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In this article we will discuss about:- 1. Introduction to Ralstonia Solanacearum 2. Race and Biovar Classification of Ralstonia Solanacearum 3. DNA Based Classification 4. Phylotype Classification 5. Ralstonia Solanacearum as a Species Complex 6. Diversity 7. Horizontal Gene Transfers 8. Conclusions, Predictions and Speculations.
Contents:
- Introduction to Ralstonia Solanacearum
- Race and Biovar Classification of Ralstonia Solanacearum
- DNA Based Classification of Ralstonia Solanacearum
- Phylotype Classification of Ralstonia Solanacearum
- Ralstonia Solanacearum as a Species Complex
- Diversity of Ralstonia Solanacearum
- Horizontal Gene Transfers of Ralstonia Solanacearum
- Conclusions, Predictions and Speculations of Ralstonia Solanacearum
1. Introduction to Ralstonia Solanacearum:
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Almost all plant-pathogenic bacteria are Gram-negative bacilli, usually associated with the pseudomonads or enterics. Bacterial disease symptoms in plants are described by a number of terms such as spots, blights, soft rots, wilts, and galls.
Bacterial wilts mainly affect herbaceous plants, bacteria invades the xylem vessels, where they multiply, interfering with the movement of water and inorganic nutrients and results in death of the plants due to wilt.
The bacteria commonly degrade xylem vessel and even cause the vessels to rupture. Once the walls have ruptured, the bacteria spread to the adjacent parenchyma tissues, where they continue to multiply. In some wilts, bacteria ooze to the surface of the stems or leaves through cracks formed over cavities filled with cellular debris, gums and bacteria.
However, bacteria do not reach the surface of the plant until the plant has been killed by the disease. Wilt of alfalfa and bean plants are caused by species of Clavibacter; bacterial wilt of cucurbits, such as squashes and watermelons are caused by Erwinia tracheiphila the black rot of crucifers such as cabbage is caused by Xanthomonas campestris.
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The most economically-important wilt of plants is caused by Ralstonia solanacearum.
Identity Name:
Ralstonia solanacearum, Yabuuchi (1995)
Synonyms:
Pseudomonas solanacearum, Smith 1914; Burkholderia solanacearum, Yabuuchi 1992; many other synonyms in Literature.
Taxonomic position:
Bacteria, Gracilicutes, Proteobacteria β subdivision.
Ralstonia solanacearum (Pseudomonas solanacearum) (E.F. Smith) as a species has an extremely wide host range, but different pathogenic varieties (races) within the species may show more restricted host ranges, Several hundred species of tropical, subtropical and warm temperature plants are susceptible to one or other races of Ralstonia solanacearum causing heavy losses in many economically important crops.
The species is highly heterogeneous and complex. It is a major constraint in the production of many important vegetables, fruit, and cash crops. It is a devastating, soil- borne plant pathogen with a global distribution and an unusually wide host range, it causes wilt in over 450 host species in 54 botanical families which may give the pathogen an evolutionary advantage and that number of new species continues to increase.
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The bacterial wilt disease is wide spread, affecting many solanaceous vegetable crops in India, especially Karnataka, which is one of the major vegetable crops growing state in India. The plants with typical symptoms showing leaf drooping followed by wilting of whole plants within a few days, leading to total plant collapse (Fig. 27.1A & 27. IB) some plants produce adventitious roots on stem.
The vascular bundle of such affected plants shows brown vascular discolouration and browning of the pith in the lower stem (Fig. 27.2). For a quick field diagnostic identification of Ralstonia solanacearum and to distinguish bacterial wilt from vascular wilts caused by fungal pathogens, bacterial streaming from infected plant material can be used.
A stem section is cut from a plant with vascular discoloration using a sharp knife or razor blade.
The stem section is placed against the inside wall of a water-filled clear beaker or test tube so that the end of the section just touches the water surface. Milky white strands containing bacteria and extracellular polysaccharide will stream from the cut ends of the xylem (Fig. 27.3) as described by Danks and Barker (2000), thus revealing the presence of bacteria.
