Occurrence and incidence of phytoplasmas of the 16SrII-D subgroup on solanaceous and cucurbit crops in Egypt

Symptoms reminiscent of phytoplasma infection were observed in four provinces (governorates) of Egypt in fields of eggplants, tomato plants and squash. Diseased plants exhibited stunting, leaf yellows and flower development abnormalities. PCR amplification of 16SrDNA with phytoplasma-specific primer pairs confirmed the phytoplasma presence. Sequencing and phylogenetic analysis indicated that all phytoplasmas had the same partial 16SrDNA sequence, assigning them to the 16SrII-D phytoplasma subgroup. Disease incidence was about 1% among the 20 squash fields surveyed and equally varied from 4% to 15% in the 20 eggplant fields and in the 40 tomato fields inspected. The widespread distribution of this phytoplasma in annual solanaceous and cucurbit crops suggests a wider plant host range including wild plants that could act as reservoir and insist on the need for a insect vector survey. A finer genetic differentiation of Egyptian 16SrII-D phytoplasma strains from different geographical origins and different host plants should help to better trace such epidemics.     

Molecular identification,characterization and transmission of phytoplasmas associated with sesame phyllody in Turkey

Phyllody is a destructive disease of sesame in Turkey. The disease has been causing significant economic losses by stunting the plants and altering their floral parts into leafy structures with no capsule and hence no seeds in sesame fields of the country. This research was undertaken to examine symptomatology, etiology, taxonomy and transmission of two recently discovered phyllody phytoplasmas infecting sesame in Turkey. Direct and nested PCR amplifications of 16S rRNA gene with the phytoplasma-specific universal primers P1/P7 and R16F2n/R2, respectively were employed for identification of the phytoplasmas associated with sesame phyllody. Phytoplasma-specific PCR amplicons of 1.8 kb and 1.2 kb were amplified only from symptomatic sesame plants and insect vector samples. Sequencing of the PCR amplicons and computer simulated restriction fragment length polymorphism analysis allowed classification of the phytoplasmas with pigeon pea witches’-broom (16SrIX-C) and peanut witches’-broom (16SrII-D) groups. The sequence homology and phylogenetic analyses further confirmed this classification. Among the insects collected from the sesame fields, the leafhopper Orosius orientalis Matsumara (Syn: O. albicinctus Distant) was the only vector proven to transmit the sesame phyllody phytoplasmas from diseased to healthy sesame plants in transmission assays. The results demonstrated that the 16SrIX-C and 16SrII-D group phytoplasmas were the agent of sesame phyllody and O. orientalis was the vector insect of the disease in Turkey.     

蚕豆和豌豆锈病病原菌的分子鉴定

为明确我国热带和亚热带地区蚕豆Vicia faba和豌豆Pisum sativum锈病的病原菌种类,通过致病性测定和ITS序列系统发育分析对来自我国云南省玉溪市的4份豌豆锈菌分离物及云南、广西、重庆和四川省(区、市)的5份蚕豆锈菌分离物进行系统鉴定。结果显示,分离自豌豆的锈菌WX1分离物对蚕豆和豌豆均具有高致病性,在侵染叶片上产生大量锈子器;分离自蚕豆的锈菌CX3分离物仅对蚕豆具有高致病性,能在叶片上产生大量夏孢子,而对豌豆的致病性相对较低,仅产生少量的夏孢子堆;分离物WX1和CX3对小扁豆和鹰嘴豆不具有致病性。基于ITS序列系统发育分析表明,所有不同寄主来源的蚕豆单胞锈菌分离物均聚类于一个系统发育组,但分离自蚕豆和豌豆的分离物分别聚类在不同的亚组。表明分离自云南省玉溪市豌豆上的蚕豆单胞锈菌Uromyces viciae-fabae应为豌豆专化型,定名为U. viciae-fabae ex P. sativaum,而来源于云南、广西、重庆和四川省(区、市)的蚕豆锈病病原菌为蚕豆专化型U. viciae-fabae ex V. faba。     

A mild strain of broad bean mottle virus from faba bean (Vicia faba L.) in the Sudan

A new strain of broad bean mottle virus, isolated from faba bean (Vicia faba L.) in the Sudan, is described. It differs considerably from known isolates by its nearly symptomless infection of faba bean in spite of high concentrations of the virus in infected plants. It does not differ from regular isolates in gel-diffusion serology, light and electron microscopy, host range and symptoms in major hosts other than faba bean. It may constitute a potential threat to other food legumes in the region.     

