scholarly journals Viability, Stability and Biocontrol Activity in Planta of Specific Ralstonia Solanacearum Bacteriophages After Their Conservation Prior to Commercialization and Use

2021 ◽  
Author(s):  
Belén Álvarez ◽  
Laura Gadea-Pallás ◽  
Alejandro Rodríguez ◽  
Begonya Vicedo ◽  
Àngela Figàs-Segura ◽  
...  
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Reference Strain ◽  
Specific Activity ◽  
Experimental Period ◽  
In Planta ◽  
Tomato Plants ◽  
Preservation Method ◽  
Biocontrol Activity ◽  
Susceptible Tomato

Ralstonia solanacearum is a pathogen that causes bacterial wilt producing severe damage in staple solanaceous crops. Traditional control has low efficacy and/or environmental impact. Recently, the bases of a new biotechnological method by lytic bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 with specific activity against R. solanacearum were established. However, some aspects remain unknown, such as the survival and maintenance of the lytic activity after submission to a preservation method as the lyophilization. To this end, viability and stability of lyophilized vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 and their capacity for bacterial wilt biocontrol have been determined against one pathogenic Spanish reference strain of R. solanacearum in susceptible tomato plants in different conditions and making use of various cryoprotectants. The assays carried out have shown satisfactory results with respect to the viability and stability of the bacteriophages after the lyophilization process, maintaining high titres throughout the experimental period, also with respect to the capacity of the bacteriophages for the biological control of bacterial wilt, controlling this disease in more than 50% of the plants. The results offer good prospects for the use of lyophilization as a conservation method for the lytic bacteriophages of R. solanacearum in view of their commercialization as biocontrol agents.

scholarly journals Ralstonia solanacearum Needs Motility for Invasive Virulence on Tomato

2001 ◽  
Vol 183 (12) ◽  
pp. 3597-3605 ◽  
Author(s):  
Julie Tans-Kersten ◽  
Huayu Huang ◽  
Caitilyn Allen
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Soft Agar ◽  
Cell Protein ◽  
In Planta ◽  
Tomato Plants ◽  
Bacterial Wilt Disease ◽  
Virulence Assay ◽  
Flagellar Filament

ABSTRACT Ralstonia solanacearum, a widely distributed and economically important plant pathogen, invades the roots of diverse plant hosts from the soil and aggressively colonizes the xylem vessels, causing a lethal wilting known as bacterial wilt disease. By examining bacteria from the xylem vessels of infected plants, we found thatR. solanacearum is essentially nonmotile in planta, although it can be highly motile in culture. To determine the role of pathogen motility in this disease, we cloned, characterized, and mutated two genes in the R. solanacearum flagellar biosynthetic pathway. The genes for flagellin, the subunit of the flagellar filament (fliC), and for the flagellar motor switch protein (fliM) were isolated based on their resemblance to these proteins in other bacteria. As is typical for flagellins, the predicted FliC protein had well-conserved N- and C-terminal regions, separated by a divergent central domain. The predicted R. solanacearum FliM closely resembled motor switch proteins from other proteobacteria. Chromosomal mutants lackingfliC or fliM were created by replacing the genes with marked interrupted constructs. Since fliM is embedded in the fliLMNOPQR operon, the aphAcassette was used to make a nonpolar fliM mutation. Both mutants were completely nonmotile on soft agar plates, in minimal broth, and in tomato plants. The fliC mutant lacked flagella altogether; moreover, sheared-cell protein preparations from the fliC mutant lacked a 30-kDa band corresponding to flagellin. The fliM mutant was usually aflagellate, but about 10% of cells had abnormal truncated flagella. In a biologically representative soil-soak inoculation virulence assay, both nonmotile mutants were significantly reduced in the ability to cause disease on tomato plants. However, the fliC mutant had wild-type virulence when it was inoculated directly onto cut tomato petioles, an inoculation method that did not require bacteria to enter the intact host from the soil. These results suggest that swimming motility makes its most important contribution to bacterial wilt virulence in the early stages of host plant invasion and colonization.

scholarly journals Using the Ralstonia solanacearum Tat Secretome To Identify Bacterial Wilt Virulence Factors

2007 ◽  
Vol 73 (12) ◽  
pp. 3779-3786 ◽  
Author(s):  
Enid T. Gonz�lez ◽  
Darby G. Brown ◽  
Jill K. Swanson ◽  
Caitilyn Allen
Keyword(s):  
Virulence Factors ◽  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Acid Tolerance ◽  
Bioinformatic Analysis ◽  
Wild Type ◽  
In Planta ◽  
Tomato Plants ◽  
Tat System

