Understanding the rhizodeposited carbon (C) dynamics of winter wheat ( Triticum aestivum L.), is crucial for soil fertility and C sequestration. Pot-grown winter wheat was pulse labelled with 14 CO 2 at the key growth stages. 14 C in the shoots, roots and soil was measured at 5 or 2 days after 14 C-labelling (DAL 5/2) at each growth stage and at harvest. The 14 C in the shoots increased from 4% of the net 14 C recovered (shoots + roots + soil) during tillering to 53% at harvest. Approximately 14–34% of the net 14 C recovered was incorporated into the soil. Allocation of photosynthesized C was extrapolated from the pot experiment to field condition, assuming a planting density of 1.8 million plants ha −1 . The estimated C input to the soil was 1.7 t C ha −1 , and 0.7 t C ha −1 of root residues was retained after wheat harvest; both values were higher than those previously reported (0.6 and 0.4 t C ha −1 , respectively). Our findings highlight that C tracing during the entire crop season is necessary to quantify the temporal allocation of photosynthesized C, especially the contribution to soil carbon in intensified farming system.

Degradation of toxins by microorganisms is a promising approach for detoxification of agricultural products. Here, a bacterial strain, Sphingomonas S3-4, that has the ability to degrade the mycotoxin deoxynivalenol (DON) was isolated from wheat fields. Incubation of Fusarium -infected wheat grains with S3-4 completely eliminated DON. In S3-4 DON is catabolized into compounds with no detectable phytotoxicity, 3-oxo-DON and 3-epi-DON, via two sequential reactions. Comparative analysis of genome sequences from two DON-degrading strains, S3-4 and Devosia D17, and one non-DON-degrading strain, Sphingobium S26, combined with functional screening of a S3-4 genomic BAC library led to the discovery that a novel aldo/keto reductase superfamily member, AKR18A1, is responsible for oxidation of DON into 3-oxo-DON. DON-degrading activity is completely abolished in a mutant S3-4 strain where the AKR18A1 gene is disrupted. Recombinant AKR18A1 protein expressed in Escherichia coli catalyzed the reversible oxidation/reduction of DON at a wide range of pH values (7.5 to 11) and temperatures (10 to 50 °C). The S3-4 strain and recombinant AKR18A1 also catabolized zearalenone and the aldehydes glyoxal and methyglyoxal. The S3-4 strain and the AKR18A1 gene are promising agents for the control of Fusarium pathogens and detoxification of mycotoxins in plants and in food/feed products.

Globally, the trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) are among the most widely distributed mycotoxins that contaminate small grain cereals. In this study, a bacterial consortium, PGC-3, with de-epoxydation activity was isolated from soil by an in situ soil enrichment method. Screening of 14 soil samples that were sprayed with DON revealed that 4 samples were able to biotransform DON into de-epoxydized DON (dE-DON). Among these, the PGC-3 consortium showed the highest and most stable activity to biotransform DON into dE-DON and NIV into dE-NIV. PGC-3 exhibited de-epoxydation activity at a wide range of pH (5–10) and temperatures (20–37 °C) values under aerobic conditions. Sequential subculturing with a continued exposure to DON substantially reduced the microbial population diversity of this consortium. Analyses of the 16S rDNA sequences indicated that PGC-3 comprised 10 bacterial genera. Among these, one species, Desulfitobacterium, showed a steady increase in relative abundance, from 0.03% to 1.55% (a 52-fold increase), as higher concentrations of DON were used in the subculture media, from 0 to 500 μg/mL. This study establishes the foundation to further develop bioactive agents that can detoxify trichothecene mycotoxins in cereals and enables for the characterization of detoxifying genes and their regulation.

