Status of Phytotoxins Isolated from Necrotrophic Fungi Causing Diseases on Grain Legumes.

Fungal phytotoxins can be defined as secondary metabolites toxic to host plants and are believed to be involved in the symptoms developed of a number of plant diseases by targeting host cellular machineries or interfering with host immune responses. As any crop, legumes can be affected by a number of fungal diseases, causing severe yield losses worldwide. In this review, we report and discuss the isolation, chemical, and biological characterization of fungal phytotoxins produced by the most important necrotrophic fungi involved in legume diseases. Their possible role in plant-pathogen interaction and structure-toxicity relationship studies have also been reported and discussed. Moreover, multidisciplinary studies on other prominent biological activity conducted on reviewed phytotoxins are described. Finally, we explore the challenges in the identification of new fungal metabolites and their possible applications in future experiments.

Genetic Diversity and Population Structure of a Wide Pisum spp. Core Collection.

Peas (Pisum sativum) are the fourth most cultivated pulses worldwide and a critical source of protein in animal feed and human food. Developing pea core collections improves our understanding of pea evolution and may ease the exploitation of their genetic diversity in breeding programs. We carefully selected a highly diverse pea core collection of 325 accessions and established their genetic diversity and population structure. DArTSeq genotyping provided 35,790 polymorphic DArTseq markers, of which 24,279 were SilicoDArT and 11,511 SNP markers. More than 90% of these markers mapped onto the pea reference genome, with an average of 2787 SilicoDArT and 1644 SNP markers per chromosome, and an average LD50 distance of 0.48 and 1.38 Mbp, respectively. The pea core collection clustered in three or six subpopulations depending on the pea subspecies. Many admixed accessions were also detected, confirming the frequent genetic exchange between populations. Our results support the classification of Pisum genus into two species, P. fulvum and P. sativum (including subsp. sativum, arvense, elatius, humile, jomardii and abyssinicum). In addition, the study showed that wild alleles were incorporated into the cultivated pea through the intermediate P. sativum subsp. jomardii and P. sativum subsp. arvense during pea domestication, which have important implications for breeding programs. The high genetic diversity found in the collection and the high marker coverage are also expected to improve trait discovery and the efficient implementation of advanced breeding approaches.

BTH and BABA induce resistance in pea against rust (Uromyces pisi) involving differential phytoalexin accumulation.

MAIN CONCLUSION: Systemic acquired resistance elicitors, BTH and BABA, reduce rust penetration in pea through phytoalexins pathway but differing in their mode of action. It has been previously shown that rust (Uromyces pisi) infection can be reduced in pea (Pisum sativum) by exogenous applications of systemic acquired resistance elicitors such as BTH and BABA. This protection is known to be related with the induction of the phenolic pathway but the particular metabolites involved have not been determined yet. In this work, we tackled the changes induced in phytoalexin content by BTH and BABA treatments in the context of the resistance responses to pea rust. Detailed analysis through high-performance liquid chromatography (HPLC) showed qualitative and quantitative differences in the content, as well as in the distribution of phytoalexins. Thus, following BTH treatment, we observed an increase in scopoletin, pisatin and medicarpin contents in all, excreted, soluble and cell wall-bound fraction. This suggests fungal growth impairment by both direct toxic effect as well as plant cell wall reinforcement. The response mediated by BTH was genotype-dependent, since coumarin accumulation was observed only in the resistant genotype whereas treatment by BABA primed phytoalexin accumulation in both genotypes equally. Exogenous application to the leaves of scopoletin, medicarpin and pisatin lead to a reduction of the different fungal growth stages, confirming a role for these phytoalexins in BTH- and BABA-induced resistance against U. pisi hampering pre- and postpenetration fungal stages.

Optimization of inoculum production of Stemphylium botryosum for large-scale resistance screening of lentils.

