海南甘蔗病毒病的调查及其病原分子鉴定

 海南是我国重要的甘蔗生产省份之一,但其甘蔗主要种植区感染病毒的种类和数量尚不十分清楚,且海南甘蔗花叶病病原病毒缺乏分子水平的系统鉴定。为明确海南甘蔗病毒病的种类、数量、分布及危害情况,本研究拟建立较为完整的甘蔗病毒病检测技术体系,对海南甘蔗病毒病展开调查,为甘蔗抗病毒基因工程及健康种苗发展提供参考。     

Sugarcane yellow leaf virus and sugarcane yellows phytoplasma: elimination by tissue culture

Yellow leaf syndrome (YLS) is a recently reported disease of sugarcane, characterized by yellowing of the leaves. Two pathogens: a virus, Sugarcane yellow leaf virus (SCYLV); and a phytoplasma, sugarcane yellows phytoplasma (SCYP), are associated with the disease. The use of tissue culture was investigated as a means to eliminate both SCYLV and SCYP from exotic varieties undergoing quarantine in Mauritius. Of 43 varieties in quarantine, 28 were infected with SCYLV and 19 with SCYP when checked by RT–PCR and nested PCR, respectively. Seventeen varieties were coinfected with both pathogens. Thirty infected varieties were induced to form callus in vitro using leaf rolls as explants. After two subcultures, 19 varieties were successfully regenerated and tested for SCYLV and SCYP. No pathogen could be detected in any regenerated plantlets. All the regenerated plants remained free from both SCYLV and SCYP over a period of 1 year in the glasshouse, confirming that the pathogens had been eliminated by tissue culture.     

Susceptibility of sugarcane, plantation weeds and grain cereals to infection by Sugarcane yellow leaf virus and selection by sugarcane breeding in Hawaii

Hawaiian commercial sugarcane cultivars (Saccharum spp.), noble canes (S. officinarum), robust canes (S. robustum) and wild relatives of sugarcane (S. spontaneum and Erianthus arundinaceus) were tested by tissue blot immunoassay to determine whether they were infected by Sugarcane yellow leaf virus (SCYLV). Two-thirds of the commercial hybrids and noble canes were infected and therefore classified as SCYLV-susceptible, in contrast to the wild cane relatives where less than one third of the varieties were infected. The pedigree list of commercial, registered cultivars showed that 80% of cultivars were SCYLV-susceptible and that also 75–90% of the progeny of resistant (female) parents were susceptible (male parents are mostly unknown because of polycross breeding). In contrast, a cross between a resistant S. robustum and a susceptible S. officinarum cultivar yielded 85% resistant progeny clones, which indicated that SCYLV-resistance is a dominant trait. It is concluded that the breeding program selected against SCYLV-resistance with the result that 80% of the newly bred cultivars were susceptible. Exceptional was the period between 1950 and 1970, in which 50% of the newly-bred clones were resistant. This is the period in which SCYLV had entered Hawaii. Weed grasses and cereal grasses which grew in or next to sugarcane fields were not infected by SCYLV. Thus SCYLV does not spread from infected sugarcane plants to adjacent grasses or cereals under field conditions, although cereal grasses can be infected experimentally.     

我国新育成甘蔗品种(系)对甘蔗线条花叶病毒和高粱花叶病毒的抗性评价

甘蔗花叶病是中国蔗区危害最严重的病毒病,利用抗病品种是控制该病害最经济有效的方法。本研究以中国蔗区甘蔗花叶病的2种主要病原甘蔗线条花叶病毒分离物(SCSMV-JP1,Gen Bank登录号JF488064)和高粱花叶病毒分离物(Sr MV-HH,Gen Bank登录号DQ530434)为接种毒源,采用人工切茎接种和RT-PCR检测相结合方法,于2015年、2016年2次对中国近年选育的71个优良甘蔗新品种(系)进行了双抗SCSMV和Sr MV鉴定与评价。结果表明:71个优良甘蔗新品种(系)中,对SCSMV表现高抗到中抗的有24个,占33.8%,感病到高感的有47个,占66.2%;对Sr MV表现高抗到中抗的有27个,占38.03%,感病到高感的有44个,占61.97%。综合分析结果显示,福农30号、福农36号、闽糖01-77、桂糖02-467、柳城05-129、粤甘34号、粤甘40号、粤糖55号、粤糖96-86、粤糖00-318、赣蔗02-70、云蔗03-258、云蔗04-241、云蔗05-51、云蔗06-80等15个优良新品种(系)双抗SCSM V和Sr M V 2种病毒,占21.13%,其中粤甘34号、粤糖55号、云蔗03-258、云蔗05-51、云蔗06-80等5个优良新品种(系)对2种病毒均表现为高抗,占7.04%,。研究结果明确了71个甘蔗优良新品种(系)对甘蔗花叶病2种主要致病病原的抗性,筛选出双抗SCSMV和Sr MV的甘蔗优良新品种(系)15个,为生产用种选择和有效防控甘蔗花叶病提供了科学依据。     

