Interaction of a hydroxyproline-rich glycoprotein from tobacco callus with potential pathogens

A hydroxyproline-rich glycoprotein was isolated from tobacco (Nicotiana tabacum L.) callus tissue cultures by an acidic-ethanol extraction procedure and purified to about 95% homogeneity by ion exchange chromatography on carboxymethyl cellulose. This glycoprotein agglutinated cells of an avirulent strain (B-1) of the bacterial pathogen Pseudomonas solanacearum but not its parental, virulent isolate (K-60). Bacterial lipopolysaccharide (from K-60 strain) inhibited this agglutination. The tobacco glycoprotein also agglutinated zoospores of both compatible and incompatible races of Phytophthora parasitica var. nicotianae. Although 34 potential haptens were tested, no low-molecular-weight carbohydrate that inhibited bacterial or fungal agglutination was found. The agglutination activity of the tobacco glycoprotein was sensitive to pronase and sodium periodate. The apparent molecular weight of the glycoprotein was 120,000. The protein moiety was basic (12% lysine and 5% histidine) and contained 38% hydroxyproline. The carbohydrate moiety comprised 26% (by weight) of the glycoprotein, and contained 87% arabinose, 8% galactose, and 5% glucose. The glycoprotein labeled with fluorescein isothiocyanate bound significantly better to the avirulent isolate (B-1) of P. solanacearum than to the virulent strain (K-60). Binding to the avirulent cells was inhibited by incubation in a higher ionic strength medium (e.g. 0.2 m NaCl). The labeled glycoprotein also bound to cystospores and mycelia of both races of P. parasitica var. nicotianae. This fungal-glycoprotein interaction was inhibited by the lipopolysaccharide from strain K-60 and by higher ionic strength conditions.     

Quinolobactin, a New Siderophore of Pseudomonas fluorescens ATCC 17400, the Production of Which Is Repressed by the Cognate Pyoverdine

Transposon mutant strain 3G6 of Pseudomonas fluorescens ATCC 17400 which was deficient in pyoverdine production, was found to produce another iron-chelating molecule; this molecule was identified as 8-hydroxy-4-methoxy-quinaldic acid (designated quinolobactin). The pyoverdine-deficient mutant produced a supplementary 75-kDa iron-repressed outer membrane protein (IROMP) in addition to the 85-kDa IROMP present in the wild type. The mutant was also characterized by substantially increased uptake of ⁵⁹Fe-quinolobactin. The 75-kDa IROMP was produced by the wild type after induction by quinolobactin-containing culture supernatants obtained from the pyoverdine-negative mutant or by purified quinolobactin. Conversely, adding purified wild-type pyoverdine to the growth medium resulted in suppression of the 75-kDa IROMP in the pyoverdine-deficient mutant; however, suppression was not observed when Pseudomonas aeruginosa PAO1 pyoverdine, a siderophore utilized by strain 3G6, was added to the culture. Therefore, we assume that the quinolobactin receptor is the 75-kDa IROMP and that the quinolobactin-mediated iron uptake system is repressed by the cognate pyoverdine.     

Neither Indole Acetic Acid nor Bacteriocin is Apparently Involved in the in vitro Antagonism between the Virulent and the Avirulent Strains of Pseudomonas solanacearum

An avirulent strain of Pseudomonas solanacearum could inhibit the growth of its virulent parent on L-tryptophan-containing glycerol nutrient agar (TGNA) medium. It was, also, capable of inhibiting, though to a less degree, Corynebacterium fascians and Pseudomonas marginata, out of five other bacterial species tested. While P. marginata was partially inhibited by the avirulent strain it was totally insensitive to indole-3-acetic acid (IAA) up to a concentration of 300 μg/ml. Additionally, Erwinia carotovora var. atroseptica which was totally unaffected by the avirulent strain showed a spectrum of sensitivity to IAA concentrations close to that of the virulent strain. No DNA, RNA, or IAA could ever be detected in the inhibition area and, thus, it is almost certain that the inhibiting agent produced by the avirulent strain is not IAA as was previously speculated. This inhibiting agent was insensitive to autoclaving and to the enzymes, pronase, trypsin, DNAse, and RNAse. P. solanacearum bacteriocin was detected by polyacrylamide gel electrophoresis in the medium near the avirulent growth line and not throughout the inhibition area. This supports the conclusion that bacteriocin alone cannot be held responsible for the inhibition phenomenon observed and leaves the nature of this inhibiting agent unknown.     

