Proteome changes in leaves of Brassica napus L. as a result of Sclerotinia sclerotiorum challenge

Sclerotinia stem rot, caused by the necrotrophic fungal pathogen Sclerotinia sclerotiorum, is a serious disease of canola (Brassica napus L.). To increase the understanding of the B. napus- S. sclerotiorum interaction, proteins potentially involved in mediating this interaction were identified and characterized. Upon infection of canola leaves by S. sclerotiorum, necrosis of host leaves was observed by 12 h and rapidly progressed during the later time points. These morphological observations were supported by microscopic study performed at different time points after pathogen challenge. Leaf proteins were extracted and analyzed by 2-DE, which revealed the modulation of 32 proteins (12 down- and 20 up-regulated). The identities of these proteins were established by ESI-q-TOF MS/MS and included proteins involved in photosynthesis and metabolic pathways, protein folding and modifications, hormone signaling, and antioxidant defense. Gene expression analysis of selected genes was performed by qRT-PCR, whereas the elevated levels of the antioxidant enzymes peroxidase and superoxide dismutase were validated by enzyme assays. To the authors' best knowledge, this is the first proteomics-based investigation of B. napus-S. sclerotiorum interaction, and the roles of many of the proteins identified are discussed within the context of this pathosystem.     

The response of a high-yielding canola hybrid to sulfur fertilization in three contrasting Saskatchewan soils

Abstract

Canola (Brassica napus) is the primary oilseed crop in western Canada; however, it is often grown on sulfur (S)-deficient soils. Moreover, canola has a high S demand compared to cereals and, therefore, is particularly sensitive to S deficiency. This study examined the growth and nutrient uptake responses of a high-yielding canola hybrid cultivar to S fertilization when grown on three contrasting soils differing in S fertility, with and without the addition of fertilizer S. The soils were collected from three soil-climatic zones within Saskatchewan (Brown, Black, and Gray) and three different fertilizer S forms were used: ammonium sulfate (AS); ammonium thiosulfate (ATS); and a composite fertilizer containing nitrogen (N), phosphorus (P), and S (NPS; 50-50 blend of sulfate (SO₄) salt and elemental S). Sulfur fertilization increased the canola biomass, along with plant uptake of N, P, and S on all three soils. Fertilizer S use efficiency (i.e. recovery) ranged from 11-75%. For all three soils, the general trend among fertilizer S forms for biomass, nutrient uptake, and fertilizer use efficiency was AS?>?ATS?>?NPS. The greatest differences were observed with the Gray soil, which had the poorest S fertility. Residual soil SO₄ after harvest was greater for ATS and NPS; reflecting continued oxidation of thiosulfate and elemental S to SO₄. Principal component analysis demonstrated the importance of tissue N:S ratio as a key diagnostic measurement related to canola growth and nutrient uptake in S-deficient soils.     

Potential fungal inhibition by immobilized hydrolytic enzymes from Trichoderma asperellum

The use of cell wall degrading enzymes from Trichoderma asperellum immobilized on biodegradable support is an alternative for food packaging. In this study, hydrolytic enzymes produced by T. asperellum were tested as a fungal growth inhibitor, in free form or immobilized on a biodegradable film composed of cassava starch and poly(butylene adipate-co-terephtalate) (PBAT). The inhibitory activity was tested against Aspergillus niger , Penicillium sp., and Sclerotinia sclerotiorum , microorganisms that frequently degrade food packaging. The use of chitin as carbon source in liquid medium induced T. asperellun to produce N-acetylglucosaminidase, β-1,3-glucanase, chitinase, and protease. The presence of T. asperellun cell wall degradating enzymes (T-CWD) immobilized by adsorption or covalent attachment resulted in effective inhibition of fungal growth. The enzymatic activity of T-CWD was stronger on S. sclerotiorum than on the Aspergillus or Penicillum isolates tested. These results suggest that T-CWD can be used in a free or immobilized form to suppress fungi that degrade food packaging.     

