Integrated analysis of transcriptomics and defense-related phytohormones to discover hub genes conferring maize Gibberella ear rot caused by Fusarium Graminearum.

BACKGROUND: Gibberella ear rot (GER) is one of the most devastating diseases in maize growing areas, which directly reduces grain yield and quality. However, the underlying defense response of maize to pathogens infection is largely unknown. RESULTS: To gain a comprehensive understanding of the defense response in GER resistance, two contrasting inbred lines 'Nov-82' and 'H10' were used to explore transcriptomic profiles and defense-related phytohormonal alterations during Fusarium graminearum infection. Transcriptomic analysis revealed 4,417 and 4,313 differentially expressed genes (DEGs) from the Nov-82 and H10, respectively, and 647 common DEGs between the two lines. More DEGs were obviously enriched in phenylpropanoid biosynthesis, secondary metabolites biosynthesis, metabolic process and defense-related pathways. In addition, the concentration of the defense-related phytohormones, jasmonates (JAs) and salicylates (SAs), was greatly induced after the pathogen infection. The level of JAs in H10 was more higher than in Nov-82, whereas an opposite pattern for the SA between the both lines. Integrated analysis of the DEGs and the phytohormones revealed five vital modules based on co-expression network analysis according to their correlation. A total of 12 hub genes encoding fatty acid desaturase, subtilisin-like protease, ethylene-responsive transcription factor, 1-aminocyclopropane-1-carboxylate oxidase, and sugar transport protein were captured from the key modules, indicating that these genes might play unique roles in response to pathogen infection, CONCLUSIONS: Overall, our results indicate that large number DEGs related to plant disease resistance and different alteration of defensive phytohormones were activated during F. graminearum infection, providing new insight into the defense response against pathogen invasion, in addition to the identified hub genes that can be further investigated for enhancing maize GER resistance.

Genome-wide association study and molecular marker development for susceptibility to Gibberella ear rot in maize.

KEY MESSAGES: Sixty-nine quantitative trait nucleotides conferring maize resistance to Gibberella ear rot were detected, including eighteen novel loci. Four candidate genes were predicted, and four kompetitive allele-specific PCR markers were developed. Maize Gibberella ear rot (GER), caused by Fusarium graminearum, is one of the most devastating diseases in maize-growing regions worldwide. Enhancing maize cultivar resistance to this disease requires a comprehensive understanding of the genetic basis of resistance to GER. In this study, 334 maize inbred lines were phenotyped for GER resistance in five environments and genotyped using the Affymetrix CGMB56K SNP Array, and a genome-wide association study of resistance to GER was performed using a 3V multi-locus random-SNP-effect mixed linear model. A total of 69 quantitative trait nucleotides (QTNs) conferring resistance to GER were detected, and all of them explained individually less than 10% of the phenotypic variation, suggesting that resistance to GER is controlled by multiple minor-effect genetic loci. A total of 348 genes located around the 200-kb genomic region of these 69 QTNs were identified, and four of them (Zm00001d029648, Zm00001d031449, Zm00001d006397, and Zm00001d053145) were considered candidate genes conferring susceptibility to GER based on gene expression patterns. Moreover, four kompetitive allele-specific PCR markers were developed based on the non-synonymous variation of these four candidate genes and validated in two genetic populations. This study provides useful genetic resources for improving resistance to GER in maize.

Genome-Wide Association Study Discovers Novel Germplasm Resources and Genetic Loci with Resistance to Gibberella Ear Rot Caused by Fusarium graminearum.

