An Innovative Approach to Study Ralstonia solanacearum Pathogenicity in 6 to 7 Days Old Tomato Seedlings by Root Dip Inoculation

In this study, we report Ralstonia solanacearum pathogenicity in the early stages of tomato seedlings by an innovative root inoculation method. Pathogenicity assays were performed under gnotobiotic conditions in microfuge tubes by employing only 6- to 7-day-old tomato seedlings for root inoculation. Tomato seedlings inoculated by this method exhibited the wilted symptom within 48 h and the virulence assay can be completed in 2 weeks. Colonization of the wilted seedlings by R. solanacearum was confirmed by using gus staining as well as fluorescence microscopy. Using this method, mutants in different virulence genes such as hrpB, phcA, and pilT could be clearly distinguished from wild-type R. solanacearum. The method described here is economic in terms of space, labor, and cost as well as the required quantity of bacterial inoculum. Thus, the newly developed assay is an easy and useful approach for investigating virulence functions of the pathogen at the seedling stage of hosts, and infection under these conditions appears to require pathogenicity mechanisms used by the pathogen for infection of adult plants.

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Effect of Biological Control Antagonists Adsorbed on Chitosan Immobilized Silica Nanocomposite on Ralstonia solanacearum and Growth of Tomato Seedlings

Advances in Research ◽

10.9734/air/2016/22742 ◽

2016 ◽

Vol 6 (3) ◽

pp. 1-23 ◽

Cited By ~ 4

Author(s):

Gatahi Dennis ◽

Wanyika Harrison ◽

Kihurani Agnes ◽

Gatebe Erastus

Keyword(s):

Biological Control ◽

Ralstonia Solanacearum ◽

Tomato Seedlings ◽

Silica Nanocomposite

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Biocontrol of Ralstonia solanacearum by Treatment with Lytic Bacteriophages

Applied and Environmental Microbiology ◽

10.1128/aem.02847-10 ◽

2011 ◽

Vol 77 (12) ◽

pp. 4155-4162 ◽

Cited By ~ 95

Author(s):

Akiko Fujiwara ◽

Mariko Fujisawa ◽

Ryosuke Hamasaki ◽

Takeru Kawasaki ◽

Makoto Fujie ◽

...

Keyword(s):

Cell Density ◽

Ralstonia Solanacearum ◽

Experimental Period ◽

The Other ◽

Bacterial Cells ◽

Effective Prevention ◽

Tomato Plants ◽

Content Type ◽

Tomato Seedlings ◽

Lower Cell Density

ABSTRACTRalstonia solanacearumis a Gram-negative bacterium and the causative agent of bacterial wilt in many important crops. We treatedR. solanacearumwith three lytic phages: φRSA1, φRSB1, and φRSL1. Infection with φRSA1 and φRSB1, either alone or in combination with the other phages, resulted in a rapid decrease in the host bacterial cell density. Cells that were resistant to infection by these phages became evident approximately 30 h after phage addition to the culture. On the other hand, cells infected solely with φRSL1 in a batch culture were maintained at a lower cell density (1/3 of control) over a long period. Pretreatment of tomato seedlings with φRSL1 drastically limited penetration, growth, and movement of root-inoculated bacterial cells. All φRSL1-treated tomato plants showed no symptoms of wilting during the experimental period, whereas all untreated plants had wilted by 18 days postinfection. φRSL1 was shown to be relatively stable in soil, especially at higher temperatures (37 to 50°C). Active φRSL1 particles were recovered from the roots of treated plants and from soil 4 months postinfection. Based on these observations, we propose an alternative biocontrol method using a unique phage, such as φRSL1, instead of a phage cocktail with highly virulent phages. Using this method, φRSL1 killed some but not all bacterial cells. The coexistence of bacterial cells and the phage resulted in effective prevention of wilting.

