Functional Characterization of Ubiquitination Genes in the Interaction of Soybean—Heterodera glycines

Ubiquitination is a kind of post-translational modification of proteins that plays an important role in plant response to biotic and abiotic stress. The response of soybean GmPUB genes to soybean cyst nematode (SCN, Heterodera glycines) infection is largely unknown. In this study, quantitative real-time PCR (qRT-PCR) was performed to detect the relative expression of 49 GmPUB genes in susceptible cultivar William 82 and resistant cultivar Huipizhi after SCN inoculation. The results show that GmPUB genes responded to cyst nematode infection at 1 day post-inoculation (dpi), 5 dpi, 10 dpi and 15 dpi. The expression levels of GmPUB16A, GmPUB20A, GmCHIPA, GmPUB33A, GmPUB23A and GmPUB24A were dramatically changed during SCN infection. Furthermore, functional analysis of these GmPUB genes by overexpression and RNAi showed that GmPUB20A, GmPUB33A and GmPUB24A negatively regulated soybean resistance under SCN stress. The results from our present study provide insights into the complicated molecular mechanism of the interaction between soybean and SCN.     

新颖杀线虫种衣剂——Poncho/Votiv0

新型种衣剂Poncho/Votivo由杀虫剂噻虫胺和生物杀线虫剂坚强芽孢杆菌组成。它可防治玉米、棉花、大豆、甜菜、高粱的肾形线虫、根瘤线虫、大豆孢囊线虫、金针虫、小地老虎、种蝇、蛴螬、初期蚜虫、葡萄肖叶甲、越冬豆叶甲虫和玉米种子蛆等。它对线虫具有高效性、持效性和高的安全性。     

多功能大豆重迎茬专用肥的研究与应用

针对造成重迎茬大豆减产的两个主要原因 ,即以大豆孢囊线虫为主病虫危害及耕层土壤速效钾、速效锌、有效硼单一消耗 ,而研制开发的肥药结合多功能大豆重迎茬专用肥 (10 - 12 - 8)可显著提高单产 ,改善品质 ,抑制大豆孢囊线虫、根腐病及地下害虫的危害 ,满足重迎茬大豆不同生育阶段对 N,P,K,Mg等大中量及 Zn,B,Mn等微量营养元素的需要。试验示范结果表明 :大豆重迎茬专用肥比农民习惯施肥 (磷酸二铵 +尿素 +氯化钾 )平均增产2 3.5 % ,增产 385 .5 kg/ hm2 ,增收 6 15 .6元 / hm2 ,比等量的磷酸二铵增产 7.8% ,增产 177.1kg/ hm2 ,增收 2 40元 /hm2     

Generation and Identification of the Number of Copies of Exogenous Genes and the T-DNA Insertion Site in SCN-Resistance Transformation Event ZHs1-2

Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) causes an estimated economic loss of about USD 3 billion each year in soybean (Glycine max L.) production worldwide. Overexpression of resistance genes against SCN provides a powerful approach to develop SCN resistance cultivars in soybean. The clarification of molecular characterization in transformation events is a prerequisite for ecological risk assessment, food safety, and commercial release of genetically modified crops. Here, we generated transgenic events harboring the BCN (beet cyst nematode) resistance Hs1^pro−1 gene using the Agrobacterium-mediated method in soybean, evaluated their resistance to SCN infection, and clarified the molecular characterization of one of the transformation events. Five independent and stable inheritable transformation events were generated by an Agrobacterium-mediated transformation method. SCN resistance tests showed the average number of developed females per plant and female index (FI) in T4 ZHs1-1, ZHs1-2, ZHs1-3, ZHs1-4, and ZHs1-5 transformation events were significantly lower than that in the nontransgenic control. Among these, the ZHs1-2 transformation event had the lowest number of developed females per plant and FI. Southern hybridization showed the exogenous target Hs1^pro−1 gene was inserted in one copy and the Bar gene was inserted two copies in the ZHs1-2 transformation event. The exogenous T-DNA fragment was integrated in the reverse position of Chr02: 5351566–5231578 (mainly the Bar gene expression cassette) and in the forward position of Chr03: 17083358–17083400 (intact T-DNA, including Hs1^pro−1 and Bar gene expression cassette) using a whole genome sequencing method (WGS). The results of WGS method and Southern hybridization were consistent. All the functional elements of exogenous T-DNA fragments were verified by PCR using specific primer pairs in the T5 and T6 ZHs1-2 transformation events. These results demonstrated that the overexpression of Hs1^pro−1 gene enhanced SCN resistance, and provide an important reference for the biosafety assessment and the labeling detection in transformation event ZHs1-2.     

