GsMAPK4, a positive regulator of soybean tolerance to salinity stress

Salt stress is one of the major factors affecting plant growth and yield in soybean under saline soil condition. Despite many studies on salinity tolerance of soybean during the past few decades, the detailed signaling pathways and the signaling molecules for salinity tolerance regulation have not been clarified. In this study, a proteomic technology based on two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify proteins responsible for salinity tolerance in soybean plant. Real-time quantitative PCR (qRT-PCR) and Western blotting (WB) were used to verify the results of 2-DE/MS. Based on the results of 2-DE and MS, we selected glucosyltransferase (GsGT4), 4-coumarate, coenzyme A ligase (Gs4CL1), mitogen-activated protein kinase 4 (GsMAPK4), dehydration responsive element binding protein (GsDREB1), and soybean cold-regulated gene (GsSRC1) in the salinity tolerant soybean variety, and GsMAPK4 for subsequent research. We transformed soybean plants with mitogen-activated-protein kinase 4 (GsMAPK4) and screened the resulting transgenics soybean plants using PCR and WB, which confirmed the expression of GsMAPK4 in transgenic soybean. GsMAPK4-overexpressed transgenic plants showed significantly increased tolerance to salt stress, suggesting that GsMAPK4 played a pivotal role in salinity tolerance. Our research will provide new insights for better understanding the salinity tolerance regulation at molecular level.     

Sublethal effects of sulfoxaflor on the fitness of two species of wheat aphids,Sitobion avenae(F.) and Rhopalosiphum padi(L.)

Sitobion avenae(F.) and Rhopalosiphum padi(L.) are two important pests of wheat in China. They typically coexist in fields during the late period of wheat growth. Sulfoxaflor is a novel sulfoximine insecticide that demonstrates broad-spectrum efficacy, especially in targeting sap-feeding insects. This study was carried out to investigate the sublethal effects of sulfoxaflor on the development, longevity, and reproduction of two species of wheat aphids. Our results showed that sublethal concentrations of sulfoxaflor did not cause significant effects on the fecundity or the longevity of the parent generation(F_0 generation) of either S. avenae or R. padi. However, it caused transgenerational sublethal effects. For S. avenae, adult longevity of F_1 generation was significantly decreased. No significant differences were observed on the population parameters of S. avenae in the F_1 generation. For R. padi, the adult preoviposition period(APOP) and the total preoviposition period(TPOP) of F_1 generation were significantly reduced. The mean generation time(T) was significantly reduced in the R. padi F_1 generation. What's more, the intrinsic rate of increase(r_m) and the finite rate of increase(λ) were significantly increased in the R. padi F_1 generation. Taken together, these results suggest that exposure to the LC_(25) of sulfoxafl had no effects on the parent generation of S. avenae or R. padi, but it reduced adult longevity of S. avenae as a negative effect and increased the r_m and λ of R. padi in the first progeny generation, which may have an impact on the population dynamics of R. padi.     

A rapid,simple, and sensitive immunoagglutination assay with silica nanoparticles for serotype identification of Pseudomonas aeruginosa

An agglutination test based on colored silica nanoparticles (colored SiNps) was established to detect serotypes of Pseudomonas aeruginosa. Monodisperse colored SiNps were used as agglutination test carriers. The colored SiNps were prepared through reverse microemulsion with reactive dyes, sensitized with 11 kinds of mono-specific antibodies against P. aeruginosa, and denoted as IgG-colored SiNps. Eleven kinds of IgG-colored SiNps were individually mixed with P. aeruginosa on a glass slide. Different serotypes of P. aeruginosa could be identified by agglutination test with evident agglutination. The P. aeruginosa could be detected in a range from 3.6 × 10⁵ to 3.6 × 10¹² cfu mL^?1. This new agglutination test was confirmed to be a specific, sensitive, fast, easy-to-perform, and cost-efficient tool for the routine diagnosis of P. aeruginosa.     

