Movement of aphid-transmitted Sugarcane yellow leaf virus (ScYLV) within and between sugarcane plants

Sugarcane yellow leaf virus (ScYLV) is distributed worldwide and has been shown to be the cause of the disease sugarcane yellow leaf syndrome (YLS). This study was an investigation of the transmission and spread of ScYLV in Hawaii. Several aphids are known to transmit the virus, but investigation of infestation and transmission efficiency showed Melanaphis sacchari to be the only vector important for field spread of the disease. The initial multiplication of ScYLV in a virus-free plant occurred exclusively in very young sink tissues. When a single leaf was inoculated on a plant, that leaf and all older leaves remained virus-free, based on tissue-blot immunoassay, whereas meristems and all subsequently formed new leaves became infected. Therefore, only after those leaves which had already developed before inoculation had been shed, did the complete plant contain ScYLV. Spread of the viral infection to neighbouring plants in the plantation fields via aphids was relatively slow and in the range of a few metres per year. No indication of long-distance transfer could be seen. This indicates that it may be possible to produce and use virus-free seed cane for planting of high-yielding but YLS-susceptible cultivars.     

甘蔗黄叶病毒复制酶基因的克隆及序列分析

 The gene of RNA-dependent RNA polymerase (RdRP) was cloned by RT-PCR from Sugarcane yellow leaf virus-Fuzhou isolate (CHN-FJ1) and then cloned into pMD18-T vector. The sequence showed that the fragment comprised 1 212 nucleotides including part gene of ORF1 and ORF2. The ORF2 was involved the RdRP gene consisted of 995 nucleotides and encoded putative protein of 331 amino acids. Compared the nucleo-tide sequence and encoded putative protein of CHN-FJ1 with the other isolates from different countries, they shared the homology above 92.0%. Phylogenetic tree suggested that the sixteen isolates were classified into four types according to the amino acid sequence of the RdRP. One of the groups contained CHN-FJ1 and the other isolates from China, American, Brazil, Australia and Colombia;however, there was the closest relation between CHN-FJ1 and BRA-YL1 isolate from Brazil.     

Carbon dioxide assimilation by virus-free sugarcane plants and by plants which were infected by Sugarcane Yellow Leaf Virus

Infection of sugarcane by Sugarcane Yellow Leaf Virus (SCYLV) remains mostly asymptomatic. The assimilation rates and the water relations parameters were measured to test whether the plants are already inflicted by the infection. The assimilation rate and the stomatal conductance were 10–30% higher in virus-free plants than in infected plants. The observed differences were significant in young potted plants, not in older field plants. Virus-free leaves had a higher bundle sheath leakiness and a lower ¹³C discrimination rate than infected leaves. The water relations parameters of SCYLV-infected asymptomatic plants showed resemblance to those of salinity- and drought-stressed plants.     

Molecular detection of sugarcane striate virus and sugarcane white streak virus and their prevalence in the Miami World Collection of sugarcane and related grasses

Viruses in the genus Mastrevirus (family Geminiviridae), including those infecting sugarcane, have natural geographical ranges almost exclusively restricted to Africa and the Indian Ocean islands off the African coast. Only sugarcane white streak virus (SWSV) in Barbados and sugarcane striate virus (SStrV) in Florida and Guadeloupe are known to infect a few sugarcane varieties in the Western Hemisphere. In this study, PCR assays were developed to detect these two viruses in sugarcane. Five hundred and seventy-one DNA samples from Saccharum species and interspecific hybrids from the Miami World Collection of sugarcane and related grasses were tested for the presence of SStrV and SWSV by PCR. No variety was found infected by SWSV but SStrV was detected in 19 varieties. PCR data were confirmed by sequencing amplified fragments (248 bp). These fragments shared 93%–100% nucleotide identity with SStrV sequences from the GenBank database. SStrV isolates were distributed in six phylogenetic groups, including the four strains of the virus. Most varieties infected by SStrV originated from Asia, thus confirming a previous hypothesis stating that this virus originated from this continent. Absence of SStrV in commercial sugarcane in Florida also suggested that this virus has not been spread in this location, while infected plants have been present for several decades.     

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.     