Soybean miR159-GmMYB33 Regulatory Network Involved in Gibberellin-Modulated Resistance to Heterodera glycines

Soybean cyst nematode (SCN, Heterodera glycines) is an obligate sedentary biotroph that poses major threats to soybean production globally. Recently, multiple miRNAome studies revealed that miRNAs participate in complicated soybean-SCN interactions by regulating their target genes. However, the functional roles of miRNA and target genes regulatory network are still poorly understood. In present study, we firstly investigated the expression patterns of miR159 and targeted GmMYB33 genes. The results showed miR159-3p downregulation during SCN infection; conversely, GmMYB33 genes upregulated. Furthermore, miR159 overexpressing and silencing soybean hairy roots exhibited strong resistance and susceptibility to H. glycines, respectively. In particular, miR159-GAMYB genes are reported to be involve in GA signaling and metabolism. Therefore, we then investigated the effects of GA application on the expression of miR159-GAMYB module and the development of H. glycines. We found that GA directly controls the miR159-GAMYB module, and exogenous GA application enhanced endogenous biologically active GA₁ and GA₃, the abundance of miR159, lowered the expression of GmMYB33 genes and delayed the development of H. glycines. Moreover, SCN infection also results in endogenous GA content decreased in soybean roots. In summary, the soybean miR159-GmMYB33 module was directly involved in the GA-modulated soybean resistance to H. glycines.     

Nematode-antagonistic trichothecenes from Fusarium equiseti

A strain of the fungus Fusarium equiseti isolated from soybean cyst nematode secretes nematode-antagonistic compounds. Bioassay-guided fractionation of an extract of the culture broth was undertaken to identify the compounds. Fractions were assayed for activity against a root-knot nematode (Meloidogyne incognita), a plant pathogen that attacks the roots of numerous plant species. Two trichothecene compounds were isolated that inhibited egg hatch and immobilized second-stage juveniles of this nematode: 4,15-diacetoxy-12,13-epoxy-3,7-dihydroxytrichothec-9-en-8-one (4,15-diacetylnivalenol) and 4,15-diacetoxy-12,13-epoxy-trichothec-9-en-3-ol ( diacetoxyscirpenol). This is the first published report of these compounds affecting plant-parasitic nematodes.     

Zinc-mediated hatching of eggs of soybean cyst nematode,Heterodera glycines

Egg hatching of the soybean cyst nematode,Heterodera glycines, was not affected by millimolar concentrations of calcium sulfate or calcium chloride. However, zinc chloride and zinc sulfate caused strong and moderate increases in hatching, respectively. The inhibitors of calcium transport, ruthenium red and lanthanum chloride, and calcium ionophore A23187 had no effect on hatching in the presence or absence of 3 mM zinc chloride. Selected chelators decreased the zinc-induced hatching ofH. glycines eggs. Eggs exhibited a formation constant with zinc between 5.5 and 11.2. The addition of zinc chloride after chelation with EDTA and rinsing caused expected hatching rates. Concentrations of calcium chloride, manganese chloride, and magnesium chloride had no effect on hatching of eggs in zinc chloride, but reduced hatching at higher concentrations, possibly by osmotic influences. Hatching of eggs was increased as the time of exposure to zinc chloride was increased and was maximal at 28 °C and a pH of 5.3. Picrolonic acid, a known hatching stimulant, increasedH. glycines hatching, while sodium metavanadate had no effect. Analysis of seasonal hatching during 1981–1982 in untreated control eggs indicated that hatching was most pronounced in May.     

