Whitefly hijacks a plant detoxification gene that neutralizes plant toxins.

Plants protect themselves with a vast array of toxic secondary metabolites, yet most plants serve as food for insects. The evolutionary processes that allow herbivorous insects to resist plant defenses remain largely unknown. The whitefly Bemisia tabaci is a cosmopolitan, highly polyphagous agricultural pest that vectors several serious plant pathogenic viruses and is an excellent model to probe the molecular mechanisms involved in overcoming plant defenses. Here, we show that, through an exceptional horizontal gene transfer event, the whitefly has acquired the plant-derived phenolic glucoside malonyltransferase gene BtPMaT1. This gene enables whiteflies to neutralize phenolic glucosides. This was confirmed by genetically transforming tomato plants to produce small interfering RNAs that silence BtPMaT1, thus impairing the whiteflies' detoxification ability. These findings reveal an evolutionary scenario whereby herbivores harness the genetic toolkit of their host plants to develop resistance to plant defenses and how this can be exploited for crop protection.

A new Bowman-Birk type protease inhibitor regulated by MeJA pathway in maize exhibits anti-feedant activity against the Ostrinia furnacalis.

Jasmonic acid (JA), an important plant hormone, plays a crucial role in defending against herbivorous insects. In this study, we have identified a new Bowman-Birk type protease inhibitor (BBTI) protein in maize that is regulated by the JA pathway and exhibits significant antifeedant activity, which is notably induced by exogenous Methyl Jasmonate and Ostrinia furnacalis feeding treatments. Bioinformatics analysis revealed significant differences in the BBTI protein among different maize inbred lines, except for the conserved domain. Prokaryotic and eukaryotic expression systems were constructed and expressed, and combined with bioassays, it was demonstrated that the antifeedant activity of BBTI is determined by protein modifications and conserved domains. Through RT-qPCR detection of BBTI and JA regulatory pathway-related genes' temporal expression in different maize inbred lines, we identified the regulatory mechanism of BBTI synthesis under the JA pathway. This study successfully cloned and identified the MeJA-induced anti-feedant activity gene BBTI and conducted functional validation in different maize inbred lines, providing valuable insights into the response mechanism of insect resistance induced by the plant JA pathway. The increased expression of the anti-feedant activity gene BBTI through exogenous MeJA induction may offer a potential new strategy for mediating plant defense against Lepidoptan insects.

Salicylic acid inducing the expression of maize anti-insect gene SPI: a potential control strategy for Ostrinia furnacalis.

BACKGROUND: Due to being rooted in the ground, maize (Zea mays L.) is unable to actively escape the attacks of herbivorous insects such as the Asian corn borer (Ostrinia furnacalis). In contrast to the passive damage, plants have evolved defense mechanisms to protect themselves from herbivores. Salicylic acid, a widely present endogenous hormone in plants, has been found to play an important role in inducing plant resistance to insects. In this study, we screened and identified the insect resistance gene SPI, which is simultaneously induced by SA and O. furnacalis feeding, through preliminary transcriptome data analysis. The functional validation of SPI was carried out using bioinformatics, RT-qPCR, and heterologous expression protein feeding assays. RESULTS: Both SA and O. furnacalis treatment increased the expression abundance of SA-synthesis pathway genes and SPI in three maize strains, and the upregulation of SPI was observed strongly at 6 hours post-treatment. The expression of SPI showed a temporal relationship with SA pathway genes, indicating that SPI is a downstream defense gene regulated by SA. Protein feeding assays using two different expression vectors demonstrated that the variation in SPI protein activity among different strains is mainly due to protein modifications. CONCLUSIONS: Our research results indicate that SPI, as a downstream defense gene regulated by SA, is induced by SA and participates in maize's insect resistance. The differential expression levels of SPI gene and protein modifications among different maize strains are one of the reasons for the variation in insect resistance. This study provides new insights into ecological pest control in maize and valuable insights into plant responses to SA-induced insect resistance.

Maize Endophytic Plant Growth-Promoting Bacteria Peribacillus simplex Can Alleviate Plant Saline and Alkaline Stress.