Smith (1896) was the first to describe the shape and size of the bacterium. He reported the bacterium was rod shaped measuring 0.5 x 1.5 mm when 48 hours culture was stained with methyl violet. White, irregular and fluidal colonies with blood red colouration in the centre were isolated (Fig. 27.4A, B) from diseased tissues plated on triphenyl tetrazolium chloride medium (TZC).
Electron microscopic studies of Ralstonia solanacearum showed that the bacterium was rod shaped and lophotrichously flagellated with one to four polar flagella (Fig. 27.5).
Several workers also confirmed that the Ralstonia solanacearum is rod shaped lophotrichous flagellated.
2. Race and Biovar Classification of Ralstonia Solanacearum:
Bacterial wilt caused by Ralstonia solanacearum was first reported at the end of the 19th century on potato, tobacco, tomato and groundnut in Asia, southern USA and South America. The bacterium was described for the first time as Bacillus solanacearum by Smith (1896).
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In the years following, at least five pathogenic races and five biovars were discriminated. The race and biovar classification has gained wide acceptance for Ralstonia solanacearum.
The racial pattern system, groups the strains of Ralstonia solanacearum on the basis of their ability to infect different host plants, viz., Race- 1 occurs in tropical areas all over the world and attacks tobacco, tomato, potato, aubergine, diploid banana and many other (solanaceous) crops and weeds. It has a high optimum temperature (35°C, as do race 2, 4 and 5).
Race-2 occurs mainly in tropical areas of South America and affects triploid bananas and Heliconia (causing so-called Moko disease), but also in the Philippines (causing so-called bugtok disease on plantains) and has a high temperature optimum (35-37°): Race-3 (potato race) occurring at higher altitudes in the tropics and in subtropical and temperate areas attacks potato, tomato, occasionally Pelargonium zonale, aubergine and capsicum, some solanaceous weeds like Solanum nigrum and Solanum dulcamara.
A number of non-solanaceous weed hosts have also been found to harbour race 3 infections, often asymptomatically. This race has a low optimum temperature (27°C) and appears to be mostly biovar 2A RFLP group 26 with a worldwide distribution, biovar 2A.RFLP group 27 (found in Chile and Colombia) or biovars 2T (sometimes also called 2N, found in tropical areas in South America).
Race-4 is particularly aggressive on gingers and Race-5 (biovar 5) is specialized on Morus. Another classification of Ralstonia solanacearum, based on RFLP and other genetic fingerprinting studies is into Division I (biovars 3, 4 and 5 originating in Asia) and II (biovars 1, 2A and 2T, originating in South America).
Subhalaxmi (1999) characterized the Sterilizia (Bird of paradise) isolate of Ralstonia solanacearum into race 1 and biotype 3 based on its pathogencity tests and ability to oxidize disaccharides and sugar alcohols. However, variation in ability to utilize sugars such as dulcitol was reported by Mathew (2002) from Kerala, they designated Ralstonia solanacearum strain on ginger as biovar IIIA.
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Prasannakumar and Khan (2004) found two ginger and one tomato strains were not able to utilize dulcitol and lactose. Hence, was designated as biovar IIIB from Kerala. At present, there is lot of controversy regarding the prevalence of strains in the various parts of the world. In India however, scanty information is available about the prevalence of biovars/races and strains in various parts of the country.
3. DNA Based Classification of Ralstonia Solanacearum:
The determination of DNA-DNA homologies has shown that relatedness between strains of this species is often less than the limit of more than 70% expected within a species. On the basis of DNA hybridisation, Ralstonia solanacearum was classified into the 16S rRNA group II, together with Burkholderia cepacia, Pseudomonas marginalis, P. caryophylli, P. syzygii and Ralstonia pickettii.
Based on 16S rRNA sequences, DNA-DNA homology values, cellular lipid and fatty acid composition, and phenotypic characteristics, a new genus Burkholderia was proposed, and this genus was later transferred to Ralstonia.
With the advent of new methods in the molecular biology in recent years, more studies on the variability at the genome level are possible. Genomic DNA analysis was carried out by Gillings and Fahy (1993) by using RFLP (Restriction Fragment length Polymorphism) and also by Cook (1989; 1991) where the technique revealed two major groups.
