Carotovora Var Research Articles

Antibacterial activity of native plants from Northwest Argentina against phytopathogenic bacteria

Extracts from aerial parts of medicinal plants from northwest Argentina were screened for antibacterial activity against the phytopathogenic strains namely CECT 124 (Pseudomonas corrugata), CECT 126 (P. syringae pv. tomato), CECT 225 (Erwinia carotovora var. carotovora), CECT 472 (Agrobacterium tumefaciens) and CECT 792 (Xanthomonas campestres pv. vesicatoria). Leaves and stems of Aspidosperma quebracho-blanco, Schinus fasciculatus, S. gracilipes, Amphilophium cynanchoides and Tecoma stans were separately extracted with solvents of increasing polarity to obtain the dichloromethane (fCH2Cl2), ethyl acetate (fEtAc) and methanol extracts (fMeOH), respectively. Among the thirty extracts tested, only fEtAc from leaves and stems of S. fasciculatus reached the IC50 against the five bacterial strains tested (IC50 = 0.9 mg/ml). The fEtAc from the leaves contained kaempferol, quercetin and agathisflavone which had moderate to strong antibacterial activity. This extract and its identified flavonoids showed synergic (CECT 124,126 and 792) or additive effects (CECT 472 and 225) in mixtures with Kocide 3000.

Antimicrobial Activity of Some Plant and Algal Extracts

The use of advanced, effective and less toxic antimicrobial agents are required for the treatment of plant pathogens. In this study, hexane and ethanol extracts of two plants (Sesbania sesban and Cymbopogon citratus) and two algae species (Spirulina platensis and Scendusmus sp.) were screened for their antifungal and antibacterial activity on eight fungi (Rhizoctonia solani, Rhizopus stolonifer, Fusarium oxysporum, Aspergillus niger, Alternaria solani, Pythium debarianum, Botrytis cinerea and Penicillium digitatum) and two species of bacteria (Agrobacterium tumefaciens and Erwinia carotovora var. carotovora). Extracts of these plants and algae showed varied levels of antifungal and antibacterial activity. It was found that, the ethanol extracts of S. sesban plant and S. platensis alga had a high activity on tested fungi. On the other hand, the ethanol extracts of Scendesmus sp. alga and S. sesban plant showed the highest antibacterial activity against tested bacteria. The ethanol extract of S. sesban was the most potent fungitoxic extract against both A. niger and A. solani fungi with effective concentration (EC50) values of 0.135 and 0.011mg/L, respectively. On contrary, Scendesmus sp. ethanol extract revealed the highest inhibitory effect against E. carotovora var. carotovora bacteria. Conference Proceeding Full Paper Abbassy et al.; IJPSS, Article no. IJPSS.2014.10.014 1367

Composition and antimicrobial activity of essential oils isolated from Egyptian plants against plant pathogenic bacteria and fungi