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Biocontrol potential of bacteriophage ɸsp1 against bacterial wilt-causing Ralstonia solanacearum in Solanaceae crops

Egyptian Journal of Biological Pest Control ◽

10.1186/s41938-021-00408-3 ◽

2021 ◽

Vol 31 (1) ◽

Author(s):

Pramila Devi Umrao ◽

Vineet Kumar ◽

Shilpa Deshpande Kaistha

Keyword(s):

Potato Tuber ◽

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Burst Size ◽

Field Application ◽

Bacteriophage Therapy ◽

Tomato Seedlings ◽

Isolation And Characterization ◽

Wide Ph Range ◽

Ph Range

Abstract Background Bacteriophages are effective biocontrol strategy as well as ecofriendly remedy for the emerging antibiotic and chemical resistance in bacterial phytopathogens such as bacterial wilt-causing Ralstonia solanacearum. One of the major challenges in the use of bacteriophage therapy for agricultural phytopathogens is maintaining their viability even during variations in pH, temperature, ultraviolet irradiation, and desiccation during field application for sustainable agriculture. Results In this study, the isolation and characterization of phage ɸsp1 for its efficacy against wilt-causing R. solanacearum performed on Solanum lycopersicum (tomato) seedlings and Solanum tuberosum (potato) tuber assay are reported. Bacteriophage was found to be viable and stable at a wide pH range (3.0–9.0) and at temperatures up to 55 °C. Phage ɸsp1 required ~15 min for adsorption and completed its life cycle in 25–30 min by host cell lysis with a burst size of ~250–300. Phage ɸsp1 eradicated 94.73% preformed R. solanacearum biofilm and inhibited biofilm formation by 73.68% as determined by the static crystal violet microtiter biofilm assay. Transmission electron microscope revealed the phage ɸsp1 to be approximately 208±15 nm in size, comprising of icosahedral head (100 ±15 nm) and tail, as belonging to Myoviridae family. Plant bioassays showed 81.39 and 87.75% reduction in pathogen count using phages ɸsp1 in potato tuber and tomato seedlings, respectively. Reversal in disease symptoms was 100% in phage-treated tuber and tomato plant (pot assay) compared to only pathogen-treated controls. Conclusion Isolated bacteriophage ɸsp1 was found to be highly host specific, effective in biofilm prevention, and capable of inhibiting bacterial wilt at low multiplicity of infection (1.0 MOI) in tomato as well as potato tuber bioassays. Phages ɸsp1 were environmentally stable as they survive at variable pH and temperature. Bacteriophage ɸsp1 shows a promise for development into a biocontrol formulation for the prevention of R. solanacearum bacterial wilt disease.

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A Streptomyces sp. NEAU-HV9: Isolation, Identification, and Potential as a Biocontrol Agent against Ralstonia solanacearum of Tomato Plants

Microorganisms ◽

10.3390/microorganisms8030351 ◽

2020 ◽

Vol 8 (3) ◽

pp. 351 ◽

Cited By ~ 4

Author(s):

Ling Ling ◽

Xiaoyang Han ◽

Xiao Li ◽

Xue Zhang ◽

Han Wang ◽

...

Keyword(s):