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。     

甘蔗黄叶病毒外壳蛋白基因克隆及其实时荧光RT-PCR检测

 甘蔗黄叶病毒（Sugarcane yellow leaf virus, SCYLV）引起的甘蔗黄叶病是一种新的全球性病毒病害。本文以YLSCPF1和YLSCPR591为引物,采用RT-PCR方法克隆了甘蔗黄叶病毒福建分离物（CHN-FJ1）外壳蛋白（CP）基因,编码196个氨基酸。分析不同地理来源的SCYLV病毒分离物cp基因核苷酸及其推导编码的氨基酸序列,同源性达95%以上。根据cp基因的保守序列,设计1对特异性引物和TaqMan探针,建立了SCYLV的TaqMan实时荧光RT-PCR方法。结果表明,检测下限为初始质粒模板DNA 1 000拷贝/μL（约3.61 fg/μL）,比常规PCR方法的灵敏度提高100倍。检测甘蔗花叶病毒、宿根矮化病菌和黑穗病菌,没有典型的扩增曲线和无Ct值。应用实时荧光RT-PCR、常规RT-PCR和组织印迹免疫杂交（TBIA）对田间甘蔗叶片样品进行检测,阳性检出率分别为100%、61.5%和69.2%,表明该方法比常规RT-PCR和TBIA具有更高的灵敏度,适合于对SCYLV的检测。     

Transmission of a sugarcane yellow leaf phytoplasma by the delphacid planthopper Saccharosydne saccharivora, a new vector of sugarcane yellow leaf syndrome

During surveys of sugarcane fields in western and central Cuba from December 2001 to March 2003, the delphacid planthopper Saccharosydne saccharivora was the most prevalent of the Auchenorrhyncha fauna surveyed. Individuals of S. saccharivora collected tested positive for the sugarcane yellow leaf phytoplasma (SCYLP). Saccharosydne saccharivora were reared in cages and used for experimental transmission studies of SCYLP. The S. saccharivora were given acquisition-access feeds of 72 h on SCYLP-infected canes collected from the field followed by an inoculation-access period of 15 days on healthy sugarcane seedlings. Symptoms of yellow leaf syndrome developed on 24 out of 36 plants, 7–12 months postinoculation. None of the 36 healthy seedlings that were inoculated with S. saccharivora fed on phytoplasma-free sugarcane developed symptoms. All phytoplasma-positive sugarcane and S. saccharivora samples showed identical RFLP patterns and had 99·89% similarity in their 16S/23S spacer-region sequences, but only 92·6–93·6% similarity with other phytoplasmas. Sequences were deposited with GenBank [accession numbers: AY725237 ( S. saccharivora ) and AY257548 (sugarcane)]. Phylogenetic analysis suggested that the phytoplasmas from sugarcane and S. saccharivora are putative members of a new 16Sr phytoplasma group. This is the first report of vector transmission of a phytoplasma associated with sugarcane yellow leaf syndrome and the first time that S. saccharivora has been shown to vector a phytoplasma.     

Impact of <Emphasis Type="Italic">Sugarcane yellow leaf virus</Emphasis> on growth and sugar yield of sugarcane

A survey revealed that Sugarcane yellow leaf virus (SCYLV) is found on all Hawaiian sugarcane plantations including those where no yellow leaf symptoms were observed. In a comparison of growth and yield between SCYLV-infected and SCYLV-free plants of the cultivar H87-4094, germination and early shoot growth of infected plants were retarded. The number of stalks per stool was reduced by 30%, biomass was reduced by 29%, and sugar yield by 26% when plants were harvested after 11 months. Yields did not decrease when plants were harvested after 2 years. Thus, SCYLV could reduce yield, even when the plants were asymptomatic. In a field test of SCYLV-susceptible (infected) and -resistant cultivars to compare growth and yield, 10 commercial cultivars (six susceptible and four resistant to SCYLV) were grown in eight fields with different climates and soils. Primary stalk length, biomass and sugar yield did not differ between susceptible and resistant cultivars under any field conditions. Thus, harmful effects of SCYLV on yield cannot be deduced by comparing different cultivars.     

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.     

Symptom expression of yellow leaf disease in sugarcane cultivars with different degrees of infection by Sugarcane yellow leaf virus

Sugarcane yellow leaf virus (ScYLV) is present in many sugarcane growing areas of the world. It is suspected to cause yellow leaf disease (formerly called YLS, yellow leaf syndrome) of sugarcane. This study investigated symptom expression in a selection of cultivars classified into three groups; ScYLV-susceptible/infected, ScYLV-resistant and intermediately infected cultivars grown in plantation fields in the islands of Hawaii. Incidence of yellow leaf symptoms was correlated, though not tightly, to the presence of ScYLV. The correlation is based on two factors: (i) only ScYLV-infected cultivars (from both susceptible and intermediate groups) showed severe symptom expression, and (ii) ScYLV-infected plants had four times higher symptom incidence than virus-free plants of the same cultivar. The yellow leaf symptom expression fluctuated, peaking at 200, 350, 500 and 600 days after planting. These symptom peaks were correlated with an increase of ScYLV content in the intermediately infected group of cultivars. No nutritional, environmental or field factor could be identified which clearly influenced symptom expression. It is speculated that the symptom expression is elicited by assimilate backup in the stalks and that the fluctuation of symptom expression is caused by the growth rhythm of mature sugarcane stalks.     