Fusarium graminearum is the fungal pathogen that causes globally important diseases of cereals and produces mycotoxins such as deoxynivalenol (DON). Owing to the dearth of available sources of resistance to Fusarium pathogens, characterization of novel genes that confer resistance to mycotoxins and mycotoxin-producing fungi is vitally important for breeding resistant crop varieties. In this study, a wheat methionyl-tRNA synthetase (TaMetRS) gene was identified from suspension cell cultures treated with DON. It shares conserved aminoacylation catalytic and tRNA anticodon binding domains with human MetRS and with the only previously characterized plant MetRS, suggesting that it functions in aminoacylation in the cytoplasm. However, the TaMetRS comprises a typical nuclear localization signal and cellular localization studies with a TaMetRS::GFP fusion protein showed that TaMetRS is localized in the nucleus. Expression of TaMetRS was activated by DON treatment and by infection with a DON-producing F. graminearum strain in wheat spikes. No such activation was observed following infection with a non-DON-producing F. graminearum strain. Expression of TaMetRS in Arabidopsis plants conferred significant resistance to DON and F. graminearum. These results indicated that this DON-activated TaMetRS gene may encode a novel type of MetRS in plants that has a role in defense and detoxification.

Plant germplasm resources with natural resistance against globally important toxigenic Fusarium are inadequate. CWP2, a Fusarium genus-specific antibody, confers durable resistance to different Fusarium pathogens that infect cereals and other crops, producing mycotoxins. However, the nature of the CWP2 target is not known. Thus, investigation of the gene coding for the CWP2 antibody target will likely provide critical insights into the mechanism underlying the resistance mediated by this disease-resistance antibody. Immunoblots and mass spectrometry analysis of two-dimensional electrophoresis gels containing cell wall proteins from Fusarium graminearum (Fg) revealed that a glyoxal oxidase (GLX) is the CWP2 antigen. Cellular localization studies showed that GLX is localized to the plasma membrane. This GLX efficiently catalyzes hydrogen peroxide production; this enzymatic activity was specifically inhibited by the CWP2 antibody. GLX-deletion strains of Fg, F. verticillioides (Fv) and F. oxysporum had significantly reduced virulence on plants. The GLX-deletion Fg and Fv strains had markedly reduced mycotoxin accumulation, and the expression of key genes in mycotoxin metabolism was downregulated. This study reveals a single gene-encoded and highly conserved cellular surface antigen that is specifically recognized by the disease-resistance antibody CWP2 and regulates both virulence and mycotoxin biosynthesis in Fusarium species.

Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co-expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T-3 to T-5 generations. Confocal microscopy revealed profoundly restricted mycelia in Fg-infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down-regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host-induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions.

种子学原有课程体系存在课时少、实验教学环节欠缺、教学方法单一、偏重理论教学、教学内容脱离实践等问题。华中农业大学在新形势下成立了种子科学与工程专业教学团队,以培养学生创新能力和实践动手能力等综合能力为目的,发挥作物学学科优势,加强种子学实验教学环节,优化实验教学内容,并就课程实验实践体系进行了探讨。

Fusarium graminearum clade species are among the main causative agents of Gibberella ear rot (GER) in maize and responsible for the various trichothecene mycotoxins accumulated in contaminated maize grains. In this study, a total of 620 isolates from diseased maize ears collected from 59 districts in 19 provinces throughout China, previously identified morphologically as Fusarium graminearum clade, was genetically characterized at the species level based on SCAR (Sequence Characterized Amplified Region) and for their potential capability of mycotoxin production using the genetic chemotyping assay. The results showed that 359 isolates were F.asiaticum (SCAR 5), which consisted of 97% nivalenol (NIV)-chemotypes, 0.8% 3-acetyldeoxynivalenol (3-ADON)-producing isolates and 2.2% 15-acetyldeoxynivalenol (15-ADON) producers, whereas the remaining 261 isolates were identified as F.graminearum sensu stricto (SCAR 1), all of which produced 15-ADON mycotoxins. This high proportion of NIV producers present in F.asiaticum is different from the chemotype patterns in F.asiaticum populations isolated from wheat and barley, where DON and its acetylated chemotypes were the predominant mycotoxins. Moreover, the majority of NIV producers (59.1%) and all the 3-ADON-producing strains were derived from the warmer regions in southern China, whereas most of the 15-ADON-producing strains (78.4%) were isolated from the colder regions in northern China. Our study is the first report of NIV chemotypes of F.asiaticum and 15-ADON chemotypes of F.graminearum sensu stricto that were associated with the GER of maize in China.