Uptake and nutrient balance of nitrogen,sulfur, and boron for optimal canola production in eastern Canada

Balanced plant nutrition is essential to achieve high yields of canola (Brassica napus L.) and get the best economic return from applied fertilizers. A field study was conducted at nine site‐years across eastern Canada to investigate the effects of nitrogen (N), sulfur (S) and boron (B) fertilization on canola nutrient uptake, nutrient balance, and their relationship to canola yields. The factorial experiment consisted of four N rates of 0 (N0), 50 (N50), 100 (N100), and 150 (N150) kg ha^?1, two S rates of 0 (S0) and 20 (S20) kg ha^?1, and three B treatments of 0 (B0), 2 kg ha^?1 at preplant (B2.0P), and 0.5 kg B ha^?1 foliar‐applied at early flowering stage (B0.5F). Each site‐year used the same experimental design and assigned treatments in a randomized complete block design with four replications. Fertilizer S application greatly improved seed yields at six out of nine site‐years, and the highest N use efficiency was in the N150+S20 treatment. Sulfur application generally increased seed S concentration, seed S removal, and plant total S uptake, while B fertilization mainly elevated straw B concentration and content, with minimal effect on seed yields. At the early flowering stage, plant tissue S ranged from 2.2 to 6.6 mg S g^?1, but the N : S ratio was over or close to the critical value of 12 in the N150+S0 combination at five site‐years. On average across nine site‐years, canola reached a plateau yield of 3580 kg ha^?1 when plants contained 197 kg N ha^?1, 33 kg S ha^?1 and 200 g B ha^?1, with a seed B content of 60 g B ha^?1. The critical N, S, and B values identified in this work and their potential for a posteriori nutrient diagnosis of canola should be useful to validate fertilizer requirements for canola production in eastern Canada.     

拮抗菌DG1的鉴定及其对大白菜菌核病菌的抑制作用研究

为减少化学农药的使用,丰富生物防治菌株资源库,在分离大白菜菌核病菌的过程中,筛选到一株对其具有抑制作用的拮抗菌,命名为DG1.本研究通过形态观察、生理生化特征检测及16S rDNA序列分析,对拮抗菌DG1进行种属鉴定;通过测量菌落直径,研究DG1发酵液及挥发性物质对大白菜菌核病菌的抑制作用;通过平板对峙法测定拮抗菌DG...     

SOURCES OF SULFUR FOR DRY MATTER,SEED YIELD,AND OIL CONCENTRATION OF CANOLA GROWN IN SULFUR DEFICIENT SOILS OF SOUTH-WESTERN AUSTRALIA

Leaching of sulfur (S) on sandy soils may limit the effectiveness of S fertilizers especially when applied at sowing. The effectiveness of S sources for canola (oil seed rape, Brassica napus L.) grown in sandy low S soils of south-western Australia is not known. This study was completed to determine the relative effectiveness of gypsum and a gypsum-based by-product from synthetic rutile processing called Canola Blue for canola grown in low S soils of the region. Canola Blue is a mixture of gypsum and elemental S, and is granulated so its effectiveness may vary from gypsum. We measured the effectiveness of the two S sources in the glasshouse for young seedling growth and for minimizing S leaching. In the four field experiments, the two S sources were evaluated for relative effects on canola seed yield and the concentration of oil in seed. Canola Blue applied at sowing was as effective as gypsum for canola growth in the glasshouse and when applied at 35 days after sowing (DAS) was as effective as gypsum for seed yield in the field. For the glasshouse study, Canola Blue when applied to the soil surface (topdressed) at 35 DAS was as effective as gypsum in achieving a rapid recovery of leaves from S deficiency symptoms and of shoot growth. Where S deficiency appears during the growing season, topdressing with Canola Blue appears to be as effective under the conditions of this experiment as was gypsum. However, the S in Canola Blue was less prone to leaching than that applied as gypsum.     

溶磷真菌促进磷素吸收和作物生长的作用研究

在盆栽条件下,利用花生、玉米、油菜在3种不同磷水平的石灰性土壤上,采用土壤耗竭性连续种植的方法,研究了溶磷青霉菌P8对吸收土壤磷素和作物生长的影响。结果表明,3种土壤上接种青霉菌P8菌剂都能提高作物的吸磷量,其生物产量比不接种显著提高,且在有效磷低的土壤上其增产幅度大于有效磷高的土壤;接种青霉菌P8在一定程度上能缓解和改善低温对作物生长和磷素吸收的不良影响。无论在有效磷高还是低的土壤,青霉菌P8都具有活化土壤磷、提高土壤磷素的利用效率和促进作物苗期生长的作用。     

