Integrated transcriptomic and lipidomic analysis provides key insights into lipid content changes during pecan (Carya illinoensis) fruit development.

Pecans [Carya illinoinensis (Wangenh.) K. Koch] are highly valued for their abundance of quality healthy lipids, positively impacting human health and making themselves a preferred choice for nutritionally rich foods. However, a comprehensive understanding of the high-resolution characteristics of pecan fruit lipid composition and its dynamic changes, as well as the transfer between embryo and pericarp during development, remains incomplete. In this study, through integrated multi-omics analysis, we observed significant spatiotemporal heterogeneity in lipid changes between the pericarp and embryo. It showed smaller fluctuations and more stable lipid levels in the pericarp while exhibiting a dynamic pattern of initially increasing and then decreasing lipid content in the embryo. In this study, a total of 52 differentially expressed genes were identified, related to fatty acid synthesis and metabolism pathways in the two tissues, with changes in oleic acid and linoleic acid composition being the primary features of the embryo. This research lays the foundation for further understanding the differential regulation mechanisms of lipid metabolism between embryo and pericarp. Overall, this study filled the knowledge gap regarding dynamic changes in pericarp lipid metabolites, provided crucial insights into the lipid metabolism network during pecan fruit development, and established a scientific basis for the genetic improvement of pecan crops.

Integrated physiological, proteomic, and metabolomic analyses of pecan cultivar 'Pawnee' adaptation to salt stress.

The pecan is a salt-alkali-tolerant plant, and its fruit and wood have high economic value. This study aimed to explore the molecular mechanisms responsible for salt stress tolerance in the pecan grown under hydroponic conditions to simulate salt stress. The results showed that the photosynthetic rate (Pn) was reduced in response to salt stress, while the intercellular carbon dioxide concentrations (Ci) increased. The response of the pecan to salt stress was measured using iTRAQ (isobaric tags for relative or absolute quantitation) and LC/MS (liquid chromatography and mass spectrometry) non-targeted metabolomics technology. A total of 198 differentially expressed proteins (65 down-regulated and 133 up-regulated) and 538 differentially expressed metabolites (283 down-regulated and 255 up-regulated) were identified after exposure to salt stress for 48 h. These genes were associated with 21 core pathways, shown by Kyoto Encyclopedia of Genes and Genomes annotation and enrichment, including the metabolic pathways involved in nucleotide sugar and amino sugar metabolism, amino acid biosynthesis, starch and sucrose metabolism, and phenylpropane biosynthesis. In addition, analysis of interactions between the differentially expressed proteins and metabolites showed that two key nodes of the salt stress regulatory network, L-fucose and succinate, were up-regulated and down-regulated, respectively, suggesting that these metabolites may be significant for adaptations to salt stress. Finally, several key proteins were further verified by parallel reaction monitoring. In conclusion, this study used physiological, proteomic, and metabolomic methods to provide an important preliminary foundation for improving the salt tolerance of pecans.

Thermal treatment using microwave irradiation for the phytosanitation of Xylella fastidiosa in pecan graftwood.

Pecan bacterial leaf scorch caused by Xylella fastidiosa is an emerging disease for the U.S. and international pecan industries and can be transmitted from scion to rootstock via grafting. With the expanse of global transportation and trade networks, phytosanitation is critical for reducing the spread of economically significant pathogens, such as X. fastidiosa. We developed and evaluated thermal treatments using microwave irradiation and microwave absorbers [sterile deionized water (dH2O) and carbon nanotubes (CNTs)] as novel disinfectant methods for remediating X. fastidiosa in pecan scions. Partial submergence of scions in dH2O or CNT dispersions resulted in the transport of microwave absorbers in the xylem tissue via transpiration but did not compromise plant health. The microwave absorbers effectively transferred heat to the scion wood to reach an average temperature range of 55-65°C. Microwave radiation exposure for 6 sec (3 sec for two iterations) of CNT- or dH2O-treated scions reduced the frequency of X. fastidiosa-positive in pecan scions without negatively affecting plant viability when compared to the control group (dH2O-treated with no microwave). The efficacy of the new thermal treatments based on microwave irradiation was comparable to the conventional hot-water treatment (HWT) method, in which scions were submerged in 46°C water for 30 min. Microwave irradiation can be employed to treat X. fastidiosa-infected scions where the conventional HWT treatment is not feasible. This study is the first report to demonstrate novel thermal treatment methods based on the microwave irradiation and microwave absorbers of dH2O and CNT as an application for the phytosanitation of xylem-inhabiting bacteria in graftwood.