Gibberella ear rot (GER) in maize caused by Fusarium graminearum is one of the most devastating maize diseases reducing grain yield and quality worldwide. The genetic bases of maize GER resistance remain largely unknown. Using artificial inoculation across multiple environments, the GER severity of an association panel consisting of 316 diverse inbred lines was observed with wide phenotypic variation. In the association panel, a genome-wide association study using a general linear model identified 69 single-nucleotide polymorphisms (SNPs) significantly associated with GER resistance at the threshold of 2.04 × 10-5, and the average phenotypic variation explained (PVE) of these SNPs was 5.09%. We also conducted a genome-wide association study analysis using a mixed linear model at a threshold of 1.0 × 10-4, and 16 significantly associated SNPs with an average PVE of 4.73% were detected. A combined general linear model and mixed linear model method obtained 10 co-localized significantly associated SNPs linked to GER resistance, including the most significant SNP (PZE-105079915) with the greatest PVE value, 9.07%, at bin 5.05 following 10 candidate genes. These findings are significant for the exploration of the complicated genetic variations in maize GER resistance. The regions and genes identified herein provide a list of candidate targets for further investigation, in addition to the elite germplasm resources that can be used for breeding GER resistance in maize.

A Combination of QTL Mapping and GradedPool-Seq to Dissect Genetic Complexity for Gibberella Ear Rot Resistance in Maize Using an IBM Syn10 DH Population.

Gibberella ear rot (GER) caused by Fusarium graminearum (teleomorph Gibberella zeae) is one of the most devastating maize diseases that reduces grain yield and quality worldwide. Utilization of host genetic resistance has become one of the most suitable strategies to control GER. In this study, a set of 246 diverse inbred lines derived from the intermated B 73 × Mo 17 doubled haploid population (IBM Syn10 DH) were used to detect quantitative trait loci (QTL) associated with resistance to GER. Meanwhile, a GradedPool-Seq (GPS) approach was performed to identify genomic variations involved in GER resistance. Using artificial inoculation across multiple environments, GER severity of the population was observed with wide phenotypic variation. Based on the linkage mapping, a total of 14 resistant QTLs were detected, accounting for 5.11 to 14.87% of the phenotypic variation, respectively. In GPS analysis, five significant single nucleotide polymorphisms (SNPs) associated with GER resistance were identified. Combining QTL mapping and GPS analysis, a peak-value SNP on chromosome 4 from GPS was overlapped with the QTL qGER4.2, suggesting that the colocalized region could be the most possible target location conferring resistance to GER. Subsequently, seven candidate genes were identified within the peak SNP, linking them to GER resistance. These findings are useful for exploring the complicated genetic variations in maize GER resistance. The genomic regions and genes identified herein provide a list of candidate targets for further investigation, in addition to the combined strategy that can be used for quantitative traits in plant species.

Impact of Gibberella Ear Rot on Grain Quality and Yield Components in Maize as Influenced by Hybrid Reaction.

The impact of Gibberella ear rot (GER; caused by Fusarium graminearum) on deoxynivalenol (DON) contamination of grain and yield components in maize were investigated using data from 30 environments in Ohio (3 years by 10 locations). Fifteen hybrids, later classified as susceptible (SU), moderately susceptible (MS), or moderately resistant (MR), based on the magnitude of differences in mean arcsine square-root-transformed GER severity (arcSEV) and log-transformed DON (logDON) relative to a reference SU check, were planted in each environment, and 10 ears per hybrid were inoculated with a spore suspension of F. graminearum. Relationships between GER severity and DON were well described by a Kono-Sugino-type nonlinear equation. Estimated parameters representing height (A) and steepness (ß) of the curves were significantly higher for SU than MS and MR hybrids but A was not significantly different between MS and MR. Results from a surrogacy analysis showed that GER was a moderate trial- and individual-level surrogate for DON. Both grain weight per ear and ear diameter decreased with increasing arcSEV but the regression slopes varied among resistance classes. The rates of reduction in both yield components per unit increase in arcSEV were significantly greater for SU than for MS and MR. An estimated 50% reduction in grain weight occurred at 62% GER severity for SU, compared with 77% severity for MS and 83% for MR. These results show that GER severity can be used as a surrogate for early estimation of DON contamination and yield loss to help guide grain handling and marketing decisions.

Mapping and Validation of a Stable Quantitative Trait Locus Conferring Maize Resistance to Gibberella Ear Rot.

