Reference Gene Selection for RT-qPCR Analysis in Maize Kernels Inoculated with Aspergillus flavus.

Resistance against infection by the fungus Aspergillus flavus Link in commercial maize (Zea mays L.) is the topic of many studies, but few studies have investigated the effects of A. flavus infection on gene expression levels in ear kernels. A crucial component of gene expression profiling by RT-qPCR is having a reliable set of reference genes that show relatively constant expression across the treatments and phenotypes under study. Currently, however, there is no published information on reference genes suitable for measuring changes in kernel gene expression levels after infection with A. flavus. Thus, in this study, six candidate reference genes (ACT1, ß-Tub2, eIF4A2, TATA, EFIα, and GAPDH) were evaluated and ranked according to their expression stability. The genes were amplified from first-strand cDNA samples synthesized from kernels of two susceptible and two resistant maize lines that were either inoculated with A. flavus or water or not inoculated. Three software packages were used to calculate and rank the stability of expression for these genesgeNorm, NormFinder, and BestKeeper. The analysis revealed that the most stable genes to normalize expression levels from maize kernels responding to A. flavus inoculation and wounding were ACT1, EFIα, and eIF4A2.

Termite Presence and Feeding on Loblolly Pine Wood Differs Among Four Root-Infecting Bluestain (ophiostomatoid) Fungal Species.

Bark beetles and root weevils can impact forests through tree death on landscape scales. Recently, subterranean termites have been linked to these beetles via the presence of bluestain fungi (Ascomycota: Ophiostomataceae), which are vectored to trees by beetles. However, only a small subset of bluestain species have been examined. Here, we tested whether termite-bluestain association patterns in the field reflect termite feeding preference in laboratory choice trials. We documented the presence of four bluestain fungi (Leptographium procerum (W.B. Kendr.), L. terebrantis (Barras & Perry), Grosmannia huntii (Rob.-Jeffr.), and G. alacris (T.A. Duong, Z.W. de Beer & M.J. Wingf.) in the roots of 2,350 loblolly pine trees in the southeastern United States and whether termites were present or absent on these roots and paired this with laboratory choice feeding trials. Termites were found 2.5-fold on tree roots with at least one bluestain fungus present than tree roots without bluestain fungi. Although termites in this study and others were associated with L. procerum, L. terebrantis, and marginally G. huntii, termites only showed preferential feeding on wood inoculated with G. huntii in laboratory trials. This suggests that increased termite presence on wood with bluestain fungi may be driven by factors other than increased wood palatability. Termites could thus disproportionately affect wood turnover rates for specific pools (e.g., bark beetle and root weevil attacked trees) and in some cases (e.g., G. huntii) accelerate wood decomposition. This study supports the growing evidence that the association between subterranean termites and bluestain fungi is spatially and taxonomically widespread.

A Study of the Gut Bacterial Community of Reticulitermes virginicus Exposed to Chitosan Treatment.

A thorough understanding of microbial communities in the gut of lower termites is needed to develop target-specific and environmentally benign wood protection systems. In this study, the bacterial community from Reticulitermes virginicus was examined by Illumina sequencing of 16S ribosomal RNA (rRNA) spanning the V3 and V4 regions. Prior to library preparation, the termites were subjected to five treatments over an 18-day period: three groups were fed on wood treated with 0.5% chitosan, 25% acetic acid, or water, the fourth group was taken directly from the original collection log, and the fifth group was starved. Metagenomic sequences were analyzed using QIIME 2 to understand the treatments' effects on the dynamics of the gut bacteria. Four dominant phyla were detected: Bacteroidetes (34.4% of reads), Firmicutes (20.6%), Elusimicrobia (15.7%), and Proteobacteria (12.9%). A significant effect of chitosan treatment was observed in two phyla; Firmicutes abundance was significantly lower with chitosan treatment when compared to other groups, while Actinobacteria was lower in unexposed and starved termites. The results suggest that chitosan treatment not only affects the structure of the microbial community in the gut, but other treatments such as starving also cause shifts in termite gut communities.

PAST: The Pathway Association Studies Tool to Infer Biological Meaning from GWAS Datasets.

In recent years, a bioinformatics method for interpreting genome-wide association study (GWAS) data using metabolic pathway analysis has been developed and successfully used to find significant pathways and mechanisms explaining phenotypic traits of interest in plants. However, the many scripts implementing this method were not straightforward to use, had to be customized for each project, required user supervision, and took more than 24 h to process data. PAST (Pathway Association Study Tool), a new implementation of this method, has been developed to address these concerns. PAST has been implemented as a package for the R language. Two user-interfaces are provided; PAST can be run by loading the package in R and calling its methods, or by using an R Shiny guided user interface. In testing, PAST completed analyses in approximately half an hour to one hour by processing data in parallel and produced the same results as the previously developed method. PAST has many user-specified options for maximum customization. Thus, to promote a powerful new pathway analysis methodology that interprets GWAS data to find biological mechanisms associated with traits of interest, we developed a more accessible, efficient, and user-friendly tool. These attributes make PAST accessible to researchers interested in associating metabolic pathways with GWAS datasets to better understand the genetic architecture and mechanisms affecting phenotypes.

