Resistance of Ascochyta rabiei isolates from chickpeas (Cicer arietinum L.) to fungicides.

Ascochyta blight is a disease that causes significant yield losses in chickpea crops in Turkey under favorable environmental conditions. The fungal pathogen Ascochyta rabiei is the causative agent of this disease. The antifungal activity of previous fungicides against A. rabiei was not effective due to the heterothallic nature of the fungus. The aim of this study was to determine the sensitivity of A. rabiei to fungicides (25.2 g kg-1 boscalid + 12.8 g kg-1 pyraclostrobin; 50 % tebuconazole + 25 % trifloxystrobin; 62.5 g L-1 propiconazole + 37.5 g L-1 azoxystrobin; 80 % thiram; 80 % kükürt (sulphur); 80 % mancozeb; 80 % maneb) under in vitro and field conditions. Pure cultures of A. rabiei were isolated from infected chickpea plants collected in Bogazlayan, Sarikaya, Sorgun, Merkez and Yerköy. A total of 14 A. rabiei isolates and 4 references were evaluated. The field test was conducted at Yozgat Bozok University, Yerköy Agricultural Application and Research Center Station. The trials began on March 14, 2021. The experimental area was divided into plots and the susceptible chickpea variety Sari98 was used for the study. Two artificial inoculations were carried out approximately on the 40th and 80th days after sowing. Twenty-four hours after inoculation, the chickpea plants were sprayed with the fungicides Nativo® WG 75, Bellis®, Dikotan® M45 and Thiovit Jet® using a handheld sprayer. In vitro testing revealed that A. rabiei was resistant to kükürt (sulphur), thiram, maneb, and mancozeb. A field study showed that the percentage of A. rabiei isolates treated with the mancozeb fungicide was between 14 and 21 % of the control. Therefore, effective disease management strategies should include not only the use of fungicides, but also alternative approaches such as the use of resistant varieties. Moreover, the study focused on phenotypic resistance and suggests that future research should investigate the genetic and molecular mechanisms underlying A. rabiei resistance to enable better resistance management.

Metabolite profiling of chickpea (Cicer arietinum) in response to necrotrophic fungus Ascochyta rabiei.

Introduction: Ascochyta blight (AB) caused by the necrotrophic fungus Ascochyta rabiei is one of the most significant diseases that limit the production of chickpea. Understanding the metabolic mechanisms underlying chickpea-A.rabiei interactions will provide important clues to develop novel approaches to manage this disease. Methods: We performed metabolite profiling of the aerial tissue (leaf and stem) of two chickpea accessions comprising a moderately resistant breeding line (CICA1841) and a highly susceptible cultivar (Kyabra) in response to one of the highly aggressive Australian A. rabiei isolates TR9571 via non-targeted metabolomics analysis using liquid chromatography-mass spectrometry. Results: The results revealed resistance and susceptibility-associated constitutive metabolites for example the moderately resistant breeding line had a higher mass abundance of ferulic acid while the levels of catechins, phthalic acid, and nicotinic acid were high in the susceptible cultivar. Further, the host-pathogen interaction resulted in the altered levels of various metabolites (induced and suppressed), especially in the susceptible cultivar revealing a possible reason for susceptibility against A.r abiei. Noticeably, the mass abundance of salicylic acid was induced in the aerial tissue of the susceptible cultivar after fungus colonization, while methyl jasmonate (MeJA) was suppressed, elucidating the key role of phytohormones in chickpea-A. rabiei interaction. Many differential metabolites in flavonoid biosynthesis, phenylalanine, Aminoacyl-tRNA biosynthesis, pentose and glucuronate interconversions, arginine biosynthesis, valine, leucine, and isoleucine biosynthesis, and alanine, aspartate, and glutamate metabolism pathways were up- and down-regulated showing the involvement of these metabolic pathways in chickpea-A. rabiei interaction. Discussion: Taken together, this study highlights the chickpea - A. rabiei interaction at a metabolite level and shows how A. rabiei differentially alters the metabolite profile of moderately resistant and susceptible chickpea accessions and is probably exploiting the chickpea defense pathways in its favour.

