The Cultured Microbiome of Pollinated Maize Silks Shifts after Infection with Fusarium graminearum and Varies by Distance from the Site of Pathogen Inoculation.

Styles transmit pollen-derived sperm nuclei from pollen to ovules, but also transmit environmental pathogens. The microbiomes of styles are likely important for reproduction/disease, yet few studies exist. Whether style microbiome compositions are spatially responsive to pathogens is unknown. The maize pathogen Fusarium graminearum enters developing grain through the style (silk). We hypothesized that F. graminearum treatment shifts the cultured transmitting silk microbiome (TSM) compared to healthy silks in a distance-dependent manner. Another objective of the study was to culture microbes for future application. Bacteria were cultured from husk-covered silks of 14 F. graminearum-treated diverse maize genotypes, proximal (tip) and distal (base) to the F. graminearum inoculation site. Long-read 16S sequences from 398 isolates spanned 35 genera, 71 species, and 238 OTUs. More bacteria were cultured from F. graminearum-inoculated tips (271 isolates) versus base (127 isolates); healthy silks were balanced. F. graminearum caused a collapse in diversity of ~20-25% across multiple taxonomic levels. Some species were cultured exclusively or, more often, from F. graminearum-treated silks (e.g., Delftia acidovorans, Klebsiella aerogenes, K. grimontii, Pantoea ananatis, Stenotrophomonas pavanii). Overall, the results suggest that F. graminearum alters the TSM in a distance-dependent manner. Many isolates matched taxa that were previously identified using V4-MiSeq (core and F. graminearum-induced), but long-read sequencing clarified the taxonomy and uncovered greater diversity than was initially predicted (e.g., within Pantoea). These isolates represent the first comprehensive cultured collection from pathogen-treated maize silks to facilitate biocontrol efforts and microbial marker-assisted breeding.

Transmitting silks of maize have a complex and dynamic microbiome.

In corn/maize, silks emerging from cobs capture pollen, and transmit resident sperm nuclei to eggs. There are > 20 million silks per U.S. maize acre. Fungal pathogens invade developing grain using silk channels, including Fusarium graminearum (Fg, temperate environments) and devastating carcinogen-producers (Africa/tropics). Fg contaminates cereal grains with mycotoxins, in particular Deoxynivalenol (DON), known for adverse health effects on humans and livestock. Fitness selection should promote defensive/healthy silks. Here, we report that maize silks, known as styles in other plants, possess complex and dynamic microbiomes at the critical pollen-fungal transmission interval (henceforth: transmitting style microbiome, TSM). Diverse maize genotypes were field-grown in two trial years. MiSeq 16S rRNA gene sequencing of 328 open-pollinated silk samples (healthy/Fg-infected) revealed that the TSM contains > 5000 taxa spanning the prokaryotic tree of life (47 phyla/1300 genera), including nitrogen-fixers. The TSM of silk tip tissue displayed seasonal responsiveness, but possessed a reproducible core of 7-11 MiSeq-amplicon sequence variants (ASVs) dominated by a single Pantoea MiSeq-taxon (15-26% of sequence-counts). Fg-infection collapsed TSM diversity and disturbed predicted metabolic functionality, but doubled overall microbiome size/counts, primarily by elevating 7-25 MiSeq-ASVs, suggestive of a selective microbiome response against infection. This study establishes the maize silk as a model for fundamental/applied research of plant reproductive microbiomes.

Occurrence and Distribution of Physiological Races of Exserohilum turcicum in Ontario, Canada.

Northern corn leaf blight (NCLB) caused by Exserohilum turcicum is the most common and economically significant fungal leaf disease of corn in Ontario, Canada. During the past 10 years in Ontario, severity and incidence of NCLB have increased, possibly owing to the appearance of new races. Several races have been identified in various parts of the world; however, information on occurrence and distribution of races in Ontario is lacking. In the current study, 677 single conidial isolates of E. turcicum were isolated from 687 symptomatic leaf samples collected between 2012 and 2016. These isolates were evaluated for pathogenicity on six corn differential inbreds (A619, A619Ht1, A619Ht2, A619Ht3, A632Htn1, and H102Htm1) under controlled environmental conditions and then grouped into 17 physiological races (0, 1, 2, 3, M, N, 12, 1M, 1N, 3M, 13M, 12N, 13N, 1MN, 12MN, 13MN, 123MN) based on the reaction of the inbreds to infection (resistant or susceptible). Four races (0, 1M, 1N, and 1MN) were most frequent, with an isolation frequency of 13, 10, 12, and 41%, respectively. Seventy-six percent of the isolates were virulent on more than one Ht resistance gene, with 2.4% (16 isolates) virulent on all five Ht resistance genes used in this study. Further analysis of the distribution of races in four regions over the years revealed that the occurrence and distribution of the races changed with time in Ontario. Overall, the frequency of virulence of the 677 isolates screened on the differentials with resistance genes Ht1, Ht2, Ht3, Htm1, and Htn1 varied from 6 to 81% (Ht1 81%, Ht2 6%, Ht3 12%, Htm1 64%, and Htn1 64%). Virulent isolates produced fewer lesions on the Htm1 differential, and smaller lesions that were slower and having less sporulation on the Htn1 differential, compared with infection of the differentials with Ht1, Ht2, and Ht3 resistance genes. Virulence frequency also changed within the four geographical regions of Ontario, with fewer isolates virulent on all resistance genes in eastern Ontario compared with southern and western Ontario. Isolates from southern Ontario had greater virulence frequency against Ht1 and Htm1, whereas isolates from western Ontario were more frequently virulent on Ht1 and Htn1. The information generated in this study on the distribution of E. turcicum races in Ontario corn will help growers to select appropriate hybrids with required resistance genes and will assist seed companies in deploying resistance genes in corn hybrids across the province or within a particular region.

