"Ermioni" a new Lemon Cultivar Resistant to Mal secco Disease (Phoma tracheiphila)

A lemon tree resistant to citrus tracheomycotic disease Mal secco (Phoma tracheiphila) found in a Greek orchard and its progenies were evaluated. Scions budded on sour orange and Volkameriana rootstocks or cuttings rooted in mist were placed under natural infections or inoculated artificially with Mal secco inoculum. No disease symptoms appeared on the original tree, while in bud-progenies only 16 out of approximately 2500 field grown trees were diseased by Mal secco. In 45 artificially inoculated trees only two were infected by the fungus. Analysis of leaf proteins using electrophoresis and further genetic control of gene/enzyme 6-Phosphogluconate dehydrogenase (6-PGD) system were carried out for the resistant selection: the Greek varieties Maglini, Adamopoulou, Karystini and the Italian Monachello and Interdonato. Protein analysis showed a close relationship of the resistant selection with var. Maglini, of which a scion is the resistant selection, while the isozyme genetic marker showed three different electrophoretic patterns, the resistant selection being in a separate pattern. Some phenotypical differences between the resistant selection and Maglini were also observed. The phenotypical differences, the resistance to Mal secco, the differences in proteins from all other varieties except Maglini and the different electrophoretic pattern of 6-PGD of this selections vs other varieties, suggest a new cultivar, for which the name “Ermioni” is proposed.     

Host-Specific Effects of Toxin from the Rough Lemon Pathotype of Alternaria alternata on Mitochondria

Host-specific toxin from the rough lemon (Citrus jambhiri Lush) pathotype of Alternaria alternata (ACR toxin) was tested for effects on mitochondria isolated from several citrus species. The toxin caused uncoupling of oxidative phosphorylation and changes in membrane potential in mitochondria from leaves of the susceptible host (rough lemon); the effects differed from those of carbonylcyanide-m-chlorophenylhydrazone, a typical protonophore. ACR toxin also inhibited malate oxidation, apparently because of lack of NAD⁺ in the matrix. In contrast, the toxin had no effect on mitochondria from citrus species (Dancy tangerine and Emperor mandarin [Citrus reticulata Blanco], and grapefruit [Citrus paradisi Macf.]) that are not hosts of the fungus. The effects of the toxin on mitochondria from rough lemon are similar to the effects of a host-specific toxin from Helminthosporium maydis (HMT) on mitochondria from T-cytoplasm maize. Both ACR and HMT toxins are highly selective for the respective host plants. HMT toxin and methomyl had no effect (toxic or protective) on the activity of ACR toxin against mitochondria from rough lemon.     

A zinc-binding citrus protein metallothionein can act as a plant defense factor by controlling host-selective ACR-toxin production

Metallothionein is a small cysteine-rich protein known to have a metal-binding function. We isolated three different lengths of rough lemon cDNAs encoding a metallothionein (RlemMT1, RlemMT2 and RlemMT3), and only RlemMT1-recombinant protein had zinc-binding activity. Appropriate concentration of zinc is an essential micronutrient for living organisms, while excess zinc is toxic. Zinc also stimulates the production of host-selective ACR-toxin for citrus leaf spot pathogen of Alternaria alternata rough lemon pathotype. Trapping of zinc by RlemMT1-recombinant protein or by a zinc-scavenging agent in the culture medium caused suppression of ACR-toxin production by the fungus. Since ACR-toxin is the disease determinant for A. alternata rough lemon pathotype, addition of RlemMT1 to the inoculum suspension led to a significant decrease in symptoms on rough lemon leaves as a result of reduced ACR-toxin production from the zinc trap around infection sites. RlemMT1-overexpression mutant of A. alternata rough lemon pathotype also produced less ACR-toxin and reduced virulence on rough lemon. This suppression was caused by an interruption of zinc absorption by cells from the trapping of the mineral by RlemMT1 and an excess supplement of ZnSO₄ restored toxin production and pathogenicity. Based on these results, we propose that zinc adsorbents including metallothionein likely can act as a plant defense factor by controlling toxin biosynthesis via inhibition of zinc absorption by the pathogen.     