我国甘蔗主要杂交亲本黄叶病病原鉴定及田间发病率

甘蔗黄叶病是一种发生普遍、危害严重的病毒病害,选育抗病品种是控制该病发生与蔓延的有效措施.利用逆转录-聚合酶链反应(RT-PCR)、组织印迹杂交免疫测定(TBIA)和抗原直接包被酶联免疫吸附(DAC-ELISA)方法对我国43份甘蔗主要杂交亲本黄叶病病原(Sugarcane yellow leaf virus,SCYLV)进行鉴定,并调查SCYLV田间自然侵染条件下甘蔗黄叶病发病率.已明确36份亲本材料感染或未感染SCYLV,其中32份鉴定结果为阳性,品种(系)感病率达88.9％.甘蔗植株田间自然发病率可分为无发病、发病率低、中等和高4个等级,方差分析表明,各等级间的植株发病率差异达极显著水平(P＜0.01).从美国引进的多数CP和HoCP系列亲本发病率较高,而多数崖城和新台糖系列亲本材料的发病率较低,可作为甘蔗抗黄叶病杂交亲本.     

Elimination of Sugarcane yellow leaf virus from infected sugarcane plants by meristem tip culture visualized by tissue blot immunoassay

Sugarcane yellow leaf virus (SCYLV), a member of the Luteoviridae , is implicated in the sugarcane disease known as yellow leaf syndrome (YLS), which is characterized by yellowing of the leaf midrib followed by leaf necrosis and possible growth suppression. YLS is distributed worldwide and susceptible cultivars are commonly infected with SCYLV. However, not all cultivars infected with SCYLV show symptoms of YLS and some cultivars that show symptoms do so sporadically. Since it is difficult to obtain virus-free plants of susceptible cultivars, it has not been possible to study the factors involved in SCYLV infection nor the effects of infection on plant growth and yield. A tissue blot immunoassay was used to visualize in vivo presence of the virus so that virus-infected and virus-free plants could be distinguished. Meristem tip cultures were used to produce virus-free plantings of six SCYLV-susceptible sugarcane cultivars. Nearly all of the regenerated sugarcane lines remained virus-free over a period of up to 4 years, whether grown in isolated fields or in the glasshouse. Experimental re-infection of the virus-free plants by viruliferous aphids demonstrated that meristem tip culture did not affect susceptibility of sugarcane to SCYLV. Improved diagnosis and production of virus-free plants of SCYLV-susceptible cultivars will facilitate research to quantify the effect of the virus on yield and to analyse the processes involved in disease development.     

甘蔗病毒病及其防治研究进展

甘蔗是最重要的糖料作物,由于其栽培过程中采用种茎无性繁殖,病毒病发生逐年加重.已知侵染甘蔗的病毒种类有甘蔗花叶病毒(Sugarcane mosaic virus,SCMV)、高粱花叶病毒(Sorghummosaic virus,SrMV)、甘蔗线条花叶病毒(Sugarcane streakmosaic virus,SCSMV)、甘蔗黄叶病毒(Sugarcane yellow leaf virus,SCYLV)、甘蔗斐济病病毒(Sugarcane Fiji disease virus,SFDV)、甘蔗线.条病毒(Sugarcane streak virus,SSV)和甘蔗杆状病毒(Sugarcane bacilliform virus,SCBV).文中简要介绍上述几种病毒的基本特性及其所致病害的发生特点,对目前甘蔗病毒病防治技术进行了评述,提出了我国甘蔗病毒研究中需要关注的若干问题.     

