Bacterial Canker of Sweet Cherry (Prunus avium) in Poland

In Polish climatic conditions cherry cankers resulting from infection by Pseudomonas morsprunorum continue development in summer, but the rate may be slower than in the period from April to June. However, from the well established, active canker developing under the dwarf shoot of the susceptible variety‘Hedelfińska’in July no P. morsprunorum were isolated. On the other hand, numerous strains of the genus Erwinia were found there which caused a hyper sensitivity reaction (HR) on tobacco leaves. From the cracks on the current-year cherry shoots, due to fresh infection and from symptomless leaves P. morsprunorum strains were isolated, always accompanied by those of the Erwinia genus in the approximate proportion 1: 1 or 1:2. The strains of Erwinia genus seemed similar to the DC and YC strains isolated by BILLING and BAKER from pear and apple trees infected by fireblight and also to the strains in group III of the bacteria isolated by CROSSE from cherry leaves. In two tests (immediately after isolation and after 4 months of storage) the strains of the Erwinia genus and 3 nonidentified isolates induced HR. At a third test, after 10 months of preservation these strains were HR-negative in contrast to the P. morsprunorum isolates. The fact that strains of the Erwinia genus inducing HR were isolated in large numbers from active cherry canker where pathogenic bacteria were not detected, may indicate that they play some role in the development of cherry tree canker.     

The ice Nucleation Activity of Pseudomonas fluorescens Migula and its Inhibition by Various Chemicals

The ice nucleation activity (INA) of three strains of Pseudomonas fluorecens, nos 553, 554 and 606, isolated by the Institute for Pathogen Diagnostics in Ascherleben, Germany, was determined. Under equal growth conditions and at given test temperatures the ice nucleation frequency spectra of the isolates differed slightly. The fraction of cells which acted as ice nuclei increased with falling temperatures. Below ?5°C the nucleation frequency rose from 10^-8 to 10^-3. Between, 0 and ?10°C only a fraction of approximately 2 to 5 × 10^-3 cells performed ice nucleation activity. Fifteen newly synthesized chemicals showed no or only a very slight intrinsic INA at ?5°C and ?7°C. The compounds were used as antinucleators against INA-exhibiting bacteria. In INA-exhibiting suspensions of isolate 553 bacterial ice nuclei were reduced after treatment with the 15 compounds. Dependent on the compounds, a nucleation frequency of ?8.32 to ?5.10 was detected at ?5°C. At ?7°C, the frequency amounted to ?7.89 to ?5.05. As the temperature was lowered to ?10°C in bacterial suspensions which were treated with 9 (of the 15) compounds, a remainder of 1.79 to 5.91 × 10^-6 cells retained ice nucleation activity. The most pronounced inhibitory effect was noted for the compounds 1989/6255, 1989/6436 and 1990/6158. In a 10-fold dilution of isolate 553 the compound 1989/6153 inhibited ice nucleation between 0 and 10°C so strongly that it was about 100 times below the control. The ‘tube-freezing’ method showed that on excised corn leaves treated with 1989/6259 and 1990/6155, the bacterial INA decreased while the super-cooling was more pronounced. ‘Frostgard’, 1986/6205, 1986/6199 and 1989/6259 inhibited most INA-exhibiting bacteria on corn seedlings. Compared to inoculated plants, a significantly higher percentage of treated plants survived at ?2 and ?3°C.     

Curium(III) Speciation Studies with Cells of a Groundwater Strain of Pseudomonas fluorescens

Ubiquitous Pseudomonads have great potential to influence the speciation and mobility of actinides in the environment. This study explores the unknown interaction between curium(III) and cell-suspensions of Pseudomonas fluorescens (CCUG 32456) isolated from the Äspö site, Sweden. The interaction between curium(III) and P. fluorescens cells was studied at trace curium(III) concentrations (0.3 μM) using time-resolved laser-induced fluorescence spectroscopy. Extraction studies have shown that the biosorption of curium(III) is a reversible process. Two Cm³⁺?P. fluorescens (CCUG 32456) species were identified, R?O?PO₃H?Cm²⁺ and R?COO?Cm²⁺, having emission maxima at 599.6 and 601.9 nm, respectively. The corresponding surface complexation constants were determined to be log β₁₁₁ = 12.7 ± 0.6 and log β₁₁₀ = 6.1 ± 0.5, respectively.     