The essential oils of eighteen Egyptian plants, namely, Artemisia judaica, A. monosperma, Callistemon viminals, Citrus aurantifolia, C. lemon, C. paradisi, C. sinensis, Cupressus macrocarpa, C. sempervirens, Myrtus communis, Origanum vulgare, Pelargonium graveolens, Rosmarinus officinalis, Syzygium cumini, Schinus molle, S. terebinthifolius, Thuja occidentalis and Vitex agnus-castus, were isolated by hydrodistillation. The chemical composition of the isolated oils was identified by gas chromatograph/mass spectrometer (GC/MS). The major constituents of the isolated oils were limonene (40.19%, 56.30%, 74.29% and 89.23% in C. aurantifolia, C. lemon, Citrus paradise and C. sinensis, respectively), α-pinene (37.88%, 35.49%, 26.16% and 17.26% in C. sempervirens, T. occidentalis, M. communis and S. cumini, respectively), 1,8-cineole (71.77% and 19.60% in C. viminals and R. officinalis), pulegone (77.45% in O. vulgare), β-thujone (49.83% in A. judaica), capillene (36.86% in A. monosperma), sabinene (14.93% in S. terebinthifolius), α-phellandrene (29.87% in S. molle), 4-terpeneol (20.29% in C. macrocarpa), trans-caryophyllene (15.19% in V. agnus-castus) and β-citronellol (35.92 in P. graveolens). The isolated oils were tested for their antimicrobial activity against the most economic plant pathogenic bacteria of Agrobacterium tumefaciens and Erwinia carotovora var. carotovora, and fungi of Alternaria alternata, Botrytis cinerea, Fusarium oxysporum and Fusarium solani. The isolated oils showed variable degree of antibacteabril activity against A. tumefaciens and E. carotovora var. carotovora. Based on minimum inhibitory concentration (MIC) values, the oils were more effective against E. carotovora var. carotovora than A. tumefaciens. The oil of T. occidentalis revealed the highest antibacterial activity among the tested oils showing the lowest MIC values of 400 and 350mg/L, on A. tumefaciens and E. carotovora var. carotovora, respectively. In mycelial growth inhibition assay, most of the essential oils showed pronounced effect and the oil of A. monosperma was the most potent inhibitor with EC50=54, 111, 106 and 148mg/L against A. alternata, B. cinerea, F. oxysporum and F. solani, respectively. On the other hand, the oils caused strong reduction in spore germination of fungi compared with control. The oils of A. judaica and A. monosperma caused the highest spore germination inhibition of F. oxysporum and their EC50 values were 69 and 62mg/L, respectively. Among the tested fungi, F. oxysporum was the most susceptible fungus to all of the tested oil except the oil of S. molle. The relationship between the antimicrobial activity and the chemical composition of the isolated oils was disclosed. The findings of the present study suggest that the isolated oils have a potential to be used as antimicrobial agents.

Screening of Antagonistic Bacterium to Control Konjac Soft Rot

Konjac soft rot is a bottleneck limiting konjac yield caused by bacterial strain of Erwinia carotovora var. carotovora. In order to control konjac soft rot, soil samples were collected, and each sample was spread on surface of a plate seeded with E. carotovora var. carotovora in advance. Strains expressing antagonistic activities were selected and then isolated with streak plate method. One bacterial strain (named Z10) was obtained from soil by this method. In field trials, strain Z10 still showed antagonistic effect against the bacterial pathogen.

Evaluation of antibacterial properties and biochemical effects of monoterpenes on plant pathogenic bacteria

The antibacterial activity of twelve monoterpenes, namely camphene, (R)-camphor, (R)-carvone, 1,8-cineole, cuminaldehyde, (S)-fenchone, geraniol, (S)-limonene, (R)-linalool, (1R,2S,5R)-menthol, myrcene and thymol was tested against two plant pathogenic bacteria Agrobacterium tumefaciens and Erwinia carotovora var. carotovora using agar dilution method. For a better understanding of monoterpenes mechanisms of action, the inhibitory effect of three monoterpenes (R)-linalool, myrcene and thymol was assessed ondehydrogenases and polyglacturonase activities. Among the tested monoterpenes, thymol, (S)-limonene and myrcene were the most potent antibacterial compounds againstA. tumefaciens with minimum inhibitory concentration (MIC) of 1000 mg/L. Thymol was also the most effective compounds against E. carotovora var. carotovora, while camphene, cunimaldhyde and 1,8-cineole were the less effective compounds against both bacteria. In biochemical studies, myrcene caused the highest inhibitory effect on dehydrogenases activity of the two tested bacteria, followed by thymol. However, thymol showed the highest inhibitory effect on polygalacturonase activity of both tested bacteria, followed by (R)-linalool. In general, there was a positive correlation between the antibacterial activity of monoterpenes and their inhibitory effects on both enzymes. This is the first report for the determination of MIC and enzymes inhibitory effects of tested monoterpenes on plant pathogenic bacteria. Key words: Monoterpenes, antibacterial activity, plant pathogenic bacteria,dehydrogenases, polyglacturonase