Distribution pattern and titer of Candidatus Liberibacter asiaticus in periwinkle (Catharanthus roseus)

Candidatus Liberibacter asiaticus (CaLas), an uncultured Gram-negative alphaproteobacterium, is the causal agent of Huanglongbing (HLB) in citrus. CaLas resides in phloem sieve tubes and has been shown to be unequally distributed in different tissues. Although HLB is a disease of citrus plants, it has been demonstrated that periwinkle can serve as an experimental host of CaLas, which can be transmitted from citrus to periwinkle via the parasitic plant dodder (Cuscuta spp.). To investigate the distribution of CaLas in various periwinkle tissues, the bacteria were transmitted from an infected periwinkle plant to healthy periwinkles by top-grafting. The movement of the inoculum and associated titer changes were observed over time in various tissues. CaLas could be detected in the leaves, main stems, and roots of infected periwinkle by conventional PCR, and in all three tissues a clear time-dependent change in CaLas titer was observed, with titer increasing soon after inoculation and then decreasing as disease symptoms became severe. The highest titer was found at 25, 35 and 35 days after inoculation in leaves, main stems and roots, respectively. The titer in leaves was much higher than in the main stems and roots at the same time point, and the spatial distribution of CaLas in the leaves, main stems and roots of infected periwinkle was uneven, similar to what has been shown in citrus. The results provide guidance for selecting the proper periwinkle tissues and sampling times for early detection of CaLas.     

The synergistic advantage of combining chloropicrin or dazomet with fosthiazate nematicide to control root-knot nematode in cucumber production

The highly-damaging root-knot nematode(Meloidogyne spp., RKN) cannot be reliably controlled using only a nematicide such as fosthiazate because of increasing pest resistance. In laboratory and greenhouse trials, we showed that chloropicrin(CP) or dazomet(DZ) synergized the efficacy of fosthiazate against RKN. The combination significantly extended the degradation half-life of fosthiazate by an average of about 1.25 times. CP or DZ with fosthiazate reduced the time for fosthiazate to penetrate the RKN cuticle compared to fosthiazate alone. CP or DZ combined with low or medium rate of fosthiazate increased the total cucumber yield, compared to the use of each product alone. A low-dose fosthiazate with DZ improved total yield more than a low dose fosthiazate with CP. Extending the half-life of fosthiazate and reducing the time for fosthiazate or fumigant to penetrate the RKN cuticle were the two features that gave the fumigant-fosthiazate combination its synergistic advantage over these products used singularly. This synergy provides the opportunity for farmers to use a low dose of fosthiazate which lowers the risk of RKN resistance. Farmers could combine DZ at 30 g m~(-2) with fosthiazate at a low rate of 0.375 g m~(-2) to control RKN and adequately control two major soil-borne diseases in cucumber greenhouses.     

Molecular,serological and biological characterization of a novel Apple stem pitting virus strain from a local pear variety grown in China

Apple stem pitting virus (ASPV) is an important causal agent of pear diseases. Nowadays, the infection status and molecular characteristics of the virus in old pear trees have never been investigated. In this study, we provide the first complete genome sequence of an ASPV isolate LYC from an over 300-year-old tree of a local Pyrus bretschneideri cultivar ‘Chili’ specifically grown at Laiyang area in China. ASPV-LYC possesses a chimeric genome consisting of 9 273 nucleotides excluding a poly(A) tail at its 3’ end and harboring a recombination region in its open reading frame (ORF1) with Aurora-1 and KL9 identified as the major and minor parents. Western blot analysis with antisera against recombinant coat proteins (CPs) of three ASPV isolates from pear indicates that ASPV-LYC is serologically related to these ASPV isolates, but with differential activities. Further biological tests on indicator plants of Pyronia veitchii show that ASPV-LYC can induce serious leaf and stem symptoms as other ASPV isolates. The results provide an important information for understanding molecular evolution of ASPV and suggest a need to prevent dissemination of the isolate among pear trees.     

First detection and complete genome of Soybean chlorotic mottle virus naturally infecting soybean in China by deep sequencing

Soybean chlorotic mottle virus (SbCMV) was first detected from soybean plants in Jiangxi Province of China by high throughput sequencing and was confirmed by PCR. The complete nucleotide sequence of NC113 was determined to be 8 210 nucleotides, and shared the highest similarity (91.7%) with sequences of SbCMV that was only reported in Japan. It encodes nine putative open reading frames (ORFs Ia, Ib and II–VIII), and contains a large intergenic region located at nucleotide 5 976–6 512 between ORFs VI and VII. Sequence analysis and phylogenetic tree indicated that NC113 is an isolate of SbCMV, and is more related to the soymoviruses Blueberry red ringspot virus (BRRSV), Peanut chlorotic streak virus (PCSV) and Cestrum yellow leaf curling virus (CmYLCV) than to other representative members in the Caulimoviridae family. Field survey of 472 legume plants from Jiangxi and Zhejiang provinces showed SbCMV was only detected from soybean in Nanchang City with a low incidence rate. This is the first report of Soybean chlorotic mottle virus identified in China.     