Interaction of virulent and non-virulent Rhodococcus equi human isolates with phagocytes, fibroblast- and epithelial-derived cells

Abstract Rhodococcus equi is a facultative, intracellular, Gram-positive coccobacillus, increasingly reported in pneumonia of AIDS-infected patients. We investigated killing resistance properties of human R. equi virulent and avirulent human strains. Avirulent β-lactam-susceptible strains had lower intracellular colony forming units after 45 min incubation in murine macrophages J774 and human monocyte-macrophage TPH-1 than those of virulent strains. Only virulent β-lactam-resistant strains persisted within macrophages for at least 18 min only. A β-lactam-resistant mutant was obtained from a β-lactam-susceptible strain after selection in a penicillin G-containing culture medium. This mutant strain, like the natural virulent strains, persisted within macrophages, harboured cell-associated appendages, produced phage-like particles and induced, after its intravenous inoculation, a chronic infection in BALB/c nude mice. Supernatant culture of virulent strains transferred partial macrophage-killing resistance properties to avirulent strains. The same supernatant was toxic for L-929, HeLa and Vero cell cultures. These supernatant effects were heat-inactivated, trypsin-inactivated and did not seem to be linked to phage-like particle presence. These data argue that virulence, β-lactam-resistance, and macrophage-killing resistance are associated in human R. equi isolates. Moreover, only virulent strains produced uncharacterized toxic factors.     

Relationship Between Ralstonia solanacearum Diversity and Severity of Bacterial Wilt Disease in Tomato Fields in China

A field survey was conducted to determine the relationship between Ralstonia solanacearum diversity and severity of bacterial wilt disease in tomato plants grown in plastic greenhouses. Both vegetative and reproductive stages of the plants were surveyed, and the symptoms were empirically categorized into five scales: 0 (asymptomatic): 1st, 2nd, 3rd and 4th. The bacterial wilt pathogen was isolated from infected plants at each disease scale; pathogenic characteristics and population densities of the bacterial strains were assessed. Two hundred and eighty‐two isolates were identified as R. solanacearum, which were divided into three pathogenic types, virulent, avirulent and interim, using the attenuation index (AI) method and a plant inoculation bioassay. Ralstonia solanacearum was detected in all asymptomatic and symptomatic tomato plants, with population numbers, ranging from 10.5 to 86.7 × 105 cfu/g. However, asymptomatic plants harboured only avirulent or interim R. solanacearum, whereas tomato plants displaying 1st or 2nd disease degree contained interim and virulent strains. Additionally, 3rd and 4th degree plants harboured only virulent strains. The disease was more severe in vegetative‐stage plants (disease severity index (DSI) 0.20) with higher total numbers of interim and virulent R. solanacearum strains than those in reproductive‐stage plants (DSI 0.12). Three pathotypes of R. solanacearum coexisted in a competitive growth system in the tomato field, and their distribution closely correlated with the severity of tomato bacterial wilt.     

Modulation of the mitogenic PHA response of carp, Cyprinus carpio L., by extracellular products of Aeromonas salmonicida

The influence of bacteria-free supernatants from cultures of atypical virulent (V234/81, auto-agglutinating. A-layer positive) and avirulent (126/68, non-agglutinating, A-layer negative) strains of A. salmonicida , obtained after different culture times in yeast-tryptone broth at 20°C, was tested on the PHA response of carp pronephric leucocytes in vitro . Supernatants from virulent cultures modulated the response, whereas avirulent supernatants had no effect. The response was enhanced (400%) by supernatant from early virulent cultures (20 h), but severely depressed (<3%) by later ones (96 h). The effects were dose-dependent. Inhibitory activity of 96-h supernatant was lost by heating (70°C, 30 min), suggesting that the inhibiting factors are all proteinaceous.
Heated 96-h supernatant was as stimulatory as early supernatant. Stimulation of leucocytes also occurred in the absence of PHA with early and heat-treated 96-h supernatants, but at a tenth of the level, suggesting that only stimulated cells (blasts) might respond to the substance(s) present in supernatants. Membrane fragments from virulent and avirulent bacteria, and purified LPS from virulent bacteria, were stimulatory with or without PHA. Endotoxin-free, heat-treated, 96-h culture supernatants were also stimulatory, suggesting that an additional mitogenic factor(s), other than LPS, is present in the supernatants. The modulating in vitro effects of extracellular products from A. salmonicida might explain the immunosuppression seen during later stages of erythrodermatitis in vivo.     