Gibberella ear rot (GER), a prevalent disease caused by Fusarium graminearum, can result in significant yield loss and carcinogenic mycotoxin contamination in maize worldwide. However, only a few quantitative trait loci (QTLs) for GER resistance have been reported. In this study, we evaluated a Chinese recombinant inbred line (RIL) population comprising 204 lines, developed from a cross between a resistant parent DH4866 and a susceptible line T877, in three field trials under artificial inoculation with F. graminearum. The RIL population and their parents were genotyped with an Affymetrix microarray CGMB56K SNP Array. Based on the genetic linkage map constructed using 1,868 bins as markers, 11 QTLs, including five stable QTLs, were identified by individual environment analysis. Joint multiple environments analysis and epistatic interaction analysis revealed six additive and six epistatic (additive × additive) QTLs, respectively. None of the QTLs could explain more than 10% of phenotypic variation, suggesting that multiple minor-effect QTLs contributed to the genetic component of resistance to GER, and both additive and epistatic effects contributed to the genetic architecture of resistance to GER. A novel QTL, qGER4.09, with the largest effect, identified and validated using 588 F2 individuals, was colocalized with genomic regions for Fusarium ear rot and Aspergillus ear rot, indicating that this genetic locus likely confers resistance to multiple pathogens and can potentially be utilized in breeding maize varieties aimed at improving the resistance not only to GER but also other ear rot diseases.

Molecular bases of multimodal regulation of a fungal transient receptor potential (TRP) channel.

Multimodal activation by various stimuli is a fundamental characteristic of TRP channels. We identified a fungal TRP channel, TRPGz, exhibiting activation by hyperosmolarity, temperature increase, cytosolic Ca(2+) elevation, membrane potential, and H2O2 application, and thus it is expected to represent a prototypic multimodal TRP channel. TRPGz possesses a cytosolic C-terminal domain (CTD), primarily composed of intrinsically disordered regions with some regulatory modules, a putative coiled-coil region and a basic residue cluster. The CTD oligomerization mediated by the coiled-coil region is required for the hyperosmotic and temperature increase activations but not for the tetrameric channel formation or other activation modalities. In contrast, the basic cluster is responsible for general channel inhibition, by binding to phosphatidylinositol phosphates. The crystal structure of the presumed coiled-coil region revealed a tetrameric assembly in an offset spiral rather than a canonical coiled-coil. This structure underlies the observed moderate oligomerization affinity enabling the dynamic assembly and disassembly of the CTD during channel functions, which are compatible with the multimodal regulation mediated by each functional module.

Gibberella moniliformis AH13 with antitumor activity, an endophytic fungus strain producing triolein isolated from Adlay (Coix lacryma-jobi: poaceae).

In this study, the isolation of an endophytic fungus from the leaves of the medicinal herb adlay (Coix lacryma-jobi L. var. ma-yuen Stapf) is reported for the first time. The fungus produced Triolein (trioleoylglycerol), a major constituent of triacylglycerols (TAGs) of adlay, in rice medium under shake-flask and bench-scale fermentation conditions. The fungus was identified as Gibberella moniliformis (Fusarium verticillioides) by its morphology and authenticated by ITS analysis (ITS1 and ITS2 regions and the intervening 5.8S rDNA region). Triolein was identified by HPLC-ELSD coupled with APCI-MS and confirmed through comparison with authentic standard. The concentration of triolein produced by G. moniliformis AH13 reached 2.536 ± 0.006 mg/g dry weight of mycelium. Moreover, the EtOAc extract of G. moniliformis AH13 showed strong antitumor activity against four types of tumor cells (A549, HCT116, MDA-MB-231, and SW1990). These results suggest that G. moniliformis AH13 in adlay has significant scientific and industrial potential to meet the pharmaceutical demands and sustainable energy requirements for TAGs in a cost-effective, easily accessible, and reproducible way and is also a potential novel source of natural antitumor bioactive agents.