Characterization of field-evolved resistance to cyantraniliprole in Bemisia tabaci MED from China

Cyantraniliprole is a novel anthranilic diamide insecticide with significant efficacy against Bemisia tabaci, an important pest insect worldwide. In this study, we conducted reversion and selection work and genetic analysis, and determined cross-resistance spectrum and synergism of cyantraniliprole resistance based on the reported population, SX population, of B. tabaci collected from Shanxi Province, China. Compared with a susceptible strain (MED-S), SX population, the field-evolved cyantraniliprole-resistant population exhibited 26.4-fold higher resistance to cyantraniliprole. In SX, a sharp decline of cyantraniliprole resistance was shown in the absence of selection. Another tested strain, SX-R, was established from SX population after successive selection with cyantraniliprole and recently developed 138.4-fold high resistance to cyantraniliprole. SX-R had no cross-resistance to abamectin, imidacloprid, thiamethoxam, sulfoxaflor, or bifenthrin. Genetic analysis illustrated that cyantraniliprole resistance in SX-R was autosomally inherited and incompletely dominant. Additionally, piperonyl butoxide (PBO) significantly inhibited cyantraniliprole resistance in the SX-R strain. In conclusion, the selection of SX with cyantraniliprole led to high resistance to cyantraniliprole which is incompletely dominant and no cross-resistance to several common types of insecticides. Enhanced oxidative metabolism is possibly involved in the resistance of SX-R, yet target-site resistance could not be excluded.     

Phenotypic characterization and genetic mapping of the dwarf mutant m34 in maize

Plant height is one of the most important agronomic traits associated with yield in maize.In this study,a gibberellins(GA)-insensitive dwarf mutant,m34,was screened from inbred line Ye478 by treatment with the chemical mutagen ethylmethanesulfonate(EMS).Compared to Ye478,m34 showed a dwarf phenotype with shorter internodes,and smaller leaf length and width,but with similar leaf number.Furthermore,m34 exhibited smaller guard cells in internodes than Ye478,suggesting that smaller cells might contribute to its dwarf phenotype.Genetic analysis indicated that the m34 dwarf phenotype was controlled by a recessive nuclear gene.An F2 population derived from a cross between m34 and B73 was used for mutational gene cloning and this gene was mapped to a chromosome region between umc2189 and umc1553 in chromosome 1 bin1.10,which harbored a previously identified dwarf gene Zm VP8.Sequencing analysis showed a nucleotide substitution(G1606 to A1606)in the sixth exon of ZmVP8,which resulted in an amino acid change(E531 to K531)from Ye478 to m34.This amino acid change resulted in anα-helix changing to aβ-sheet in the secondary protein structure and the‘SPEC’domain changed to a‘BOT1NT’domain in the tertiary protein structure.Taken together,these results suggested that m34 is a novel allelic mutant originally derived from Ye478 that is useful for further ZmVP8 functional analysis in maize.     

Field effect of Cnaphalocrocis medinalis granulovirus (CnmeGV) on the pest of rice leaffolder

Rice leaffolder, Cnaphalocrocis medinalis(Guenée), has become a major pest throughout the rice cultivating areas of China and caused severe damage to rice production. Cnaphalocrocis medinalis granulovirus(CnmeGV), a naturally occurring baculovirus, is revealed as a potential microbial agent for the pest control. Field applications of CnmeGV were conducted against rice leaffolder larvae in rice paddies. CnmeGV infected the larvae not only in the current generation but also in the successive generation, resulting in a sustained infection in the larva population for at least 48 days. Under diferent concentrations of CnmeGV(7.5×10~(11) and 1.125×10~(12) occlusion body(OB) ha~(-1)) at 30 days after spraying, larval population reduced up to 76.32% and rice leaf rolled rate kept in 15.42%. Simultaneously, CnmeGV had no impact on arthropod predators of C. medinalis, with abundances ranging from 2.39 to 3.79 per ten hills. These results revealed that CnmeGV is suitable as a bio-pesticide for rice leaffolder management in rice paddies.     

