福建甘薯病毒病病原鉴定及主要病毒多样性

【背景】病毒病是甘薯的一种重要病害,给甘薯生产带来了严重的经济损失,而生产中甘薯病毒病病原种类复杂多样。【目的】明确福建甘薯病毒病种类、分布及流行,对主要病毒进行多样性分析。【方法】从福建主要甘薯种植区采集病毒病样品,利用PCR/RT-PCR的方法对采集的样品进行病原检测,获得病毒序列,利用MEGA 6.0构建系统进化树进行遗传分析。【结果】从福建7个甘薯产区鉴定12种甘薯病毒,包括9种RNA病毒:甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)、甘薯褪绿矮化病毒(Sweet potato chlorotic stunt virus,SPCSV)、甘薯G病毒(Sweet potato virus G,SPVG)、甘薯C病毒(Sweet potato virus C,SPVC)、甘薯2号病毒(Sweet potato virus 2)、甘薯褪绿斑病毒(Sweet potato chlorotic fleck virus,SPCFV)、甘薯潜隐病毒(Sweet potato latent virus,SPLV)、甘薯轻型斑点病毒(Sweetpotatomildspeakingvirus,SPMSV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV),3种DNA病毒:甘薯卷叶病毒(Sweet potato leaf curl virus,SPLCV),甘薯无症状1号病毒(Sweet potato symptomless virus 1,SPSMV-1),甘薯杆状DNA病毒B (SPBV-B)。SPFMV、SPCSV、SPVG和SPLCV检出率最高,分别为50.28%、41.90%、35.75%和24.58%,CMV检出率最低,为2.79%,未检测到甘薯C-6病毒(SweetpotatoC-6)和甘薯轻型斑驳病毒(Sweetpotatomild mottle virus,SPMMV)。福建甘薯主要以2-6种病毒复合侵染为主,单一侵染率占14.39%,2种以上复合侵染占85.61%。福建SPFMV分离物存在EA、O和RC3种株系,SPCSV分离物存在WA1种株系,未发现EA株系,甘薯卷叶病毒分属于2个不同的株系群。【结论】福建甘薯病毒种类多样,以复合侵染为主,且存在多种株系,遗传结构复杂。     

中国甘薯地方种质资源遗传多样性分析

利用形态农艺性状标记对来自17个省份的176份中国甘薯地方种质资源进行遗传多样性分析,结果表明形态标记并未按照这些地方种质的来源地聚类,176份种质可以划分为5大类群,发现了复份保存的2对种质材料。对其中133份地方种质资源的品质特征进行比较和统计分析,筛选到高干物率、高淀粉种质11份,其中广东地方种质爆皮王的干物率和鲜薯淀粉含量最高,分别为34.87%和23.32%;鲜薯可溶性糖≥4.00%的材料11份,其中来自云南的腾冲本地种可溶性糖含量最高,为5.22%;筛选高蛋白材料2份。同时利用品质性状标记将133份地方种质划分为5大类群,第Ⅰ类群主要为干物率、淀粉含量和粗蛋白含量低,可溶性糖含量高的14份种质;第Ⅱ类群主要为可溶性糖含量较高、干物率和粗蛋白含量较低的28份种质;第Ⅲ类群由可溶性糖含量最高的腾冲本地种1份种质构成;第Ⅴ类群由干物率及鲜薯淀粉含量都很高的4份广东品种构成;剩余的86份种质组成第Ⅳ类群,各项表现不等。通过对我国甘薯地方种质资源遗传多样性的探索,为甘薯育种选配亲本提供参考,同时促进我国甘薯种质资源研究利用和种质创新。     

