Characterization of Pseudomonas chlororaphis from Theobroma cacao L. rhizosphere with antagonistic activity against Phytophthora palmivora (Butler).

AIM: To isolate and characterize rhizobacteria from Theobroma cacao with antagonistic activity against Phytophthora palmivora, the causal agent of the black pod rot, which is one of the most important diseases of T. cacao. METHODS AND RESULTS: Among 127 rhizobacteria isolated from cacao rhizosphere, three isolates (CP07, CP24 and CP30) identified as Pseudomonas chlororaphis, showed in vitro antagonistic activity against P. palmivora. Direct antagonism tested in cacao detached leaves revealed that the isolated rhizobacteria were able to reduce symptom severity upon infection with P. palmivora Mab1, with Ps. chlororaphis CP07 standing out as a potential biocontrol agent. Besides, reduced symptom severity on leaves was also observed in planta where cacao root system was pretreated with the isolated rhizobacteria followed by leaf infection with P. palmivora Mab1. The production of lytic enzymes, siderophores, biosurfactants and HCN, as well as the detection of genes encoding antibiotics, the formation of biofilm, and bacterial motility were also assessed for all three rhizobacterial strains. By using a mutant impaired in viscosin production, derived from CP07, it was found that this particular biosurfactant turned out to be crucial for both motility and biofilm formation, but not for the in vitro antagonism against Phytophthora, although it may contribute to the bioprotection of T. cacao. CONCLUSIONS: In the rhizosphere of T. cacao, there are rhizobacteria, such as Ps. chlororaphis, able to protect plants against P. palmivora. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a theoretical basis for the potential use of Ps. chlororaphis CP07 as a biocontrol agent for the protection of cacao plants from P. palmivora infection.

Investigation of the mutagenic and antimutagenic effects of Origanum compactum essential oil and some of its constituents.

In the present study, the chemical composition of Origanum compactum essential oil was determined by gas chromatography and mass spectrometry, and its mutagenic and antimutagenic activities were investigated by the somatic mutation and recombination test (SMART) in Drosophila melanogaster. No significant increase in the number of somatic mutations was observed with the essential oil tested using both the standard (ST) and high bio-activation (HB) cross. In order to investigate the antimutagenic effect of the essential oil, we have tested the effect on the indirect-acting mutagen urethane (URE), as well as the direct-acting mutagen methyl methanesulfonate (MMS). O. compactum essential oil showed a strong inhibitory effect against URE-induced mutagenicity, especially with the HB cross. However, only a weak inhibitory effect on the mutagenicity induced by MMS was observed. These results suggest that the detected antimutagenicity could be mediated by an inhibitory effect on metabolic activation. The essential oil was fractionated to identify the components responsible of the suppressing effect detected. Seven fractions were obtained: two of them showed the most potent inhibitory effect against URE-induced mutagenicity and were further fractionated. The sub-fractions obtained from the second chromatographic fractionation were tested for their antimutagenic activity, together with carvacrol and thymol. The highest antimutagenic effect obtained with the sub-fractions was similar to the effect of the crude essential oil, as well as to the effect of carvacrol alone. These results suggest the absence of a synergic antimutagenic effect between the components of O. compactum essential oil and indicate that carvacrol was the most active oil component.

Leafy gall formation by Rhodococcus fascians.

Rhodococcus fascians infects a wide range of plants, initiating the formation of leafy galls that consist of centers of shoot amplification and shoot growth inhibition. R. fascians is an epiphyte but it also can establish endophytic populations. Bacterial signals involved in symptom development initiate de novo cell division and shoot meristem formation in differentiated tissues. The R. fascians signals exert activities that are distinct from mere cytokinin effects, and the evidence points to a process that adopted cytokinin biosynthetic enzymes to form derivatives with unique activity. Genes implicated in leafy gall formation are located on a linear plasmid and are subject to a highly controlling, complex regulatory network, integrating autoregulatory compounds and environmental signals. Leafy galls are considered as centers with specific metabolic features, a niche where populations of R. fascians experience a selective advantage. Such "metabolic habitat modification" might be universal for gall-inducing bacteria.

Specific binding sites on human phagocytic blood cells for Gly-Leu-Phe and Val-Glu-Pro-Ile-Pro-Tyr, immunostimulating peptides from human milk proteins.

