Phenotyping and Exploitation of Kompetitive Allele-Specific PCR Assays for Genes Underpinning Leaf Rust Resistance in New Spring Wheat Mutant Lines.

Leaf rust (Puccinia triticina Eriks) is a wheat disease causing substantial yield losses in wheat production globally. The identification of genetic resources with permanently effective resistance genes and the generation of mutant lines showing increased levels of resistance allow the efficient incorporation of these target genes into germplasm pools by marker-assisted breeding. In this study, new mutant (M3 generation) lines generated from the rust-resistant variety Kazakhstanskaya-19 were developed using gamma-induced mutagenesis through 300-, 350-, and 400-Gy doses. In field trials after leaf rust inoculation, 75 mutant lines showed adult plant resistance. These lines were evaluated for resistance at the seedling stage via microscopy in greenhouse experiments. Most of these lines (89.33%) were characterized as resistant at both developmental stages. Hyperspectral imaging analysis indicated that infected leaves of wheat genotypes showed increased relative reflectance in visible and near-infrared light compared to the non-infected genotypes, with peak means at 462 and 644 nm, and 1936 and 2392 nm, respectively. Five spectral indexes, including red edge normalized difference vegetation index (RNDVI), structure-insensitive pigment index (SIPI), ratio vegetation index (RVSI), water index (WI), and normalized difference water index (NDWI), demonstrated significant potential for determining disease severity at the seedling stage. The most significant differences in reflectance between susceptible and resistant mutant lines appeared at 694.57 and 987.51 nm. The mutant lines developed were also used for the development and validation of KASP markers for leaf rust resistance genes Lr1, Lr2a, Lr3, Lr9, Lr10, and Lr17. The mutant lines had high frequencies of "a" resistance alleles (0.88) in all six Lr genes, which were significantly associated with seedling resistance and suggest the potential of favorable haplotype introgression through functional markers. Nine mutant lines characterized by the presence of "b" alleles in Lr9 and Lr10-except for one line with allele "a" in Lr9 and three mutant lines with allele "a" in Lr10-showed the progressive development of fungal haustorial mother cells 72 h after inoculation. One line from 300-Gy-dosed mutant germplasm with "b" alleles in Lr1, Lr2a, Lr10, and Lr17 and "a" alleles in Lr3 and Lr9 was characterized as resistant based on the low number of haustorial mother cells, suggesting the contribution of the "a" alleles of Lr3 and Lr9.

Diversity of Expression Patterns of Lr34, Lr67, and Candidate Genes towards Lr46 with Analysis of Associated miRNAs in Common Wheat Hybrids in Response to Puccinia triticina Fungus.

Leaf rust caused by Puccinia triticina (Pt) is one of the most dangerous diseases causing significant losses in common wheat crops. In adult plants resistant to rust, a horizontal adult plant resistance (APR) type is observed, which protects the plant against multiple pathogen races and is distinguished by greater persistence under production conditions. Crucial pleiotropic slow-rust genes such as Lr34, Lr46, Lr67, and Lr68, in combination with other genes of lesser influence, continue to increase durable resistance to rust diseases. Based on our previous results, we selected four candidate genes for Lr46 out of ten candidates and analysed them for expression before and after inoculation by P. triticina. As part of our study, we also investigated the expression patterns of miRNA molecules complementary to Lr34 and the candidate genes. The aim of the study was to analyse the expression profiles of candidate genes for the Lr46 gene and the Lr34 and Lr67 genes responsible for the differential leaf-rust resistance of hybrid forms of the F1 generation resulting from crosses between the Glenlea cultivar and cultivars from Polish breeding companies. In addition, the expression of five miRNAs (tae-miR9653b, tae-miR5384-3p, tae-miR9780, tae-miR9775 and tae-miR164), complementary to Lr34, and selected candidate genes were analysed using stem-loop RT-PCR and ddPCR. Biotic stress was induced in adult plants by inoculation with Pt fungal spores, under controlled conditions. Plant material was collected before and 6, 12, 24, and 48 h after inoculation (hpi). Differences in expression patterns of Lr34, Lr67, and candidate genes (for Lr46) were analysed by qRT-PCR and showed that gene expression changed at the analysed time points. Identification of molecular markers coupled to the Lr genes studied was also carried out to confirm the presence of these genes in wheat hybrids. qRT-PCR was used to examine the expression levels of the resistance genes. The highest expression of Lr46/Yr29 genes (Lr46-Glu2, Lr46-RLK1, Lr46-RLK2, and Lr46-RLK3) occurred at 12 and 24 hpi, and such expression profiles were obtained for only one candidate gene among the four genes analysed (Lr46-Glu2), indicating that it may be involved in resistance mechanisms of response to Pt infection.