The viviparous-1 (Vp1) gene from maize encodes a transcription factor involved in abscisic acid (ABA) signaling that is associated with seed dormancy and preharvest sprouting (PHS). Mis-splicing of wheat homologous Vp1 transcripts has been considered the main factor causing PHS sensitivity in wheat. A maize Vp1 gene including its promoter and coding sequence was used for Agrobacterium tumefaciens mediated transformation of an elite wheat cultivar, Zheng9023. Plants expressing the Vp1 gene displayed a genetically stable, significantly enhanced seed dormancy and PHS tolerance. A significant reduction of α-amylase activity of mature grains was detected in all the transgenic wheat plants. Furthermore, quantitative real-time polymerase chain reactions revealed that more transcripts for the genes involved in VP1/ABA signaling, such as the genes coding for wheat ABA-insensitivity, malate oxidoreductase, peroxiredoxin, and late embryogenesis abundant proteins, were concomitantly accumulated during seed development in the transgenic wheat plants compared with the non-transgenic Zheng9023. These results indicated a highly functional compensation of the Vp1 gene in wheat by an alien homolog from maize, providing a promising approach to breed wheat cultivars with improved tolerance for PHS through a genetic engineering process.

Chloroplast transformation in plants has many advantages over nuclear transformation.Proteins in chloroplasts can be expressed at high levels with proper folding and disulfide bonds as the cells of higher plants contain a large number of chloroplast genomes.Multiple genes can be co-expressed in chloroplast genomes.Furthermore,chloroplast genes are inherited in a strictly maternal fashion in most angiosperm plant species,and this minimizes the possibility of out-crossing transgenes to related weeds or species.In addition,gene silencing,position effects and random integration have not been reported in chloroplast transformation.Although chloroplast transformation is very attractive,this technology is not as widely used as nuclear transformation.It has been mostly focused on 16 plants species,especially tobacco in which many proteins has been expressed including vaccines and antibodies.In this review we briefly summarize the rationales,methodologies,applications,bottlenecks and prospects of this promising genetic engineering technology for chloroplasts.

To develop a wheat genetic transformation system that is not restricted by seasons,this work investigated the optimized conditions and methods for induction and regeneration of multiple shoots directly derived from shoot apical meristem tissues in wheat.The study included the comparisons of three germination media,four induction media,three induction times and three rooting media with three elite wheat cultivars.The results indicated that MB5 basal medium without any hormones was suitable for germination of wheat mature embryos.MB5 basal medium plus 1.0 mg/L TDZ and 0.5 mg/L IBA produced the highest rate of multiple shoot induction.Numbers of multiple shoots increased with the increase of induction time.The suitable time for multiple shoot induction is not more than 30 days.There was no difference among the wheat cultivars in terms of induction rate and number of multiple shoots.The best medium for rooting was 1/2 MS basal medium plus 1.0 mg/L IBA.Regenerated plants derived form multiple shoots showed normal flowering and seeds.These results provide useful methods for regeneration and genetic transformation based on multiple shoots of wheat.

A protocol for chloroplast transformation of an elite rapeseed cultivar (Brassica napus L.) was developed based on optimized conditions for callus induction and regeneration from cotyledonary tissues. Comparison of six different media with three elite cultivars showed that B5 medium plus 3 mg/l AgNO(3) supplemented with 0.6 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-furfurylaminopurine was optimal for callus formation and maintenance without differentiation, while the medium suitable for regeneration was B5 medium supplemented with 1 mg/l 6-benzylaminopurine, 1 mg/l 6-furfurylaminopurine and 0.5 mg/l alpha-naphthaleneacetic acid. A rapeseed-specific chloroplast transformation vector was constructed with the trnI and trnA sequences amplified from the rapeseed chloroplast genome using two primers designed according to Arabidopsis homologs. The aadA gene was used as a selection marker regulated by the ribosome-binding site from the bacteriophage T7 gene 10L, the tobacco 16S rRNA promoter and the psbA terminator. After bombardment, cotyledonary segments were cultured for callus formation on media containing 10 mg/l spectinomycin and regeneration was carried out on medium with 20 mg/l spectinomycin. Heteroplasmic plastid transformants were isolated. An overall efficiency for the chloroplast transformation was one transplastomic plant per four bombarded plates. Southern blot analyses demonstrated proper integration of the target sequence into the rapeseed chloroplast genome via homologous recombination. The expression of the aadA gene was confirmed by Northern blot analysis. Analysis of T1 transplastomic plants revealed that the transgenes integrated into the chloroplast were inheritable with a ratio of about 8%. These results suggest that rapeseed may be a suitable crop for chloroplast transformation with cotyledons as explants under appropriate conditions.

