Use of Polyclonal Antibodies to Identify Mycoplasma-like organisms (MLOs) From the Sudan and from Thailand

Rabbit polyclonal antibodies prepared against faba bean phyllody MLO from the Sudan reacted with its homologous antigen and with extracts of Catharanthus roseus experimentally infected with the same or a related MLO from Crotalaria saltiana showing symptoms of phyllody disease, as well as with extracts of naturally MLO-infected C. saltiana growing in the field in the Sudan. The antibodies also reacted positively with extracts of C. roseus experimentally infected with Crotalaria juncea phyllody MLO and soybean phyllody MLO from Thailand. Polyclonal antibodies prepared against an MLO associated with witches' broom disease in C. juncea reacted positively in ELISA tests with homologous antigen extracts from naturally infected C. juncea as well as with extracts from experimentally infected C. roseus and with extracts prepared from Sesamum indicum plants with phyllody symptoms growing in Thailand. There was no reaction between these antibodies and extracts from C. roseus plants infected with the MLOs associated with C. juncea phyllody or with soybean phyllody. No cross reactions were observed among the antigens and antibodies of the two MLO groups by immunoflorescence, ELISA or western blotting. However, the molecular weight of the principal protein antigen, determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting was the same for both types of MLO. Serologically-similar MLOs thus occur in the Sudan and in Thailand, where they are associated with phyllody symptoms in C. saltiana and faba bean and with C. juncea and soybean, respectively. A second, serologically distinct MLO group was also found infecting C. juncea and S. indicum in Thailand but MLOs from this group have not yet been identified in crops from the Sudan.     

Use of Polyclonal Antibodies to Identify Mycoplasma-like organisms (MLOs) From the Sudan and from Thailand

Rabbit polyclonal antibodies prepared against faba bean phyllody MLO from the Sudan reacted with its homologous antigen and with extracts of Catharanthus roseus experimentally infected with the same or a related MLO from Crotalaria saltiana showing symptoms of phyllody disease, as well as with extracts of naturally MLO-infected C. saltiana growing in the field in the Sudan. The antibodies also reacted positively with extracts of C. roseus experimentally infected with Crotalaria juncea phyllody MLO and soybean phyllody MLO from Thailand. Polyclonal antibodies prepared against an MLO associated with witches' broom disease in C. juncea reacted positively in ELISA tests with homologous antigen extracts from naturally infected C. juncea as well as with extracts from experimentally infected C. roseus and with extracts prepared from Sesamum indicum plants with phyllody symptoms growing in Thailand. There was no reaction between these antibodies and extracts from C. roseus plants infected with the MLOs associated with C. juncea phyllody or with soybean phyllody. No cross reactions were observed among the antigens and antibodies of the two MLO groups by immunoflorescence, ELISA or western blotting. However, the molecular weight of the principal protein antigen, determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting was the same for both types of MLO. Serologically-similar MLOs thus occur in the Sudan and in Thailand, where they are associated with phyllody symptoms in C. saltiana and faba bean and with C. juncea and soybean, respectively. A second, serologically distinct MLO group was also found infecting C. juncea and S. indicum in Thailand but MLOs from this group have not yet been identified in crops from the Sudan.     

Occurrence,Molecular Characterization and Vector Transmission of a Phytoplasma Associated with Rapeseed Phyllody in Iran