Antibacterial Activity ◽

Ralstonia Solanacearum ◽

Actinomycin D ◽

Biological Control Agent ◽

Sequence Similarity ◽

Morphological Characteristics ◽

Rrna Gene ◽

Tomato Seedling ◽

Genus Streptomyces ◽

Tomato Seedlings

Ralstonia solanacearum is an important soil-borne bacterial plant pathogen. In this study, an actinomycete strain named NEAU-HV9 that showed strong antibacterial activity against Ralstonia solanacearum was isolated from soil using an in vitro screening technique. Based on physiological and morphological characteristics and 98.90% of 16S rRNA gene sequence similarity with Streptomyces panaciradicis 1MR-8T, the strain was identified as a member of the genus Streptomyces. Tomato seedling and pot culture experiments showed that after pre-inoculation with the strain NEAU-HV9, the disease occurrence of tomato seedlings was effectively prevented for R. solanacearum. Then, a bioactivity-guided approach was employed to isolate and determine the chemical identity of bioactive constituents with antibacterial activity from strain NEAU-HV9. The structure of the antibacterial metabolite was determined as actinomycin D on the basis of extensive spectroscopic analysis. To our knowledge, this is the first report that actinomycin D has strong antibacterial activity against R. solanacearum with a MIC (minimum inhibitory concentration) of 0.6 mg L−1 (0.48 μmol L−1). The in vivo antibacterial activity experiment showed that actinomycin D possessed significant preventive efficacy against R. solanacearum in tomato seedlings. Thus, strain NEAU-HV9 could be used as BCA (biological control agent) against R. solanacearum, and actinomycin D might be a promising candidate for a new antibacterial agent against R. solanacearum.

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Ralstonia solanacearum Virulence in Cotyledon Stage Tomato Seedlings

Modern Concepts & Developments in Agronomy ◽

10.31031/mcda.2020.07.000664 ◽

2020 ◽

Vol 7 (3) ◽

Author(s):

Ray SK

Keyword(s):

Ralstonia Solanacearum ◽

Tomato Seedlings ◽

Cotyledon Stage

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Suppressing Bacterial Wilt (Ralstonia solanacearum) by Grafting Tomato Seedlings Grown on Six Comparable Media

Jurnal Perlindungan Tanaman Indonesia ◽

10.22146/jpti.51430 ◽

2020 ◽

Vol 24 (1) ◽

pp. 68

Author(s):

Anggita Rahmawati ◽

Triwidodo Arwiyanto

Keyword(s):

Rice Husk ◽

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Dry Mass ◽

Control Technique ◽

Tomato Seedling ◽

Tomato Seedlings ◽

Progress Curve ◽

The Media ◽

Resistant Rootstock

Bacterial wilt caused by Ralstonia solanacearum is an important disease of tomatoes. The grafting method with a resistant rootstock variety is a reliable control technique and has been carried out in many countries. Healthy tomato seedling are needed for grafting so this study was aimed to find a good seedling medium to grow the upper stem and rootstock of tomato for grafting and to know the effect of seed media on the intensity of bacterial wilt). Tomato seeds were grown in coir and rice husk charcoal with ratio A (10:0), B (9:1), C (8:2), D (7:3), E (6:4), F (5:5) (coir : rice husk charcoal, v/v). Seedlings were watered regularly and after seedlings aged 21 days, the grafting was conducted. Agronomic observations were justified by measuring germination style, plant height, leaf area, fresh mass and dry mass of plants at 21 days after planting. Tomato seedlings were grafted by using H-7996 variety as a rootstock and Servo variety as an upper stem. Tomato seedlings were transplanted into polybags and then inoculated with R. solanacearum (108 CFU/ml). The disease intensity and AUDPC (Area Under Disease Progress Curve) were recorded. The results revealed that the media with a ratio of 8:2 (coir : rice husk charcoal, v/v) is the best medium for growing tomatoes and the grafting treatment could reduce the disease intensity of bacterial wilt by 40% compared with non grafted treatment and reduce 16% disease intensity compared to the grafted treatment with seedling sown in other ratio media at the last observation.

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Compost Amendments Based on Vinegar Residue Promote Tomato Growth and Suppress Bacterial Wilt Caused by Ralstonia Solanacearum

Pathogens ◽

10.3390/pathogens9030227 ◽

2020 ◽

Vol 9 (3) ◽

pp. 227 ◽

Cited By ~ 5

Author(s):

Mingming He ◽

Mohammad Shah Jahan ◽

Yu Wang ◽

Jin Sun ◽

Sheng Shu ◽

...