Genome-wide identification of lncRNAs during hickory (Carya cathayensis) flowering.

Non-coding RNAs with lengths greater than 200 bp are known as long non-coding RNAs (lncRNAs), and these RNAs play important role in gene regulation and plant development. However, to date, little is known regarding the role played by lncRNAs during flowering in hickory (Carya cathayensis). Here, we performed whole transcriptome RNA-sequencing of samples from hickory female and male floral buds, in which three samples (H0311PF, H0318PF, and H0402PF) represent pre-flowering, flowering, and post-flowering, respectively, while eight male samples collected from May 8th to June 13th as this time course are the key stage for male floral bud differentiation. We identified 2163 lncRNAs in hickory during flowering, containing 213 intronic, 1488 intergenic, and 462 antisense lncRNAs. We noticed that 510 and 648 lncRNAs were differentially expressed corresponding to female and male floral buds, respectively. And some of the lncRNAs were in a tightly tissue-specific or stage-specific manner. To further understand the roles of the lncRNAs, we predicted the function of the lncRNAs in cis- and trans-acting modes. The results showed that 924 lncRNAs were cis-correlated with 1536 protein-coding genes, while 1207 lncRNAs co-expressed (trans-acting) with 7432 protein-coding genes (R > 0.95 or R < - 0.95). These lncRNAs were all enriched in flower development-associated biological processes, i.e., circadian rhythm, vernalization response, response to gibberellin, inflorescence development, floral organ development, etc. To further understand the relationships between lncRNAs and floral-core genes, we build a co-expressing lncRNA-mRNA flowering network. We classified these floral genes into different pathway (photoperiod, vernalization, gibberellin, autonomous, and sucrose pathway) according to their particular functions. We found a set of lncRNAs that preferentially expressed in these pathways. The network showed that some lncRNAs (i.e., XLOC_038669, XLOC_017938) functioned in a particular flowering time pathway, while others (i.e., XLOC_011251, XLOC_04110) were involved in multiple pathway. Furthermore, some lncRNAs (i.e., XLOC_038669, XLOC_009597, and XLOC_049539) played roles in single or multiple pathways via interaction with each other. This study provides a genome-wide survey of hickory flower-related lncRNAs and will contribute to further understanding of the molecular mechanism underpinning flowering in hickory.

Isolation and Characterization of Three Chalcone Synthase Genes in Pecan (Carya illinoinensis).

Phenolics are a group of important plant secondary metabolites that have been proven to possess remarkable antioxidant activity and to be beneficial for human health. Pecan nuts are an excellent source of dietary phenolics. In recent years, many studies have focused on the separation and biochemical analysis of pecan phenolics, but the molecular mechanisms of phenolic metabolism in pecans have not been fully elucidated, which significantly hinders quality breeding research for this plant. Chalcone synthase (CHS) plays crucial roles in phenolic biosynthesis. In this study, three Carya illinoinensisCHSs (CiCHS1, CiCHS2, and CiCHS3), were isolated and analyzed. CiCHS2 and CiCHS3 present high expression levels in different tissues, and they are also highly expressed at the initial developmental stages of kernels in three pecan genotypes. A correlation analysis was performed between the phenolic content and CHSs expression values during kernel development. The results indicated that the expression variations of CiCHS2 and CiCHS3 are significantly related to changes in total phenolic content. Therefore, CiCHSs play crucial roles in phenolic components synthesis in pecan. We believe that the isolation of CiCHSs is helpful for understanding phenolic metabolism in C. illinoinensis, which will improve quality breeding and resistance breeding studies in this plant.