Transcriptome analysis of resistant and susceptible Medicago truncatula genotypes in response to spring black stem and leaf spot disease.

Ascochyta blights cause yield losses in all major legume crops. Spring black stem (SBS) and leaf spot disease is a major foliar disease of Medicago truncatula and Medicago sativa (alfalfa) caused by the necrotrophic fungus Ascochyta medicaginicola. This present study sought to identify candidate genes for SBS disease resistance for future functional validation. We employed RNA-seq to profile the transcriptomes of a resistant (HM078) and susceptible (A17) genotype of M. truncatula at 24, 48, and 72 h post inoculation. Preliminary microscopic examination showed reduced pathogen growth on the resistant genotype. In total, 192 and 2,908 differentially expressed genes (DEGs) were observed in the resistant and susceptible genotype, respectively. Functional enrichment analysis revealed the susceptible genotype engaged in processes in the cell periphery and plasma membrane, as well as flavonoid biosynthesis whereas the resistant genotype utilized calcium ion binding, cell wall modifications, and external encapsulating structures. Candidate genes for disease resistance were selected based on the following criteria; among the top ten upregulated or downregulated genes in the resistant genotype, upregulated over time in the resistant genotype, hormone pathway genes, plant disease resistance genes, receptor-like kinases, contrasting expression profiles in QTL for disease resistance, and upregulated genes in enriched pathways. Overall, 22 candidate genes for SBS disease resistance were identified with support from the literature. These genes will be sources for future targeted mutagenesis and candidate gene validation potentially helping to improve disease resistance to this devastating foliar pathogen.

Comparison of different screening methods for the selection of Ascochyta blight disease on chickpea (Cicer arietinum L.) genotypes.

Chickpea (Cicer arietinum L.) is the second most important edible food grain legume, widely grown all over the world. However, the cultivation and production of chickpea are mainly affected by the Ascochyta blight (AB) disease, which causes losses of up to 100% in areas with high humidity and warm temperature conditions. Various screening methods are used in the selection of chickpea genotypes for resistance to AB disease. These methods are natural field condition (NFC), artificial epidemic field condition (AEC), marker-assisted selection (MAS), and real-time PCR (RT-PCR). The study was conducted with 88 chickpea test genotypes between the 2014 and 2016 growing seasons. The results of the screening were used to sort the genotypes into three categories: susceptible (S), moderately resistant (MR), and resistant (R). Using MAS screening, 13, 21, and 54 chickpea genotypes were identified as S, MR, and R, respectively. For RT-PCR screening, 39 genotypes were S, 31 genotypes were MR, and 18 genotypes were R. In the AEC method for NFC screening, 7, 17, and 64 genotypes were S, MR, and R, while 74 and 6 genotypes were S and MR, and 8 genotypes were R-AB disease. As a result of screening chickpea genotypes for AB disease, it was determined that the most effective method was artificial inoculation (AEC) under field conditions. In the study, Azkan, ICC3996, Tüb-19, and Tüb-82 were determined as resistant within all methods for Pathotype 1.

Identification of Pathogen-Specific Novel Sources of Genetic Resistance Against Ascochyta Blight and Identification of Their Underlying Genetic Control.

Global chickpea production is restricted by Ascochyta blight caused by the necrotrophic fungi Ascochyta rabiei. Developing locally adapted disease-resistant cultivars is an economically and environmentally sustainable approach to combat this disease. However, the lack of genetic variability in cultivated chickpeas and breeder-friendly markers poses a significant challenge to Ascochyta blight-resistant breeding efforts in chickpeas. In this study, we screened the mini-core germplasm of Cicer reticulatum against a local pathotype of A. rabiei. A modified mini-dome screening approach resulted in the identification of five accessions showing a high level of resistance. The mean disease score of resistant accessions ranged between 1.75 ± 0.3 and 2.88 ± 0.4 compared to susceptible accessions, where the mean disease score ranged between 3.59 ± 0.62 and 8.86 ± 0.14. Genome-wide association study revealed a strong association on chromosome 5, explaining ∼58% of the phenotypic variance. The underlying region contained two candidate genes (Cr_14190.1_v2 and Cr_14189.1_v2), the characterization of which showed the presence of a DNA-binding domain (cl28899 and cd18793) in Cr_14190.1_v2 and its orthologs in C. arietinum, whereas Cr_14190.1_v2 carried an additional N-terminal domain (cl31759). qPCR expression analysis in resistant and susceptible accessions revealed ∼3- and ∼110-fold higher transcript abundance for Cr_14189.1 and Cr_14190.1, respectively.

