Cotton leafroll dwarf disease: An enigmatic viral disease in cotton

Taxonomy: Cotton leafroll dwarf virus (CLRDV) is a member of the genus Polerovirus, family Solemoviridae. Geographical Distribution: CLRDV is present in most cotton-producing regions worldwide, prominently in North and South America. Physical Properties : The virion is a nonenveloped icosahedron with T = 3 icosahedral lattice symmetry that has a diameter of 26–34 nm and comprises 180 molecules of the capsid protein. The CsCl buoyant density of the virion is 1.39–1.42 g/cm³ and S_20w is 115–127S. Genome: CLRDV shares genomic features with other poleroviruses; its genome consists of monopartite, single-stranded, positive-sense RNA, is approximately 5.7–5.8 kb in length, and is composed of seven open reading frames (ORFs) with an intergenic region between ORF2 and ORF3a. Transmission: CLRDV is transmitted efficiently by the cotton aphid (Aphis gossypii Glover) in a circulative and nonpropagative manner. Host: CLRDV has a limited host range. Cotton is the primary host, and it has also been detected in different weeds in and around commercial cotton fields in Georgia, USA. Symptoms: Cotton plants infected early in the growth stage exhibit reddening or bronzing of foliage, maroon stems and petioles, and drooping. Plants infected in later growth stages exhibit intense green foliage with leaf rugosity, moderate to severe stunting, shortened internodes, and increased boll shedding/abortion, resulting in poor boll retention. These symptoms are variable and are probably influenced by the time of infection, plant growth stage, varieties, soil health, and geographical location. CLRDV is also often detected in symptomless plants. Control: Vector management with the application of chemical insecticides is ineffective. Some host plant varieties grown in South America are resistant, but all varieties grown in the United States are susceptible. Integrated disease management strategies, including weed management and removal of volunteer stalks, could reduce the abundance of virus inoculum in the field.     

Global alteration of microRNAs and transposon-derived small RNAs in cotton (Gossypium hirsutum) during Cotton leafroll dwarf polerovirus (CLRDV) infection

Small RNAs (sRNAs) are a class of non-coding RNAs ranging from 20- to 40-nucleotides (nts) that are present in most eukaryotic organisms. In plants, sRNAs are involved in the regulation of development, the maintenance of genome stability and the antiviral response. Viruses, however, can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of sRNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs). To understand the impact of viral infection on the plant sRNA pathway, we deep sequenced the sRNAs in cotton leaves infected with Cotton leafroll dwarf virus (CLRDV), which is a member of the economically important virus family Luteoviridae. A total of 60 putative conserved cotton miRNAs were identified, including 19 new miRNA families that had not been previously described in cotton. Some of these miRNAs were clearly misregulated during viral infection, and their possible role in symptom development and disease progression is discussed. Furthermore, we found that the 24-nt heterochromatin-associated siRNAs were quantitatively and qualitatively altered in the infected plant, leading to the reactivation of at least one cotton transposable element. This is the first study to explore the global alterations of sRNAs in virus-infected cotton plants. Our results indicate that some CLRDV-induced symptoms may be correlated with the deregulation of miRNA and/or epigenetic networks.     

Improvement in enzymatic desizing of starched cotton cloth using yeast codisplaying glucoamylase and cellulose-binding domain

To utilize glucoamylase-displaying yeast cells for enzymatic desizing of starched cotton cloth, we constructed yeast strains that codisplayed Rhizopus oryzae glucoamylase and two kinds of Trichoderma reesei cellulose-binding domains (CBD1, CBD of cellobiohydrolase I (CBHI); and CBD2, CBD of cellobiohydrolase II (CBHII)). In this study, we aimed to obtain a high efficiency of enzymatic desizing of starched cotton cloth. Yeast cells that codisplayed glucoamylase and CBD had higher activity on starched cotton cloth than yeast cells that displayed only glucoamylase. Glucoamylase and double CBDs (CBD1 and CBD2) codisplaying yeast cells exhibited the highest activity ratio (4.36-fold), and glucoamylase and single CBD (CBD1 or CBD2) codisplaying yeast cells had higher relative activity ratios (2.78- and 2.99-fold, respectively) than glucoamylase single-displaying cells. These results indicate that the glucoamylase activity of glucoamylase-displaying cells would be affected by the binding ability of CBD codisplayed on the cell surface to starched cotton cloth. These novel strains might play useful roles in the enzymatic desizing of starched cotton cloth in the textile industry.     

