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

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.

2021 ◽  
Author(s):  
Ebrahim Osdaghi

Abstract Ralstonia solanacearum is included in the A2 (high risk) list of quarantine organisms by the European and Mediterranean Plant Protection Organization (EPPO). EPPO Code for R. solanacearum is RALSSO, while the phytosanitary categorization of the species in EPPO A2 list is no.58, EU: I/A2 (EPPO, 2018). Bacterial wilt disease was first reported in southern USA in the late nineteenth century on tomato plants (Smith, 1896). Infected plant materials (e.g. potato tubers) transmit the pathogen over long distances; hence, quarantine inspections and plant sanitary practices are the cornerstone of disease management (EPPO, 2018). R. solanacearum strains in the race 3 group are a select agent under the US Agricultural Bioterrorism Protection Act of 2002 (USDA, 2005). Peculiarly, the organism, if not yet already present in North America in pelargonium (Strider et al., 1981), was introduced with cuttings of this host by American companies producing these cuttings for their markets in countries like Kenya and Guatemala (Norman et al., 1999, 2009; Kim et al., 2002; Williamson et al., 2002; O'Hern, 2004). A similar situation led to introductions of the pathogen from Kenya into some northern European nurseries. Once the source (contaminated surface water) was recognized and proper control measures (use of deep soil water, disinfection of cutting producing premises and replacement of mother stock), the problem was solved and the disease in greenhouses eradicated (Janse et al., 2004). Similarly race 1 has been introduced into greenhouses with ornamental plants (rhizomes, cuttings or fully grown plants) such as Epipremnum, Anthurium, Curcuma spp. and Begonia eliator from tropical areas (Norman and Yuen, 1998, 1999; Janse et al., 2006; Janse, 2012). Introduction can and did occur from Costa Rica and the Caribbean, Indonesia, Thailand and South Africa. However, this idea of placing pathogens on bioterrorist list for unclear and perhaps industry-driven reasons and its effects, is strongly opposed in a recent publication from leading phytobacteriologists. This is because R. solanacearum is an endemic pathogen, causing endemic disease in most parts of its geographic occurrence, moreover normal quarantine regulations are already in place where the disease is not present or only sporadically and are thought to be more efficient and less damaging to trade and research than placing this pathogen on select agent lists and treating it as such (Young et al., 2008). Peculiarly, it has been used in the control of a real invasive species, the weed kahili ginger (Hedychium gardenarium) in tropical forests in Hawaii. This is not without risks because strains occurring on this weed host were thought to be non-virulent, but later appeared to be virulent on many edible and ornamental ginger species as well (Anderson and Gardner, 1999; Paret et al., 2008). Another threat for these countries could be strains belonging to race 1, biovar 1 (phylotype I) that have already been reported from field-grown potatoes in Portugal (Cruz et al., 2008).