Leveraging GWAS data to identify metabolic pathways and networks involved in maize lipid biosynthesis.

Maize (Zea mays mays) oil is a rich source of polyunsaturated fatty acids (FAs) and energy, making it a valuable resource for human food, animal feed, and bio-energy. Although this trait has been studied via conventional genome-wide association study (GWAS), the single nucleotide polymorphism (SNP)-trait associations generated by GWAS may miss the underlying associations when traits are based on many genes, each with small effects that can be overshadowed by genetic background and environmental variation. Detecting these SNPs statistically is also limited by the levels set for false discovery rate. A complementary pathways analysis that emphasizes the cumulative aspects of SNP-trait associations, rather than just the significance of single SNPs, was performed to understand the balance of lipid metabolism, conversion, and catabolism in this study. This pathway analysis indicated that acyl-lipid pathways, including biosynthesis of wax esters, sphingolipids, phospholipids and flavonoids, along with FA and triacylglycerol (TAG) biosynthesis, were important for increasing oil and FA content. The allelic variation found among the genes involved in many degradation pathways, and many biosynthesis pathways leading from FAs and carbon partitioning pathways, was critical for determining final FA content, changing FA ratios and, ultimately, to final oil content. The pathways and pathway networks identified in this study, and especially the acyl-lipid associated pathways identified beyond what had been found with GWAS alone, provide a real opportunity to precisely and efficiently manipulate high-oil maize genetic improvement.

Indirect effects of bark beetle-generated dead wood on biogeochemical and decomposition processes in a pine forest.

Bark beetle outbreaks are increasing in frequency and intensity, generating massive inventories of dead trees globally. During attacks, trees are pre-inoculated with ophiostomatoid fungi via bark beetles, which has been shown to increase termite presence and feeding. These events may, in turn, alter biogeochemical cycles during decomposition. We examined these relationships by experimentally inoculating dead wood with bluestain fungi in a temperate pine forest. Across ten replicate plots, eight 0.5 m-long logs were inoculated with Ophiostoma minus and eight with distilled water. Half of the logs from each inoculation treatment were covered from above with a mesh cage barrier to exclude aboveground beetles while permitting access by belowground decomposers. After 1 year, significant increases in mass (34%) and decreases in moisture content (- 17%) were observed across all treatments, but no consistent changes in density were evident. C concentrations were 12% greater in bark when barriers were present and 17% greater in sapwood when barriers and inoculation fungi were absent. N concentrations were 16% greater in bark for fungal-inoculated logs and 27% greater when barriers were present. C:N ratios in A horizon soils under fungal-inoculated logs were 12% greater. Furthermore, termites were present fourfold more in fungal-inoculated logs than controls and the presence of termites was associated with 6% less C in sapwood and 11% more N in both sapwood and heartwood. Together these results suggest dead wood generated via bark beetle attacks has different biogeochemical responses during initial decomposition phases, which could have implications for the C status in forests following bark beetle outbreaks.

Genome-Wide Association and Metabolic Pathway Analysis of Corn Earworm Resistance in Maize.

Maize ( L.) is a staple crop of economic, industrial, and food security importance. Damage to the growing ears by corn earworm [ (Boddie)] is a major economic burden and increases secondary fungal infections and mycotoxin levels. To identify biochemical pathways associated with native resistance mechanisms, a genome-wide association analysis was performed, followed by pathway analysis using a gene-set enrichment-based approach. The gene-set enrichment exposed the cumulative effects of genes in pathways to identify those that contributed the most to resistance. Single nucleotide polymorphism-trait associations were linked to genes including transcription factors, protein kinases, hormone-responsive proteins, hydrolases, pectinases, xylogluconases, and the flavonol synthase gene (in the maysin biosynthesis pathway). The most significantly associated metabolic pathways identified included those that modified cell wall components, especially homogalacturonan, wax esters, and fatty acids; those involved in antibiosis, especially 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), flavonoids, and phenolics; and those involved in plant growth, including N uptake and energy production. The pathways identified in this study, and especially the cell wall-associated pathways, identified here for the first time, provide clues to resistance mechanisms that could guide the identification of new resistant ideotypes and candidate genes for creation of resistant maize germplasm via selection of natural variants or gene editing.

Termiticidal activity of chitosan against the subterranean termites Reticulitermes flavipes and Reticulitermes virginicus.