A new SNP haplotype associated with blue disease resistance gene in cotton (Gossypium hirsutum L.)

Resistance to cotton blue disease (CBD) was evaluated in 364 F_2.3 families of three populations derived from resistant variety ‘Delta Opal’. The CBD resistance in ‘Delta Opal’ was controlled by one single dominant gene designated Cbd. Two simple sequence repeat (SSR) markers were identified as linked to Cbd by bulked segregant analysis. Cbd resides at the telomere region of chromosome 10. SSR marker DC20027 was 0.75 cM away from Cbd. DC20027 marker fragments amplified from 3 diploid species and 13 cotton varieties whose CBD resistance was known were cloned and sequenced. One single nucleotide polymorphism (SNP) was identified at the 136th position by sequence alignment analysis. Screening SNP markers previously mapped on chromosome 10 identified an additional 3 SNP markers that were associated with Cbd. A strong association between a haplotype based on four SNP markers and Cbd was developed. This demonstrates one of the first examples in cotton where SNP markers were used to effectively tag a trait enabling marker-assisted selection for high levels of CBD resistance in breeding programs.     

The cotton rat model of respiratory viral infections

Development of successful vaccines against human infectious diseases depends on using appropriate animal models for testing vaccine efficacy and safety. For some viral infections the task is further complicated by the frequently changing genetic make-up of the virus, as in the case of influenza, or by the existence of the little-understood phenomenon of vaccine-enhanced disease, as in the case of respiratory syncytial virus (RSV). The cotton rat Sigmodon hispidus has been used for years as an excellent small animal model of the RSV vaccine-enhanced disease. Recently, using cotton rats, we have demonstrated that vaccination against another paramyxovirus, human metapneumovirus (hMPV), can also lead to vaccine-enhanced disease. In addition to the study of paramyxoviruses, S. hispidus presents important advantages for the study of orthomyxoviruses such as influenza. The cotton rat is susceptible to infection with unadapted human influenza strains, and heterosubtypic immunity to influenza can be evoked in S. hispidus. The mechanisms of influenza, RSV, and hMPV pathogenesis and immunity can now be investigated in the cotton rat with the development of species-specific reagents for this animal model.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.     

Induction and establishment of somatic embryogenesis in elite Indian cotton cultivar (Gossypium hirsutumL. cv Khandwa-2)

Embryogenesis in cotton is a difficult task due its genome dependency. We used 3 cotton cultivars (Khandwa-2, G. Cot. 10, and BC-68–2) and Coker-312 as control for regeneration. Efficient somatic embryogenesis was induced in agronomically important Indian cotton cultivars, Khandwa-2 and G. Cot. 10. For callusing in all the cultivars, different media combinations were tried. Embryogenesis was initiated on a hormone-free MS medium (MSB). For embryo maturation and recovery excess of L-glutamine and l-asparagine were used. Khandwa-2 somatic embryos were successfully regenerated into plants. However, no plantlet was obtained in case of G. Cot. 10. Callus induction was also observed in BC-68–2 but there was no embryogenesis observed. The study indicated that the medium and genotype significantly effects embryogenesis. An efficient protocol is described here for regenerating plants via somatic embryogenesis in an elite Indian cotton cultivar Khandwa-2.     

Sequences regulating tropism of human immunodeficiency virus type 1 for brain capillary endothelial cells map to a unique region on the viral genome.