BACKGROUND: Stemphylium blight incited by Stemphylium botryosum poses a significant threat to lentil crops worldwide, inducing severe defoliation and causing substantial yield losses in susceptible varieties under favorable conditions. While some moderate levels of resistance have been identified within lentil germplasm, a low number of resistant cultivars are available to farmers. Adding to the common constraints of resistance breeding, a notable challenge is generating a sufficient number of spores for large-scale screenings, which are essential for pinpointing additional sources of resistance for integration into breeding programs. Therefore, there is a pressing need to improve existing screening methods and tailor them for large-scale material selection. In pursuit of this objective, a protocol for the efficient production of fungal material has been adapted. RESULTS: Optimization of fungal material production was successfully achieved by comparing the use of fungal mycelia and spores. Spore production was found to be optimal when produced on solid V8-PDA(hi) medium, while liquid Richard's medium was identified as superior for mycelium yield. Furthermore, a refined screening method was developed by evaluating the resistance of six lentil accessions to stemphylium blight. This assessment included the use of either fungal mycelia (at densities ranging from 1 to 5 g L- 1) or spores (with densities ranging from 5 × 104 to 2 × 105 conidia mL- 1) under three different relative humidity levels (from 50 to 100%). Both humidity levels and inoculum dose significantly influenced the final disease rating (DR) and the relative Area Under the Disease Progress Curve (rAUDPC). Differences among genotypes in final symptom severity (DR) became more pronounced after inoculation with inoculum densities of 5 g L- 1 of mycelium or of 105 and 2 × 105 conidia mL- 1 of spore under 100% relative humidity. Given the challenges associated with the large-scale production of S. botryosum spores, inoculations with 5 g L- 1 of mycelium is highly recommended as a practical alternative for conducting mass-scale screenings. CONCLUSIONS: The findings from this study underscore the critical importance of maintaining high level of humidity during inoculation and disease progression development for accurately assessing resistance to stemphylium blight. The optimization of mycelial production for suspension inoculation emerges as a more reliable and efficient approach for conducting large-scale screening to assess germplasm resistance against stemphylium blight in lentil crops.

Identification and Characterization of Resistance to Rust in Lentil and Its Wild Relatives.

Lentil rust is a major disease worldwide caused by Uromyces viciae-fabae. In this study, we screened a large germplasm collection of cultivated lentils (Lens culinaris ssp. culinaris) and its wild relatives, both in adult plants in the field with a local rust isolate during 2 seasons and in seedlings under controlled conditions with four fungal isolates of worldwide origin. The main results from our study were the following: (1) a significant number of accessions with resistance based on hypersensitive reaction (reduced Infection Type (IT)) were identified in cultivated lentil and in L. ervoides, L. nigricans and L.c. orientalis. The IT scores showed a clear isolate-specific response suggesting race-specificity, so each fungal isolate might be considered a different race. Resistance was identified against all isolates what might be the basis to develop a standard differential set that should be a priority for rust definition and monitoring. (2) Interestingly, although at lower frequency than in L. ervoides and L. nigricans, the hypersensitive response was also observed within cultivated lentil, with accession 1561 (L.c. culinaris) displaying resistance to the four isolates making this accession a valuable ready-to-use resource for lentil resistance breeding. Resistance to all other rust isolates was also available within L.c. culinaris in an isolate-specific manner. Accession 1308 (L. ervoides) showed resistance against all isolates tested, as well as a reduced number of accessions belonging to other wild Lens species. (3) In addition, our screenings allowed the identification of several accessions with partial resistance (reduced Disease Severity (DS) despite high IT). Adult Plant Resistance resulting in reduced severity in adult plants in the field, despite high susceptibility in seedlings, was more frequently identified in L.c. culinaris, but also in L. nigricans and L.c. orientalis.

Uncovering Phytotoxic Compounds Produced by Colletotrichum spp. Involved in Legume Diseases Using an OSMAC-Metabolomics Approach.