Seal (1992) divided the species into three fingerprint groups based on tRNA consensus primers. Other studies applying rDNA-PCR described different combinations of primers, allowing PCR amplification of Ralstonia solanacearum division I (biovars 3, 4 and 5) and division II (biovars 1, N2 and 2) including the Blood Disease Bacterium (BDB) and P. syzygii, or amplification of division II only except for biovar 1, 2, or N2 isolates from Indonesia, P. syzygii and the BDB.
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Thwaites (1999) investigated the genetic relationship between strains of Ralstonia solanacearum which cause the moko and bugtok vascular wilt diseases in Musa spp., by using random decamer primers (RAPD).
Prasannakumar and Khan, (2004) grouped 57 stains from India as racel and biovar 3 and found that all the isolates collected from different host and different geographic regions were into seven clusters based on RAPD. Other alternative methods of genomic fingerprinting are AP-PCR (arbitrary primers), and AFLP (Amplified Fragment length Polymorphism).
The latter is based on amplification of digested DNA fragment and was also used by Van Der Wolfed (1998) to fingerprint the Ralstonia solanacearum genome. Another alternative approach established restriction endonuclease profiles of Ralstonia solanacearum genomic DNA, with the use of rare- cutting restriction endonucleases and resolution of the macrofragments by Pulsed-Field Gel Electrophoresis (RC-PFGE).
Bacterial chromosomes contain multiple interspersed repetitive sequences that occupy intergenic regions at sites dispersed throughout the genome. The well characterized prokaryotic repetitive element is the REP (Repetitive Extragenic Palindromic) sequence, also called PUs (Palindromic Units).
A palindromic unit (PU) was originally defined in 1982 as a palindromic DNA element present several times in four intergenic regions of bacterial operons. Many roles for REP sequences have been suggested. It has been suggested therefore, that REP sequences may represent a selfish sequence which is maintained by gene conservation.
A second family of conserved sequences is the ERIC (Enterobacterial Repetitive Intergenic Consensus) sequence, which has no similarity to the REP sequence. Nevertheless, some features of ERIC and REP sequences are similar and they may be maintained in the bacterial genome by similar mechanisms.
The repetitive sequence based polymerase chain reaction (rep-PCR) was introduced by Versalovic (1991; 1994). De Bruijn (1992) used REP and ERIC-PCR to fingerprint the genome of Rhizobium meliloti and other soil bacteria. Furthermore, rep-PCR-mediated genomic fingerprinting has been applied to plant pathogenic bacteria at pathovar and subspecies level.
A third group of highly conserved DNA sequences located within intergenic regions of the chromosome was called BOX elements. From 5′ to 3′, BOX elements are located in the immediate vicinity of genes whose product has been implicated at some stage in the process of genetic transformation or in virulence of Streptococcus pneumoniae, raising the intriguing possibility that BOX sequences are regulatory elements shared by several coordinately controlled genes, including competence- specific and virulence related genes.
Specific genomic fingerprints by means of rep-PCR were obtained by Louws (1994). Rep-PCR-based fingerprinting of bacterial pathogens of Musa spp. produced groupings broadly concurrent with those obtained from RAPDs.
Genetic diversity of Ralstonia solanacearum race 3 in Kenya was studied by means of ERIC and BOX elements along with RC-PFGE. These fingerprinting techniques added to AFLP were also used by Van Der Wolf (1998) to analyse Ralstonia solanacearum race 3 in west Europe.
Race 1 indigenous to the French, West Indies was characterized by bacteriocin typing and two genomic finger-printings: RC-PFGE and rep-PCR. Tsuchiya and Horita (1998) described the genetic diversity of Ralstonia solanacearum strains in Japan, based on RFLP and rep-PCR. By using the same techniques, Raymondo (1998) could analyse the genetic variation of Ralstonia solanacearum strains infecting banana.
Biogeographic studies of races 1 and 3 by genomic fingerprinting were undertaken by Smith (1998) with macro-restriction of genomic DNA resolved by pulsed-field gel electrophoresis (MR-PFGE) and PCR with primer sets to concerned bacterial repetitive DNA motifs (ERIC and BOX elements). Gadewar (2002) studied diversity in seven Indian isolates using PFGE and found dissimilar restriction fragment patterns.