Two infectious molecular clones of human immunodeficiency virus type 1, NL4-3 and JR-CSF, differ in their abilities to productively infect human brain capillary endothelial (HBCE) cells. The phenotypes of recombinants between these two molecular strains were examined to identify viral sequences responsible for the difference in HBCE cell tropism between the two parental strains. Our results indicate that HBCE cell tropism maps to a region that encompasses the C1 region of env and includes overlapping reading frames for the accessory genes vpr, vpu, tat, and rev. This region was unique for HBCE cell tropism and did not cosegregate with either macrophage or T-cell line tropism. However, several recombinant clones displayed dual tropism for both HBCE cells and macrophages. These endothelial cell- and macrophage-tropic strains may have a unique pathogenic advantage by entering the brain via HBCE cells and subsequently infecting microglial cells with high efficiency, leading to the induction of human immunodeficiency virus dementia.     

Detection of somaclonal variation of cotton (Gossypium hirsutum) using cytogenetics, flow cytometry and molecular markers

Two protocols of plant regeneration for cotton were adopted in this study, namely, 2, 4-D and kinetin hormone combination and IBA and kinetin hormone combination. Twenty-eight embryogenic cell lines via somatic embryogenesis and 67 regenerated plants from these embryogenic calli were selected and used for random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), chromosomal number counting, and flow cytometric analysis. The roles of RAPD and SSR markers in detecting somaclonal variation of cotton (Gossypium hirsutum L.) were evaluated. Two cluster analyses were performed to express, in the form of dendrograms, the relationships among the hormone combinations and the genetic variability. Both DNA-based techniques were able to amplify all of the cell clones and regenerated plantlets genomes and relative higher genetic variation could be detected in the culture type with 2, 4-D and kinetin hormone combination. The result suggested that 2, 4-D and kinetin hormone combination could induce relative high somaclonal variation and RAPD and SSR markers are useful in detecting somaclonal variation of regenerated cotton plants via somatic embryogenesis. Chromosome number counting and flow cytometry analysis revealed that the number of chromosomes and ploidy levels were nearly stable in all regenerated plants except two regenerated plantlets (lost 4 and 5 chromosomes, respectively) which meant that cytological changes were not correlated with the frequency of RAPD and SSR polymorphisms. This result also might mean that the cell lines with variation of chromosome numbers were difficult to regenerate plants.     

胚轴切断法诱导棉株对棉铃虫生长发育的影响

通过室内饲喂棉铃虫的方法,测定了诱导棉株对棉铃虫幼虫发育历期、幼虫体重和肾重的影响。结果表明,不加菌处理和加菌处理的诱导抗性棉株可分别使棉铃虫幼虫发育历期延长０．５和３ｄ,幼虫体重（５龄）降低１１．４５％和１９．６０％,蛹重降低６．５４％和１０．８１％。用诱导棉株叶片饲养８日龄棉铃虫幼虫３天后,菌诱导和无菌诱导分别使棉铃虫的相对生长率降低２２．９％和１７．２％,相对取食量降低２６．１％和２１．４％     

湖北棉区转Bt基因棉对棉铃虫的控制作用

2000-2001年通过田间系统调查,表明转Bt基因棉（品种为GK19）在湖北江汉平原棉区对棉铃虫抗性稳定。试验设3个处理：转Bt基因棉化防田（使用化学农药控制害虫）、转Bt基因棉自控田（依靠天敌控制害虫）及常规棉对照田（利用综合防治措施控制害虫）。从棉铃虫的第2代到第5代整个发生期内,即使在不进行化学防治的情况下,棉铃虫在Bt棉田的发生量也保持在极低的水平(最高百株虫量为12头)。室内饲养结果表明,转Bt基因棉对棉铃虫的生长发育（幼虫体重、蛹重）有较为明显的影响,使6龄幼虫体重减少25.6%,蛹重减少18.2%。棉铃虫幼虫取食转Bt基因棉组织后,发育迟缓,相对于常规棉喂养的整个发育历期延长17 d,使棉铃虫在田间的危害减少至少一个世代。另外,接虫试验表明,棉铃虫幼虫在常规棉上的取食时间是转Bt基因棉株上的6.1倍,极大地减轻了棉铃虫的危害程度。     