Response to Xanthomonas campestris pv. vesicatoria in tomato involves regulation of ethylene receptor gene expression

Although ethylene regulates a wide range of defense-related genes, its role in plant defense varies greatly among different plant-microbe interactions. We compared ethylene's role in plant response to virulent and avirulent strains of Xanthomonas campestris pv. vesicatoria in tomato (Lycopersicon esculentum Mill.). The ethylene-insensitive Never ripe (Nr) mutant displays increased tolerance to the virulent strain, while maintaining resistance to the avirulent strain. Expression of the ethylene receptor genes NR and LeETR4 was induced by infection with both virulent and avirulent strains; however, the induction of LeETR4 expression by the avirulent strain was blocked in the Nr mutant. To determine whether ethylene receptor levels affect symptom development, transgenic plants overexpressing a wild-type NR cDNA were infected with virulent X. campestris pv. vesicatoria. Like the Nr mutant, the NR overexpressors displayed greatly reduced necrosis in response to this pathogen. NR overexpression also reduced ethylene sensitivity in seedlings and mature plants, indicating that, like LeETR4, this receptor is a negative regulator of ethylene response. Therefore, pathogen-induced increases in ethylene receptors may limit the spread of necrosis by reducing ethylene sensitivity.     

Lipopolysaccharide-Defective Mutants of the Wilt Pathogen Pseudomonas solanacearum

Lipopolysaccharide (LPS)-defective mutants of Pseudomonas solanacearum were used to test the hypothesis that differences in LPS structure are associated with the ability or inability of different strains to induce a hypersensitive response (HR) in tobacco. To obtain these mutants, LPS-specific bacteriophage of P. solanacearum were isolated and used to select phage-resistant mutants of the virulent, non-HR-inducing strain K60. The LPS of 24 of these mutants was purified and compared with that of K60 and its HR-inducing variant, B1. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, LPS from K60 and other smooth strains separated into many evenly spaced bands that migrated slowly, whereas LPS from B1 and most phage-resistant strains separated into one to three bands that migrated rapidly. Carbohydrate analysis showed that the LPS of the phage-resistant strains lacked O-antigen sugars (rhamnose, xylose, and N-acetylglucosamine) and could be grouped into (i) those that had all core sugars (rhamnose, glucose, heptose, and 2-keto-3-deoxyoctonate), (ii) those that had no core rhamnose, and (iii) those that lacked all core sugars except for 2-keto-3-deoxyoctonate. The LPS composition of 10 of the rough, phage-resistant mutants was similar to that of the HR-inducing strain, B1, yet none of them induced the HR. Only 2 of 13 mutant strains tested caused wilting of tobacco, and these had rough LPS but produced large amounts of extracellular polysaccharide, unlike most LPS-defective mutants. The evidence did not support the hypothesis that the initial interaction between rough LPS and tobacco cell walls is the determining factor in HR initiation.     

Tropical Strains of Ralstonia solanacearum Outcompete Race 3 Biovar 2 Strains at Lowland Tropical Temperatures

Bacterial wilt, caused by members of the heterogenous Ralstonia solanacearum species complex, is an economically important vascular disease affecting many crops. Human activity has widely disseminated R. solanacearum strains, increasing their global agricultural impact. However, tropical highland race 3 biovar 2 (R3bv2) strains do not cause disease in tropical lowlands, even though they are virulent at warm temperatures. We tested the hypothesis that differences in temperature adaptation and competitive fitness explain the uneven geographic distribution of R. solanacearum strains. Using three phylogenetically and ecologically distinct strains, we measured competitive fitness at two temperatures following paired-strain inoculations of their shared host, tomato. Lowland tropical strain GMI1000 was only weakly virulent on tomato under temperate conditions (24°C for day and 19°C for night [24/19°C]), but highland tropical R3bv2 strain UW551 and U.S. warm temperate strain K60 were highly virulent at both 24/19°C and 28°C. Strain K60 was significantly more competitive than both GMI1000 and UW551 in tomato rhizospheres and stems at 28°C, and GMI1000 also outcompeted UW551 at 28°C. The results were reversed at cooler temperatures, at which highland strain UW551 generally outcompeted GMI1000 and K60 in planta. The superior competitive index of UW551 at 24/19°C suggests that adaptation to cool temperatures could explain why only R3bv2 strains threaten highland agriculture. Strains K60 and GMI1000 each produced different bacteriocins that inhibited growth of UW551 in culture. Such interstrain inhibition could explain why R3bv2 strains do not cause disease in tropical lowlands.     