Genus- and Isolate-Specific Real-Time PCR Quantification of <Emphasis Type="Italic">Erwinia</Emphasis> on Leaf Surfaces of English Oaks (<Emphasis Type="Italic">Quercus robur</Emphasis> L.)

In order to quantify pathogenic epiphytic bacteria on leaf surfaces of the important European forest tree Quercus robur without time-intensive cultivation and separation of microorganisms, methods were developed to selectively quantify DNA copy numbers of the genus Erwinia in DNA isolated from the leaf surface. By using the combination of the two different real-time PCR techniques SYBR®-Green and TaqMan®, methods were developed not only to allow quantification of the total DNA copy number of Erwinia on the oak leaf surface, but also to distinguish between two significantly different groups of Erwinia strains. In the present work, these techniques were successfully applied to quantify the copy number of the genus Erwinia and its subgroups compared with the total bacteria number in DNA samples extracted from the upper leaf surface of English oaks collected on the 4^th of June 2001 (Julian day 155). Received: 24 June 2002 / Accepted: 25 October 2002     

Halorubrum rubrum sp. nov., an extremely halophilic archaeon from a Chinese salt lake

Two halophilic archaeal strains, YC87^T and YCA11, were isolated from Yuncheng salt lake in Shanxi, China. Cells of the two strains were observed to be pleomorphic rod-shaped, stained Gram-negative and produced red-pigmented colonies. Strain YC87^T was able to grow at 20–50 °C (optimum 37 °C), at 1.4–4.8 M NaCl (optimum 2.1 M NaCl), at 0.05–1.0 M MgCl₂ (optimum 0.3 M MgCl₂) and at pH 6.0–9.0 (optimum pH 7.0) while strain YCA11 was able to grow at 20–50 °C (optimum 37 °C), at 2.1–4.8 M NaCl (optimum 3.1 M NaCl), at 0.01–0.7 M MgCl₂ (optimum 0.1 M MgCl₂) and at pH 6.0–9.0 (optimum pH 7.5). The cells of both isolates were observed to lyse in distilled water. The minimum NaCl concentrations that prevented cell lysis were determined to be 8 % (w/v) for strain YC87^T and 12 % (w/v) for strain YCA11. The major polar lipids of the two strains were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and one major glycolipid chromatographically identical to sulfated mannosyl glucosyl diether; another major glycolipid and trace amounts of several unidentified lipids were also detected. The 16S rRNA gene sequences of the two strains were 99.8 % identical, showing 93.2–98.2 % similarity to members of the genus Halorubrum of the family Halobacteriaceae. The rpoB′ gene similarity between strains YC87^T and YCA11 was 99.3 % and showed 87.5–95.2 % similarity to the closest relative members of the genus Halorubrum. The DNA G+C content of strains YC87^T and YCA11 were determined to be 64.9 and 64.5 mol%, respectively. The DNA–DNA hybridization value between strain YC20^T and strain YC77 was 87 % and the two strains showed low DNA–DNA relatedness with Halorubrum cibi JCM 15757^T and Halorubrum aquaticum CGMCC 1.6377^T, the most related members of the genus Halorubrum. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strains YC87^T and YCA11 represent a novel species of the genus Halorubrum, for which the name Halorubrum rubrum sp. nov. is proposed. The type strain is YC87^T (=CGMCC 1.12124^T = JCM 18365^T).     

Genotypic differences in the uptake and distribution of radiocaesium in plants

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).     

Statistical evaluation and modeling of cheap substrate-based cultivation medium of Chlorella vulgaris to enhance microalgae lipid as new potential feedstock for biolubricant

Chlorella vulgaris (C. vulgaris) microalga was investigated as a new potential feedstock for the production of biodegradable lubricant. In order to enhance microalgae lipid for biolubricant production, mixotrophic growth of C. vulgaris was optimized using statistical analysis of Plackett–Burman (P-B) and response surface methodology (RSM). A cheap substrate-based medium of molasses and corn steep liquor (CSL) was used instead of expensive mineral salts to reduce the total cost of microalgae production. The effects of molasses and CSL concentration (cheap substrates) and light intensity on the growth of microalgae and their lipid content were analyzed and modeled. Designed models by RSM showed good compatibility with a 95% confidence level when compared to the cultivation system. According to the models, optimal cultivation conditions were obtained with biomass productivity of 0.123 g L^?1 day^?1 and lipid dry weight of 0.64 g L^?1 as 35% of dry weight of C. vulgaris. The extracted microalgae lipid presented useful fatty acid for biolubricant production with viscosities of 42.00 cSt at 40°C and 8.500 cSt at 100°C, viscosity index of 185, flash point of 185°C, and pour point of ?6°C. These properties showed that microalgae lipid could be used as potential feedstock for biolubricant production.     