Antibacterial screening of some essential oils, monoterpenoids and novel N-methyl carbamates based on monoterpenoids against Agrobacterium tumefaciens and Erwinia carotovora

A comprehensive evaluation of 13 plant essential oils namely: caraway, chenopodium, cinnamon, clove, eucalyptus, garlic, geranium, lemon, matrecary, peppermint, rose, rosemary and thyme and 14 of their monoterpenoidal constituents: borneol, camphor, carvacrol, carveol, carvone (R and S), chlorothymol, cineol, cinnamaldehyde, citronellol, eugenol, geraneol, menthol, and thymol was investigated for their antibacterial activity against the two phytopathogenic bacteria, Agrobacterium tumefaciens and Erwinia carotovora var carotovora. Trials for improving the antibacterial activity by either structure modification or mixing with the three synergists: piperonyl butoxide, Triton X-100 and Tween 20 were achieved. New N-methyl carbamate derivatives based on monoterpenoids were introduced. Cinnamon, clove, chenopodium, caraway, rosemary and thyme oils proved to possess good antibacterial activity. Thymol, chlorothymol, and carvacrol were potent against the tested bacteria and quite close to the effect of the standard. Conversion of menthol into its carbamate derivative gave good enhancement in the bactericidal activity at all concentrations compared to menthol itself. Also, the carbamate derivatives of β-citronellol and borneol improved the bactericidal activity against E. carotovora, but decreased it against A. tumefaciens. Synergistic antibacterial activity of the tested monoterpenoids was observed when combined with each of the three synergists particularly with Triton X-100.

Affinity purification of a siderophore that exhibits an antagonistic effect against soft rot bacterium

Bacterial colonies were isolated from different Egyptian soil samples. From these isolates, one bacterial species was found to produce siderophore. Using classical and biochemical identification methods, the siderophore producing isolate was identified as Pseudomonas fluorescens. Based on the affinity of siderophores for metal ions, an affinity chromatography system was designed for the purification of the siderophore in one step. It was possible to isolate 25 mg siderophore per liter of culture media. The purified siderophore was found to exist in two forms of approximately 30 and 90 kD. They are believed to be polymers of several siderophore molecules. Both forms were found to be active against the pathogen Erwinia carotovora var. carotovora, the causal bacteria of soft rot disease on potato tubers. The advantage of this method over other purification methods is that it uses metal ion so it can be applied for the purification of the known types of siderophores. Moreover, the purification is based on affinity chromatography, so the siderophore purity state permits several biotechnological applications without further treatments.

Serodiagnosis of onion soft rot bacterium (Erwinia carotovora var. carotovora) from onion seeds

Studies on the detection of the seed-borne nature of bacterium revealed that bacterial oozing from seed contains Erwinia carotovora var. carotovora and it was confirmed by biochemical studies. Polyclonal antibodies were raised against unfractionated protein of E. carotovora var. carotovora by immunizing in New Zealand white rabbit. DAS-ELISA was carried out to determine the titre value of antibody against antigen from different genotypes at 405 nm absorbance. The studies on the seed infection by soft rot bacteria revealed that the higher titre value compared to disease-free seed materials. Western blot analysis confirmed the presence of 40 kDa bacterial protein, which was isolated from onion seed.

Some Biochemical Changes in Carrot Roots Induced by Erwinia carotovora var. carotovora