ABSTRACT To identify secreted virulence factors involved in bacterial wilt disease caused by the phytopathogen Ralstonia solanacearum, we mutated tatC, a key component of the twin-arginine translocation (Tat) secretion system. The R. solanacearum tatC mutation was pleiotropic; its phenotypes included defects in cell division, nitrate utilization, polygalacturonase activity, membrane stability, and growth in plant tissue. Bioinformatic analysis of the R. solanacearum strain GMI1000 genome predicted that this pathogen secretes 70 proteins via the Tat system. The R. solanacearum tatC strain was severely attenuated in its ability to cause disease, killing just over 50% of tomato plants in a naturalistic soil soak assay where the wild-type parent killed 100% of the plants. This result suggested that elements of the Tat secretome may be novel bacterial wilt virulence factors. To identify contributors to R. solanacearum virulence, we cloned and mutated three genes whose products are predicted to be secreted by the Tat system: RSp1521, encoding a predicted AcvB-like protein, and two genes, RSc1651 and RSp1575, that were identified as upregulated in planta by an in vivo expression technology screen. The RSc1651 mutant had wild-type virulence on tomato plants. However, mutants lacking either RSp1521, which appears to be involved in acid tolerance, or RSp1575, which encodes a possible amino acid binding protein, were significantly reduced in virulence on tomato plants. Additional bacterial wilt virulence factors may be found in the Tat secretome.

scholarly journals First Report of Bacterial Wilt of Tomato (Solanum lycopersicum) Caused by Ralstonia solanacearum in Benin

Plant Disease ◽  
2009 ◽  
Vol 93 (5) ◽  
pp. 549-549 ◽  
Author(s):  
R. Sikirou ◽  
F. Beed ◽  
V. Ezin ◽  
G. Gbèhounou ◽  
S. A. Miller ◽  
...  
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Rural Areas ◽  
Reference Strain ◽  
Plant Protection ◽  
Tomato Production ◽  
Tetrazolium Chloride ◽  
Tomato Plants ◽  
First Report ◽  
Ecological Zones

In June 2004, wilted tomatoes with no foliar yellowing were observed in Ouègbo, Atlantique District, Benin. The cut tomato stems released whitish bacterial ooze. Longitudinal sections of most stems showed brown vascular discoloration. Twenty symptomatic tomato plants were collected from 10 fields and exported to the Institute of Plant Disease and Plant Protection, Leibniz Universität Hannover, Germany. Bacteria were isolated on triphenyl tetrazolium chloride (TTC) medium (2) and three of the nine bacterial isolates that resembled Ralstonia solanacearum (colonies with red center and whitish periphery) and reference strain ToUdk (race 1 biovar 3; N. Thaveechai, Kasetsart University, Bangkok, Thailand) were used for pathogenicity tests. Five 4-week-old tomato plants cv. Tohounvi, grown in individual plastic pots (14 × 16 cm) containing sterilized field soil, were inoculated with each of the four isolates individually by soil drenching with 30 ml of the test cultures at 108 CFU/ml. Control plants were treated with 30 ml of sterile water. All plants were incubated in a glasshouse at 30°C. All plants inoculated with the isolates from Benin wilted 4 days after inoculation with symptoms similar to those observed in the field. Plants inoculated with the reference strain wilted 7 to 11 days after inoculation. Control plants treated with water remained healthy. R. solanacearum was recovered from the 20 symptomatic plants on TTC medium. The identity of the strains in comparison with the reference strain was confirmed by PCR with species-specific primers 759/760, which produced a single 281-bp fragment (3). Because similar symptoms were being increasingly reported by farmers across Benin and linked with reduced tomato yields, a disease survey was undertaken by IITA in 2006 and 2007. Wilted tomato plants were found across all agro-ecological zones of Benin (3 to 72% of plants per field). Isolates were recovered from the southeastern districts of Adja-Ouèrè, Sakété, Adjohoun, and Dangbo, the southwestern districts of Klouékanmè and Athiémé, the southern districts of Toffo and Bohicon, the central districts of Dassa and Savè, and the northern districts of Malanville and Karimama. Identification of R. solanacearum was confirmed following inoculation of tomato, production of characteristic wilting symptoms, recovery of the pathogen on TTC medium, and positive identification with ELISA kits (Pathoscreen Rs; Agdia Inc., Elkhart, IN). To our knowledge, this is the first report of R. solanacearum infecting tomato in Benin. Tomato is the most cultivated vegetable crop in Benin and important to the livelihood of many people in peri-urban and rural areas. Understanding that the cause of the observed crop losses is R. solanacearum may lead to implementation of management strategies such as deployment of disease-resistant cultivars or grafting tomatoes onto bacterial wilt-resistant rootstocks (1). References: (1) P. Aggarwal et al. Indian J. Agric. Sci. 78:379, 2008. (2) A. Kelman. Phytopathology 44:693, 1954. (3) N. Opina et al. Asian Pac. J. Mol. Biol. Biotechnol. 5:19, 1997.