They also observed plasticity in Ralstonia solanacearum genome by change in molecular weight of plasmids. It was concluded that both genomic plasticity and genetic exchange appear to contribute towards creation of large scale diversity in Ralstonia solanacearum (Fig. 27.6).
4. Phylotype Classification of Ralstonia Solanacearum:
Ralstonia solanacearum was placed on the USA’s list of potential food safety threats and agro-terrorism agents and EPPO A2 quarantine organism (OEPP/EPPO, 1978). Ralstonia solanacearum and its close relatives, the blood disease bacterium and R. syzygii, constitute a species complex, a diverse group of related isolates that represent more than one species.
From last decade, knowledge on the genetic diversity and the phylogenetic relationships within the Ralstonia solanacearum complex has indicated that phenotypically based schemes (race and biovar) are not sufficient to encompass the diversity of strains represented in the species.
Prior and Fegan (2005) developed a hierarchical classification system based upon phylogenetic analysis of sequence data of four molecular markers. This classification reported four monophyletic clusters of strains, termed phylotypes, related to the geographical origin of the strains. The phylotyping scheme is highly discriminatory, flexible, additive, and allows prediction of new properties of strains.
The development of a classification scheme which is representative of the organismal evolutionary relationships should be of benefit to the interdisciplinary “Global Ralstonia solanacearum Projects Initiative” (GRASP IN) to explore and survey phylogenetic diversity and to reassess resistance sources in the Solanaceae.
Institutions like INRA, CIRAD, CNRS, AVRDC, IRAD, CIAT and CIP are willing to develop such a collaborative network. Phylogenetic information will be cross referenced with additional criteria to allow nomination of an international set of Ralstonia solanacearum strains representative of the genetic and phenotypic diversity within this species complex.
In future, this collection could be reduced to a working collection of strains carrying the highest aggressiveness to challenge bacterial wilt resistance sources in solanaceous plants assembled for the GRASP IN at Reunion Is., under the leadership of CIRAD-INRA. Three hundred additional strains were phylotyped.
As expected, all but two exotic strains representative of the Taiwan population were from phylotype I; strains newly isolated from major agro-ecological zones in Cameroon were 50% phylotype I, 30% phylotype II and 20% phylotype III; a selection of CIP strains from South America, Asia and Africa were 90% phylotype II and 10% phylotype III.
The mutS and egl partial gene sequences from all strains were added to the Arb databases maintained at Reunion Is to up-date the additive phylogenic trees. The topology of Arb- computed trees was not changed and they reported additional clusters of strains with high bootstrap values within the Asiatic phylotype I (Bv 3, 4, and 5), the Ralstonia solanacearum potato pathogens (race 3) of phylotype II, and the African phylotype III (Bv 1 and 2T).
Phylogenetic information was crossed with additional criterion to nominate an international set of 32 strains representing the Core-Rs collection, a unique attempt to consider the genetic and phenotypic diversity within the Ralstonia solanacearum species complex. Core-Rs were reduced to 10 strains carrying the highest aggressiveness to challenge bacterial wilt resistance sources in tomato, eggplant and pepper assembled for the GRASP IN Reunion Is.
Despite the great international effort allocated for breeding and mapping QTLs for resistance to bacterial wilt, the successes are limited since the efficiency of the resistances used so far may have failed due to the strong interaction between plant, bacteria and environment.
Internationally recognized breeders and plant geneticists contributed to nominate the Core-TEP, a reference collection assembled to encompass resistance sources identified in Tomato, Eggplant and Pepper (TEP), based on their genetic relatedness (syntenic genome species) and origins.
It is anticipated that studying interactions between the Core-Rs and the Core-TEP in artificial and natural environment (multi local trials with a consortium of private breeders worldwide) will provide a better understanding of resistance and tolerance proprieties to bacterial wilts in response to various genotypes and pathotypes of Ralstonia solanacearum.
5. Ralstonia Solanacearum as a Species Complex:
A species complex is defined as a cluster of closely related isolates whose individual members may represent more than one species. The term “species complex” was first applied to Ralstonia solanacearum by Gillings and Fahy (1994) to reflect the phenotypic and genotypic variation within the species.