Purification and characterization of Xanthomonas campestris pv. glycines exopolysaccharide

The exopolysaccharides (EPS) of virulent and avirulent strains of Xanthomonas campestris pv. glycines, causal agent of bacterial pustule disease of soybean, and one strain of the soybean non-pathogen X. c. pv. campestris were isolated, purified, and their compositions compared. EPS produced by X. c. pv. glycines in a completely defined medium appears to be identical to the well-characterized EPS produced by X. c. pv. campestris (commonly referred to as xanthan gum). The EPS of all strains was composed of the carbohydrates glucose, mannose and glucuronic acid with acetyl and pyruvyl substituents present. Permethylation analyses indicated EPS preparations had identical hexose substitution patterns. Avirulent strains of X. c. pv. glycines produced as much or more acidic EPS as did virulent strains in vitro. None of the EPS preparations were active as elicitors of the soybean pterocarpanoid phytoalexin glyceollin as determined by a soybean cotyledon bioassay.     

Gentamicin-Containing Peptone-Yeast Extract Medium for Cocultivation of Hartmannella vermiformis ATCC 50256 and Virulent Strains of Legionella pneumophila

We evaluated the use of peptone-yeast extract (PY) medium, different strains of Hartmannella vermiformis, and gentamicin in a coculture system to improve the discrimination of virulent and avirulent strains of Legionella pneumophila. H. vermiformis ATCC 50256 was unique among four strains of H. vermiformis, in that it multiplied equally well in Medium 1034 and PY medium (Medium 1034 without fetal calf serum, folic acid, hemin, and yeast nucleic acid and with a 50% reduction of peptone). However, both a virulent strain of L. pneumophila and its avirulent derivative strain multiplied in cocultures when PY medium was used. The multiplication of this avirulent strain was greatly reduced by incorporating gentamicin (1 (mu)g/ml) into the cocultivation system. Five virulent-avirulent sets of L. pneumophila strains were then tested for multiplication in cocultures with H. vermiformis ATCC 50256 and the gentamicin-containing PY medium. Only the virulent strains multiplied. The modified cocultivation system can discriminate between virulent and avirulent strains of L. pneumophila.     

Role of Trehalose Synthesis in Ralstonia syzygii subsp. indonesiensis PW1001 in Inducing Hypersensitive Response on Eggplant (Solanum melongena cv. Senryo-nigou)

Ralstonia syzygii subsp. indonesiensis (Rsi, former name: Ralstonia solanacearum phylotype IV) PW1001, a causal agent of potato wilt disease, induces hypersensitive response (HR) on its non-host eggplant (Solanum melongena cv. Senryo-nigou). The disaccharide trehalose is involved in abiotic and biotic stress tolerance in many organisms. We found that trehalose is required for eliciting HR on eggplant by plant pathogen Rsi PW1001. In R. solanacearum, it is known that the OtsA/ OtsB pathway is the dominant trehalose synthesis pathway, and otsA and otsB encode trehalose-6-phosphate (T6P) synthase and T6P phosphatase, respectively. We generated otsA and otsB mutant strains and found that these mutant strains reduced the bacterial trehalose concentration and HR induction on eggplant leaves compared to wild-type. Trehalose functions intracellularly in Rsi PW1001 because addition of exogenous trehalose did not affect the HR level and ion leakage. Requirement of trehalose in HR induction is not common in R. solanacearum species complex because mutation of otsA in Ralstonia pseudosolanacearum (former name: Ralstonia solanacearum phylotype I) RS1002 did not affect HR on the leaves of its non-host tobacco and wild eggplant Solanum torvum. Further, we also found that each otsA and otsB mutant had reduced ability to grow in a medium containing NaCl and sucrose, indicating that trehalose also has an important role in osmotic stress tolerance.     