Bacterial degradation of styrene in waste gases using a peat filter

A biofiltration process was developed for styrene-containing off-gases using peat as filter material. The average styrene reduction ratio after 190 days of operation was 70% (max. 98%) and the mean styrene elimination capacity was 12 g m⁻³ h⁻¹ (max. 30 g m⁻³ h⁻¹). Efficient styrene degradation required addition of nutrients to the peat, adjustment of the pH to a neutral level and efficient control of the humidity. Maintenance of the water balance was easier in a down-flow than in an up-flow process, the former consequently resulting in much better filtration efficiency. The optimum operation temperature was around 23 °C, but the styrene removal was still satisfactory at 12 °C. Seven different bacterial isolates belonging to the genera Tsukamurella, Pseudomonas, Sphingomonas, Xanthomonas and an unidentified genus in the γ group of the Proteobacteria isolated from the microflora of active peat filter material were capable of styrene degradation. The isolates differed in their capacity to decompose styrene to carbon dioxide and assimilate it to biomass. No toxic intermediate degradation products of styrene were detected in the filter outlet gas or in growing cultures of isolated bacteria. The use of these isolates in industrial biofilters is beneficial at low styrene concentrations and is safe from both the environmental and public health points of view. Received: 30 May 1997 / Received revision: 22 August 1997 / Accepted: 25 August 1997     

Isolation,Selection and Characterization of Root-Associated Rock Phosphate Solubilizing Bacteria in Moroccan Wheat (Triticum aestivum L.)

Abstract

Phosphorus (P) is the most important macronutrient next to nitrogen for the growth and development of plants. But often unavailable for plants because of its high reactivity with many soil constituents. Thus, the use of phosphate solubilizing bacteria (PSBs) as biofertilizers seems to be an effective way to resolve the soluble phosphorus availability in soil. The present study was conducted to isolate and characterize rock PSB associated with the rhizosphere of wheat (Triticum aestivum L.) from fourteen different wheat-growing sites of Meknes region in Morocco. A total of one hundred ninety-eight (198) rock PSBs were isolated employing NBRIP medium amended with rock phosphate (RP), out of which five strains (A17, A81, B26, B106, and B107) were selected for their strong ability to dissolve RP and were tested in vitro for plant growth-promoting (PGP) traits including production of indole acetic acid (IAA), siderophores, hydrogen cyanide (HCN), and antifungal activity, as well as their response to the effect of extrinsic and intrinsic stress. The 16S rRNA gene sequencing and phylogenetic analysis identified these isolates belong to four genera, Pantoea, Pseudomonas, Serratia, and Enterobacter. The phosphate solubilization index (SI) of selected isolates ranged between 2.3 and 2.7, and the amount of solubilized phosphorus in the liquid medium varied from 59.1 to 90.2 µg mL^?1. HPLC analysis revealed that all the selected isolates produced multiple organic acids (oxalic, citric, gluconic succinic, and fumaric acids) from glucose under aerobic conditions. Except for the A81 strain, all selected isolates were able to produce IAA ranging between 2.9 and 21.2 µg mL^?1. The isolates A17, B26, and B107 showed the ability to produce siderophores ranging from 79.3 to 20.8% siderophore units. Only two strains (A17 and B26) were able to produce HCN. All selected isolates showed good resistance against different environmental stresses like 10–50?°C temperature, 0.5–2?g L^?1 salt concentration and 4.5–9?pH range, and against different antibiotics. The antagonistic effect showed that among the five selected strains, only two strains (B26 and A17) were able to suppress the growth of tested fungi. This study clearly indicates that our selected rock PSBs can be used as biofertilizers for grain crops after studying their interaction with the host crop and field evaluation.     

