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.     

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.     

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.     

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.     

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).     

Determination of the Antigenic Protein Size Associated with Faba Bean Phyllody MLO by Using (SDS-PAGE) Electrophoresis and Immunotransfer

The antigenic proteins of MLO associated with faba bean phyllody (FBP) occurring in the Sudan were studied by using SDS-PAGE electrophoresis followed either by silver nitrate staining or by transferring to nitrocellulose membrane probed with specific polyclonal antiserum. No remarkable differences between healthy and FBP infected plants were observed when the gel was colored with silver nitrate. In contrast, after probing the transferred membrane with the specific polyclonal FBP antisera, band formation was only detected with FBP infected plants. These results were treated through an image analyser using a BIOLAB logicial. The analysed proteins measure approximately 18,000 and 36,000 daltons with regard to the protein molecular weight niarkers used (Bio-Rad). Possibility of the existence of a dimer is discussed. The localization of the bands is the same whatever the origins of FBP: Vicia faba or Crotalaria saltiana. However, the partial purification of the MLO including differential centrifugations followed by precipitation with polyethylene glycol (PEG) and passage through a column of Sepharose 4B were found to be essential for having clear electrophoretic profiles.     

Nonradioactive Screening Method for Isolation of Disease-Specific Probes To Diagnose Plant Diseases Caused by Mycoplasmalike Organisms

DNA fragments of tomato big bud (BB) mycoplasmalike organism (MLO) in diseased periwinkle plants (Catharanthus roseus L.) were cloned to pSP6 plasmid vectors and amplified in Escherichia coli JM83. A nonradioactive method was developed and used to screen for MLO-specific recombinants. Cloned DNA probes were prepared by nick translation of the MLO recombinant plasmids by using biotinylated nucleotides. The probes all hybridized with nucleic acid from BB MLO-infected, but not healthy, plants. Results from dot hybridization analyses indicated that several MLOs, e.g., those of Italian tomato big bud, periwinkle little leaf, and clover phyllody, are closely related to BB MLO. The Maryland strain of aster yellows and maize bushy stunt MLOs are also related to BB MLO. Among the remaining MLOs used in this study, Vinca virescence and elm yellows MLOs may be very distantly related, if at all, to BB MLO. Potato witches' broom, clover proliferation, ash yellows, western X, and Canada X MLOs are distantly related to BB MLO. Southern hybridization analyses revealed that BB MLO contains extrachromosomal DNA that shares sequence homologies with extrachromosomal DNAs from aster yellows and periwinkle little leaf MLOs.     

Production and characteristics of antisera to Spiroplasma citri and clover phyllody-associated antigens derived from plants

Antisera were produced to clover phyllody- and Spiroplasma citri-associated antigens partially purified from infected Vinca rosea plants. Separate antisera were made to ‘membrane fraction’ (MF) preparations comprising the resuspended pellet obtained by high speed centrifugation, and to ‘soluble fraction’ (SA) preparations, comprising the supernatant from high speed centrifugation concentrated by freeze-drying. All antisera showed considerable activity against normal plant antigens but after cross-absorption with extracts of healthy plants the MF antisera were used in F(ab')₂based ELISA tests to detect S. citri- or clover phyllody-associated antigens in infected plants. The ‘clover phyllody’ antiserum also reacted specifically with extracts of clover plants with phyllody, and with naturally-infected strawberry plants showing symptoms of green petal disease. Both the ‘clover phyllody’ and S. citri antisera were specific for their respective homologous antigens. No cross-reactions were observed in heterologous tests or between either antiserum and extracts of V. rosea infected with various MLOs obtained from different host plants.     

Genetic Interrelatedness among Clover Proliferation Mycoplasmalike Organisms (MLOs) and Other MLOs Investigated by Nucleic Acid Hybridization and Restriction Fragment Length Polymorphism Analyses

DNA was isolated from clover proliferation (CP) mycoplasmalike organism (MLO)-diseased periwinkle plants (Catharanthus roseus (L.) G. Don.) and cloned into pSP6 plasmid vectors. CP MLO-specific recombinant DNA clones were biotin labeled and used as probes in dot hybridization and restriction fragment length polymorphism analyses to study the genetic interrelatedness among CP MLO and other MLOs, including potato witches'-broom (PWB) MLO. Results from dot hybridization analyses indicated that both a Maryland strain of aster yellows and a California strain of aster yellows are distantly related to CP MLO. Elm yellows, paulownia witches'-broom, peanut witches'-broom, loofah witches'-broom, and sweet potato witches'-broom may be very distantly related, if at all, to CP MLO. A new Jersey strain of aster yellows MLO, tomato big bud MLO, clover phyllody MLO, beet leafhopper-transmitted virescence MLO, and ash yellows MLO are related to CP MLO, but PWB MLO is the most closely related. Similarity coefficients derived from restriction fragment length polymorphism analyses revealed that PWB and CP MLOs are closely related strains and thus provided direct evidence of their relatedness in contrast to reliance solely on biological characterization.     