High-throughput diagnostic markers for foliar fungal disease resistance and high oleic acid content in groundnut.

BACKGROUND: Foliar diseases namely late leaf spot (LLS) and leaf rust (LR) reduce yield and deteriorate fodder quality in groundnut. Also the high oleic acid content has emerged as one of the most important traits for industries and consumers due to its increased shelf life and health benefits. RESULTS: Genetic mapping combined with pooled sequencing approaches identified candidate resistance genes (LLSR1 and LLSR2 for LLS and LR1 for LR) for both foliar fungal diseases. The LLS-A02 locus housed LLSR1 gene for LLS resistance, while, LLS-A03 housed LLSR2 and LR1 genes for LLS and LR resistance, respectively. A total of 49 KASPs markers were developed from the genomic regions of important disease resistance genes, such as NBS-LRR, purple acid phosphatase, pentatricopeptide repeat-containing protein, and serine/threonine-protein phosphatase. Among the 49 KASP markers, 41 KASPs were validated successfully on a validation panel of contrasting germplasm and breeding lines. Of the 41 validated KASPs, 39 KASPs were designed for rust and LLS resistance, while two KASPs were developed using fatty acid desaturase (FAD) genes to control high oleic acid levels. These validated KASP markers have been extensively used by various groundnut breeding programs across the world which led to development of thousands of advanced breeding lines and few of them also released for commercial cultivation. CONCLUSION: In this study, high-throughput and cost-effective KASP assays were developed, validated and successfully deployed to improve the resistance against foliar fungal diseases and oleic acid in groundnut. So far deployment of allele-specific and KASP diagnostic markers facilitated development and release of two rust- and LLS-resistant varieties and five high-oleic acid groundnut varieties in India. These validated markers provide opportunities for routine deployment in groundnut breeding programs.

Development and identification of a novel wheat-Thinopyrum ponticum disomic substitution line DS5Ag(5D) with new genes conferring resistance to powdery mildew and leaf rust.

BACKGROUND: Powdery mildew (caused by Blumeria graminis f. sp. tritici (Bgt)) and leaf rust (caused by Puccinia triticina (Pt)) are prevalent diseases in wheat (Triticum aestivum L.) production. Thinopyrum ponticum (2n = 10x = 70, EeEeEbEbExExStStStSt) contains genes that confer high levels of resistance to these diseases. RESULTS: An elite wheat-Th. ponticum disomic substitution line, DS5Ag(5D), was developed in the Bainong Aikang 58 (AK58) background. The line was assessed using genomic in situ hybridization (GISH), oligo-nucleotide probe multiplex (ONPM) fluorescence in situ hybridization (FISH), and molecular markers. Twenty eight chromosome-specific molecular markers were identified for the alien chromosome, and 22 of them were co-dominant. Additionally, SNP markers from the wheat 660 K SNP chip were utilized to confirm chromosome identification and they provide molecular tools for tagging the chromosome in concern. The substitution line demonstrated high levels of resistance to powdery mildew throughout its growth period and to leaf rust at the adult stage. Based on the resistance evaluation of five F5 populations between the substitution lines and wheat genotypes with different levels of sensitivity to the two diseases. Results showed that the resistance genes located on 5Ag confered stable resistance against both diseases across different backgrounds. Resistance spectrum analysis combined with diagnostic marker detection of known resistance genes of Th. ponticum revealed that 5Ag contained two novel genes, Pm5Ag and Lr5Ag, which conferred resistance to powdery mildew and leaf rust, respectively. CONCLUSIONS: In this study, a novel wheat-Th. ponticum disomic substitution line DS5Ag(5D) was successfully developed. The Th. ponticum chromosome 5Ag contain new resistance genes for powdery mildew and leaf rust. Chromosomic-specific molecular markers were generated and they can be used to track the 5Ag chromosome fragments. Consequently, this study provides new elite germplasm resources and molecular markers to facilitate the breeding of wheat varieties that is resistant to powdery mildew and leaf rust.

Genome-wide analysis of R2R3-MYB transcription factors in poplar and functional validation of PagMYB147 in defense against Melampsora magnusiana.