抗速灭威单链抗体基因在巴斯德毕赤酵母中的表达及性质研究

选用巴斯德毕赤酵母（Pichia pastoris）系统表达特异性抗速灭威单链抗体（scFv）基因,以为速灭威特异性抗体的大量制备奠定基础。设计引物扩增阳性克隆scFv基因,亚克隆至表达载体pPICZαC,获得重组酵母表达质粒pPICZαC-scFv,线性化pPICZαC-scFv并高效电转P.pastoris（X-33）,对转化子进行抗性梯度筛选得到一株高效表达的X-33-Pp-SMW-12-6菌株。对获得的菌株先后进行表达条件的优化、优化条件下的诱导表达及单链抗体性质研究。结果表明：P.pastoris-scFv的质量接近其亲本E.coli-scFv的质量,X-33-Pp-SMW12-6在优化条件下的表达产量达28mg/L,比未优化前的产量提高了约8mg/L,经纯化后抗体纯度可达85%以上。因此,利用P.pastoris表达系统制备抗速灭威单链抗体比细菌表达系统更有效、更经济。     

Volatile compounds emitted by sclerotia of Sclerotinia minor,Sclerotinia sclerotiorum,and Sclerotium rolfsii

Volatile compounds emitted by sclerotia of Sclerotinia minor, Sclerotinia sclerotiorum, and Sclerotium rolfsii were identified by solid phase microextraction followed by gas chromatography and mass spectometry. Both S. minor and S. sclerotiorum emitted 2-methylenebornane and 2-methylisoborneol. In addition, S. minor emitted mesityl oxide, gamma-butyrolactone, cis- and trans-linalool oxide, linalool, and trans-nerolidol. S. sclerotiorum emitted 2-methyl-2-bornene, 1-methylcamphene, and a diterpene with a molecular weight of 272. Sclerotium rolfsii did not emit any of these compounds but did emit delta-cadinene and cis-calamenene. Chemicals emitted by S. minor and S. sclerotiorum were tested to determine if they could stimulate germination of conidia of Sporidesmium sclerotivorum, a mycoparasite on sclerotia of Sclerotinia spp. Chemicals were tested at 1 part per billion to 100 parts per million, both in direct contact with conidia and near, but not in, physical contact. None of the chemicals alone nor a combination of all chemicals induced germination of conidia of S. sclerotivorum.     

Significant Nitrogen by Sulfur Interactions Occurred for Canola Grain Production and Oil Concentration in Grain on Sandy Soils in the Mediterranean-Type Climate of Southwestern Australia

The nitrogen (N) by sulfur (S) interaction for canola (Brassica napus L.) grain production and oil concentration in grain has been quantified in temperate climates, but it is not known if these results also apply to sandy soils common in the Mediterranean-type climate of southwestern Australia where canola is now a major crop. Seventeen field experiments were undertaken with canola in the region during 1994 to 2005 in which 4 rates of both N (0–138 kg N/ha) and S (0–34 kg S/ha) were applied. Significant grain yield responses to applied N occurred in all experiments and the responses increased as more S was applied. Grain yield responses to applied S only occurred when N was applied and tended to increase as more N was applied. When no S was applied the two largest rates of N applied, 69 and 138 kg N/ha, induced S deficiency reducing grain yields. The oil concentration in grain tended to decrease as more N was applied and increased as more S was applied, particularly when the two largest rates of N were applied. Consequently significant N × S interactions were obtained in all experiments for grain production and in 15 experiments for oil concentration in grain.     

Nitrogen and Sulfur Effects on Macro- and Micronutrient Contents in Canola (Brassica napus L.) Grown on Acidic Soils of the Western Cape Province of South Africa

Soil pH and nutrient contents influence the uptake and utilization of nutrients required for plant growth. Soil characteristics in the canola-growing areas of the Western Cape Province of South Africa are often very variable. Hence, the major aim of this research was to determine the effects of soil and climatic differences as experienced at different localities on macro- and micronutrient contents and uptake in canola plants fertilized with different nitrogen (N) and sulfur (S) application rates. Plants fertilized with 0, 15, and 30 kg S ha^?1 in combination with N rates of 0 and 90 kg ha^?1 were sampled at 90 days after planting (DAP) (flowering stage) at Altona, Elsenburg, Langgewens, Roodebloem, and Welgevallen localities in the Western Cape during 2009 2010, and 2011. Nutrient content in canola plants were affected by locality and interactions between locality and N application rates. Sulfur content within the plants remained less than the sufficient quantity of 0.5%, and a dilution effect on elemental concentration in canola especially at greater N rates (plus lack of sufficient S) is apparently evident from the results. The results also reveal that canola S application should match S adsorption capacity of the low pH soils of the Western Cape.     