浙江省甘薯种质资源的品质鉴定与聚类分析

开展甘薯种质资源的品质鉴定评价,可以系统地了解种质的营养价值、食用品质和加工性能,为甘薯生产的品种选择和种质资源的育种利用提供依据。对2017-2019年浙江省第三次全国农作物种质资源普查与收集行动中征集的62份甘薯种质进行了干物率、胡萝卜素含量、生薯和熟薯可溶性糖含量以及食味的测定,并进行了品质性状的主成分和系统聚类分析。结果表明,在参试种质中,有高干物率品种24份,胡萝卜素含量≥5 mg/100 g·FW的品种5份,生薯可溶性糖含量超过水果甘薯六十日的品种7份,熟薯麦芽糖含量超过迷你甘薯心香的品种6份。生薯和熟薯均含有果糖、葡萄糖、蔗糖和麦芽糖。在生薯中蔗糖含量最高,平均含量为27.00 mg/g·FW,占可溶性糖的47.49%。在熟薯中麦芽糖含量最高,麦芽糖与可溶性糖含量高度相关(r=0.925)。食味与熟薯可溶性糖和还原糖含量、胡萝卜素含量呈极显著正相关,与生薯可溶性糖含量呈显著正相关。主成分F1、F2、F3、F4分别与食用品质、淀粉和油炸薯片加工性能、蔗糖含量有关,可以代表94.15%的甘薯品质变量信息。系统聚类分析表明,参试种质的品质类型可以分为Ⅰ、Ⅱ、Ⅲ3个类群和Ⅰ-1、Ⅰ-2、Ⅱ-1、Ⅱ-2、Ⅲ-1、Ⅲ-2 6个亚群,依用途可以简要归类为:淀粉型23份,粮饲兼用型19份,优质食用型16份,油炸薯片型4份。总之,浙江省甘薯种质资源以淀粉型和粮饲兼用型为主,品质类型丰富,在鲜食及淀粉、全粉、薯脯、油炸薯片加工等用途上有较好的利用价值。     

中国甘薯种质资源研究现状及发展战略

中国保存的甘薯种质资源约2000份,绝大部分进行了抗性鉴定评价及营养成分含量测定。甘薯种质资源的保存方式有田间种质圃和试管苗库。作为种质利用的资源材料有地方品种、引进品种、育成品种(系)、突变体和近缘野生种等。根据我国甘薯种质资源研究现状,本提出了未来研究重点,包括调整资源收集重点、建立完整的资源保存体系、深入开展鉴定评价、建立核心种质库、进行种质创新等。     

甘薯丛枝病植原体的PCR检测

以报道的植原体 (Phytoplasma)16SrDNA基因保守序列为依据,设计合成了两对引物对R16mF2/ R16mR2和R16F2/ R16R2,以甘薯丛枝病（SPWB）带病植株的叶脉中提取的DNA为模板,应用聚合酶链式反应（PCR）技术和巢式PCR（Nested- PCR）技术对甘薯丛枝病病原进行分子检测。结果表明PCR扩增出了1.5　kb的特异片段,在PCR基础上的巢式PCR扩增出了1.2　kb的特异片段。灵敏度实验显示该方法所需PCR模板DNA量为0.1073　ng/μl,在PCR基础上的巢式PCR可以将灵敏度提高约10000倍,所需模板DNA仅为0.01073　pg/μl,在甘薯丛枝病的检测中是一种快速、灵敏、可靠的方法。     