Crop Science is an applied science directly serving agricultural production.Based on the analysis on practical teaching status quo of Crop Science,this paper illustrates the problems in practical teaching and the significance of the practical teaching reform.This paper also puts forward the general idea and a series of specific measures on practical teaching reform of Crop Science:such as establishing new teaching idea;integrating small courses of different professional courses,such as crop cultivation,crop breeding and so on into the big course of crop science practice;specifying and integrating different practical teaching content and establishing a common teaching resources platform as well as introducing the new course content and method.After the reform,the teaching system may become more effective to use teaching time and teaching resources so that the practical teaching contents can be integrated and student's practical ability and innovative thinking ability can be improved.

Based on the amino acid sequence of a single-chain antibody specific for zearalenone mycotoxin and codon usage in E.coli,a variable heavy chain(VH) and a variable light chain(VL) were synthesized by PCR to create a single-chain(scFv) antibody gene,ZEN2,that was linked by a(Gly4Ser)2 linker.The scFv antibody gene,ZEN2,was further ligated into an alkaline phosphatase(AP) sequence to construct a fusion protein,ZEN2 scFv-AP that was cloned into pET vector.E.coli strain BL21(DE3) was transformed with the pET plasmid containing ZEN2 scFv-AP and used for the expression of the fusion protein upon induction by isopropylthio-β-D-galactoside(IPTG).The bacterially expressed soluble fusion protein was purified by affinity chromatography.SDS-PAGE and Western blot analyses of the purified protein detected a 75 ku protein band.Enzyme-linked immunosorbent assay(ELISA) revealed an enzymatic activity for the alkaline phosphatase in the fusion protein ZEN2 scFv-AP.These results would serve as a foundation for the development of a rapid,accurate and cheap method for the detection of zearalenone mycotoxin via ELISA.

作物学是一门直接为农业生产服务的应用科学,实践课程是培养学生应用理论知识并提高分析问题与解决问题能力的关键环节.本文在对作物学实践教学的发展及现状进行总结分析的基础上,提出了实践教学工作中存在的问题,以及与现代科学技术接轨进一步改进提升的重要性.

Fusarium graminearum clade pathogens cause Fusarium head blight (FHB) or scab of wheat and other small cereal grains, producing different kinds of trichothecene mycotoxins that are detrimental to human and domestic animals. Type B trichothecene mycotoxins such as deoxynivalenol, 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON) and nivalenol (NIV) are the principal Fusarium mycotoxins reported in China, as well as in other countries. A genomic polymerase chain reaction (PCR) to predict chemotypes was developed based on the structural gene sequences of Tri13 genes involved in trichothecene mycotoxin biosynthesis pathways. A single pair of primers derived from the Tri13 genes detected a 583 bp fragment from 15-AcDON-chemotypes, a 644 bp fragment from 3-AcDON-chemotypes and an 859 bp fragment from NIV-producing strains. Fusarium strains from China, Nepal, USA and Europe were identified by this method, revealing their mycotoxin chemotypes identical to that obtained by chemical analyses of HPLC or GC/MS and other PCR assays. The mycotoxin chemotype-specific fragments were amplified from a highly variable region located in Tri13 genes with three deletions for 15-AcDON-chemotypes, two deletions for 3-AcDON-chemotypes and no deletion for NIV-producers. This PCR assay generated a single amplicon and thus should be more reliable than other PCR-based assays that showed the absence or presence of a PCR fragment since these assays may generate false-negative results. The results with strains from several different countries as well as from different hosts further indicated that this method should be globally applicable. This is a rapid, reliable and cost-effective method for the identification of type B trichothecene mycotoxin chemotypes in Fusarium species and food safety controls.

