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.     

Association of Mycoplasmalike Organisms (MLOs) with Mild Type of Hydrangea virescence: A Study with 60–1000 nm Thick Sections

The study of the agent associated with the mild type of Hydrangea virescence in France involved three steps, with the aid of transmission electron microscope (TEM). In the first step, we observed the presence of polymorphic procaryotes in thephloem sieve tubes of diseased plants and their absence from corresponding healthy plant parts. The procaryotes were detected in the areas suspected in 1000 nm thick sections stained with thionin-acridine orange. In the next step, the ultrastructure of their unit membrane was studied at magnifications higher than 100 000. The two osmiophilic layers of the membrane were 6 nm distant and no preliminary parietal shape was detected. These observations on ultrathin (60 nm) sections allowed us to classify the, particles into the class “Mollicutes”. The third step involving the examination of 350–1000 nm thick sections revealed the absence of spiral forms. The TEM observations are consistent with the hypothesis that the agents associated with the mild type of Hydrangea virescence observed in France should be included within the MLO group. A method specially adjusted to the fixation of MLO inside sieve tubes has been mentioned.     

Attachment of the Flavescence doree pathogen (MLO) to leafhopper vectors and other insects

Flavescence doree (FD) is an important yellows disease of grapevine, caused by mycoplasma-like-organism (MLO) and is transmitted in the field by the leafhopper Scaphoideus titanus Ball. It can be transmitted in the laboratory between Vicia faba test plants by the leafhopper, Euscelidius variegatus Kbm. A technique to identify a specific attachment system between the MLO and the leafhopper vectors was developed. In this method, called “Double Dot”, extracts of macerated healthy whole insects or organs applied to a support membrane or cryosections of healthy whole leafhoppers, are incubated with a MLO-enriched extract from FD-infected V. faba or FD-infected E. variegatus. Attached MLO cells were identified by immunolabelling using FD-MLO specific monoclonal antibodies. Attachment of MLO cells was obtained on extracts of healthy S. titanus and E. variegatus and on tissues such as salivary glands, hemolymph and alimentary tract. On cryosections, MLO attachment was obtained on acini IV and V of the salivary glands and on some acini III, on the ventriculus of the alimentary tract, and on the abdomen fat bodies. “Double dot” experiments were done using other insect species, and MLO cells attachment was obtained on most MLO-vector insects but also on insects from a few non-vector species.     

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.     

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).     

Arabidopsis mlo3 mutant plants exhibit spontaneous callose deposition and signs of early leaf senescence

Key message

Arabidopsis thaliana mlo3 mutant plants are not affected in pathogen infection phenotypes but—reminiscent of mlo2 mutant plants—exhibit spontaneous callose deposition and signs of early leaf senescence.

Abstract

The family of Mildew resistance Locus O (MLO) proteins is best known for its profound effect on the outcome of powdery mildew infections: when the appropriate MLO protein is absent, the plant is fully resistant to otherwise virulent powdery mildew fungi. However, most members of the MLO protein family remain functionally unexplored. Here, we investigate Arabidopsis thaliana MLO3, the closest relative of AtMLO2, AtMLO6 and AtMLO12, which are the Arabidopsis MLO genes implicated in the powdery mildew interaction. The co-expression network of AtMLO3 suggests association of the gene with plant defense-related processes such as salicylic acid homeostasis. Our extensive analysis shows that mlo3 mutants are unaffected regarding their infection phenotype upon challenge with the powdery mildew fungi Golovinomyces orontii and Erysiphe pisi, the oomycete Hyaloperonospora arabidopsidis, and the bacterial pathogen Pseudomonas syringae (the latter both in terms of basal and systemic acquired resistance), indicating that the protein does not play a major role in the response to any of these pathogens. However, mlo3 genotypes display spontaneous callose deposition as well as signs of early senescence in 6- or 7-week-old rosette leaves in the absence of any pathogen challenge, a phenotype that is reminiscent of mlo2 mutant plants. We hypothesize that de-regulated callose deposition in mlo3 genotypes might be the result of a subtle transient aberration of salicylic acid-jasmonic acid homeostasis during development.

     

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.     