BACKGROUND: Chitosan is a derivative form of chitin, which is the major component of exoskeletons of arthropods and the cell walls of fungi. The antimicrobial activity of chitosan against lepidopterans, aphids, fungi and bacteria has been extensively investigated, but only one report on the termiticidal effect of chitosan on termites has been published. In this study, we examined the termiticidal activity of chitosan by exposing single colonies of Reticulitermes flavipes (Kollar) and Reticulitermes virginicus Banks to wood treated with six different concentrations of chitosan solutions. Termite mortality and percent mass loss of wood samples after exposure to termites for 4 weeks were calculated. RESULTS: High termite mortality (≥ 94%) occurred during exposure of R. flavipes termites to chitosan-treated wood with ≥38 mg g-1 treatment concentrations (≥ 2% chitosan), while <50% termite mortality was observed at lower treatment concentrations (11-15 mg g-1 ; 0.5% and 1% chitosan). For R. virginicus, 100% mortality was observed at all levels of treatment concentrations. A decrease in the percent mass loss of the wood sample was apparent in samples treated with solutions with an increasing chitosan concentration, with a significant difference (P < 0.05) between lower and higher treatment concentrations. Treatment retention in wood samples upon leaching was also determined and showed retention levels of between 0 and 30 mg g-1 chitosan retention. CONCLUSION: This study investigated the exposure of subterranean termites to chitosan as a wood preservative. The results show that chitosan treatments at sufficiently high loadings could protect wood against termites, preferably under non-leaching conditions. © 2018 Society of Chemical Industry.

Characterization of the maize lipoxygenase gene family in relation to aflatoxin accumulation resistance.

Maize (Zea mays L.) is a globally important staple food crop prone to contamination by aflatoxin, a carcinogenic secondary metabolite produced by the fungus Aspergillus flavus. An efficient approach to reduce accumulation of aflatoxin is the development of germplasm resistant to colonization and toxin production by A. flavus. Lipoxygenases (LOXs) are a group of non-heme iron containing dioxygenase enzymes that catalyze oxygenation of polyunsaturated fatty acids (PUFAs). LOX derived oxylipins play critical roles in plant defense against pathogens including A. flavus. The objectives of this study were to summarize sequence diversity and expression patterns for all LOX genes in the maize genome, and map their effect on aflatoxin accumulation via linkage and association mapping. In total, 13 LOX genes were identified, characterized, and mapped. The sequence of one gene, ZmLOX10, is reported from 5 inbred lines. Genes ZmLOX1/2, 5, 8, 9, 10 and 12 (GRMZM2G156861, or V4 numbers ZM00001D042541 and Zm00001D042540, GRMZM2G102760, GRMZM2G104843, GRMZM2G017616, GRMZM2G015419, and GRMZM2G106748, respectively) fell under previously published QTL in one or more mapping populations and are linked to a measurable reduction of aflatoxin in maize grains. Association mapping results found 28 of the 726 SNPs tested were associated with reduced aflatoxin levels at p ≤ 9.71 x 10-4 according to association statistics. These fell within or near nine of the ZmLOX genes. This work confirms the importance of some lipoxygenases for resistance to aflatoxin accumulation and may be used to direct future genetic selection in maize.

Using genome-wide associations to identify metabolic pathways involved in maize aflatoxin accumulation resistance.

BACKGROUND: Aflatoxin is a potent carcinogen that can contaminate grain infected with the fungus Aspergillus flavus. However, resistance to aflatoxin accumulation in maize is a complex trait with low heritability. Here, two complementary analyses were performed to better understand the mechanisms involved. The first coupled results of a genome-wide association study (GWAS) that accounted for linkage disequilibrium among single nucleotide polymorphisms (SNPs) with gene-set enrichment for a pathway-based approach. The rationale was that the cumulative effects of genes in a pathway would give insight into genetic differences that distinguish resistant from susceptible lines of maize. The second involved finding non-pathway genes close to the most significant SNP-trait associations with the greatest effect on reducing aflatoxin in multiple environments. Unlike conventional GWAS, the latter analysis emphasized multiple aspects of SNP-trait associations rather than just significance and was performed because of the high genotype x environment variability exhibited by this trait. RESULTS: The most significant metabolic pathway identified was jasmonic acid (JA) biosynthesis. Specifically, there was at least one allelic variant for each step in the JA biosynthesis pathway that conferred an incremental decrease to the level of aflatoxin observed among the inbred lines in the GWAS panel. Several non-pathway genes were also consistently associated with lowered aflatoxin levels. Those with predicted functions related to defense were: leucine-rich repeat protein kinase, expansin B3, reversion-to-ethylene sensitivity1, adaptor protein complex2, and a multidrug and toxic compound extrusion protein. CONCLUSIONS: Our genetic analysis provided strong evidence for several genes that were associated with aflatoxin resistance. Inbred lines that exhibited lower levels of aflatoxin accumulation tended to share similar haplotypes for genes specifically in the pathway of JA biosynthesis, along with several non-pathway genes with putative defense-related functions. Knowledge gained from these two complementary analyses has improved our understanding of population differences in aflatoxin resistance.

