Tissue culture and Agrobacterium-mediated genetic transformation of the oil crop sunflower.

Sunflower is one of the four major oil crops in the world. 'Zaoaidatou' (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle, high yield, and high resistance to abiotic stress. However, the ability to tolerate adervesity is limited. Therefore, in this study, we used the retention line of backbone parent ZADT as material to establish its tissue culture and genetic transformation system for new variety cultivating to enhance resistance and yields by molecular breeding. The combination of 0.05 mg/L IAA and 2 mg/L KT in MS was more suitable for direct induction of adventitious buds with cotyledon nodes and the addition of 0.9 mg/L IBA to MS was for adventitious rooting. On this basis, an efficient Agrobacterium tumefaciens-mediated genetic transformation system for ZADT was developed by the screening of kanamycin and optimization of transformation conditions. The rate of positive seedlings reached 8.0%, as determined by polymerase chain reaction (PCR), under the condition of 45 mg/L kanamycin, bacterial density of OD600 0.8, infection time of 30 min, and co-cultivation of three days. These efficient regeneration and genetic transformation platforms are very useful for accelerating the molecular breeding process on sunflower.

Pangenome characterization and analysis of the NAC gene family reveals genes for Sclerotinia sclerotiorum resistance in sunflower (Helianthus annuus).

BACKGROUND: Sunflower (Helianthus annuus) is one of the most important economic crops in oilseed production worldwide. The different cultivars exhibit variability in their resistance genes. The NAC transcription factor (TF) family plays diverse roles in plant development and stress responses. With the completion of the H. annuus genome sequence, the entire complement of genes coding for NACs has been identified. However, the reference genome of a single individual cannot cover all the genetic information of the species. RESULTS: Considering only a single reference genome to study gene families will miss many meaningful genes. A pangenome-wide survey and characterization of the NAC genes in sunflower species were conducted. In total, 139 HaNAC genes are identified, of which 114 are core and 25 are variable. Phylogenetic analysis of sunflower NAC proteins categorizes these proteins into 16 subgroups. 138 HaNACs are randomly distributed on 17 chromosomes. SNP-based haplotype analysis shows haplotype diversity of the HaNAC genes in wild accessions is richer than in landraces and modern cultivars. Ten HaNAC genes in the basal stalk rot (BSR) resistance quantitative trait loci (QTL) are found. A total of 26 HaNAC genes are differentially expressed in response to Sclerotinia head rot (SHR). A total of 137 HaNAC genes are annotated in Gene Ontology (GO) and are classified into 24 functional groups. GO functional enrichment analysis reveals that HaNAC genes are involved in various functions of the biological process. CONCLUSIONS: We identified NAC genes in H. annuus (HaNAC) on a pangenome-wide scale and analyzed S. sclerotiorum resistance-related NACs. This study provided a theoretical basis for further genomic improvement targeting resistance-related NAC genes in sunflowers.

Morphological and Molecular Characterization of Bacterial Pathogens Associated with Leaf Mottle of Sunflower in Northern Kazakhstan.

Leaf mottle is a serious disease in the common sunflower (Helianthus annuus L.), which affects plant growth and development and seed quality and yield. Over the past few years, the North Kazakhstan region, a sunflower-producing area in Kazakhstan, has been seriously affected by leaf mottle. Since 2021, symptomatic leaves have been collected from production areas of this base to determine the pathogens causing sunflower foliar diseases. One hundred bacterial strains were isolated, and two genera and five species were identified based on morphological characteristics, molecular genetics, and phylogenetic analysis (16S gene region). The genus Bacillus was represented by four species: Bacillus subtilis, B. megaterium, B. amyloliquefaciens, and B. flexus. The genus Paenibacillus was represented by one species, P. peoriae. Pathogenicity experiments showed that B. subtilis, B. megaterium, B. flexus, and P. peoriae could cause leaf mottle disease symptoms. However, disease symptoms caused by B. flexus were highly similar to those observed on infected leaves under natural conditions in the field. Therefore, these bacterial isolates were found to be the primary pathogens causing sunflower leaf mottle, and B. flexus was the most common and virulent pathogen in this study. In addition, this is the first report of B. megaterium, B. flexus, and P. peoriae as pathogens associated with sunflower leaf mottle in Kazakhstan.

