitory effects on the growth of C. fragariae at all tested concentrations. Our results indicate that these active natural compounds, i.e., (3), (4), (7), and (8), could be scaffolds for the production of more active pesticides with better physicochemical properties.

Molecular Targets of Herbicides and Fungicides─Are There Useful Overlaps for Fungicide Discovery?

New fungicide modes of action are needed for fungicide resistance management strategies. Several commercial herbicide targets found in fungi that are not utilized by commercial fungicides are discussed as possible fungicide molecular targets. These are acetyl CoA carboxylase, acetolactate synthase, 5-enolpyruvylshikimate-3-phosphate synthase, glutamine synthase, phytoene desaturase, protoporphyrinogen oxidase, long-chain fatty acid synthase, dihydropteroate synthase, hydroxyphenyl pyruvate dioxygenase, and Ser/Thr protein phosphatase. Some of the inhibitors of these herbicide targets appear to be either good fungicides or good leads for new fungicides. For example, some acetolactate synthase and dihydropteroate inhibitors are excellent fungicides. There is evidence that some herbicides have indirect benefits to certain crops due to their effects on fungal crop pathogens. Using a pesticide with both herbicide and fungicide activities based on the same molecular target could reduce the total amount of pesticide used. The limitations of such a product are discussed.

Synthesis, Herbicidal Activity, and Structure-Activity Relationships of O-Alkyl Analogues of Khellin and Visnagin.

Khellin and visnagin furanochromones were recently reported as potential new bioherbicides with phytotoxic activities comparable to those of some commercially available herbicides. In this study, we examined the effect of O-alkylation and O-arylalkylation of both khellin and visnagin on its effect on herbicidal and antifungal activity. Synthetic analogues included O-demethyl khellin and visnagin, acetylated O-demethyl khellin and visnagin, O-benzylated demethyl khellin and visnagin, four O-demethyl alkylated khellin analogues, and six O-demethyl alkylated visnagin analogues, many of which are reported here for the first time. Both acetate analogues of khellin and visnagin indicated more activity as herbicides on Lemna pausicostata than visnagin, with IC50 values of 71.7 and 77.6 µM, respectively. Complete loss of activity for all O-alkyl analogues with a carbon chain length of greater than 14 carbons was observed. The O-demethyl butylated visnagin analogue was the most active compound with an IC50 of 47.2 µM against L. pausicostata. O-Demethyl ethylated analogues of both khellin and visnagin were as effective as khellin. In the antifungal bioautography bioassay against Colletotrichum fragariae at 100 µg, the only active O-alkyl and O-arylalkyl analogues were O-ethylated, O-butylated, and O-benzylated visnagin analogues with zones of inhibition of 10, 9, and 9 mm, respectively, an effect comparable to that of visnagin and khellin.

Regioselective Claisen-Schmidt Adduct of 2-Undecanone from Houttuynia cordata Thunb as Insecticide/Repellent against Solenopsis invicta and Repositioning Plant Fungicides against Colletotrichum fragariae.

The U.S. Department of Agriculture (USDA) has established research programs to fight the phytopathogen Colletotrichum fragariae and the invasive red imported fire ant, Solenopsis invicta. C. fragariae is known to cause anthracnose disease in fruits and vegetables, while S. invicta is known for its aggressive behavior and painful stings and for being the cause of significant damage to crops, as well as harm to humans and animals. Many plants have been studied for potential activity against C. fragariae and S. invicta. Among the studied plants, Houttuynia cordata Thunb has been shown to contain 2-undecanone, which h is known for its antifungal activity against Colletotrichum gloesporioides. Based on the mean amount of sand removed, 2-undecanone showed significant repellency at 62.5 µg/g, similar to DEET (N,N-diethyl-meta-toluamide), against S. invicta. The 2-Undecanone with an LC50 of 44.59 µg/g showed toxicity against S. invicta workers. However, neither H. cordata extract nor 2-undecanone had shown activity against C. fragariae despite their known activity against C. gloesporioides, which in turn motivates us in repositioning 2-undecanone as a selected candidate for a Claisen-Schmidt condensation that enables access to several analogs (2a-f). Among the prepared analogs, (E)-1-(3-methylbenzo[b]thiophen-2-yl)dodec-1-en-3-one (2b) and (E)-1-(5-bromothiophen-2-yl)dodec-1-en-3-one (2f) showed promising activity against C. fragariae, revealing a distinctive structural activity relationship (SAR). The generated analogs revealed a clear regioselectivity pattern through forming the C=C alkene bond at position C-1. These data open the window for further lead optimization and product development in the context of managing C. fragariae and S. invicta.