Quinolobactin, a new siderophore of Pseudomonas fluorescens ATCC 17400, the production of which is repressed by the cognate pyoverdine

Transposon mutant strain 3G6 of Pseudomonas fluorescens ATCC 17400 which was deficient in pyoverdine production, was found to produce another iron-chelating molecule; this molecule was identified as 8-hydroxy-4-methoxy-quinaldic acid (designated quinolobactin). The pyoverdine-deficient mutant produced a supplementary 75-kDa iron-repressed outer membrane protein (IROMP) in addition to the 85-kDa IROMP present in the wild type. The mutant was also characterized by substantially increased uptake of (59)Fe-quinolobactin. The 75-kDa IROMP was produced by the wild type after induction by quinolobactin-containing culture supernatants obtained from the pyoverdine-negative mutant or by purified quinolobactin. Conversely, adding purified wild-type pyoverdine to the growth medium resulted in suppression of the 75-kDa IROMP in the pyoverdine-deficient mutant; however, suppression was not observed when Pseudomonas aeruginosa PAO1 pyoverdine, a siderophore utilized by strain 3G6, was added to the culture. Therefore, we assume that the quinolobactin receptor is the 75-kDa IROMP and that the quinolobactin-mediated iron uptake system is repressed by the cognate pyoverdine.     

Interaction of Pseudomonas solanacearum with Suspension-Cultured Tobacco Cells and Tobacco Leaf Cell Walls In Vitro

Attachment of radiolabeled Pseudomonas solanacearum cells to suspension-cultured tobacco cells and tobacco leaf cell walls was measured in vitro by a filtration technique that allowed separation of attached and unattached bacteria. An avirulent strain (B1) attached more rapidly to suspension-cultured cells than did the virulent parent strain (K60), and B1 attachment was less sensitive to inhibition by high ionic strength than was K60. Attachment of B1 bacteria to suspension-cultured cells and to leaf cell walls was comparable (50 to 70%), but only a small proportion (10 to 20%) of K60 bacteria attached to leaf cell walls under optimal conditions. With high bacterial populations (10⁸ bacteria per ml), attachment of K60 to suspension-cultured cells was greatly reduced. Attachment of both strains was completely inhibited by pretreating bacterial cells with heat (41°C) or azide and was partially inhibited by EDTA and kanamycin. The mechanism of attachment is not known, but ionic forces may be involved.     

Responses of halophytes to high salinities and low water potentials

Agrobacterium tumefaciens can induce tumors on thin slices which are excised from Jerusalem artichoke (Helianthus tuberosus) tubers and grown in culture on medium containing minerals and a carbon source. A comparative study was made of the kinetics of cell division in slices under three conditions: (a) slices which were untreated and showed only spontaneous (wound-induced) cell divisions; (b) slices treated with indoleacetic acid at several concentrations; and (c) slices treated with virulent or avirulent bacteria. The earliest spontaneous cell divisions were completed (as detected by the appearance of new daughter cell pairs) by about 3 hours. These cells divide only once. In indoleacetic acid-treated tissue, more cells divide, with the first cell pairs being detected slightly earlier than in slices not subjected to the hormone. The number of cells which divide is roughly proportional to auxin concentration. Tissue treated with virulent bacteria showed only the pattern of spontaneous cell division until about 72 hours, after which another burst of cell division commenced and continued indefinitely. The bacteria-induced growths produced the unusual amino acids which are characteristic of crown gall tumors. The percentage of slices with tumors was sharply reduced if certain avirulent A. tumefaciens strains were applied prior to virulent strains.     

phlF− mutant of Pseudomonas fluorescens J2 improved 2,4-DAPG biosynthesis and biocontrol efficacy against tomato bacterial wilt