Different fungal species belonging to the Colletotrichum genus cause anthracnose disease in a range of major crops, resulting in huge economic losses worldwide. Typical symptoms include dark, sunken lesions on leaves, stems, or fruits. Colletotrichum spp. have synthesized, in vitro, a number of biologically active and structurally unusual metabolites that are involved in their host's infection process. In this study, we applied a one strain many compounds (OSMAC) approach, integrated with targeted and non-targeted metabolomics profiling, to shed light on the secondary phytotoxic metabolite panels produced by pathogenic isolates of Colletotrichum truncatum and Colletotrichum trifolii. The phytotoxicity of the fungal crude extracts was also assessed on their primary hosts and related legumes, and the results correlated with the metabolite profile that arose from the different cultural conditions. To the best of our knowledge, this is the first time that the OSMAC strategy integrated with metabolomics approaches has been applied to Colletotrichum species involved in legume diseases.

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.

Identification and Characterization of Novel Sources of Resistance to Rust Caused by Uromyces pisi in Pisum spp.

Pea rust is a major disease worldwide caused by Uromyces pisi in temperate climates. Only moderate levels of partial resistance against U. pisi have been identified so far in pea, urging for enlarging the levels of resistance available for breeding. Herein, we describe the responses to U. pisi of 320 Pisum spp. accessions, including cultivated pea and wild relatives, both under field and controlled conditions. Large variations for U. pisi infection response for most traits were observed between pea accessions under both field and controlled conditions, allowing the detection of genotypes with partial resistance. Simultaneous multi-trait indexes were applied to the datasets allowing the identification of partial resistance, particularly in accessions JI224, BGE004710, JI198, JI199, CGN10205, and CGN10206. Macroscopic observations were complemented with histological observations on the nine most resistant accessions and compared with three intermediates and three susceptible ones. This study confirmed that the reduced infection of resistant accessions was associated with smaller rust colonies due to a reduction in the number of haustoria and hyphal tips per colony. Additionally, a late acting hypersensitive response was identified for the first time in a pea accession (PI273209). These findings demonstrate that screening pea collections continues to be a necessary method in the search for complete resistance against U. pisi. In addition, the large phenotypic diversity contained in the studied collection will be useful for further association analysis and breeding perspectives.

Anthraquinones and their analogues as potential biocontrol agents of rust and powdery mildew diseases of field crops.

BACKGROUND: Rusts and powdery mildews are severe fungal diseases of major crops worldwide, including cereals and legumes. They can be managed by chemical fungicide treatments, with negative consequences as environmental pollution and risk for human and animal health. Bioactive natural products could be the safest alternative for pest control. The family of anthraquinones, as well as analogue compounds containing an anthraquinone moiety or some modified anthraquinone rings, has been reported to exhibit certain antibiotic activity. Thus, the potential antifungal activity of some anthraquinones isolated from Ascochyta lentis, was assayed in this study for their effectiveness to reduce rust and powdery mildew diseases on pea and oat. Their effect on fungal development was macro- and microscopically assessed on inoculated leaves, and compared to the control achieved by the chemical fungicide (Tetraconazol 12.5% and Azoxystrobin 25%). In addition, the most promising compound was also tested at different concentrations in inoculated whole plants in order to evaluate its preventive and curative potential against fungal infection. RESULTS: All metabolites studied strongly reduced the development of rust and powdery mildews in both pea and oat, being pachybasin and lentiquinone C the most effective ones in hampering fungal spore germination and appressoria formation. Some of them also affected post-penetration events reducing colony size and number of haustoria per colony. Results were confirmed for pachybasin in whole plants assays, showing an efficacy similar to the commercial fungicide to control fungal diseases, both in preventive and curative applications. CONCLUSIONS: Some fungal anthraquinones and close metabolites, especially pachybasin, could be very promising molecules with effective potential as antifungal agents against both rust and powdery mildew of both pea and oat. Some structure activity-relationships feature have also been evaluated. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Identification of potential candidate genes controlling pea aphid tolerance in a Pisum fulvum high-density integrated DArTseq SNP-based genetic map.