Species-specific COI primers for rapid identification of a globally significant invasive pest,the cassava mealybug Phenacoccus manihoti Matile-Ferrero

The globally invasive cassava mealybug Phenacoccus manihoti Matile-Ferrero is a pernicious pest of cassava,and its recent introduction into Asia has raised considerable alarm.To slow or prevent further invasion,an accurate,simple,and developmental-stage-independent detection method for P.manihoti is required.In the present study,a PCR method based on a species-specific mitochondrial DNA cytochrome oxidase I(SS-COI)marker was developed for rapid identification of P.manihoti.One pair of SS-COI primers(PMSSZW-1F and PMSSZW-1R)was designed based on sequence variations in the COI gene among P.manihoti and related mealybug species.Specificity of the primer pair was validated on 21 closely related species.Sensitivity tests were performed on four immature developmental stages and female adults.Efficacy tests demonstrated that at the relatively low concentration of(135.2±14.7)pgresuspended DNA,the specific fragment was detected in all replicates.Furthermore,the SS-COI primer pair was assayed on three populations of P.manihoti from major exporting countries of cassava.The PCR assay was proved to be a rapid,simple,and reliable molecular measure for the identification of P.manihoti.This tool will be useful for quarantine,monitoring,and management of this invasive pest.     

Effect of exogenous GA_3 on flowering quality,endogenous hormones,and hormone-and flowering-associated gene expression in forcing-cultured tree peony(Paeonia suffruticosa)

Gibberellins(GAs) promote flowering in the forcing-cultured tree peony(Paeonia suffruticosa), however, the mechanism of regulating flowering is not fully understood. In this study, exogenous GA_3 was applied to five-year-old Luoyang Hong plants to explore responses in terms of endogenous hormones, flowering quality, and the hormone-and flowering-associated gene expression. Exogenous GA_3 application significantly promoted flower bud development and new branch growth, as well as improved flowering quality. Exogenous GA_3 application also stimulated the synthesis of endogenous GA_3 and indole-3-acetic acid(IAA) but reduced abscisic acid(ABA) levels. To further elucidate the regulatory mechanism, eight genes for GA biosynthesis and signaling, including Ps CPS, Ps KS, Ps GA_3 ox, Ps GA2 ox, Ps GID1 b, Ps GID1 c, Ps DELLA, and Ps GID2 were cloned for the first time, and sequence analysis was also performed. The results suggested that all the cloned genes have conserved structure as each homologous gene reported in the other species. Phylogenetic trees constructed by the each cloned gene showed that the phylogenetic evolutionary relationship of P. suffruticosa was closely related to Vitis vinifera. The expression patterns of the above genes, and genes for ABA and IAA biosynthetic and signaling, and the flowering time were also investigated. Most of the above genes showed higher expression in the control buds than those in the GA_3 treated buds at six developmental stages, whereas the expression levels of PsSOC1 and PsSPL9 were up-regulated by GA_3 treatment. The results also showed that the GA-biosynthetic and signaling pathways are conserved in tree peony, and the PsCPS, PsGA_3 ox, PsGA2 ox, PsGID1, PsDELLA, and PsGID2 genes are necessary for feedback regulation of GAs. Furthermore, hormone changes promoted PsSOC1 and PsSPL9 expression, and repressed PsSVP expression, which contributed to the improvement flowering quality in tree peony of forcing culture.     

Comparison of phenolic profiles and antioxidant activities in skins and pulps of eleven grape cultivars(Vitis vinifera L.)

Eleven grape cultivars were analysed to explore the variety differences of fresh grape phenolic profiles. The results showed that free phenolics were predominant in grape skins and pulps, and showed the higher antioxidant activities than bound. In 11 cultivars, Muscat Kyoho extracts had the highest total phenolic content in skins(10.525 mg GAE g~(–1) FW) and pulps(1.134 mg GAE g~(–1) FW), and exhibited the highest DPPH radical scavening capacity(EC_(50)=11.7 μg mL~(–1)) and oxygen radical absorbance capacity(ORAC) value(190.57 μmol TE g~(–1) FW) of free phenolic in skin. In addition, the most abundant phenolics in grape skins were found to be flavonoids such as kaempferol in Kyoho skin(541.2 μg g~(–1) FW), rutin, catechin and epicatechin in Muscat Kyoho skin(262.3, 86.3 and 70.0 μg g~(–1) FW, respectively). Furthermore, the principal component analysis showed a strong difference of phenolic profiles with the cultivars, existing forms and distributions. Pearson correlation coefficient analysis showed a significant linear correlation between total phenolic content and antioxidant activity(P0.05). Therefore, both skins and pulps were rich sources of bioactive phenolic compounds, and Muscat Kyoho was the ideal source among all samples.     