Symptoms of rapeseed phyllody were observed in rapeseed fields of Fars, Ghazvin, Isfahan, Kerman and Yazd provinces in Iran. Circulifer haematoceps leafhoppers testing positive for phytoplasma in polymerase chain reaction (PCR) successfully transmitted a rapeseed phyllody phytoplasma isolate from Zarghan (Fars province) to healthy rapeseed plants directly after collection in the field or after acquisition feeding on infected rapeseed in the greenhouse. The disease agent was transmitted by the same leafhopper from rape to periwinkle, sesame, stock, mustard, radish and rocket plants causing phytoplasma‐type symptoms in these plants. PCR assays using phytoplasma‐specific primer pair P1/P7 or nested PCR using primers P1/P7 followed by R16F2n/R2, amplified products of expected size (1.8 and 1.2 kbp, respectively) from symptomatic rapeseed plants and C. haematoceps specimens. Restriction fragment length polymorphism analysis of amplification products of nested PCR and putative restriction site analysis of 16S rRNA gene indicated the presence of aster yellows‐related phytoplasmas (16SrI‐B) in naturally and experimentally infected rapeseed plants and in samples of C. haematoceps collected in affected rapeseed fields. Sequence homology and phylogenetic analysis of 16S rRNA gene confirmed that the associated phytoplasma detected in Zarghan rapeseed plant is closer to the members of the subgroup 16SrI‐B than to other members of the AY group. This is the first report of natural occurrence and characterization of rapeseed phyllody phytoplasma, including its vector identification, in Iran.     

Mycoplasma-like Organisms in Phloem Elements of Ammobium alatum

Using transmission electron and fluorescent microscopes, Mycoplasma-like organisms (MLOs) were found in phloem cells of stems and leaves of Ammobium alatum. The diameter of these organisms ranged from 0.12–0.73 μm and averaged 0.4 μm. Symptoms induced by MLOs included chlorosis and reddening of leaves and winged stems, plant stunting and flower phyllody. This is the first report of MLOs associated with disease symptoms in A. alatum.     

Phyllody Disease of Crotalaria saltiana in the Sudan: Transmission to Catharanthus roseus and Detection of MLOs by Fluorescence and Electron Microscopy

Phyllody disease of Crotalaria saltiana Andr. first noted in the Sudan in 1962, was recently observed in many localities in the Gezira province in the central region of the country. Diseased plants generally exhibited stunting and excessive proliteration of lateral shoots (witches' broom growth) with small and chlorotic leaves. Morphological transformations of flowers were the most striking symptoms. Floral segments showed various stages of virescence and phyllody as a part of a complete transformation of floral buds into leafy branches. The Crotalaria phyllody agent was transmitted by grafting to faba bean (Vicia faba L.) and with dodder from the latter to periwinkle (Catharanthus roseus). The symptoms reproduced in C. roseus resembled those induced in it by the faba bean phyllody MLO (mycoplasma-like organism), suggesting a close relationship between the two agents. Fluorescence and electron microscopy were used to detect and characterize MLO in diseased plants. Fluorescence reactions in sieve tube elements were observed in sections stained with the DNA-binding fluorochrome Bisbenzimid H 33258. Electron microscope observations in corresponding zones permitted the visualization of wall-less pleiomorphic MLOs confined to sieve tube elements of the phloem tissues of diseased plants.     

Genetic diversity and vector transmission of phytoplasmas associated with sesame phyllody in Iran

During 2010–14 surveys in the major sesame growing areas of Fars, Yazd and Isfahan provinces (Iran), genetic diversity and vector transmission of phytoplasmas associated with sesame phyllody were studied. Virtual RFLP, phylogenetic, and DNA homology analyses of partial 16S ribosomal sequences of phytoplasma strains associated with symptomatic plants revealed the presence of phytoplasmas referable to three ribosomal subgroups, 16SrII-D, 16SrVI-A, and 16SrIX-C. The same analyses using 16S rDNA sequences from sesame phyllody-associated phytoplasmas retrieved from GenBank database showed the presence of phytoplasmas clustering with strains in the same subgroups in other Iranian provinces including Bushehr and Khorasan Razavi. Circulifer haematoceps and Orosius albicinctus, known vectors of the disease in Iran, were tested for transmission of the strains identified in this study. C. haematoceps transmitted 16SrII-D, 16SrVI-A, and 16SrIX-C phytoplasmas, while O. albicinctus only transmitted 16SrII-D strains. Based on the results of the present study and considering the reported presence of phytoplasmas belonging to the same ribosomal subgroups in other crops, sesame fields probably play an important role in the epidemiology of other diseases associated with these phytoplasmas in Iran.     