High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants

This protocol describes a method for high-frequency recovery of transgenic soybean, bean and cotton plants, by combining resistance to the herbicide imazapyr as a selectable marker, multiple shoot induction from embryonic axes of mature seeds and biolistics techniques. This protocol involves the following stages: plasmid design, preparation of soybean, common bean and cotton apical meristems for bombardment, microparticle-coated DNA bombardment of apical meristems and in vitro culture and selection of transgenic plants. The average frequencies (the total number of fertile transgenic plants divided by the total number of bombarded embryonic axes) of producing germline transgenic soybean and bean and cotton plants using this protocol are 9, 2.7 and 0.55%, respectively. This protocol is suitable for studies of gene function as well as the production of transgenic cultivars carrying different traits for breeding programs. This protocol can be completed in 7-10 months.     

液体地膜覆盖对棉花根系生长发育的影响

基于棉田可持续发展的思想,利用茚三酮法、钼蓝法及土壤双向切片法,研究了液体地膜覆盖对棉花根系生长发育的影响。结果表明,覆盖棉花前期根系生长发育加快,表现为根系活力增强,根系干重较大,但覆盖不利于棉花根系下扎,土壤深层根系衰减较快。与塑料地膜覆盖相比,液体地膜覆盖增强根系吸收与合成能力的效应在棉花各生育阶段均较明显,根系在土壤内分布较为合理,土壤深层根系衰减较慢,更有利于棉株均衡生长发育,防止棉花早衰。在棉花生产上,采用液体地膜覆盖栽培是一项可行技术．     

华北地区棉铃虫对转Bt基因抗虫棉抗性适应的模拟模型

通过对华北地区耕作制度和生态系统的了解,在充分考虑种群遗传学、生物学和人为操纵因子等三大因素的基础上,建立了一个预测棉铃虫对转Bt基因抗虫棉抗性适应的模拟模型。在华北地区典型的耕作制度下,如果所有棉田均为Bt棉,则Bt棉的预期寿命为7年;如果只有春播棉为Bt棉(约占棉田总面积的70%),则其寿命为10年。模型的灵敏度分析表明, Bt棉的使用寿命随抗性基因的显性度、初始抗性频率、Bt棉所占比例等因素的增长而迅速缩短。当Bt棉表达的杀虫蛋白量恰好全部杀死敏感基因型(G_SG_S)个体时,Bt棉的预期寿命最短。由于国外采用的“高剂量/庇护所”抗性治理策略不适用于棉铃虫及华北棉区的耕作制度,我国需要加强对其它抗性治理措施(如转双基因抗虫棉)的研究与应用。     

新疆棉花黄萎病棉株与健康棉株内生古菌定量及其时空变化规律

【背景】现今棉花黄萎病严重阻碍棉花的稳定高产,妨碍棉花产业的发展。在生物防治中内生菌潜力巨大,但关于内生古菌含量在棉花黄萎病棉株的变化规律鲜有报道。【目的】研究不同生育期以及不同植棉地区黄萎病棉株和健康棉株内生古菌的分类学信息和数量变化规律。【方法】采用Miseq高通量测序和taqMan探针实时荧光定量PCR技术,对新疆棉花黄萎病棉株、健康棉株不同生育期和不同典型生态区的内生古菌进行定量分析。【结果】内生古菌在新疆各采样地和不同生育期的棉花黄萎病棉株、健康棉株内的群落组成相似。在不同生育期,新疆黄萎病、健康棉株内生古菌数量呈先增加后减少然后趋于平缓的趋势,蕾期达到最高值。在不同地区,新疆黄萎病棉株内生古菌数量在北疆地区最高,其次是东疆地区,最后是南疆地区。健康棉株则是南疆地区最高,东疆次之,北疆最低。【结论】新疆黄萎病棉株、健康棉株内生古菌数量在不同的生育期以及不同空间存在较大差异,整体变化趋势显著,可为后续研究提供相关理论支撑。     

Functional analysis of a reproductive organ predominant expressing promoter in cotton plants