MAIN CONCLUSION: Transcription of PagMYB147 was induced in poplar infected by Melampsora magnusiana, and a decline in its expression levels increases the host's susceptibility, whereas its overexpression promotes resistance to rust disease. Poplars are valuable tree species with diverse industrial and silvicultural applications. The R2R3-MYB subfamily of transcription factors plays a crucial role in response to biotic stresses. However, the functional studies on poplar R2R3-MYB genes in resistance to leaf rust disease are still insufficient. We identified 191 putative R2R3-MYB genes in the Populus trichocarpa genome. A phylogenetic analysis grouped poplar R2R3-MYBs and Arabidopsis R2R3-MYBs into 33 subgroups. We detected 12 tandem duplication events and 148 segmental duplication events, with the latter likely being the main contributor to the expansion of poplar R2R3-MYB genes. The promoter regions of these genes contained numerous cis-acting regulatory elements associated with response to stress and phytohormones. Analyses of RNA-Seq data identified a multiple R2R3-MYB genes response to Melampsora magnusiana (Mmag). Among them, PagMYB147 was significantly up-regulated under Mmag inoculation, salicylic acid (SA) and methyl jasmonate (MeJA) treatment, and its encoded product was primarily localized to the cell nucleus. Silencing of PagMYB147 exacerbated the severity of Mmag infection, likely because of decreased reactive oxygen species (ROS) production and phenylalanine ammonia-lyase (PAL) enzyme activity, and up-regulation of genes related to ROS scavenging and down-regulation of genes related to PAL, SA and JA signaling pathway. In contrast, plants overexpressing PagMYB147 showed the opposite ROS accumulation, PAL enzyme activity, SA and JA-related gene expressions, and improved Mmag resistance. Our findings suggest that PagMYB147 acts as a positive regulatory factor, affecting resistance in poplar to Mmag by its involvement in the regulation of ROS homeostasis, SA and JA signaling pathway.

The F-box protein encoding genes of the leaf-rust fungi Puccinia triticina: genome-wide identification, characterization and expression dynamics during pathogenesis.

The F-box proteins in fungi perform diverse functions including regulation of cell cycle, circadian clock, development, signal transduction and nutrient sensing. Genome-wide analysis revealed 10 F-box genes in Puccinia triticina, the causal organism for the leaf rust disease in wheat and were characterized using in silico approaches for revealing phylogenetic relationships, gene structures, gene ontology, protein properties, sequence analysis and gene expression studies. Domain analysis predicted functional domains like WD40 and LRR at C-terminus along with the obvious presence of F-box motif in N-terminus. MSA showed amino acid replacements, which might be due to nucleotide substitution during replication. Phylogenetic analysis revealed the F-box proteins with similar domains to be clustered together while some sequences were spread out in different clades, which might be due to functional diversity. The clustering of Puccinia triticina GG705409 with Triticum aestivum TaAFB4/TaAFB5 in a single clade suggested the possibilities of horizontal gene transfer during the coevolution of P. triticina and wheat. Gene ontological annotation categorized them into three classes and were functionally involved in protein degradation through the protein ubiquitination pathway. Protein-protein interaction network revealed F-box proteins to interact with other components of the SCF complex involved in protein ubiquitination. Relative expression analysis of five F-box genes in a time course experiment denoted their involvement in leaf rust susceptible wheat plants. This study provides information on structure elucidation of F-box proteins of a basidiomycetes plant pathogenic fungi and their role during pathogenesis.

Breaking the association between gametocidal gene(s) and leaf rust resistance gene (LrS2427) in Triticum aestivum-Aegilops speltoides derivative by gamma irradiation.

Utilization of crop wild relatives of wheat can be very effective in building the genetic diversity to cater to the evolving strains of disease pathogens. Aegilops speltoides is a rich source of rust resistance genes however transferring those to wheat genome can be tedious due to co-transfer and preferential transmission of undesirable genes causing gametocidal activity. Such an unholy association was observed in Triticum aestivum-Ae. speltoides derivative line Sel. 2427 which possess the broad-spectrum leaf rust seedling resistance gene (LrS2427). Molecular analysis based on 35 K wheat breeder's array revealed the maximum percentage of Ae. speltoides genome introgression on homoeologous group 2. In situ hybridization studies revealed the presence of S genome in Sel. 2427, showing six translocations on four chromosomes. Karyotyping using repetitive probe (AAG)6 revealed that the two chromosomes involved are 2D and 2B. Genic regions causing gametocidal activity were identified by dissecting it into component traits and QTLs on 2D and 2B chromosomes were revealed in case of the trait seed shrivelling index. To break the inadvertent association of LrS2427 with gametocidal genes, F1(Agra Local X Sel. 2427) seeds were irradiated with gamma rays and stable leaf rust resistant mutants lacking gametocidal activity were developed. These mutants showed resistance to different races of leaf rust pathogen and showed superior agronomic performance as well. These mutants could be a great resource in wheat improvement for utilization of the leaf rust resistance gene LrS2427 without any yield penalty. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01491-8.