RNA-Seq Reveals Flavonoid Biosynthesis-Related Genes in Pecan ( Carya illinoinensis) Kernels.

Pecan ( Carya illinoinensis) is an important tree nut throughout the world. The high concentration of flavonoid in its kernels makes it an excellent food with health benefits. However, the molecular basis of flavonoid biosynthesis in pecan remains unclear, which hinders quality breeding in this plant. Therefore, in order to find the crucial genes involved in flavonoid biosynthesis, the changes in flavonoid profiles and the transcriptomes of pecan kernels at four developmental stages (late water, gel, dough, and mature stages) were analyzed. As a result, the highest levels of total phenolic, condensed tannin, and flavan-3-ols were observed at the "late water stage". Catechin was the most abundant flavan-3-ol at different development stages. In total, 64 773 unigenes were obtained, and 46 924 (72.44%) unigenes were annotated. After differentially expressed gene (DEG) analysis, 12 750 unique DEGs were identified. Flavonoid-related DEGs of 36 structural genes and eight MYBs were obtained. The structural gene set contained three PALs, three CHSs, two CHIs, one F3H, two F3'Hs, two F3'5'Hs, one DFR, one ANS, two LARs, and two ANRs. The expression patterns of most of the structural genes were consistent with the changes in flavonoid profiles during kernel development. We believe that this RNA-Seq data set will provide valuable resources for unraveling the molecular mechanism of flavonoid metabolism in pecan and will significantly promote genetic studies and quality breeding in this plant.

[Spatial Variation of Heavy Metals in Soils and Its Ecological Risk Evaluation in a Typical Carya cathayensis Production Area].

In recent years, the problem of soil pollution has become more and more serious. The problem of soil heavy metal pollution and its related human health risks has become a hot spot at home and abroad. Carya cathayensis is a unique high-grade woody nut and oil tree from China, and there are few reports on heavy metal pollution in Carya cathayensis plantation soils. Therefore, in order to study the spatial variability of heavy metals and the risk of pollution in Carya cathayensis soil and to promote the sustainable development of the Carya cathayensis industry, Lin'an, a typical Carya cathayensis plantation area, was selected for this study. A total of 188 soil samples were collected from the study area. We systematically studied the spatial heterogeneity of soil heavy metal content in the study area based on GIS technology, geostatistics, Moran's I, and other spatial analysis methods. The single factor pollution index method, the Nemerow comprehensive pollution index method, and the potential ecological risk assessment method were used to evaluate the heavy metal pollution in the study area. The results indicated that the mean content of soil cadmium (Cd), copper (Cu), zinc (Zn), lead (Pb), nickel (Ni), and chromium (Cr) were 0.37, 40.76, 87.61, 30.10, 28.33, 56.57 mg·kg-1, respectively. The average values of Cd and Cu were 1.33 and 2.87 times of the background values, respectively, and the average content of other heavy metals did not exceed the background values. The results of the single factor Nemerow pollution index and potential ecological risk assessment methods showed that heavy metals in the study area exceeded the soil background values in some samples, and the second grade standard of soil environmental quality was exceed for Cd, Cu, Zn, Pb, and Ni in 31.38%, 31.38%, 2.65%, 0.53%, and 17.02% of the samples, respectively. This indicated that the soils in the study area had different accumulation characteristics for Cd, Cu, Zn, Pb, Ni, and Cr, and the local soil had reached pollution levels for Cd, Cu, Zn, Pb, and Ni. Among them, Cd was the most serious, reaching the degree of strong ecological damage, followed by Cu. In general, the heavy metal contents indicated a moderate degree of ecological damage. Based on the analysis of the semi-variance function, the Cd, Cu, and Ni in the soil were best fit with exponential models, the Zn and Pb were better fit with the Gaussian model, and Cr was consistent with the spherical model. Cd, Cu, Pb, Ni, and Cr had the strong spatial autocorrelation, with Nugget/Sill ratios of 12.1%, 4.6%, 14.9%, 2.6%, and 11.2%, respectively, while the Nugget/Sill ratio of Zn was 48.8%, indicating a medium spatial autocorrelation. Moran's I and Kriging interpolation results found that the heavy metals Cd, Cu, Zn, Pb, Ni, and Cr all had obvious spatial distribution patterns and local spatial aggregation phenomena. The high values of heavy metals in soils were mainly found in Taiyang, Daoshi, Qingliangfeng, Heqiao, and Tuankou, and the probability of the risk for contamination by Cd and Cu was higher in the study area. The high values of Cd, Cu, Zn, Ni, and Cr were mainly related to mining, while Pb was closely related to the application of potassium.

