Lianyang Bai

Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae).

Background Accurate evaluation of gene expression requires normalization relative to the expression of reliable reference genes. Expression levels of “classical” reference genes can differ, however, across experimental conditions. Although quantitative real-time PCR (qRT-PCR) has been used extensively to decipher gene function in the sweetpotato whitefly Bemisia tabaci, a world-wide pest in many agricultural systems, the stability of its reference genes has rarely been validated. Results In this study, 15 candidate reference genes from B. tabaci were evaluated using two Excel-based algorithms geNorm and Normfinder under a diverse set of biotic and abiotic conditions. At least two reference genes were selected to normalize gene expressions in B. tabaci under experimental conditions. Specifically, for biotic conditions including host plant, acquisition of a plant virus, developmental stage, tissue (body region of the adult), and whitefly biotype, ribosomal protein L29 was the most stable reference gene. In contrast, the expression of elongation factor 1 alpha, peptidylprolyl isomerase A, NADH dehydrogenase, succinate dehydrogenase complex subunit A and heat shock protein 40 were consistently stable across various abiotic conditions including photoperiod, temperature, and insecticide susceptibility. Conclusion Our finding is the first step toward establishing a standardized quantitative real-time PCR procedure following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guideline in an agriculturally important insect pest, and provides a solid foundation for future RNA interference based functional study in B. tabaci.

Isolation, Identification and Characteristics of an Endophytic Quinclorac Degrading Bacterium Bacillus megaterium Q3

In this study, we isolated an endophytic quinclorac-degrading bacterium strain Q3 from the root of tobacco grown in quinclorac contaminated soil. Based on morphological characteristics, Biolog identification, and 16S rDNA sequence analysis, we identified strain Q3 as Bacillus megaterium. We investigated the effects of temperature, pH, inoculation size, and initial quinclorac concentration on growth and degrading efficiency of Q3. Under the optimal degrading condition, Q3 could degrade 93% of quinclorac from the initial concentration of 20 mg/L in seven days. We analyzed the degradation products of quinclorac using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The major degradation products by Q3 were different from those of previously identified quinclorac degrading strains, which suggests that Q3 may employ new pathways for quinclorac degradation. Our indoor pot experiments demonstrated that Q3 can effectively alleviate the quinclorac phytotoxicity in tobacco. As the first endophytic microbial that is capable of degrading quinclorac, Q3 can be a good bioremediation bacterium for quinclorac phytotoxicity.

Echinochloa crus-galli genome analysis provides insight into its adaptation and invasiveness as a weed

Barnyardgrass (Echinochloa crus-galli) is a pernicious weed in agricultural fields worldwide. The molecular mechanisms underlying its success in the absence of human intervention are presently unknown. Here we report a draft genome sequence of the hexaploid species E. crus-galli, i.e., a 1.27 Gb assembly representing 90.7% of the predicted genome size. An extremely large repertoire of genes encoding cytochrome P450 monooxygenases and glutathione S-transferases associated with detoxification are found. Two gene clusters involved in the biosynthesis of an allelochemical 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and a phytoalexin momilactone A are found in the E. crus-galli genome, respectively. The allelochemical DIMBOA gene cluster is activated in response to co-cultivation with rice, while the phytoalexin momilactone A gene cluster specifically to infection by pathogenic Pyricularia oryzae. Our results provide a new understanding of the molecular mechanisms underlying the extreme adaptation of the weed.Barnyardgrass is a problematic agricultural weed. Here, via genomic analysis, Guo et al. identify genes potentially underlying its success, including a gene cluster activated in response to co-cultivation with rice that leads to synthesis of the allelochemical DIMBOA.

Cloning and characterization of a GABA receptor from Plutella xylostella (Lepidoptera: Plutellidae).

Abstract A full-length cDNA, with an open reading frame (ORF) of 1,449 bp, encoding a subunit of the γ-aminobutyric acid (GABA)-activated chloride channel was isolated from Plutella xylostella (L.) (Lepidoptera: Plutellidae) (GenBank accession no. EF156251). The subunit gene encoded a 483-amino acid polypeptide that showed 84% sequence identity with DmRdl subunit (U02042) (Drosophila melanogaster resistant to dieldrin). When expressed in Xenopus laevis oocytes, the subunit assembled as a functional homomeric complex activated by GABA and abamectin in a dose-dependent manner. The EC50 value of GABA was 0.49 mM (0.41–0.58) (n = 5). However, the responses to abamectin were very robust, with an EC50 of 4.85 μM (4.02–5.89) (n = 6), indicating that abamectin was >100-fold more potent in activating chloride currents than GABA. The results suggest that this subunit is vital to the formation of a functional channel and contains the binding site of abamectin.