新鲜猪沼液和牛沼液对农作物病原真菌抑制作用的比较研究

本试验在实验室条件下,研究大中型沼气工程猪沼和牛沼两种沼液原液及其离心上清液对大豆尖孢镰刀菌、大豆菌核病菌、小麦纹枯病菌、小麦根腐病菌、水稻纹枯病菌、玉米大斑病菌和玉米小斑病菌等7种农作物病原真菌的抑制作用。结果表明：猪沼原液和牛沼原液对其中的大豆尖孢镰刀菌、大豆菌核病菌、小麦纹枯病菌、小麦根腐病菌和水稻纹枯病菌5种病原真菌具有较好的抑制作用,其菌丝生长抑制率均在72%以上,但对玉米大斑病菌和玉米小斑病菌的菌丝生长基本没有抑制作用;相比之下,猪沼和牛沼离心上清液对以上5种病菌的菌丝生长抑制作用明显减弱,除猪沼离心上清液对大豆菌核病菌的菌丝生长抑制率大于70%以外,试验中猪沼和牛沼离心上清液对实验病原菌的菌丝生长抑制率基本都在60%以下。试验结果显示猪沼液和牛沼液对农作物病原真菌具有潜在的植物病害防治作用,为养殖场大中型沼气工程沼液应用新技术的开发提供科学依据。     

Increasing expression of an anti-picloram single-chain variable fragment (ScFv) antibody and resistance to picloram in transgenic tobacco (Nicotiana tabacum)

Systematic research involving four chimeric gene constructions designed to express the same anti-picloram single-chain variable fragment (scFv) antibody is described. Agrobacterium-mediated transformation produced at least 25 transgenic tobacco plants with each of these, and the number of T-DNA loci in each plant was determined using kanamycin-resistance segregation assays. The relative amounts of active and total scFv in each plant were evaluated using quantitative enzyme-linked immunosorbent assay and immunoblot technologies, respectively. No significant differences in scFv activity were found among the four groups of single-locus plants, although the 35S/M construct was found to produce significantly more total anti-picloram scFv than the other three constructs. A dose-response bioassay involving T(1) seedlings from several of the highest expressers of active scFv demonstrated resistance to a constant exposure of picloram at 5 x 10(-)(8) M. Other approaches for increasing antibody-based herbicide resistance are discussed, as further improvements are needed before practical application of this technology.     

Immunoassay for wheat processing quality: utilization of a sandwich assay incorporating an immobilized single-chain fragment.

A single-chain fragment (scFv) was engineered from a monoclonal antibody to high molecular weight glutenin subunits (HMW-GS), wheat flour polypeptides that play a major role in determining the mixing- and extension strength-related properties of dough and its subsequent baking performance. The scFv was expressed in a thioredoxin mutant Escherichia coli strain that allows disulfide bond formation in the cytoplasm and incorporated into a diagnostic test for wheat quality. Although the scFv lacks the more highly conserved antibody constant regions usually involved with immobilization, it was able to be directly immobilized to a polystyrene microwell solid phase without chemical or covalent modification of the protein or solid phase and utilized as a capture antibody in a double-antibody (two-site) immunoassay. In the sandwich assay, increasing HMW-GS concentrations produced increasing assay color, and highly significant correlations were obtained between optical densities obtained in the ELISA using the scFv and the content of large glutenin polymers in flours as well as measures of dough strength as measured by resistance to dough extension in rheological testing. The assay using the scFv was able to be carried out at lower flour sample extract dilutions than that required for a similar assay utilizing a monoclonal capture antibody. This research shows that engineered antibody fragments can be utilized to provide superior assay performance in two-site ELISAs over monoclonal antibodies and is the first application of an engineered antibody to the analysis of food processing quality.     