Determining the optimum mixture of three essential oils for potato sprout suppression at room temperature storage.

As a staple crop, potatoes (Solanum tuberosum) play an important role in meeting daily caloric needs. To ensure adequate supplies for year-round consumption, potato quality must be maintained throughout lengthy storage periods. Towards this end, potato sprouting during storage must be minimized. Due to changing regulations regarding chemical means of potato sprout suppression, increased focus has turned to alternative products including essential oils (EO) as sprout suppressants in recent years. The complex composition of various EOs promises numerous options for sprout suppression. Furthermore, blends of several EOs may achieve enhanced sprout suppressant properties if synergistic interactions are present. We evaluated Syzygium aromaticum, Artemisia herba-alba, and Laurus nobilis EOs and blends thereof as sprout suppressants in potato cultivar Ranger Russet stored at room temperature and also tested for their antifungal activity against Colletotrichum fragariae, a causal organism of anthracnose disease in strawberries including other vegetables and fruits. A. herba-alba EO was an effective sprout suppressant when used alone and suppressed sprouting over the 90-day storage period. Interactions between A. herba-alba and S. aromaticum affected sprout length whereas interactions between A. herba-alba and L. nobilis EOs affected sprout number. An optimum blend of 50% - 82.31% A. herba-alba, 17.69% - 50% L. nobilis, and 0% - 1.01% S. aromaticum EOs could more effectively minimize tuber sprout length and number than any of the three whole EOs used alone. Among these three EOs, only S. aromaticum EO showed antifungal activity against C. fragariae in bioautography assay. These results exhibit the potential of EOs blends as a novel tactic in potato sprout suppression as well as potential natural product-based fungicides in managing C. fragariae.

Mutations in a barley cytochrome P450 gene enhances pathogen induced programmed cell death and cutin layer instability.

Disease lesion mimic mutants (DLMMs) are characterized by the spontaneous development of necrotic spots with various phenotypes designated as necrotic (nec) mutants in barley. The nec mutants were traditionally considered to have aberrant regulation of programmed cell death (PCD) pathways, which have roles in plant immunity and development. Most barley nec3 mutants express cream to orange necrotic lesions contrasting them from typical spontaneous DLMMs that develop dark pigmented lesions indicative of serotonin/phenolics deposition. Barley nec3 mutants grown under sterile conditions did not exhibit necrotic phenotypes until inoculated with adapted pathogens, suggesting that they are not typical DLMMs. The F2 progeny of a cross between nec3-γ1 and variety Quest segregated as a single recessive susceptibility gene post-inoculation with Bipolaris sorokiniana, the causal agent of the disease spot blotch. Nec3 was genetically delimited to 0.14 cM representing 16.5 megabases of physical sequence containing 149 annotated high confidence genes. RNAseq and comparative analysis of the wild type and five independent nec3 mutants identified a single candidate cytochrome P450 gene (HORVU.MOREX.r2.6HG0460850) that was validated as nec3 by independent mutations that result in predicted nonfunctional proteins. Histology studies determined that nec3 mutants had an unstable cutin layer that disrupted normal Bipolaris sorokiniana germ tube development.

Genetic characterization of melon accessions in the U.S. National Plant Germplasm System and construction of a melon core collection.

Melon (C. melo L.) is an economically important vegetable crop cultivated worldwide. The melon collection in the U.S. National Plant Germplasm System (NPGS) is a valuable resource to conserve natural genetic diversity and provide novel traits for melon breeding. Here we use the genotyping-by-sequencing (GBS) technology to characterize 2083 melon accessions in the NPGS collected from major melon production areas as well as regions where primitive melons exist. Population structure and genetic diversity analyses suggested that C. melo ssp. melo was firstly introduced from the centers of origin, Indian and Pakistan, to Central and West Asia, and then brought to Europe and Americas. C. melo ssp. melo from East Asia was likely derived from C. melo ssp. agrestis in India and Pakistan and displayed a distinct genetic background compared to the rest of ssp. melo accessions from other geographic regions. We developed a core collection of 383 accessions capturing more than 98% of genetic variation in the germplasm, providing a publicly accessible collection for future research and genomics-assisted breeding of melon. Thirty-five morphological characters investigated in the core collection indicated high variability of these characters across accessions in the collection. Genome-wide association studies using the core collection panel identified potentially associated genome regions related to fruit quality and other horticultural traits. This study provides insights into melon origin and domestication, and the constructed core collection and identified genome loci potentially associated with important traits provide valuable resources for future melon research and breeding.