Development of Real-Time and Conventional PCR Assays for Identifying a Newly Named Species of Root-Lesion Nematode (Pratylenchus dakotaensis) on Soybean

A rapid and accurate PCR-based method was developed in this study for detecting and identifying a new species of root-lesion nematode (Pratylenchus dakotaensis) recently discovered in a soybean field in North Dakota, USA. Species-specific primers, targeting the internal transcribed spacer region of ribosomal DNA, were designed to be used in both conventional and quantitative real-time PCR assays for identification of P. dakotaensis. The specificity of the primers was evaluated in silico analysis and laboratory PCR experiments. Results showed that only P. dakotaensis DNA was exclusively amplified in conventional and real-time PCR assays but none of the DNA from other control species were amplified. Detection sensitivity analysis revealed that the conventional PCR was able to detect an equivalent to 1/8 of the DNA of a single nematode whereas real-time PCR detected an equivalent to 1/32 of the DNA of a single nematode. According to the generated standard curve the amplification efficiency of the primers in real-time PCR was 94% with a R² value of 0.95 between quantification cycle number and log number of P. dakotaensis. To validate the assays to distinguish P. dakotaensis from other Pratylenchus spp. commonly detected in North Dakota soybean fields, 20 soil samples collected from seven counties were tested. The PCR assays amplified the DNA of P. dakotaensis and discriminated it from other Pratylenchus spp. present in North Dakota soybean fields. This is the first report of a species-specific and rapid PCR detection method suitable for use in diagnostic and research laboratories for the detection of P. dakotaensis.     

Non-destructive Determination of High Oleic Acid Content in Single Soybean Seeds by Near Infrared Reflectance Spectroscopy

Soybean [Glycine max (L.) Merr] with increased oleic acid is desirable to improve oxidative stability and functionality of soybean seed oil. Recently, soybean genotypes with high oleic acid (≥70 %) were developed by breeding programs. Efficient and effective identification of high oleic acid soybean genotypes using non-destructive near infrared reflectance (NIR) on whole seeds would greatly enhance progress in breeding programs. The objective of this study was to develop a calibration equation for NIR determination of high oleic acid from single soybean seeds. A total of 600 intact, single F₂ seeds were scanned by NIR. Spectral data were collected between 400 and 2,500 nm at 2 nm intervals. The relationship between NIR spectral patterns of each soybean seed and its oleic acid content was examined. The best predicted equations for oleic acid were selected on the basis of minimizing the standard error of cross-validation and increasing the coefficient of determination. Validation demonstrated that the equations for determining total oleic acid and over 50 % oleic acid content had high predictive ability (r ² = 0.91 and r ² = 0.99, respectively). To validate the newly developed equation, F₂ seeds from a different genetic background were tested. Again, high oleic acid from single soybean seeds was accurately predicted from various genetic backgrounds. Therefore, applying the calibration equations to NIR will be useful to rapidly and efficiently select high oleic acid soybean genotypes in breeding programs.     

The Plant Sesquiterpene Nootkatone Efficiently Reduces Heterodera schachtii Parasitism by Activating Plant Defense

Plant parasitic nematodes, including the beet cyst nematode Heterodera schachtii, constitute a devastating problem for crops worldwide. The limited availability of sustainable management options illustrates the need for new eco-friendly control means. Plant metabolites represent an invaluable source of active compounds for the discovery of such novel antagonistic agents. Here, we evaluated the impact of eight plant terpenoids on the H. schachtii parasitism of Arabidopsis thaliana. None of the metabolites affected the plant development (5 or 10 ppm). Nootkatone decreased the number of adult nematodes on A. thaliana to 50%, with the female nematodes being smaller compared to the control. In contrast, three other terpenoids increased the parasitism and/or female size. We discovered that nootkatone considerably decreased the number of nematodes that penetrated A. thaliana roots, but neither affected the nematode viability or attraction to plant roots, nor triggered the production of plant reactive oxygen species or changed the plant’s sesquiterpene profile. However, we demonstrated that nootkatone led to a significant upregulation of defense-related genes involved in salicylic and jasmonic acid pathways. Our results indicate that nootkatone is a promising candidate to be developed into a novel plant protection agent acting as a stimulator of plant immunity against parasitic nematodes.     

GmWAK1, Novel Wall-Associated Protein Kinase,Positively Regulates Response of Soybean to Phytophthora sojae Infection