Estimation of the toxicity of sulfadiazine to Daphnia magna using negligible depletion hollow-fiber liquid-phase microextraction independent of ambient pH

The toxicity of ionizable organic compounds to organisms depends on the pH, which therefore affects risk assessments of these compounds. However, there is not a direct chemical method to predict the toxicity of ionizable organic compounds. To determine whether hollow-fiber liquid-phase microextraction (HF-LPME) is applicable for this purpose, a three-phase HF-LPME was used to measure sulfadiazine and estimate its toxicity to Daphnia magna in solutions of different pH. The result indicated that the sulfadiazine concentrations measured by HF-LPME decreased with increasing pH, which is consistent with the decreased toxicity. The concentration immobilize 50% of the daphnids (EC50) in 48 h calculated from nominal concentrations increased from 11.93 to 273.5 mg L−1 as the pH increased from 6.0 to 8.5, and the coefficient of variation (CV) of the EC50 values reached 104.6%. When calculated from the concentrations measured by HF-LPME (pH 12 acceptor phase), the EC50 ranged from 223.4 to 394.6 mg L−1, and the CV decreased to 27.60%, suggesting that the concentrations measured by HF-LPME can be used to estimate the toxicity of sulfadiazine irrespective of the solution pH.

Effects of abamectin selection on the genetic differentiation within Plutella xylostella (Lepidoptera: Plutellidae) based on amplified fragment length polymorphism.

Abstract  Diamondback moth, Plutella xylostella (L.) is one of the most serious insect pests for its remarkable ability to develop resistance to virtually every insecticide that has been used against it. In the present study, amplified fragment length polymorphism (AFLP) is used to study the genetic differentiation as well as the effects of abamectin‐selection on population genetic differentiation for P. xylostella. A dendrogram was constructed from the matrix of genetic distances using the STATISTICA software (Version 4.5) and unweighted pair‐group method with arithmetic averages (UPGMA). The data demonstrated that compared to the susceptible strain (ABM‐s), the heterozygosity in the abamectin‐resistant strain (ABM‐r) decreased with the increased selection pressure and resistant level. When the resistance ratio was below 4.3, there was no significant differentiation at the genome DNA level. When the resistance ratio reached 5.8, a fairly significant differentiation began to appear, and when the resistance ratio reached and exceeded 8.1, there appeared a significant genetic differentiation. The results suggest that abamectin selection is associated with increased genetic polymorphism in P. xylostella.

Influence of Environmental Factors on Seed Germination and Emergence of Asia Minor Bluegrass (Polypogon fugax)

Asia minor bluegrass (AmB) is a major weed impacting rapeseed production in Dongting Lake District, China. Growth chamber experiments were conducted to determine the influence of environmental factors on germination and emergence of AmB. The optimum constant temperature for germination was around 20 C. Seeds showed germination percentages above 60% under 22/15 and 24/19 C day/night temperature regimes. Seeds could germinate in the dark, but light exposure significantly enhanced the germination percentage. More than 50% of seeds germinated over a pH range between 4 and 10. Seeds were highly sensitive to osmotic stress, and germination was completely inhibited at an osmotic potential of −0.4 MPa, indicating that it was favored by a moist environment. Increasing salinity reduced germination of AmB seeds from 58% at 0 mM to 13% at 80 mM NaCl. The highest seedling emergence (62%) was observed when seeds were placed on the soil surface, and no seedlings emerged from seeds placed at a depth of 5 cm. This work shows that the climate and soil conditions in Dongting Lake District are suitable for AmB seed germination and that no-till fields, where seeds remain on the soil surface, promote the successful establishment of the weed. Nomenclature: Asia minor bluegrass, Polypogon fugax Nees ex Steud.; rapeseed, Brassica napus L. Polypogon fugax (AmB) es una maleza importante que impacta la producción de colza en el distrito Lago Dongting, China. Se realizaron experimentos en cámaras de crecimiento para determinar la influencia de factores ambientales en la germinación y emergencia de AmB. La temperatura constante óptima para la germinación fue alrededor de 20 C. Las semillas mostraron porcentajes de germinación superiores a 60% con regímenes de temperatura de 22/15 y 24/19 C día/noche. Las semillas pudieron germinar en la oscuridad, pero la exposición a la luz mejoró significativamente el porcentaje de germinación. Más del 50% de las semillas germinaron en un rango de pH entre 4 y 10. Las semillas fueron muy sensibles al estrés osmótico y la germinación fue completamente inhibida a un potencial osmótico de −0.4 MPa, indicando que la germinación fue favorecida en un ambiente húmedo. El aumentar la salinidad redujo la germinación de las semillas de AmB desde 58% con 0mM a 13% con 80 mM NaCl. La mayor emergencia de plántulas (62%) fue observada cuando las semillas fueron puestas sobre la superficie del suelo, y ninguna plántula emergió cuando las semillas fueron puestas a una profundidad de 5 cm. Este trabajo muestra que el clima y las condiciones de suelo en el distrito Lago Dongting son favorables para la germinación de semillas de AmB y que los campos con labranza cero, donde la semilla permanece sobre la superficie del suelo, promueven el establecimiento exitoso de esta maleza.