The combination of multiple plant growth promotion and hydrolytic enzyme producing rhizobacteria and their effect on Jerusalem artichoke growth improvement.

Rhizobacteria are well recognized for their beneficial multifunctions as key promoters of plant development, suppressing pathogens, and improving soil health. In this study, experiments focused on characterizing the plant growth promotion (PGP) and extracellular hydrolase production traits of rhizobacteria, and their impact on Jerusalem artichoke growth. A total of 50 isolates proved capable of either direct PGP or hydrolase-producing traits. Two promising strains (Enterobacter cloacae S81 and Pseudomonas azotoformans C2-114) showed potential on phosphate and potassium solubilization, IAA production, and 1-aminocyclopropane-1-carboxylic acid deaminase activity and hydrolase production. A hydrolase-producing strain (Bacillus subtilis S42) was able to generate cellulase, protease, amylase, ß-glucosidase, and phosphatase. These three selected strains also gave positive results for indirect PGP traits such as siderophore, ammonia, oxalate oxidase, polyamine, exopolysaccharide, biofilm, motility, and tolerance to salinity and drought stress. Colonization was observed using a scanning electron microscope and rhizobacteria appeared at the root surface. Interestingly, inoculation with consortia strains (S42, S81, and C2-114) significantly increased all plant parameters, including height, biomass, root (length, surface, diameter, and volume), and tuber fresh weight. Therefore, we recommend that potential consortia of PGP and hydrolase-producing rhizobacteria be employed as a biofertilizer to improve soil and boost crop productivity.

Mutation and sequencing-based cloning and functional studies of a rust resistance gene in sunflower (Helianthus annuus).

Rust, caused by the fungus Puccinia helianthi Schwein., is one of the most devastating diseases of sunflower (Helianthus annuus L.), affecting global production. The rust R gene R11 in sunflower line HA-R9 shows broad-spectrum resistance to P. helianthi virulent races and was previously mapped to an interval on sunflower chromosome 13 encompassing three candidate genes annotated in the XRQr1.0 reference genome assembly. In the current study, we combined ethyl methane sulfonate (EMS) mutagenesis with targeted region capture and PacBio long-read sequencing to clone the R11 gene. Sequencing of a 60-kb region spanning the R11 locus from the R11 -HA-R9 rust-resistant line and three EMS-induced susceptible mutants facilitated the identification of R11 and definition of induced mutations. The R11 gene is predicted to have a single 3996-bp open reading frame and encodes a protein of 1331 amino acids with CC-NBS-LRR domains typical of genes conferring plant resistance to biotrophic pathogens. Point mutations identified in the R11 rust-susceptible mutants resulted in premature stop codons, consistent with loss of function leading to rust susceptibility. Additional functional studies using comparative RNA sequencing of the resistant line R11 -HA-R9 and R11 -susceptible mutants revealed substantial differences in gene expression patterns associated with R11 -mediated resistance at 7 days post-inoculation with rust, and uncovered the potential roles of terpenoid biosynthesis and metabolism in sunflower rust resistance.

Genome-Wide Association Mapping in Sunflower (Helianthus annuus) Reveals Common Loci and Putative Candidate Genes for Resistance to Diaporthe gulyae and D. helianthi Causing Phomopsis Stem Canker.

