Detection and molecular characterization of sugarcane grassy shoot phytoplasma in Vietnam

Sugarcane is an important cash crop in Vietnam and has been widely promoted at national and provincial level. In 2006, a new disease was discovered in sugarcane in the Nghean Tate&Lyle Sugar Mill in Nghean Province in north-central Vietnam. The key symptoms were the formation of green grassy shoots around the base of mature stools. We applied nested polymerase chain reaction (PCR) using P1/P7 and R16F2n/R16R2 for detection and characterization of phytoplasma from the symptomatic tissues. PCR products of the expected size (approx. 1200?bp) were obtained from the 16S rDNA of the phytoplasma. The restriction fragment length polymorphism profiles indicated that all samples were infected by the same phytoplasma. Phylogenetic analysis confirmed that the SCGS phytoplasma from Vietnam belong to the 16SrXI group, formerly Rice Yellow Dwarf group.     

Identification of potential vectors of the coconut lethal disease phytoplasma

Lethal disease (LD) is a lethal yellowing-type disease of coconuts associated with phytoplasmas in Tanzania, but the insect vector for it has not yet been identified. In this study, the auchenorrynchous insects in LD-infected coconut fields were surveyed to determine potential vectors for the disease. No significant correlation was found between disease incidence and numbers of insects collected from the field, possibly reflecting the unknown incubation period for the disease. However, analysis of more than 5000 individual insects by the polymerase chain reaction (PCR), using LD-specific primers derived from the phytoplasma 16S rRNA gene, revealed PCR products of the correct size from eight individuals of Diastrombus mkurangai and four of Meenoplus spp. When digested with restriction endonucleases, fragments of the same size as the LD phytoplasma were obtained. No PCR products were detected in any of the other insect species tested. These results implicate D. mkurangai and Meenoplus spp. as probable vectors of the LD phytoplasma.     

Nested PCR assay for detection of sugarcane grassy shoot phytoplasma in the leafhopper vector Deltocephalus vulgaris: a first report

Nymphs of Deltocephalus vulgaris , the leafhopper vector of sugarcane grassy shoot (SCGS) disease, fed on SCGS-infected and healthy sugarcane leaves, and SCGS-infected and healthy plant tissue of sugarcane cv. CoLk 8102, were examined by nested PCR using phytoplasma-specific rRNA operon primers for detection of the SCGS phytoplasma. Samples of SCGS-infected plants with symptoms and SCGS-exposed D. vulgaris nymphs yielded SCGS-exclusive DNA bands when nested PCR was performed. Negative results were obtained when symptomless plant host and unexposed insect vector samples devoid of phytoplasma DNA templates were used. Such a reliable molecular tool for the precise detection of SCGS phytoplasma in the D. vulgaris population would help forecast the potential of secondary spread of SCGS in a susceptible sugarcane variety, and may facilitate control of the disease.     

云南蔗区发现由植原体引起的检疫性病害甘蔗白叶病

By used of nested-PCR, the 16S rDNA sequence of phytoplasma associated with sugarcane white leaf (SCWL) in 48 suspected SCWL samples from Baoshan and Lincang of Yunnan were amplified with two primer pairs MLOX/ MLOY and P1/P2. The sequencing showed that the fragment size of 17 Baoshan suspected SCWL samples were all 210 bp and their sequences were all identical (GenBank: KC662509); the fragment size of 10 Lincang suspected SCWL samples were all 202 bp and their sequences were all identical (GenBank: KF431837). The Blast result indicated that the sequences obtained in this study were derived from the 16S-23S ISR intergenic spacer region of phytoplasma that causes SCWL and were highly homologous (99.05%-100% similarity) to the corresponding genome region registered in GenBank. Plant height, stalk diameter, millable stalk rate and single stalk weight were significantly reduced by infection of SCWL,which caused destructive damage to sugarcane.     

人工培养液在植原体昆虫介体筛选中的适用性

研究了一种人工培养液对各种常见的昆虫(主要是叶蝉)的亲和性和适用性.结果表明,该人工培养液适于本试验中大多数昆虫的人工饲养.用此方法,悬钩子广头叶蝉Macropsis.ftscula Zetterstedt和桤树广头叶蝉Oncopsis alniSchrank分别被再次确认为悬钩子矮化植原体和桤树黄化植原体的传播介体;田旋花麦蜡蝉Hyalesthes obsoletus Signoret再次被确认为葡萄黄化(stolbur)植原体的传播介体.此前,上述三种叶蝉已被传统的人工接种方法鉴定为相应植原体的传播介体.危害桤树的河谷树叶蝉Allygus modestus Scott尽管虫体DNA检测结果经常为阳性,但迄今其人工培养液的检测结果都是阴性,因此,我们认为河谷树叶蝉不是桤树黄化植原体的传播介体.Eppendorf管人工培养液饲养法不仅适用于潜在的植原体介体昆虫的筛选鉴定,而且可用于介体昆虫的生物防治研究.此外,本研究首次发现自然感染了葡萄上的一种被德国人称为"Vergi-lungskrankheit"植原体(AY组)的草地脊冠叶蝉Aprodes makarovi Zachvatkin.     