Carrot is a rich source of nutrients. Carrots contains carotene and lycopene, which gives bright color to the roots. The quality of the carrots was assessed based on the carotene, lycopene, and other biochemical constituents such as sugars, starch, and protein. To study the effect of various isolates of the Erwinia carotovora var. carotovora on the above biochemical constituents, the pathogens were inoculated and the contents were analyzed separately at 1, 2, 3, 4, and 5 days after inoculation. The contents of ß-carotene increased significantly due to all the three isolates of the pathogen and the Coimbatore isolate recorded highest of 36.03%. The same trend was also observed in the lycopene content, with 93.55% increase over control. The contents of total and reducing sugars were found to significantly increase due to inoculation with the pathogen. The starch content showed a decreasing trend in all the isolates tested. The maximum reduction of 62.98% was observed in the roots inoculated with Coimbatore isolate. The protein content showed a decreasing trend up to 5th day of inoculation, and further reduction of about 25.45% was recorded with Coimbatore isolate on the 5th day. The total phenol content in the roots of carrot decreased significantly, and reached the least on 5th day due to the infection by all the three isolates and the maximum reduction of 22.79% was observed in roots treated with Coimbatore isolate.

Antimicrobial activity of glycosidase inhibitory protein isolated from Cyphomandra betacea Sendt. fruit

Broad-spectrum antimicrobial activity of an invertase inhibitory protein (IIP) isolated from Cyphomandra betacea ripe fruits is documented. Minimal inhibitory concentration (MIC) values were determined by agar macrodilution and broth microdilution assays. This IIP inhibited the growth of xylophagous and phytopatogenic fungi ( Ganoderma applanatum, Schizophyllum commune, Lenzites elegans, Pycnoporus sanguineous, Penicillium notatum, Aspergillus niger, Phomopsis sojae and Fusarium mango) and phytopathogenic bacteria ( Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae pv. syringae and Erwinia carotovora var carotovora). The IIP concentration required to completely inhibit the growth of all studied fungi ranged from 7.8 to 62.5 μg/ml. Phytopatogenic bacteria were the most sensitive, with MIC values between 7.8 and 31.25 μg/ml. Antifungal and antibacterial activities can be associated with their ability to inhibit hydrolytic enzymes. Our results indicate the possible participation of IIP in the plant defense mechanism and its potential application as a biocontrol agent against phytopathogenic fungi and bacteria.

Hydrogen peroxide regulates elicitor PB90-induced cell death and defense in non-heading Chinese cabbage

PB90, a protein elicitor secreted by Phytophthora boehmeriae, can induce a hypersensitive response and systemic acquired resistance in tobacco. In this study, we first developed a novel plant-elicitor interaction system between PB90 and non-heading Chinese cabbage, and then reported PB90-induced programmed cell death and defense regulated by hydrogen peroxide (H 2O 2) in the plant leaves. The PB90-induced cell death shared common morphological characteristics of apoptosis with mammalian cell death, including rapid chromatin condensation and cytoplasmic shrinkage, suggesting that cell death is a programmed occurrence. At the same time, elicitor treatment strongly activated and enhanced phenylalanine ammonia-lyase (PAL) activity. PB90 treatment also resulted in enhanced PR-1 and Chitinase expression, as evaluated by a reverse transcriptase–polymerase chain reaction, concomitant with enhanced resistance of plants to infection by Colletotrichum higginsianum and Erwinia carotovora var. carotovora. Moreover, a PB90-induced oxidative burst, the result of H 2O 2 accumulation over approximately 3 h, preceded PB90-triggered cell death by 12 h post-infiltration with PB90. Infiltration with antioxidants (DTT, glutathione, and ascorbic acid) and DPI, an inhibitor of NADPH oxidase, into leaves 30 min before treatment with PB90 significantly suppressed PB90-induced cell death and expression of PR-1 and Chitinase. We therefore, provided evidence that a PB90-induced H 2O 2 burst triggered programmed cell death and defense in non-heading Chinese cabbage.