Two immunostimulating peptides were isolated from human milk proteins by enzymatic digestion, the tripeptide GLF and the hexapeptide VEPIPY. These peptides increased the phagocytosis of human and murine macrophages and protected mice against Klebsiella pneumoniae infection. The present study showed that this activity may be correlated to the presence of specific binding sites on human blood phagocytic cells. The receptor molecules implicated were different for the two peptides. [3H]GLF specifically bound to PMNL and monocytes, whereas [3H]VEPIPY only bound to monocytes. The leukemic promyelocytic cell line HL-60 differentiated into granulocytes or into macrophages (depending on inducer used) coroborated these results. Specific binding of [3H]GLF on plasma membrane preparations of human PMNL (20 degrees C) was saturable and Scatchard analysis indicated two classes of binding sites: high-affinity sites of Kd 2.3 +/- 1.0 nM and Bm 60 +/- 9 fmol/mg protein and low-affinity sites of Kd 26.0 +/- 3.5 nM and Bm 208 +/- 45 fmol/mg protein. [3H]GLF binding was inhibited in a concentration-dependent manner by various analogous peptides, such as LLF, GLY, LLY and RGDGLF, but not by RGD, RGDS, VEPIPY and the chemotactic peptide f-Met-Leu-Phe (f-MLF). Only at high concentrations the direct analog MLF competed with labeled GLF. An important inhibitory effect was also observed with C1q component of the complement whereas C3 and BSA were uneffective. Specific binding of [3H]VEPIPY on monocyte membranes (20 degrees C) was saturable and Scatchard analysis was consistent with one class of binding sites of Kd 3.7 +/- 0.3 nM and Bm 150 +/- 6 fmol/mg protein.

The plant pathogen Rhodococcus fascians colonizes the exterior and interior of the aerial parts of plants.

Rhodococcus fascians is a plant-pathogenic bacterium that causes malformations on aerial plant parts, whereby leafy galls occur at axillary meristems. The colonization behavior on Nicotiana tabacum and Arabidopsis thaliana plants was examined. Independent of the infection methods, R. fascians extensively colonized the plant surface where the bacteria were surrounded by a slime layer. R. fascians caused the collapse of epidermal cells and penetrated intercellularly into the plant tissues. The onset of symptom development preceded the extensive colonization of the interior. The meristematic regions induced by pathogenic strain D188 were surrounded by bacteria. The nonpathogenic strain, D188-5, colonized the exterior of the plant equally well, but the linear plasmid (pFiD188) seemed to be involved in the penetration efficiency and colonization of tobacco tissues.

Immunoaffinity chromatography for the sample pretreatment of Taxus plant and cell extracts prior to analysis of taxanes by high-performance liquid chromatography.

The application of immunoaffinity chromatography for the purification of Taxus plant and cell extracts prior to the HPLC analysis is described. Polyclonal antibodies raised against 10-deacetylbaccatin III (10-DAB III), paclitaxel's main precursor in plant, were characterised by enzymed-linked immunosorbent assay. Immunoglobulins from selected antisera were immobilised on CNBr-activated Sepharose 4B. The immunoaffinity column was used for the purification of plant and plant cell culture extracts prior to their analysis by HPLC. Immunoaffinity chromatography enabled the selective concentration of taxoids and enhanced sample clean-up.

[Bone lesions in Gardner's syndrome: A report on two cases (author's transl)]. / Lésions osseuses du syndrome de Gardner. A propos de deux cas.

Gardner's syndrome consists of the classical triad of bone and soft tissue tumors associated with colon polyps. The two cases reported were familial affections and the typical signs were more or less absent, the characteristic features being the bone lesions.

[Diagnostic and therapeutic thoracoscopy in 83 cases of chronic pleurisy]. / Thoracoscopie diagnostique et thérapeutique dans 83 cas de pleurésies chroniques.

From May 1983 to June 1985 the authors performed a thoracoscopy as a diagnostic and therapeutic objective in 83 patients with chronic recurrent pleurisy; amongst these patients, 69 had know intra or extra thoracic cancer and the other 14 presented with an isolated pleurisy. Eight other patients had pleural carcinomatosis proven by needle biopsy and had pleural talc introduced by thoracoscopy. The examination was performed under local anaesthesia with neuroleptanalgesia; a rigid 7 mm diameter thoracoscope was used; the biopsies were performed uniquely on the parietal pleura using tropical forceps. The outcome was uncomplicated with no deaths nor any serious complications due to the method. The sensitivity of the pleural biopsy was 91.5% or 76 positive biopsies out of 83. 73 biopsies were metastatic and 3 were tuberculous pleurisies. The macroscopic appearance was strongly suggestive of malignancy in 78 patients and appeared inflammatory in 13, of whom 9 cases were positive on biopsy. The authors performed a pleural talcage in every case; a lasting pleural adhesion was obtained in 66 patients but regrettably there were 14 failures, the effusion recurring after removal of the drain. The authors stress the reduced number of incidents of pleural effusion, which confirms the good tolerance of pleural endoscopy.