Structural variation and dynamics of the nuclear ribosomal intergenic spacer region in key members of the Gibberella fujikuroi species complex.

The intergenic spacer (IGS) region of the ribosomal DNA was cloned and sequenced in eight species within the Gibberella fujikuroi species complex with anamorphs in the genus Fusarium, a group that includes the most relevant toxigenic species. DNA sequence analyses revealed two categories of repeated elements: long repeats and short repeats of 125 and 8 bp, respectively. Long repeats were present in two copies and were conserved in all the species analyzed, whereas different numbers of short repeat elements were observed, leading to species-specific IGS sequences with different length. In Fusarium subglutinans and Fusarium nygamai, these differences seemed to be the result of duplication and deletion events. Here, we propose a model based on unequal crossing over that can explain these processes. The partial IGS sequence of 22 Fusarium proliferatum isolates was also obtained to study variation at the intraspecific level. The results revealed no differences in terms of number or pattern of repeated elements and detected frequent gene conversion events. These results suggest that the homogenization observed at the intraspecific level might not be achieved primarily by unequal crossing-over events but rather by processes associated with recombination such as gene conversion events.

Mitochondrial carnitine-dependent acetyl coenzyme A transport is required for normal sexual and asexual development of the ascomycete Gibberella zeae.

Fungi have evolved efficient metabolic mechanisms for the exact temporal (developmental stages) and spatial (organelles) production of acetyl coenzyme A (acetyl-CoA). We previously demonstrated mechanistic roles of several acetyl-CoA synthetic enzymes, namely, ATP citrate lyase and acetyl-CoA synthetases (ACSs), in the plant-pathogenic fungus Gibberella zeae. In this study, we characterized two carnitine acetyltransferases (CATs; CAT1 and CAT2) to obtain a better understanding of the metabolic processes occurring in G. zeae. We found that CAT1 functioned as an alternative source of acetyl-CoA required for lipid accumulation in an ACS1 deletion mutant. Moreover, deletion of CAT1 and/or CAT2 resulted in various defects, including changes to vegetative growth, asexual/sexual development, trichothecene production, and virulence. Although CAT1 is associated primarily with peroxisomal CAT function, mislocalization experiments showed that the role of CAT1 in acetyl-CoA transport between the mitochondria and cytosol is important for sexual and asexual development in G. zeae. Taking these data together, we concluded that G. zeae CATs are responsible for facilitating the exchange of acetyl-CoA across intracellular membranes, particularly between the mitochondria and the cytosol, during various developmental stages.

Isolation and characterization of Gibberella intermedia CA3-1, a novel and versatile nitrilase-producing fungus.

Nitrilase-mediated biocatalysis has attracted substantial attention for its application in carboxylic acid production in recent years. In the present study, the fungus CA3-1 was isolated and identified as Gibberella intermedia based on its morphology, its 18S ribosomal DNA (rDNA), and internal transcribed spacer (ITS) sequences. The enzymatic properties of G. intermedia resting cells were determined, and the optimum activity was achieved at 40 °C with pH 7.6. The half-lives of the nitrilase at 30, 40, and 50 °C were 231.1, 72.9, and 6.4 h, respectively. This Gibberella nitrilase showed a wide substrate spectrum with high specificity for heterocyclic and aliphatic nitriles. It remained extremely active in 5% propanol. The presence of Ag(+), Hg(2+), and excess substrate inhibited the nitrilase activity, whereas Fe(2+), Mn(2+), and Li(+) improved enzyme activity. 3-Cyanopyridine (50 mM) was hydrolyzed into nicotinic acid within 30 min, whereas only <5% of nicotinamide was detected. The results show that this fungal nitrilase is a promising candidate for commercial application in nicotinic acid production.

Effectiveness of introgression of resistance loci for Gibberella ear rot from two European flint landraces into adapted elite maize (Zea mays L.).