Mechanism of resistance to ricin toxin in selected mouse lymphoma cell lines

The role of impaired toxin uptake in conferring cellular resistance to the plant toxin RCA_II (ricin) has been examined using a murine BW5 147 lymphoma line and a toxin-resistant variant (BW5 147 Ric^R.3) selected by repeated exposure to RCA_II. The toxin-resistant variant is 250 times more resistant to RCA_II in long-term growth experiments and 1,000 times more resistant in short-term protein synthesis assays. Experiments with ferritin-conjugated ¹²⁵I-labeled RCA_II (ferritin-¹²⁵I-RCA_II) indicated that toxin binding to sensitive and resistant cells is similar at low toxin concentrations where maximum differential cytotoxicity occurs but that major difference exist with respect to toxin uptake. In sensitive cells toxin is internalized via endocytosis, and as seen previously in other systems subsequent rupture of some of the toxin-containing endocytotic vesicles releases toxin into the cytoplasm, where it inhibits protein synthesis. The process of toxin transfer to the cytoplasm is presumed to account for the one-hour lag before toxin-induced inhibition of protein synthesis can be detected. Endocytotic uptake of toxin is impaired in resistant BW5147Ric^R.3 cells, and they are unaffected by toxin concentrations that inhibit protein synthesis and kill sensitive parental cells. Killing of resistant cells at low toxin concentrations was accomplished by encapsulating RCA_II into lipid vesicles capable of fusing with the plasma membrane. Direct introduction of toxin into resistant cells using lipid vesicles as carriers produced rapid inhibition (< 15 min) of protein synthesis and eliminated the lag in toxin action seen in sensitive cells exposed to free toxin. These findings are discussed in relation to the mechanism of toxin action and proposals that toxin activity requires structural modification of the toxin molecule at the cell surface before transport into the cell.     

Mal3, the Fission Yeast Homologue of the Human APC-interacting Protein EB-1 Is Required for Microtubule Integrity and the Maintenance of Cell Form

Through a screen designed to isolate novel fission yeast genes required for chromosome segregation, we have identified mal3⁺. The mal3-1 mutation decreased the transmission fidelity of a nonessential minichromosome and altered sensitivity to microtubule-destabilizing drugs. Sequence analysis revealed that the 35-kD Mal3 is a member of an evolutionary conserved protein family. Its human counterpart EB-1 was identified in an interaction screen with the tumour suppressor protein APC. EB-1 was able to substitute for the complete loss of the mal3⁺ gene product suggesting that the two proteins might have similar functions. Cells containing a mal3 null allele were viable but showed a variety of phenotypes, including impaired control of cell shape. A fusion protein of Mal3 with the Aequorea victoria green fluorescent protein led to in vivo visualization of both cytoplasmic and mitotic microtubule structures indicating association of Mal3 with microtubules. The absence of Mal3 protein led to abnormally short, often faint cytoplasmic microtubules as seen by indirect antitubulin immunofluorescence. While loss of the mal3⁺ gene product had no gross effect on mitotic spindle morphology, overexpression of mal3⁺ compromised spindle formation and function and led to severe growth inhibition and abnormal cell morphology. We propose that Mal3 plays a role in regulating the integrity of microtubules possibly by influencing their stability.     

Oligomerization of Clostridium perfringens Epsilon Toxin Is Dependent upon Caveolins 1 and 2

