Genetic diversity in the coconut lethal yellowing disease phytoplasmas of East Africa

DNA primers, based on the ribosomal sequences of lethal yellowing-type disease (LYD) phytoplasmas, were used to analyse genetic variation within the lethal yellowing-type diseases of coconut in East Africa. Samples were collected from palms in Kenya, Mozambique and high, medium and low disease incidence areas of Tanzania. The mollicute-specific primer pair P1 and P6 amplified a 1.5 kbp product from all diseased palms and no product from symptomless palms, indicating that phytoplasmas were associated with all of these diseases. However, the Rohde forward and Rohde reverse primers (a second rRNA primer pair designed to detect East African LYD-associated phytoplasmas) only amplified products from Tanzanian and Kenyan diseased palms and not from those of Mozambique. Conversely, primers Ghana 813 and AK-SR, designed for specific detection of coconut-associated phytoplasmas in West Africa, amplified products only from the Mozambique palms, indicating that the phytoplasma associated with LYD in Mozambique is more closely related to those from West Africa. This was supported by restriction enzyme digestion of PCR products. DNA sequencing of PCR products from phytoplasmas within Tanzania revealed no detectable differences in the rDNA sequences of isolates from high, medium and low incidence areas.     

Characterization of phytoplasmas detected in Chinaberry trees with symptoms of leaf yellowing and decline in Bolivia

Polymerase chain reaction (PCR) assays were used to detect phytoplasmas in foliage samples from Chinaberry ( Melia azedarach ) trees displaying symptoms of yellowing, little leaf and dieback in Bolivia. A ribosomal coding nuclear DNA (rDNA) product (1·8 kb) was amplified from one or more samples from seven of 17 affected trees by PCR employing phytoplasma-universal rRNA primer pair P1/P7. When P1/P7 products were reamplified using nested rRNA primer pair R16F2n/R16R2, phytoplasmas were detected in at least one sample from 13 of 17 trees with symptoms. Restriction fragment length polymorphism (RFLP) analysis of P1/P7 products indicated that trees CbY1 and CbY17 harboured Mexican periwinkle virescence (16SrXIII)-group and X-disease (16SrIII)-group phytoplasmas, respectively. Identification of two different phytoplasma types was supported by reamplification of P1/P7 products by nested PCR employing X-disease-group-specific rRNA primer pair R16mF2/WXint or stolbur-group-related primer pair fSTOL/rSTOL. These assays selectively amplified rDNA products of 1656 and 579 bp from nine and five trees with symptoms, respectively, of which two trees were coinfected with both phytoplasma types. Phylogenetic analysis of 16S rDNA sequences revealed Chinaberry yellows phytoplasma strain CbY17 to be most similar to the chayote witches'-broom (ChWBIII-Ch10) agent, a previously classified 16SrIII-J subgroup phytoplasma. Strain CbY1 resembled the Mexican periwinkle virescence phytoplasma, a 16SrXIII-group member. The latter strain varied from all known phytoplasmas composing group 16SrXIII. On this basis, strain CbY1 was assigned to a new subgroup, 16SrXIII-C.     

High diversity,expanding populations and purifying selection in phytoplasmas causing coconut lethal yellowing in Mozambique

In this study, the putative phytoplasma species causing coconut lethal yellowing disease in Mozambique and Tanzania were characterized. The 16S rRNA and secA genes were sequenced. Phylogenetic analysis revealed that Mozambican coconut phytoplasmas belong to three different types: ‘Candidatus Phytoplasma palmicola’ 16SrXXII‐A, a second strain that was previously isolated in Tanzania and Kenya (16SrIV‐C), and a third strain that was different from all known lethal yellowing phytoplasma species. The third strain potentially represents a novel species and is closely related to pine phytoplasma. Co‐infection with ‘Ca. Phytoplasma pini’‐related and ‘Ca. Phytoplasma palmicola’ 16SrXXII‐A strains was observed. Furthermore, sequence variation in ‘Ca. Phytoplasma palmicola’ at the population level was consistent with purifying selection and population expansion.     