Soil salinization is currently one of the main abiotic stresses that restrict plant growth. Plant endophytic bacteria can alleviate abiotic stress. The aim of the current study was to isolate, characterize, and assess the plant growth-promoting and saline and alkaline stress-alleviating traits of Peribacillus simplex M1 (P. simplex M1) isolates from maize. One endophytic bacterial isolate, named P. simplex M1, was selected from the roots of maize grown in saline-alkali soil. The P. simplex M1 genome sequence analysis of the bacteria with a length of 5.8 Mbp includes about 700 genes that promote growth and 16 antioxidant activity genes that alleviate saline and alkaline stress. P. simplex M1 can grow below 400 mM NaHCO3 on the LB culture medium; The isolate displayed multiple plant growth-stimulating features, such as nitrogen fixation, produced indole-3-acetic acid (IAA), and siderophore production. This isolate had a positive effect on the resistance to salt of maize in addition to the growth. P. simplex M1 significantly promoted seed germination by enhancing seed vigor in maize whether under normal growth or NaHCO3 stress conditions. The seeds with NaHCO3 treatment exhibited higher reactive oxygen species (ROS) levels than the maize in P. simplex M1 inoculant on maize. P. simplex M1 can colonize the roots of maize. The P. simplex M1 inoculant plant increased chlorophyll in leaves, stimulated root and leaf growth, increased the number of lateral roots and root dry weight, increased the length and width of the blades, and dry weight of the blades. The application of inoculants can significantly reduce the content of malondialdehyde (MDA) and increase the activity of plant antioxidant enzymes (Catalase (CAT), Superoxide Dismutase (SOD), and Peroxidase (POD)), which may thereby improve maize resistance to saline and alkaline stress. Conclusion: P. simplex M1 isolate belongs to plant growth-promoting bacteria by having high nitrogen concentration, indoleacetic acid (IAA), and siderophore, and reducing the content of ROS through the antioxidant system to alleviate salt alkali stress. This study presents the potential application of P. simplex M1 as a biological inoculant to promote plant growth and mitigate the saline and alkaline effects of maize and other crops.

GDSL in Lilium pumilum (LpGDSL) Confers Saline-Alkali Resistance to the Plant by Enhancing the Lignin Content and Balancing the ROS.

In order to explore the response mechanism of Lilium pumilum (L. pumilum) to saline-alkali stress, we successfully cloned LpGDSL (GDSL lipase, Gly-Asp-Ser-Leu) from L. pumilum. The qRT-PCR results indicated that the LpGDSL expression was higher in the leaves of L. pumilum, and the expression of the LpGDSL reached the highest level at 12 h in leaves under 11 mM H2O2, 200 mM NaCl, 25 mM Na2CO3, and 20 mM NaHCO3. The bacteriophage overexpressing LpGDSL was more tolerant than the control under different NaHCO3 contents. Overexpressed and wild-type plants were analyzed for phenotype, chlorophyll content, O2- content, H2O2 content, lignin content, and so on. Overexpressed plants had significantly higher resistance than the wild type and were less susceptible to saline-alkali stress. The yeast two-hybrid and BiFC assays demonstrated the existence of an interaction between LpGDSL and LpBCP. The yeast one-hybrid assay and transcriptional activation assay confirmed that B3 transcription factors could act on LpGDSL promoters. Under saline-alkali stress, L. pumilum will promote the expression of LpGDSL, which will then promotes the accumulation of lignin and the scavenging of reactive oxygen species (ROS) to reduce its damage, thus improving the saline-alkali resistance of the plant.

MAPK-directed activation of the whitefly transcription factor CREB leads to P450-mediated imidacloprid resistance.