Impact of Sugarcane yellow leaf virus on Sugarcane Yield and Juice Quality in Réunion Island

Sugarcane yellow leaf virus (SCYLV) was first detected in sugarcane of Réunion Island in 1997. A field experiment was undertaken to assess the potential impact of this virus on sugarcane production. The agronomic characteristics of SCYLV-infected plants were compared to those of virus-free plants of three sugarcane cultivars (R570, R577 and R579) which occupy more than 90% of the cultivated sugarcane area on Réunion Island. In the plant crop, significant losses in stalk weight (28%) and in sugar content (11%) were detected for cultivar R577, but not for either of the two other cultivars. In the first ratoon crop, yield reduction was detected for cultivar R577 (37%), but also for cultivar R579 (19%). Cultivar R577 also showed significant losses in sugar content (12%) due to reduced amount and quality of extracted cane juice. No yield reduction was found for cultivar R570, although stalk height and diameter were reduced in SCYLV-infected canes of this cultivar in the first ratoon crop. Leaf yellowing was observed at harvest of plant and ratoon crops when sugarcane was no longer irrigated, and 10–59% of symptomatic stalks could be attributed to the presence of SCYLV. The most severe yellowing symptoms were related to infection of sugarcane by the virus.     

Phylogenetic and recombination analysis of sorghum isolates of Sugarcane yellow leaf virus

Recombination has played an important role in evolution and genetic diversity of Sugarcane yellow leaf virus (SCYLV) isolates sequenced to date. This study found that three newly sequenced SCYLV sorghum isolates from the USA underwent intraspecies recombination. No statistical significance on probable progeny–parent relationships involving SCYLV sorghum isolates were found in possible interspecies recombination with 18 members of the Luteoviridae family. Sorghum isolates deposited in the GenBank database under accession numbers KT960995, KT960996 and KT960997 were phylogenetically closely related to SCYLV genotypes IND, CUB and CHN1, all members of phylogroup II. Networked relationships among the sorghum isolates showed that numerous incompatibilities occurred in the sequences. These conflicting signals were probably due to recombination, especially in KT960997, which was heavily impacted by recombination. The KT960997 accession was positioned on a distinct branch compared to other members of phylogroup II, suggesting that it has probably emerged as a new genotype. Future studies on molecular evolution may reveal further insights into the adaptation capacity of these SCYLV lineages to new environments.     

植物诱抗剂AHO联合茎尖培养脱除马铃薯S病毒和马铃薯Y病毒

研究了植物诱抗剂3-丙酮基-3-羟基羟吲哚(3-acetonyl-3-hydroxyoxindole, AHO)联合茎尖培养从试管苗中脱除马铃薯S病毒(PVS)?马铃薯Y病毒(PVY)的方法和效率?取PVS侵染的‘定薯3号’和‘定薯4号’以及PVY侵染的‘靖薯3号’和‘靖薯4号’的壮芽, 茎尖剥离后培养至4~5个叶片, 用100 mg/L植物诱抗剂 AHO水剂喷施试管苗, 每隔2 d喷施一次, 共3次, 末次喷施2 d后取茎尖剥离培养, 获得再生试管苗?用电子显微镜负染色?ELISA?荧光定量RT-PCR检测再生试管苗的带病毒情况?结果显示, AHO对4个品种的马铃薯试管苗生长无影响, 用AHO处理后再茎尖剥离培养, 脱毒率均高于未处理的对照; 检测结果还显示AHO处理的马铃薯再生苗的带毒量也低于未处理的对照, 且随处理次数增加带毒量下降?研究结果表明, 利用植物诱抗剂AHO联合茎尖剥离培养方法可以提高脱除PVS?PVY的效率, 获得无病毒核心苗?     