Immunolabeling of grapevine flavescence dorée MLO in salivary glands of Euscelidius variegatus: a light and electron microscopy study

Flavescence dorée (FD), a grapevine yellows disease, is caused by a mycoplasma-like organism (MLO). A colloidal gold indirect immunolabeling technique identified MLO in salivary glands of a vector leafhopper, Euscelidius variegatus. After aldehyde fixation, tissue samples were prepared by cryoultramicrotomy or embedding in acrylic resins. Double fixation with aldehydes and osmium retroxide, followed by embedding in epon, was also performed. Thin or semi-thin serial sections were treated with polyclonal anti-FD-MLO rabbit antibodies, then with gold-conjugated anti-rabbit IgG. Labeling was revealed using the silver enhancement technique for light microscopy. MLO in frozen thin sections of glands were efficiently labeled. Optimal results were obtained with 4% paraformaldehyde-0.1% glutaraldehyde fixation and low-temperature embedding in LR White resin. Both scattered MLO and unusual dense forms of MLO were easily detected with the electron-dense gold probe. This method distinguished MLO from other membrane-limited bodies and provided a good tool for studying infection in large regions of FD-infected tissues by light microscopy.     

Enhancement of the periodic acid — Schiff (PAS) and periodic acid — thiocarbohydrazide — silver proteinate (PA-TCH-SP) reaction in LR White sections

Summary LR White is a well-suited resin for the demonstration of carbohydrates with the PAS or PA-TCH-SP reaction in semithin and ultrathin sections. The intensity of these reactions can be greatly enhanced by using 3 steps in tissue preparation, either singly or in combination:1) The PAS reaction in semithin sections turns out stronger afterpartial (70% ethanol) than complete (100% ethanol)dehydration of the tissue before its transfer to 100% LR White.2)Silver enhancement of the PA-TCH-SP reaction product can simply be effected by physical development of ultrathin sections (PA-TCH-SP-SE reaction). Least precipitates are formed in this procedure, when sections are mounted on uncoated gold grids, processed for cytochemistry, and thinly coated with carbon in the end.3) The use ofhot silver proteinate (50° C) plus strong silver enhancement (15–20 min silver lactate developer) reveals minute concentrations of TCH-labelled aldehyde groups in the tissue that do not react with silver proteinate at room temperature.-Silver enhancement and the use of hot silver proteinate do not depend on LR White, but may also be applied to ultrathin sections of tissue embedded in other resins.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

A critical role for Arabidopsis MILDEW RESISTANCE LOCUS O2 in systemic acquired resistance

Members of the MILDEW RESISTANCE LOCUS O (MLO) gene family confer susceptibility to powdery mildews in different plant species, and their existence therefore seems to be disadvantageous for the plant. We recognized that expression of the Arabidopsis MLO2 gene is induced after inoculation with the bacterial pathogen Pseudomonas syringae, promoted by salicylic acid (SA) signaling, and systemically enhanced in the foliage of plants exhibiting systemic acquired resistance (SAR). Importantly, distinct mlo2 mutant lines were unable to systemically increase resistance to bacterial infection after inoculation with P. syringae, indicating that the function of MLO2 is necessary for biologically induced SAR in Arabidopsis. Our data also suggest that the close homolog MLO6 has a supportive but less critical role in SAR. In contrast to SAR, basal resistance to bacterial infection was not affected in mlo2. Remarkably, SAR‐defective mlo2 mutants were still competent in systemically increasing the levels of the SAR‐activating metabolites pipecolic acid (Pip) and SA after inoculation, and to enhance SAR‐related gene expression in distal plant parts. Furthermore, although MLO2 was not required for SA‐ or Pip‐inducible defense gene expression, it was essential for the proper induction of disease resistance by both SAR signals. We conclude that MLO2 acts as a critical downstream component in the execution of SAR to bacterial infection, being required for the translation of elevated defense responses into disease resistance. Moreover, our data suggest a function for MLO2 in the activation of plant defense priming during challenge by P. syringae.     

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.     

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.     

Mycoplasma-like Organisms Associated with Little Leaf Disease of Eucalyptus microtheca Muell.

A little leaf disease has been observed in several plants of Eucalyptus microtheca Muell. grown in Wad Medani area, Sudan. The disease is characterized by general plant stunting, reduction in diameter and height of trunks of affected trees, yellowing and reduction in leaf size, shortening of internodes together with excessive proliferation of axillary shoots. Electron microscope observations on ultrathin sections have revealed the presence of pleomorphic bodies in the sieve elements of diseased but not of healthy plants. These bodies possess DNA strands, ribosome-like granules and a three-layered unit membrane well defined at high magnifications (> 100,000) suggesting a mollicute nature of these bodies. Studies using semi-thin sections revealed the absence of helical forms. Thus, a mycoplasma-like organism (MLO) is suspected to be the agent responsible for the disease. The results are discussed with reference to the economic and environmental importance of this tree species in the Sudanand, the possible threat of the disease to its cultivation.     