Keyword(s):

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Microbial Populations ◽

H2o2 Production ◽

Microbial Activities ◽

Growth Environment ◽

Soil Microbial ◽

Tomato Seedlings ◽

Vinegar Residue ◽

Tomato Bacterial Wilt

Tomato bacterial wilt caused by Ralstonia solanacearum (RS) is one of the most devastating soil-borne diseases, and compost is to be considered as a resource-saving and environment-friendly measure to control the disease. Herein, a pot experiment was implemented to explore the effects of vinegar residue matrix amendments on the growth performances of tomato seedlings and to examine the suppression ability against bacterial wilt under vinegar residue substrate (VRS), and peat substrate (Peat) with RS inoculation. The results revealed that VRS effectively suppressed the disease incidence of bacterial wilt, increased the number of bacteria and actinomycetes, decreased fungi populations, promoted soil microbial populations and microbial activities, enhanced the growths of tomato seedlings, and modulated defense mechanism. In addition, VRS efficiently inhibited the oxidative damage in RS inoculated leaves via the regulation of excess reactive oxide species (O2•− and H2O2) production, lessening of malondialdehyde (MDA) content, and causing less membrane injury; resulting in enhancements of antioxidants enzymes activities accompanying with modulating their encoding gene expression. The transcription levels of NPR1, PIN2, PR1b, ACO1, EDS1, PR1B, MAPK3, PIN2, and RRS1 were also modulated with the pathogens inoculated in tomato leaves both in VRS and Peat treatments, which indicated that systemic-acquired resistance possesses cross-talk between salicylic acid, jasmonic acid, and the ethylene-dependent signaling pathway. Besides, the RS inoculation significantly inhibited the growth of tomato seedlings, and all growth indices of plants grown in VRS were considerably higher than those produced in Peat. Taken together, VRS represents a new strategy to control tomato bacterial wilt through boosting the soil microbial populations and microbial activities. Furthermore, VRS promotes the plant immune response to provide a better growth environment for plants surviving in disease conditions.

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Effect of Calcium Nutrition on Disease Development and Latent Infection of Bacterial Wilt in Grafted Tomato Seedlings

HortScience ◽

10.21273/hortsci.32.3.445b ◽

1997 ◽

Vol 32 (3) ◽

pp. 445B-445

Author(s):

H. Yamazaki ◽

S. Kikuchi ◽

T. Hoshina ◽

T. Kimura

Keyword(s):

Nutrient Solution ◽

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Latent Infection ◽

Disease Incidence ◽

Selective Medium ◽

Disease Development ◽

Tomato Seedlings ◽

Latently Infected ◽

Resistant Rootstock

To control bacterial wilt of tomato, grafting with resistant rootstocks widely prevails in Japan, but the disease has recently occurred even on grafted plants. Concerning this breakdown of resistance, the experiments were conducted to investigate the effect of Ca nutrition on disease development and latent infection of bacterial wilt in grafted tomato seedlings. Three levels of Ca (0.4, 4.4, or 20.4 mm) in a nutrient solution were applied to grafted seedlings (scion: `Momotarou', resistant rootstock: `Hawaii 7998') grown in a phytotron. One week after the Ca treatment, seedlings were inoculated with a 108 cfu/ml suspension of Ralstonia solanacearum by a stem puncture at the basal stem of the rootstock. The disease incidence was recorded for 21 days. In a second experiment, xylem exudates were collected from decapitated scions of the Ca-treated seedlings 5 days after inoculation. Populations of the pathogen in the exudates were counted by plating on a selective medium. Colonies isolated were reinoculated to susceptible seedlings to check the virulence. The high Ca treatment increased leaf and stem Ca contents, and significantly reduced the disease incidence. While Ca concentrations in the xylem exudates increased with the high Ca treatment, the populations of the pathogen in the exudates were high (>109 cfu/ml) even at the high Ca treatment. All the colonies isolated were virulent. These results showed that grafted tomato seedlings treated with a high Ca concentration were highly resistant to bacterial wilt, but latently infected.

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