Development of early maturity maize hybrids for resistance to fusarium and aspergillus ear rots and their associated mycotoxins

Maize is mainly affected by two fungal pathogens, Fusarium verticillioides and Aspergillus flavus, causing Fusarium ear rot (FER) and Aspergillus ear rot (AER), respectively. Both fungi are of concern to stakeholders as they affect crop yield and quality, contaminating maize grains with the mycotoxins fumonisins and aflatoxins. The easiest strategy to prevent pre-harvest contamination by F. verticillioides and A. flavus is to develop maize hybrids resistant to FER and AER, as well as to their associated mycotoxins. The objective of this investigation was to test 46 F1 hybrids, originated from different Italian, US and Canadian breeding groups, for these important traits and their agronomic performances. All hybrids were planted and artificially inoculated with toxigenic strains of F. verticillioides and A. flavus at two locations in 2017, and the best performing 17 out of 46 were also tested in 2018. Ear rots were present in all hybrids in 2017 and 2018, with percentages ranging from 6.50 to 49.50%, and 5.50 to 45.53%, for FER and AER, respectively. Seven hybrids (PC8, PC15, PC9, PC11, PC14, PC34 and PC17) presented the lowest levels of both diseases considering the overall locations and growing seasons, and three of these (PC8, PC11 and PC14) were also amongst the least mycotoxin contaminated hybrids in 2017. The inbred lines used in hybrid production may provide additional sources of resistance suitable in breeding programs targeting multiple pathogens and their mycotoxins

CHARACTERIZING HETEROSIS IN A SET OF RECOMBINANT INTERCROSSES (RIXS) DEVELOPED FROM A MULTIPARENTAL MAIZE POPULATION

The exploitation of heterosis is key in modern maize breeding to capture the superior performance of heterozygous genotypes. Here, we developed a recombinant intercross (RIX) population in maize by crossing pairs of multiparental MAGIC recombinant inbred lines (RILs) to evaluate the heterosis across mosaics of eight maize haplotypes. Field phenotyping was performed on 400 RIX genotypes considering 11 agronomic traits as well as the resistance to Fusarium Ear Rot (FER), caused by Fusarium verticillioides (Sacc.) Nirenberg, in 2018 and 2019 in Piacenza, northern Italy. Phenotypic data showed a broad range of diversity in production and phenology traits thanks to the high level of allelic diversity available in parental genomes. The heterotic response of agronomic traits was computed based on RIL values as mid parent heterosis (MPH) and best parent heterosis (BPH). Both heterosis measures showed different magnitudes for different traits, with higher level of heterosis in yield and lower in flowering time, suggesting effects from partial dominance to over-dominance. No correlation was observed between phenotypic performance and heterozygosity level of RIXs for most of the agronomic traits. A preliminary quantitative trait locus (QTL) mapping detected a number of significant associations with agronomic traits across all chromosomes. The RIX collection showed a moderate heritability of FER resistance, and QTL were associated to this trait as well. The allelic effect estimates by our mapping model indicated the presence of minor effect QTL with relatively small additive effects on disease resistance in both years. Our findings confirm the usefulness of the RIX population to decipher heterotic loci in maize and support utilizing this resource in future to accelerate crop improvement