Transcriptome analysis of pecan seeds at different developing stages and identification of key genes involved in lipid metabolism.

Pecan is an economically important nut crop tree due to its unique texture and flavor properties. The pecan seed is rich of unsaturated fatty acid and protein. However, little is known about the molecular mechanisms of the biosynthesis of fatty acids in the developing seeds. In this study, transcriptome sequencing of the developing seeds was performed using Illumina sequencing technology. Pecan seed embryos at different developmental stages were collected and sequenced. The transcriptomes of pecan seeds at two key developing stages (PA, the initial stage and PS, the fast oil accumulation stage) were also compared. A total of 82,155 unigenes, with an average length of 1,198 bp from seven independent libraries were generated. After functional annotations, we detected approximately 55,854 CDS, among which, 2,807 were Transcription Factor (TF) coding unigenes. Further, there were 13,325 unigenes that showed a 2-fold or greater expression difference between the two groups of libraries (two developmental stages). After transcriptome analysis, we identified abundant unigenes that could be involved in fatty acid biosynthesis, degradation and some other aspects of seed development in pecan. This study presents a comprehensive dataset of transcriptomic changes during the seed development of pecan. It provides insights in understanding the molecular mechanisms responsible for fatty acid biosynthesis in the seed development. The identification of functional genes will also be useful for the molecular breeding work of pecan.

Genome-wide analysis of miRNAs in Carya cathayensis.

BACKGROUND: MicroRNA (miRNA) plays an important role in plant development regulation. Hickory is an economically important plant in which the amount of flowering determines its production. RESULTS: Here, 51 conserved miRNAs, which belong to 16 families and 195 novel miRNAs were identified in hickory genome. For each conserved miRNA family, we used sequences from hickory and other plants to construct a phylogenetic tree, which shows that each family has members in hickory. Some of the conserved miRNA families (i.e., miR167 and miR397) have more members in hickory than in other plants because of gene expansion. MiR166 exhibited tandem duplication with three copies being observed. Many members of these conserved miRNA families were detected in hickory flowers, and the expression patterns of target genes were opposite to those of the related miRNAs, indicating that miRNAs may have important functions in floral regulation of hickory. CONCLUSIONS: Taken together, a comprehensive analysis was conducted to identify miRNAs produced in hickory flower organs, demonstrating functional conservation and diversity of miRNA families among hickory, Arabidopsis, grape, and poplar.

Control of Pecan Weevil With Microbial Biopesticides.