Molecular identification and prevalence of Acidovorax avenae subsp. avenae causing red stripe of sugarcane in China

Red stripe caused by the bacterium Acidovorax avenae subsp. avenae (Aaa) is a disease of sugarcane that is distributed worldwide. In this study, 108 sugarcane leaf samples were collected in 2013–2016 from nine sugarcane‐growing regions in China. Aaa was detected by PCR with specific and novel primers from the 16S–23S rDNA internal transcribed spacer region in 81 of 84 (96%) leaves with red stripe symptoms and in 20 of 24 (83%) leaves without symptoms. Furthermore, Aaa was detected in all nine sampling locations representing six sugarcane‐producing provinces in China. The 101 amplified fragments were cloned and sequenced. The size of the nucleotide sequences varied from 436 to 454 bp and the sequence identity ranged from 89.2% to 100%, suggesting a significant genetic variation among Aaa strains from China. Five major restriction fragment length polymorphism (RFLP) profiles were obtained by in silico and polyacrylamide gel electrophoresis analyses of the PCR products digested with HindIII and EcoRI. The causal agent of sugarcane red stripe was also successfully isolated from a diseased plant and its pathogenicity confirmed by inoculation of healthy sugarcane plantlets and reproduction of disease symptoms. The data showed that Aaa is currently widespread in China, suggesting that control methods should be implemented to limit the impact of red stripe on sugarcane production.     

云南甘蔗杆状病毒的分子检测及其对甘蔗品质和产量的影响

 Sugarcane bacilliform virus(SCBV) was detected by PCR from sugarcane showing chlorosis and mottle symptom from Kaiyuan, Yunnan Province.Part sequence of replicase gene of the isolate SCBV-Kaiyuan was determined.Sequence analysis indicated that the 589 bp of SCBV-Kaiyuan shared identities of 73.2%-74.0% and 83.1%-84.1% at nucleotide and amino acid levels with SCBV-Australia respectively, 66.7%-68.4% and 65.6%-67.7% with SCBV-Morocco.The quality and yield of the sugarcane infected with SCBV-Kaiyuan was also investigated.The juice extraction, sucrose content, gravity purity and average stalk weight were decreased 1.55%, 1.24%, 2.22% and 0.26 kg in plants infected with SCBV-Kaiyuan, but reducing sugar was increased by 0.21% in infected plants.     

利用RNA干扰介导抗病性获得兼抗四种病毒的转基因马铃薯

为获得兼抗马铃薯X病毒(Potato virus X,PVX)、马铃薯Y病毒(Potato virus Y,PVY)、马铃薯卷叶病毒(Potato leaf roll virus,PLRV)和马铃薯潜隐花叶病毒(Potato virus S,PVS)4种病毒的转基因马铃薯新材料,分别以这4种病毒全长CP基因为模板,通过设计PCR引物和亚克隆获得4种病毒CP基因相对保守区段的基因片段,并将其拼接成融合基因,以载体pHANNIBAL和pBI121为基础,构建RNA干扰(RNA interference,RNAi)载体,利用农杆菌介导的转基因体系进行马铃薯遗传转化,并对获得的转基因马铃薯进行病毒抗性检测。结果表明,所获得的融合基因片段RH1和RH2,酶切鉴定分别得到长度为1 200 bp的条带,与预期片段相符;构建了含pdk内含子和RH1、RH2融合基因的RNAi植物表达载体,经Bam H I/Sac I双酶切,获得长度约3 200 bp的片段,表明RNAi植物表达载体pBI121-pRH构建成功;转化易感病毒马铃薯品种陇薯11号,PCR检测和PCRSouthern杂交分析表明融合基因已整合到陇薯11号马铃薯基因组中;抗病性检测显示4株转基因马铃薯植株对4种病毒均免疫。表明利用RNAi可筛选出抗多种病毒的转基因马铃薯新种质。     

甘蔗黄叶病及其病原分子生物学研究进展

甘蔗黄叶病是由甘蔗黄叶病毒(Sugarcane yellow leaf virus,SCYLV)引起的一种病毒病害,在全球主要甘蔗种植国家或地区普遍发生,危害日益严重。SCYLV经甘蔗蚜虫以持久性方式传播,在寄主植株内主要侵染韧皮部组织。该病毒起源于黄症病毒科属间基因重组,被国际病毒分类命名委员会确认为黄症病毒科马铃薯卷叶病毒属成员。文章综述了甘蔗黄叶病毒生物学特征、病害发生和危害、病原鉴定和检测、分子进化和遗传多样性、基因组结构和基因功能以及抗病转基因等方面研究进展,并对甘蔗黄叶病抗病育种和防治措施作了讨论。     