Genetic Analysis of Partially Resistant and Susceptible Chickpea Cultivars in Response to Ascochyta rabiei Infection.

The molecular mechanism involved in chickpea (Cicer arietinum L.) resistance to the necrotrophic fungal pathogen Ascochyta rabiei is not well documented. A. rabiei infection can cause severe damage in chickpea, resulting in significant economic losses. Understanding the resistance mechanism against ascochyta blight can help to define strategies to develop resistant cultivars. In this study, differentially expressed genes from two partially resistant cultivars (CDC Corinne and CDC Luna) and a susceptible cultivar (ICCV 96029) to ascochyta blight were identified in the early stages (24, 48 and 72 h) of A. rabiei infection using RNA-seq. Altogether, 3073 genes were differentially expressed in response to A. rabiei infection across different time points and cultivars. A larger number of differentially expressed genes (DEGs) were found in CDC Corinne and CDC Luna than in ICCV 96029. Various transcription factors including ERF, WRKY, bHLH and MYB were differentially expressed in response to A. rabiei infection. Genes involved in pathogen detection and immune signalings such as receptor-like kinases (RLKs), Leucine-Rich Repeat (LRR)-RLKs, and genes associated with the post-infection defence response were differentially expressed among the cultivars. GO functional enrichment and pathway analysis of the DEGs suggested that the biological processes such as metabolic process, response to stimulus and catalytic activity were overrepresented in both resistant and susceptible chickpea cultivars. The expression patterns of eight randomly selected genes revealed by RNA-seq were confirmed by quantitative PCR (qPCR) analysis. The results provide insights into the complex molecular mechanism of the chickpea defence in response to the A. rabiei infection.

Comparative Analysis of Secondary Metabolites Produced by Ascochyta fabae under In Vitro Conditions and Their Phytotoxicity on the Primary Host, Vicia faba, and Related Legume Crops.

Ascochyta blight, caused by Ascochyta fabae, poses a significant threat to faba bean and other legumes worldwide. Necrotic lesions on stems, leaves, and pods characterize the disease. Given the economic impact of this pathogen and the potential involvement of secondary metabolites in symptom development, a study was conducted to investigate the fungus's ability to produce bioactive metabolites that might contribute to its pathogenicity. For this investigation, the fungus was cultured in three substrates (Czapek-Dox, PDB, and rice). The produced metabolites were analyzed by NMR and LC-HRMS methods, resulting in the dereplication of seven metabolites, which varied with the cultural substrates. Ascochlorin, ascofuranol, and (R)-mevalonolactone were isolated from the Czapek-Dox extract; ascosalipyrone, benzoic acid, and tyrosol from the PDB extract; and ascosalitoxin and ascosalipyrone from the rice extract. The phytotoxicity of the pure metabolites was assessed at different concentrations on their primary hosts and related legumes. The fungal exudates displayed varying degrees of phytotoxicity, with the Czapek-Dox medium's exudate exhibiting the highest activity across almost all legumes tested. The species belonging to the genus Vicia spp. were the most susceptible, with faba bean being susceptible to all metabolites, at least at the highest concentration tested, as expected. In particular, ascosalitoxin and benzoic acid were the most phytotoxic in the tested condition and, as a consequence, expected to play an important role on necrosis's appearance.

Genomic data of two chickpea populations sharing a potential Ascochyta blight resistance region.