Evidence from multiple studies suggests that Clostridium perfringens ε-toxin is a pore-forming toxin, assembling into oligomeric complexes in the plasma membrane of sensitive cells. In a previous study, we used gene-trap mutagenesis to identify mammalian factors contributing to toxin activity, including caveolin-2 (CAV2). In this study, we demonstrate the importance of caveolin-2 and its interaction partner, caveolin-1 (CAV1), in ε-toxin-induced cytotoxicity. Using CAV2-specific shRNA in a toxin-sensitive human kidney cell line, ACHN, we confirmed that cells deficient in CAV2 exhibit increased resistance to ε-toxin. Similarly, using CAV1-specific shRNA, we demonstrate that cells deficient in CAV1 also exhibit increased resistance to the toxin. Immunoprecipitation of CAV1 and CAV2 from ε-toxin-treated ACHN cells demonstrated interaction of both CAV1 and -2 with the toxin. Furthermore, blue-native PAGE indicated that the toxin and caveolins were components of a 670 kDa protein complex. Although ε-toxin binding was only slightly perturbed in caveolin-deficient cells, oligomerization of the toxin was dramatically reduced in both CAV1- and CAV2-deficient cells. These results indicate that CAV1 and -2 potentiate ε-toxin induced cytotoxicity by promoting toxin oligomerization – an event which is requisite for pore formation and, by extension, cell death.     

Characterization of a selenocystine-resistant carrot cell line : alterations in cystine and sulfate uptake

A selenocystine-resistant carrot cell line, C-1, was isolated from a haploid carrot (Daucus carota) cell culture, HA. The C-1 variant takes up cystine, but not cysteine, more slowly than does HA. The selenocystine resistance is maintained in culture in the absence of selection and is expressed in regenerated plants. Results based on chromatographic separation of sulfur metabolites from cells fed with [³⁵S]cystine suggest a block either in the uptake or reduction of cystine in the variant. Both lines can grow on cystine as sole sulfur source. Growth of the HA line on cystine suppressed the development of sulfate uptake capacity (Furner, Sung 1982 Proc Natl Acad Sci USA 79: 1149-1153), while cystine-grown C-1 cells have high levels of sulfate uptake capacity.

We suggest that the C-1 line, grown on cystine, accumulates an insufficient quantity of some sulfur metabolite, which is involved in the control of sulfate uptake, to suppress the uptake. C-1 grown on cystine is more sensitive than HA to growth inhibition by the sulfate analog selenate.

     

Treatment of oral thrush in HIV/AIDS patients with lemon juice and lemon grass (Cymbopogon citratus) and gentian violet

PurposeThe purpose of the study was to investigate the safety and efficacy of lemon juice and lemon grass (Cymbopogon citratus) in the treatment of oral thrush in HIV/AIDS patients when compared with the control group using gentian violet aqueous solution 0.5%. Oral thrush is a frequent complication of HIV infection.In the Moretele Hospice, due to financial constraints, the treatment routinely given to patients with oral thrush is either lemon juice directly into the mouth or a lemon grass infusion made from lemon grass (Cymbopogon citratus) grown and dried at the hospice. These two remedies have been found to be very efficacious therefore are used extensively. Gentian violet, the first line medication for oral thrush in South Africa, is not preferred by the primary health clinic patients due to the visible purple stain which leads them to being stigmatized as HIV-positive. Cymbopogon citratus and Citrus limon have known antifungal properties.MethodsThe study design was a randomised controlled trial. Ninety patients were randomly assigned to one of three groups: gentian violet, lemon juice or lemon grass. Inclusion criteria included being HIV-positive with a diagnosis of oral thrush. The study period was 11 days and patients were followed up every second day. International ethical principles were adhered to during the study.ResultsOf the 90 patients, 83 completed the study. In the intention-to-treat analysis, none of the p-values were significant therefore the null hypothesis could not be rejected. In the analysis of the participants who actually completed the trial, the lemon juice showed better results than the gentian violet aqueous solution 0.5% in the treatment of oral thrush in an HIV-positive population (p<0.02). The null hypothesis in terms of the lemon grass and gentian violet could also be rejected on the basis of the Chi-square test and the likelihood ratio test (p<0.05).ConclusionsThough the patient population was small, the use of lemon juice and lemon grass for the treatment of oral candidiasis in an HIV population was validated by the randomised controlled trial.     