Association of phytoplasmas with the decline of European hazel in southern Italy

In the Campania region of southern ltaly. commercial orchards of European hazel ( Corylus avellana ) are severely affected by yellowing and decline. To determine whether phytoplasmas are associated with the disorder, stem samples from diseased trees were examined using polymerase chain reaction assays. No visible products were obtained by amplification of sample DNA with universal and group-specific phytoplasma primers. However, when the products obtained with universal primers were re-amplified with nested primers that were specific for the fruit tree phytoplasmas of the apple proliferation group, most samples tested positively. Restriction site analysis revealed that the trees were infected with the apple proliferation, pear decline, and European stone fruit yellows phytoplasmas in about the same proportion. Some of the trees were doubly infected with one of the fruit tree phytoplasmas and the aster yellows agent. Most of the infected trees were also identified by hybridization of the products obtained in the initial amplification with suitable oligonucleotide probes.     

Characterization and classification of phytoplasmas from wild and cultivated plants by RFLP and sequence analysis of ribosomal DNA

Restriction fragment length polymorphism and sequence analysis of PCR-amplified ribosomal DNA were used to identify and classify phytoplasmas associated with diseases of various wild and cultivated plants. The diseases examined were either not known before or the presumable causal agents were not yet identified and characterized or were only known from other geographic areas. New diseases examined were those causing virescence and phyllody of Bunias orientalis and Cardaria draba. Both were associated with strains of the aster yellows phytoplasma. The same type of aster yellows phytoplasma was also found to be associated with yellows and phyllody diseases of Portulaca oleracea, Stellaria media, Daucus carota ssp. sativus, and Cyclamen persicum. In German and French DNA samples from diseased Trifolium repens, the clover phyllody phytoplasma was identified, which could clearly be distinguished from other phytoplasmas of the aster yellows group. Strains of the stolbur phytoplasma were detected in big bud-affected tomatoes and almost exclusively in Convolvulus arvensis. In Cirsium arvense and Picris echioides two distinct phytoplasmas were identified which showed relationship to the sugarcane white leaf phytoplasma group but may represent a new group or subgroup. In Conyza (syn.: Erigeron) canadensis a phytoplasma of the X-disease group was detected. A strain from Gossypium hirsutum showed the same restriction profiles as the faba bean phyllody phytoplasma.     

Ecological implications from a molecular analysis of phytoplasmas involved in an aster yellows epidemic in various crops in Texas

ABSTRACT In the spring of 2000, an aster yellows (AY) epidemic occurred in carrot crops in the Winter Garden region of southwestern Texas. A survey revealed that vegetable crops, including cabbage, onion, parsley, and dill, and some weeds also were infected by AY phytoplasmas. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis of PCR-amplified phytoplasma 16S rDNA were employed for the detection and identification of phytoplasmas associated with these crops and weeds. Phytoplasmas belonging to two subgroups, 16SrI-A and 16SrI-B, in the AY group (16SrI), were predominantly detected in infected plants. Carrot, parsley, and dill were infected with both subgroups. Onion and three species of weeds (prickly lettuce, lazy daisy, and false ragweed) were predominantly or exclusively infected by subgroup 16SrI-A phytoplasma strains, while cabbage was infected by subgroup 16SrI-B phytoplasmas. Both types of phytoplasmas were detected in three leafhopper species, Macrosteles fascifrons, Scaphytopius irroratus, and Ceratagallia abrupta, commonly present in this region during the period of the epidemic. Mixed infections were very common in individual carrot, parsley, and dill plants and in individual leafhoppers. Sequence and phylogenetic analyses of 16S rDNA and ribosomal protein (rp) gene sequences indicated that phytoplasma strains within subgroup 16SrI-A or subgroup 16SrI-B, detected in various plant species and putative insect vectors, were highly homogeneous. However, based on rp sequences, two rpI subgroups were identified within the subgroup 16SrI-A strain cluster. The majority of subgroup 16SrI-A phytoplasma strains were classified as rp subgroup rpI-A, but phytoplasma strains detected in one onion sample and two leafhoppers (M. fascifrons and C. abrupta) were different and classified as a new rp subgroup, rpI-N. The degree of genetic homogeneity of the phytoplasmas involved in the epidemic suggested that the phytoplasmas came from the same pool and that all three leafhopper species may have been involved in the epidemic. The different phytoplasma population profiles present in various crops may be attributed to the ecological constraints as a result of the vector-phytoplasma-plant three-way interaction.     