BACKGROUND: Pea (Pisum sativum) is one of the most important temperate grain legumes in the world, and its production is severely constrained by the pea aphid (Acyrthosiphon pisum). Wild relatives, such as P. fulvum, are valuable sources of allelic diversity to improve the genetic resistance of cultivated pea species against A. pisum attack. To unravel the genetic control underlying resistance to the pea aphid attack, a quantitative trait loci (QTL) analysis was performed using the previously developed high density integrated genetic linkage map originated from an intraspecific recombinant inbred line (RIL) population (P. fulvum: IFPI3260 × IFPI3251). RESULTS: We accurately evaluated specific resistance responses to pea aphid that allowed the identification, for the first time, of genomic regions that control plant damage and aphid reproduction. Eight QTLs associated with tolerance to pea aphid were identified in LGs I, II, III, IV and V, which individually explained from 17.0% to 51.2% of the phenotypic variation depending on the trait scored, and as a whole from 17.0% to 88.6%. The high density integrated genetic linkage map also allowed the identification of potential candidate genes co-located with the QTLs identified. CONCLUSIONS: Our work shows how the survival of P. fulvum after the pea aphid attack depends on the triggering of a multi-component protection strategy that implies a quantitative tolerance. The genomic regions associated with the tolerance responses of P. fulvum during A. pisum infestation have provided six potential candidate genes that could be useful in marker-assisted selection (MAS) and genomic assisted breeding (GAB) after functional validation in the future. © 2019 Society of Chemical Industry.

Identification of quantitative trait loci (QTL) controlling resistance to pea weevil (Bruchus pisorum) in a high-density integrated DArTseq SNP-based genetic map of pea.

Pea weevil (Bruchus pisorum) is a damaging insect pest affecting pea (Pisum sativum) production worldwide. No resistant cultivars are available, although some levels of incomplete resistance have been identified in Pisum germplasm. To decipher the genetic control underlying the resistance previously identify in P. sativum ssp. syriacum, a recombinant inbred line (RIL F8:9) population was developed. The RIL was genotyped through Diversity Arrays Technology PL's DArTseq platform and screened under field conditions for weevil seed infestation and larval development along 5 environments. A newly integrated genetic linkage map was generated with a subset of 6,540 markers, assembled into seven linkage groups, equivalent to the number of haploid pea chromosomes. An accumulated distance of 2,503 cM was covered with an average density of 2.61 markers cM-1. The linkage map allowed the identification of three QTLs associated to reduced seed infestation along LGs I, II and IV. In addition, a QTL for reduced larval development was also identified in LGIV. Expression of these QTLs varied with the environment, being particularly interesting QTL BpSI.III that was detected in most of the environments studied. This high-saturated pea genetic map has also allowed the identification of seven potential candidate genes co-located with QTLs for marker-assisted selection, providing an opportunity for breeders to generate effective and sustainable strategies for weevil control.

Impact of fungal and plant metabolites application on early development stages of pea powdery mildew.

BACKGROUND: Pea powdery mildew incited by Erysiphe pisi represents a major constraint for pea crops worldwide. Crop protection is largely based on chemical control, although recently a renewed interest in the discovery of natural products as alternatives to synthetic fungicides application has emerged. Thus, 12 bioactive plant and fungal metabolites belonging to different class of natural compounds were evaluated, together with a commercial fungicide, at different concentrations on detached pea leaves for their potential to inhibit spore germination and subsequent stages of fungal growth. The most effective metabolites were tested at different concentrations in planta under controlled conditions to evaluate the level of control achieved by treatments before, concurrently and after pathogen inoculation. Pathogen development was macroscopically scored on whole plants as percentage of disease severity and area under the disease progress curve. RESULTS: Cavoxin, inuloxin C and sphaeropsidin A strongly inhibited E. pisi germination and haustoria formation and reduced colony size. This effect was dose dependent. These results were further confirmed in whole plants by spraying the metabolites on plant leaves for preventive or curative control, which reduced fungal developmental of E. pisi at levels comparable with those obtained by application of the fungicide. CONCLUSIONS: Cavoxin, inuloxin C and sphaeropsidin A have potential as alternatives to synthetic fungicides for the control of crop pathogens of economic importance such as powdery mildew. © 2019 Society of Chemical Industry.