The evolution of insect resistance to pesticides poses a continuing threat to agriculture and human health. While much is known about the proximate molecular and biochemical mechanisms that confer resistance, far less is known about the regulation of the specific genes/gene families involved, particularly by trans-acting factors such as signal-regulated transcription factors. Here we resolve in fine detail the trans-regulation of CYP6CM1, a cytochrome P450 that confers resistance to neonicotinoid insecticides in the whitefly Bemisia tabaci, by the mitogen-activated protein kinase (MAPK)-directed activation of the transcription factor cAMP-response element binding protein (CREB). Reporter gene assays were used to identify the putative promoter of CYP6CM1, but no consistent polymorphisms were observed in the promoter of a resistant strain of B. tabaci (imidacloprid-resistant, IMR), which overexpresses this gene, compared to a susceptible strain (imidacloprid-susceptible, IMS). Investigation of potential trans-acting factors using in vitro and in vivo assays demonstrated that the bZIP transcription factor CREB directly regulates CYP6CM1 expression by binding to a cAMP-response element (CRE)-like site in the promoter of this gene. CREB is overexpressed in the IMR strain, and inhibitor, luciferase, and RNA interference assays revealed that a signaling pathway of MAPKs mediates the activation of CREB, and thus the increased expression of CYP6CM1, by phosphorylation-mediated signal transduction. Collectively, these results provide mechanistic insights into the regulation of xenobiotic responses in insects and implicate both the MAPK-signaling pathway and a transcription factor in the development of pesticide resistance.

Residue dynamics of florpyrauxifen-benzyl and its effects on bacterial community structure in paddy soil of Northeast China.

Florpyrauxifen-benzyl is an herbicide that has been developed in recent years. Its degradation mode in paddy soil environments is not clear. In this study, the degradation dynamics in paddy soil and water were studied by ultrahigh-performance liquid chromatography. Microbial degradation was the main degradation pathway. Using third-generation high-throughput sequencing technology, the changes in the soil bacterial community structure were studied. After 30 days of application, compared with the control group (F0), the abundance of Sphingomonas, Lysobacter, and Flavisolibacter in the recommended and repeated application groups (F1, F5 and F10) increased significantly, and uncultured bacterium and Terrimonas decreased significantly. Compared with the F0 and F1 groups, the species diversity of the F0 and F1 groups showed a significant increase over time. The species diversity of the F5 and F10 groups decreased significantly on Days 5 and 15. On Day 30, the recovery even exceeded that of the control group. Luteimonas and five other genera were positively correlated with herbicide residues, and Pseudolabrys and two other genera were negatively correlated. Repeated application showed a significant effect on the structure of the soil bacterial community, mainly showing a trend of a significant decrease in the initial stage and gradual recovery in the later stage. The results will guide the safe and rational use of florpyrauxifen-benzyl and provide a scientific basis for florpyrauxifen-benzyl dynamic supervision of environmental pollution and protection of black soil in Northeast China.

Genome-Wide Analysis of Carboxylesterases (COEs) in the Whitefly, Bemisia tabaci (Gennadius).

The whitefly (Bemisia tabaci), an important invasive pest that causes severe damage to crops worldwide, has developed resistance to a variety of insecticides. Carboxylesterases (COEs) are important multifunctional enzymes involved in the growth, development, and xenobiotic metabolism of insects. However, systematic studies on the COEs of B. tabaci are scarce. Here, 42 putative COEs in different functional categories were identified in the Mediterranean species of B. tabaci (B. tabaci MED) based on a genome database and neighbor-joining phylogeny. The expression patterns of the COEs were affected by the development of B. tabaci. The expression levels of six COEs were positively correlated with the concentration of imidacloprid to which B. tabaci adults were exposed. The mortality of B. tabaci MED adults fed dsBTbe5 (67.5%) and dsBTjhe2 (58.4%) was significantly higher than the adults fed dsEGFP (41.1%) when treated with imidacloprid. Our results provide a basis for functional research on COEs in B. tabaci and provide new insight into the imidacloprid resistance of B. tabaci.

RNA Interference to Control Asian Corn Borer Using dsRNA from a Novel Glutathione-S-Transferase Gene of Ostrinia furnacalis (Lepidoptera: Crambidae).