The pecan weevil, Curculio caryae (Horn) (Coleoptera: Curculionidae), is a key pest of pecans Carya illinoinensis ([Wangenh.] K. Koch) (Fagales: Juglandaceae). Control recommendations rely on broad spectrum chemical insecticides. Due to regulatory and environmental concerns, effective alternatives for C. caryae control must be sought for pecan production in conventional and organic systems. We explored the use of microbial biopesticides for control of C. caryae in Georgia pecan orchards. Three experiments were conducted. The first investigated an integrated microbial control approach in an organic system at two locations. Three microbial agents, Grandevo (based on byproducts of the bacterium Chromobacterium subtsugae Martin, Gundersen-Rindal, Blackburn & Buyer), the entomopathogenic nematode Steinernema carpocapsae (Weiser), and entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin, were applied to each treatment plot (0.6 ha) at different times during the season. A second experiment compared the effects of S. carpocapsae and B. bassiana applied as single treatments relative to application of both agents (at different times); survival of C. caryae was assessed approximately 11 mo after larvae were added to pots sunk in an organic pecan orchard. In a conventional orchard (with 1.0 ha plots), the third experiment compared Grandevo applications to a commonly used regime of chemical insecticides (carbaryl alternated with a pyrethroid). All experiments were repeated in consecutive years. The combined pest management tactic (experiment 1) reduced C. caryae infestation relative to non-treated control plots in both locations in 2014 and one of the two locations in 2015 (the other location had less than 1% infestation). In experiment 2, no differences among combined microbial treatments, single-applied microbial treatments or different numbers of application were observed, yet all microbial treatments reduced C. caryae survival relative to the control. In the third experiment, both Grandevo and standard chemical insecticide applications resulted in lower weevil infestation than the control (both years) and there was no difference between the insecticide treatments in 2014 although the chemical insecticide regime had slightly lower infestation in 2015. These results provide evidence that microbial biopesticides can substantially reduce pecan weevil infestations in organic and nonorganic systems.

Functional diversity response to hardwood forest management varies across taxa and spatial scales.

Contemporary forest management offers a trade-off between the potential positive effects of habitat heterogeneity on biodiversity, and the potential harm to mature forest communities caused by habitat loss and perforation of the forest canopy. While the response of taxonomic diversity to forest management has received a great deal of scrutiny, the response of functional diversity is largely unexplored. However, functional diversity may represent a more direct link between biodiversity and ecosystem function. To examine how forest management affects diversity at multiple spatial scales, we analyzed a long-term data set that captured changes in taxonomic and functional diversity of moths (Lepidoptera), longhorned beetles (Coleoptera: Cerambycidae), and breeding birds in response to contemporary silvicultural systems in oak-hickory hardwood forests. We used these data sets to address the following questions: how do even- and uneven-aged silvicultural systems affect taxonomic and functional diversity at the scale of managed landscapes compared to the individual harvested and unharvested forest patches that comprise the landscapes, and how do these silvicultural systems affect the functional similarity of assemblages at the scale of managed landscapes and patches? Due to increased heterogeneity within landscapes, we expected even-aged silviculture to increase and uneven-aged silviculture to decrease functional diversity at the landscape level regardless of impacts at the patch level. Functional diversity responses were taxon-specific with respect to the direction of change and time since harvest. Responses were also consistent across patch and landscape levels within each taxon. Moth assemblage species richness, functional richness, and functional divergence were negatively affected by harvesting, with stronger effects resulting from uneven-aged than even-aged management. Longhorned beetle assemblages exhibited a peak in species richness two years after harvesting, while functional diversity metrics did not differ between harvested and unharvested patches and managed landscapes. The species and functional richness of breeding bird assemblages increased in response to harvesting with more persistent effects in uneven- than in even-aged managed landscapes. For moth and bird assemblages, species turnover was driven by species with more extreme trait combinations. Our study highlights the variability of multi-taxon functional diversity in response to forest management across multiple spatial scales.

Trap Height Affects Capture of Lady Beetles (Coleoptera: Coccinellidae) in Pecan Orchards.