The Genetic Relationship Between Mycoplasma-like Organisms Causing Diseases in the Sudan and Different Continents Revealed by Polymerase Chain Reaction (PCR) Amplification of the 16S rRNA Gene Followed by Restriction Fragment Length Polymorphism Analysis

The genetic relationship between faba bean (Vicia faba L.) phyllody and other mycoplasma-like organism (MLO) diseases has been studied by amplification of the conserved region of the 16S rRNA gene followed by restriction fragment length polymorphism (RFLP) analysis using Alu I restriction endonuclease. The restriction patterns produced by faba bean phyllody MLO were smilar to that of Crotalaria saltiana phyllody MLO which persists throughout the year in the Sudan. These, and serological results clearly confirmed that C. saltiana is a reservoir of faba bean phyllody MLO in the Sudan. Moreover, restriction patterns have also shown that MLOs of other diseases have the same RFLP fragment pattern as faba bean phyllody MLO, including C. juncea witches'broom (Thailand) and tomato big-bud (Australia), which differs from the other selected MLO diseases (Gladiolus aster yellow, clover phyllody and yellow decline of lavender, aqll from France). Fragment patterns also revealed the existence of genetically diverse MLO strains in the Sudan. Faba bean phyllody may be placed in group III including WX, apricot chlorotic leaf roll, golden flaveswcence dorée of grapevine, plum leptonecrosis of Prunus salciana, peachy yellow leaf roll, sunnhemp phyllody from Thailand, and blueberry witches' broom.     

Mycoplasma-like bodies inSolanum laciniatum plants infected with potato witches’ broom disease

Mycoplasma-like organisms (MLO) spread from the infectious grafts intoSolanum laciniatum Ait. stock plants relatively slowly. MLO were present in all sprouts ofS. laciniatum four weeks after grafting, but the infected plants remained under glasshouse conditions mostly symptomless and flowered normally and formed fruits like healthy plants. The growth of plants with infectious tomato grafts was identical with the controls but that of plants with infectious tobacco (Nicotiana glauca Grah.) grafts was expressively stimulated. The first flower symptoms appeared onS. laciniatum plants with tomato grafts after five and half months and on.S. laciniatum plants with tobacco grafts after seven months of graft symbiosis. Electron micrographs of ultrathin sections showed the presence of MLO in sieve tubes of potiols and midribs of the infected but symptomless plants. In the phloem parenchyma cells of the witches’ broom diseased plants, highly ordered crystals were occasionally found lying in a microbody surrounded by a membrane. The possible reasons of the disease latency are discussed.     

Studies of Polyclonal Antibodies for the Detection of MLOs Associated with Faba Bean (Vicia faba L.) Using Different ELISA Methods and Dot-blot

Polyclonal antibodies were raised in rabbits against MLO associated with faba bean (Vicia. faba L.) phyllody which exists in the Sudan. Two indirect ELISA methods were able to detect the MLO antigens. In the former, the whole antigen was directly coated onto plates, while in the second, only the F(ab')₂, fragments of the IgG were used to coat the ELISA plates. Higher detectable efficiency was obtained when the F(ab')₂ method was used. Moreover the obtainable antiserum was found to exhibit a high degree of specificity through which the MLO associated with faba bean phyllody in the Sudan, are serologically differentiated from other isolates of MLO existing in the Sudan as well as European MLO isolates maintained at Versailles, and Spiroplasma citri, causal agent of Citrus Stubborn Disease. The positive reactions obtained with this antiserum against MLO phyllody naturally existing in the Sudan on Crotalaria saltiana and some Catharanthus roseus demonstrate that these plants are potential reservoirs of the disease in the Sudan. The same antiserum was used in order to distinguish healthy and diseased plant preparations using the membrane ELISA method (dot-blot).     