COMPARING FERTILIZER PHOSPHORUS REQUIREMENTS OF CANOLA,LUPIN, AND WHEAT

Increases in yield due to applications of phosphorus (P) (0, 5, 10, 15, 20, and 40 kg P/ha) applied as single (ordinary) superphosphate were measured for canola (Brassica napus), lupin (Lupinus angustifolius) and wheat (Triticum aestivum) in a field experiment on a deep sandy soil near Esperance, south-western Australia (WA). There are no data comparing the P requirements of these species grown at the same time, which was done by determining the amount of P required to produce 90% of the maximum yield for each species. The amount of P required was about 50% less for canola than wheat and about 10% more for lupin than wheat (60% more than canola). For each amount of P applied, the concentration of P in shoots and grain was greater for canola, followed by lupin and then wheat, suggesting that canola and lupin roots were better at accessing soil P than wheat. The critical concentration of P (diagnostic) required for 90% maximum yield of dried shoots measured in September was about 2.3 g/kg P for wheat, 2.8 g/kg P for lupin, and 3 g/kg P for canola. Similar critical values were obtained when P concentration in the shoots was related to grain yield (prognostic).     

菊花菌核病病原菌鉴定、抗性筛选与防治

菌核病是菊花生产中的一种重要病害.为明确菊花菌核病病原菌的种类、品种抗性差异和有效的防治药剂,本研究采用组织分离法对病原菌进行分离培养,结合形态学特征、生物学特征和分子生物学研究进行鉴定,并筛选了33个切花菊品种以及7种杀菌剂.结果 表明,从感病植株上分离得到的病原菌能够重新感染菊花,并且再次分离得到的菌株与原菌株相同...     

Effects of two new siderophore-producing rhizobacteria on growth and iron content of maize and canola plants

Siderophore-producing rhizobacteria beneficially affect plant growth by providing available iron to plants. In this study, bacteria were isolated from the rhizosphere of canola (Brassica napus L.) plants grown in the central fields in Iran, for the presence of siderophore-producing bacteria. A total of 45 distinct isolates were found to produce siderophore using qualitative chrome azurol sulfonate (CAS)-agar assay. Of them, ten isolates, based on the highest halo diameter/colony diameter ratios, were selected to quantify the rate of siderophore production using CAS-liquid assay. A variety of biochemical assays was used to determine the type(s) of siderophores produced by each of the ten isolates. The best isolates, based on production of the highest rates of either hydroxamates or carboxylates, were identified and used in further studies. Based on 16S ribosomal ribonucleic acid (rRNA) sequence analysis and a variety of phenotypic properties, the isolates were identified as Micrococcus yunnanensis YIM 65004 (T) and Stenotrophomonas chelatiphaga LPM-5 (T). We also studied the plant growth-promoting effect of the most promising isolates (YIM 65004 and LPM-5) on canola and maize plants under greenhouse conditions. The results of this study showed that M. yunnanensis and S. chelatiphaga increased gain weight and iron (Fe) content of roots and shoots significantly, in comparison with control, indicating beneficial effects of these rhizobacteria on plant growth and development. This study reports M. yunnanensis and S. chelatiphaga, as new records for Iran. The latter is reported for the first time from plant (canola) rhizosphere. Besides, the ability of both M. yunnanensis and S. chelatiphaga to produce siderophores is documented for the first time.     

Glyphosate-resistant and -susceptible soybean (Glycine max) and canola (Brassica napus) dose response and metabolism relationships with glyphosate