There is scarce information regarding the vertical stratification of predaceous Coccinellidae in tall trees. Although numerous studies have been done in orchards and forests, very few studies have assessed the occurrence of predaceous Coccinellidae high in tree canopies. The objective of this study was to examine the abundance of Coccinellidae at different heights in mature pecan, Carya illinoinensis (Wangenh.) K. Koch, orchards with tall trees. From spring through late fall during 2013 and 2014, yellow pyramidal Tedders traps were suspended in the pecan canopy at 6.1 and 12.2 m, in addition to being placed on the ground (0 m). The exotic species Harmonia axyridis and Coccinella septempunctata accounted for a high percentage of trap capture during this study. Except for Olla v-nigrum, low numbers of native species (Hippodamia convergens, Coleomegilla maculata, Cycloneda munda, Scymnus spp., and Hyperaspis spp.) were captured. However, significantly more were captured in ground traps rather than in canopy traps with the exception of O. v-nigrum. Similar to most native species, significantly more C. septempunctata were captured in ground traps than canopy traps. This contrasts sharply with H. axyridis captured similarly at all trap heights. The ability to exploit resources across vertical strata, unlike many intraguild predators, may be an underestimated factor helping to explain the invasiveness of H. axyridis.

Morphological and molecular characterization of Cladosporium cladosporioides species complex causing pecan tree leaf spot.

The objective of this study was to characterize species of the Cladosporium cladosporioides complex isolated from pecan trees (Carya illinoinensis) with symptoms of leaf spot, based on morphological and molecular approaches. Morphological attributes were assessed using monosporic cultures on potato dextrose agar medium, which were examined for mycelial growth, sporulation, color, and conidia and ramoconidia size. Molecular characterization comprised isolation of DNA and subsequent amplification of the translation elongation factor 1α (TEF-1α) region. Three species of the C. cladosporioides complex were identified: C. cladosporioides, Cladosporium pseudocladosporioides, and Cladosporium subuliforme. Sporulation was the most important characteristic differentiating species of this genus. However, morphological features must be considered together with molecular analysis, as certain characters are indistinguishable between species. TEF-1αcan be effectively used to identify and group isolates belonging to the C. cladosporioides complex. The present study provides an important example of a methodology to ascertain similarity between isolates of this complex causing leaf spot in pecan trees, which should facilitate future pathogenicity studies.

Analysis of pecan cultivars Mahan and Western in East China.

Pecan (Carya illinoensis) has been introduced to East China for over one hundred years, but its planting is still only occurring at a small scale. The key limiting factor is its low yield. To enhance the yield pecan in East China, two pecan cultivars, Mahan and Western, were examined. Twenty traits describing phasic development, yield, nut quality, and cultural practice were investigated. We found that pecan cultivar Mahan gives a higher yield and nut quality than cultivar Western. We recommend interplanting of cultivar Pawnee to act as a pollinator tree. Appropriate cultivation practices that can be implemented to enhance fruit yield of cultivars Mahan and Western include soil-applied paclobutrazol (PBZ) at certain concentrations, pinching, and supplementary pollination. For example, the addition of 1.25 g/m2 of PBZ inhibits pecan branch growth and stimulates short bearing branches, which promotes fruit yield. We found that soil-applied PBZ reached optimal performance 82 days after application. A pinching length of 40 cm resulted in a fruit yield increase. In addition, grafting and transplantation may promote male flowering, but delays female flowering. These cultural practices may provide insights that can be used to improve pecan cultivation in East China.

Identification of microRNAs differentially expressed involved in male flower development.