5-mC methylation study of sORFs in 3'UTR of transcription factor JUNGBRUNNEN 1-like during leaf rust pathogenesis in wheat.

BACKGROUND: JUB1, a NAC domain containing hydrogen peroxide-induced transcription factor, plays a critical role in plant immunity. Little is known about how JUB1 responds to leaf rust disease in wheat. Recent discoveries in genomics have also unveiled a multitude of sORFs often assumed to be non-functional, to argue for the necessity of including them as potential regulatory players of translation. However, whether methylation on sORFs spanning the 3'UTR of regulatory genes like JUB1 modulate gene expression, remains unclear. METHODS AND RESULTS: In this study, we identified the methylation states of two sORFs in 3'UTR of a homologous gene of JUB1 in wheat, TaJUB1-L, at cytosine residues in CpG, CHH and CHG sites at different time points of disease progression in two near-isogenic lines of wheat (HD2329), with and without Lr24 gene during leaf rust pathogenesis. Here, we report a significant demethylation of the CpG dinucleotides occurring in the sORFs of the 3'UTR in the resistant isolines after 24 h post-infection. Also, the up-regulated gene expression observed through RT-qPCR was directly proportional to the demethylation of the CpG sites in the sORFs. CONCLUSIONS: Our findings indicate that TaJUB1-L might be a positive regulator in providing tolerance during leaf rust pathogenesis and cytosine methylation at 3'UTR might act as a switch for its expression control. These results enrich the potential benefit of conventional methylation assay techniques for unraveling the unexplored enigma in epigenetics during plant-pathogen interaction in a cost-effective and confidentially conclusive manner.

Characterization of Quantitative Trait Loci for Leaf Rust Resistance in the Uzbekistani Wheat Landrace Teremai Bugdai.

Leaf rust, caused by Puccinia triticina, is a major cause of wheat yield losses globally, and novel leaf rust resistance genes are needed to enhance wheat leaf rust resistance. Teremai Bugdai is a landrace from Uzebekistan that is highly resistant to many races of P. triticina in the United States. To unravel leaf rust resistance loci in Teremai Bugdai, a recombinant inbred line (RIL) population of Teremai Bugdai × TAM 110 was evaluated for response to P. triticina race Pt54-1 (TNBGJ) and genotyped using single nucleotide polymorphism (SNP) markers generated by genotyping-by-sequencing (GBS). Quantitative trait loci (QTL) analysis using 5,130 high-quality GBS-SNPs revealed three QTLs, QLr-Stars-2DS, QLr-Stars-6BL, and QLr.Stars-7BL, for leaf rust resistance in two experiments. QLr-Stars-2DS, which is either a new Lr2 allele or a new resistance locus, was delimited to an ∼19.47-Mb interval between 46.4 and 65.9 Mb on 2DS and explained 31.3 and 33.2% of the phenotypic variance in the two experiments. QLr-Stars-6BL was mapped in an ∼84.0-kb interval between 719.48 and 719.56 Mb on 6BL, accounting for 33 to 36.8% of the phenotypic variance in two experiments. QLr.Stars-7BL was placed in a 350-kb interval between 762.41 and 762.76 Mb on 7BL and explained 4.4 to 5.3% of the phenotypic variance. Nine GBS-SNPs flanking these QTLs were converted to kompetitive allele specific PCR (KASP) markers, and these markers can be used to facilitate their introgression into locally adapted wheat lines.

Characterization of Quantitative Trait Loci for Leaf Rust Resistance from CI 13227 in Three Winter Wheat Populations.