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.     

低纬高原甘蔗白叶病植原体传播方式测定分析

甘蔗白叶病(Sugarcane white leaf,SCWL)是由16SrXI组植原体引起的甘蔗重要病害[1],主要通过带病蔗种传播,还可在田间通过叶蝉自然传播[2,3].该病1954年首次在泰国发现[4],现已在印度、巴基斯坦、斯里兰卡、菲律宾、缅甸、越南等国普遍发生,给当地蔗糖业造成巨大经济损失[5,6].201...     

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].     

Identification of a phytoplasma causing yellows of Monarda

Monarda yellows occurring in southern Alberta was found to be associated with a phytoplasma. Using two pairs of universal primers, 16S ribosomal DNA fragments (about 1.5 and 1.2 kb) were amplified separately by polymerase chain reaction (PCR) from DNA samples that had been extracted from infected monarda. No such DNA bands were observed using DNA samples from uninfected monarda. The DNA fragment (1.2 kb) amplified by nested-PCR was analysed and compared with western aster yellows (AY27, Canada), eastern aster yellows (EAY, USA), French hydrangea aster yellows (AYHF), Belgium hydrangea aster yellows (AYHB), clover proliferation (CP, Canada) and potato witches'-broom (PWB, Canada) by means of restriction fragment length polymorphism (RFLP) using endonucleases Alu I, Mse I, Hpa II, Sau 3AI, Kpn I and Rsa I. The results showed that monarda yellows phytoplasma belongs to the aster yellows subclade and is different from CP and PWB. This is the first report of aster yellows phytoplasma infecting monarda.     

国外引进甘蔗材料白叶病植原体巢式PCR检测及其序列分析

为有效防止引起重要危险性检疫病害甘蔗白叶病(SCWL)的植原体病原随引进甘蔗材料侵入我国,确保我国甘蔗生产安全,本研究对从缅甸、菲律宾、法国和泰国引进的22个甘蔗材料进行了SCWL植原体巢式PCR检测,并对阳性样品的巢式PCR产物进行了测序及序列分析。结果表明,18个甘蔗材料呈SCWL植原体阳性,阳性检出率为81.8%。所有SCWL阳性样品的16S-23S基因间隔区片段长210bp,与GenBank中已有的其他SCWL植原体分离物(登录号HQ917068、AB646271)的同源性为99.8%~100%,并在系统发育树中聚为一个类群。根据SCWL的巢氏PCR检测及其序列分析结果,对呈阳性的材料及时进行了集中销毁处理。     

竹小叶病植原体的分子鉴定

 2008年在杨凌采集到具有典型植原体侵染症状的菲白竹,应用植原体16S rRNA基因的通用引物对R16mF2／R16mR1和R16F2n／R16R2对其进行检测,巢式PCR得到约1.2 kb的特异性片段。对扩增片段进行测序并进行系统进化树分析,结果表明,该病原属于翠菊黄化组(Candidatus Phytoplasma asteris),与该组成员同源性均在98%以上。随后用16Sr Ⅰ组和Ⅴ组特异引物对R16（Ⅰ）F1／R16（Ⅰ）R1和R16（Ⅴ）F1／R16（Ⅴ）R1也证明其属于翠菊黄化组,RFLP 分析表明该植原体属于16SrⅠ-B亚组。植原体侵染菲白竹在中国属首次报道。     

Detection and molecular characterization of phytoplasma associated with chickpea phyllody disease in south India

Chickpea (Cicer arietinum L.) plants showing typical symptoms of infection by a phytoplasma that causes phyllody disease have been commonly observed in recent years in parts of south India. The symptoms included pale green leaves, bushy appearance due to excessive stunting of shoots, reduced internodal length and excessive axillary proliferation. The causal agent of the phyllody disease was identified based on symptoms, amplification of 16S rDNA of the phytoplasma by polymerase chain reaction (PCR) from infected samples, as well as by sequencing and phylogenetic analysis. First round PCR and nested-PCR protocols were standardized for improved efficiency and reliability of the diagnostic protocols. Using the primers P1/P7 and R16F2n/R16R2, 1,800?bp and 1,200?bp size products were amplified in first round PCR and nested-PCR protocols, respectively. The PCR product was cloned and sequenced and compared with the reference phytoplasma sequences from the database (NCBI). The Indian chickpea phyllody phytoplasma 16S rDNA sequences shared the highest nucleotide identity (>98%) with the 16S rII group phytoplasma candidates, also infecting chickpea from Australia and Pakistan. This is the first report of a phytoplasma of the 16SrII-group infecting chickpea from India. The genetic similarities and the potential threat of this new disease to chickpea cultivation in India are discussed.     