scholarly journals Interactions with Hosts at Cool Temperatures, Not Cold Tolerance, Explain the Unique Epidemiology of Ralstonia solanacearum Race 3 Biovar 2

2009 ◽  
Vol 99 (10) ◽  
pp. 1127-1134 ◽  
Author(s):  
Annett Milling ◽  
Fanhong Meng ◽  
Timothy P. Denny ◽  
Caitilyn Allen
Keyword(s):  
North American ◽  
Ralstonia Solanacearum ◽  
Potato Tubers ◽  
In Planta ◽  
Plant Host ◽  
Tomato Plants ◽  
North American Strain ◽  
Tropical Highlands ◽  
American Strain ◽  
Warm Temperate

Most strains of the bacterial wilt pathogen Ralstonia solanacearum are tropical, but race 3 biovar 2 (R3bv2) strains can attack plants in temperate zones and tropical highlands. The basis of this distinctive ecological trait is not understood. We compared the survival of tropical, R3bv2, and warm-temperate North American strains of R. solanacearum under different conditions. In water at 4°C, North American strains remained culturable the longest (up to 90 days), whereas tropical strains remained culturable for the shortest time (≈40 days). However, live/dead staining indicated that cells of representative strains remained viable for >160 days. In contrast, inside potato tubers, R3bv2 strain UW551 survived >4 months at 4°C, whereas North American strain K60 and tropical strain GMI1000 were undetectable after <70 days in tubers. GMI1000 and UW551 grew similarly in minimal medium at 20 and 28°C and, although both strains wilted tomato plants rapidly at 28°C, UW551 was much more virulent at 20°C, killing all inoculated plants under conditions where GMI100 killed just over half. Thus, differences among the strains in the absence of a plant host were not predictive of their behavior in planta at cooler temperatures. These data indicate that interaction with plants is required for expression of the temperate epidemiological trait of R3bv2.

scholarly journals Selection and Inheritance of Tomato Resistance against Ralstonia solanacearum

2019 ◽  
Vol 23 (1) ◽  
pp. 61
Author(s):  
Isna Maulida ◽  
Rudi Hari Murti ◽  
Triwidodo Arwiyanto
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Dominant Gene ◽  
Tomato Plants ◽  
Breeding Lines ◽  
Back Cross ◽  
Inheritance Patterns ◽  
Randomized Design ◽  
Resistant Varieties ◽  
Resistant Lines

Ralstonia solanacearum is a plant pathogen causes wilting which is a major obstacle in the cultivation of tomato plants. In plant breeding, knowledge of the source of resistance genes and inheritance patterns is important in the development of bacterial wilt resistant varieties. This study aimed to obtain bacterial wilt resistant lines and to find out the inheritance pattern of tomato resistance to bacterial wilt. Selection of resistant plant involved the selected breeding lines from irradiation and crossing collections of the Genetic Laboratory, Faculty of Agriculture, Universitas Gadjah Mada. Introduced lines of H-7996 and F1 Permata and Timoti were used as a control. H-7996 as resistant parents and GM2 as susceptible parents, and their offspring include F1 GM2 x H-7996, F1 reciprocal, F2, Back Cross 1 (F1 x GM2), and Back Cross 2 (F1 x H-7996) used in testing inheritance patterns. Inoculation was carried out 1 week after planting by pouring 100 ml of water suspension of R. solanacarum (108  cfu/ml) on the roots. Completely Randomized Design (CRD) was used in this experiment. The scoring observation was carried out every week for one month. This study showed that Permata as a control was the most resistant, while Timoti and H-7996 were medium resistant. The CLN, G6, G8, and G7 lines were susceptible medium, yet only G8 and G7 with the smallest percentage of disease intensity and not significantly different than Timoti. The resistance gene to bacterial wilt on H-7996 was controlled by genes in the cell nucleus with additive-dominant gene action. Resistance to bacteria has a moderate level of heritability.

scholarly journals Resistance and Susceptibility of Arabidopsis thaliana to Bacterial Wilt Caused by Ralstonia solanacearum