Phytophthora root rot is a destructive soybean disease worldwide, which is caused by the oomycete pathogen Phytophthora sojae (P. sojae). Wall-associated protein kinase (WAK) genes, a family of the receptor-like protein kinase (RLK) genes, play important roles in the plant signaling pathways that regulate stress responses and pathogen resistance. In our study, we found a putative Glycine max wall-associated protein kinase, GmWAK1, which we identified by soybean GmLHP1 RNA-sequencing. The expression of GmWAK1 was significantly increased by P. sojae and salicylic acid (SA). Overexpression of GmWAK1 in soybean significantly improved resistance to P. sojae, and the levels of phenylalanine ammonia-lyase (PAL), SA, and SA-biosynthesis-related genes were markedly higher than in the wild-type (WT) soybean. The activities of enzymatic superoxide dismutase (SOD) and peroxidase (POD) antioxidants in GmWAK1-overexpressing (OE) plants were significantly higher than those in in WT plants treated with P. sojae; reactive oxygen species (ROS) and hydrogen peroxide (H₂O₂) accumulation was considerably lower in GmWAK1-OE after P. sojae infection. GmWAK1 interacted with annexin-like protein RJ, GmANNRJ4, which improved resistance to P. sojae and increased intracellular free-calcium accumulation. In GmANNRJ4-OE transgenic soybean, the calmodulin-dependent kinase gene GmMPK6 and several pathogenesis-related (PR) genes were constitutively activated. Collectively, these results indicated that GmWAK1 interacts with GmANNRJ4, and GmWAK1 plays a positive role in soybean resistance to P. sojae via a process that might be dependent on SA and involved in alleviating damage caused by oxidative stress.     

Pellioditis pellio: Substituted phenolic compounds as chemoattractants

Vanillin, vanillic acid, and other related compounds are chemoattractants either for nematodes or arthropods. We tested vanillin, vanillic acid, and 2,4-dihydroxybenzaldehyde in an in vitro bioassay system for their ability to attractPellioditis pellio males and females. Females were not attracted to any of the three compounds tested. Males responded to vanillin and vanillic acid, but not to 2,4-dihydroxybenzaldehyde. Males responded to vanillin and vanillic acid when these compounds were present at 10^–5 M concentration. Vanillin at 10^–3 and 10^–7 M concentrations was not attractive.     

Effects of three phenolic acids on chlorophyll content and growth of soybean and grain sorghum seedlings

Experiments were designed to test the hypothesis that interference with chlorophyll metabolism may be one mechanism of inhibition of plant growth in allelopathic interactions. Effects of ferulic,p-coumaric, and vanillic acids on soybean and grain sorghum growth and chlorophyll content were quantified and compared after seedlings were treated with these compounds in a nutrient culture. Following a 6-day treatment cycle, dry weights of soybean seedlings were reduced by both 10^–3 M and 5 × 10^–4 M treatments of ferulic,p-coumaric and vanillic acids. Soybean weight reductions in each case were paralleled by a significant reduction in the concentration (g Chl/mg dry wt) of chlorophylls a and b and total chlorophyll in the unifoliate leaves. Sorghum seedling growth was also reduced by each of the compounds at the 5 × 10^–4 M level, but leaf chlorophyll concentration was not below that of control plants.     

Statistical optimization of process parameters for the production of vanillic acid by solid‐state fermentation of groundnut shell waste using response surface methodology

BACKGROUND: Vanillic acid is a flavoring agent and also serves as precursor for vanillin production. Culture medium and fermentation condition for the single step production of vanillic acid from Phanerochaete chrysosporium using lignocellulosic waste as a substrate under solid state fermentation (SSF) were optimized using response surface methodology. RESULTS: The process parameters were chosen by borrowing methodology, and L‐asparagine, pH and moisture content of the solid medium during SSF were identified as the most significant variables. The optimum value of selected variable and their mutual interactions were determined by response surface methodology. The result demonstrated that a yield of 73.58 mg vanillic acid g^?1 substate was predicted under optimum conditions (L‐asparagine 5.98 mmol L^?1 (2.37 mg g^?1 groundnut shell), pH of solid medium 4.51 and moisture content 74.83%). The predicted response was experimentally validated and resulted in a maximum vanillic acid yield of 73.69 mg g^?1 after 8 days of SSF. CONCLUSION: The optimization of fermentation variables resulted in a maximum 10‐fold increase in vanillic acid yield compared with that observed under sub‐optimal conditions (from 7.2 mg g^?1 to 73.69 mg g^?1). Copyright © 2011 Society of Chemical Industry     

大豆秸秆酶水解及L-乳酸发酵

用纤维素酶对氨预处理后的大豆秸秆进行酶水解,利用纤维素酶的作用使纤维素、半纤维素水解为可溶性糖,继而研究了用干酪乳杆菌及清酒乳杆菌进行L-乳酸发酵,通过微生物发酵将生成的可溶性糖转化为用于生产具有可生物降解性的聚乳酸塑料的原料乳酸,实现可再生资源的充分利用.结果表明,实验条件下,5%的大豆秸秆经酶水解后,还原糖浓度为242.25 mg•g^-1,纤维素糖化率为51.22%.清酒乳杆菌、干酪乳杆菌及该两种混合菌种发酵酶解液所得L-乳酸的转化率分别为 48.27%、56.42%和71.05%.     