Genome Analysis and Development of a Multiplex TaqMan Real-Time PCR for Specific Identification and Detection of Clavibacter michiganensis subsp. nebraskensis.

The reemergence of the Goss's bacterial wilt and blight disease in corn in the United States and Canada has prompted investigative research to better understand the genome organization. In this study, we generated a draft genome sequence of Clavibacter michiganensis subsp. nebraskensis strain DOAB 395 and performed genome and proteome analysis of C. michiganensis subsp. nebraskensis strains isolated in 2014 (DOAB 397 and DOAB 395) compared with the type strain, NCPPB 2581 (isolated over 40 years ago). The proteomes of strains DOAB 395 and DOAB 397 exhibited a 99.2% homology but had 92.1 and 91.8% homology, respectively, with strain NCPPB 2581. The majority (99.9%) of the protein sequences had a 99.6 to 100% homology between C. michiganensis subsp. nebraskensis strains DOAB 395 and DOAB 397, with only four protein sequences (0.1%) exhibiting a similarity <70%. In contrast, 3.0% of the protein sequences of strain DOAB 395 or DOAB 397 showed low homologies (<70%) with the type strain NCPPB 2581. The genome data were exploited for the development of a multiplex TaqMan real-time polymerase chain reaction (PCR) tool for rapid detection of C. michiganensis subsp. nebraskensis. The specificity of the assay was validated using 122 strains of Clavibacter and non-Clavibacter spp. A blind test and naturally infected leaf samples were used to confirm specificity. The sensitivity (0.1 to 1.0 pg) compared favorably with previously reported real-time PCR assays. This tool should fill the current gap for a reliable diagnostic technique.

Quantitative trait loci mapping for Gibberella ear rot resistance and associated agronomic traits using genotyping-by-sequencing in maize.

KEY MESSAGE: Unique and co-localized chromosomal regions affecting Gibberella ear rot disease resistance and correlated agronomic traits were identified in maize. Dissecting the mechanisms underlying resistance to Gibberella ear rot (GER) disease in maize provides insight towards more informed breeding. To this goal, we evaluated 410 recombinant inbred lines (RIL) for GER resistance over three testing years using silk channel and kernel inoculation techniques. RILs were also evaluated for agronomic traits like days to silking, husk cover, and kernel drydown rate. The RILs showed significant genotypic differences for all traits with above average to high heritability estimates. Significant (P < 0.01) but weak genotypic correlations were observed between disease severity and agronomic traits, indicating the involvement of agronomic traits in disease resistance. Common QTLs were detected for GER resistance and kernel drydown rate, suggesting the existence of pleiotropic genes that could be exploited to improve both traits at the same time. The QTLs identified for silk and kernel resistance shared some common regions on chromosomes 1, 2, and 8 and also had some regions specific to each tissue on chromosomes 9 and 10. Thus, effective GER resistance breeding could be achieved by considering screening methods that allow exploitation of tissue-specific disease resistance mechanisms and include kernel drydown rate either in an index or as indirect selection criterion.

Draft Genome Sequence of Clavibacter michiganensis subsp. nebraskensis Strain DOAB 397, Isolated from an Infected Field Corn Plant in Manitoba, Canada.

In 2014, the pathogen Clavibacter michiganensis subsp. nebraskensis was isolated from symptomatic corn leaves in Manitoba, Canada. We report the draft genome sequence of C. michiganensis subsp. nebraskensis DOAB 397, consisting of 3.059 Mb with 73.0% G+C content, 2,922 predicted protein-coding sequences, 45 tRNAs, 3 rRNAs, and 37 pseudogenes.

Environmental factors related to fungal infection and fumonisin accumulation during the development and drying of white maize kernels.