Exogenous application of melatonin improves eradication of apple stem grooving virus from the infected in vitro shoots by shoot tip culture

Production and maintenance of virus-free planting materials is pivotal for the control of viral diseases. The present study attempted to test exogenous application of melatonin for eradication of apple stem grooving virus (ASGV) from virus-infected in vitro shoots of apple cultivar Gala. Exogenous application of 15 μm melatonin to the shoot proliferation medium significantly increased the number of shoots and shoot length. The level of endogenous indole-3-acetic acid (IAA) was the highest in the shoots proliferating on the shoot proliferation medium containing 15 μm melatonin. Shoot regrowth levels were significantly higher in shoot tips of the virus-infected shoots cultured for 4 weeks on this medium than the control. In addition, culture of shoot tips of the virus-infected in vitro shoots proliferated for 4 weeks on this medium resulted in 95% of shoots being virus-free, while no virus-free shoots were obtained in shoot tips of the virus-infected shoots cultured without melatonin. Analyses by microtissue direct RT-PCR and RT-qPCR showed that ASGV concentration decreased in shoot tips of the virus-infected shoots proliferating on the medium containing 15 μm melatonin for 4 weeks. Virus localization showed that exogenous application of melatonin enlarged the virus-free area in the virus-infected shoot tips. These data provide explanations as to why exogenous application of melatonin can efficiently eradicate ASGV. Exogenous application of melatonin provides an alternative means for plant virus eradication and has the potential to produce virus-free plants.     

Viral dieback of carrot and other umbelliferae caused by the Anthriscus strain of parsnip yellow fleck virus,and its distinction from carrot motley dwarf

Viral dieback of carrot, chervil, coriander, dill and wild Umbelliferae is described. Disease incidence in carrot crops grown for seed is often high but low in ware carrot. There is no secondary spread in carrot crops.The causal virus was identified as theAnthriscus strain of parsnip yellow fleck virus (PYFV) transmitted byCavariella aegopodii from cow parsley(Anthriscus sylvestris). Nicotiana benthamiana was practically indespensable for isolation of PYFV by sap transmission from plants with viral dieback.No immunity was found in 12 carrot cultivars or in wild carrot. Disease control with a systemic insecticide had limited effect.Carrot red leaf virus and carrot mottle virus were commonly found in carrot, but they did not cause dieback symptoms. Cucumber mosaic virus, parsnip mosaic virus and a virus resembling that of carrot yellow leaf were occasionally isolated from carrot. Symptoms due to mycoplasma were also observed.Samenvatting Bij de zaadteelt van peen is in ons land reeds lang een schadelijke, vroeg in het seizoen optredende instervingsziekte bekend als voorjaarsziekte of het zwart. Planten vallen op door necrose van jonge spruiten (insterving). Soms gaat meer dan de helft van het gewas verloren. Voor consumptie geteelde peen wordt echter nauwelijks aangetast. De ziekte is nu ook gevonden bij dille, kervel, koriander en wilde schermbloemigen.Uit zieke planten en ook vaak uit symptoomloze fluitekruidplanten werd een virus geïsoleerd waarmee de insterving kon worden gereproduceerd. Het werd herkend als de fluitekruid-(ofAnthriscus-)stam van pastinakegeelvlekvirus (PYFV) op grond van waardplanten, symptomen, serologie en overdracht doorCavariella aegopodii met als onmisbare helper hetAnthriscus-vergelingsvirus (AYV), dat ook in fluitekruid voorkomt. Het gebruik vanNicotiana benthamiana als toetsplant maakte isolatie uit planten met virusinsterving mogelijk. Voor de ziekte wordt nu de naam virusinsterving van schermbloemigen voorgesteld.Peenroodbladigheid veroorzaakt door peenroodbladvirus, dat meestal samengaat met peenvlekkenvirus, bleek ook algemeen voor te komen. Deze twee virussen spelen geen rol bij het veroorzaken van virusinsterving, zoals wel werd aangenomen. Beide ziekten zijn geheel verschillend in symptomatologie en epidemiologie. Incidenteel werden komkommermozaïekvirus, pastinakemozaïekvirus en een virus gelijkend op peengeelbladvirus in aangetroffen. Ook werd eenmaal een aan een mycoplasma toe te schrijven ziekte geconstateerd.Virusinsterving bleek epidemiologisch te kunnen worden verklaard door de massale jaarlijkse migratie vanC. aegopodii in het voorjaar, waarbij PYFV van fluitekruid naar peen en andere schermbloemigen wordt verspreid. Door onvatbaarheid van peen voor het helpervirus (AYV) treedt in dit gewas geen secundaire verspreiding op.In geen van 12 peenrassen en wilde peen werd resistentie aangetroffen. Toepassing van een systemisch insekticide bleek in eerder onderzoek slechts een beperkt effect te hebben. Peenzaadteelt in gebieden met minder bladluizen, zoals het noorden des lands, lijkt aan te bevelen, maar verder lijkt de ziekte niet te bestrijden.Work in partial fulfillment of requirements for master's training at Agricultural University, Wageningen