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.     

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.     

Wissadula Proliferation in Jamaica II. Psyllid transmission and antibiotic therapy

Field symptoms of Wissadula proliferation (WP) in Jamaica were reproduced on W. periplocifolia (L.) C. Presl. ex Thwaites test plants, using field-collected or laboratory reared and infected Paracarsidara concolor Crawford. This psyllid naturally is associated with W. periplocifolia in the field. Diseased plants contained phloem-restricted prokaryotes, most of which resembled the phloem-restricted rickettsialike organisms (RLO) known to be associated with a small group of plant diseases. They were bound by a “cell wall” composed of a double unit membrane separated by an electron-lucent layer. A small proportion of the prokaryotes, which were bound by only one recognizable unit membrane and were ill-defined at the periphery, were difficult to distinguish from mycoplasmalike organisms (MLO). Penicillin, applied as a soil drench of 200—400 %mUg/ml caused remission of symptoms of the psyllid-infected test plants 6—17 days after first effective application, with the concomitant disappearance of all of the structures in the resulting new growth. Since MLO are not known to be penicillin-sensitive, it is proposed that the “MLO” were in fact, poorly preserved RLO and it is suggested that RLO may be the aetiological agent of WP. Achromycin also caused symptom remission in WP at a soil drench of 100—400 %mUg/ml, but was generally slightly slower-acting than penicillin, eliciting a response in 12—23 days. Both antibiotics could produce lengthy remissions; neither seemed curative since two penicillin- and two achromycin-treated plants redeveloped symptoms after cessation of treatment. WP was not transmitted by two cicadellids, Protalebra maculata Baker and Scaphytopius fuliginosus Osborn. P. concolor did not transmit disease to test plants of Abutilon hulseanum (Torr. & A. Gray) Torr, ex. Chapm. which was affected by a proliferation disease (Abutilon proliferation), symptomatologically similar to, and which occurred in the same locations, as WP. WP and AP thus may be aetiologically distinct. The putative RLO aetiology of WP provides evidence against Wissadula being an alternate host of the MLO-associated coconut lethal yellowing (CLY) disease in Jamaica, a fact reinforced by negative psyllid transmission tests to CLY-susceptible palms. The results for WP are discussed in relation to other diseases possibly caused by phloem-restricted RLO.     

Histopathology of Apple Proliferation in Malus Taxa and Hybrids of Different Susceptibility

Malus taxa and hybrids (“taxa”) grafted with M. pumila cv.‘Golden Delicious’differ significantly in their susceptibility to apple proliferation which is caused by a mycoplasma-like organism (MLO). These differences are correlated with the severity of anatomical aberrations and the numbers of MLOs in the phloem. The roots of declining trees of highly susceptible taxa with a mortality of more than 50 % are characterized by extensive phloem necrosis and the depletion of starch. MLOs are either not detectable or are present in low numbers, or the population appears degenerate when viewed by fluorescence microscopy. In comparable trees of a hybrid of M. sieboldii×M. pumila which shows a high recovery rate, both phloem necrosis and starch depletion are less pronounced, and the MLO numbers are low or the organisms are not detectable. Decline-tolerant taxa such as M. silvestris or M. pumila×M. baccata are little affected. Their phloem conditions and starch content do not differ significantly from that of healthy trees. However, the MLO titer is high. The histopathology of the scion cultivar of all groups examined is rather similar to that of the roots of the decline-tolerant taxa. Only in a late stage of decline, phloem necrosis increases while starch content and MLO numbers decrease in the scions grafted onto highly susceptible stockls.     

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.     

The Role of Non-Vectored Soil Transmission as a Primary Source of Infection by Pepper Mild Mottle and Cucumber Mosaic Viruses in Glasshouse-Grown Capsicum in Australia

Polyclonal rabbit antibodies, prepared against partially purified mycoplasmalike-organisms (MLO) from Grapevine Flavescence Dorée-infected plant extracts, were used to detect MLO in situ in excised salivary glands of the experimental vector Euscelidius variegates Kbm by an indirect immunofluorescent staining technique. Glands were examined from the fourth to the tenth week after the start of MLO acquisition. MLO detection was compared using this method, the transmission test and the ELISA technique applied to insects corpses after removal of salivary glands.