Diaporthe gulyae and D. helianthi cause Phomopsis stem canker of sunflower (Helianthus annuus L.) in the United States. Because Phomopsis stem canker did not gain importance until the disease epidemic in 2010, limited studies were conducted to understand the genetic basis of sunflower resistance to D. gulyae and D. helianthi. The objectives of this study were to evaluate the United States Department of Agriculture cultivated accessions for resistance to D. gulyae and D. helianthi as well as to utilize genome-wide association studies (GWAS) to identify quantitative trait loci (QTLs) and putative candidate genes underlying those loci common to both organisms. For each fungus, 213 accessions were screened in a complete randomized design in the greenhouse and the experiment was repeated once. Six plants per accession were inoculated with a single isolate of D. gulyae or D. helianthi at four to six true leaves using the mycelium-contact inoculation method. At 15 days (D. gulyae) and 30 days (D. helianthi) postinoculation, accessions were evaluated for disease severity and compared with the susceptible confection inbred PI 552934. GWAS identified 28 QTLs common to the two fungi, and 24 genes overlapped close to these QTLs. Additionally, it was observed that the resistance QTLs derived mainly from landraces rather than from wild species. Seventeen putative candidate genes associated with resistance to D. gulyae or D. helianthi were identified that may be related to plant-pathogen interactions. These findings advanced our understanding of the genetic basis of resistance to D. gulyae and D. helianthi and will help develop resources for genomics-assisted breeding.

Application of malrstone-based conditioner and plantation of Jerusalem artichoke improved properties of saline-alkaline soil in Inner Mongolia.

Soil salinization is a critical environmental issue restricting agricultural production. Inner Mongolia is one of the areas with severe land salinization in China. This study aimed to investigate the effects of conditioning agent (containing marlstone and a range of enzymes) and cultivating Jerusalem artichoke on saline soils in Inner Mongolia. The effects of conditioner (0, 0.06 and 0.18 kg/m2) on soil physical, chemical and biological properties, including soil carbon fractions and microbiota in saline soils planted with Jerusalem artichoke, were characterized. The results showed that soil salinity was reduced significantly after cultivating Jerusalem artichoke and declined also after the conditioner addition. The application of conditioner increased the content of DOC (dissolved organic carbon), HFOC (heavy fraction organic carbon) and the content of aggregates >0.25 mm compared to the soil planted with Jerusalem artichoke alone. The relative abundance of halophilic bacteria such as Thioalkalivibrio and Thiohalobacter was greater in the CK (non-treated control). By contrast, the relative abundance of microorganisms with the carbon assimilation and nitrogen fixation capacities, such as Cyanobacteria and Rhodovulum, was greater in the conditioner-treated and Jerusalem artichoke-planted treatments. The planting of Jerusalem artichoke reduced soil salinity, increased soil organic carbon fractions, improved soil structure, and altered the soil microbial community, with the application of the conditioning agent enhancing these positive changes. The co-occurrence network structure of "Jerusalem artichoke-conditioner-saline soil-soil microorganism" was established, which provided scientific basis for Jerusalem artichoke-conditioner to improve saline soil.

Genome-Wide Association Studies in Sunflower: Towards Sclerotinia sclerotiorum and Diaporthe/Phomopsis Resistance Breeding.

Diseases caused by necrotrophic fungi, such as the cosmopolitan Sclerotinia sclerotiorum and the Diaporthe/Phomopsis complex, are among the most destructive diseases of sunflower worldwide. The lack of complete resistance combined with the inefficiency of chemical control makes assisted breeding the best strategy for disease control. In this work, we present an integrated genome-wide association (GWA) study investigating the response of a diverse panel of sunflower inbred lines to both pathogens. Phenotypic data for Sclerotinia head rot (SHR) consisted of five disease descriptors (disease incidence, DI; disease severity, DS; area under the disease progress curve for DI, AUDPCI, and DS, AUDPCS; and incubation period, IP). Two disease descriptors (DI and DS) were evaluated for two manifestations of Diaporthe/Phomopsis: Phomopsis stem canker (PSC) and Phomopsis head rot (PHR). In addition, a principal component (PC) analysis was used to derive transformed phenotypes as inputs to a univariate GWA (PC-GWA). Genotypic data comprised a panel of 4269 single nucleotide polymorphisms (SNP), generated via genotyping-by-sequencing. The GWA analysis revealed 24 unique marker-trait associations for SHR, 19 unique marker-trait associations for Diaporthe/Phomopsis diseases, and 7 markers associated with PC1 and PC2. No common markers were found for the response to the two pathogens. Nevertheless, epistatic interactions were identified between markers significantly associated with the response to S. sclerotiorum and Diaporthe/Phomopsis. This suggests that, while the main determinants of resistance may differ for the two pathogens, there could be an underlying common genetic basis. The exploration of regions physically close to the associated markers yielded 364 genes, of which 19 were predicted as putative disease resistance genes. This work presents the first simultaneous evaluation of two manifestations of Diaporthe/Phomopsis in sunflower, and undertakes a comprehensive GWA study by integrating PSC, PHR, and SHR data. The multiple regions identified, and their exploration to identify candidate genes, contribute not only to the understanding of the genetic basis of resistance, but also to the development of tools for assisted breeding.