Hydraulic Resistance of Rough Lemon Roots

A pressure chamber technique was used to estimate hydraulic root resistance in rough lemon (Citrus jambhiri Lush.) seedlings. The effect of previous water stress on hydraulic root resistance was evaluated. A factorial 3 × 3 design with four replications was established with potted rough lemon seedlings in a growth chamber. Three water-stress treatments were applied by watering at intervals of 1, 2 and 3 days, and root resistance measurements were made after 6, 12 and 18 days of treatment. Plants that had experienced mild and severe water stress (irrigation interval of 2 and 3 days, respectively) had higher hydraulic root resistances after several drying cycles than those plants irrigated daily. Additional cycles had no significant effect. The increase in root resistance was not due to decreased root growth but apparently to changes in the permeability of the root cell membranes or to increased suberin deposition in the cell walls of the cortical cells. In a short-term experiment (1 h), temperature strongly affected water flow through rough lemon roots in the range 5 to 35°C. Temperature influenced root membrane permeability, since reduced blow could not be explained by changes in water viscosity.     

Plant pathotoxins from Alternaria citri: The major toxin specific for rough lemon plants

A pathotype of the fungus Alternaria citri that attacks rough lemon plants produces several toxins in culture which specifically damage rough lemon and Rangpur lime plants. The major toxin produced, Toxin I, was by far the most potent compound (ED₅₀ = 30 ng/ml). Five other minor toxins were active at ED₅₀ levels greater than 1 μg/ml. On the basis of mass, ¹H and ¹³C NMR spectra and decoupling studies of Toxin I and derivative, Toxin I is a 19 carbon polyalcohol with an α-dihydropyrone ring. The γ-dihydropyrone tautomer was less predominant. Culture filtrates of A. citri also contained a biologically inactive, partially analogous, component possessing a tetrahydropyran ring. It probably arises from decarboxylation of Toxin I. Toxin I was highly specific and did not affect nonhost plants at 10 000 times the concentrations affecting rough lemon.     

Modification of the cell surface with neuraminidase increases the sensitivities of cells to diphtheria toxin and Pseudomonas aeruginosa exotoxin.

When cell lines that are susceptible to diphtheria toxin, such as human FL cells, were treated with C. perfringens neuraminidase their sensitivities to the toxin were increased. The sensitivities of the cells to the toxin were also increased by treatment with neuraminidase from Arthrobacter ureafaciens or HVJ (Sendai virus). Neuraminidase did not have this effect on a toxin-resistant cell line. It also did not increase the cytotoxic effect of a large concentration of fragment A of diphtheria toxin, which lacks the moiety of the toxin molecule that binds to the cell membrane. Neuraminidase from C. perfringens or HVJ also increased the sensitivity of cells to ricin toxin. Furthermore, neuraminidase from C. perfringens or A. ureafaciens increased the sensitivity of cells to Pseudomonas aeruginosa exotoxin (PA toxin), but in this case neuraminidase from HVJ did not have a similar effect.     

Continuous cell propagation using low-charge microcarriers

Microcarrier cell culture technology has been extended by the finding that two mammalian epithelial cell lines can be continuously subcultured by simple bead-to-bead transfer in normal medium in which calcium concentrations have been reduced. Data are reported which show that the hamster ovary line CHO-Kl and the monkey kidney line LLC-MK₂ can be subcultured simply by adding fresh microcarriers to the stirred suspension culture. Thirteen generations of continuous exponential growth are demonstrated with two such subcultures for the CHO-Kl cells and with four such subcultures for the LLC-MK₂ cells. Cell generation times were unchanged by this subculturing approach compared to standard subculturing procedure using trypsin to remove cells from surfaces. We have applied this technique to the production of vesicular stomatitis virus (VSV) from CHO-Kl cells. Viral yields were comparable (less than twofold difference) in microcarrier cultures which were subcultured via bead-to-bead transfer or by the standard means of removing cells from microcarriers with trypsin.     

