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.     

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).     

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.     

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.     

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.     

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.     

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.     

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.     

Mycoplasma-like Organisms in Narcissus sp.

Abstract The use of electron and optical fluorescent microscope confirmed the presence of mycoplasma-like organisms (MLO) in Narcissus sp. plants showing leaf chlorosis, phyllody and degenerated flowers. Unidentified multivesicular membrane bounded bodies were also found in some sieve tubes of the diseased plants.     

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.     

Detection of Mycoplasmalike Organism of Paulownia Witches′-Broom with DAPI Fluorescence Microscopy

Paulownia witches’-broom infected by mycoplasmalike organism (MLO) has been developed several cytochemical methods for diagnosis. These methods all based on the special stain reactions or abnormal fluorescence in groups of infected sieve elements as a diseased symptom,. not really on the direct detection of MLO under light microscope. This paper deals with the demonstration of MLO specific white fluorescence after DAPI staining with GMA sections of diseased young stems. Such fluorescence was absent in sections from health plants. The results were confirmed by the ulrrastrueture of MLO and the structure of sieve elements showing from PAS-TBO stained GMA sections. The described method may not only be used in accurate diagnosis of MLO diseased in different plants, but is also worth in the studies of MLO distribution in plants, MLO dynamics in plant resting stage and MLO transmission to support the theoretical basis for protection.     

Occurrence of Didymella fabae,the Teleomorph of Ascochyta fabae,on Faba Bean Straw in Spain

Pseudothecia of Didymella fabae, the teleomorph of Ascochyta fabae, were first observed on faba bean (Vicia faba) debris in Spain during autumn 1995. Most pseudothecia were mature by December–February. The ascospores gave rise to typical cultures of A. fabae, and conidia from these cultures caused ascochyta blight symptoms on inoculated faba bean plants. Placing straw‐bearing pseudothecia over the plants to allow ascospore discharge also resulted in typical ascochyta blight symptoms. Pseudothecia maturity and discharge of ascospores from the infested faba bean straw overlapped with the vegetative stage of the faba bean crop, which occurs in southern Spain during winter as the crop is sown in autumn and harvested in spring. These observations indicate that ascospores may serve as primary inoculum for the disease.     

Transmission of a New Mycoplasma-Like Organism (MLO) from Cuscuta odorata (Ruiz et Pav.) to Herbaceous Plants and Attempts to its Elimination in the Vector

A new MLO-type, originating from a holoparasite plant Cuscuta odorata (Ruiz et Pav.) causing stunting and reduction of flower- and leaf size on Catharanthus roseus (L.) G. Don. (HEINTZ 1986) was transmitted to Apium graveolens L., Plantago major L., Bellis perennis L. and Cirsium japonicum hybrid. The observed symptoms on the test plants probably caused by the MLO have not yet been described in the literature. The symptomatology on these herbaceous plants gives further data in order to classify the MLO as a new one which is named Guscuta latent MLO (Cl-MLO). Attempts to transmit the pathogens by the leafboppers Euscelidius variegates (Kirschbaum) and Euscelis lineolatus (Brullé) failed. It also was impossible to elimmate the MLO completely from Cuscuta odorata by heat treatment and antibiotic application. However, we succeeded in eliminating the pathogens from Catharanthus roseus by heat treatment.     

Fluorescence and Electron Microscopy of Cynodon dactylon Affected with a White Leaf Disease in the Sudan

A disease characterized by complete chlorosis of plant foliage was recently observed in Cynodon dactylon L. naturally growing in central Sudan. Severely affected plants were stunted with short internodes and small inward folded leaves. Examination of sections from paraffin embedded leaf and rhizome fractions, stained with a DNA-binding fluorochrome Bisbenzimid H 33258, and observed in a fluorescent microscope revealed abnormal amounts of DNA material in the phloem sieve tubes of diseased plants. More intense fluorescence was also observed in the xylem region and was shown to be due to the presence of a rod-shaped bacterium. No such anomalies were observed in healthy plants. Electron microscopy of ultrathin sections prepared from phloem zones, reacting with fluorescence in light microscopy revealed the presence of pleomorphic prokaryotes in the sieve tube elements of such zones. No helical forms were observed in semi-thin sections from the same plant material, suggesting the MLO nature of these prokaryotes.     

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 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.     

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.     

Occurrence of Mycoplasma-like Organisms in Parenchyma Cells of Cuscuta odorata (Ruiz et Pav.)

In shoots of the dodder Cuscuta odorata mycoplasma-like organisms (MLO) were observed to occur in nearly all mature sieve elements. Their frequency within the sieve elements varied from a few organisms to high concentrations. In addition, MLO, were found in other cell types. By identifying these cells and investigating the location in the vascular tissue three different types of cells infected with MLO could be distinguished: (1) phloem parenchyma cells, (2) parenchyma cells separating the five vascular bundles of C. odorata and (3) cells in the outer region of the vascular system next to the cells looking like an endodermis. Within the cells of the types 2 and 3, MLO occurred in large numbers and at different morphological structures., Therefore, we assume that the MLO multiply in these cells.     

Effects of mycorrhizae and chitin-hydrolysing microbes on Vicia faba

The effect of Streptomyces albovinaceus (S-22) and Bacillus sp. (B1) on the growth response, nodulation, nutrition and nitrogenase activities of faba bean (Vicia faba) varieties infected with Glomus mosseae under pot conditions in sterile soil amended with chitin was studied. The growth, nodulation, nutrients content and nitrogenase activity of mycorrhiza-treated plants of Giza-667 were significantly increased compared to untreated ones. Such increases were related to the increase in mycorrhizal root infection. Amendment of soil with chitin alone reduced the growth, nodulation, total nitrogen contents and nitrogenase activities of mycorrhiza-treated faba bean plants (Giza-667) compared to untreated plants. Inoculation of plants with S. albovinaceus or Bacillus sp. significantly increased the level of mycorrhizal roots infection, but addition of chitin to the soil in combination with Bacillus sp. reduced the mycorrhizal infection of faba bean roots. Highest phosphorus contents of faba bean Giza-667 were recorded after G. mosseae inoculation in the presence of all treatments. Similar results were observed for the other varieties. The microbial populations were significantly increased in rhizospheres amended with chitin. Such increases were not in response to the mycorrhizal inoculation. Generally, the microflora of faba bean rhizospheres was increased after treatment with G. mosseae in the absence of chitin amendment alone compared with non-mycorrhizal rhizospheres.