Base Composition of the DNA of Mycoplasma-Like Organisms Associated with Various Plant Diseases

DNA was isolated from periwinkle plants infected by various mycoplasma-like organisms (MLOs), and from apple trees affected by apple proliferation. The DNA of the causal agents was separated from the host plant DNA by repeated bisbenzimide-CsCl buoyant density gradient centrifugation which resulted in highly enriched MLO DNA bands characterized by a lower buoyant density than that of the host DNA. The MLO DNAs were hydrolyzed to free bases which were determined by HPLC. The analyses revealed a similar low G + C content as found in the DNAs of several culturable mycoplasmas and spiroplasmas. The values of the DNA of the agents of the diseases investigated were as follows: European aster yellows 23.0, periwinkle virescence 23.5, apple proliferation 23.7, rape virescence 24.2, and phyllody of Diplotaxis erucoidcs 26.2 mol % G + C, respectively. Methylated bases were detected in low amounts only.     

Transmission Characteristics of the Clover Phyllody Agent by Dodder*

The results obtained in investigations on the biological characteristics of the clover phyllody agent (CPA) in respect to its transmission by the experimental vector Cuscuta campestris Younk, using Catbarantus roseus L. as constant host, are reported. Transmission efficiency of CPA was comparable whether the “stable bridge” or the “cut strand” of dodder method was adopted. The acquisition and inoculation threshold was between 4 and 6 days. The efficiency of transmission became greater by lengthening the test period up to 15 days. In cuttings of dodder maintained in water after recision, the maximum retention of infectivity, in respect to transmission capacity of CPA, was 28 days. It was proved that CPA invades C. campestris in a persistent manner. In comparative tests between CPA and APA (apple proliferation agent) it was demonstrated that the former is more efficiently transmitted than the latter. Moreover there is a much higher pathogenic effect of APA than CPA in dodder; in fact C. campestris as well as C. subinclusa Dur. and Hilg. develops poorly and shows deformations when growing on AP infected periwinkles. The basic distinguishable symptoms of the two diseases in C. roseus are: virescence and phyllody for CP; small but never virescent flowers for AP. MLOs have been detected, by electron microscopy, in sieve elements of C. roseus and C. campestris infected by CPA.     

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.     

Etiology and Transmission of Sesame Phyllody in Iran

Phyllody is a destructive disease of sesame (Sesamum indicum L.) in Iran. The major symptoms of the disease are floral virescence, phyllody and proliferation. Other symptoms which sometimes accompany the disease are yellowing, cracking of seed capsules, germination of seeds in the capsules and formation of dark exudates on the foliage. Light microscopy of hand-cut sections of sesame and colza (Brassica napus L. cv. Oro) stems treated with Dienes' stain showed blue areas in the phloem region of phyllody infected plants. Mycoplasma-like bodies were found in the sieve cells of infected sesame stems when thin sections were examined m an electron microscope. Sesame phyllody was successfully transmitted from sesame to sesame by grafting. Among various leafhoppers collected in sesame fields only Neoaliturus haematoceps transmitted the disease. This is the first report on the identification of a Mycoplasma-like organism (MLO) as the cause of sesame phyllody and N. haematoceps as an MLO vector in Iran. In host range studies using the leafhopper vector, only B. napus cv. Oro, Lepidium sativum, Catharanthus roseus, Lactuca sp. and Portulaca oleracea, but not 17 other species, developed symptoms. The species of vector and host range of MLO indicate that sesame phyllody in Iran is different from that reported in India and Upper Volta.     

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.     

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.     

Preparation of mycoplasma immunogens from plants and a comparison of polyclonal and monoclonal antibodies made against primula yellows MLO-associated antigens

A method is described for obtaining from plants partially purified preparations of mycoplasma-like organisms (MLO) which are suitable for use as immunogens for polyclonal or monoclonal antibody production, and as antigens for directly coating ELISA plates. Using this method a mouse monoclonal antibody to primula yellows MLO was prepared, and its characteristics compared with those of primula yellows polyclonal antibodies from rabbits and also against polyclonal antibodies made to similar preparations of European aster yellows MLO. No serological distinction was obtained between any of the homologous or heterologous combinations of antibody and MLO preparation using ELISA, fluorescence microscopy with FITC-labelled antibodies, or immunoprobes of western blots of partially purified MLO preparations. By contrast, there were no cross-reactions between the primula or aster yellows antibodies or MLO preparations and preparations of clover phyllody or tomato big bud MLOs or their respective polyclonal antibodies. The primula yellows MLO monoclonal and polyclonal antibodies, and also the European aster yellows MLO polyclonal antibodies, all appeared to recognize only a single major antigen of approximate M, = 22 400 daltons. Some possible explanations for the apparent specificity of the polyclinic antisera for a single antigen, and the relevance to MLO preparation procedures are discussed.     