Sampling for adults of the planthopper Myndus crudus a vector of lethal yellowing of palms 1

Abstract

Tests were made using coloured sticky traps to determine trap efficacy in catching adult Myndus crudus Van Duzee (Homoptera, Cixiidae) under field conditions. Blue traps caught more M. crudus adults than any of the other colour traps tested. Blue traps caught adults of both sexes during the day and night although significantly more adults were caught during the day. A sampling bias was shown in that blue traps caught significantly more males than expected from the field population. Blue sticky traps were more effective at sampling adult M. crudus populations than previous sampling techniques based on host plant attractiveness and should be considered for use in future sampling.     

Detection of lethal yellowing phytoplasma in embryos from coconut palms infected with Cape St Paul wilt disease in Ghana

This study investigated the potential of seed transmission of Cape St. Paul wilt disease (CSPWD) in coconuts. PCR amplification was used to assess the distribution of phytoplasmas in parts of West African Tall (WAT) palms infected with CSPWD. Employing phytoplasma universal primer pair P1/P7 in standard PCR, or followed with a nested PCR using CSPWD–specific primer pair G813f/AwkaSR, phytoplasma infection was detected in the trunks, peduncles, spikelets, male and female flowers of four infected WAT coconut palms. Through nested PCR, phytoplasma was also detected in four of 19 embryo DNA samples extracted individually from fruits harvested from three of the four infected palms and was confirmed as CSPWD by cloning and sequencing. Subsequently, CSPWD phytoplasma was again detected in five of 33 embryos from nine infected palms, and in one of eight fruits from two symptomless palms. Fruits from infected palms recorded higher percentage germinations in two field nurseries (average of 71·0%) compared to fruits from healthy palms (average of 57·6%), and matured fruits that had dropped from infected palms showed the same levels of germination as those harvested directly from the palms. This indicates that infected fruits retain the ability to germinate whether harvested or dropped. No phytoplasmas were detected in any of the resulting seedlings and plantlets obtained through embryo in-vitro culture. Therefore, although phytoplasma DNA can be detected in embryos, there is as yet no evidence that the pathogen is seed transmitted through to the seedling to cause disease in progeny palms.     

Comparison of phytoplasmas infecting winter oilseed rape in the Czech Republic with Italian Brassica phytoplasmas and their relationship to the aster yellows group

Winter oilseed rape grown in several areas in South Bohemia showed symptoms of stunting, leaf reddening and extensive malformation of floral parts. Phytoplasmas were consistently observed by using electron microscopy only in phloem tissue of symptomatic plants. DNA isolated from infected and healthy control plants was used in PCR experiments. Primer pairs R16F2/R2, P1/P7 and rpF2/R2, amplifying, respectively, 16S rDNA, 16S rDNA plus spacer region and the beginning of the 23S and ribosomal protein gene L22 specific for phytoplasmas, were used. According to RFLP and sequence analyses of PCR products, the phytoplasma from rape was classified in the aster yellows phytoplasma group, subgroup 16SrI-B. The PCR products from the Czech phytoplasma-infected rape also had RFLP profiles identical to those of phytoplasma strains from Italian Brassica . This first molecular characterization of phytoplasmas infecting rape compared with strains from Brassica does not, however, clearly indicate differences among isolates of the same 16SrI-B subgroup. Further studies on other chromosomal DNA portions could help the research on host specificity or on geographical distribution of these phytoplasmas.     