Survial and Management of onion soft rot caused by Erwinia carotovera var. carotovora

Studies on the nature and extent of bacterial inoculum in storage godowns of onion revealed the occurrence of E. carotovora var carotovora in rootlets, gunny bags, godown sweeping and leaf debris and infected bulb in the field which may serve as source of inoculum. E. carotovora var carotovora survived better in the combination of sterilized soil + leaves + bulbs, which retained more number of bacteria even after six months of inoculation. Half of the inoculum load was detected in the unsterilized soil + leaves + bulbs. Studies on the detection of seed-borne nature of bacterium revealed that bacterial oozing from seed contains E. carotovora var. carotovora. Polyclonal antibodies were raised against unfractionated protein of E. carotovora var. carotovora. Indirect ELISA was carried out to detect the antigen and it was found that 1:1000 dilution of antibodies was necessary to detect the antigen. Seed treatment with Pseudomonas fluorescens FP7 was effective in checking the seed-borne nature of E. carotovora var. carotovora besides increasing vigour index of seedlings. Formalin at two per cent concentration also checked the growth of E. carotovora var. carotovora.

Biochemical methods for the detection of Erwinia carotovora varcarotovora from onion seeds

Erwinia carotovora var. caotovora causes soft rot of onion bulbs in storage.. Three genotypes (N 53, L3V1T2C2, ACSP1) were tested for the presence of soft rot bacterium in seed by roll towel and blotter paper method. A higher per centage of oozing was observed in ACSP1 (81.0 and 83.2 per cent). Studies on morphological character revealed that the causal agent was E. cartovora var carotovora. The biochemical tests. were carried out to identify the causal agents. The bacterium was identified as Erwinia carotovora var. carotovora and pathogenicity was proved. The seed borne nature of E. carotovora var. carotovora was proved.

Inhibition of Hydrolytic Enzyme Activities and Plant Pathogen Growth by Invertase Inhibitors

The invertase inhibitory protein isolated from Cyphomandra betacea Sendt and Solanum tuberosum inhibited the invertase activity from different species, genera and even plant family. Furthermore, proteinaceous inhibitors are not invertase specific; fungal, bacterial and higher plant enzymes including polygalacturonase, pectinase, pectin lyase, α - l -arabinofuranosidase and β -glucosidase are also shown to be inhibited. Both inhibitors exhibited an in vitro antibacterial action against phytopathogenics strains of Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae and Erwinia carotovora var carotovora.

Bactericide-like effect of Lupinus alkaloids

The bactericidal effect of lupine alkaloids against Pseudomonas syringae P.V. phaseolicola; Pseudomonas syringae P.V. tomato; Pseudomonas putida; Erwinia carotovora var. carotovora was tested. Crude alkaloids extracts from Lupinus albus (L.) and Lupinus luteus (L.) containing mainly lupanine and lupinine, respectively, were assayed at different concentrations. The individual bactericidal effects of sparteine and gramine were also analyzed. The results indicated that lupinine showed the highest bactericidal effect on the four bacteria studied, as concentrations between 5 and 10 mM are needed to stop the bacteria growth completely. Gramine could be also considered effective in the control of P. phaseolicola and P. tomato. In both cases the growth is completely inhibited by concentrations lower than 10 mM. The possible bactericidal effect of Lupinus alkaloids is discussed.

Populations of endophytic bacteria which influence host-resistance to Erwinia-induced bacterial soft rot in potato tubers

The ability of endophytic bacteria to influence Erwinia carotovora var. atroseptica (Eca) growth and disease development was examined in potatoes. Bacterial populations isolated from within the tubers of five potato (Solanum tuberosum L.) cultivars (Kennebec, Butte, Green Mountain, Russet Burbank and Sebago) showed antibiosis toward Eca in an in vitro assay. Sebago was host to the highest percentage of bacterial isolates inhibiting Eca growth in vitro (49.5%), followed by Green Mountain (33.3%), Kennebec (29.3%), Russet Burbank (12.9%) and Butte (1.8%). Of these, Curtobacterium luteum was the most common species. Few endophytic bacteria from Butte were inhibitory to Erwinia; all were from Pantoea agglomerans. Significantly higher populations of Erwinia-inhibiting bacteria were recovered from Kennebec (1.89 × 106 cfu fresh weight tuber tissue) as compared to the other cultivars; the lowest populations were recovered from Butte (0.01 × 106 cfu per g fresh weight tuber tissue). Published levels of cultivar disease resistance to blackleg did not correspond to actual bacterial soft rot development (induced by Eca) in an in vivo (tuber) assay. However, bacterial soft rot development was negatively correlated with the density of tuber populations of endophytic bacteria found able to inhibit Eca growth in vitro (R=−0.879, p=0.05).