Leafhopper transmission of Rhynchosia little leaf, a disease associated with mycoplasma-like organisms in Jamaica

Little leaf disease of Rhynchosia minima (RLL) in Jamaica is reported for the first time. The presence of phloem-restricted MLO in diseased but not healthy plants, the remission of symptoms induced in RLL-affected plants with soil drenches of tetracycline, but not penicillin, and the transmission of disease-associated MLO to R. minima test plants, suggests that RLL has an MLO aetiology. RLL is vectored by the cicadellid leafhopper Ollarianus balli, for which R. minima represents the specific field host. Healthy colonies of O. balli produced from eggs oviposited on the RLL-immune weed Asystasia gangetica suggest that RLL is not transovarially transmitted. O. balli acquired the RLL agent after access to infected plants for 5 days (shorter feeds were not tried), and there was a maximum latent period in the leafhopper of 21 days. Of the O. balli collected from heavily-infected field stands of RLL, 35% transmitted the disease, while, of those reared on RLL in captivity for 14–16 days, 56% transmitted. Male and female O. balli transmitted equally efficiently, while nymphs were less frequent vectors. O. balli also infected Cajanus cajan, an important small scale subsistence crop in Jamaica, and Catharanthus roseus. It did not, however, transmit coconut lethal yellowing (CLY) disease to test palms after natural or deliberate acquisition-feeding on RLL, acquisition-feeding on CLY-affected palms, or, after injection with CLY-affected phloem exudate. There was thus no evidence that RLL is related to CLY or that O. balli can act as a vector of CLY.     

Molecular Cloning, Detection of Chromosomal DNA of the Mycoplasmalike Organism (MLO) Associated with Faba Bean (Vicia faba L.) Phyllody by Southern Blot Hybridization and the Polymerase Chain Reaction (PCR)

DNA from faba bean (Vicia faba L.) phyllody-diseased periwinle plants was separated from host plant DNA by bisbenzimid-CsCl buoyant-density gradient centrifugation. The mycoplasmalike organism (MLO) DNA was used for the construction of DNA probes. Two probes, 1.45 and 1.35 kbp, were selected and used for the detection of MLO DNA associated with faba pean (FBP) and for assessing the genetic relatedness of FBP-MLO with other mollicutes. The 1.45 kbp DNA probe hybridized with all MLO strains and, with Spiroplasma citri. The 1.35 kbp DNA probe specifically detected the MLO associated with FBP. Moreover, a specific primer pair (E1 and E2) selected from the partially sequenced 1.35 kbp probe allowed amplification of the 1.35 kbp fragment. DNA amplification was obtained also with Crotaltiana saltiana phyllody (Sudan), C. juncea, witches' broom (Thailand), and tomato big-bud (Australia), but no amplification was obtained in the cases of the healthy control, C. roseus phyllody (isolate n⁰) from Sudan, clover phyllody, Gladiolus aster yellow and yellow decline of lavender from France. The very strong signal observed in the case of FBP and C. saltiana phyllody agrees with previous results indicating that FBP and C. saltiana phyllody are caused by an identical MLO, and hence, C. saltiana acts as a reservoir of FBP-MLO in the Sudan. The weak signal obtained in the case of C. juncea witches' broom and tomáto big-bud indicates partial nucleotide homology. The major interest of this primer pair is the low quantity (as little as 100 pg) of the total DNA of diseased plant required for the detection of the FBP disease and the possibility of detecting genetic relatedness with other MLOs.     

Identification,occurrence, incidence and transmission of phytoplasma associated with Petunia violacea witches’ broom in Iran

A petunia witches’ broom (PvWB) disease, characterized by phyllody, virescence, witches’ broom, little leaf and yellowing, was observed in municipal lands and parks in Bandar Abbas, Hormozgan province, Iran. The disease was present with an average incidence of 20%. PCR and sequencing analysis carried out on selected samples from symptomatic plants showed the presence of a phytoplasma associated with the disease. The molecular comparison of the 16S ribosomal gene indicated 99% sequence identity with the one of “Candidatus Phytoplasma australasia”. This phytoplasma was transmitted to healthy petunia plants under experimental conditions by the leafhopper Orosius albicinctus that was then demonstrated to be a vector of this phytoplasma.     