Leaf rust is a widespread foliar wheat disease causing substantial yield losses worldwide. Slow rusting is "adult plant" resistance that significantly slows epidemic development and thereby reduces yield loss. Wheat accession CI 13227 was previously characterized as having slow-rusting resistance. To validate the quantitative trait loci (QTLs) and develop diagnostic markers for slow rusting resistance in CI 13227, a new population of recombinant inbred lines of CI 13227 × Everest was evaluated for latent period, final severity, area under the disease progress curve, and infection type in greenhouses and genotyped using genotyping-by-sequencing. Four QTLs were identified on chromosome arms 2BL, 2DS, 3BS, and 7BL, explaining 6.82 to 28.45% of the phenotypic variance for these traits. Seven kompetitive allele-specific polymorphism markers previously reported to be linked to the QTLs in two other CI 13227 populations were validated. In addition, the previously reported QLr.hwwg-7AL was remapped to 2BL (renamed QLr.hwwg-2BL) after adding new markers in this study. Phenotypic data showed that the recombinant inbred lines harboring two or three of the QTLs had a significantly longer latent period. QLr.hwwg-2DS on 2DS showed a major effect on all rust resistance traits and was finely mapped to a 2.7-Mb interval by two newly developed flanking markers from exome capture. Three disease-resistance genes and two transporter genes were identified as the putative candidates for QLr.hwwg-2DS. The validated QTLs can be used as slow-rusting resistance resources, and the markers developed in this study will be useful for marker-assisted selection.

Impact of ontogenic changes on the dynamics of a fungal crop disease model motivated by coffee leaf rust.

Ontogenic resistance has been described for many plant-pathogen systems. Conversely, coffee leaf rust, a major fungal disease that drastically reduces coffee production, exhibits a form of ontogenic susceptibility, with a higher infection risk for mature leaves. To take into account stage-dependent crop response to phytopathogenic fungi, we developed an SEIR-U epidemiological model, where U stands for spores, which differentiates between young and mature leaves. Based on this model, we also explored the impact of ontogenic resistance on the sporulation rate. We computed the basic reproduction number [Formula: see text], which classically determines the stability of the disease-free equilibrium. We identified forward and backward bifurcation cases. The backward bifurcation is generated by the high sporulation of young leaves compared to mature ones. In this case, when the basic reproduction number is less than one, the disease can persist. These results provide useful insights on the disease dynamics and its control. In particular, ontogenic resistance may require higher control efforts to eradicate the disease.

Synergistic dispersal of plant pathogen spores by jumping-droplet condensation and wind.

Plant pathogens are responsible for the annual yield loss of crops worldwide and pose a significant threat to global food security. A necessary prelude to many plant disease epidemics is the short-range dispersal of spores, which may generate several disease foci within a field. New information is needed on the mechanisms of plant pathogen spread within and among susceptible plants. Here, we show that self-propelled jumping dew droplets, working synergistically with low wind flow, can propel spores of a fungal plant pathogen (wheat leaf rust) beyond the quiescent boundary layer and disperse them onto neighboring leaves downwind. An array of horizontal water-sensitive papers was used to mimic healthy wheat leaves and showed that up to 25 spores/h may be deposited on a single leaf downwind of the infected leaf during a single dew cycle. These findings reveal that a single dew cycle can disperse copious numbers of fungal spores to other wheat plants, even in the absence of rain splash or strong gusts of wind.

Biocontrol of Hemileia vastatrix, the Causal Agent of Coffee Leaf Rust, by Paenibacillus sp. NMA1017.

Coffee leaf rust (CLR), caused by Hemileia vastatrix, is considered a highly important phytosanitary problem in Mexico. Currently, there are few microorganisms used as biocontrol alternatives to chemical control of CLR in organic coffee fields in Mexico. This study evaluates the use of Paenibacillus sp. NMA1017 as a biocontrol agent to inhibit the development of H. vastatrix in in vitro and in vivo (greenhouse) experiments. Hemileia vastatrix urediniospores were placed on water agar plates, and then Paenibacillus sp. NMA1017 was inoculated simultaneously or 8 h later. Urediniospores germination rate was reduced by 94% when the NMA1017 strain was inoculated simultaneously with the urediniospores and reduced by 38% when NMA1017 was inoculated 8 h later. Experiments with 8-month-old Bourbon coffee plants that were infected with H. vastatrix showed that disease incidence was reduced by 38, 90, and 50% when NMA1017 was applied 8 days before, simultaneously, or 8 days after the application of H. vastatrix, respectively. Paenibacillus sp. NMA1017 also reduced the severity of CLR on the leaves by up to 62%. The germination urediniospores of other rust pathogens such as Puccinia sorghi (maize leaf rust), Puccinia triticina (wheat leaf rust), Puccinia graminis f. sp. tritici (black stem rust of wheat), Uromyces striatus (alfalfa leaf rust), and Phragmidium sp. (rosebush leaf rust) were also inhibited. Use of the potential biocontrol agent Paenibacillus sp. NMA1017 might help reduce the application of chemical fungicides for the control of CLR, making coffee a more sustainable crop and providing management options for organic coffee growers.