1998 ◽  
Vol 88 (4) ◽  
pp. 330-334 ◽  
Author(s):  
Chang-Hsien Yang ◽  
Gan-Der Ho
Keyword(s):  
Arabidopsis Thaliana ◽  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Bacterial Colonization ◽  
Plant Diseases ◽  
Infection Site ◽  
Bacterial Isolation ◽  
Tomato Plants ◽  
Different Strains ◽  
Growth Of Bacteria

Tomato bacterial wilt caused by Ralstonia solanacearum is a model system for studying plant-bacterial interactions, because it is genetically one of the best characterized plant diseases. We demonstrate here that four different strains of R. solanacearum, two from radishes (Rd4 and Rd15) and two from tomato (Ps21 and Ps95), can infect 27 different ecotypes of Arabidopsis thaliana, causing different responses. All ecotypes tested were highly susceptible to strain Rd15, which caused symptoms similar to those observed in tomato plants. For example, leaf drooping and discoloration developed just 3 days after inoculation, and plants completely wilted within 1 week. Strains Rd4 and Ps95 were less infectious than Rd15. With these two strains, a variety of disease responses were observed among different ecotypes at 2 weeks after inoculation; both susceptible and resistant ecotypes of A. thaliana were identified. Ps21 was the least infectious of the four strains and caused almost no symptoms in any of the ecotypes of Arabidopsis tested. Direct bacterial isolation and plant skeleton hybridization analysis from infected plants indicated that bacterial colonization was correlated with the severity of symptoms. Growth of bacteria was limited to the infection site in resistant plants, whereas the bacteria spread throughout susceptible plants by 1 week after inoculation.

scholarly journals Utilization of Filamentous Phage ϕRSM3 to Control Bacterial Wilt Caused by Ralstonia solanacearum

Plant Disease ◽  
2012 ◽  
Vol 96 (8) ◽  
pp. 1204-1209 ◽  
Author(s):  
Hardian S. Addy ◽  
Ahmed Askora ◽  
Takeru Kawasaki ◽  
Makoto Fujie ◽  
Takashi Yamada
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Plant Protection ◽  
Control Measures ◽  
Filamentous Phage ◽  
Pr Genes ◽  
Efficient Tool ◽  
Tomato Plants ◽  
Pathogenesis Related ◽  
Infected Cells

The wide host range of Ralstonia solanacearum, causal agent of bacterial wilt, and its ability to survive for long periods in the environment restrict the effectiveness of cultural and chemical control measures. The use of phages for disease control is a fast-expanding trend of plant protection with great potential to replace chemical measures. The filamentous phage ϕRSM3 that infects R. solanacearum strains and inactivates virulence on plants is a potential agent for controlling bacterial wilt in tomato. We demonstrated that inoculation of ϕRSM3-infected cells into tomato plants did not cause bacterial wilt. Instead, ϕRSM3-infected cells enhanced the expression of pathogenesis-related (PR) genes, including PR-1a, PR-2b, and PR7, in tomato plants. Moreover, pretreatment with ϕRSM-infected cells protect tomato plants from infection by virulent R. solanacearum strains. The effective dose of ϕRSM3-infected cells for disease prevention was determined to be approximately 105 CFU/ml. Because the ϕRSM3-infected cells can grow and continue to produce infectious phage particles under appropriate conditions, ϕRSM phages may serve as an efficient tool to control bacterial wilt in crops.

scholarly journals First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Plectranthus amboinicus in Martinique

Plant Disease ◽  
2021 ◽  
Author(s):  
Peninna Deberdt ◽  
Gilles Cellier ◽  
Régine Coranson-Beaudu ◽  
Mathis Delmonteil--Girerd ◽  
Joanye Canguio ◽  
...  
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Disease Incidence ◽  
Bacterial Suspension ◽  
Diagnostic Pcr ◽  
Tomato Plants ◽  
First Report ◽  
Initial Symptoms ◽  
Plectranthus Amboinicus ◽  
Negative Controls