Niger Seed (Guizotia abyssinica), a New Host of ‘Candidatus Phytoplasma asteris’ in Iran

Shrubs of niger seed with phyllody and internode elongation symptoms suggestive of phytoplasma infections occurred in the central regions of Iran. Phytoplasma was detected by polymerase chain reaction (PCR) and nested PCR amplifications using phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2. Using aster yellows group–specific primer pair rp(I)F1A/rp(I)R1A, a fragment of 1212 bp of the rp genes was amplified from DNA samples of infected plants. Random fragment length polymorphism (RFLP) analyses of R16F2n/R16R2‐amplified products using the CfoI restriction enzyme confirmed that Iranian niger seed phyllody phytoplasma is associated with aster yellows group phytoplasmas. Sequence analyses of the partial rp genes fragment indicated that the Iranian niger seed phyllody phytoplasma, which was collected from central regions of Iran, is related to ‘Candidatus Phytoplasma asteris’. This is the first report of a phytoplasma infecting the niger seed plant.     

Dodder Transmission of Pear Decline, European Stone Fruit Yellows, Rubus Stunt, Picris echioides Yellows and Cotton Phyllody Phytoplasmas to Periwinkle

The pear decline, European stone fruit yellows and rubus stunt agents as well as the phytoplasmas causing Picris echioides (bristly oxtongue) yellows and cotton (Gossypium hirsutum) phyllody, respectively, were transmitted from naturally infected plants to the experimental host Catharanthus roseus (periwinkle) via dodder (Cuscuta spp.) bridges. The identities of the dodder-transmitted phytoplasmas were confirmed by restriction length fragment polymorphism analysis of polymerase chain reaction-amplified ribosomal DNA. On the basis of restriction profiles the cotton phyllody agent could be differentiated from the phytoplasma causing faba bean phyllody, a disease previously thought to be induced by the same organism as cotton phyllody.     

MLO-Infected Weeds in the Vineyards of North-western Italy

Ten species of herbaceous plants and shrubs with MLO symptoms such as stunting, bushing, yellowing, reddening, virescence or phyllody were collected in both grapevine yellows (GY)-infected and uninfected vineyards. By electron microscopy, MLOs were found in the sieve tubes of eight species. The presence of MLOs in Picris echioides is demonstrated for the first time. The possible role of MLO-infected weeds in the spread of GY disease is discussed.     

Sensitive detection of mycoplasmalike organisms in field-collected and in vitro propagated plants of Brassica, Hydrangea and Chrysanthemum by polymerase chain reaction

A polymerase chain reaction (PCR) protocol, previously designed for amplification of a DNA fragment from aster yellows mycoplasmalike organism (MLO), was employed to investigate the detection of MLO DNA in field-collected and in vitro micropropagated plants. PCR with template DNA extracted from symptomatic, naturally-infected samples of Brassica, Chrysanthemum and Hydrangea, each yielded a DNA band corresponding to 1.0 Kbp. However, no DNA product was observed when either infected Ranunculus (with phyllody disease) or Gladiolus with (symptoms of ‘germs fins’) was used as source of template nucleic acid for PCR; further experiments indicated absence of target DNA in the case of Ranunculus and the presence of substances in Gladiolus which inhibited the PCR. The MLO-specific DNA was detected by PCR using less than 95 pg of total nucleic acid (equivalent to total nucleic acid from 1.9, ug tissue) in the case of field-collected Hydrangea and less than 11.4 pg of nucleic acid (equivalent to total nucleic acid from 19 ng of tissue) in the case of field-collected Brassica. The findings illustrate highly sensitive detection of MLOs in both field-grown and in vitro micropropagated infected plants.     