广西甘薯病毒病的病原病毒种类检测

甘薯病毒病是广西甘薯的主要病害之一。为明确为害广西甘薯的病毒种类,到广西南宁、玉林、崇左、北海、宜州和桂林等市的甘薯种植区采集疑似甘薯病毒病样品,共采集到127个甘薯样品,用为害甘薯的12种病毒的引物,通过RT-PCR或PCR技术检测,并对扩增产物进行测序和所获序列的Blast分析。结果表明:为害广西甘薯的病毒种类有11种,包括10种RNA病毒:甘薯羽状斑驳病毒(sweet potato feathery mottle virus,SPFMV)、甘薯G病毒(sweet potato virus G,SPVG)、甘薯褪绿矮化病毒(sweet potato chlorotic stunt virus,SPCSV)、甘薯轻型斑点病毒(sweet potato mild speckling virus,SPMSV)、甘薯C病毒(sweet potato virus C,SPVC)、黄瓜花叶病毒(cucumber mosaic virus,CMV)、甘薯褪绿斑病毒(sweet potato chlorotic fleck virus,SPCFV)、甘薯脉花叶病毒(sweet potato vein mosaic virus,SPVMV)、甘薯潜隐病毒(sweet potato latent virus,SPLV)、甘薯轻型斑驳病毒(sweet potato mild mottle virus,SPMMV),1种DNA病毒:甘薯卷叶病毒(sweet potato leaf curl virus,SPLCV)。在127份甘薯样品中,有48个样品检测到SPFMV,占37.79%;31个样品检测到SPVG,占24.40%;29个样本检测到SPLCV,占22.83%;27个样本检测到SPCSV,占21.30%,其他被检测到的甘薯病毒占20.00%之下。检测到病毒的88个样品中,有40个样品仅检测到一种病毒,即SPFMV、SPVG、SPCSV、SPMSV、CMV、SPLCV单独侵染。48个样品为2种或2种以上的病毒复合侵染。病毒复合侵染时,甘薯症状表现复杂,有矮化、花叶、皱缩、叶脉突起等症状。其中,检测到SPCSV的甘薯植株,表现出严重的矮化症状。     

PCR相关技术在植物病毒检疫检测中的应用

PCR和建立在PCR基础上的分子生物学技术以其灵敏、快速、简便等优点广泛应用于植物病毒的检测。阐述反转录聚合酶链式反应、免疫捕捉反转录PCR、PCR-单链构型多态性、实时荧光定量PCR、差异显示PCR和巢式PCR等相关技术的原理及其在植物病毒检测中的应用现状,以期为我国植物病毒的检疫检测提供有益参考。     

基因库中离体保存甘薯种质遗传稳定性研究

用形态标记、生化标记和蛋白分子标记等三个水平检测国家基因库中离体保存10年的甘薯种质遗传稳定性。结果表明：长期试管保存的甘薯种质,大多数在农艺性状、POD、MDH和叶可溶性蛋白电泳图谱上与其原种基本相同。只有少数品种在EST、或α-Amy酶谱出现差异。这就意味着离体保存对甘薯种质的遗传产生影响,并且认为POD、MDH和叶可溶性蛋白电泳图谱不适宜作为鉴定指标;而EST和α-Amy两种同工酶谱可用于检测甘薯离体种质遗传稳定性的生化指标。从22份种质的大田和试管两种生长状态下的生化指标测试结果显示,在这两种生长状态下,遗传稳定的种质,均与原种相同;遗传产生差异的种质,差异均能表达,只是因生长条件不同,酶谱表达方式有别。     

甘薯薯干高淀粉资源的鉴定及综合评价

通过对332份新征集甘薯品种资源薯干粗淀粉含量的分析测定,鉴定得到58份高淀粉种质资源。通过烘干率测定,得到36份兼具高干的高淀粉种质资源。通过抗病性、时逆性鉴定,得到5份兼抗多病的优异高淀粉品性种质资源,5份兼具耐旱性的高淀粉种质资源,29份兼具耐贮性的高淀粉种质资源。     

甘薯近缘野生种的抗病性鉴定与新型种间杂种的获得

为了发掘甘薯近缘种抗茎线虫病、病毒病基因资源,改良栽培种抗性,拓宽甘薯遗传基础,本文利用田间自然病圃对30份甘薯近缘野生种进行抗茎线虫病鉴定,利用硝化纤维素膜-酶联免疫(NCM-ELISA)对20份野生种进行了抗病毒病筛选.以筛选到的抗性材料为父本、栽培种徐薯18为母本,采用生长调节剂处理方法进行种间杂交,结果得到8份高抗、10份抗茎线虫病野生种,5份高抗、8份抗病毒病材料;获得4个新型种间杂种.经过染色体计数和ISSR分子标记鉴定,证实了所得杂种的真实性.表明甘薯近缘野生材料中存在着栽培种所需要的抗性基因,通过有性杂交手段可以获得含野生种染色体组的种间杂种新类型,为渐渗野生抗性基因起到"桥梁"作用.     