Presence of Quorum-Sensing-Mediated Gene Regulation in Pathogenic Ice-Nucleation-Active (INA) Bacteria

Nine out of a total of 20 pathogenic ice-nucleation-active bacteria, with different levels of inducible INA, were tested and found positive for their ability to synthesize quorum-sensing (QS) signals. The bacteria were isolated from willow plants and belonged to the genera Bacillus, Erwinia, Pseudomonas and Sphingomonas. As reporter bacteria, to detect the homoserine lactone (HSL) autoinducer, Agrobacterium tumefaciens, Chromobacterium violaceum, Pseudomonas aeroginosa, Aeromonas hydrophila and Vibrio fischeri strains were used. We thus provide evidence that pathogenic ice-nucleation bacteria with inducible INA produce QS signals that in other bacteria have been shown to be in the control of genes of importance for pathogenicity.     

Oxidative degradation of Amaranth dye by a new genus Kerstersia sp.

A dye-decolorizing bacterium was isolated from a coconut coir sample and identified as a new genus Kerstersia sp. by various biochemical tests and 16S rRNA gene sequencing. This bacterium was capable of degrading sulfonated azo dye Amaranth aerobically at 40?°C and pH 7.0. Tests conducted on intracellular crude enzyme extract identified an oxygen insensitive azoreductase. The optimum dye-decolorizing activity at pH 7.0 and 40?°C for the decolorization of dye was 0.091?U mL^?1 (μ_max 0.522?mg h^?1). The K_s 104.51?μM^?1 has been evaluated by plotting Lineweaver–Burk plot for the Amaranth dye. The dye degraded products were extracted and characterized by TLC, diazotization and Carbylamines test, which indicated that Amaranth was biotransformed into non-toxic aromatic metabolite without amine group.     

In vitro salt and thermal tolerance of fungal endophytes of Nicotiana spp. growing in arid regions of north-western Australia

Abstract

Endophytic fungal strains isolated from indigenous Nicotiana plants naturally growing in dry and hot regions of north-western Australia were characterised based on their tolerance to salinity and temperature. Sixty-eight fungal isolates were tested on eight levels (0.5 M, 1.0 M, 1.5 M, 2.0 M, 2.5 M, 3.0 M, 3.5 M and 4.0 M) of five different of salts solutions NaCl, KCl, MgCl₂, CaCl₂ and MgSO₄ and at various temperatures (25–50?°C). The salt adaptation test indicated that the fungal strains namely Aspergillus niger (E-202), A. ochraceous-A (E-134), Aurantiporus sp. (E-135), Cladosporium halotolerance (E-128), Pleurostomophora richardsiae (E-13) and Trichoderma sp. (E-185.1) were tolerant to higher concentrations of various salts. The most growth-limiting salt turned out to be MgCl₂ followed by the chaotrope CaCl₂. Responses to temperature tolerance revealed that most fungi tested could grow at 30?°C. About 50% all the fungi did not show any growth when the temperature was raised above 30?°C. When the temperature was raised up to 50?°C all the fungi failed to grow but the fungus Rasamsonia piperina (E-172). Endophyte strains identified could be promising candidates for future research in investigating the fungus–plant interactions and their roles in plant adaptation to inhospitable environments.     

Nonlabens marina sp. nov., a novel member of the Flavobacteriaceae isolated from the Pacific Ocean

Two aerobic, Gram-negative, orange pigmented and irregular rod-shaped bacteria, designated S1-05 and S1-08^T, were isolated from seawater from the Pacific Ocean. Phylogenetic analysis based on their 16S rRNA gene sequences revealed that the novel isolates could be affiliated with the genus Nonlabens of the family Flavobacteriaceae. The strains S1-05 and S1-08^T shared 100 % pairwise sequences similarity with each other and showed less than 96.8 % similarity with the cultivated members of the genus Nonlabens. The novel isolates are phenotypically and physiologically different from strains described previously. The strains were found to be non-motile, oxidase positive, catalase positive and hydrolyzed gelatin and aesculin. The G+C contents of the DNA were determined to 41.4 and 41.7 mol% and MK-6 the predominant menaquinone. Anteiso-C15:0 and iso-C15:0 were found to be the major two cellular fatty acids. On the basis of polyphasic taxonomic studies, it was concluded that strains S1-05 and S1-08^T represent a novel species within the genus Nonlabens, for which the name Nonlabens marina sp. nov. is proposed. The type strain of N. marina is S1-08^T (=KCTC 23432^T = NBRC 107738^T).     