Effect of the Nanoclay Treated Streptomyces sp. UTMC 3136 as a Bioformulation on the Growth of Helianthus annuus.

Biofertilizers based on plant growth-promoting actinobacteria are used as potential alternatives to chemical fertilizers for sustainable agricultural systems. However, successful application of PGPA to agricultural land is challenging. The present study was an attempt to develop and evaluate the effect of a low-cost biofertilizer named NCTS (nanoclay-treated-Streptomyces) based on Streptomyces sp. UTMC 3136 spores amalgamated in a hybrid material of nanoclay Na-montmorillonite K10-glycerol-water substrate. In addition, the effect of NCTS on sunflower growth was investigated. In vivo tests showed a statistically significant increase in the agronomic characteristics of sunflowers treated with NCTS. Characterization of NCTS by FTIR, Raman spectroscopy, and scanning electron microscopy testified to the structural alignment and good adhesion of NCTS components. The viability of NCTS was 100% after 72 h of storage at 4 °C. Overall, the present study attempted to validate the efficacy of the formulation of Streptomyces sp. UTMC 3136 in nanoclay for growth improvement of sunflower. It was the first study to show the administration of PGPA in combination with nanomaterials as a growth enhancing biofertilization agent for sunflower.

Microbe- plant interaction as a sustainable tool for mopping up heavy metal contaminated sites.

BACKGROUND: Phytoremediation is a green technology that removes heavy metal (HM) contamination from the environment by using HM plant accumulators. Among soil microbiota, plant growth promoting bacteria (PGPR) have a role influencing the metal availability and uptake. METHODS: This current study evaluates the plant growth promoting qualities of microbial flora isolated from rhizosphere, plant roots, and marine aquatic HMs polluted environments in Alexandria through several biochemical and molecular traits. Metal contents in both collected soils and plant tissues were measured. Transcript levels of marker genes (HMA3 and HMA4) were analyzed. RESULTS: Three terrestrial and one aquatic site were included in this study based on the ICP-MS identification of four HMs (Zn, Cd, Cu, and Ni) or earlier reports of HMs contamination. Using the VITEK2 bacterial identification system, twenty-two bacteria isolated from these loci were biochemically described. Pseudomonas and Bacillus were the most dominant species. Furthermore, the soil microbiota collected from the most contaminated HMs site with these two were able to enhance the Helianthus annuus L. hyper-accumulation capacity significantly. Specifically, sunflower plants cultivated in soils with HMs adapted bacteria were able to accumulate about 1.7-2.5-folds more Zn and Cd in their shoots, respectively. CONCLUSION: The influence of PGPR to stimulate crop growth under stress is considered an effective strategy. Overall, our findings showed that plants cultivated in HMs contaminated sites in the presence of PGPR were able to accumulate significant amounts of HMs in several plant parts than those cultivated in soils lacking microbiota.

Antimicrobial Activity of Sunflower (Helianthus annuus) Seed for Household Domestic Water Treatment in Buhera District, Zimbabwe.

Various plants have been used by humans for a very long time, and the uses vary, including food, medicine, toothpaste, dyes, food preservatives, water treatment, and beer brewing, among others. For food preservation and water treatment, the plant must have antimicrobial properties which are biocidal. For this research, extracts were obtained from sunflower (Helianthus annuus) seeds. The extracts were assessed for the presence of antimicrobial properties against three groups of bacteria, including faecal coliforms, total coliforms, and Escherichia coli (E. coli). Dosages of ground sunflower seeds ranging from 0.5 g to 4 g were administered to the three bacterial species and their susceptibilities to the antimicrobial agents were measured and recorded. The results indicate the presence of antimicrobial properties in sunflower. The antimicrobial activities were more effective on E. coli, with an average zone of inhibition of 12 mm with a 3 g dosage of sunflower seed extract. This was followed by total coliforms (11 mm) and lastly faecal coliforms (11 mm). These findings suggested that sunflower seeds proved to be potentially effective in treating water against microbial contaminants.