Hickory (Carya cathayensis Sarg.) is one of the most economically important woody trees in eastern China, but its long flowering phase delays yield. Our understanding of the regulatory roles of microRNAs (miRNAs) in male flower development in hickory remains poor. Using high-throughput sequencing technology, we have pyrosequenced two small RNA libraries from two male flower differentiation stages in hickory. Analysis of the sequencing data identified 114 conserved miRNAs that belonged to 23 miRNA families, five novel miRNAs including their corresponding miRNA*s, and 22 plausible miRNA candidates. Differential expression analysis revealed 12 miRNA sequences that were upregulated in the later (reproductive) stage of male flower development. Quantitative real-time PCR showed similar expression trends as that of the deep sequencing. Novel miRNAs and plausible miRNA candidates were predicted using bioinformatic analysis methods. The miRNAs newly identified in this study have increased the number of known miRNAs in hickory, and the identification of differentially expressed miRNAs will provide new avenues for studies into miRNAs involved in the process of male flower development in hickory and other related trees.

SVP-like MADS-box protein from Carya cathayensis forms higher-order complexes.

To properly regulate plant flowering time and construct floral pattern, MADS-domain containing transcription factors must form multimers including homo- and hetero-dimers. They are also active in forming hetero-higher-order complexes with three to five different molecules. However, it is not well known if a MADS-box protein can also form homo-higher-order complex. In this study a biochemical approach is utilized to provide insight into the complex formation for an SVP-like MADS-box protein cloned from hickory. The results indicated that the protein is a heterogeneous higher-order complex with the peak population containing over 20 monomers. Y2H verified the protein to form homo-complex in yeast cells. Western blot of the hickory floral bud sample revealed that the protein exists in higher-order polymers in native. Deletion assays indicated that the flexible C-terminal residues are mainly responsible for the higher-order polymer formation and the heterogeneity. Current results provide direct biochemical evidences for an active MADS-box protein to be a high order complex, much higher than a quartermeric polymer. Analysis suggests that a MADS-box subset may be able to self-assemble into large complexes, and thereby differentiate one subfamily from the other in a higher-order structural manner. Present result is a valuable supplement to the action of mechanism for MADS-box proteins in plant development.

Analysis of pecan nut (Carya illinoinensis) unsaponifiable fraction. Effect of ripening stage on phytosterols and phytostanols composition.

Changes in 4-desmethylsterol, 4-monomethylsterol, 4,4-dimethylsterol and phytostanol composition were quantitatively and qualitatively investigated during the ripening of three varieties of Tunisian-grown pecan nuts (Mahan, Moore and Burkett). These components have many health benefits, especially in lowering LDL-cholesterol and preventing heart disease. The phytosterol composition of whole pecan kernel was quantified by Gas Chromatography-Flame Ionisation Detection (GC-FID) and identified by Gas Chromatography-Mass Spectrometry (GC-MS). Fifteen phytosterols and one phytostanol were quantified. The greatest amount of phytosterols (2852.5mg/100g of oil) was detected in Mahan variety at 20 weeks after the flowering date (WAFD). Moore had the highest level of phytostanols (7.3mg/100g of oil) at 20 WAFD. Phytosterol and phytostanol contents showed a steep decrease during pecan nut development. Results from the quantitative characterisation of pecan nut oils revealed that ß-sitosterol, Δ5-avenasterol, and campesterol were the most abundant phytosterol compounds at all ripening stages.

Use of transcriptome sequencing to understand the pistillate flowering in hickory (Carya cathayensis Sarg.).