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

AbstractAn 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.

Production of volatile metabolites in potatoes infected byErwinia carotovoravar.carotovoraandE. carotovoravar.atroseptica

Gas chromatography of headspace atmospheres was used to evaluate the production of metabolic volatiles by small lots of stored potato tubers (Solanum tuberosum) infected with either Erwinia carotovora var. carotovora or Erwinia carotovora var. atroseptica bacteria. The total concentration of volatiles in the headspace of the plastic bags in which the potatoes were maintained increased exponentially as infection by both E. carotovora varieties developed. Temperature-related differences in the growth rates of the E. carotovora varieties were reflected in minor differences in the patterns of volatiles from the infected material. There were no detectable differences in the range of volatile metabolites identified in the headspace profiles of the E. carotovora infections and only one metabolite was produced at a significantly different rate by the two host-pathogen combinations.

Interaction ofErwinia spp. andFusarium roseum ‘Sambucinum’ on the Russet Burbank potato

Laboratory and field studies with the Russet Burbank (RB) potato provide evidence for synergism betweenErwinia carotovora var. atroseptica (Ea) andFusarium roseunt ‘Sambucinum’ (Fs). When these pathogens were inoculated together, the severity of tuber rot was significantly greater than when either pathogen was inoculated separately. Similarly, these pathogens interacted to reduce yield. When both organisms were uniformly applied to puncture wounds on potato seed (inoculum suspension consisted of 108 cells/ml Ea and 105 cells/ml Fs), the total yield was reduced by 46% and U.S. #1 yield by 53%. These reductions occurred even though blackleg symptoms (caused by Ea) were negligible (< 1%). In contrast, inoculations withErwinia carotovora var.carotovora (Ec) and Fs did not interact to reduce potato yield. Potato yields were also not influenced when these pathogens (Ea, Ec, Fs) were separately inoculated.Verticillium wilt (Verticillium dahliae) was significantly less when tubers were inoculated with either Ea, Ec, Fs, Ea + Fs, or Ec + Fs than with uninoculated tubers.

Variations in Protein Content Due to Proteolytic Enzyme Activities and Their Influence on Susceptor Resistant Response in Maize — Erwinia carotovora var. chrysanthemi Host-Pathogen System

Summary The susceptibility of maize varieties ‘Vijay’, ‘Popcorn’, and ‘Swan’ towards the three isolates ‘Columbia’, ‘Hawaii’, and ‘Wisconsin’ of Erwinia carotovora var. chrysanthemi was due to increase or decline of protein content, enhanced proteolytic enzyme activities, and increase or diminution of total amino acid output in stalk- and leaf-tissues of middle- and old-age-group of plants. The interaction of the three activities was at the summit 16 d after infection in all the three age groups of plants. The heightened proteolytic enzyme activity with concurrent release of amino components as nutrients sustained susceptibility. In younger tissues of these cultivars such a response was maintained through restricted release of amino acids, specially those which were inhibitory to the 3 isolates of the pathogen. This was apparent especially in variety ‘G.S.2’, where protein accumulated in infected tissues with simulate lessening of excreted amino compounds. Here the protein incorporated in itself all such free amino acids which acted as inhibitors. The resistance in young stalk- and leaf-tissues of ‘Pant-7421’ variety and young and mature tissues of ‘Pool’ variety was engineered through decline in the total content of nutritionally utilizable free amino acids. The resistant response of middle- and old-age-group of tissues of variety ‘Pant’ and the middle aged tissue of variety ‘Pool’ was conducted through diminution of protein, accompanied with vigorous proteolytic enzyme activity and high level of total amino acid output, which acted as repressor to the bacterium during host-pathogen interplay.