European flint landraces are a major class of maize possessing favorable alleles for improving host resistance to Gibberella ear rot (GER) disease which reduces yield and contaminates the grains with mycotoxins. However, the incorporation of these landraces into breeding programs requires a clear understanding of the effectiveness of their introgression into elite materials. We evaluated 15 pre-selected doubled haploid (DH) lines from two European flint landraces, "Kemater Landmais Gelb" (KE) and "Petkuser Ferdinand Rot" (PE), together with two adapted elite flint lines and seven standard lines for GER severity as the main trait, and several adaptation traits (plant height, days to silking, seed-set, plant vigor) across four environments. From this evaluation, three KE DH lines and one PE DH line, with the lowest GER severity, were selected and used as donor parents that were crossed with the two adapted and GER susceptible flint lines (Flint1 and Flint2) to develop six bi-parental DH populations with 34-145 DH lines each. Each DH population was evaluated across two locations. Correlations between GER severity, which was the target trait, and adaptation traits were weak (-0.02 to 0.19). GER severity of lines from PE landrace was on average 2-fold higher than lines from KE landrace, indicating a clear superiority of the KE landrace lines. Mean GER severity of the DH populations was 39.4-61.0% lower than the adapted elite flint lines. All KE-derived DH populations were on average more resistant (27.0-36.7%) than the PE-derived population (51.0%). Highly resistant lines (1.3-5.2%) were found in all of the populations, suggesting that the DH populations can be successfully integrated into elite breeding programs. The findings demonstrate that selected KE landrace lines used as donors were effective in improving GER resistance of the adapted elite inbreds.

Causes and consequences of variability in peptide mating pheromones of ascomycete fungi.

The reproductive genes of fungi, like those of many other organisms, are thought to diversify rapidly. This phenomenon could be associated with the formation of reproductive barriers and speciation. Ascomycetes produce two classes of mating type-specific peptide pheromones. These are required for recognition between the mating types of heterothallic species. Little is known regarding the diversity or the extent of species specificity in pheromone peptides among these fungi. We compared the putative protein-coding DNA sequences of the 2 pheromone classes from 70 species of Ascomycetes. The data set included previously described pheromones and putative pheromones identified from genomic sequences. In addition, pheromone genes from 12 Fusarium species in the Gibberella fujikuroi complex were amplified and sequenced. Pheromones were largely conserved among species in this complex and, therefore, cannot alone account for the reproductive barriers observed between these species. In contrast, pheromone peptides were highly diverse among many other Ascomycetes, with evidence for both positive diversifying selection and relaxed selective constraint. Repeats of the α-factor-like pheromone, which occur in tandem arrays of variable copy number, were found to be conserved through purifying selection and not concerted evolution. This implies that sequence specificity may be important for pheromone reception and that interspecific differences may indeed be associated with functional divergence. Our findings also suggest that frequent duplication and loss causes the tandem repeats to experience "birth-and-death" evolution, which could in fact facilitate interspecific divergence of pheromone peptide sequences.

Functional analyses of regulators of G protein signaling in Gibberella zeae.

Regulators of G protein signaling (RGS) proteins make up a highly diverse and multifunctional protein family that plays a critical role in controlling heterotrimeric G protein signaling. In this study, seven RGS genes (FgFlbA, FgFlbB, FgRgsA, FgRgsB, FgRgsB2, FgRgsC, and FgGprK) were functionally characterized in the plant pathogenic fungus, Gibberella zeae. Mutant phenotypes were observed for deletion mutants of FgRgsA and FgRgsB in vegetative growth, FgFlbB and FgRgsB in conidia morphology, FgFlbA in conidia production, FgFlbA, FgRgsB, and FgRgsC in sexual development, FgFlbA and FgRgsA in spore germination and mycotoxin production, and FgFlbA, FgRgsA, and FgRgsB in virulence. Furthermore, FgFlbA, FgRgsA, and FgRgsB acted pleiotropically, while FgFlbB and FgRgsC deletion mutants exhibited a specific defect in conidia morphology and sexual development, respectively. Amino acid substitutions in Gα subunits and overexpression of the FgFlbA gene revealed that deletion of FgFlbA and dominant active GzGPA2 mutant, gzgpa2(Q207L), had similar phenotypes in cell wall integrity, perithecia formation, mycotoxin production, and virulence, suggesting that FgFlbA may regulate asexual/sexual development, mycotoxin biosynthesis, and virulence through GzGPA2-dependent signaling in G. zeae.