Enzyme-linked immunosorbent assay for the detection and the semi-quantitative determination of taxane diterpenoids related to taxol in Taxus sp. and tissue cultures.

An ELISA-assay for the detection and the semi-quantitative determination of taxane diterpenoids structurally related to taxol found in Taxus sp. has been developed. The antiserum was raised in rabbits using a 2'-succinyltaxol-bovine serum albumin conjugate as immunogen. The working range of the assay was from 1 to 100 ng of taxol. In order to improve the production of taxol, preliminary experiments have been performed on crude extracts of several Taxus sp.; the results indicate that the present immunoassay is an useful tool for the rapid screening of species, varieties or individual plants out of a large population as well as for tissue cultures analysis.

A role for the miR396/GRF network in specification of organ type during flower development, as supported by ectopic expression of Populus trichocarpa miR396c in transgenic tobacco.

The MIR396 family, composed of ath-miR396a and ath-miR396b in Arabidopsis, is conserved among plant species and is known to target the Growth-Regulating Factor (GRF) gene family. ath-miR396 overexpressors or grf mutants are characterised by small and narrow leaves and show embryogenic defects such as cotyledon fusion. Heterologous expression of ath-miR396a has been reported in tobacco and resulted in reduction of the expression of three NtGRF genes. In this study, the precursor of the Populus trichocarpa ptc-miR396c, with a mature sequence identical to ath-miR396b, was expressed under control of the CaMV35S promoter in tobacco. Typical phenotypes of GRF down-regulation were observed, including cotyledon fusion and lack of shoot apical meristem (SAM). At later stage of growth, transgenic plants had delayed development and altered specification of organ type during flower development. The third and fourth whorls of floral organs were modified into stigmatoid anthers and fasciated carpels, respectively. Several NtGRF genes containing a miR396 binding site were found to be down-regulated, and the cleavage of their corresponding mRNA at the miR396 binding site was confirmed for two of them using RACE-PCR analysis. The data obtained agree with the functional conservation of the miR396 family in plants and suggest a role for the miR396/GRF network in determination of floral organ specification.

Endophytic fungi from leaves of Centella asiatica: occurrence and potential interactions within leaves.

Fungal endophytes were isolated from leaves of Centella asiatica (Apiaceae) collected at Mangoro (middle eastern region of Madagascar, 200 km from Antananarivo). Forty- five different taxa were recovered. The overall foliar colonization rate was 78%. The most common endophytes were the non-sporulating species 1 (isolation frequency IF 19.2%) followed by Colletotrichum sp.1 (IF 13.2%), Guignardia sp. (IF 8.5%), Glomerella sp. (IF 7.7%), an unidentified ascomycete (IF 7.2%), the non-sporulating species 2 (IF 3.7%) and Phialophora sp. (IF 3.5%). Using sequences of the ribosomal DNA internal transcribed spacer (ITS) regions, major endophytes (IF > 7%) were identified as xylariaceous taxa or as Colletotrichum higginsianum, Guignardia mangiferae and Glomerella cingulata. Results from in vitro fungal disk experiments showed a strong inhibitory activity of the xylariaceous non-sporulating species 1 against G. mangiferae and C. higginsianum and of C. higginsianum against G. mangiferae. This can be explained by antagonism between dominant taxa.

An unusual root tip formation in hairy root culture of Hyoscyamus muticus.

Hairy root cultures of Hyoscyamus muticus were established using Agrobacterium rhizogenes ATCC 15834. In one out of 8 clones established, an unusual root tip formation was observed after transfer of cultures from half-strength Murashige and Skoog (1962) to White's medium (1939). This phenomenon was associated with the production of a fine brownish cell suspension culture. Hairy root development resumed after transfer of the root tips from White to half-strength Murashige and Skoog medium. After plating the isolated brownish cells on hormone-free half-strength Murashige and Skoog or White solid medium, callus proliferation was observed, and then redifferentiation of hairy roots occurred. The polymerase chain reaction analysis of the H. muticus hairy root (clone Z2) revealed that only the tl region of the T-DNA was integrated. The growth and the production of five tropane alkaloids by this clone were examined.