Effect of Salicylic Acid Treatment on Disease Resistance to Coleosporium zanthoxyli in Chinese Pepper (Zanthoxylum armatum).

The fungus Coleosporium zanthoxyli causes leaf rust in Chinese pepper (Zanthoxylum armatum). To investigate the control effect of elicitor treatment on leaf rust in this species, the impact of salicylic acid (SA) on the spores and growth of C. zanthoxyli and the induced resistance to leaf rust by Z. armatum were analyzed, and the possible defense mechanisms involved in SA induction were evaluated. The results showed that SA had no effect on C. zanthoxyli spore germination and growth; however, rust resistance was induced in Z. armatum. The optimal SA treatment concentration was 0.4 mg/ml, and the relative cure effect reached 44.56%. SA-induced disease resistance was evident for up to 10 days, while the optimal induction interval was 48 h after stimulation. Consistent with the induced resistance, H2O2, total protein, total phenol, and lignin concentrations and polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), superoxide dismutase (SOD), and catalase (CAT) activities were significantly increased with the SA treatment, whereas the malondialdehyde content was significantly decreased. In addition, exogenous SA promoted defense-related enzyme activities, including those of POD, CAT, and PAL, and increased H2O2, lignin, and endogenous SA contents. Furthermore, SA induced the expression of SA signaling pathway genes such as ZaPR1 and ZaNPR1, and silencing ZaPR1 enhanced the sensitivity of Z. armatum to leaf rust. Our results demonstrated that 0.4 mg/ml SA priming increased the activities of CAT, POD, and PAL, elevated the contents of H2O2, lignin, and endogenous SA, and upregulated the expression of the SA-related gene ZaPR1, thereby enhancing the resistance of Z. armatum to leaf rust.

Mapping of quantitative trait loci for leaf rust resistance in the wheat population 'Xinmai 26/Zhoumai 22'.

Leaf rust, caused by the fungal pathogen Puccinia triticina (Pt), is one of the major and dangerous diseases of wheat, and has caused serious yield loss of wheat worldwide. Here, we investigated adult-plant resistance (APR) to leaf rust in a recombinant inbred line (RIL) population derived from 'Xinmai 26' and 'Zhoumai 22' over 3 years. Linkage mapping for APR to leaf rust revealed four quantitative trait loci (QTL) in this RIL population. Two QTL, QLr.hnau-2BS and QLr.hnau-3BS were contributed by 'Zhoumai22', whereas QLr.hnau-2DS and QLr.hnau-5AL were contributed by 'Xinmai 26'. The QLr.hnau-2BS covering a race-specific resistance gene Lr13 showed the most stable APR to leaf rust. Overexpression of Lr13 significantly increased APR to leaf rust. Interestingly, we found that a CNL(coiled coil-nucleotide-binding site-leucine-rich repeat)-like gene, TaCN, in QLr.hnau-2BS completely co-segregated with leaf rust resistance. The resistant haplotype TaCN-R possessed half the sequence of the coiled-coil domain of TaCN protein. Lr13 strongly interacted with TaCN-R, but did not interact with the full-length TaCN (TaCN-S). In addition, TaCN-R was significantly induced after Pt inoculation and changed the sub-cellular localization of Lr13 after interaction. Therefore, we hypothesized that TaCN-R mediated leaf rust resistance possibly by interacting with Lr13. This study provides important QTL for APR to leaf rust, and new insights into understanding how a CNL gene modulates disease resistance in common wheat.

Synthesis of bis(ylidene) cyclohexanones and their antifungal activity against selected plant pathogenic fungi.