The Barley HvWRKY6 Transcription Factor Is Required for Resistance Against Pyrenophora teres f. teres.

Barley is an important cereal crop worldwide because of its use in the brewing and distilling industry. However, adequate supplies of quality malting barley are threatened by global climate change due to drought in some regions and excess precipitation in others, which facilitates epidemics caused by fungal pathogens. The disease net form net blotch caused by the necrotrophic fungal pathogen Pyrenophora teres f. teres (Ptt) has emerged as a global threat to barley production and diverse populations of Ptt have shown a capacity to overcome deployed genetic resistances. The barley line CI5791 exhibits remarkably effective resistance to diverse Ptt isolates from around the world that maps to two major QTL on chromosomes 3H and 6H. To identify genes involved in this effective resistance, CI5791 seed were γ-irradiated and two mutants, designated CI5791-γ3 and CI5791-γ8, with compromised Ptt resistance were identified from an M2 population. Phenotyping of CI5791-γ3 and -γ8 × Heartland F2 populations showed three resistant to one susceptible segregation ratios and CI5791-γ3 × -γ8 F1 individuals were susceptible, thus these independent mutants are in a single allelic gene. Thirty-four homozygous mutant (susceptible) CI5791-γ3 × Heartland F2 individuals, representing 68 recombinant gametes, were genotyped via PCR genotype by sequencing. The data were used for single marker regression mapping placing the mutation on chromosome 3H within an approximate 75 cM interval encompassing the 3H CI5791 resistance QTL. Sequencing of the mutants and wild-type (WT) CI5791 genomic DNA following exome capture identified independent mutations of the HvWRKY6 transcription factor located on chromosome 3H at ∼50.7 cM, within the genetically delimited region. Post transcriptional gene silencing of HvWRKY6 in barley line CI5791 resulted in Ptt susceptibility, confirming that it functions in NFNB resistance, validating it as the gene underlying the mutant phenotypes. Allele analysis and transcript regulation of HvWRKY6 from resistant and susceptible lines revealed sequence identity and upregulation upon pathogen challenge in all genotypes analyzed, suggesting a conserved transcription factor is involved in the defense against the necrotrophic pathogen. We hypothesize that HvWRKY6 functions as a conserved signaling component of defense mechanisms that restricts Ptt growth in barley.

Characterization of genes required for both Rpg1 and rpg4-mediated wheat stem rust resistance in barley.

BACKGROUND: Puccinia graminis f. sp. tritici (Pgt) race TTKSK and its lineage pose a threat to barley production world-wide justifying the extensive efforts to identify, clone, and characterize the rpg4-mediated resistance locus (RMRL), the only effective resistance to virulent Pgt races in the TTKSK lineage. The RMRL contains two nucleotide-binding domain and leucine-rich repeat (NLR) resistance genes, Rpg5 and HvRga1, which are required for resistance. The two NLRs have head-to-head genome architecture with one NLR, Rpg5, containing an integrated C-terminal protein kinase domain, characteristic of an "integrated sensory domain" resistance mechanism. Fast neutron mutagenesis of line Q21861 was utilized in a forward genetics approach to identify genetic components that function in the RMRL or Rpg1 resistance mechanisms, as Q21861 contains both genes. A mutant was identified that compromises both RMRL and Rpg1-mediated resistances and had stunted seedling roots, designated required for P. graminis resistance 9 (rpr9). RESULTS: The rpr9 mutant generated in the Q21861 background was crossed with the Swiss landrace Hv584, which carries RMRL but contains polymorphism across the genome compared to Q21861. To map Rpr9, a Hv584 x rpr9 F6:7 recombinant inbred line (RIL) population was developed. The RIL population was phenotyped with Pgt race QCCJB. The Hv584 x rpr9 RIL population was genotyped with the 9 k Illumina Infinium iSelect marker panel, producing 2701 polymorphic markers. A robust genetic map consisting of 563 noncosegregating markers was generated and used to map Rpr9 to an ~ 3.4 cM region on barley chromosome 3H. The NimbleGen barley exome capture array was utilized to capture rpr9 and wild type Q21861 exons, followed by Illumina sequencing. Comparative analysis, resulting in the identification of a 1.05 Mbp deletion at the chromosome 3H rpr9 locus. The identified deletion contains ten high confidence annotated genes with the best rpr9 candidates encoding a SKP1-like 9 protein and a F-box family protein. CONCLUSION: Genetic mapping and exome capture rapidly identified candidate gene/s that function in RMRL and Rpg1 mediated resistance pathway/s. One or more of the identified candidate rpr9 genes are essential in the only two known effective stem rust resistance mechanisms, present in domesticated barley.