2021 ◽  
Author(s):  
Ebrahim Osdaghi

Abstract Ralstonia solanacearum is included in the A2 (high risk) list of quarantine organisms by the European and Mediterranean Plant Protection Organization (EPPO). EPPO Code for R. solanacearum is RALSSO, while the phytosanitary categorization of the species in EPPO A2 list is no.58, EU: I/A2 (EPPO, 2018). Bacterial wilt disease was first reported in southern USA in the late nineteenth century on tomato plants (Smith, 1896). Infected plant materials (e.g. potato tubers) transmit the pathogen over long distances; hence, quarantine inspections and plant sanitary practices are the cornerstone of disease management (EPPO, 2018). R. solanacearum strains in the race 3 group are a select agent under the US Agricultural Bioterrorism Protection Act of 2002 (USDA, 2005). Peculiarly, the organism, if not yet already present in North America in pelargonium (Strider et al., 1981), was introduced with cuttings of this host by American companies producing these cuttings for their markets in countries like Kenya and Guatemala (Norman et al., 1999, 2009; Kim et al., 2002; Williamson et al., 2002; O'Hern, 2004). A similar situation led to introductions of the pathogen from Kenya into some northern European nurseries. Once the source (contaminated surface water) was recognized and proper control measures (use of deep soil water, disinfection of cutting producing premises and replacement of mother stock), the problem was solved and the disease in greenhouses eradicated (Janse et al., 2004). Similarly race 1 has been introduced into greenhouses with ornamental plants (rhizomes, cuttings or fully grown plants) such as Epipremnum, Anthurium, Curcuma spp. and Begonia eliator from tropical areas (Norman and Yuen, 1998, 1999; Janse et al., 2006; Janse, 2012). Introduction can and did occur from Costa Rica and the Caribbean, Indonesia, Thailand and South Africa. However, this idea of placing pathogens on bioterrorist list for unclear and perhaps industry-driven reasons and its effects, is strongly opposed in a recent publication from leading phytobacteriologists. This is because R. solanacearum is an endemic pathogen, causing endemic disease in most parts of its geographic occurrence, moreover normal quarantine regulations are already in place where the disease is not present or only sporadically and are thought to be more efficient and less damaging to trade and research than placing this pathogen on select agent lists and treating it as such (Young et al., 2008). Peculiarly, it has been used in the control of a real invasive species, the weed kahili ginger (Hedychium gardenarium) in tropical forests in Hawaii. This is not without risks because strains occurring on this weed host were thought to be non-virulent, but later appeared to be virulent on many edible and ornamental ginger species as well (Anderson and Gardner, 1999; Paret et al., 2008). Another threat for these countries could be strains belonging to race 1, biovar 1 (phylotype I) that have already been reported from field-grown potatoes in Portugal (Cruz et al., 2008).


Plant Disease ◽  
2009 ◽  
Vol 93 (5) ◽  
pp. 549-549 ◽  
Author(s):  
R. Sikirou ◽  
F. Beed ◽  
V. Ezin ◽  
G. Gbèhounou ◽  
S. A. Miller ◽  
...  

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.


2001 ◽  
Vol 183 (12) ◽  
pp. 3597-3605 ◽  
Author(s):  
Julie Tans-Kersten ◽  
Huayu Huang ◽  
Caitilyn Allen

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.


2019 ◽  
Vol 23 (1) ◽  
pp. 61
Author(s):  
Isna Maulida ◽  
Rudi Hari Murti ◽  
Triwidodo Arwiyanto

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.


1998 ◽  
Vol 88 (4) ◽  
pp. 330-334 ◽  
Author(s):  
Chang-Hsien Yang ◽  
Gan-Der Ho

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.


2001 ◽  
Vol 14 (2) ◽  
pp. 214-224 ◽  
Author(s):  
Véronique Pautot ◽  
Frances M. Holzer ◽  
Josette Chaufaux ◽  
Linda L. Walling

Tomato plants constitutively express a neutral leucine aminopeptidase (LAP-N) and an acidic LAP (LAP-A) during floral development and in leaves in response to insect infestation, wounding, and Pseudomonas syringae pv. tomato infection. To assess the physiological roles of LAP-A, a LapA-antisense construct (35S:asLapA1) was introduced into tomato. The 35S:asLapA1 plants had greatly reduced or showed undetectable levels of LAP-A and LAP-N proteins in healthy and wounded leaves and during floral development. Despite the loss of these aminopeptidases, no global changes in protein profiles were noted. The 35S:asLapA1 plants also exhibited no significant alteration in floral development and did not impact the growth and development of Manduca sexta and P. syringae pv. tomato growth rates during compatible or incompatible infections. To investigate the mechanism underlying the strong induction of LapA upon P. syringae pv. tomato infection, LapA expression was monitored after infection with coronatine-producing and -deficient P. syringae pv. tomato strains. LapA RNA and activity were detected only with the coronatine-producing P. syringae pv. tomato strain. Coronatine treatment of excised shoots caused increases in RNAs for jasmonic acid (JA)-regulated wound-response genes (LapA and pin2) but did not influence expression of a JA-regulated pathogenesis-related protein gene (PR-1). These results indicated that coronatine mimicked the wound response but was insufficient to activate JA-regulated PR genes.


Plant Disease ◽  
2021 ◽  
Author(s):  
Peninna Deberdt ◽  
Gilles Cellier ◽  
Régine Coranson-Beaudu ◽  
Mathis Delmonteil--Girerd ◽  
Joanye Canguio ◽  
...  

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.


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