Oncogene overexpression and de novo drug-resistance in human prostate cancer cells

We have isolated a variant [PC3(R)] of the human prostate PC3 tumor cell line which showed resistance to several anticancer drugs. Studies to evaluate the mechanisms of resistance to anticancer drugs in the PC3(R) cell line indicated that mdrl was not overexpressed. Studies also indicated that activities of topo I and topo II were not different in these cell lines, nor was there any difference in the formation of drug-induced KCl-SDS precipitable complexes, including that topoisomerases were not involved in the development of resistance in PC3(R) cells. While the activity of glutathione S-transferase and total glutathione levels were also similar in these cell lines, the glutathione peroxidase activity in PC3(R) cells was 5-fold lower than in PC3 cells. ] Furthermore, proto-oncogene expression for c-myc, and H-ras was significantly higher in resistant cell than in sensitive cells, indicating that the amplication of early response genes may play a role in the emergence of de novo resistance in PC3(R) cells.     

Citrus Tree Decline Caused by Pratylenchus coffeae

The pathogenic effects of Pratylenchus coffeae on growth and yield of tangelo (Citrus paradisi × C. reticulata) scions grafted on rough lemon (C jambhiri), sour orange (C. aurantium) and ''Cleopatra'' mandarin (C. reticulata) rootstocks were evaluated under field conditions for 4 years. Pratylenchus coffeae on inoculated trees increased to significantly damaging population densities on rough lemon rootstock the second year, on sour orange the third and on Cleopatra mandarin the fourth year after planting. Mean growth reduction of P. coffeae-infected trees after 4 years was 80, 77 and 49%, respectively, for the three rootstocks. Noninoculated trees on rough lemon and sour orange rootstocks yielded significantly more fruit than comparable inoculated trees. Natural migration of P. coffeae occurred horizontally on roots for a distance of 4.5 m.     

Mechanisms for Bt Toxin Resistance and Increased Chemical Pesticide Susceptibility in Cry1Ac10-resistant Cultured Insect Cells

Cabbage looper moth (Trichoplusia ni) cell line BTI-Tn-5B1-4 (TnH5) has developed high-level resistance (>1000 fold) by the selection of Bt Cry1Ac10 toxin. In order to examine mechanisms of resistance to Cry1Ac10 toxin (biological pesticide), both general esterase activities and cell tolerance to osmotic lysis were compared between non-selected Cry1Ac10-susceptible Trichoplusia ni cell line TnH5-S and Cry1Ac10-resistant Trichoplusia ni cell line TnH5-R selected by Bt Cry1Ac10. The Cry1Ac10-resistant TnH5-R cells had lower general esterase activity than the non-selected TnH5-S cells, and the esterase isozyme bands for the Cry1Ac10-resistant TnH5-R cells were much weaker than that for the non-selected TnH5-S cells. Both activated Cry1Ac10 toxin and multi-toxin from Bacillus thuringiensis subsp. aizawai GC-91 (an engineering bacterium) could not inhibit the esterase activity both in the Cry1Ac10-susceptible and Cry1Ac10-resistant cells, but two chemical pesticides, chlopyrifos and methomyl, could greatly inhibit the esterase activities both in the TnH5-R and TnH5-S cells. On the other hand, cell tolerance to osmotic lysis caused by hypotonic solution for the Cry1Ac10-resistant TnH5-R cells was higher than that for the non-selected TnH5-S cells (2.5×). Based on these results, we made the following conclusions. The general esterase activities in the Cry1Ac10-resistant TnH5-R cells was not related to Bt Cry1Ac10 resistance, but the susceptibility to the two tested chemical pesticides increased in TnH5-R cells because of their lower esterase activity. The increase of cell tolerance to osmotic lysis for the Cry1Ac10-resistant TnH5-R cells may be one of the mechanisms for Bt toxin resistance because midgut cells of insects are also disrupted by an osmotic lysis caused by Bt toxin.     