Chickpea (Cicer arietinum L.) is one of the most important crops worldwide and a valuable nutritional source. The availability of data from different genotypes and populations is important for the comprehension of the biology and trait control of chickpea. Tissue from young leaves was collected from adult plants and sequenced using an Illumina HiSeq X platform, which provided sequencing data for a total of 169 individuals from two different populations. Furthermore, functional annotation was performed with BLAST2GO software in a candidate region for resistance to Ascochyta blight, a devastating disease that produces huge yield reductions if the growth conditions are optimal for the fungus. A total of 273 different genes in a region spanning ∼4.67 Mb in chromosome 4 were fully annotated. The raw DNA sequences and functional annotation data can be reused by the scientific community for the analysis of different agronomic traits of interest in chickpea.

Didymella fabae Punith.: mating type occurrence, distribution and phenotyping of the anamorph Ascochyta fabae Speg. in Tunisia.

Faba bean ascochyta blight, caused by Ascochyta fabae Speg. (teleomorph: Didymella fabae Punith.), is one of the most devastating diseases of the crop. It can cause yield losses that reach 95% in conducive weather conditions. Surveys were carried out in five regions of Tunisia: Beja, Bizerte, Jendouba, Kef and Tunis-Cap Bon. A total of 513 fungal isolates were collected from 2011 to 2013. A molecular characterization was conducted to identify the mating type of each individual using a mating type specific PCR. Results revealed that the two mating types MAT1-2 and MAT1-1 coexisted in all surveyed regions. An imbalance in favor of MAT1-2 was observed particularly in Bizerte and Jendouba regions (sex ratio was 18:85 and 32:80, respectively). Moreover, morphological and pathogenic characterization of 122 isolates among the collection revealed a significant variability in conidia type (one celled or two celled conidia) frequency, in conidia mean size and in aggressiveness toward Badii faba bean cultivar (incubation period, IP; percentage necrotic leaf area, S; and area under disease progression curve, AUDPC). A principal component analysis (PCA) performed on morphologically studied parameters (frequency of conidia cell number and conidia mean size) identified three groups of isolates based on morphological traits: one celled (1C) and two celled (2C) conidia rates, one celled and two celled conidia length and width (1L, 1W, 2L and 2W, respectively). A second PCA using aggressiveness parameters (IP: Incubation period, S1, S4 and S9: percentage of necrotic leaf area respectively 5, 20 and 45 days after inoculation) identified three distinct pathogenic groups: poorly pathogenic AG1, moderately pathogenic AG2 and highly pathogenic AG3. Morphological and pathogenic groups and mating type data were used to conduct a multiple factorial correspondence analysis (MFCA) which revealed a correlation between the variables studied. Five groups were identified, each associated with a morphological and pathogenic trait and mating type. The most pathogenic group belonged to MAT1-2 suggesting that in locations where MAT1-2 is prevalent the epidemic risk is more important.

Ascochyta blight in North Dakota field pea: the pathogen complex and its fungicide sensitivity.

Worldwide, Ascochyta blight is caused by a complex of host-specific fungal pathogens, including Ascochyta pisi, Didymella pinodes, and Didymella pinodella. The application of foliar fungicides is often necessary for disease management, but a better understanding of pathogen prevalence, aggressiveness, and fungicide sensitivity is needed to optimize control. Leaf and stem samples were obtained from 56 field pea production fields in 14 counties in North Dakota from 2017 to 2020 and isolates were collected from lesions characteristic of Ascochyta blight. Based on fungal characteristics and sequencing the ITS1-5.8S-ITS2 region, 73% of isolates were confirmed to be D. pinodes (n = 177) and 27% were A. pisi (n = 65). Across pathogens, aggressiveness was similar among some isolates in greenhouse assays. The in vitro pyraclostrobin sensitivity of all D. pinodes isolates collected from 2017 to 2020 was lower than that of the three baseline isolates. Sensitivity of 91% of A. pisi isolates collected in 2019 and 2020 was lower than the sensitivity of two known sensitive isolates. Resistance factors (Rf) from mean EC50 values of pyraclostrobin baseline/known sensitive isolates to isolates collected from 2017 to 2020 ranged from 2 to 1,429 for D. pinodes and 1 to 209 for A. pisi. In vitro prothioconazole sensitivity of 91% of D. pinodes isolates collected from 2017 to 2020 was lower than the sensitivity of the baseline isolates and 98% of A. pisi isolates collected from 2019 to 2020 was lower than the sensitivity of the known sensitive isolates. Prothioconazole Rf ranged from 1 to 338 for D. pinodes and 1 to 127 for A. pisi. Based on in vitro results, 92% of D. pinodes and 98% of A. pisi isolates collected displayed reduced-sensitivity/resistance to both fungicides when compared to baseline/known sensitive isolates. Disease control under greenhouse conditions of both pathogens provided by both fungicides was significantly lower in isolates determined to be reduced-sensitive or resistant in in vitro assays when compared to sensitive. Results reported here reinforce growers desperate need of alternative fungicides and/or management tools to fight Ascochyta blight in North Dakota and neighboring regions.