Functional analyses of two acetyl coenzyme A synthetases in the ascomycete Gibberella zeae.

Acetyl coenzyme A (acetyl-CoA) is a crucial metabolite for energy metabolism and biosynthetic pathways and is produced in various cellular compartments with spatial and temporal precision. Our previous study on ATP citrate lyase (ACL) in Gibberella zeae revealed that ACL-dependent acetyl-CoA production is important for histone acetylation, especially in sexual development, but is not involved in lipid synthesis. In this study, we deleted additional acetyl-CoA synthetic genes, the acetyl-CoA synthetases (ACS genes ACS1 and ACS2), to identify alternative acetyl-CoA production mechanisms for ACL. The ACS1 deletion resulted in a defect in sexual development that was mainly due to a reduction in 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol production, which is required for perithecium development and maturation. Another ACS coding gene, ACS2, has accessorial functions for ACS1 and has compensatory functions for ACL as a nuclear acetyl-CoA producer. This study showed that acetate is readily generated during the entire life cycle of G. zeae and has a pivotal role in fungal metabolism. Because ACSs are components of the pyruvate-acetaldehyde-acetate pathway, this fermentation process might have crucial roles in various physiological processes for filamentous fungi.

Effect of simulated space gravity environment on Gibberella moniliformis EZG0807.

To determine the feasibility of inducing mutation for Gibberella moniliformis EZG0807 with a superconducting magnet, this paper investigated the effects of this instrument on the filamentous fungus G. moniliformis EZG0807. The superconducting magnet could simulate space gravity environment from hypo-gravity (0 g) to hyper-gravity (2 g). After G. moniliformis EZG0807 was exposed to the superconducting magnet for 72 h, the morphological observation, agar diffusion method, and amplified fragment length polymorphism were performed to detect the mutagenic effects in the aspect of morphology, the activity of metabolites, and genomic DNA, respectively. The mutant strain M7212 in 1 g (16 T) was different from the control in the morphology, showing no activity against the four tested bacteria Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Proteus vulgaris, and lost a size of 675 bp band on the genomic DNA. These results indicated that the superconducting magnet could be used to induce mutation for G. moniliformis EZG0807, which enabled improving the production of G. moniliformis EZG0807 and providing an effective approach for fungal breeding.

Fusarium tupiense sp. nov., a member of the Gibberella fujikuroi complex that causes mango malformation in Brazil.

Fusarium tupiense, the main causal agent of mango malformation in Brazil, is described through a combination of morphological, biological and molecular markers. This new species belongs to the Gibberella fujikuroi species complex (GFSC) and has an anamorph morphologically similar to Fusarium mangiferae and F. sterilihyphosum. F. tupiense can be differentiated from other species in the G. fujikuroi species complex on the basis of sexual crosses, amplified fragment length polymorphism (AFLP) markers and partial sequences of the tef1 and tub2 genes. Female fertility for field isolates of F. tupiense appears to be low. PCR with primers specific for the mating type (MAT) alleles and sexual crosses identified this species as heterothallic with two idiomorphs. Female-fertile tester strains were developed for the identification of field strains of this species through sexual crosses.

New PCR assays for the identification of Fusarium verticillioides, Fusarium subglutinans, and other species of the Gibberella fujikuroi complex.