A large number of isolates from the Fusarium graminearum clade representing all regions in China with a known history of Fusarium head blight (FHB) epidemics in wheat were assayed using PCR to ascertain their trichothecene mycotoxin chemotypes and associated phylogenetic species and geographical distribution. Of the 299 isolates assayed, 231 are from F. asiaticum species lineage 6, which produce deoxynivalenol and 3-acetyldeoxynivalenol (3-AcDON); deoxynivalenol and 15-acetyldeoxynivalenol (15-AcDON); and nivalenol and 4-acetylnivalenol (NIV) mycotoxins, with 3-AcDON being the predominant chemotype. Ninety-five percent of this species originated from the warmer regions where the annual average temperatures were above 15 degrees C, based on the climate data of 30 y during 1970-1999. However, 68 isolates within F. graminearum species lineage 7 consisted only of 15-AcDON producers, 59% of which were from the cooler regions where the annual average temperatures were 15 degrees C or lower. Identification of a new subpopulation of 15-AcDON producers revealed a molecular distinction between F. graminearum and F. asiaticum that produce 15-AcDON. An 11-bp repeat is present in F. graminearum within their Tri7 gene sequences but is absent in F. asiaticum, which could be directly used for differentiating the two phylogenetic species of the F. graminearum clade.

选择来自我国12省市有代表性的101株禾谷镰刀菌菌株,比较它们在PDA培养基上的表型性状以及在CMS液中的产孢量;并于田间小麦开花期,用单花剪滴法接种3个抗赤霉病性不同的小麦品种,鉴定其致病力,用SAS软件进行统计分析.结果表明,菌落扩展速度不同的菌株间,其致病力差异显著;相关分析证实,菌落扩展速度与致病力之间呈极显著线性正相关(P＝0.000 3),菌丝生长状况与致病力之间呈极显著非线性相关(P＝0.008 3),而基质颜色和产孢量与致病力没有相关性.这些结果说明,禾谷镰刀菌菌落的扩展速度及菌丝生长状况与致病力之间具有共同的遗传基础或遗传相关性.

综述了近年来国内外关于小麦赤霉菌主要致病菌种禾谷镰刀菌毒素的形成与致病力的关系、毒素生物检测及免疫化学检测方法、脱毒技术及毒素代谢途径的分子生物学研究的一些进展.

由镰刀菌引起的赤霉病是危害小麦、大麦、燕麦、黑麦等禾谷类作物的一种重要病害,广泛分布于世界温暖潮湿地区.在我国赤霉病发病区,禾谷镰刀菌(Fusarium graminearum Schwabe)是主要致病菌. 我们选用小麦感病品种安农8455为材料,用蘸不同禾谷镰刀菌滤纸片接种其幼芽,接种后3 d即可在芽基部见到褐色病斑,随后褐色病斑逐渐增大.利用病菌特异引物进行PCR,证实接种菌确已侵入幼芽,而且菌株间致病力差异极显著,与田间花期接种结果高度一致. 1材料和方法 1.1供试菌株和小麦品种供试禾谷镰刀菌菌株均来自我国主要赤霉病区的病穗样本,小麦品种为安农8455. 1.2 接种鉴定方法室内芽期接种采用蘸有菌液的小滤纸片包住剪去少许的芽尖,对照则用蘸有无菌水的小滤纸片处理,置于25℃、相对湿度95%培养.7 d后测量小麦幼苗基部褐色病斑的长度;田间花期接种采用单小花定位定量孢子悬浮液注射接种,按徐雍皋(1982)等的方法进行病情分级和菌株致病力分析. 1.3 DNA提取及PCR反应按Aljanabi(1997)等的方法提取菌丝DNA;接种7 d后取样,按Sharp(1988)等的方法提取植物DNA.PCR引物及反应条件参照Doohan(1998)等的方法.