Glutathione-S-transferases (GST) comprise a multifunctional protein superfamily, which plays important roles as detoxifiers and antioxidants in insects. The GST in Asian corn borer has not been previously characterized. In this study, we cloned, characterized, and expressed the complete GST genes from the midgut of Asian corn borer. Furthermore, we designed htL4440-OfGST vector to exploit this gene for RNA interference (RNAi) strategy to control this pest. A complete GST cDNA sequence in Asian corn borer was obtained by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends technology. The gene was 887bp in length and contained a 705bp open reading frame and 5' UTR and 3' UTR lengths of 89 and 93bp, respectively. The putative sequence encoded a putative 234 amino acid residue peptide and had a predicted molecular weight of ~26kDa. The GST protein of Asian corn borer is hydrophilic and may have a 30 amino acid signal peptide with a cleavage site between L30 and K31. A recombination vector pET28a-OfGST was constructed for purification and antibody preparation. Western blotting analysis showed that this protein reached the maximum expression level around 24 h in Asian corn borer larvae fed the plant toxin 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one. A second vector, htL4440-OfGST, was constructed to generate the dsRNA of the GST gene. A larval feeding bioassay showed that the expressed dsRNA significantly reduced the detoxification ability of Asian corn borer larvae and increased mortality rate up to 54%. Our data indicated that GST plays very important roles in detoxifying in Asian corn borer and can be used as an RNAi method to control this pest in the field.

Proteomics-based identification of midgut proteins correlated with Cry1Ac resistance in Plutella xylostella (L.).

The diamondback moth, Plutella xylostella (L.), is a worldwide pest of cruciferous crops and can rapidly develop resistance to many chemical insecticides. Although insecticidal crystal proteins (i.e., Cry and Cyt toxins) derived from Bacillus thuringiensis (Bt) have been useful alternatives to chemical insecticides for the control of P. xylostella, resistance to Bt in field populations of P. xylostella has already been reported. A better understanding of the resistance mechanisms to Bt should be valuable in delaying resistance development. In this study, the mechanisms underlying P. xylostella resistance to Bt Cry1Ac toxin were investigated using two-dimensional differential in-gel electrophoresis (2D-DIGE) and ligand blotting for the first time. Comparative analyses of the constitutive expression of midgut proteins in Cry1Ac-susceptible and -resistant P. xylostella larvae revealed 31 differentially expressed proteins, 21 of which were identified by mass spectrometry. Of these identified proteins, the following fell into diverse eukaryotic orthologous group (KOG) subcategories may be involved in Cry1Ac resistance in P. xylostella: ATP-binding cassette (ABC) transporter subfamily G member 4 (ABCG4), trypsin, heat shock protein 70 (HSP70), vacuolar H(+)-ATPase, actin, glycosylphosphatidylinositol anchor attachment 1 protein (GAA1) and solute carrier family 30 member 1 (SLC30A1). Additionally, ligand blotting identified the following midgut proteins as Cry1Ac-binding proteins in Cry1Ac-susceptible P. xylostella larvae: ABC transporter subfamily C member 1 (ABCC1), solute carrier family 36 member 1 (SLC36A1), NADH dehydrogenase iron-sulfur protein 3 (NDUFS3), prohibitin and Rap1 GTPase-activating protein 1. Collectively, these proteomic results increase our understanding of the molecular resistance mechanisms to Bt Cry1Ac toxin in P. xylostella and also demonstrate that resistance to Bt Cry1Ac toxin is complex and multifaceted.

Glutathione S-transferases are involved in thiamethoxam resistance in the field whitefly Bemisia tabaci Q (Hemiptera: Aleyrodidae).

The whitefly, Bemisia tabaci, has developed a high level of resistance to thiamethoxam, a second generation neonicotinoid insecticide that has been widely used to control this pest. In this study, we assessed the level of cross-resistance, the activities of detoxifying enzymes, and the expression profiles of 23 glutathione S-transferase (GST) genes in a thiamethoxam-resistant ant and -susceptible strain of Bemisia tabaci Q. The thiamethoxam-resistant strain showed a moderate level of cross-resistance to another nicotinoid insecticide imidacloprid, a low level of cross-resistance to acetamiprid and nitenpyram, and no significant cross-resistance to abamectin and bifenthrin. Among detoxifying enzymes, only GSTs had significantly higher activity in the resistant strain than in the susceptible strain. Seven of 23 GST genes were over-expressed in the resistant strain relative to the susceptible strain. Using the technology of RNA interference to knockdown a GST gene (GST14), the results showed that silencing GST14 increased the mortality of whiteflies to thiamethoxam in Bemisia tabaci.