Diverse conserved domains and a positively selected site in the sugarcane streak mosaic virus P1 protein are essential for RNA silencing suppression and protein stability

RNA silencing is one of the conserved antiviral mechanisms in plants, and viruses encode RNA silencing suppressors (RSS) to overcome host RNA silencing and facilitate virus infection. Sugarcane streak mosaic virus (SCSMV; species Sugarcane streak mosaic virus, genus Poacevirus, family Potyviridae) is a major causal agent of sugarcane mosaic disease in many countries in Asia, including China. In this study, we used Agrobacterium co-infiltration to show that the SCSMV P1 protein, rather than the helper component-proteinase (HC-Pro), functions as a strong RSS to suppress local RNA silencing in Nicotiana benthamiana. Mutational analysis indicated that the 15 amino acids (aa; aa 1–15) of the SCSMV P1 N-terminus were not important for RNA silencing suppression, but rather another 15 aa domain (aa 108–122) containing a conserved motif (LFR/KNKQAYIST) was essential for efficient silencing suppression by P1. In addition to the 15 aa (aa 344–358) domain in the P1 N-terminus, another 15 aa domain (aa 65–79) of P1, containing the LXKA motif and one conserved aa (D78), were associated with P1 protein stability. Furthermore, substituting the histidine (H263) residue in P1 with threonine (H263T) or alanine (H263A) also affected P1 protein stability. Notably, the H263 residue is both a positively selected site and part of the serine protease catalytic triad (HDS). Taken together, our data demonstrate that SCSMV P1, and not HC-Pro, plays a functional role in suppressing RNA silencing, and also show that some conserved motifs and a positivelyselected site in the P1 protein are associated with RSS activity and protein stability.     

Variation in virus populations and growth characteristics of two sugarcane cultivars naturally infected by Sugarcane yellow leaf virus in different geographical locations

Two sugarcane cultivars (R570 and SP71-6163) naturally infected by Sugarcane yellow leaf virus (SCYLV) were each imported from several geographical locations into a sugarcane yellow leaf-free environment (Montpellier, France). Plants were grown as plant cane for 5–6 months and the experiment was repeated for three consecutive years (2003–2005) in a greenhouse. Several sugarcane-growth and disease characteristics were monitored to identify variation in pathogenicity of SCYLV. Depending on their geographical origin, sugarcane cvs R570 and SP71-6163 were infected by SCYLV genotypes BRA-PER or REU, or a mixture of the two. Severity of symptoms did not vary between plants of cv. R570, but variation in disease severity between plants of cv. SP71-6163 from different geographical locations suggested the occurrence of pathogenic variants of SCYLV. For each sugarcane cultivar, differences in stalk length, number of stalk internodes, virus titre in the top visible dewlap leaf, and percentage of infection of leaf and stalk phloem vessels were also found between plants from different geographical origins. However, these differences were not always reproducible from one year to another, suggesting occurrence of different plant responses to SCYLV isolates under varying environmental conditions.     

Survey of granulosis virus infection in sugarcane borers,Chilo infuscatellus Snellen and C. sacchariphagus indicus (Kapur) in India

Abstract

A survey was undertaken to observe the pevalence of granulosis viruses infecting sugarcane shoot borer Chilo infuscatellus Snellen and internode borer C. sacchariphagus indicus (Kapur) in Tamil Nadu State and Union Territory of Pondicherry in India. Nine districts in Tamil Nadu and Pondicherry had the occurrence of shoot borer granulosis virus in all the locations surveyed ranging from 2.6 to 14.6%. In internode borerthe mortality varied from 3.8 to 31.5% in the eight districts of Tamil Nadu and Pondicherry.     

甘蔗白叶病植原体巢式PCR检测方法的建立及初步应用

甘蔗白叶病(sugarcane white leaf,SCWL)是由植原体引起的重要甘蔗病害[1],广泛分布在印度、泰国等许多国家[1,2].我国甘蔗产区的栽培品种也有SCWL的发生[3].甘蔗是无性繁殖作物,植原体可通过繁殖种苗进行传播,台湾斑纹叶蝉(Matsumuratetlix hiroglyhious)通过咬食感染甘蔗植株的韧皮部可引起该病害[4].     