Genome wide association study of genes controlling resistance to Didymella rabiei Pathotype IV through genotyping by sequencing in chickpeas (Cicer arietinum).

Ascochyta blight (AB) is a major disease in chickpeas (Cicer arietinum L.) that can cause a yield loss of up to 100%. Chickpea germplasm collections at the center of origin offer great potential to discover novel sources of resistance to pests and diseases. Herein, 189 Cicer arietinum samples were genotyped via genotyping by sequencing. This chickpea collection was phenotyped for resistance to an aggressive Turkish Didymella rabiei Pathotype IV isolate. Genome-wide association studies based on different models revealed 19 single nucleotide polymorphism (SNP) associations on chromosomes 1, 2, 3, 4, 7, and 8. Although eight of these SNPs have been previously reported, to the best of our knowledge, the remaining ten were associated with AB resistance for the first time. The regions identified in this study can be addressed in future studies to reveal the genetic mechanism underlying AB resistance and can also be utilized in chickpea breeding programs to improve AB resistance in new chickpea varieties.

First report of Ascochyta rabiei infections on endemic Turkish populations of Cicer bijugum and C. turcicum.

Ascochyta rabiei causes Ascochyta blight disease on C. arietinum as well as on annual C. reticulatum, C. pinnatifidum and C. judaicum and perennial C. montbretii, C. isauricum, C. ervoides species (Can et al. 2007; Frenkel et al. 2007; Ozkilinc et al. 2019; Peever et al. 2007; Tekin et al. 2018). During field survey studies carried out on annual Cicer spp. in June 2022, concentric ring-shaped lesions were observed on the stems and leaves of C. bijugum in Mardin province and C. turcicum in Elazig province. Cicer reticulatum and C. arietinum plants were also found in the location where C. bijugum was found. No disease symptoms were observed in other Cicer species, while C. bijugum had 32% disease incidence. The disease incidence among the C. turcicum population was 37.3 %, and no chickpea cultivation area was found near it. Diseased plant parts were surface sterilized, placed on ½ potato dextrose agar (PDA) and incubated at 24±2 oC in 12 hours light/dark photoperiod. Each symptomatic plant was considered as one isolate. Monosporic isolates were obtained and the same colony morphology developed from all plant parts of C. turcicum and C. bijugum. Spores were oblong and spore sizes were 10.73±0.62 µm (n=15) in length and 3.60±0.25 (n=15) µm in width, 10.64±0.98 (n=15) µm in length and 3.00±0.26 (n=15) µm in width for isolates obtained from C. turcicum and C. bijugum, respectively. Amplicons for all 40 isolates were generated with mating type (MAT) primers, and the ITS region was amplified and sequenced by using the ITS4 and ITS5 primers (Peever et al. 2007). For the MAT primers, a 700 bp amplicon was observed for all the 20 isolates obtained from C. bijugum conferring to MAT1.1 idiomorph. In contrast, for the isolates obtained from C. turcicum 14 isolates had a 700 bp amplicon for MAT1.1 and 6 isolates had a 500 bp amplicon for MAT1.2, thus representing both idiomorphs in a ~2:1 ratio. BLAST analysis of the ITS sequences showed 100% homology with the reference ITS sequences for A. rabiei except for 23 SVRC CT 09/22 and 23 SVRC CT 22/22 isolates showing 99.81 % similarity. All sequences were submitted to GenBank (OP967923, OP967924, OP967925, OP967926 and OP967927 for A. rabiei isolates from C. turcicum; OP981072, OP981073 and OP981074 for A. rabiei isolates from C. bijugum). A phylogenetic tree was constructed using MEGAX software and the Neighbor-Joining method, using the ITS sequences of A. rabiei, other Ascochyta spp. and Colletotrichum gloeosporioides. The A. rabiei isolates from C. turcicum and C. bijugum clustered together with A. rabiei sequences from the NCBI (Kumar et al. 2018). Twelve-day-old C. bijugum and C. turcicum seedlings were inoculated with 5 x 105 spore/mL concentration of spores from 5 C. turcicum and 3 C. bijugum isolates and put in plastic bags for 24 hours (Can et al. 2007). Pathogenicity tests were carried out in triplicate pots with four plants each for each isolate in a controlled climate chamber at 24±2 oC, 70% humidity under 12 hours light/dark conditions. The first symptoms were observed within 7 days after inoculation (dai) and severe Ascochyta blight symptoms developed on all plants by 21 dai. Cicer bijugum and C. turcicum are endemic Cicer species exhibiting narrow distribution in the Southeastern region of Republic of Türkiye. As a major biotic stress source, A. rabiei could be an important threat to Cicer spp (Abbo et al. 2003). To our knowledge, this is the first report of A. rabiei from C. bijugum and C. turcicum species.