In Southern Europe where whole maize kernels are ground and used for making bread and other food products, infection of the kernels by Fusarium verticillioides and subsequent fumonisin contamination pose a serious safety issue. The influence of environmental factors on this fungal infection and mycotoxin accumulation as the kernel develops has not been fully determined, especially in such food grade maize. The objectives of the present study were to determine which environmental factors may contribute to kernel invasion by F. verticillioides and fumonisin accumulation as kernels develop and dry in naturally infected white maize. Three maize hybrids were planted at two different sowing dates and kernel samples were collected 20, 40, 60, 80 and 100 days after silking. The percentage of kernels infected, and ergosterol and fumonisin contents were recorded for each sampling. F. verticillioides was the most prevalent species identified as the kernels developed. Temperature and moisture conditions during the first 80 days after silking favored natural kernel infection by F. verticillioides rather than by Aspergillus or Penicillium species. Fumonisin was found in kernels as early as 20 days after silking however significant fumonisin accumulation above levels acceptable in the EU did not occur until after physiological maturity of the kernel indicating that kernel drying in the field poses a high risk. Our results suggest that this could be due to increasing kernel damage by insects that favor fungal development, such as the damage by the moth Sitotroga cerealella, and to the occurrence of stress conditions for F. verticillioides growth that could trigger fumonisin biosynthesis, such as exposure to suboptimal temperatures for growth simultaneously with low water activity.

Role of hydroxycinnamic acids in the infection of maize silks by Fusarium graminearum Schwabe.

In the current study, the hydroxycinnamic acids in silks of diverse maize inbred lines differing in Fusarium resistance were determined at several times after inoculation with Fusarium graminearum or sterile water as control. The main objective was to determine the possible relationship between the hydroxycinnamic acid changes in silks and ear rot resistance. Several changes in the cell-wall-bound hydroxycinnamic acid concentrations were observed after inoculation with F. graminearum, although these changes were not directly correlated with genotypic resistance to this fungus. Ester-bound ferulic acid decreased, probably due to degradation of hemicellulose by hydrolytic enzymes produced by Fusarium spp., while p-coumaric acid and diferulates showed slight increases that, in conjunction, did not result in delayed F. graminearum progression through the silks. It is important to note that the decrease of ferulic acid in the F. graminearum treatment was faster in susceptible than in resistant genotypes, suggesting a differential hemicellulose degradation in silk tissues. Therefore, the ability of the maize genotypes to slow down that process through hemicellulose structural features or xylanase inhibitors needs to be addressed in future studies.

Inhibition of non-enzymatic glycation by silk extracts from a Mexican land race and modern inbred lines of maize (Zea mays).

Non-enzymatic glycation and the accumulation of advanced glycation end products (AGEs) are associated with various disease states, including complications of diabetes and aging. Secondary metabolites from several plant species are known to inhibit non-enzymatic glycation and the formation of AGEs, including flavonoids found in the style (silk) of Zea mays (maize). Thirteen modern maize inbreds and one land race were tested for in vitro inhibition of non-enzymatic glycation of bovine serum albumin. Many of the tested extracts exhibited inhibitory activity, in particular the newest inbreds, which were bred for resistance to gibberella ear rot (Fusarium graminearum) and common smut (Ustilago maydis). The most active maize genotype (CO441), displaying an IC50 of 9.5 microg/mL, was more effective than aminoguanidine, a known inhibitor of glycation. Zapalote chico, a land race with high maysin content, showed only moderate inhibitory activity compared with the modern maize genotypes. Antiglycation activity was highly correlated with the total phenolic content of silk extracts and mildly correlated with resistance to certain fungal infections. The results identify modern resistant and high phenolic maize inbreds as promising candidates for the development of natural AGE inhibitors for the prevention and treatment of diabetic complications and the degenerative effects of aging.

Phenolics in maize genotypes differing in susceptibility to Gibberella stalk rot (Fusarium graminearum Schwabe).

The relationship between phenolic compounds and maize pith resistance to Fusarium graminearum, the causal agent of Gibberella stalk rot, was investigated. The phenolic acid profiles in the stalks of six maize inbred lines of varying susceptibility were evaluated from silking to grain maturity. Four different fractions of phenolic compounds were extracted from inoculated and non-inoculated (control) pith tissues: insoluble cell-wall-bound, free, soluble ester-bound, and soluble glycoside-bound phenolics. Analysis by HPLC revealed that p-coumaric acid and ferulic acid were the most abundant compounds in the soluble and cell-wall-bound fractions. The quantity of free, glycoside-bound, and ester-bound phenolics in the pith was lower than the level required for the inhibition of Fusarium growth or mycotoxins production; however, significant negative correlations between diferulic acid contents in the cell walls and disease severity ratings 4 days after inoculation were found. The results indicated that future studies should focus on the levels of diferulic acids during the early infection process. Diferulates may play a role in genotypic resistance of maize to Gibberella stalk rot as preformed barriers to infection.