Evaluation of Housekeeping Genes for Quantitative Real-Time PCR Analysis of Bradysia odoriphaga (Diptera: Sciaridae).

The soil insect Bradysia odoriphaga (Diptera: Sciaridae) causes substantial damage to Chinese chive. Suitable reference genes in B. odoriphaga (Bradysia odoriphaga) have yet to be identified for normalizing target gene expression among samples by quantitative real-time PCR (qRT-PCR). This study was focused on identifying the expression stability of 12 candidate housekeeping genes in B. odoriphaga under various experiment conditions. The final stability ranking of 12 housekeeping genes was obtained with RefFinder, and the most suitable number of reference genes was analyzed by GeNorm. The results revealed that the most appropriate sets of internal controls were RPS15, RPL18, and RPS18 across developmental phases; RPS15, RPL28, and GAPDH across temperatures; RPS15 and RPL18 across pesticide treatments; RSP5, RPS18, and SDHA across photoperiods; ACTb, RPS18, and RPS15 across diets; RPS13 and RPL28 across populations; and RPS15, ACTb, and RPS18 across all samples. The use of the most suitable reference genes versus an arbitrarily selected reference gene resulted in significant differences in the analysis of a target gene expression. HSP23 in B. odoriphaga was found to be up-regulated under low temperatures. These results will contribute to the standardization of qRT-PCR and will also be valuable for further research on gene function in B. odoriphaga.

Identification and Characterization of the Gene CYP340W1 from Plutella xylostella and Its Possible Involvement in Resistance to Abamectin.

Abamectin has been used to control the diamondback moth, Plutella xylostella (P. xylostella), which is a major agricultural pest that can rapidly develop resistance against insecticides including abamectin. Although cytochrome P450 has been confirmed to play an important role in resistance in P. xylostella, the specific P450 genes associated with the resistance are unclear. The full-length cDNA of the cytochrome P450 gene CYP340W1 was cloned and characterized in the present study. The cDNA assembly yielded a sequence of 1929 bp, containing the open reading frame (ORF) 1491 bp and encodes a 496-amino acid peptide. CYP340W1 was expressed in all P. xylostella developmental stages but its expression level was highest in larvae and especially in the heads of larvae. The expression of CYP340W1 was significantly higher in an abamectin-resistant strain (ABM-R) than in its susceptible counterpart (ABM-S). In addition, expression of CYP340W1 was increased when the ABM-R strain was exposed to abamectin. When injected into third-stage ABM-R larvae, CYP340W1 dsRNA significantly reduced CYP340W1 expression at 6 h and reduced expression by 83% at 12 h. As a consequence of RNAi, the mortality of the injected abamectin-resistant larvae increased after a 48-h exposure to abamectin. The results indicate that the overexpression of CYP340W1 plays an important role in abamectin resistance in P. xylostella.

MYB4 in Lilium pumilum affects plant saline-alkaline tolerance.

Lilium pumilum DC (L. pumilum DC) plays an important role in the rational utilization of salinized soil. To explore the molecular mechanism of salt-tolerant L. pumilum, the LpMYB4 was cloned. LpMYB4 close relationship with Bambusa emeiensis and Zea mays MYB4 throughout the phylogenetic tree construction. LpMYB4 protein was found to be localized in the nucleus. Prokaryotic and eukaryotic bacterial solution resistance experiments proved that the exogenous introduction of LpMYB4 made the overexpression strains obtain better survival ability under saline-alkaline stress. Compared with wild-type plants, tobacco plants overexpressing LpMYB4 had better growth and lower leaf wilting and lodging, the content of chlorophyll was higher, the content of hydrogen peroxide and superoxide anion was lower, the activity of peroxidase and superoxide dismutase was higher and the relative conductivity was lower under saline-alkaline stress. The analysis of seed germination and seedling resistance of transgenic plants under salt stress showed that LpMYB4 transgenic seeds were more tolerant to salt stress during germination and growth. Yeast two-hybrid and two-luciferase complementation experiments showed that LpMYB4 interacted with yeast two-hybrid and LpGPX6. The analysis of the role of LpMYB4 in improving plant saline-alkali resistance is helpful to the transformation of plant germplasm resources and has great significance for agriculture and sustainable development.