New subgroup 16SrIII-Y phytoplasmas associated with false-blossom diseased cranberry (Vaccinium macrocarpon) plants and with known and potential insect vectors in New Jersey

The identity of the presumed phytoplasmal pathogen associated with cranberry false-blossom disease has never been fully clarified. In the present study a molecular-based procedure was employed to determine the identity of the phytoplasma. Tissues of cranberry plants exhibiting cranberry false-blossom symptoms were collected from multiple bogs on each of three randomly selected commercial cranberry farms in New Jersey. Leafhoppers, including the known vector Limotettix vaccinii (Van Duzee) (=Scleroracus vaccinii, Euscellis striatulus) and the sharp-nosed leafhopper Scaphytopius magdalensis (Provancher), a known vector of blueberry stunt disease, were collected from two different farms in New Jersey. Nested PCR assays and RFLP analysis of 16S rRNA gene sequences were employed for the detection and identification of the associated phytoplasmas. All of 20 cranberry plants sampled and five out of 14 batches of leafhoppers tested positive for phytoplasma. Virtual RFLP and sequence analyses revealed that all the associated phytoplasmas were members or variants of a new subgroup, 16SrIII-Y. Phylogenetic analysis of 16S rRNA sequences indicated that cranberry false-blossom phytoplasma strains represented a lineage distinct from other 16SrIII subgroups. This is the first report confirming that a new phytoplasma (designated as a new subgroup 16SrIII-Y) is associated with cranberry false-blossom disease and associated with both leafhopper species in New Jersey.     

Molecular characterization of the phytoplasmas associated with toon trees and periwinkle in India

In November 2008 in Himachal Pradesh and Chandigarh regions in India, toon trees and periwinkles were observed to have formed short internodes, small leaves and witches’-broom symptoms, typical of phytoplasma infection. The symptomatic toon and periwinkle samples were tested with universal PCR tests, and the 16S rRNA, rplB-rpsC, secA and secY genes were sequenced. The causal agents belonged to subgroup 16SrI-B of ‘Candidatus Phytoplasma asteris’, based on 16S rDNA, ribosomal protein gene, secA and secY phylogenetic analysis.     

Association of aster yellows subgroup 16SrI-B phytoplasmas with a disease of Rehmannia glutinosa var. purpurea

A new phytoplasma disease of Rehmannia glutinosa var. purpurea was observed in the Czech Republic in 1998. Infected plants showing severely proliferating shoots, leaves reduced in size with vein clearing and chlorosis, shortened internodes and virescent petals died in advanced stages of the disease. Electron microscopy examination of the ultra-thin sections revealed the presence of numerous polymorphic bodies in phloem tissue of leaf midribs and petioles. The disease was successfully transmitted from infected plant via a dodder bridge into periwinkle ( Catharanthus roseus ). The phytoplasma aetiology of this disease was further confirmed by polymerase chain reaction (PCR) using universal primers R16F2/R16R2. Restriction fragment length polymorphism (RFLP) analysis of amplification products indicated the presence of aster yellows related phytoplasmas (16SrI-B) in naturally infected samples of R. glutinosa var . purpurea and in symptomatic periwinkle after dodder transmission of the agent. A comparison of the amplified sequence with 17 sequences available in the GenBank confirmed the classification of the phytoplasma in the subgroup 16SrI-B. This is the first report of natural occurrence of phytoplasma-associated disease in R. glutinosa var. purpurea.     

Broad bean mottle virus in Morocco; variability,interaction with food legume species,and seed transmission in faba bean,pea, and chickpea

Biological indexing of faba-bean samples collected during an earlier virus survey in Morocco revealed variation in symptom severity among isolates of broad bean mottle virus (BBMV). When seven selected isolates from Morocco and three from Algeria, Sudan, and Tunisia were further compared, they could be divided into mild, severe, and intermediate isolates, according to their pathogenicity on a number of food-legume genotypes tested. The Moroccan isolate SN1 and the Sudanese SuV256 were very mild, and deviant also in their effect onGomphrena globosa, whereas the Tunisian TV75-85 and the Moroccan VN5 were virulent. Representative isolates were indistinguishable, however, in coat-protein molecular weights, and they reacted similarly to the antisera to a Moroccan and a Syrian isolate in electro-blot immunoassay.Promising ICARDA breeding lines and accessions—ten each of pea and lentil, nine of chickpea, and twelve of faba bean-were all found vulnerable (susceptible and sensitive) to all isolates. Within each food-legume species, vulnerability varied from high to moderate, and no immunity was detected. Virus concentrations in faba-bean lines suggest that isolates differ in virulence rather than in aggressiveness, and that the differences in vulnerability among the lines are due to differences in sensitivity rather than in susceptibility.When pooled seed samples were germinated and seedlings were tested for BBMV in DASELISA, the virus was found seed-transmitted in faba bean, chickpea, and pea at transmission rates of ca 1.2, 0.9, and 0.1%, respectively. This is the first report on seed transmission of BBMV in faba bean, when occurring on its own, and the first record of such seed transmission in chickpea and pea.     