Role of PGPR on the physiology of sunflower irrigated with produced water containing high total dissolved solids (TDS) and its residual effects on soil fertility.

The present study was conducted to evaluate the bioremediation potential of plant growth-promoting rhizobacteria (PGPR) PGPR isolates from high total dissolved solids (TDS) bearing produced water on the water quality, soil physicochemical properties and growth and physiology of sunflower irrigated with high TDS bearing produced water having salinity level 130 times higher above seawater and also containing traces of oil and grease. Seeds of sunflower hybrid Parsun 3 were soaked for 3-4 h prior to sowing in 72 h old culture of PGPR strains W1 and W2 isolated from high TDS bearing polluted water. The control plants were irrigated with 90% diluted TDS water supplemented with 5 ml LB media. Whereas, the inoculated plants were irrigated with 90% diluted TDS water supplemented with 5 ml PGPR inocula.in LB media. The plants were grown under natural conditions. The 16S rRNA sequence analyses identified the isolate W1 bearing 100% similarity with the plant growth-promoting rhizobacteria (PGPR) Ralstonia pickettii and W2 bearing 99.7% similarity with Brevibacillus invocatus. Both the isolate were catalase and oxidase positive. The Ralstonia pickettii and Brevibacillus invocatus treatments decreased the EC and TDS values significantly such that the EC and TDS values of 90% diluted TDS water were 29 times and 19 times higher than tap water. Sodium adsorption ratio (SAR), organic matter, nitrogen, potassium, magnesium and carbon content were 1.96, 1.10, 2.28 1.20, 6.63 and 1.00 times greater than control in the rhizosphere soil of Ralstonia pickettii inoculated plants irrigated with high TDS bearing water There were significant increases in plant growth, sugar, flavonoids and phenolics, chlorophyll b, total chlorophyll, carotenoids content and activities of superoxide dismutase, catalase and peroxidase in plants inoculated with Ralstonia pickettii and Brevibacillus invocatus. The flavonoids, phenolics and proline contents were 0.54, 0.72 and 0.30 times higher in Ralstonia pickettii inoculated plants. Shoot/root dry weight ratio was about (50%) lower than control in Ralstonia pickettii and Brevibacillus invocatus treatments. Ralstonia pickettii was more effective than Brevibacillus invocatus to combat oxidative and osmotic stresses. It is inferred that the high TDS bearing produced water from oil factory harbor Plant growth-promoting rhizobacteria (PGPR) having the potential to combat high salinity stress in plants when used as bioinoculant. The broth culture containing the bacteria may be supplemented with the saline water used for irrigation as it provides nutrients for the growth and proliferation of bacteria present in the saline water and hence the synergistic action of bacterial inocula with the indigenous bacteria present in saline water may better alleviate osmotic and oxidative stresses of plants encountered under salinity stress. The residual effect of Ralstonia pickettii on organic matter and Ca, Mg, K and P content of the rhizosphere soil was notably higher for succeeding crops. Novelty statement This is the first report demonstrating that rhizobacteria can proliferate in water containing salinity higher above seawater in addition to oil grease and TSS. Their efficiency to reduce TDS can be augmented by an exogenous supply of LB broth culture of PGPR isolated from the polluted water. These indigenous rhizobacteria when used as bioinoculant on the plant can act as plant growth promoters as well as bioremediation of salinity effects.

Identification of lead-resistant rhizobacteria of Carthamus tinctorius and their effects on lead absorption of Sunflower.