Effects of rice straw biochar on methanogenic bacteria and metabolic function in anaerobic digestion.

Anaerobic digestion technology mitigates agricultural organic waste pollution, thereby alleviating the energy crisis. Biochar materials increase the utilisation rate of biomass resources and promote the enrichment and growth of microorganisms. Biochar is an effective exogenous additive that stabilises the anaerobic digestion, improves anaerobic digestion efficiency and gas production. Herein, biochar materials were prepared from rice straw utilising the sequencing batch anaerobic digestion process. The biochar microstructure was characterised by scanning electron microscopy (SEM) and Brunauer-Emmett-Teller (BET) analysis, and microbial succession and metabolic pathways were analysed using 16S rRNA sequencing to reveal the molecular mechanisms. Rice straw biochar addition increased gas production during anaerobic fermentation. SEM revealed that numerous cocci and microbacteria became agglomerated and attached to the surface and pores of biochar, which was revealed by BET analysis to be a good habitat for microorganisms. After anaerobic digestion, the specific surface area and total pore volume of biochar decreased. 16S rRNA gene sequencing showed that biochar affected the abundance of certain bacteria and archaea. Biochar had no obvious effect on the function of bacterial flora but inhibited carbohydrate metabolism by bacteria and glycan biosynthesis and metabolism by archaea in the anaerobic fermentation system while promoting lipid metabolism by archaea. Biochar addition inhibited acetic acid production in the anaerobic fermentation system and promoted methane production based on hydrogen and carbon dioxide levels.

Identification, characterization, and expression of a novel P450 gene encoding CYP6AE25 from the Asian corn borer, Ostrinia furnacalis.

An allele of the cytochrome P450 gene, CYP6AE14, named CYP6AE25 (GenBank accession no. EU807990) was isolated from the Asian com borer, Ostrinia fumacalis (Guenée) (Lepidoptera: Pyralidae) by RT-PCR. The cDNA sequence of CYP6AE25 is 2315 bp in length and contains a 1569 nucleotides open reading frame encoding a putative protein with 523 amino acid residues and a predicted molecular weight of 59.95 kDa and a theoretical pI of 8.31. The putative protein contains the classic heme-binding sequence motif F××G×××C×G (residues 451-460) conserved among all P450 enzymes as well as other characteristic motifs of all cytochrome P450s. It shares 52% identity with the previously published sequence of CYP6AE14 (GenBank accession no. DQ986461) from Helicoverpa armigera. Phylogenetic analysis of amino acid sequences from members of various P450 families indicated that CYP6AE25 has a closer phylogenetic relationship with CYP6AE14 and CYP6B1 that are related to metabolism of plant allelochemicals, CYP6D1 which is related to pyrethroid resistance and has a more distant relationship to CYP302A1 and CYP307A1 which are related to synthesis of the insect molting hormones. The expression level of the gene in the adults and immature stages of O. furnacalis by quantitative real-time PCR revealed that CYP6AE25 was expressed in all life stages investigated. The mRNA expression level in 3(rd) instar larvae was 12.8- and 2.97-fold higher than those in pupae and adults, respectively. The tissue specific expression level of CYP6AE25 was in the order of midgut, malpighian tube and fatty body from high to low but was absent in ovary and brain. The analysis of the CYP6AB25 gene using bioinformatic software is discussed.

[Isolation, identification and soil remediation of fomesafen-degrading strain FB8].