Detection of the mycoplasma-like organism associated with lethal yellowing disease of palms in Florida by polymerase chain reaction

DNA amplification by polymerase chain reaction (PCR) was used specifically to detect the mycoplasma-like organism (MLO) associated with lethal yellowing disease of palms in Florida. For PCR, a pair of oligonucleotide primers was synthesized according to partial sequences of a cloned 1·3 kbp fragment of lethal yellowing MLO-specific genomic DNA isolated from a diseased windmill palm ( Trachycarpus fortunei ). A DNA product of about 1 kbp was specifically amplified by PCR in reaction mixtures containing template DNA derived from either heart, inflorescence or leaf tissues of lethal yellowing-affected palms. PCR performed for 35 cycles with as little as 5 pg of DNA template, in some instances, was sufficient consistently to amplify the same lethal yellowing MLO DNA product from hearts of 11 species comprising 30 symptomatic palms. Similar reliable and reproducible detection of the lethal yellowing MLO in palm inflorescence spikelets was also achieved after 35 cycles of PCR. When template DNA for PCR was derived from tissues of the the most immature emerging leaf, a 40-cycle reaction was sufficient for consistent foliar detection of the pathogen in all coconut palms including palms with earliest visible symptoms of disease.     

Evidence for the physical integrity of flavescence dorée phytoplasmas purified by immunoaffinity from infected plants or leafhoppers and the plant pathogenicity of phytoplasmas from leafhoppers

Phytoplasmas were extracted from flavescence dorée-infected broadbean ( Vicia faba ) using a vacuole isolation medium, and were immunoaffinity purified from infected leafhoppers. Purified phytoplasmas from both host sources were immunolabelled and observed under the electron microscope. The infectivity of the purified phytoplasmas from leafhoppers was checked by injecting healthy leafhoppers which were then allowed to feed on healthy V. faba seedlings. The appearance of typical symptoms in these plants, and the positive results obtained in ELISA with extracts of some of the injected leafhoppers and with symptomatic V. faba , indicated that the purified phytoplasmas had retained their infectivity and had multiplied in the injected leafhoppers which had become infective. These results support a previous report that phytoplasmas purified by immunoaffinity chromatography are well preserved.     

Phylogenetic relationships among Flavescence dorée strains and related phytoplasmas determined by heteroduplex mobility assay and sequence of ribosomal and Nonribosomal DNA

Heteroduplex mobility assay (HMA) and DNA sequencing were performed on Flavescence dorée (FD) phytoplasma strains and related phytoplasmas belonging to the elm yellows group. Part of the ribosomal RNA gene operon and a nonribosomal DNA region were utilized for phylogenetic analyses. Two FD strains, FD92 and FD-D, detected in France and Italy, respectively, were identical in both DNA fragments, confirming previous results. Other FD strains were all very similar and most closely resembled ALY, an Italian alder phytoplasma. Phytoplasmas associated with German Palatinate grapevine yellows were shown to form a distinct subcluster, also different from the elm yellows phytoplasma subcluster. Strain disparities revealed by HMA and sequence data were mostly in agreement, highlighting the utility of HMA in differentiation and classification of phytoplasmas belonging to the same ribosomal RNA group.     

刺山柑(Capparis spinosa)总RNA不同提取方法的比较

以野生刺山柑叶片为材料,采用70%丙酮抽提研磨材料,结合TRIZOL,CTAB,SDS 3种常见的RNA提取方法提取总RNA,通过RNA获得率、纯度、电泳图谱以及RT-PCR反应等分析,初步确立了一种有效的提取刺山柑叶片总RNA的改良SDS法.该方法提取的RNA A<,260>/A<,280>值在1.88～1.94之间,电泳图谱完整,具有产量高、时间短、成本低等特点,所得的RNA可以用于RT-PCR和cDNA文库构建以及Northern杂交等后续实验.     