Gene expression analysis of copper tolerance and wood decay in the brown rot fungus Fibroporia radiculosa.

High-throughput transcriptomics was used to identify Fibroporia radiculosa genes that were differentially regulated during colonization of wood treated with a copper-based preservative. The transcriptome was profiled at two time points while the fungus was growing on wood treated with micronized copper quat (MCQ). A total of 917 transcripts were differentially expressed. Fifty-eight of these genes were more highly expressed when the MCQ was protecting the wood from strength loss and had putative functions related to oxalate production/degradation, laccase activity, quinone biosynthesis, pectin degradation, ATP production, cytochrome P450 activity, signal transduction, and transcriptional regulation. Sixty-one genes were more highly expressed when the MCQ lost its effectiveness (>50% strength loss) and had functions related to oxalate degradation; cytochrome P450 activity; H(2)O(2) production and degradation; degradation of cellulose, hemicellulose, and pectin; hexose transport; membrane glycerophospholipid metabolism; and cell wall chemistry. Ten of these differentially regulated genes were quantified by reverse transcriptase PCR for a more in-depth study (4 time points on wood with or without MCQ treatment). Our results showed that MCQ induced higher than normal levels of expression for four genes (putative annotations for isocitrate lyase, glyoxylate dehydrogenase, laccase, and oxalate decarboxylase 1), while four other genes (putative annotations for oxalate decarboxylase 2, aryl alcohol oxidase, glycoside hydrolase 5, and glycoside hydrolase 10) were repressed. The significance of these results is that we have identified several genes that appear to be coregulated, with putative functions related to copper tolerance and/or wood decay.

Short-read sequencing for genomic analysis of the brown rot fungus Fibroporia radiculosa.

The feasibility of short-read sequencing for genomic analysis was demonstrated for Fibroporia radiculosa, a copper-tolerant fungus that causes brown rot decay of wood. The effect of read quality on genomic assembly was assessed by filtering Illumina GAIIx reads from a single run of a paired-end library (75-nucleotide read length and 300-bp fragment size) at three different stringency levels and then assembling each data set with Velvet. A simple approach was devised to determine which filter stringency was "best." Venn diagrams identified the regions containing reads that were used in an assembly but were of a low-enough quality to be removed by a filter. By plotting base quality histograms of reads in this region, we judged whether a filter was too stringent or not stringent enough. Our best assembly had a genome size of 33.6 Mb, an N50 of 65.8 kb for a k-mer of 51, and a maximum contig length of 347 kb. Using GeneMark, 9,262 genes were predicted. TargetP and SignalP analyses showed that among the 1,213 genes with secreted products, 986 had motifs for signal peptides and 227 had motifs for signal anchors. Blast2GO analysis provided functional annotation for 5,407 genes. We identified 29 genes with putative roles in copper tolerance and 73 genes for lignocellulose degradation. A search for homologs of these 102 genes showed that F. radiculosa exhibited more similarity to Postia placenta than Serpula lacrymans. Notable differences were found, however, and their involvements in copper tolerance and wood decay are discussed.

Genetics and fitness costs of cyromazine resistance in the house fly (Diptera: Muscidae).

The genetic basis of cyromazine resistance was investigated in the house fly, Musca domestica L. The ED-R strain, which was collected in Mississippi and selected further in the laboratory, was 116.5-fold resistant compared with the laboratory susceptible strain, OR-S. The SEL strain, which was created by crossing ED-R with OR-S followed by three cycles of reselection and backcrossing to OR-S, was 84.7-fold resistant relative to the susceptible strain. Mortality data from reciprocal crosses of resistant and susceptible flies indicated that resistance was autosomal and not influenced by maternal effects. The relative position of probit lines from the parental strains and reciprocal crosses showed that resistance was expressed as an incompletely dominant trait with D = 0.30 and 0.32 for ED-R and SEL, respectively. To determine the number of genes involved, models of one, two, three, four, and five loci were used to compare observed and expected mortality of F1ED-R x susceptible backcross. Resistance was best described by a polygenic model of three loci when equal and additive effects of loci were assumed. Another approach, which was based on phenotypic variances, showed that nE, or the minimum number of freely segregating genetic factors for ED-R, equaled 3.07. ED-R showed greater reductions in fitness compared with SEL independent of the presence or absence of sublethal concentrations of cyromazine. These data suggested that reduced fitness was not due to deleterious pleiotropic effects of the resistance genes themselves but arose from other loci in the ED-R genotype.