引起甘蔗花叶病的病原分子生物学进展

花叶病是最主要的甘蔗病毒病害之一,在全球种植甘蔗的国家或地区普遍发生,可导致甘蔗产量下降,糖分减少,给甘蔗生产带来严重的经济损失。引起甘蔗花叶病的病毒主要有甘蔗花叶病毒(Sugarcane mosaic virus,SCMV)、高粱花叶病毒(Sorghum mosaic virus,Sr MV)和甘蔗条纹花叶病毒(Sugarcane streak mosaic virus,SCSMV)。本文综述了这3种病毒的生物学特性、鉴定与检测、基因组结构与基因功能、遗传变异与分子进化等方面的研究进展,并讨论了对甘蔗花叶病的生态防控措施。     

广东甘蔗黄叶病田间调查及病原病毒的分子检测

 广东省粤北和粤西蔗区多个县市的田间甘蔗上观察到甘蔗黄叶病(Sugarcane yellow leaf disease,SYLD)典型症状,目前该病仅局部分布,但部分田块病株率为5%~80%,发病品种有青皮果蔗、黑皮果蔗、新台糖系列品种、粤糖79/177和粤糖93/159等。采集发病田间显症叶片、无症叶片和在病叶上取食的甘蔗绵蚜(Ceratovacuna lanigera)样品,抽提总RNA,以基于甘蔗黄叶病毒(Sugarcane yellow leaf virus,SCYLV) CP基因序列的特异引物进行一步RT-PCR和巢式PCR扩增,并对扩增产物进行核苷酸序列测定和BLAST比对。结果显示,RT-PCR及巢式PCR产物核苷酸序列与分离自巴西的SCYLV B1株系相应区段同一率为100%;一步RT-PCR可从约70%的显症叶片样品中检测到SCYLV,而病田中的无症叶片样品以及在病叶上取食的单头甘蔗绵蚜样品需经巢式PCR扩增方可检测到SCYLV,阳性率分别为1%~5%和83%。本研究表明,广东省栽培甘蔗已受到SCYLV侵染,甘蔗绵蚜携带SCYLV。     

侵染扶桑的烟草花叶病毒分离物鉴定

从表现叶斑驳症状的扶桑病株上获得一病毒分离物,电镜下可见约300 nm×18 nm的杆状粒子,其与烟草花叶病毒抗血清呈明显的阳性反应,dsRNA约为6.4 kbp。根据烟草花叶病毒(tobacco.mosaic virus,TMV)的RNA序列设计引物,进行RT-PCR检测,扩增出约800 bp的预期特异片段。将PCR产物连接pMD18-T载体,转化大肠杆菌DH5α,得到了含有目的片段的重组子。序列分析表明,与周雪平等报道的序列(GenBank AJ011933.1)同源性达99％。通过生物学、病毒粒子观察、血清学以及分子生物学实验结果,确定该病毒分离物为TMV。     

Occurrence of two races of Puccinia kuehnii causing orange rust of sugarcane in Florida

Orange rust of sugarcane caused by Puccinia kuehnii was first reported in Florida in 2007. Since then, several sugarcane cultivars that were resistant during the initial epidemics became susceptible within a few years. These shifts in resistance were attributed to the evolution of the pathogen and appearance of new races. To study the variation in virulence of P. kuehnii, healthy leaf pieces of sugarcane cultivars susceptible to orange rust were brush inoculated with isolates of P. kuehnii collected from susceptible cultivars in the field. After inoculation, leaf pieces were placed in an incubator and disease severity based on the number of rust uredinia was determined 2 weeks postinoculation. Isolates of P. kuehnii collected from sugarcane cultivar CP 89-2143, which only showed severe symptoms of orange rust starting in 2011–2012, produced 300%–500% more uredinia on CP 89-2143 than the isolates collected from cultivar CL 85-1040 that has been susceptible since 2007. Sugarcane cultivar CL 85-1040 exhibited high and equivalent numbers of uredinia regardless of the inoculated isolate of the pathogen. These data support the occurrence of pathogenic specialization within P. kuehnii and the existence of at least two races of this pathogen in Florida. Analysis of amplified fragment-length polymorphism among isolates of P. kuehnii from cultivars CP 89-2143 and CL 85-1040 differing in resistance to orange rust revealed genetic variation among rust uredinia. However, this variation was not associated with a specific sugarcane cultivar, suggesting that pathogenic variation was not linked to major, but rather to small genetic changes within the genome of P. kuehnii.