A High-Density Integrated DArTseq SNP-Based Genetic Map of Pisum fulvum and Identification of QTLs Controlling Rust Resistance.

Pisum fulvum, a wild relative of pea is an important source of allelic diversity to improve the genetic resistance of cultivated species against fungal diseases of economic importance like the pea rust caused by Uromyces pisi. To unravel the genetic control underlying resistance to this fungal disease, a recombinant inbred line (RIL) population was generated from a cross between two P. fulvum accessions, IFPI3260 and IFPI3251, and genotyped using Diversity Arrays Technology. A total of 9,569 high-quality DArT-Seq and 8,514 SNPs markers were generated. Finally, a total of 12,058 markers were assembled into seven linkage groups, equivalent to the number of haploid chromosomes of P. fulvum and P. sativum. The newly constructed integrated genetic linkage map of P. fulvum covered an accumulated distance of 1,877.45 cM, an average density of 1.19 markers cM-1 and an average distance between adjacent markers of 1.85 cM. The composite interval mapping revealed three QTLs distributed over two linkage groups that were associated with the percentage of rust disease severity (DS%). QTLs UpDSII and UpDSIV were located in the LGs II and IV respectively and were consistently identified both in adult plants over 3 years at the field (Córdoba, Spain) and in seedling plants under controlled conditions. Whenever they were detected, their contribution to the total phenotypic variance varied between 19.8 and 29.2. A third QTL (UpDSIV.2) was also located in the LGIVand was environmentally specific as was only detected for DS % in seedlings under controlled conditions. It accounted more than 14% of the phenotypic variation studied. Taking together the data obtained in the study, it could be concluded that the expression of resistance to fungal diseases in P. fulvum originates from the resistant parent IFPI3260.

Inhibition of early development stages of rust fungi by the two fungal metabolites cyclopaldic acid and epi-epoformin.

BACKGROUND: Rusts are a noxious group of plant diseases affecting major economically important crops. Crop protection is largely based on chemical control. There is a renewed interest in the discovery of natural products as alternatives to synthetic fungicides for control. In this study we tested two fungal metabolites, namely cyclopaldic acid and epi-epoformin, for their effectiveness in reducing early stages of development of two major rust fungi from the genera Puccinia and Uromyces, P. triticina and U. pisi. Spore germination and appressorium formation were assessed on pretreated detached leaves under controlled conditions. Cyclopaldic acid and epi-epoformin were also tested in infected plants in order to evaluate the level of control achieved by treatments both before and after inoculation. RESULTS: Cyclopaldic acid and epi-epoformin were strongly effective in inhibiting fungal germination and penetration of both rust species studied. This effect was not dose dependent. These results were further confirmed in planta by spraying the metabolites on plant leaves, which reduced fungal developmental of U. pisi and P. triticina at values comparable with those obtained by application of the fungicide. CONCLUSION: Our results further demonstrate the potential of fungal metabolites as natural alternatives to synthetic fungicides for the control of crop pathogens of economic importance as rusts. © 2016 Society of Chemical Industry.

Clarification on Host Range of Didymella pinodes the Causal Agent of Pea Ascochyta Blight.