AIMS: Using rhizobacteria as plant growth-promoting agents for improving heavy-metal phytoremediation processes in contaminated soil has attracted a lot of attention mainly because of their eco-friendliness. The aim of this study was the evaluation of lead phytoremediation by Carthamus tinctorius improved with the isolated and molecularly identified lead-resistant rhizobacteria. METHODS AND RESULTS: Rhizobacteria were isolated from C. tinctorius root and was identified using macroscopic and microscopic characteristics, biochemical testing and PCR. Then, the indole acetic acid production and phosphate-solubilizing activity were determined. Finally, the amount of lead in the plant was measured by atomic absorption method. Five strains of Bacillus cereus, Bacillus muralis, Bacillus sp., Pseudomonas fluorescens and Brevibacterium frigoritolerans with the ability of mineral phosphate solubilizing, high levels of indole acetic acid production and resistance to lead were isolated from the rhizosphere of C. tinctorius. The amount of produced indole acetic acid and the level of phosphate solubilizing by the isolates were 7.1-69.54 µg ml-1 and 91-147.3 µg ml-1 respectively. Lead assimilation in aerial part of safflower ranged from 925 to 2175 ppm. P. fluorescens and B. cereus strains had the highest effect on Lead assimilation with 2175 and 1862 ppm respectively. CONCLUSIONS: The results showed that different bacterial treatments influenced the rate of lead absorption by C. tinctorius exposed to lead stress. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of rhizosphere isolates of C. tinctorius can improve phytoremediation capability and lead absorption in lead-contaminated soil.

Multiple Species of Asteraceae Plants Are Susceptible to Root Infection by the Necrotrophic Fungal Pathogen Sclerotinia sclerotiorum.

The necrotrophic fungal pathogen Sclerotinia sclerotiorum can cause disease on numerous plant species, including many important crops. Most S. sclerotiorum-incited diseases of crop plants are initiated by airborne ascospores produced when fungal sclerotia germinate to form spore-bearing apothecia. However, basal stalk rot of sunflower occurs when S. sclerotiorum sclerotia germinate to form mycelia within the soil, which subsequently invade sunflower roots. To determine whether other plant species in the Asteraceae family are susceptible to root infection by S. sclerotiorum, cultivated sunflower (Helianthus annuus L.) and seven other Asteraceae species were evaluated for S. sclerotiorum root infection by inoculation with either sclerotia or mycelial inoculum. Additionally, root susceptibility of sunflower was compared with that of dry edible bean and canola, two plant species susceptible to S. sclerotiorum but not known to display root-initiated infections. Results indicated that multiple Asteraceae family plants are susceptible to S. sclerotiorum root infection after inoculation with either sclerotia or mycelium. These observations expand the range of plant hosts susceptible to S. sclerotiorum root infection, elucidate differences in root inoculation methodology, and emphasize the importance of soilborne infection to Asteraceae crop and weed species.

Comparative study of microbial structure and functional profile of sunflower rhizosphere grown in two fields.

BACKGROUND: Microbial communities inhabiting the rhizosphere play pivotal roles in determining plant health and yield. Manipulation of the rhizosphere microbial community is a promising means to enhance the productivity of economically viable and important agricultural crops such as sunflower (Helianthus annuus). This study was designed to gain insights into the taxonomic and functional structures of sunflower rhizosphere and bulk soil microbiome at two different locations (Sheila and Itsoseng) in South Africa. RESULTS: Microbial DNA extracted from the sunflower rhizosphere and bulk soils was subjected to next-generation sequencing using 16S amplicon sequencing technique. Firmicutes, Actnobacteria and Proteobacteria predominated sunflower rhizosphere soils. Firmicutes, Cyanobacteria, Deinococcus-Thermus and Fibrobacteres were positively influenced by Na+ and clay content, while Actinobacteria, Thaumarchaeota, Bacteroidetes, Planctomycetes, Aquificae and Chloroflexi were positively influenced by soil resistivity (Res) and Mg2+. The community-level physiological profiling (CLPP) analysis showed that the microbial communities in SHR and ITR used the amino acids tryptophan and malic acid efficiently. The metabolisms of these carbon substrates may be due to the dominant nature of some of the organisms, such as Actinobacteria in the soils. CONCLUSION: The CLPP measurements of soil from sunflower rhizosphere were different from those of the bulk soil and the degree of the variations were based on the type of carbon substrates and the soil microbial composition. This study has shown the presence of certain taxa of rhizobacteria in sunflower rhizosphere which were positively influenced by Na+ and Mg2+, and taxa obtained from SHR and ITR were able to effectively utilized tryptophan and malic acid. Many unclassified microbial groups were also discovered and it is therefore recommended that efforts should further be made to isolate, characterize and identify these unclassified microbial species, as it might be plausible to discover new microbial candidates that can further be harnessed for biotechnological purpose.