Combined use of Bacillus subtilis strain B‐001 and bactericide for the control of tomato bacterial wilt

BACKGROUND Tomato bacterial wilt caused by Ralstonia solanacearum poses a serious threat to tomato production. However, no effective control measures are available. In this study, the bactericide Saisentong was combined with an effective biological control agent, Bacillus subtilis B-001, to control tomato bacterial wilt under greenhouse and field conditions. RESULTS Growth of B-001 in vitro was unaffected by Saisentong. In greenhouse experiments, the combined application of B-001 and Saisentong via root irrigation or spray resulted in better disease control compared with either agent alone. In two field trials, at a Saisentong concentration of 400 or 500 mg kg-1 , the combined treatment was more effective than expected and showed a synergistic effect. A lower concentration of Saisentong (200 or 300 mg kg-1 ) in combination with B-001 resulted in an antagonistic effect. However, disease control was significantly greater compared with either treatment alone. CONCLUSION The combination of Saisentong and B-001 effectively controls tomato bacterial wilt. The integrated strategy represents a promising new tool to control this disease.

Two Coumarins with Safener Activity from Rhizoma et Radix Notopterygii

Abstract Rhizoma et Radix Notopterygii is often used as a traditional medicine in China. In our recent work, we found that the ethanol crude extract of Rhizoma et Radix Notopterygii could reduce phytotoxicity of acetochlor on rice. The crude extract of Rhizoma et Radix Notopterygii was isolated and purified by activity-guided fractionation. Two coumarins, isopimpinellin and 5-methoxypsoralen (5-MOP) were identified, and their bioactivity was tested in a growth chamber. The results showed that the two coumarins increased herbicide tolerance of rice shoots, and 5-MOP demonstrated better protection than isopimpinellin. The treatment of 5-MOP at 50 g ai ha−1 recovered the shoot height of rice from 42.6% (treated with acetochlor only) to 81.6% of the control treated without acetochlor, whereas the phytocidal activity of acetochlor on barnyardgrass was not impaired by 5-MOP. Further study suggested that 5-MOP increases herbicide tolerance of rice by enhancing the glutathione S-transferase level of activity in rice. Our findings suggest that isopimpinellin and 5-MOP have the potential to be applied as safeners for rice. Nomenclature: Acetochlor, 2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)acetamide; isopimpinellin, 2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)acetamide; methoxypsoralen, 4-methoxyfuro[3,2-g]chromen-7-one; Rhizoma et Radix Notopterygii; barnyardgrass, Echinochloa crus-galli (L.) Beauv.; rice; Oryza sativa L. Resumen Rhizoma et Radix Notopterygii es comúnmente usado como una medicina tradicional en China. En nuestro reciente trabajo encontramos que el extracto crudo con ethanol de Rhizoma et Radix Notopterygii pudo reducir la fitotoxicidad de acetochlor en arroz. El extracto crudo de Rhizoma et Radix Notopterygii fue aislado y purificado por medio del fraccionamiento guiado por actividad. Dos coumarins, isopimpinellin y 5-methoxypsoralen (5-MOP) fueron identificadas, y se evaluó su bioactividad en una cámara de crecimiento. Los resultados mostraron que estas dos coumarins aumentaron la tolerancia al herbicida de tejidos aéreos de arroz, y 5-MOP demostró una mejor protección que isopimpinellin. El tratamiento de 5-MOP a 50 g ai ha−1 recuperó la altura del arroz de 42.6% (tratado con sólo acetochlor) a 81.6% al compararse con el testigo tratado sin acetochlor, mientras que la actividad herbicida de acetochlor en Echinochloa crus-galli no fue afectada por 5-MOP. Estudios adicionales sugirieron que 5-MOP incrementa la tolerancia del arroz al herbicida al mejorar el nivel de actividad de glutathione-S-transferase en arroz. Nuestros descubrimientos sugieren que isopimpinellin y 5-MOP tienen el potencial de ser aplicados como antídotos en arroz.

Photocatalytic degradation of acephate in pak choi, Brassica chinensis, with Ce-doped TiO2

The photocatalytic degradation of acephate was investigated using Ce-doped TiO2 (TiO2/Ce) hydrosol. In contrast to previous research conducted under artificial light in the laboratory, this study investigated the decomposition of acephate in a field trial. The results show that acephate can be efficiently degraded by the TiO2/Ce system under natural field conditions; the degradation efficiency was affected by the dosage of the photocatalyst and acephate. The optimum dosage of TiO2/Ce was 2400 g a.i.ha−1, and the photodegradation efficiency of acephate reached 93.5% after 20 h at an acephate dosage of 675 g a.i.ha−1. Ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) analysis detected and identified four degradation products—methamidophos, phosphorothioic acid O,O,S-trimethyl ester, S-methyl methanethiosulfonate and phosphorous acid—that were formed during the TiO2/Ce photodegradation of acephate. Based on the structural identification of the degradation products, a probable photodegradation pathway was proposed, and the first decomposition step may be the cleavage of the C‒N bond of acephate. Subsequently, the P‒S and P‒O bonds may be oxidized gradually or simultaneously to complete the mineralization.