Fusarium verticillioides and Fusarium subglutinans are important fungal pathogens of maize and other cereals worldwide. In this study, we developed PCR-based protocols for the identification of these pathogens targeting the gaoB gene, which codes for galactose oxidase. The designed primers recognized isolates of F. verticillioides and F. subglutinans that were obtained from maize seeds from several producing regions of Brazil but did not recognize other Fusarium spp. or other fungal genera that were either obtained from fungal collections or isolated from maize seeds. A multiplex PCR protocol was established to simultaneously detect the genomic DNA from F. verticillioides and F. subglutinans. This protocol could detect the DNA from these fungi growing in artificially or naturally infected maize seeds. Another multiplex reaction with a pair of primers developed in this work combined with a pre-existing pair of primers has allowed identifying F. subglutinans, F. konzum, and F. thapsinum. In addition, the identification of F. nygamai was also possible using a combination of two PCR reactions described in this work, and another described in the literature.

Novel Insights into the Inheritance of Gibberella Ear Rot (GER), Deoxynivalenol (DON) Accumulation, and DON Production.

Gibberella ear rot (GER) is an important fungal ear pathogen of maize that causes ear rot and toxin contamination. Most previous works have only dealt with the visual symptoms, but not with the toxins of GER. As food and feed safety rankings depend on toxin contamination, including deoxynivalenol (DON), without toxins, nothing can be said about the risks involved in food and feed quality. Therefore, three susceptible, three medium-susceptible, and three medium-resistant mother lines were crossed with three testers with differing degrees of resistance and tested between 2017-2020. Two plot replicates and two fungal strains were used separately. The highest heterosis was found at the GER% with a 13% increase across 27 hybrids, including 7 hybrids showing negative heterosis (a higher hybrid performance above the parental mean), with a variance ranging between 63.5 and -55.4. For DON, the mean heterosis was negative at -35%, and only 10 of the 27 hybrids showed a positive heterosis. The mean heterosis for DON contamination, at 1% GER, was again negative (-19.6%, varying between 85% and 224%). Only 17 hybrids showed heterosis, while that of the other 17 was rated higher than the parental mean. A positive significant correlation was found only for GER% and DON; the other factors were not significant. Seven hybrids were identified with positive (2) or negative (5) heterosis for all traits, while the rest varied. For DON and GER, only 13 provided identical (positive or negative) heteroses. The majority of the hybrids appeared to diverge in the regulation of the three traits. The stability of GER and DON (variance across eight data sets) did not agree-only half of the genotypes responded similarly for the two traits. The genetic background for this trait is unknown, and there was no general agreement between traits. Thus, without toxin analyses, the evaluation of food safety is not possible. The variety in degrees of resistance to toxigenic fungi and resistance to toxin accumulation is an inevitable factor.

An ABC transporter and a cytochrome P450 of Nectria haematococca MPVI are virulence factors on pea and are the major tolerance mechanisms to the phytoalexin pisatin.

The fungal plant pathogen Nectria haematococca MPVI produces a cytochrome P450 that is responsible for detoxifying the phytoalexin pisatin, produced as a defense mechanism by its host, garden pea. In this study, we demonstrate that this fungus also produces a specific ATP-binding cassette (ABC) transporter, NhABC1, that enhances its tolerance to pisatin. In addition, although both mechanisms individually contribute to the tolerance of pisatin and act as host-specific virulence factors, mutations in both genes render the fungus even more sensitive to pisatin and essentially nonpathogenic on pea. NhABC1 is rapidly induced after treatment with pisatin in vitro and during infection of pea plants. Furthermore, NhABC1 was able to confer tolerance to the phytoalexin rishitin, produced by potato. NhABC1 appears to be orthologous to GpABC1 of the potato pathogen Gibberella pulicaris and, along with MoABC1 from Magnaporthe oryzae, resides in a phylogenetically related clade enriched with ABC transorters involved in virulence. We propose that NhABC1 and the cytochrome P450 may function in a sequential manner in which the energy expense from pisatin efflux by NhABC1 releases the repression of the cytochrome P450, ultimately allowing pisatin tolerance by two mechanisms. These results demonstrate that a successful pathogen has evolved multiple mechanisms to overcome these plant antimicrobial compounds.