Identification and molecular characterization of a phytoplasma associated with sunflower in Argentina

Sunflower (Helianthus annuus L.) plants showing capitulum with virescence, phyllody and flower malformation, shortened internodes and abnormal branches were found in a field in Pedro Luro (Buenos Aires province, Argentina). Pleomorphic bodies resembling phytoplasmas were observed in sieve tube elements of symptomatic plants but not in healthy ones. DNA from all symptomatic sunflower plants analysed yielded, in direct PCR with phytoplasma universal primers P1/P7 and R16F2n/R2, fragments of expected size 1.8 kb and 1.2 kb, respectively. The phytoplasma associated with the disease, was named Sunflower Phyllody (SunPhy). Real and putative RFLP of the 16S rDNA showed the affiliation of SunPhy to 16SrIII (X-disease group), subgroup J. The 16S rDNA sequence from SunPhy showed the highest identity (99 %) with 16SrIII members and the phylogenetic tree confirmed a closer relationship to subgroup J of the 16SIII ribosomal group. This is the first report of a phytoplasma related to the 16SrIII group affecting sunflower.     

Identification and characterization of two phytoplasma subgroups (16SrXI-D and 16SrXIV-A) associated with lychee (Litchi chinensis Sonn) in India

Journal of Plant Diseases and Protection - Leaf rolling, necrosis and apical bud proliferation symptoms were recorded on lychee plants at Bhatpar Rani (Deoria district) and Gorakhpur (Gorakhpur...     

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.     

Incidence of Bois Noir phytoplasma in different viticulture regions of Spain and Stolbur isolates distribution in plants and vectors

The incidence of Bois Noir (BN) disease in grapevine plots, the population of the vector Hyalesthes obsoletus, and the distribution of stolbur phytoplasma isolates were studied over a 4 year period in five regions of Spain. BN incidence in affected plots ranged from 1 to 75 %. A study of the H. obsoletus population indicated that individuals of this insect vector were identified in most of the sampled plots with low populations, from 0.25–5 individuals per aspiration. The population peaks of H. obsoletus were reached at different dates between June 6th and July 24th, depending on the sampled zone and the year. The percentage of H. obsoletus individuals carrying the phytoplasma showed an average of 55 %. In Aragon, this percentage rose to 76 %. The strain tuf-b of stolbur phytoplasma was the most prevalent type in grapevine plants and H. obsoletus, except in grapevine plants from La Rioja where the strain tuf-a was detected in most plants. A study of the vmp1 gene revealed the presence of four different isolates in grapevine plants and H. obsoletus.     

Multilocus genes based characterization of phytoplasma strains associated with Mexican and French marigold species in India

European Journal of Plant Pathology - During the winters of 2018 to 2020, witches’ broom, phyllody, flat stem, little leaf, yellowing and stunting symptoms were recorded in Mexican...     

甘蔗新品系对甘蔗黑穗病菌的抗性鉴定与评价

本研究采用浸渍接种和注射接种方法,对华南农业大学和湛江市农业科学研究院联合育成的20个甘蔗新品系进行黑穗病抗性鉴定。结果表明:浸渍接种鉴定的10个甘蔗新品系中,抗性水平达中抗及以上的品系有2个,分别为‘13177’(HR)和‘13113’(MR),占供试品系的20%,抗性水平为中感及以下的有8个,占80%。经3个不同致病力菌株注射接种鉴定的10个供试甘蔗新品系中,对Ss16菌株抗性水平达中抗及以上的供试品系有3个,分别为‘A6-13111’(HR)、‘A6-13115’(HR)和‘A13-1396’(MR),占供试品系的30%,中感及以下有7个,占70%;对Ss25菌株抗性水平达中抗及以上的品系有1个,为‘A6-13111’(HR),占供试品系的10%,中感及以下有9个,占90%;对Ss47菌株抗性水平达中抗及以上的品系有2个,分别为‘A3-1320’(HR)和‘A6-13111’(MR),占供试品系的20%,抗性水平为中感及以下有8个,占80%。综合评价结果显示,甘蔗新品系‘A6-13111’和‘13117’对甘蔗黑穗病具有良好的抗性。