我国几种植物植原体的快速分子鉴别与鉴定的研究

 选用桑萎缩病(Mulberry dwarf,MD)、枣疯病(Jujube witches'-broom,JWB)、酸枣丛枝病(Wild jujube witches'-broom,WJWB)、泡桐丛枝病(Paulownia witches'-broom,PaWB)和苦楝丛枝病(Chinaberry tree witches'-broom,CWB)5种不同植物植原体和来源于3个不同地区PaWB和JWB材料进行16S rDNA和23S rDNA PCR扩增、异源双链迁移率分析(HMA)、PCR产物的RFLP分析和16S rDNA的克隆和测序等比较研究,建立了一种快速确定未知植原体种类和分类地位的分子鉴别与鉴定优化程序;并可对田间采集的各种植物植原体样品进行快速鉴定和鉴别。16S rDNA PCR产物HMA分析结果显示,JWB与CWB、MD和PaWB皆可形成明显的异源杂交双链;而CWB、MD和PaWB植原体之间未能形成异源双链。JWB和PaWB不同地区样品之间、JWB和WJWB之间也未发现异源杂交双链的形成。而23S rDNA PCR产物HMA分析则可以将MD与PaWB区分开。进一步对未知分类地位的CWB序列测定及与其它植原体16S rDNA的RFLP和同源性比较结果显示,CWB与PaWB同源性为99.5%,其中与MD的同源性高达99.7%,因而应将CWB归为翠菊黄花组16Sr I-B,16Sr I-B (rp-B)。     

EP-1与溴敌隆对长爪沙鼠野生种群增长的控制作用

2010年3—10月,在内蒙古鄂尔多斯荒漠草原分别设置不育剂试验区、毒饵试验区和对照区3个样区,采用春季一次性足量投饵、按月铗捕统计种群相对数量的方法,比较了不育剂EP-1和毒饵溴敌隆对长爪沙鼠Meriones unguiculatus野生种群数量增长的控制作用。结果显示,EP-1改变了 长爪沙鼠种群的年龄结构,不育剂区与毒饵区成体组成差异显著(F_{2, 14}=5.89,P<0.05),降低了长爪沙鼠幼体出生率和全年种群密度,全年幼体数量和总体种群数量均呈下降趋势(R₃²=0.8552,R₄²=0.5126)。EP-1对长爪沙鼠种群增长有持续控制作用,而溴敌隆对长爪沙鼠种群的有效杀灭作用只持续3个月,仅是短期控制。     

Disease progression of a lethal decline caused by the 16SrIV‐D phytoplasma in Florida palms

Recently, a new phytoplasma was discovered in Hillsborough County in the state of Florida, USA. This phytoplasma belongs to the 16SrIV taxonomic group and is classified as subgroup D. It is the causal agent of lethal bronzing disease (LBD) of palm. Since the discovery of LBD in 2006, the disease has spread throughout much of the state. In 2014 and 2015, stands of cabbage palm and queen palms that had been present at the University of Florida's Fort Lauderdale Research and Education Center in Davie, FL began showing symptoms of LBD. After confirming the presence of the LBD phytoplasma in initially infected palms by nested PCR and RFLP analysis, all palms were systematically sampled over the period of 1 year to monitor and quantify disease spread. A total of 30 cabbage palms were tested monthly by qPCR, with five testing positive on the first sample date. By the end of the study period, 16 cabbage palms had died from the infection. A total of 16 queen palms were surveyed, with three palms initially testing positive. By the end of the study, four queen palms had tested positive and died from the infection. To the authors’ knowledge, this study is the first to document and quantify spread of palm‐infecting phytoplasmas. This data provides important insights into the ecology of palm‐infecting phytoplasmas and highlights the impact that the movement of infective insects can pose to established stands of palms.