抗甘薯黑斑病优异种质资源的筛选与评价

通过对1107份甘薯种质资源抗黑斑病鉴定,筛选出高抗品种8份,占鉴定品种总数的0.7%;抗病品种128份,占11.6%.并进一步对这些抗性资源的兼抗病性、抗逆性、产量及品质进行分析研究,从中筛选出了兼抗两种或两种以上病害的优异种质资源若干份,以期为育种和生产提供依据.     

微波消解-FAAS法测定普通甘薯和紫甘薯中的金属元素

采用微波消解法处理普通甘薯和紫甘薯样品,运用火焰原子吸收光谱法测定其中的K、Ca、Mg、Fe、Mn、Zn、Cu 7种对人体有益的金属元素含量。结果表明,普通甘薯和紫甘薯中K、Ca、Fe、Mg元素含量较高,Mn、Zn、Cu元素含量较低,且7种金属元素含量在两者之间存在一定的差异,各元素在紫甘薯中的含量均比普通甘薯中含量高。方法的加标回收率介于98.5%～103.2%,相对标准偏差(RSD)不大于3.14%。可为普通甘薯与紫甘薯的品质评价提供理论参考。     

Ipomoea crinkle leaf curl caused by a whitefly-transmitted gemini-like virus

A whitefly-transmitted infectious agent, associated with geminate particles, induced distinct symptoms on several Ipomoea species, but not on I. batatas cv. Georgia Jet. The virus was transmitted by Bemisia argentifolii in a persistent manner and by grafting, but not mechanically. No transmission to species outside Ipomoea was obtained. Extracts from infected Ipomoea plants hybridised with a bean yellow mosaic virus riboprobe and a tomato yellow leaf curl virus riboprobe, although not so strongly as hybridisation of these riboprobes with extracts from plants infected with the homologous viruses. Based on host range, we consider this virus to be distinct from sweet potato leaf curl virus reported from the Far East, and propose it be named “Ipomoea crinkle leaf curl virus” (ICLCV).     

Agroinfection of sweet potato by vacuum infiltration of an infectious sweepovirus

Sweepovirus is an important monopartite begomovirus that infects plants of the genus Ipomoea worldwide. Development of artificial infection methods for sweepovirus using agroinoculation is a highly efficient means of studying infectivity in sweet potato. Unlike other begomoviruses, it has proven difficult to infect sweet potato plants with sweepoviruses using infectious clones. A novel sweepovirus, called Sweet potato leaf curl virus-Jiangsu (SPLCV-JS), was recently identified in China. In addition, the infectivity of the SPLCV-JS clone has been demonstrated in Nicotiana benthamiana. Here we describe the agroinfection of the sweet potato cultivar Xushu 22 with the SPLCV-JS infectious clone using vacuum infiltration. Yellowing symptoms were observed in newly emerged leaves. Molecular analysis confirmed successful inoculation by the detection of viral DNA. A synergistic effect of SPLCV-JS and the heterologous betasatellite DNA-β of Tomato yellow leaf curl China virus isolate Y10 (TYLCCNV-Y10) on enhanced symptom severity and viral DNA accumulation was confirmed. The development of a routine agroinoculation system in sweet potato with SPLCV-JS using vacuum infiltration should facilitate the molecular study of sweepovirus in this host and permit the evaluation of virus resistance of sweet potato plants in breeding programs.     

甘薯生物技术研究进展

新兴的生物技术为甘薯这一古老的农作物带来了新的发展契机。细胞大规模培养、体细胞融合、基因转导等技术的研究和应用 ,可望从根本上改变甘薯传统的生产和育种模式。本文综合近年来国内外甘薯体细胞胚胎发生、原生质体培养和基因工程等方面的研究进展 ,对影响甘薯体胚发生体系及原生质体再生体系建立的诸多因素进行了详细论述 ,讨论了甘薯基因工程研究的应用潜力和目前存在的一些问题。     