CHARACTERIZATION AND VALORIZATION OF MAIZE LANDRACES FROM VALLE D'AOSTA

During 1949-1950 in Italy begun a formal investigation to characterize maize (Zea mays L.) cultivation. In 1954, started a project for the sampling of all Italian maize landraces; this work ended with the collection of 562 different accessions collected in all regions with the exception of Valle d’Aosta, even if historical cultivation of maize in this Region is well documented. In Italy maize landraces have been extensively grown until the mid of the XX century when the cultivation of hybrid took place due to their significant agronomic performances. Despite that, being Valle d’Aosta a mountain region where intensive maize cultivation never started, it was possible to preserve the presence of some landraces. These local materials, which are still cultivated, mainly at domestic level, have high importance from a genetic and historical point of view. Recently, 5 maize landraces from Valle d’Aosta and 2 landraces from the adjacent Canavese (Piedmont) have been collected and subjected to historic, morphologic and genetic characterization. These landraces were named after the sampling location as it follows: Arnad, Arnad-Crest, Chatillon, Entrebin, Perloz, Bianco Canavese, and Rostrato Canavese. Firstly, on these 6 varieties the historic characterization has been carried out. Information and photographs have been searched in local archives and this was crucial to prove their long presence in all the sampling sites under study. From this historic reconstruction, the variety Entrebin resulted as the one that is better historically characterized. To study the variability and differentiation of landraces from Valle d’Aosta, the genetic characterization was performed by the means of 10 SSR markers tested on 20 samples from each landrace. This study highlighted a significant genetic variability among the landraces and, especially, a good level of differentiation between the accessions under investigation. This last result may be explained by the long reproductive isolation experienced by these materials. Complete morphological characterization is actually ongoing. Preliminary morphological observations revealed that these landraces have, generally, flint kernels with the exception of Bianco Canavese (dent) whose color is variable from white (Bianco Canavese) to dark red (Chatillon). Arnad landrace showed 8 kernel rows, probably being an Eight-rowed Flint while the others presented more rows, like many Derived Races. Interestingly, Perloz and Rostrato Canavese showed kernels with an apical beak which was more pronounced in the latter. This suggest that these two landraces belong to the “Rostrata” group, which is common in mountain areas. The present work confirms the importance of mountain areas in conserving biodiversity and increases the rich Italian maize germplasm with materials well adapted to marginal areas. Such new genetic variability may be used to breed new materials for a more resilient agriculture

Loss of zmlipoxygenase4 decreases fusarium verticillioides resistance in maize seedlings

Fusarium verticillioides is one of the most relevant fungal species in maize responsible for ear, stalk and seedling rot, as well as the fumonisin contamination of kernels. Plant lipoxygenases (LOX) synthesize oxylipins that play a crucial role in the regulation of defense mechanisms against pathogens and influence the outcome of pathogenesis. To better uncover the role of these signaling molecules in maize resistance against F. verticillioides, the functional characterization of the 9\u2010LOX gene, ZmLOX4, was carried out in this study by employing mutants carrying Mu insertions in this gene (named as UFMulox4). In this regard, the genotyping of five UFMulox4 identified the mutant UFMu10924 as the only one having an insertion in the coding region of the gene. The impact of ZmLOX4 mutagenesis on kernel defense against F. verticillioides and fumonisin accumulation were investigated, resulting in an increased fungal susceptibility compared to the inbred lines W22 and Tzi18. Moreover, the expression of most of the genes involved in the LOX, jasmonic acid (JA) and green leaf volatiles (GLV) pathways, as well as LOX enzymatic activity, decreased or were unaffected by fungal inoculation in the mutant UFMu10924. These results confirm the strategic role of ZmLOX4 in controlling defense against F. verticillioides and its influence on the expression of several LOX, JA and GLV genes

Unravelling the genetic basis of Fusarium resistance in different maize populations

Fungal infection by Fusarium verticillioides is cause of substantial reductions in maize yield and grain quality worldwide. Developing natural resistance in maize genotypes is an effective way to achieve sustainable control of F. verticillioides in the field, and breeding for resistance may be accelerated by identifying genes and loci responsible for natural disease resistance. Significant advances have been made in the development of transcriptomic, genetic and genomic information for maize, F. verticillioides moulds, and their interactions over recent years. Several quantitative trait loci (QTL) and single-nucleotide polymorphism markers for resistance to Fusarium deriving from QTL mapping and genome-wide association studies have been described in three different maize populations: 1. Bi-parental population; 2. Association mapping panel; 3. Multi-parent Advanced Generation Inter Crosses (MAGIC). To guide the identification of candidate genes within the identified QTL, transcriptomic and sequencing information have been exploited. Promising candidate genes associated with disease resistance and pathogen related-mechanisms at the Fusarium resistant loci have been identified on maize chromosomes 4, 5 and 7. Many of the identified candidates genes offer hints to key metabolic pathways that may have a significant effect on reducing Fusarium infection. Measuring Fusarium resistance in open field could confirm and support their direct use in maize breeding either through crosses or genome editing approaches

