Detection of Mycoplasmalike Organisms In Situ by Indirect Immunofluorescence Microscopy

Rocha, Aline

doi:10.1094/phyto-76-864

Cited by 21 publications

(11 citation statements)

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“…All or some of these may be responsible for supplying the plants with fixed N (27,37). Moreover, in addition to the culturable diazotrophs associated with rice, a substantial molecular diversity of N 2 -fixing bacteria has been detected in field-grown rice based on retrieval of nifH or nifD gene fragments from root DNA (12,63,64). However, the contribution of the bacteria externally associated with rice is insufficient to sustain a high yield (39).…”

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Endophytic Colonization of Rice by a Diazotrophic Strain of Serratia marcescens

et al. 2001

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Six closely related N 2 -fixing bacterial strains were isolated from surface-sterilized roots and stems of four different rice varieties. The strains were identified as Serratia marcescens by 16S rRNA gene analysis. One strain, IRBG500, chosen for further analysis showed acetylene reduction activity (ARA) only when inoculated into media containing low levels of fixed nitrogen (yeast extract). Diazotrophy of IRBG500 was confirmed by measurement of 15 N 2 incorporation and by sequence analysis of the PCR-amplified fragment of nifH. To examine its interaction with rice, strain IRBG500 was marked with gusA fused to a constitutive promoter, and the marked strain was inoculated onto rice seedlings under axenic conditions. At 3 days after inoculation, the roots showed blue staining, which was most intense at the points of lateral root emergence and at the root tip. At 6 days, the blue precipitate also appeared in the leaves and stems. More detailed studies using light and transmission electron microscopy combined with immunogold labeling confirmed that IRBG500 was endophytically established within roots, stems, and leaves. Large numbers of bacteria were observed within intercellular spaces, senescing root cortical cells, aerenchyma, and xylem vessels. They were not observed within intact host cells. Inoculation of IRBG500 resulted in a significant increase in root length and root dry weight but not in total N content of rice variety IR72. The inoculated plants showed ARA, but only when external carbon (e.g., malate, succinate, or sucrose) was added to the rooting medium.

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“…To reduce cross-reactions with plant material, the diluted IRBG500 antibody was first cross-absorbed overnight with a rice seedling homogenate prepared as described by da Rocha et al (12). The homogenate was prepared by grinding surface-sterilized seedlings in liquid nitrogen and then washing with acetone to remove the chlorophyll.…”

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Endophytic Colonization of Rice by a Diazotrophic Strain of Serratia marcescens

et al. 2001

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“…Immunofluorescence methods recently reported by some workers (ROCHA et al 1986) provided sensitive means of detecting MLO in such conditions. The present paper reports further improvements in the technique and its application for tomato stolbur and faba-bean phyllody.…”

Section: Resumementioning

confidence: 99%

“…(1983) have used a simple extraction method and ELISA to successfully detect MLOs and spiroplasmas in diseased plant tissues. Following this report, other workers were able to prepare antisera against some plant MLOs (KIRKPATRICK and GARROT 1984, ROCHA et al 1986. Methods other than ELISA such as indirect immunofluorescence were successfully used to detect MLOs, both in plant and insect tissues (LIN andCHEN 1986, BOUDON-PADIEU et al 1986).…”

Section: Resumementioning

confidence: 99%

In situ Detection of MLOs for Solanaceae Stolbur and Faba Bean Phyllody by Indirect Immunofluorescence

Mt¹,

Dafalla²,

Demazeau³

et al. 1989

Journal of Phytopathology

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MLOs of faba bean phyllody and stolbur diseases were detected by a simple sensitive indirect immunofluorescence technique. Antisera prepared against membrane fractions of diseased plants were used with both 40 ,urn-thick sections obtained with a freezing microtome and with 7 ,urn-thick sections prepared from aldehyde-fixed paraffin-embedded plant materials. The technique is highly sensitive, simple to practice and utilizes small quantities of reagents. Specific MLO labeling was obtained using antisera with low titers and containing high proportions of anti-plant antibodies. The ease of using sections from fresh plant material suggests the potential use of the technique for routine indexing and screening of MLO infections. Immunofluorescence on sections from paraffin-embedded plant tissues improved the visualization of labeled MLOs, factilitated the accessibility of antigens, reduced non-specific labeling and allowed more precise localization and interpretation of immunofluorescent reactions. ZusammenfassungIn situ-Nachweis von Mycoplasrna-ahnlichen Organismen (MLOs)-Erreger von Stolbur bei Nachtschattengewachsen und Phyllodie bei Feldbohnen -durch indirekte Immunfluoreszenz

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“…A substantial molecular diversity of N 2 fixing bacteria has been detected in field grown rice based on retrieval of nif H or nif D gene fragments from root DNA (da Rocha et al, 1986). Since the nif H gene only occurs in nitrogen fixing microorganisms, it has been used to monitor the presence of these diazotrophs, for example, in pure cultures (Frank et al, 1998), in soil (Widmer et al, 1999) and plants.…”

Section: Introductionmentioning

confidence: 99%

Identification of nif genes of heterotrophic and endophytic diazotrophs associated with rice (Oryza sativa L.,) by targeted DNA finger printing

Sugitha¹,

Kumar²

2009

Afr. J. Biotechnol.

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Heterotrophic and endophytic diazotrophs were isolated from rhizosphere soil, rhizoplane, roots and stems of different rice varieties. A total of thirteen isolates obtained were subjected to acetylene reduction assay (ARA) and eight isolates recorded significant amount of nitrogenase activity in a range of 31.65 to 91.95 nmoles of ethylene mg -1 cells h -1. Targeted PCR fingerprinting using nif H primers generated specific DNA band of approximately 750 bp, confirming the presence of nif genes in these isolates. Two isolates of heterotrophic diazotrophs, HDM 7 and HDT 1, generated multiple bands ranging from 500 -1000 bp.

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Detection of Mycoplasmalike Organisms In Situ by Indirect Immunofluorescence Microscopy

Cited by 21 publications

References 0 publications

Endophytic Colonization of Rice by a Diazotrophic Strain of Serratia marcescens

Endophytic Colonization of Rice by a Diazotrophic Strain of Serratia marcescens

In situ Detection of MLOs for Solanaceae Stolbur and Faba Bean Phyllody by Indirect Immunofluorescence

Identification of nif genes of heterotrophic and endophytic diazotrophs associated with rice (Oryza sativa L.,) by targeted DNA finger printing

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