Botrytis cinerea, Rhizoctonia solani and Hemileia vastatrix are three species of phytopathogenic fungi behind major crop losses worldwide. These have been selected as target models for testing the fungicide potential of a series of bis(ylidene) cyclohexanones. Although some compounds of this chemical class are known to have inhibitory activity against human pathogens, they have never been explored for the control of phytopathogens until now. In the present work, bis(ylidene) cyclohexanones were synthesized through simple, fast and low-cost base- or acid-catalyzed aldol condensation reaction and tested in vitro against B. cinerea, R. solani and H. vastatrix. bis(pyridylmethylene) cyclohexanones showed the highest activity against the target fungi. When tested at 200 nmol per mycelial plug against R. solani., these compounds completely inhibited the mycelial growth, and the most active bis(pyridylmethylene) cyclohexanone compound had an IC50 of 155.5 nmol plug-1. Additionally, bis(pyridylmethylene) cyclohexanones completely inhibited urediniospore germination of H. vastatrix, at 125 µmol L-1. The most active bis(pyridylmethylene) cyclohexanone had an IC50 value of 4.8 µmol L-1, which was estimated as approximately 2.6 times lower than that found for the copper oxychloride-based fungicide, used as control. Additionally, these substances had a low cytotoxicity against the mammalian Vero cell line. Finally, in silico calculations indicated that these compounds present physicochemical parameters regarded as suitable for agrochemicals. Bis(ylidene) cyclohexanones may constitute promising candidates for the development of novel antifungal agents for the control of relevant fungal diseases in agriculture.

Modeling the Impact of Proportion, Sowing Date, and Architectural Traits of a Companion Crop on Foliar Fungal Pathogens of Wheat in Crop Mixtures.

Diversification of cropping systems is a lever for the management of epidemics. However, most research to date has focused on cultivar mixtures, especially for cereals, even though crop mixtures can also improve disease management. To investigate the benefits of crop mixtures, we studied the effect of different crop mixture characteristics (i.e., companion proportion, sowing date, and traits) on the protective effect of the mixture. We developed a SEIR (Susceptible, Exposed, Infectious, Removed) model of two damaging wheat diseases (Zymoseptoria tritici and Puccinia triticina), which were applied to different canopy components, ascribable to wheat and a theoretical companion crop. We used the model to study the sensitivity of disease intensity to the following parameters: wheat-versus-companion proportion, companion sowing date and growth, and architectural traits. For both pathogens, the companion proportion had the strongest effect, with 25% of companion reducing disease severity by 50%. However, changing companion growth and architectural traits also significantly improved the protective effect. The effect of companion characteristics was consistent across different weather conditions. After decomposing the dilution and barrier effects, the model suggested that the barrier effect is maximized for an intermediate proportion of companion crop. Our study thus supports crop mixtures as a promising strategy to improve disease management. Future studies should identify real species and determine the combination of host and companion traits to maximize the protective effect of the mixture. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

High-Resolution Melting-Based Marker Development for Wheat Leaf Rust Resistance Gene Lr34.

Deploying adult plant resistance (APR) against rust diseases is an important breeding objective of most wheat-breeding programs. The gene Lr34 is an effective and widely deployed broad-spectrum APR gene in wheat against leaf rust fungus Puccinia triticina. Various molecular markers have been developed for Lr34, but they either require post-PCR handling processes or are not economical. Herein, we developed a high-resolution melting (HRM)-based diagnostic assay for Lr34 based on a 3-bp 'TTC' deletion in exon 11 of the resistant allele. The susceptible cultivar Thatcher (Tc) and the near-isogenic Thatcher line (RL6058) with Lr34 yielded distinct melting profiles and were differentiated with high reproducibility. For further validation, all three copies of Lr34 were cloned in plasmid vectors, and HRM analysis using individual and combination (equimolar mixture of three copies) homoeologs yielded distinct melting profiles. An additional layer of genotyping was provided by a LunaProbe assay. The allele-specific probes successfully distinguished the homoeologs but not Tc and RL6058. Furthermore, the practical deployment of the HRM assay was tested by running the marker on a set of breeding lines. When compared with a kompetitive allele-specific PCR (KASP) Lr34 assay, the HRM assay had similar genotyping results and was able to accurately differentiate the resistant and susceptible breeding lines. However, our HRM assay was unable to detect the heterozygote. To our knowledge, this is the first report of an HRM assay for genotyping a wheat rust resistance gene.

Sensitivity of Puccinia triticina f. sp. tritici from China to Triadimefon and Resistance Risk Assessment.