Heat Inactivation of Listeria monocytogenes on Pecans, Macadamia Nuts, and Sunflower Seeds.

This project was undertaken to determine the kinetic parameters of thermal inactivation of Listeria monocytogenes on pecans, macadamia nuts, and sunflower seeds subjected to heat treatments simulating industry processes. Five strains were grown in nonselective medium, mixed, and resuspended before inoculating macadamia nuts, pecans, and sunflower seeds (6 to 9 Log CFU/g). Redried inoculated pecans and macadamia nuts were heated in an oven at a temperature range of 90 to 140°C. Unshelled sunflower seeds were heated in sunflower seed oil. The thermal inactivation was determined by measuring viable cell counts using standard microbiological methods. Average count data were fit to the log-linear model, and thermal-death kinetics were calculated. On pecans, the viable Listeria counts were reduced by 3 and 3.5 Log CFU/g after 40 min at 110°C and 8 min at 140°C, respectively. On macadamia nuts, the L. monocytogenes population was reduced by 5 Log CFU/g after 20 min at 120°C. Unshelled sunflower seeds were subjected to heat treatment via a hot-oil bath. On sunflower seeds, >7 Log CFU/g reductions were observed after 15 min at 120°C. The thermal resistance (D value) for inactivation on pecans at 140°C was 3.1 min and on macadamia nuts at 120°C was 4.4 min. The inactivation of L. monocytogenes was influenced by the kind of nut or seed. These results suggest that L. monocytogenes has a relatively high thermal tolerance. The findings from this study will contribute to the assessment of the effectiveness of heat treatment for control of this pathogen on nuts and seeds. IMPORTANCE Listeria monocytogenes is a major concern for the food industry in ready-to-eat (RTE) foods. In recent years, large-scale recalls have occurred with contaminated sunflower seeds and macadamia nuts that triggered product withdrawals. These events stress the importance of understanding Listeria's ability to survive heat treatments in these low-water activity foods. Nuts and seeds are subjected to a variety of thermal treatments typically referred as roasting. To date, no listeriosis outbreak has been linked to nuts and seeds, but the recent recognition that this pathogen can be detected in commercial products stresses the need for research on thermal treatments. The characterization of heat inactivation kinetics at temperatures typically used during roasting processes will be very beneficial for validation studies. This manuscript reports inactivation rates of L. monocytogenes strains inoculated onto macadamia nuts, sunflower seeds, and pecan halves subjected to temperatures between 90 and 140°C.

Bacterial community structure of the sunflower (Helianthus annuus) endosphere.

Agrochemical applications on farmland aim to enhance crop yield; however, the consequence of biodiversity loss has caused a reduction in ecological functions. The positive endosphere interactions and crop rotation systems may function in restoring a stable ecosystem. Employing culture-independent techniques will help access the total bacteria community in the sunflower endosphere. Limited information is available on the bacteria diversity in sunflower plants cultivated under different agricultural practices. Hence, this study was designed to investigate the endophytic bacterial community structure of sunflower at the growing stage. Plant root and stem samples were sourced from two locations (Itsoseng and Lichtenburg), for DNA extraction and sequenced on the Illumina Miseq platform. The sequence dataset was analyzed using online bioinformatics tools. Saccharibacteria and Acidobacteria were dominant in plant roots, while the stem is dominated by Proteobacteria, Bacteriodetes, and Gemmatimonadetes across the sites. Bacterial genera, Acidovorax, Flavobacterium, Hydrogenophaga, and Burkholderia-Paraburkhoderia were found dominant in the root, while the stem is dominated by Streptomyces. The diverse bacterial community structure at phyla and class levels were significantly different in plant organs across the sites. The influence of soil physical and chemical parameters analyzed was observed to induce bacterial distribution across the sites. This study provides information on the dominant bacteria community structure in sunflowers at the growing stage and their predictive functions, which suggest their future exploration as bioinoculants for improved agricultural yields.