The successful development and application of transgenic Bt cotton is a milestone of cotton produc-tion in China[1]. However, the CaMV35s promoter is commonly used for driving Bt gene expression in transgenic cotton plants. During infection, the CaMV35S promoter can direct the synthesis of 35RNA [2]. From the aspect of bio-security, it would be more secure and compatible if the promoter of cot-ton plants could be utilized for transgene expression[3]. In addition, studies showed that unde…     

棉花抗虫工程菌对棉铃虫的离体及活体生物测定

棉花抗虫工程菌是以能在棉株体内定殖的优势内生细菌Bacillus cereus (Bc9102)为宿主菌,将Bt kurstaki的δ-内毒素基因cryIA©整合到其染色体上形成的内生工程菌。以棉铃虫Helicoverpa armigera为供试昆虫的离体生测结果表明：在同一剂量水平上,HE-1、HE-2、ME14-2、ME14-3、MK14-1等工程菌株对棉铃虫幼虫的毒力高于或等于Bt野生菌株HD-73,浓度最高时这几个工程菌处理的棉铃虫死亡率分别为96.7%、83.3%、93.3%、83.3%、80.0%,而HD-73为80.0%,死亡率与浓度呈正相关。在用上述工程菌接种棉花的活体生测中,注射处理和喷雾处理法杀虫效果均优于浸种处理法。以HE-1为例,注射、喷雾、浸种处理4周后棉铃虫校正死亡率分别达到90.0%、76.0%和23.3%。工程菌对棉铃虫的生长发育也有抑制作用。     

Biodegradation and plant protection from the herbicide 2,4-D by plant-microbial associations in cotton production systems

A significant "biosafening" protection of plants from the effect of 2,4-D in plant-microbial associations has been demonstrated in this study. The 2,4-D-degrading plasmid, pJP4 was transferred into Rhizobium sp. CB1024, which nodulates Dolichos lablab, and Azospirillum brasilense Sp7 carrying a nifA-lacZ gene marker, which can colonize cotton roots. Both transconjugants degraded 2,4-D in pure culture via cometabolism up to 50 mug mL(-1). When the transconjugants were inoculated onto Dolichos lablab and cotton, respectively, such plants were resistant to this herbicide when the nutrient solution was treated with 2,4-D up to 10 mug mL(-1) for Dolichos lablab and 0.5 mug mL(-1) for cotton. Plants inoculated with wild-type strains were dead (Dolichos lablab) or dying (cotton). Because cotton is more sensitive to herbicides, only incomplete protection of plants was achieved with the transconjugant. Improving the effect of colonization of Azospirillum on cotton roots may be critical for a complete degradation and plant protection. The transconjugant of Rhizobium sp. CB1024 was still able to nodulate Dolichos lablab, N(2)-fixing activity was only slightly affected. Other pesticide-degrading capacities may also be inserted into those plant-associated bacterial strains for the degradation of these chemicals by plant-microbial associations. Whether such systems will be successful when applied in the field with competition from other bacteria is being examined. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 513-519, 1997.     

Endogenous Moloney leukemia virus in nonviremic Mov substrains of mice carries defects in the proviral genome

Substrains of mice carrying Moloney murine leukemia virus as a Mendelian gene (Mov locus) have been derived previously. Some of these strains, i.e., Mov-3 and Mov-9, develop viremia, whereas others, i.e., Mov-2, Mov-7, and Mov-10, do not regularly activate virus. We previously have molecularly cloned the respective Mov loci and shown that proviral clones derived from the different viral loci were either infectious (Mov-3, Mov-9) or failed to induce infectious virus (Mov-2, Mov-7, Mov-10) in a transfection assay. To analyze the sites affecting infectivity of the latter clones, complementation assays, in vitro recombinations, and marker rescue experiments were performed. Our results show that the three endogenous Moloney murine leukemia virus clones derived from Mov-2, Mov-7, and Mov-10 carry different mutations in the gag-pol region of the proviral genome. No inhibitory effect of flanking mouse sequences on provirus infectivity was observed.