Wheat leaf rust, caused by Puccinia triticina f. sp. tritici (Pt), is distributed widely in wheat-producing areas and results in serious yield losses worldwide. In China, leaf rust has been largely controlled with a demethylation inhibitor (DMI) fungicide, triadimefon. Although high levels of fungicide resistance in pathogens have been reported, no field failure of wheat leaf rust to DMI fungicides has been reported in China. A resistance risk assessment of triadimefon to Pt was investigated in the present study. The sensitivity of 197 Pt isolates across the country to triadimefon was determined, and the density distribution of EC50 values (concentration at which mycelial growth is inhibited by 50%) showed a continuous multimodal curve because of the extensive use of this fungicide in wheat production, with a mean value of 0.46 µg/ml. The majority of the tested Pt isolates were sensitive to triadimefon, whereas 10.2% developed varying degrees of resistance. Characterization of parasitic fitness revealed that the triadimefon-resistant isolates exhibited strong adaptive traits in urediniospore germination rate, latent period, sporulation intensity, and lesion expansion rate. No correlation was observed between triadimefon and tebuconazole and hexaconazole, which have the similar mode of action, or pyraclostrobin and flubeneteram, which have different modes of action. Overexpression of the target gene Cyp51 led to the triadimefon resistance of Pt. The risk of resistance to triadimefon in Pt may be low to moderate. This study provided important data for fungicide resistance risk management against wheat leaf rust.

First report of Pseudocerradoa paullula causing aroid leaf rust on Swiss cheese plant Monstera deliciosa in South Carolina, USA.

In February 2023, two Monstera deliciosa Liebm. (Araceae) plants with typical symptoms of leaf rust disease were detected at a grocery store in Oconee Co., South Carolina. Symptoms included chlorotic leaf spots and abundant brownish uredinia, mainly on the adaxial surface of more than 50% of leaves. The same disease was detected on 11 out of 481 M. deliciosa plants in a greenhouse at a plant nursery located in York Co., South Carolina, in March 2023. The first plant sample detected in February was used for morphological characterization, molecular identification, and pathogenicity confirmation of the rust fungus. Urediniospores were densely aggregated, globose, golden to golden brown in color, and measured 22.9 to 27.9 µm (aver. 26.0 ± 1.1 µm; n=50) in diameter with wall thickness at 1.3 to 2.6 µm (aver. 1.8 ± 0.3 µm; n=50). Telia were not observed. These morphological traits aligned with those of Pseudocerradoa paullula (basionym: Puccinia paullula; Ebinghaus et al. 2022; Sakamoto et al. 2023; Sydow and Sydow 1913; Urbina et al. 2023). Genomic DNA was extracted from urediniospores collected from the naturally infected plant sample and used for PCR amplification and DNA sequencing of the large subunit (LSU) genetic marker with primers LRust1R and LR3 (Vilgalys and Hester 1990; Beenken et al. 2012). The LSU sequence of the rust fungus in South Carolina (GenBank accession: OQ746460) is 99.9% identical to that of Ps. paullula voucher BPI 893085 (763/764 nt.; KY764151), 99.4% identical to that of voucher PIGH 17154 in Florida, USA (760/765 nt.; OQ275201), and 99% identical to that of voucher TNS-F-82075 in Japan (715/722 nt.; OK509071). Based on its morphological and molecular characteristics, the causal agent was identified as Ps. paullula. This pathogen identification was also corroborated by the U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Plant Pathogen Confirmatory Diagnostics Laboratory in Laurel, Maryland. To confirm the fungus's pathogenicity on M. deliciosa and M. adansonii Schott (Sakamoto et al. 2023), three plants of each Monstera species were inoculated by spraying with a suspension of urediniospores collected from the original plant sample (1 × 106 spores per ml; approx. 40 ml per plant). Three non-inoculated control plants of each host species were treated with deionized water in the same manner. Plants were placed in a plastic tray with wet paper towels to maintain moisture. The tray was placed at 22C for an 8-h photoperiod and covered for five days to facilitate infection. On 25 days after inoculation, abundant spots bearing urediniospores were produced on all leaves of inoculated M. deliciosa plants. A few uredinia were observed on two of the three inoculated M. adansonii plants. All non-inoculated control plants remained asymptomatic. Morphological features of urediniospores collected from inoculated plants matched those of Ps. paullula used as the inoculum. Aroid leaf rust on Monstera plants was officially reported in Australia, China, Japan, Malaysia, Philippines, and Florida, USA (Shaw 1991; Sakamoto et al. 2023; Urbina et al. 2023). This is the first report of Ps. paullula causing this disease on M. deliciosa in South Carolina, USA. Monstera species are popular indoor and landscape plants. Potential impact and regulatory responses regarding Ps. paullula, a newly introduced and rapidly spreading pathogen in the USA, warrant further evaluation and discussion.