OBJECTIVE: In order to find new strains to degrade fomesafen in contaminated soil, we isolated and identified a high-efficiency degrading bacterium from polluted soil. The degrading characteristics and remediation ability of the strain were also studied. METHODS: Characteristics of morphological, physiological, biochemical and 16S rRNA sequence were applied to identify the strain. The optimum growth conditions were obtained by studying the effect of environmental factors such as fomesafen concentration, primary pH and temperature on the strain. The strain's remediation ability to fomesafen-polluted soil was verified by sensitive crop and target weeds bioassay in pot soil. RESULTS: A high-efficiency degrading strain FB8 that used fomesafen as sole carbon source was isolated from soybean field suffering fomesafen in Heilongjiang province. It was initially identified as a member of the genus Pseudomonas. The strain could degrade 86.75% of 500 mg/L fomesafen within 96 h. Its optimal growth conditions were determined as follows: 500 mg/L fomesafen, primary pH between 6.0 and 8.0, and at 35 to 37 degrees C. The strain could remedy the sensitive crop maize and sorghum biomass after treating for 30 d for soil contaminated with 5 mg/kg of fomesafen. CONCLUSION: A fomesafen-degrading strain FB8 was selected from fomesafen-contaminated soil in Heilongjiang Province. The strain was closely related to Pseudomonas mendocina. The strain was a suitable candidate for bioremediation of fomesafen-contaminated soil.

Selection and Validation of Reference Genes For qRT-PCR Analysis of Rhopalosiphum padi (Hemiptera: Aphididae).

Rhopalosiphum padi (L.) (Hemiptera: Aphididae) is an important cosmopolitan pest in cereal crops. Reference genes can significantly affect qRT-PCR results. Therefore, selecting appropriate reference genes is a key prerequisite for qRT-PCR analyses. This study was conducted to identify suitable qRT-PCR reference genes in R. padi. We systematically analyzed the expression profiles of 11 commonly used reference genes. The ΔCt method, the BestKeeper, NormFinder, geNorm algorithms, and the RefFinder online tool were used to evaluate the suitability of these genes under diverse experimental conditions. The data indicated that the most appropriate sets of reference genes were ß-actin and GAPDH (for developmental stages), AK and TATA (for populations), RPS18 and RPL13 (for tissues), TATA and GAPDH (for wing dimorphism), EF-1α and RPS6 (for antibiotic treatments), GAPDH and ß-actin (for insecticide treatments), GAPDH, TATA, RPS18 (for starvation-induced stress), TATA, RPS6, and AK (for temperatures), and TATA and GAPDH (for all conditions). Our study findings, which revealed the reference genes suitable for various experimental conditions, will facilitate the standardization of qRT-PCR programs, while also improving the accuracy of qRT-PCR analyses, with implications for future research on R. padi gene functions.

Effects of Atrazine on Chernozem Microbial Communities Evaluated by Traditional Detection and Modern Sequencing Technology.

Atrazine is a long residual herbicide commonly used in maize fields. Although atrazine can effectively control weeds and improve crop yield, long-term application leads to continuous pollution in the agricultural ecological environment, especially in the soil ecosystem, and its impact on soil microorganisms is still not clear. Four methods were used in the experiment to clarify the effect of atrazine on the bacterial populations of cultivated soil layers of chernozem in a cold region in different periods: high-performance liquid chromatography (HPLC), colorimetry, microplate, and high-throughput sequencing. The level of residual atrazine in cold chernozem decreased from 4.645 to 0.077 mg/kg soil over time, and the residue gradually leached into deep soil and then decreased after accumulating to a maximum value. Atrazine significantly affected the activities of urease and polyphenol oxidase activity in the soil layers at different periods but had no significant effect on sucrase and phosphatase activity. Atrazine significantly reduced the diversity of microbial carbon source utilization and total activity in soil layers of 0-10 and 20-30 cm but only reduced the diversity of microbial carbon source utilization in the 10-20 cm layer. Atrazine had no significant effect on bacterial populations (10-12 phyla, 29-34 genera), but had a slight effect on the relative abundance of various groups. Atrazine significantly reduced the diversity of bacterial populations in cultivated soil layers of chernozem in a cold region, and the diversity of bacterial populations decreased with decreased residue. This lays a foundation for guiding the safe use of herbicides on farmland in Northeast China.