Studies on Taxonomic Relationships of Mycoplasma–like Organisms by Southern blot Analysis

Chromosomal DNA fragments from the mycoplasma-like organisms (MLOs) associated with American aster yellows, apple proliferation, clover phyllody, and vaccinium witches' broom were cloned. Several MLO-specific fragments from each of these four isolates and a sequence from the 16S rRNA gene of an aster yellows MLO were used in Southern blot hybridizations to investigate the taxonomic relationships of 26 pathologically and geographically diverse MLOs. These MLOs were divided into four categories according to the symptoms induced in periwinkle. Genotypically, these isolates represented four groups (16S RFLP groups) of a classification based on restriction fragment length polymorphisms (RFLP) and sequencing data of the 16S rRNA gene. Probes from three isolates of one symptom category hybridized with isolates from all symptom categories. This result indicates that classification of MLOs by symptomatology does often not coincide with genetic relationships. The hybridization results confirmed the findings, of the 16S RFLP classification that most MLOs from herbaceous plants, especially those inducing virescence in periwinkle, are interrelated. These isolates, which were assigned to one 16S RFLP group, could be further differentiated in this study. Itcould be shown that aster yellows, clover phyllody, stolbur, and safflower phyllody and sandal spike are caused by distinct MLOs. The MLOs associated with apple proliferation, vaccinium witches' broom, and witches' broom of lime as well as two isolatesfrom, stone fruits could also be recognized as distinct organisms.     

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.     

Host Status of Crotalaria juncea,Sesamum indicum,Dolichos lablab,and Elymus glaucus to Meloidogyne javanica

Reproduction of Meloidogyne javanica on Crotalaria juncea PI 207657 and cv. Tropic Sun, Sesamum indicum, Dolichos lablab, and Elymus glaucus was assessed using a root-gall index, a reproductive index obtained by dividing the final population of juveniles (J2) in soil by the initial J2 population (Pf/Pi), and the number of J2 per gram of root recovered from roots by mist chamber extraction. Lycopersicon esculentum (cv. UC 204 C) was included as a susceptible host. The root-gall index and soil reproductive index were poor indicators of the host status of our test plants as compared with mist chamber extraction of J2 from roots. Lycopersicon esculentum had a mean root-gall index of 7.8. Some plants of S. indicum and E. glaucus had a few galls and other plants had none, with mean root-gall indices of 1.6 and 0.8, respectively. No galls were observed in C. juncea and D. lablab. Lycopersicon esculentum had the highest mean soil Pf/Pi value (mean = 1.93), while in C. juncea and some replicates of S. indicum no soil J2 were found. Even though some replicates had no galls, all replicates supported nematode reproduction. The mean numbers of J2 per gram root after 5 days of mist extraction were 447.7, 223.3, 165.5, 96.9, 42.3, and 41.9 for D. lablab, L. esculentum, E. glaucus, S. indicum, and C. juncea PI 207657 and cv. Tropic Sun, respectively. Accurate assessment of nematode resistance was influenced by sampling time and the nematode extraction technique used. Individual plants of both C. juncea and S. indicum supported nematode reproduction to some extent; however, both C. juncea and S. indicum have potential as cover crops to reduce M. javanica numbers.     

Amplification of plasmid DNA to detect plant pathogenic mycoplasmalike organisms

The polymerase chain reaction (PCR) was employed to develop a specific assay for plant pathogenic mycoplasmalike organisms (MLOs). A cloned fragment of a plasmid from a severe strain of western aster yellows (AY)-MLO was sequenced to identify oligonucleotide primers for PCR. Amplified DNA fragments of the predicted size were obtained from DNA extracted from plants and insects infected with pear decline MLO, beet leafhopper-transmitted virescence agent, elm yellows MLO and several AY-MLO strains. No amplification occurred from healthy leafhopper or plant DNA. The PCR-based assay was over 500 times more sensitive than a _tilized_tion-based assay which _tilized a cloned AY plasmid fragment as a probe. With the PCR-based assay, MLOs could be detected using DNA samples of leafhoppers that were only crushed and boiled in buffer. Amplification of the target DNA was confirmed by digestion of the PCR product with Mbo I which yielded predicted sized fragments for all MLO strains except Bradford AY and eastern AY. Sequencing the PCR product from elm yellows and eastern AY-MLOs revealed greater than 90% homology, and the failure to restrict the PCR product with Mbo I was due to a single base change in the restriction endonuclease site. The ability of the assay to detect a wide range of MLOs with minimal sample preparation and high sensitivity will be useful in epidemiological studies of MLO-caused diseases.