BACKGROUND: Different from herbaceous plants, the woody plants undergo a long-period vegetative stage to achieve floral transition. They then turn into seasonal plants, flowering annually. In this study, a preliminary model of gene regulations for seasonal pistillate flowering in hickory (Carya cathayensis) was proposed. The genome-wide dynamic transcriptome was characterized via the joint-approach of RNA sequencing and microarray analysis. RESULTS: Differential transcript abundance analysis uncovered the dynamic transcript abundance patterns of flowering correlated genes and their major functions based on Gene Ontology (GO) analysis. To explore pistillate flowering mechanism in hickory, a comprehensive flowering gene regulatory network based on Arabidopsis thaliana was constructed by additional literature mining. A total of 114 putative flowering or floral genes including 31 with differential transcript abundance were identified in hickory. The locations, functions and dynamic transcript abundances were analyzed in the gene regulatory networks. A genome-wide co-expression network for the putative flowering or floral genes shows three flowering regulatory modules corresponding to response to light abiotic stimulus, cold stress, and reproductive development process, respectively. Totally 27 potential flowering or floral genes were recruited which are meaningful to understand the hickory specific seasonal flowering mechanism better. CONCLUSIONS: Flowering event of pistillate flower bud in hickory is triggered by several pathways synchronously including the photoperiod, autonomous, vernalization, gibberellin, and sucrose pathway. Totally 27 potential flowering or floral genes were recruited from the genome-wide co-expression network function module analysis. Moreover, the analysis provides a potential FLC-like gene based vernalization pathway and an 'AC' model for pistillate flower development in hickory. This work provides an available framework for pistillate flower development in hickory, which is significant for insight into regulation of flowering and floral development of woody plants.

Effects of entomopathogenic fungus species, and impact of fertilizers, on biological control of pecan weevil (Coleoptera: Curculionidae).

The pecan weevil, Curculio caryae (Horn), is a key pest of pecan, Carya illinoinensis (Wangenh.) K. Koch. Prior research indicated the potential for use of Hypocreales fungi to suppress C. caryae. We compared the efficacy of two fungal spp., Beauveria bassiana (GHA strain) and Metarhizium brunneum (F52), in their ability to cause C. caryae mortality. The fungus, B. bassiana, was applied to trunks of pecan trees (a method previously shown to be effective in C. caryae suppression) and efficacy was compared with M. brunneum applied to the ground or to the trunk with or without SoyScreen Oil as an ultraviolet protecting agent. Results indicated B. bassiana to be superior to M. brunneum regardless of application method; consequently, the potential for applying B. bassiana to control C. caryae was explored further. Specifically, the impact of different fertilizer regimes (as used by pecan growers) on the persistence of B. bassiana (GHA) in soil was determined. B. bassiana was applied to soil in a pecan orchard after one of several fertilizer treatments--i.e., ammonium nitrate, crimson clover, poultry litter, clover plus poultry litter, and a no-fertilizer control. B. bassiana persistence up to 49 d in 2009 and 2010 was assessed by plating soil onto selective media and determining the number of colony forming units, and by baiting soil with a susceptible host, Galleria mellonella (L.). Fertilizer treatments did not impact B. bassiana persistence. We conclude that standard fertilizers for nitrogen management, when applied according to recommended practices, are unlikely to negatively impact survival of B. bassiana in pecan orchards when the fungus is applied for C. caryae suppression during weevil emergence. Additional research on interactions between entomopathogenic fungi and fertilizer amendments (or other tree nutrition or soil management practices) is merited.

Discovery and profiling of novel and conserved microRNAs during flower development in Carya cathayensis via deep sequencing.

Hickory (Carya cathayensis Sarg.) is an economically important woody plant in China, but its long juvenile phase delays yield. MicroRNAs (miRNAs) are critical regulators of genes and important for normal plant development and physiology, including flower development. We used Solexa technology to sequence two small RNA libraries from two floral differentiation stages in hickory to identify miRNAs related to flower development. We identified 39 conserved miRNA sequences from 114 loci belonging to 23 families as well as two novel and ten potential novel miRNAs belonging to nine families. Moreover, 35 conserved miRNA*s and two novel miRNA*s were detected. Twenty miRNA sequences from 49 loci belonging to 11 families were differentially expressed; all were up-regulated at the later stage of flower development in hickory. Quantitative real-time PCR of 12 conserved miRNA sequences, five novel miRNA families, and two novel miRNA*s validated that all were expressed during hickory flower development, and the expression patterns were similar to those detected with Solexa sequencing. Finally, a total of 146 targets of the novel and conserved miRNAs were predicted. This study identified a diverse set of miRNAs that were closely related to hickory flower development and that could help in plant floral induction.