Experiments were conducted to determine (1) dose response of glyphosate-resistant (GR) and -susceptible (non-GR) soybean [Glycine max (L.) Merr.] and canola (Brassica napus L.) to glyphosate, (2) if differential metabolism of glyphosate to aminomethyl phosphonic acid (AMPA) is the underlying mechanism for differential resistance to glyphosate among GR soybean varieties, and (3) the extent of metabolism of glyphosate to AMPA in GR canola and to correlate metabolism to injury from AMPA. GR50 (glyphosate dose required to cause a 50% reduction in plant dry weight) values for GR (Asgrow 4603RR) and non-GR (HBKC 5025) soybean were 22.8 kg ae ha-1 and 0.47 kg ha-1, respectively, with GR soybean exhibiting a 49-fold level of resistance to glyphosate as compared to non-GR soybean. Differential reduction in chlorophyll by glyphosate was observed between GR soybean varieties, but there were no differences in shoot fresh weight reduction. No significant differences were found between GR varieties in metabolism of glyphosate to AMPA, and in shikimate levels. These results indicate that GR soybean varieties were able to outgrow the initial injury from glyphosate, which was previously caused at least in part by AMPA. GR50 values for GR (Hyola 514RR) and non-GR (Hyola 440) canola were 14.1 and 0.30 kg ha-1, respectively, with GR canola exhibiting a 47-fold level of resistance to glyphosate when compared to non-GR canola. Glyphosate did not cause reduction in chlorophyll content and shoot fresh weight in GR canola, unlike GR soybean. Less glyphosate (per unit leaf weight) was recovered in glyphosate-treated GR canola as compared to glyphosate-treated GR soybean. External application of AMPA caused similar injury in both GR and non-GR canola. The presence of a bacterial glyphosate oxidoreductase gene in GR canola contributes to breakdown of glyphosate to AMPA. However, the AMPA from glyphosate breakdown could have been metabolized to nonphytotoxic metabolites before causing injury to GR canola. Injury in GR and non-GR canola from exogenous application of AMPA was similar.     

Crop Response to Elemental S and Sulfate-S Sources on S-Deficient Soils in the Parkland Region of Alberta and Saskatchewan

Six field experiments were conducted on sulfur-(S) deficient Gray Luvisol (Typic Cryoboralf) and Dark Gray Chernozem (Typic Cryoborolls) soils at various locations in Alberta and Saskatchewan to evaluate the effectiveness of elemental S (ES-90, ES-95, Urea S, Fine S Powder), sulfate-S (ammonium sulfate, sodium sulfate), and elemental S + sulfate-S (Sulgro-68, Agrium Plus) fertilizers, and their rates and placement methods on seed yield and S uptake of canola and barley, and forage dry matter yield of pure bromegrass and bromegrass-alfalfa mixed stands. There was a substantial increase in yield and/or S uptake from sulfate-S application in most experiments. This indicated that the soils used in these experiments were deficient in plant-available S, especially for canola. In the initial year, the performance of elemental S fertilizers was usually inferior to sulfate-S fertilizers. Among the elemental S fertilizers, the ES-95 tended to produce greater yield and S uptake than ES-90. In general, there was no difference in seed yield between early (May 1) and late (May 23) spring applications for the elemental S (Urea S). The results indicated that from the second or third year of initiating annual applications the effectiveness of some elemental S fertilizers almost approached the level of sulfate-S fertilizers. Residual effect of S application lasted for at least three years. Overall, the findings suggest that correction of a severe S deficiency by elemental S alone may be risky in the short term, and thus addition of some sulfate-S in the initial 1 or 2 years to supplement the sulfate-S from elemental S is advisable. Increased exposure of elemental S fertilizers to oxidation either by incorporation and broadcast methods or by application ahead of plant use time tends to improve crop response.     

盐胁迫油菜根际中含假单胞菌（Pseudomonas spp.1）的氨基环丙烷-1-羧酸 (ACC) 脱氨基酶特征研究

When exposed to biotic or abiotic stress conditions,plants produce ethylene from its immediate precursor 1-aminocyclopropane-1-carboxylate(ACC),leading to retarded root growth and senescence.Many plant growth-promoting rhizobacteria contain the enzyme ACC deaminase and this enzyme can cleave ACC to form α-ketobutyrate and ammonium,thereby lowering levels of ethylene.The aim of this study was to isolate and characterize ACC deaminase-producing bacteria from the rhizosphere of salt-stressed canola(Brassica napus L.).Out of 105 random bacterial isolates,15 were able to utilize ACC as the sole source of nitrogen.These 15 isolates were also positive for indole acetic acid(IAA) production.Phylogenetic analysis based on partial 16 S rDNA sequences showed that all isolates belonged to fluorescent Pseudomonas spp.In the canola rhizosphere investigated in this study,Pseudomonas fluorescens was the dominant ACC deaminase-producing species.Cluster analysis based on BOX-A1R-based repetitive extragenic palindromic-polymerase chain reaction(BOX-PCR) patterns suggested a high degree of genetic variability in ACC deaminase-producing P.fluorescens strains.The presence of indigenous ACC-degrading bacteria in the rhizosphere of canola grown in saline soils indicates that these bacteria may contribute to salinity tolerance.