Genomics-assisted genetics of complex regions from chickpea chromosome 4 reveals two candidate genes for Ascochyta blight resistance.

Ascochyta blight (AB) disease caused by the fungus Ascochyta rabiei is a major threat to global chickpea production. Molecular breeding for improved AB resistance requires the identification of robust fine-mapped QTLs/candidate genes and associated markers. Earlier, we identified three QTLs (qABR4.1, qABR4.2, and qABR4.3) for AB resistance on chickpea chromosome 4 by employing multiple quantitative trait loci sequencing strategy on an intra-specific (FLIP84-92C x PI359075) and an inter-specific (FLIP84-92C x PI599072) crosses derived recombinant inbred lines. Here, we report the identification of AB resistance providing candidate genes under the fine mapped qABR4.2 and qABR4.3 genomic region by combining genetic mapping, haplotype block inheritance, and expression analysis. The qABR4.2 region was narrowed down from 5.94 Mb to ∼800 kb. Among 34 predicted gene models, a secreted class III peroxidase encoding gene showed higher expression in AB-resistant parent after A. rabiei conidia inoculation. Under qABR4.3, we identified a frame-shift mutation in a cyclic nucleotide-gated channel CaCNGC1 gene leading to the truncated N-terminal domain in resistant accession of chickpea. The extended N-terminal domain of CaCNGC1 interacts with chickpea calmodulin. Thus, our analysis has revealed narrowed genomic regions and their associated polymorphic markers, namely CaNIP43 and CaCNGCPD1. These co-dominant markers significantly associate with AB resistance on qABR4.2 and qABR4.3 regions. Our genetic analysis revealed that the presence of AB-resistant alleles at two major QTLs (qABR4.1 and qABR4.2) together provide AB resistance in the field while minor QTL qABR4.3 determines the degree of resistance. The identified candidate genes and their diagnostic markers will assist in the biotechnological advancement and introgression of AB resistance into locally adapted chickpea varieties used by farmers.

Five Regions of the Pea Genome Co-Control Partial Resistance to D. pinodes, Tolerance to Frost, and Some Architectural or Phenological Traits.