Overexpression of G10-EPSPS in soybean provides high glyphosate tolerance

Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)-mediated pathway of shikimic acid. The screening of glyphosate-resistant EPSPS gene is a major means for the development of new genetically modified glyphosate-resistant transgenic crop. Currently, the main commercialized glyphosate-resistant soybean contains glyphosate-resistant gene CP4-EPSPS. In this study, a G10-EPSPS gene was reported providing glyphosate resistance in Zhongdou 32. Here, G10-EPSPS gene was introduced into soybeans through Agrobacterium-mediated soybean cotyledon node. PCR, Southern blotting, semi-quantitative RT-PCR, qRT-PCR, and Western blotting were used, and the results revealed that G10-EPSPS had been integrated into the soybean genome and could be expressed steadily at both mRNA and protein levels. In addition, glyphosate resistance analysis showed that the growth of transgenic soybean had not been affected by concentrations of 900 and 2 700 g a.e. ha~(–1) of glyphosate. All the results indicated that G10-EPSPS could provide high glyphosate resistance in soybeans and be applied in production of glyphosate-resistant soybean.     

Genetic dissection of hexanol content in soybean seed through genome-wide association analysis

Hexanol is a major compound contributing to the off-flavors(the bean-like odor) of soybean derived soymilk. The most effective way to reduce the off-flavors of soymilk is the screening and utilization of soybean cultivars with improved hexanol content. However, no genome-wide genetic analysis for this particular trait has been conducted to date. The objective of the present study was to dissect the genetic basis of hexanol content in soybean seed through genome-wide association analysis(GWAS). A total of 105 soybean accessions were analyzed for hexanol content in a three-year experiments and genotyped by sequencing using the specific locus amplified fragment sequencing(SLAF-seq) approach. A total of 25 724 single nucleotide polymorphisms(SNPs) were obtained with minor allele frequencies(MAF)5%. GWAS showed that 25 quantitative trait nucleotides(QTNs) were significantly associated with the hexanol concentration in soybean seed. These identified QTNs distributed on different genomic regions of the 15 chromosomes. A total of 91 genes were predicted as candidate genes underlying the seed hexanol level and six candidates were predicted possibly underlying the seed hexanol by gene based association. In this study, GWAS has been proven to be an effective way to dissect the genetic basis of the hexanol concentration in multiple genetic backgrounds. The identified beneficial alleles and candidate genes might be valuable for the improvement of marker-assisted breeding efficiency for low hexanol level and help to explore possible molecular mechanisms underlying hexanol content in soybean seed.     

Dynamic changes of root proteome reveal diverse responsive proteins in maize subjected to cadmium stress

Toxic symptoms and tolerance mechanisms of heavy metal in maize are well documented. However, limited information is available regarding the changes in the proteome of maize seedling roots in response to cadmium(Cd) stress. Here, we employed an i TRAQ-based quantitative proteomic approach to characterize the dynamic alterations in the root proteome during early developmental in maize seedling. We conducted our proteomic experiments in three-day seedling subjected to Cd stress, using roots in four time points. We identified a total of 733, 307, 499, and 576 differentially abundant proteins after 12, 24, 48, or 72 h of treatment, respectively. These proteins displayed different functions, such as ribosomal synthesis, reactive oxygen species homeostasis, cell wall organization, cellular metabolism, and carbohydrate and energy metabolism. Of the 166 and 177 proteins with higher and lower abundance identified in at least two time points, 14 were common for three time points. We selected nine proteins to verify their expression using quantitative real-time PCR. Proteins involved in the ribosome pathway were especially responsive to Cd stress. Functional characterization of the proteins and the pathways identified in this study could help our understanding of the complicated molecular mechanism involved in Cd stress responses and create a list of candidate gene responsible for Cd tolerance in maize seeding roots.     

Understanding the biochar's role in ameliorating soil acidity

Extensive acidic soils, which suffer from accelerated soil acidification, are found in southern China. Soil acidity, aluminum toxicity, and nutrient deficiencies severely limited crop productivity in acidic soils. It has been widely reported that crop residue biochars can ameliorate acidic soils and increase crop productivity. Here, we summarized the positive effects and mechanisms involved in the correction of soil acidity, the alleviation of aluminum toxicity and the increase of soil pH buffering capacity by crop residue biochars. The carbonate, oxygen-containing functional groups and silicates in biochars are the major components responsible for their efficacy in amending acidic soils and resisting soil re-acidification. We conclude that application of crop residue biochars may be a better option than traditional liming to ameliorate acidic soils. Nonetheless, further researches into soil acidification are still required to address some issues that are controversial and poorly understood.