Anti-salmonella properties of kefir yeast isolates: An in vitro screening for potential infection control

The rise of antibiotic resistance has increased the need for alternative ways of preventing and treating enteropathogenic bacterial infection. Various probiotic bacteria have been used in animal and human. However, Saccharomyces boulardii is the only yeast currently used in humans as probiotic. There is scarce research conducted on yeast species commonly found in kefir despite its claimed potential preventative and curative effects. This work focused on adhesion properties, and antibacterial metabolites produced by Kluyveromyces lactis and Saccharomyces unisporus isolated from traditional kefir grains compared to Saccharomyces boulardii strains. Adhesion and sedimentation assay, slide agglutination, microscopy and turbidimetry assay were used to analyze adhesion of Salmonella Arizonae and Salmonella Typhimurium onto yeast cells. Salmonella growth inhibition due to the antimicrobial metabolites produced by yeasts in killer toxin medium was analyzed by slab on the lawn, turbidimetry, tube dilution and solid agar plating assays. Alcohol and antimicrobial proteins production by yeasts in killer toxin medium were analyzed using gas chromatography and shotgun proteomics, respectively. Salmonella adhered onto viable and non-viable yeast isolates cell wall. Adhesion was visualized using scanning electron microscope. Yeasts-fermented killer toxin medium showed Salmonella growth inhibition. The highest alcohol concentration detected was 1.55%, and proteins with known antimicrobial properties including cathelicidin, xanthine dehydrogenase, mucin-1, lactadherin, lactoperoxidase, serum amyloid A protein and lactotransferrin were detected in yeasts fermented killer medium. These proteins are suggested to be responsible for the observed growth inhibition effect of yeasts-fermented killer toxin medium. Kluyveromyces lactis and Saccharomyces unisporus have anti-salmonella effect comparable to Saccharomyces boulardii strains, and therefore have potential to control Salmonella infection.     

Enterotoxin-producing Bacteroides fragilis (ETBF) Strains in Stool Samples Submitted for Testing ofClostridium difficile and its Toxins

Fifty faecal samples of patients suspected of having diarrhoea associated with Clostridium difficile were studied. Toxins of C. difficile were tested in vivo directly from the faecal sample using Toxin Detection Kits (Oxoid) to detect toxin A and primers for detection genes of Toxin A and B in a PCR test. The same samples were tested for B. fragilis enterotoxin gene directly from the faecal sample using special primers and a PCR test. Samples were inoculated onto selective media for C. difficile (CCCA) and B. fragilis (BBE) for isolation of bacteria.In vitro Toxin A of C. difficile in culture was tested using a C. difficile toxin A immunoassay (Oxoid, U.K. test and Toxin B of C. difficile was tested by using the McCoy cell line. C. difficile toxin A and B genes were determined in DNA of isolated strains using special primers and a PCR reaction. The enterotoxin production in B. fragilis strains was tested on the human carcinoma cell line HT29/C1. The presence of fragilysin gene was detected using a special pair of primers and a PCR reaction. Toxinogenic strains of C. difficile and enterotoxigenic Bacteroides fragilis (ETBF) strains were isolated from the same samples.     

The Influence of Chemical Composition of Commercial Lemon Essential Oils on the Growth of Candida Strains

Candida yeasts are saprophytes naturally present in the environment and forming colonies on human mucous membranes and skin. They are opportunistic fungi that cause severe and even fatal infections in immunocompromised individuals. Several essential oils, including eucalyptus, pine, cinnamon and lemon, have been shown to be effective against Candida strains. This study addresses the chemical composition of some commercial lemon essential oils and their antifungal potential against selected Candida yeast strains. Antifungal potential and minimum inhibitory concentrations were determined for six commercial lemon essential oils against five Candida yeast strains (Candida albicans 31, Candida tropicalis 32, Candida glabrata 33, Candida glabrata 35 and Candida glabrata 38). On the basis of the GCMS analysis, it was found that the tested lemon essential oils had different chemical compositions, but mostly, they contained almost exclusively terpenes and oxygenated terpenes. The tests show that antifungal potential of lemon essential oils against Candida yeast strains was related to the high content of monoterpenoids and the type of Candida strains. From six tested commercial oils, only four (ETJA, Vera-Nord, Avicenna-Oil and Aromatic Art) shows antifungal potential against three Candida species (C. albicans, C. tropicalis and C. glabrata). Vera-Nord and Avicenna-Oil show the best activity and effectively inhibit the growth of the C. albicans strain across the full range of the concentrations used. Our study characterises lemon essential oils, which could be used as very effective natural remedies against candidiasis caused by C. albicans.