Electron microscopy and molecular identification of phytoplasmas associated with strawberry green petals in the Czech Republic

The presence of phytoplasma inFragaria ananassa x Duch cv Senga Sengana showing strawberry green petals symptoms was observed by electron microscopy of phloem tissue. No phytoplasmas were found in asymptomatic strawberry plants used as controls. Nucleic acids extracted from these plants were used in nested-PCR assays with primers amplifying 16S rRNA sequences specifie for phytoplasmas. Bands of 1.2 kb were obtained and the subsequent nested-PCR with specific primers and RFLP analyses allowed to classify the detected phytoplasmas in the aster yellows group (16SrI). They belonged to the subgroup I-C of which type strain is clover phyllody phytoplasma.     

5种仓储豆象的分子鉴定和检测

本研究以在检疫中经常被截获的5种豆象为研究对象,通过对GenBank中5种豆象mtDNA COⅠ序列的查询,并进行序列比对,共设计了30对引物,通过筛选获得5对能分别鉴定5种豆象的特异引物,并用50、25、10、5、1、0.5、0.1、0.05ng/μL 8个不同浓度系列的豆象DNA模板来检测灵敏度,结果表明,豌豆象和四纹豆象特异引物对的灵敏度为0.1ng/μL,蚕豆象、菜豆象和绿豆象特异引物对的灵敏度为0.5ng/μL。该方法可用于快速准确地鉴定5种豆象,对于豆象类害虫的检测监测具有重要意义。     

Diverse phytoplasmas associated with leguminous crops in Russia

A large scale survey of diseased legume plants (mainly clover and alfalfa in the Fabaceae family) was conducted from 2009 to 2013 in four Economic Regions of Russia, Northern (Arkhangelsk and Vologda oblast), Central (Moscow oblast), Volga (Samara oblast) and West Siberian (Novosibirsk oblast). The majority of infected clover plants exhibited symptoms typical of clover phyllody (CPh), clover yellow edge (CYE), or clover proliferation (CP), and infected alfalfa plants exhibited symptoms typical of alfalfa witches’-broom (AWB). Of 161 symptomatic plants from 22 different legume species, 103 tested positive for phytoplasma infection. Phytoplasmas belonging to four groups and six subgroups were detected, of which 31.1% were group 16SrI, with the majority belonging to subgroup 16SrI-C- (causal agent of CPh disease), two belonging to 16SrI-B and two group 16SrI phytoplasmas not identified to the subgroup level;47.6% were group 16SrIII, with the majority belonging to subgroup 16SrIII-B or 16SrIII-B variant (causal agent of CYE disease), and one strain belonging to16SrIII-F; 8.7% were subgroup 16SrVI-A (causal agent of CP and AWB diseases); 9.7% were subgroup 16SrXII-A (causal agent of AWB disease); and 2.9% were mixed infected with subgroups 16SrIII-B and 16SrI-C. The predominant phytoplasma species detected varied by region. In the Northern and Central Regions, the majority of the phytoplasmas detected belonged to subgroups 16SrI-C and 16SrIII-B. In the West Siberian and the Volga Regions, the phytoplasmas predominately detected belonged to subgroups 16SrVI-A and 16SrXII-A, respectively. Subgroup 16SrIII-F was detected in a single plant in the West Siberian Region and a mixed infection of 16SrIII-B and 16SrI-C was detected in three plants, one in the Northern Region and two in the Central Region. Eleven species of insects of the order Hemiptera, suborder Auchenorrhyncha, were collected from leguminous plants in the Moscow oblast of the Central Region. Euscelis incisus and Aphrodes bicinctus were the most prevalent species and may be potential phytoplasma vectors in the Central Region.     