CHARACTERIZATION AND CONSERVATION OF EMILIA ROMAGNA MAIZE LANDRACES

The project aims at the characterization and conservation of regional maize landraces. Genetic material, consisting of 32 accessions, was retrieved both from recent surveys in Emilia Romagna, 11 accessions, and from the germplasm bank of CREA-MAC (Bergamo), 21 accessions. In particular, these last samples derive from the whole Italian collection of maize landraces carried out in the 50\u2019s by Brandolini and Fenaroli. In the first two years of the project, we carried out the landrace census and field characterization of the different materials according to UPOV guidelines and prepared, for each accession, a descriptive sheet. For each landrace, 100 seeds were sown and, from each plant, leaf samples were collected for DNA analysis. Approximately 2,000 plants, around 60 plants per landrace, were sampled. Each landrace is maintained in field by controlled randomintermating. For genetic analyses, 80 SSR markers were tested to identify the 10 most polymorphic to be used for the genetic characterization of all samples. . In parallel to these activities, an agronomic trial has been set up with the 32 landraces on four replicates to investigate production potential and resistance to mycotoxigenic fungi, with a special focus on Fusarium verticillioides. Considering that landraces can be exploited in conditions of low input agriculture, one of the objectives of the project is the identification of the best performing landraces suitable for cultivation nowadays (with a particular interest at mountain and disadvantaged areas). With this purpose, seeds, obtained from the propagation carried out in the first year, were distributed to partner farms and fields have been set-up in different hilly and mountainous areas of the region Emilia Romagna. These fields are presently underway with the double aim to evaluate the landraces in low input conditions and to propagate seeds for the future cultivation. The work was supported by PSR 2014- 2020, Emilia Romagna Region

A genome-wide association study to understand the effect of Fusarium verticillioides infection on seedlings of a maize diversity panel

Fusarium verticillioides, which causes ear, kernel and stem rots, has been reported as the most prevalent species on maize worldwide. Kernel infection by F. verticillioides results in reduced seed yield and quality as well as fumonisin contamination, and may affect seedling traits like germination rate, entire plant seedling length and weight. Maize resistance to Fusarium is a quantitative and complex trait controlled by numerous genes with small effects. In the present work, a Genome Wide Association Study (GWAS) of traits related to Fusarium seedling rot was carried out in 230 lines of a maize association population using 226,446 SNP markers. Phenotypes were scored on artificially infected kernels applying the rolled towel assay screening method and three traits related to disease response were measured in inoculated and not-inoculated seedlings: plant seedling length (PL), plant seedling weight (PW) and germination rate (GERM). Overall, GWAS resulted in 42 SNPs significantly associated with the examined traits. Two and eleven SNPs were associated with PL in inoculated and not-inoculated samples, respectively. Additionally, six and one SNPs were associated with PW and GERM traits in not-inoculated kernels, and further nine and thirteen SNPs were associated to the same traits in inoculated kernels. Five genes containing the significant SNPs or physically closed to them were proposed for Fusarium resistance, and 18 out of 25 genes containing or adjacent to significant SNPs identified by GWAS in the current research co-localized within QTL regions previously reported for resistance to Fusarium seed rot, Fusarium ear rot and fumonisin accumulation. Furthermore, linkage disequilibrium analysis revealed an additional gene not directly observed by GWAS analysis. These findings could aid to better understand the complex interaction between maize and F. verticillioides

Breeding for resistance to Fusarium infection in maize

Fungal infection by Fusarium verticillioides causes substantial reductions in maize yield and grain quality worldwide. Developing natural resistance in maize genotypes is an effective way to achieve sustainable control of F. verticillioides in the field, and breeding for resistance may be accelerated by identifying genes and loci responsible for natural disease resistance. Significant advances have been made in the development of transcriptomic, genetic and genomic information for maize, F. verticillioides moulds, and their interactions over recent years. Several quantitative trait loci (QTL) and single-nucleotide polymorphism markers for resistance to Fusarium deriving from QTL mapping and genome-wide association studies have been described in three different maize populations: 1. Bi-parental population; 2. Association mapping panel; 3. Multi-parent Advanced Generation Inter Crosses (MAGIC). To guide the identification of candidate genes within the identified QTL, transcriptomic and sequencing information have been exploited. Promising candidate genes associated with disease resistance and pathogen related-mechanisms at the Fusarium resistant loci have been identified on maize chromosomes 4, 5 and 7. Many of the identified candidates\u2019 genes offer hints to key metabolic pathways that may have a significant effect on reducing Fusarium infection. Measuring Fusarium resistance in open field could confirm and support their direct use in maize breeding either through crosses or genome editing approaches