营养保健甘薯汁制备工艺的优化

目的：优化营养保健甘薯汁的制备工艺。方法：本研究以甘薯为原料,在加酶量、作用时间、反应温度、pH及底物浓度五个单因素试验的基础上采用响应面分析法,以甘薯浆中还原糖量为评价指标,对耐高温α-淀粉酶酶解甘薯浆中淀粉的最佳工艺进行了研究,并利用统计学方法建立了耐高温α-淀粉酶酶解甘薯浆中淀粉的二次多项数学模型。结果：最佳酶解条件为：加酶量480U／g,作用时间90min,反应温度77℃,pH值6．0,底物浓度2．6g／10ml。结论：在最佳酶解条件下,甘薯中还原糖最大估计值为13．97345％。实测值为（13．968±0．05）％。     

甘薯丛枝病植原体的PCR检测

以报道的植原体（Phytoplasma)16SrDNA基因保守序列为依据,设计合成了两对引物对R16mF2/R16mR2和R16F2／R16R2,以甘薯丛枝病（SPWB）带病植株的叶脉中提取的DNA为模板,应用聚合酶链式反应（PCR）技术和巢式PCR（Nested-PCR)技术对甘薯丛枝病病原进行分子检测。结果表明PCR扩增出了1．5kb的特异片段,在PCB基础上的巢式PCR扩增出了1．2kb的特异片段,灵敏度实验显示该方法所需PCR模板DNA量为0．1073ng/ul在PCR的基础上的巢度PCR可以将灵敏度提高约10000倍,所需模板DNA仅为0．01073pg/ul,在甘薯丛枝病的检测中是一种快速,灵敏,可靠的方法。     

Sweet Potato Virus Disease in Sub-Saharan Africa: Evidence that Neglect of Seedlings in the Traditional Farming System Hinders the Development of Superior Resistant Landraces

Sweet potato virus disease (SPVD) occurs in sweet potato at all localities on the perimeter of Lake Victoria areas surveyed in Uganda and Tanzania, and was particularly common in Kagera District in Tanzania and in Rukungiri District in Uganda. All fields were planted with landraces and the most important control practices, as perceived by farmers, were the planting of cuttings derived from only symptomless parents and destroying diseased plants. Although SPVD-resistant landraces were available, they were perceived by most farmers to have poor and late yields. Most farmers considered that their greatest need was new, more acceptable, SPVD-resistant genotypes. Few farmers had seen either sweet potato seeds (15%) or sweet potato seedlings (11%) and, of those that had, most had ignored them. The lack of seedlings and their neglect by farmers is likely to be hindering the evolution of more acceptable, SPVD-resistant landraces, and is probably responsible for SPVD being a long-term disease problem.     

Synergistic interactions of begomoviruses with Sweet potato chlorotic stunt virus (genus Crinivirus) in sweet potato (Ipomoea batatas L.)

Three hundred and ninety‐four sweet potato accessions from Latin America and East Africa were screened by polymerase chain reaction (PCR) for the presence of begomoviruses, and 46 were found to be positive. All were symptomless in sweet potato and generated leaf curling and/or chlorosis in Ipomoea setosa. The five most divergent isolates, based on complete genome sequences, were used to study interactions with Sweet potato chlorotic stunt virus (SPCSV), known to cause synergistic diseases with other viruses. Co‐infections led to increased titres of begomoviruses and decreased titres of SPCSV in all cases, although the extent of the changes varied notably between begomovirus isolates. Symptoms of leaf curling only developed temporarily in combination with isolate StV1 and coincided with the presence of the highest begomovirus concentrations in the plant. Small interfering RNA (siRNA) sequence analysis revealed that co‐infection of SPCSV with isolate StV1 led to relatively increased siRNA targeting of the central part of the SPCSV genome and a reduction in targeting of the genomic ends, but no changes to the targeting of StV1 relative to single infection of either virus. These changes were not observed in the interaction between SPCSV and the RNA virus Sweet potato feathery mottle virus (genus Potyvirus), implying specific effects of begomoviruses on RNA silencing of SPCSV in dually infected plants. Infection in RNase3‐expressing transgenic plants showed that this protein was sufficient to mediate this synergistic interaction with DNA viruses, similar to RNA viruses, but exposed distinct effects on RNA silencing when RNase3 was expressed from its native virus, or constitutively from a transgene, despite a similar pathogenic outcome.