First record of an entomopathogenic fungus of tomato leafminer,Tuta absoluta (Meyrick) in Tanzania

Tuta absoluta (Meyrick) is a destructive pest causing adverse environmental and economic effects globally. Management of T. absoluta by synthetic chemicals is of diminutive achievement due to resistance trick of the pest. Harnessing of biocontrols is currently fascinating and efficient for management of the tremendous pests. This study isolated an entomopathogenic fungus from dead larvae of T. absoluta on tomato leaves from farmer’s field Tanzania. Two Aspergillus fungal isolates were isolated and tested against larvae and adults of T. absoluta under conditions of 19.5°C and 50%RH, 30.4°C and 70% RH, respectively, in the laboratory. Sequence analysis of Aspergillus isolates that we referred as A-Tz1 and A-Tz2 were analysed with the BLAST program available at the National Center for Biological Information (NCBI). The two isolates (A-Tz1 and A-Tz2) showed 100% similarity with Aspergillus oryzae of GeneBank accession numbers EF121337.1 and MG519722.1. Such similarity implied that both A-Tz1 and A-Tz2 isolates were A. oryzae. To confirm pathogenicity, bioassays were conducted against the larvae, pupa and adults of T. absoluta. Results showed that at 1.0?×?10⁸?conidia/mL, fungal isolates caused up to 70% larval mortality 3 days post inoculation and consequently inhibited pupation by 84.5% and adult emergence by 74.4%. The identified A. oryzae reduced the life span of adult T. absoluta up to 5 days post inoculation at 1.0?×?10⁸?conidia/mL whereas in control T. absoluta survived up to 25 days. This study found that isolates of Aspegillus species have the potential to be used as active ingrediennt for the formulation of a new bioinjections for application by farmers to control the pest in fields.     

Nitratireductor shengliensis sp. nov., Isolated from an Oil-Polluted Saline Soil

Two Gram-negative, non-motile, short-rod-shaped bacterial isolates, designated 110399^T and 110248, were isolated from an oil-polluted saline soil in Shengli Oilfield, Eastern China. The two strains shared 99.9 % 16S rRNA gene sequence similarity with the DNA–DNA relatedness value being 80.0 %. They were both capable to grow at 20–40 °C, pH 7–9, and 1–9 % (w/v) NaCl with the optimum growth happened at 30 °C, pH 8, and 2–6 % (w/v) NaCl. The phylogenetic analysis based on 16S rRNA gene sequences revealed that the two strains were members of Nitratireductor and most closely related to Nitratireductor pacificus pht-3B^T and N. basaltis J3^T with the 16S rRNA gene sequence similarities being 97.1 and 97.0 %. The DNA–DNA relatedness between the novel strains and two type strains were below 27 ± 7 %. The strains 110399^T and 110248 also differed from N. pacificus and N. basaltis in nitrate reduction, salt tolerance, enzyme activities, and utilization of carbon sources. The major cellular fatty acids of strain 110399^T were C_19:0ω8c cyclo (10.5 %) and Summed Feature 8 (C_18:1ω7c and/or C_18:1ω6c, 41.5 %) which are typical in the genus Nitratireductor. The predominant ubiquinone was Q-10. The genome DNA G+C content of strain 110399^T and 110248 was 61.1 and 61.7 mol%. On the basis of genetic, phenotypic, and chemotaxonomic analyses, strains 110399^T and 110248 represent a novel species within the genus Nitratireductor, for which the name Nitratireductor shengliensis sp. nov. is proposed. The type strain is 110399^T (=CGMCC 1.12519^T = LMG 27405^T).     

Reverse Line Blot Macroarray for Simultaneous Detection and Characterization of Four Biological Warfare Agents

The need for a rapid detection and characterization of biowarfare (BW) agents cannot be over emphasized. With diverse array of potential BW pathogen available presently, rapid identification of the pathogen is crucial, so that specific therapy and control measures can be initiated. We have developed a multiplex polymerase chain reaction based reverse line blot macroarray to simultaneously detect four pathogens of BW importance viz. Bacillus anthracis, Yersinia pestis, Brucella melitensis and Burkholderia pseudomallei. The multiplex PCR utilizes 14 pairs of primers targeting 18 specific markers. These markers include genes which are genus specific, species-specific chromosomal sequences and virulence markers of plasmid origin. The assay was evaluated on various human, environment and animal isolates. The assay w successful in simultaneous detection and characterization of isolates of the four pathogens on as a single platform with sensitivity ranging from 0.3 pg to 0.3 ng of genomic DNA. The assay was able to detect 5 × 10² cfu/ml for B. anthracis, 8 × 10² cfu/ml for Yersinia sp., 1.4 × 10² cfu/ml for B. melitensis and 4 × 10² cfu/ml for B. pseudomallei.     