Identification of phytoplasmas associated with sesame phyllody disease in southeastern Iran

Phyllody disease is a threat to sesame production in Kerman province, southeastern Iran. RFLP analysis of PCR products of phytoplasma-specific 16S rRNA gene (1.8 kb) and phylogenetic analyses of 16S-23S rDNA spacer region (SR) sequence indicated that the predominant agent associated with sesame phyllody in Kerman province is a phytoplasma with 100% similarity with eggplant big bud, and peanut witches’-broom phytoplasmas, members of “Candidatus Phytoplasma aurantifolia” from Iran and China, respectively. Among the samples tested, only one strain (SPhSr1), had a unique RFLP profile and its SR was 100% similar in nucleotide sequence with the phytoplasma carried by Orosius albicinctus and Helianthus annus witches’-broom phytoplasma from Iran, members of “Ca. Phytoplasma trifolii”. Virtual RFLP patterns of SPhJ2 (representative of the predominant PCR-RFLP profiles) SR sequence were identical to those of peanut witches’-broom phytoplasma (16SrII-A, JX871467). However, SPhSr1 SR sequence patterns resemble (99.7%) those of vinca virescence phytoplasma (16SrVI-A, AY500817).     

Correlation between Light and Electron Microscopic Observations and Identification of Mycoplasmalike Organisms using Consecutive 350 nm Thick Sections

Serial sections of 350 nm thickness were used to make a correlation between electron and light microscopic observations. While thionin-acridine orange staining gave a positive result to detect abnormal sieve tubes of phyllody affected Phlox drummondii Hook, when observed under light microscope, the same cells revealed the presence of typical mycoplasmalike organisms (MLOs) in electron microscopic examination. Advantage of 350 nm thick sections in electron microscopy, and the utility of the technique in MLO detection have been discussed.     

TRANSMISSION AND HOST-RANGE STUDIES OF STRAWBERRY GREEN-PETAL VIRUS

The virus causing phyllody (virescence) in clover flowers was transferred by Cuscuta subinclusa to Fragaria vesca and Duchesnia indica plants which then produced symptoms of strawberry green-petal disease.
The jassid Euscelis plebejus (Fall.) in several forms, including E. lineolatus Brullé, transmitted green-petal virus from clover to clover, to and from a wide range of other hosts, and from but not to strawberry. Two viruses (or strains) were distinguished, one causing phyllody and the other witches' broom on clover; both were retained for more than 2 months by the vector, in which both had a latent period of about 30 days. Macrosteles viridigriseus (Edwards) also transmitted both viruses.
Variation in symptoms on strawberry plants infected naturally, and experimentally through dodder, suggested that two diseases have previously been grouped under the name 'green petal". It is proposed to distinguish these as ( a ) green petal caused by the virus inducing phyllody in clover, and ( b ) bronze leaf wilt caused by the clover witches' broom virus.     

Spiroplasmas are the causal agents of citrus little-leaf disease

A spiroplasma isolated from citrus with little-leaf disease was grown in a cell-free medium and injected into leafhoppers (Euscelis plebejus) Injected leafhoppers, but not those fed on infected plants, transmitted the spiroplasma to white clover (Trifolium repens cv. S100) and sweet orange (Citrus sinensis cv. Valencia). Infected clover plants were severely stunted; infected sweet orange plants showed typical symptoms of citrus little-leaf disease. The spiroplasma was detected in clover and sweet orange plants by electron microscopy; the helical morphology of the organisms was most easily recognizable in sections 150–200 nm thick. The organism was re-isolated in cell-free media both from infected plants and from injected E. plebejus. The original isolate and those re-isolated from experimentally infected clover and sweet orange appeared by morphological, cultural, biochemical and serological criteria to be identical to each other and to the R8-A2 (type) and C-189 strains of Spiroplasma citri. Serological tests and electrophoretic analysis of protein preparations indicated no relationship to Acholeplasma laidlawii, although this organism survived for at least 10 wk after injection into E. plebejus. Our results show that the causal agent of little-leaf disease is related to S. citri.