Pseudomonas fluorescens J2 can produce 2,4-diacetylphloroglucinol (2,4-DAPG) as the main antibiotic compound and effectively inhibits the wilt pathogens Ralstonia solanacearum and Fusarium oxysporum. The phlF which negatively regulates the 2,4-DAPG synthesis in strain J2 was disrupted by homologous recombination to construct a mutant strain J2-phlF⁻. The mutant J2-phlF⁻ produced much more 2,4-DAPG and showed higher inhibitory effect on R. solanacearum than the wild type strain J2 in vitro. The mutant J2-phlF⁻ also showed more colonization of tomato roots and higher inhibition to R. solanacearum in soil than wild type strain J2. The biocontrol efficiency of mutant J2-phlF⁻ was higher against tomato bacterial wilt than wild type strain J2, but the differences were not significant. However, the application of both strains with organic fertilizer improved the colonization and biocontrol efficiency against tomato bacterial wilt and mutant strain J2-phlF⁻ showed higher biocontrol efficiency against tomato bacterial wilt than wild type strain J2. Both strains, J2 and J2-phlF⁻, could also promote the growth of tomato plants.     

Attachment of Agrobacteria to Grape Cells

The presence of the Ti plasmid favorably influences the attachment of agrobacteria to grape callus cells, especially during the early stages of a 2-h incubation. Agrobacterium strains attached to a similar extent to both the crown gall-resistant cultivar (Catawba), Vitis labruscana, and the crown gall-susceptible cultivar (Chancellor), Vitis sp. Attachment of the virulent strain to grape callus cells is blocked by the avirulent strain HLB-2 in both the tissue culture cell suspension and the seedling root systems.     

Inhibition of Giardia intestinalis by Extracellular Factors from Lactobacilli: an In Vitro Study

The aim of the present work was to evaluate the effect of spent culture supernatants of different strains of lactobacilli on giardia trophozoites. The growth of Giardia intestinalis strain WB, as well as the attachment to the human intestinal epithelial cell line Caco-2, was evaluated by using proliferation and adhesion assays with radiolabeled parasites. In addition, scanning electron microscopy and flow cytometric analysis were performed. The effect of spent culture supernatants from lactobacilli was strain dependent. Lactobacillus johnsonii La1 significantly inhibited the proliferation of G. intestinalis trophozoites. Although the effect was strongly pH dependent, it was not simply due to lactic acid. According to flow cytometric analysis, trophozoites were arrested in G₁ phase but neither significant necrosis nor apoptosis could be detected. Bacterial cells or their spent culture supernatants were unable to modify trophozoite attachment to Caco-2 cells. However, trophozoites treated with spent culture supernatants had little, if any, proliferative capacity. These results suggest that La1 produces some substance(s) able to inhibit proliferation of Giardia trophozoites. Partial characterization of the factors involved in the antigiardiasic action showed that they have a low molecular mass and are inactivated by heating. On this basis, it seems worthwhile to explore how colonization of the proximal small bowel with these lactic acid bacteria could interfere with giardiasis in vivo.     

Virulent and Avirulent Strains of Babesia bovis: The Relationship Between Parasite Protease Content and Pathophysiological Effect of the Strain

A virulent strain of Babesia bovis (“L” strain) was rendered avirulent by irradiation with 35 krads with a γ source. Another virulent strain of B. bovis (“C” strain) was made avirulent by rapid blood passage through 12 splenectomised calves. Both the parent virulent and their respective avirulent strains were injected into susceptible cattle. A nonfatal disease was observed in those intact cattle that had avirulent parasites; however, a fatal disease was produced in those animals that had received virulent parasites and in splenectomised calves that had received avirulent parasites. Blood kinin levels rose and plasma kininogen levels fell significantly in those animals infected with both virulent strains. Nonsignificant changes occurred with these parameters in animals infected with avirulent parasites. Preparations of disrupted parasites were obtained from the four parasite populations. Both virulent strains contained high levels of protease. The avirulent forms contained insignificant amounts. As parasite doubling times and maximum parasitaemias were the same for all four parasite populations, we conclude that these enzymes are not obligatory for parasite multiplication in the vertebrate host. Their role in producing pathological changes in the host is discussed.     

Study of the virulence of some mutants of Beauveria brongniartii (= Beauveria tenella)

Ten lipase-negative mutants from a lipase-positive virulent strain of Beauveria brongniartii were found to be avirulent, similar to lipase-negative strains isolated from nature. Two morphological mutants and four auxotrophic mutants which were lipase positive were also avirulent. Hypotheses to explain these results are presented.