Direct Metatranscriptomic Survey of the Sunflower Microbiome and Virome.

Sunflowers (Helianthus annuus L.) are susceptible to multiple diseases in field production. In this study, we collected diseased sunflower leaves in fields located in South Dakota, USA, for virome investigation. The leaves showed visible symptoms on the foliage, indicating phomopsis and rust infections. To identify the viruses potentially associated with the disease diagnosed, symptomatic leaves were obtained from diseased plants. Total RNA was extracted corresponding to each disease diagnosed to generate libraries for paired-end high throughput sequencing. Short sequencing reads were assembled de novo and the contigs with similarities to viruses were identified by aligning against a custom protein database. We report the discovery of two novel mitoviruses, four novel partitiviruses, one novel victorivirus, and nine novel totiviruses based on similarities to RNA-dependent RNA polymerases and capsid proteins. Contigs similar to bean yellow mosaic virus and Sclerotinia sclerotiorum hypovirulence-associated DNA virus were also detected. To the best of our knowledge, this is the first report of direct metatranscriptomics discovery of viruses associated with fungal infections of sunflowers bypassing culturing. These newly discovered viruses represent a natural genetic resource from which we can further develop potential biopesticide to control sunflower diseases.

Genome-wide investigation of bHLH genes and expression analysis under different biotic and abiotic stresses in Helianthus annuus L.

The basic helix-loop-helix (bHLH) transcription factors play important roles in many processes such as plant growth, metabolism and response to biotic/abiotic stresses. Sunflower (Helianthus annuus) is a major oil crop, cultivated throughout the world. However, no systematic characterization of bHLH gene members in sunflower (HabHLH) and their functions involved in drought, cadmium tolerance and Orobanche cumana resistance has been reported yet. In this study, 183 HabHLH genes were identified and named according to their chromosomal locations. We classified these proteins into 21 subfamilies by phylogenetic tree analysis. Subsequently, DNA-binding patterns, sequence analysis, duplication analysis and gene structures were analyzed. All of the HabHLH genes were randomly distributed on 17 chromosomes, and 10 pairs of tandem duplicated genes and one pair of segmental duplicated genes were detected in the HabHLH family. Among the duplicated gene pairs, eight pairs of HabHLH genes suffer from positive selection. Moreover, qRT-PCR results revealed significant up-regulated expression of HabHLH024 gene in response to both abiotic (cadmium, drought) and biotic (Orobanche cumana) stresses, suggesting its important functions in response to different stresses. Therefore, HabHLH024 would be the potential candidate gene for the sunflower tolerance breeding.

On-field phenotypic evaluation of sunflower populations for broad-spectrum resistance to Verticillium leaf mottle and wilt.

Sunflower Verticillium Wilt and Leaf Mottle (SVW), caused by Verticillium dahliae (Kleb.; Vd), is a soil-borne disease affecting sunflower worldwide. A single dominant locus, known as V1, was formerly effective in controlling North-American Vd races, whereas races from Argentina, Europe and an emerging race from USA overcome its resistance. This emphasizes the need for identifying broad-spectrum genetic resistance (BSR) sources. Here we characterize two sunflower mapping populations (MPs) for SVW resistance: a biparental MP and the association MP from the National Institute of Agricultural Technology (INTA), under field growing conditions. Nine field-trials (FTs) were conducted in highly infested fields in the most SVW-affected region of Argentina. Several disease descriptors (DDs), including incidence and severity, were scored across four phenological stages. Generalized linear models were fitted according to the nature of each variable, adjusting mean phenotypes for inbred lines across and within FTs. Comparison of these responses allowed the identification of novel BSR sources. Furthermore, we present the first report of SVW resistance heritability, with estimates ranging from 35 to 45% for DDs related to disease incidence and severity, respectively. This study constitutes the largest SVW resistance characterization reported to date in sunflower, identifying valuable genetic resources for BSR-breeding to cope with a pathogen of increasing importance worldwide.