Evidence for reciprocal links between plant responses to biotic or abiotic stresses and architectural and developmental traits has been raised using approaches based on epidemiology, physiology, or genetics. Winter pea has been selected for years for many agronomic traits contributing to yield, taking into account architectural or phenological traits such as height or flowering date. It remains nevertheless particularly susceptible to biotic and abiotic stresses, among which Didymella pinodes and frost are leading examples. The purpose of this study was to identify and resize QTL localizations that control partial resistance to D. pinodes, tolerance to frost, and architectural or phenological traits on pea dense genetic maps, considering how QTL colocalizations may impact future winter pea breeding. QTL analysis revealed five metaQTLs distributed over three linkage groups contributing to both D. pinodes disease severity and frost tolerance. At these loci, the haplotypes of alleles increasing both partial resistance to D. pinodes and frost tolerance also delayed the flowering date, increased the number of branches, and/or decreased the stipule length. These results question both the underlying mechanisms of the joint control of biotic stress resistance, abiotic stress tolerance, and plant architecture and phenology and the methods of marker-assisted selection optimizing stress control and productivity in winter pea breeding.

Stability and suitability of genotypes and environment to Ascochyta blight of chickpea.

Ascochyta blight (AB) is a major biotic constraint to chickpea production internationally. The disease caused by the phytopathogenic fungus Ascochyta rabiei is highly favored by prolonged spells of low temperature and high humidity. The disease scenario is expected to aggravate in the near future as a result of rapidly changing climatic conditions and the emergence of fungicide-resistant pathogen strains. Tapping into host-plant resistance is the most logical way to preempt such a crisis. Presently, high levels of stable resistance against AB are yet to be identified from the chickpea gene pool. The present study was aimed at facilitating this process through multi-environment testing of chickpea genotypes. Using the GGE biplot analysis method, we could identify three genotypes, viz., ICCV 16508, ICCV 16513, and ICCV 16516, from the International Ascochyta Blight Nursery, which showed consistent moderate resistance reactions across all the tested environments. Moreover, we were able to evaluate the test locations for their suitability to support AB screening trials. Ludhiana and Palampur locations were identified as the most ideal for continual screening in the future. Controlled environment screening at the ICRISAT location offered to reduce large plant populations to small meaningful sizes through initial screening under controlled environment conditions. This study will further improve the scope of phenotyping and sources of stable resistance to be utilized in future AB resistance breeding programs.

Surfactin and Spo0A-Dependent Antagonism by Bacillus subtilis Strain UD1022 against Medicago sativa Phytopathogens.

Plant growth-promoting rhizobacteria (PGPR) such as the root colonizers Bacillus spp. may be ideal alternatives to chemical crop treatments. This work sought to extend the application of the broadly active PGPR UD1022 to Medicago sativa (alfalfa). Alfalfa is susceptible to many phytopathogens resulting in losses of crop yield and nutrient value. UD1022 was cocultured with four alfalfa pathogen strains to test antagonism. We found UD1022 to be directly antagonistic toward Collectotrichum trifolii, Ascochyta medicaginicola (formerly Phoma medicaginis), and Phytophthora medicaginis, and not toward Fusarium oxysporum f. sp. medicaginis. Using mutant UD1022 strains lacking genes in the nonribosomal peptide (NRP) and biofilm pathways, we tested antagonism against A. medicaginicola StC 306-5 and P. medicaginis A2A1. The NRP surfactin may have a role in the antagonism toward the ascomycete StC 306-5. Antagonism toward A2A1 may be influenced by B. subtilis biofilm pathway components. The B. subtilis central regulator of both surfactin and biofilm pathways Spo0A was required for the antagonism of both phytopathogens. The results of this study indicate that the PGPR UD1022 would be a good candidate for further investigations into its antagonistic activities against C. trifolii, A. medicaginicola, and P. medicaginis in plant and field studies.

Accuracy of Selection in Early Generations of Field Pea Breeding Increases by Exploiting the Information Contained in Correlated Traits.