Didymella pinodes is the principal causal agent of ascochyta blight, one of the most important fungal diseases of pea (Pisum sativum) worldwide. Understanding its host specificity has crucial implications in epidemiology and management; however, this has not been clearly delineated yet. In this study we attempt to clarify the host range of D. pinodes and to compare it with that of other close Didymella spp. D. pinodes was very virulent on pea accessions, although differences in virulence were identified among isolates. On the contrary, studied isolates of D. fabae, D. rabiei, and D. lentil showed a reduced ability to infect pea not causing macroscopically visible symptoms on any of the pea accessions tested. D. pinodes isolates were also infective to some extend on almost all species tested including species such as Hedysarum coronarium, Lathyrus sativus, Lupinus albus, Medicago spp., Trifolium spp., Trigonella foenum-graecum, and Vicia articulata which were not mentioned before as hosts of D. pinodes. On the contrary, D. lentil and D. rabiei were more specific, infecting only lentil and chickpea, respectively. D. fabae was intermediate, infecting mainly faba bean, but also slightly other species such as Glycine max, Phaseolus vulgaris, Trifolium spp., Vicia sativa, and V. articulata. DNA sequence analysis of the nuclear ribosomal internal transcribed spacer region (ITS) was performed to confirm identity of the isolates studies and to determine phylogenetic relationship among the Didymella species, revealing the presence of two clearly distinct clades. Clade one was represented by two supported subclusters including D. fabae isolates as well as D. rabiei with D. lentil isolates. Clade two was the largest and included all the D. pinodes isolates as well as Phoma medicaginis var. pinodella. Genetic distance between D. pinodes and the other Didymella spp. isolates was not correlated with overall differences in pathogenicity. Based on evidences presented here, D. pinodes is not specialized on pea and its host range is larger than that of D. fabae, D. lentil, and D. rabiei. This has relevant implications in epidemiology and control as these species might act as alternative hosts for D. pinodes.

Inhibition of Spore Germination and Appressorium Formation of Rust Species by Plant and Fungal Metabolites.

Eight fungal and plant metabolites belonging to different classes of naturally occurring compounds, a 24-oxa[14]-cytochalasan as cytochalasin B (1), a trisubstituted isocoumarin as 6-hydroxymellein (2), a tetracyclic pimarane diterpene as sphaeropsidin A (3), a chalcone as cavoxin (4), a pentasubstituted benzofuranone as cyclopaldic acid (5), a bicyclic-sesquiterpene as inuloxin A (6), a epipolythiopiperazine as gliotoxin (7) and a cyclohexene epoxide as epiepoformin (8), were tested for their effectiveness in reducing early stages of development of several major rust fungi from the genera Puccinia and Uromyces. Spore germination and appressoria formation were assessed on pre-treated detached leaves, under controlled conditions. Among the various metabolites evaluated, compounds 5 and 8 were the most effective in inhibiting fungal germination and penetration of all rust species studied at values comparable with those obtained by fungicide application, while compound 4 was phytotoxic to plant leaves at any concentration tested.

Comparative proteomic analysis of BTH and BABA-induced resistance in pea (Pisum sativum) toward infection with pea rust (Uromyces pisi).

Systemic acquired resistance (SAR) to Uromyces pisi in pea was studied by using a proteomic approach. Two-dimensional electrophoresis (2-DE) was used in order to compare the leaf proteome of two pea genotypes displaying different phenotypes (susceptible and partial resistance to the fungus), and in response to parasite infection under the effect of two inducers of SAR, BTH and BABA. Multivariate statistical analysis identified 126 differential protein spots under the experimental conditions (genotypes/treatments). All of these 126 protein spots were subjected to MALDI-TOF/TOF mass spectrometry to deduce their possible functions. A total of 50 proteins were identified using a combination of peptide mass fingerprinting (PMF) and MSMS fragmentation. Most of the identified proteins corresponded to enzymes belonging to photosynthesis, metabolism, biosynthesis, binding and defense response, whose behavior pattern was different in relation to susceptibility/resistance of the genotypes studied and to the BTH/BABA induction to pathogen response. Results obtained in this work suggested that plants could reduce their photosynthesis and other energy metabolism and enhance the production of defense-related proteins to cope the stress. On the other side, we postulated that resistance induced by the chemicals operates via different mechanisms: BABA inducer could act via phenolic biosynthesis pathway, whereas resistance provided by BTH inducer seems to be mediated by defense and stress-related proteins. The results are discussed in terms of response to rust under the effect of inducers.