Plectranthus amboinicus, commonly known as Gwo ten in the French West Indies (Martinique), is a semi-succulent perennial plant of the Lamiaceae family. This aromatic plant wich is widespread naturally throughout the tropics is of economic importance because of the therapeutic and nutritional properties attributed to its natural phytochemical compounds wich are highly valued in the pharmaceutical industry. In March 2019, wilted P. amboinicus plants intercropped with tomato plants (cv. Heatmaster) in order to reduce the insect-pest damages on tomato, were observed in a field located at the CIRAD experimental station in Lamentin, Martinique (14.663194 N, -60.999167 W). Average disease incidence of 65.74% was recorded on P. amboinicus, in 3 plots with an area of 22.04 m2. The initial symptoms observed were irregular, black, necrotic lesions on leaves. After 10 days, plants wilted and black stripes were observed on stems. Within 4 weeks, more than 50% of plants were fully wilted. Longitudinal stem sections of the wilted plants showed brown vascular discoloration. The cut stems of the wilted plants released a whitish bacterial ooze in water. In all, 108 stem sections were collected, surface disinfected with 70% ethanol and each was crushed in 2 mL of Tris-buffer, then processed for bacterial isolation by plating on modified Semi-Selective Medium from South Africa SMSA (Engelbrecht 1994). Typical Ralstonia solanacearum colonies grew on SMSA medium for 100 of the 108 samples after incubation for 48h at 28°C and were identified as Ralstonia solanacearum using diagnostic PCR with 759/760 primers (Opina et al. 1997). A phylotype-specific multiplex PCR (Fegan and Prior 2005) classified all the strains in R. solanacearum Phylotype IIA. A subset of 11 strains was selected for sequevar identification. All the strains were identified as sequevar I-39 (100% nucleotide identity with strain ANT92 - Genbank accession EF371828), by partial egl sequencing (Fegan and Prior 2005) (GenBank Accession Nos. MT314067 to MT314077). This sequevar has been reported to be widespread in the Caribbean and tropical America on vegetable crops (particularly on tomato), but not on P. amboinicus (Deberdt et al. 2014; Ramsubhag et al. 2012; Wicker et al. 2007). To fulfil Koch’s postulates, a reference strain, isolated from diseased P. amboinicus (CFBP 8733, Phylotype IIA/sequevar 39), was inoculated on 30 healthy P. amboinicus plants. A common tomato cultivar grown in Martinique (cv. Heatmaster) was also inoculated on 30 plants with the same bacterial suspension. Three-weeks-old plants of both crops grown in sterilized field soil were inoculated by soil drenching with 20 ml of a calibrated suspension (108 CFU/mL). P. amboinicus and tomato plants drenched with sterile water served as a negative controls. Plants were grown in a fully controlled environment at day/night temperatures of 30–26°C ± 2°C under high relative humidity (80%). The P. amboinicus plants started wilting 9 days after inoculation, and within four weeks 60% of the P. amboinicus plants had wilted. The tomato plants started wilting 5 days after inoculation with 62% of wilted plants within four weeks. R. solanacearum was recovered from all symptomatic plants on modified SMSA medium. No symptoms were observed and no R. solanacearum strains were isolated from negative controls plants. To our knowledge, this is the first report of R. solanacearum causing bacterial wilt on Gwo ten (P. amboinicus) in Martinique. The importance of this discovery lies in the reporting of an additional host for R. solanacearum, which can be associated with other crops as tomato crop in order to reduce the abundance of insect-pests. Further studies need to be conducted to assess the precise distribution of bacterial wilt disease on P. amboinicus in Martinique and to develop a plan of action avoiding its association with R. solanacearum host crops as tomato for reducing epidemic risk.

scholarly journals Induced ligno-suberin vascular coating and tyramine-derived hydroxycinnamic acid amides restrict Ralstonia solanacearum colonization in resistant tomato roots

2021 ◽  
Author(s):  
Anurag Kashyap ◽  
Montserrat Capellades ◽  
Weiqi Zhang ◽  
Sumithra Srinivasan ◽  
Anna Laromaine ◽  
...  
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Structural Changes ◽  
De Novo ◽  
Susceptible Variety ◽  
Hydroxycinnamic Acid ◽  
Physico Chemical ◽  
Tomato Roots ◽  
Hydroxycinnamic Acid Amides ◽  
Susceptible Tomato

The soil borne pathogen Ralstonia solanacearum is the causing agent of bacterial wilt, a devastating disease affecting major agricultural crops. R. solanacearum enters plants through the roots and reaches the vasculature, causing rapid wilting. We recently showed that tomato varieties resistant to bacterial wilt restrict bacterial movement in the plant. In the present work we go a step forward by identifying the physico-chemical nature of the barriers induced in resistant tomato roots in response to R. solanacearum. We describe that resistant tomato specifically responds to infection by assembling de novo a structural barrier at the vasculature formed by a ligno-suberin coating and tyramine-derived hydroxycinnamic acid amides (HCAAs). On the contrary, susceptible tomato does not form these reinforcements in response to the pathogen but instead displays lignin structural changes compatible with its degradation. Further, we show that overexpressing genes of the ligno-suberin pathway in a commercial susceptible variety of tomato restricts R. solanacearum movement inside the plant and slows disease progression, enhancing resistance to the pathogen. We thus propose that the induced barrier in resistant plants does not only restrict the movement of the pathogen, but may also prevent cell wall degradation by the pathogen and confer anti-microbial properties.

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