Accuracy of predicted breeding values (PBV) for low heritability traits may be increased in early generations by exploiting the information available in correlated traits. We compared the accuracy of PBV for 10 correlated traits with low to medium narrow-sense heritability (h2) in a genetically diverse field pea (Pisum sativum L.) population after univariate or multivariate linear mixed model (MLMM) analysis with pedigree information. In the contra-season, we crossed and selfed S1 parent plants, and in the main season we evaluated spaced plants of S0 cross progeny and S2+ (S2 or higher) self progeny of parent plants for the 10 traits. Stem strength traits included stem buckling (SB) (h2 = 0.05), compressed stem thickness (CST) (h2 = 0.12), internode length (IL) (h2 = 0.61) and angle of the main stem above horizontal at first flower (EAngle) (h2 = 0.46). Significant genetic correlations of the additive effects occurred between SB and CST (0.61), IL and EAngle (-0.90) and IL and CST (-0.36). The average accuracy of PBVs in S0 progeny increased from 0.799 to 0.841 and in S2+ progeny increased from 0.835 to 0.875 in univariate vs MLMM, respectively. An optimized mating design was constructed with optimal contribution selection based on an index of PBV for the 10 traits, and predicted genetic gain in the next cycle ranged from 1.4% (SB), 5.0% (CST), 10.5% (EAngle) and -10.5% (IL), with low achieved parental coancestry of 0.12. MLMM improved the potential genetic gain in annual cycles of early generation selection in field pea by increasing the accuracy of PBV.

Virulence Profiles and Genome-Wide Association Study for Ascochyta lentis Isolates Collected from Australian Lentil-Growing Regions.

Ascochyta lentis, the causal organism of Ascochyta blight (AB) of lentil (Lens culinaris), has been shown to produce an avirulence effector protein that mediates AB resistance in certain lentil cultivars. The two known forms of the effector protein were identified from a biparental mapping population between isolates that have reciprocal virulence on 'PBA Hurricane XT' and 'Nipper'. The effector AlAvr1-1 was described for the PBA Hurricane XT-avirulent isolate P94-24 and AlAvr1-2 characterized in the PBA Hurricane XT-virulent isolate AlKewell. Here, we performed a genome-wide association study to identify other loci associated with AB for a differential set of lentil cultivars from a diverse panel of isolates collected in the Australian lentil-growing regions from 2013 to 2020. The chromosome 3 AlAvr1 locus was strongly associated with the PBA Hurricane XT, 'Indianhead', and Nipper disease responses, but one other genomic region on chromosome 11 was also associated with the Nipper disease trait. Our results corroborate earlier work that identified the AlAvr1 locus for field-collected isolates that span the period before release and after widespread adoption of PBA Hurricane XT. A multiplex PCR assay was developed to differentiate the genes AlAvr1-1 and AlAvr1-2 to predict PBA Hurricane XT avirulence and pathotype designation in the diversity panel. Increasing numbers of the PBA Hurricane XT-virulent pathotype 2 isolates across that time indicate strong selection for isolates with the AlAvr1-2 allele. Furthermore, one other region of the A. lentis genome may contribute to the pathogen-host interaction for lentil AB.

Optimized High Throughput Ascochyta Blight Screening Protocols and Immunity to A. pisi in Pea.

Ascochyta blight (AB) is a destructive disease of the field pea (Pisum sativum L.) caused by necrotrophic fungal pathogens known as the AB-disease complex. To identify resistant individuals to assist AB resistance breeding, low-cost, high throughput, and reliable protocols for AB screening are needed. We tested and optimized three protocols to determine the optimum type of pathogen inoculum, the optimal development stage for host inoculation, and the timing of inoculation for detached-leaf assays. We found that different plant development stages do not affect AB infection type on peas, but the timing of inoculation affects the infection type of detached leaves due to wound-induced host defense response. After screening nine pea cultivars, we discovered that cultivar Fallon was immune to A. pisi but not to A. pinodes or the mixture of the two species. Our findings suggest that AB screening can be done with any of the three protocols. A whole-plant inoculation assay is necessary for identifying resistance to stem/node infection. Pathogen inoculation must be completed within 1.5 h post-detachment to avoid false positives of resistance for detach-leaf assays. It is essential to use a purified single-species inoculum for resistant resource screenings to identify the host resistance to each single species.