Diferulate content of maize sheaths is associated with resistance to the Mediterranean corn borer Sesamia nonagrioides (Lepidoptera: Noctuidae).

The leaf sheaths of selected inbred lines of maize (Zea mays L.) with variable levels of stem resistance to the Mediterranean corn borer Sesamia nonagrioides (Lefèvbre) were evaluated for antibiotic effect on insect development. Phytochemical analyses of leaf sheaths were conducted for cell wall phenylpropanoid content to gain a better understanding of maize-resistance mechanisms. Laboratory bioassays established that sheath tissues from different genotypes significantly affected the growth of neonate larvae. Three hydroxycinnamates, p-coumaric, trans-ferulic, and cis-ferulic acids, and three isomers of diferulic acid, 8-5', 8-O-4', and 8-5' b (benzofuran form), were identified. Significant negative correlations were found between larvae weight and diferulic acid content for six genotypes. These results are in agreement with previous studies concerning the role of cell wall structural components in stem borer resistance.

Putative role of pith cell wall phenylpropanoids in Sesamia nonagrioides (Lepidoptera: Noctuidae) resistance.

The stem borer Sesamia nonagrioides (Lefèbvre) is the most important insect pest that attacks maize, Zea mays L., in northwestern Spain. Host plant resistance to this borer was investigated in relation to the cell wall phenylpropanoids content in the pith. Eight inbred lines that differ in resistance were analyzed. Three major simple phenolic acids, p-coumaric, trans-ferulic, and cis-ferulic acids, and three isomers of diferulic acid, 8-5', 8-O-4', and 8-5'b (benzofuran form), were identified. The amount of all these compounds was correlated with the resistance level in the genotypes, with the resistant inbreds having the highest concentrations. The role of these compounds in cell wall fortification and lignification is well-documented, suggesting their possible intervention in S. nonagrioides resistance. Future studies that focus on these compounds could be useful to enhance S. nonagroides resistance.

Molecular mapping of QTLs for resistance to Gibberella ear rot, in corn, caused by Fusarium graminearum.

Gibberella ear rot, caused by the fungus Fusarium graminearum Schwabe, is a serious disease of corn (Zea mays) grown in northern climates. Infected corn is lower yielding and contains toxins that are dangerous to livestock and humans. Resistance to ear rot in corn is quantitative, specific to the mode of fungal entry (silk channels or kernel wounds), and highly influenced by the environment. Evaluations of ear rot resistance are complex and subjective; and they need to be repeated over several years. All of these factors have hampered attempts to develop F. graminearum resistant corn varieties. The aim of this study was to identify molecular markers linked to the genes for resistance to Gibberella ear rot. A recombinant inbred (RI) population, produced from a cross between a Gibberella ear rot resistant line (CO387) and a susceptible line (CG62), was field-inoculated and scored for Gibberella ear rot symptoms in the F4, F6, and F7 generations. The distributions of disease scores were continuous, indicating that resistance is probably conditioned by multiple loci. A molecular linkage map, based on segregation in the F5 RI population, contained 162 markers distributed over 10 linkage groups and had a total length of 2237 cM with an average distance between markers of 13.8 cM. Composite interval mapping identified 11 quantitative trait loci (QTLs) for Gibberella ear rot resistance following silk inoculation and 18 QTLs following kernel inoculation in 4 environments that accounted for 6.7%-35% of the total phenotypic variation. Only 2 QTLs (on linkage group 7) were detected in more than 1 test for silk resistance, and only 1 QTL (on linkage group 5) was detected in more than 1 test for kernel resistance, confirming the strong influence of the environment on these traits. The majority of the favorable alleles were derived from the resistant parent (CO387). The germplasm and markers for QTLs with significant phenotypic effects may be useful for marker-assisted selection to incorporate Gibberella ear rot resistance into commercial corn cultivars.

Long chain alkanes in silk extracts of maize genotypes with varying resistance to Fusarium graminearum.

The alkane content of the silks of nine maize genotypes was analyzed to investigate the role of silk wax in resistance to Fusarium graminearum. Silk samples were collected 2, 4, 6, and 8 days after silk emergence and divided into three sections: exposed silk, silk channel silk, and silk that is under the husk and overlying the kernels. Four major unbranched alkanes (C(25), C(27), C(29), and C(31)) and three isoalkanes (C(27i), C(29i), and C(31i)) were identified. Total alkane contents were highest in the exposed silk followed by the silk channel silk, with the lowest in the youngest silk closest to the kernels. In the silk channel and overlying kernel silks, the moderately resistant inbred CO272 consistently had the highest alkane content. None of the other inbreds with improved resistance had as high a level of alkanes as CO272, indicating that alkane content is not a major mechanism of resistance.