Mapping of barley susceptibility/resistance QTL against spot form net blotch caused by Pyrenophora teres f. maculata using RIL populations.

Spot form net blotch (SFNB) caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm) is an important disease of barley worldwide including the major barley production regions of North America. To characterize SFNB resistance/susceptibility quantitative trait loci (QTL), three recombinant inbred line (RIL) populations were developed from crosses between the malting barley cultivars, Tradition (six row) and Pinnacle (two row), and the two world barley core collection lines, PI67381 and PI84314. Tradition and Pinnacle were susceptible to many North American Ptm isolates, while PI67381 and PI84314 carry resistances to diverse Ptm isolates from across the globe. The RIL populations, Tradition/PI67381, Pinnacle/PI67381, and Pinnacle/PI84314 were genotyped using polymerase chain reaction-mediated genotype-by-sequencing single nucleotide polymorphism marker panels and phenotyped at the seedling stage with six geographically distinct Ptm isolates: FGOB10Ptm-1 (North Dakota, USA), Pin-A14 (Montana, USA), Cel-A17 (Montana, USA), SG1 (Australia), NZKF2 (New Zealand) and DEN2.6 (Denmark). The goal was to determine if the susceptible elite lines contained common susceptibility genes/QTL or if the resistant lines had common resistant genes/QTL effective against diverse Ptm isolates. The QTL analyses identified a total of 12 resistance and/or susceptibility loci on chromosomes 2H, 3H, 4H, 6H, and 7H of which three had not been previously reported. Common major QTL were detected on chromosome 2H (R2 = 14-40%) and 7H (R2 = 24-80%) in all three RIL populations, suggesting underlying genes with broad resistance specificity. The major 7H QTL was shown to be a dominant susceptibility gene in both susceptible malting barley varieties.

Association mapping of seedling resistance to spot form net blotch in a worldwide collection of barley.

Spot form net blotch (SFNB), caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata, is an important foliar disease of barley in major production regions around the world. Deployment of adequate host resistance is challenging because the virulence of P. teres f. maculata is highly variable and characterized minor-effect resistances are typically ineffective against the diverse pathogen populations. A world barley core collection consisting of 2,062 barley accessions of diverse origin and genotype were phenotyped at the seedling stage with four P. teres f. maculata isolates collected from the United States (FGO), New Zealand (NZKF2), Australia (SG1), and Denmark (DEN 2.6). Of the 2,062 barley accessions phenotyped, 1,480 were genotyped with the Illumina barley iSelect chip and passed the quality controls with 5,954 polymorphic markers used for further association mapping analysis. Genome-wide association mapping was utilized to identify and map resistance loci from the seedling disease response data and the single nucleotide polymorphism (SNP) marker data. The best among six different regression models was identified for each isolate and association analysis was performed separately for each. A total of 138 significant (-log10P value>3.0) marker-trait associations (MTA) were detected. Using a 5 cM cutoff, a total of 10, 8, 13, and 10 quantitative trait loci (QTL) associated with SFNB resistance were identified for the FGO, SG1, NZKF2, and DEN 2.6 isolates, respectively. Loci containing from 1 to 34 MTA were identified on all seven barley chromosomes with one locus at 66 to 69 cM on chromosome 2H common to all four isolates. Six distinct loci were identified by the association mapping (AM) analysis that corresponded to previously characterized SFNB resistance QTL identified by biparental population analysis (QRpt4, QRpt6, Rpt4, Rpt6, Rpt7, and a QTL on 4H that was not given a provisional gene or QTL nomenclature). The 21 putative novel loci identified may represent a broad spectrum of resistance and or susceptibility loci. This is the first comprehensive AM study to characterize SFNB resistance loci underlying broad populations of the barley host and P. teres f. maculata pathogen.