Allelopathic potential of wild oat (Avena fatua) on spring wheat (Triticum aestivum) growth

Wild oat plants may produce toxic substances that suppress the growth and development of desirable species, thus accounting for severe yield loss in infested fields. The purpose of this study was to determine the allelopathic potential of wild oat (Avena fatua) on the growth of spring wheat (Triticum aestivum var. Fieldwin) in the absence of plant competition. Wild oat and spring wheat plants were grown separately in 250-ml beakers in a sand medium. Root exudates were extracted from wild oat medium at the 1-, 2-, 3-, and 4-leaf stages of wild oat development and added to beakers containing spring wheat in temporally corresponding stages of development. Spring wheat root and leaf dry weights were measured to determine if one or more allelochemical agents were released from wild oat roots. Spring wheat leaf and root dry weights were significantly reduced by exudates from wild oat plants at the 2- and 4-leaf stages of development, respectively. Allelochemicals were isolated from wild oat root exudates at various stages of plant development. Paper chromatography analysis indicated that at least two unknown compounds were present.R _f values in benzene-acetic acid-water of the two unknown compounds (0.825 and 0.930) were similar to scopoletin (7-hydroxy-6-methoxycoumarin) and vanillic acid (4-hydroxy-3-methoxybenzoic acid), respectively. Additional tests using diazotizedp-nitraniline, ultraviolet absorption spectra, and gas chromatography analysis also indicated that the unknowns were coumarin-related compounds such as scopoletin and vanillic acid.     

Phenolic acid content of soils from wheat-no till,wheat-conventional till,and fallow-conventional till soybean cropping systems

Soil core (0–2.5 and/or 0–10 cm) samples were taken from wheat no till, wheat-conventional till, and fallow-conventional till soybean cropping systems from July to October of 1989 and extracted with water in an autoclave. The soil extracts were analyzed for seven common phenolic acids (p-coumaric, vanillic,p-hydroxybenzoic, syringic, caffeic, ferulic, and sinapic; in order of importance) by high-performance liquid chromatography. The highest concentration observed was 4 g/g soil forp-coumaric acid. Folin & Ciocalteu's phenol reagent was used to determine total phenolic acid content. Total phenolic acid content of 0- to 2.5-cm core samples was approximately 34% higher than that of the 0- to 10-cm core samples. Phenolic acid content of 0- to 2.5-cm core samples from wheat-no till systems was significantly higher than those from all other cropping systems. Individual phenolic acids and total phenolic acid content of soils were highly correlated. The last two observations were confirmed by principal component analysis. The concentrations were confirmed by principal component analysis, tions of individual phenolic acids extracted from soil samples were related to soil pH, water content of soil samples, total soil carbon, and total soil nitrogen. Indirect evidence suggested that phenolic acids recovered by the water-autoclave procedure used came primarily from bound forms in the soil samples.The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.     

Facile functionalization of soybean oil by thiol-ene photo-click reaction for the synthesis of polyfunctional acrylate

Nowadays biosource-based materials have received revitalized interest for their ability to substitute for petrochemical-based materials. In this paper, we report a facile synthetic method of soybean oil-based polyfunctional acrylate (PFA) for UV-curable materials. Specifically, rapid and highly efficient side-chain functionalization of soybean oil was achieved via photoclick thiol-ene reaction, soybean oil-based polycarboxylic acid (PCA) was thus obtained. Next, by DCC (N,N′-dicyclohexylcarbodiimide) catalyzed esterification reaction with hydroxypropyl acrylate, polyfunctional acrylate (PFA) was synthesized at room temperature. Real-FTIR result indicated that almost 100% conversion of double-bond within vegetable oil was observed within 16.7 min, yielding the soybean oil-based polycarboxylic acid quantitatively. Furthermore, the structure of PFA was confirmed by ¹H NMR and FTIR. Finally, the excellent UV-curing rate of PFA was revealed by real-FTIR.