Broomrape control in faba bean (Vicia faba L.) with glyphosate and imazaquin

Field studies were carried out to evaluate the efficacy of imazaquin and glyphosate in controlling broomrape (Orobanche crenata Forsk. and O. aegyptiaca Pers.) in faba bean (Vicia faba L.) in Northern Syria. Several rates and times of application of the herbicides were tested. Nearly complete control of broomrape was achieved with both the herbicides when they were sprayed twice (when the broomrape attachments to the faba bean roots were at tubercle stage of develoment and 15 days later) at a rate of application of 80 g a.i. ha^?1 for glyphosate and 10 g a.i. ha^?1 for imazaquin. However, the yield of faba bean did not increase with these treatments, suggesting that the herbicides at these rates were still causing some physiological disorder in the plant that counteracted the beneficial effect of broomrape control on the performance of faba bean plants.     

蚕豆马铃薯间作种植对蚕豆赤斑病的防控效果

为系统探索蚕豆赤斑病防控策略,保护农田生态环境,2010-2011连续2年在重庆市3个区县进行蚕豆与马铃薯间作试验。结果表明,蚕豆与马铃薯多样性种植能有效控制蚕豆赤斑病的发生,改善蚕豆产量构成因素,从而提高产量和经济效益。蚕豆与马铃薯间作行比为2∶2时对蚕豆赤斑病的防控效果最好,发病率与病情指数分别为46.5%和26.3,在此模式下经济效益最大,纯收入达18 454.9元/hm2。     

Molecular characterization of diverse phytoplasmas of subgroups 16SrI-A, 16SrI-B, 16SrI-L, and 16SrI-M infecting ornamental plants in Lithuania

Trade in ornamental plant species comprises a significant segment in the economies of countries in Europe, North America and Asia. Since the quality of ornamental plants is adversely affected by diseases attributed to phytoplasmas, we surveyed plant collections in botanical gardens and floriculture farms in Lithuania for phytoplasmal diseases. Seventeen ornamental species belonging to nine plant families exhibited disease symptoms including general yellowing and stunting, proliferation of shoots, phyllody, virescence and reduced size of flowers, and reddening of leaves. Analysis of the phytoplasmal 16S rRNA gene sequences amplified by PCR revealed that the plants were infected by phytoplasmas belonging to four distinct subgroups (16SrI-A, 16SrI-B, 16SrI-L, and 16SrI-M) of group 16SrI (aster yellows phytoplasma group) and indicated the presence of sequence-heterogeneous 16S rRNA genes in newly recognized strains belonging to subgroups 16Sr-L and 16SrI-M. Infections by these diverse phytoplasmas in a wide array of plant species and families suggests that unidentified, polyphagous insect vectors may actively transmit phytoplasmas threatening the Baltic region's ornamental plant industry.     

青海省不同生态区蚕豆根瘤菌16S rDNA分析

为了阐明青海省蚕豆根瘤菌的遗传多样性和亲缘关系,同时为发现和获得新的根瘤菌物种提供资源,采集和分离了青海省不同生态区蚕豆主栽品种青蚕14中的根瘤菌,并对其中分离到的6个菌株(CD1~6)进行16S rDNA鉴定分析。将6株蚕豆根瘤菌的全序列结果与NCBI中已报道的序列进行相似性比对,发现其相似度较高,其中供试菌株CD4与KF008225.1相似度最高,达到98%,其余均在95%到96%之间。6株供试菌分属4个菌属,CD1和CD2共属节细菌属,CD3属于分枝杆菌属,CD4和CD5属于快生根瘤菌属,CD6属于中慢生根瘤菌属,表明来自不同地区的蚕豆根瘤菌存在较大差异。聚类分析结果显示,蚕豆根瘤菌的6个菌株分属5个不同系统发育分支,证明青海省不同生态区蚕豆根瘤菌种类多样性较为丰富。     

宁夏马铃薯僵顶植原体的分子鉴定

通过透射电子显微镜,在从宁夏回族自治区固原市彭阳县红河镇采集的表现叶片上卷、红叶、气生薯症状的马铃薯样品叶脉韧皮部筛管细胞内观察到大量直径为500~700 nm的球形植原体粒子。以提取的感病和健康马铃薯叶片总DNA为模板,应用植原体16S rRNA基因和rp基因通用引物进行PCR扩增,从感病样品中扩增得到了长度均约为1.2 kb的片段。对获得基因核酸一致性比较分析表明,马铃薯僵顶植原体宁夏株系16S rRNA基因与‘Candidatus Phytoplasma fragariae’槭树株系(MK501642)16S rRNA基因核酸一致性最高,为99.7%,rp基因与‘Ca.P.fragariae’云南马铃薯YN-2G株系(KJ144889)rp基因核酸一致性最高,为100%;基于16S rRNA基因和rp基因构建系统进化树发现,马铃薯僵顶植原体宁夏株系与16SrⅫ-E亚组成员聚在一起。基于透射电镜观察和基因序列比较分析,证明宁夏发生的马铃薯僵顶病与植原体侵染相关,该植原体在分类地位上属于植原体16SrⅫ-E亚组。     