Biofilms isolated from washing machines from three continents and their tolerance to a standard detergent

The goal of this comparative study was to investigate biofilm forming microorganisms living in washing machines (WMs). Biofilms were sampled from 11 washing machines from four countries and three continents. Among the 94 isolated strains, 30% were potential human pathogens. Representative strains were selected and biofilm formation was evaluated with the crystal violet (CV) assay. The majority of the WM isolates formed more biofilm than their reference strains. Biofilms of P. putida WM (the largest biofilm producer) were exposed to different concentrations (0.0007–7 g l^?1) of the standard detergent IEC-A* at 30°C for 30 min and observed with confocal laser scanning microscopy. Using quantitative CVA, P. putida WM biofilm removal required higher detergent concentrations than the type strain. However, for both strains the recommended detergent concentration (7 g l^?1) was insufficient to completely clean surfaces from cell debris and exopolymeric substances.     

Polyphasic Taxonomy of Novel Actinobacteria Showing Macromolecule Degradation Potentials in Bigeum Island,Korea

Aerobic, alkaliphilic to alkalitolerant and mesophilic bacteria were isolated and characterized from soil and sediment samples collected from Bigeum Island, South Korea. The total numbers of microorganisms in the soil and sediment samples were found to be 10³–10⁵ cfu/g and 10²–10⁷ cfu/g, respectively. A total of 163 isolates were isolated and subjected to further characterization on the basis of pH, temperature and salt tolerance. Among the 163 isolates, 54 were selected based on their tolerance attributes to temperature, pH and NaCl. Out of the 54 isolates, 27 were further selected based on their multiple tolerance ability and enzyme profile and were subjected to 16S rRNA gene sequencing and phylogenetic analysis. The latter indicated that most of the Bigeum Island isolates were related to the phylum Actinobacteria. The phylogenetic tree based on 16S rRNA gene sequences placed the 27 isolates into 9 different major bacterial genera, each genus comprising pure cultures that shared ≤97% sequence identity and 18 putative novel species. Most of the strains were alkalitolerant and mesophilic, and produced biotechnologically important enzymes at alkaline pH.     

Effect of temperature,cultivars, injury of root and inoculums load of Ralstonia solanacearum to cause bacterial wilt of tomato

Bacterial wilt, caused by Ralstonia solanacearum , is responsible for severe losses in tomato crops in the world. In the present study, the effect of temperature, cultivars of tomato, injury of root system and inoculums load of R. solanacearum to cause bacterial wilt disease under control conditions was undertaken. Three strains UTT-25, HPT-3 and JHT-5 of R. solanacearum were grown at 5–40?°C in vitro to study, the effect of temperature on the growth of bacteria and maximum growth was found at 30?°C after 72?h in all the strains. Twenty-one days old seedlings of two cultivars of tomato i.e. N-5 (moderately resistant) and Pusa Ruby (highly susceptible) were transplanted into the pots and inoculated with R. solanacearum strain UTT-25 (5 × 10⁸?cfu/ml), mechanically injured and uninjured roots of the plant. The plants were allowed to grow at 20, 25, 30 and 35?°C at National Phytotron Facility, IARI, New Delhi to study the effect of temperature on intensity of bacterial wilt disease. Maximum wilt disease intensity was found 98.73 and 95.9 % in injured roots of Pusa Ruby and N-5 cultivars of tomato at 35?°C on 11th days of inoculation, respectively. However, no wilt disease was observed in both the cultivars at 20?°C up to 60?days. For detection of R. solanacearum from asymptomatic tomato plants, hrpB-based sequence primers (Hrp_rs2F and Hrp_rs2R) amplified at 323?bp was used in bio-PCR to detect R. solanacearum from crown, mid part of stem and upper parts of the plant. Another experiment was conducted to find out the inoculum potential of R. solanacearum strain UTT-25 to cause bacterial wilt in susceptible cultivar Pusa Ruby. The bacteria were inoculated at concentration of bacterial suspension 10 to 10¹⁰?cfu/ml in injured and uninjured roots of the plants separately and injured root accelerated wilt incidence and able to cause wilt disease 63.3% by 100?cfu/ml of R. solanacearum, while no disease appeared at 10?cfu/ml on the 11th day of inoculation in injured and uninjured roots of the plant.