Wheat/faba bean intercropping improves physiological and structural resistance of faba bean to fusaric acid stress

Fusarium oxysporum f. sp. fabae is the causal agent of fusarium wilt. Fusaric acid (FA), produced by F. oxysporum, plays an important role in the occurrence of disease, and intercropping is an effective measure for control of disease and for improving host resistance in plants. The objective of this study was to investigate the physiological and biochemical responses, and mechanisms of tissue structure resistance, of intercropped faba beans following exposure to different concentrations of FA. Results demonstrated that intercropping reduced the occurrence of fusarium wilt, and improved faba bean growth and yield. In addition, wheat intercropping significantly reduced red ink absorption of faba bean (33.2%), increased water content (3.1%), and increased activity of the root antioxidant enzymes peroxidase (POD) and catalase (CAT) (26.3% and 2.2.%, respectively). Furthermore, increased lignin content and callose deposition in plant vessels were observed (12.5% and 42.7%, respectively) when subjected to the highest concentration of FA stress (200 mg/L). Intercropping resulted in more intact root cell morphology, increased occurrence of intracellular vacuoles, increased cell wall thickness, and an increase in the number of mitochondria and rough endoplasmic reticulum. Intercropping alleviated the wilting effect of FA on faba bean via enhanced physiological, biochemical, and tissue structure resistance of faba bean root.     

Latent infection of asymptomatic Hawaiian sugarcane cultivars with 16SrI and 16SrXI phytoplasmas

Leaves from sugarcane were collected at the Hawaiian sugarcane breeding station and from recent and previous Hawaiian plantation fields and tested for phytoplasma by nested PCR, quantitative PCR and partly by the 16S/23S internal spacer sequence. Phytoplasmas were found in samples of 10 of the 11 tested cultivars from the station and identified as strains 16SrI phytoplasma (aster yellows phytoplasma) and 16SrXI phytoplasma (rice yellow dwarf phytoplasma). Hot water treatment could partially eliminate the phytoplasmas, but sugarcane plants in the Hawaiian plantations, which routinely use hot-water-treated seed cane cuttings, were nevertheless infected by 16SrXI phytoplasma. Samples from abandoned sugarcane plantations contained 16SrI phytoplasma or 16SrXI phytoplasma. The titre of phytoplasma was very low in all cases, i.e., at or below the detection threshold of quantitative PCR, and no difference in phytoplasma infection was observed between healthy-looking, green plants and plants that had YLS symptoms. Apparently the Hawaiian sugarcane cultivars have some kind of phytoplasma resistance under the growth conditions in Hawaii. The latent presence of phytoplasma strains calls for awareness and rigorous treatment of sugarcane setts even in cases, where YLS was so far exclusively related to the presence of Sugarcane yellow leaf virus.     

Detection and identification of phytoplasmas in yellows-diseased weeds in Italy

Yellows-diseased plants of Crepis setosa (hawksbeard), Knautia arvensis (field scabious), Convolvulus arvensis (field bindweed), Picris echioides (bristly oxtongue), Echium vulgare (blueweed) and Calendula officinalis (pot marigold) collected in central and southern Italy were examined for phytoplasma infection by means of polymerase chain reaction (PCR) technology using universal phytoplasma primers directed to ribosomal sequences. The detected phytoplasmas were characterized and differentiated using restriction fragment length polymorphism analysis of PCR-amplified DNA. The phytoplasma detected in diseased pot marigold plants was identified as a member of the aster yellows group and proved indistinguishable from a strain of the American aster yellows phytoplasma. The phytoplasma identified in diseased field bindweed plants is a putative new type of the stolbur group that differed from the typical stolbur phytoplasma. Phytoplasmas detected in diseased hawksbeard, blueweed and field scabious plants are all putative new members of the sugarcane white leaf group while the phytoplasma detected in diseased bristly oxtongue plants represents a new member of the faba bean phyllody group. For hawksbeard and field scabious this is the first report on the occurrence of phytoplasma diseases, whereas phytoplasmas infecting bristly oxtongue and blueweed have never been characterized before.