Taghavi (1996) then expanded the concept of the Ralstonia solanacearum species complex to include two closely related organisms, the blood disease bacterium (BDB) and Pseudomonas syzygii as both of these organisms were found to fall within the diversity of Ralstonia solanacearum as defined by 16S rDNA sequence analysis.
Studies of DNA-DNA homology of Ralstonia solanacearum strains have revealed that the relatedness between isolates of this species is often less than the 70% threshold level commonly expected within a species. Therefore we define Ralstonia solanacearum as a species complex.
A stable and meaningful taxonomy and nomenclature which accurately defines subspecific groups of Ralstonia solanacearum has to be the aim of taxonomists working on the Ralstonia solanacearum species complex.
Such a taxonomic system will aid plant breeders, plant pathologists and quarantine officials who require a system of classification where strains can be grouped into clusters of isolates that relate to epidemiology, pathogenicity, host range and/or geographic origin.
The taxonomic framework and methodology proposed below allows identification of subspecific groups within the Ralstonia solanacearum species complex and will improve our ability to predict the properties of Ralstonia solanacearum strains.
6. Diversity in Ralstonia Solanacearum:
Traditionally Ralstonia solanacearum has been classified into five races on the basis of differences in host range and six biovars on the basis of biochemical properties.
The work of Cook (1989) and Cook and Sequeira (1994) employing restriction fragment length polymorphism (RFLP) analysis showed that Ralstonia solanacearum can be divided into two divisions: Division-1 comprising strains belonging to biovars 3, 4 and 5, primarily isolated in Asia and Division-2 comprising strains belonging to biovars 1, 2 and N2, primarily isolated in the Americas.
Several other investigations employing molecular methods have confirmed this dichotomy within Ralstonia solanacearum. Taghavi (1996), using 16S rDNA sequence analysis, also revealed the existence of a subdivision within division 2 comprising isolates of Ralstonia solanacearum from Indonesia including the closely related organisms the blood disease bacterium (BDB) and P. syzygii.
Further sequencing of thel6S- 23S rRNA gene intergenic spacer region (ITS), the polygalacturonase gene and the endoglucanase gene has supported the existence of the two divisions and the existence of the group of strains originating in Indonesia. PCR-RFLP analysis of the hrp gene region demonstrated that certain African biovar 1 strains did not cluster with other biovar 1 isolates as expected.
An extended PCR-RFLP analysis of the hrp gene region complemented by amplified fragment length polymorphism (AFLP) and sequencing of the 16S rRNA gene has provided further support for the existence of this group of strains.
Phylogenetic analysis of the endoglucanase and hrpB genes has confirmed the presence of a group of strains originating in Africa. Hence the picture has emerged that the Ralstonia solanacearum species complex is comprised of four broad genetic groups corresponding with geographic origin.
Phylotyping Scheme: A New Scheme for Classifying Ralstonia Solanacearum:
A hierarchical classification scheme is proposed to reflect the known diversity within the Ralstonia solanacearum species complex. The scheme is outlined in Table 27.1. Under this classification system, members of the Ralstonia solanacearum species complex can be subdivided into four phylotypes corresponding to the four genetic groups identified via sequence analysis (Fig. 27.7).
A phylotype is defined as a monophyletic cluster of strains revealed by phylogenetic analysis of sequence data, in this case the ITS region, the hrpB gene and the endoglucanase gene. Phylotype I is equivalent to division 1 defined by Cook (1994).
This phylotype includes all strains belonging to biovars 3, 4, and 5; strains are isolated primarily from Asia. Phylotype II is equivalent to division 2 (1989), includes strains belonging to biovars 1, 2 and 2T isolated primarily from America.
Phylotype II contains the Ralstonia solanacearum race 3 potato pathogen, with worldwide distribution, and the race 2 banana pathogens. Phylotype III contains strains primarily isolated from Africa and surrounding islands, strains belong to biovars 1 and 2T.
Phylotype IV contains strains isolated primarily from Indonesia belonging to biovars 1, 2 and 2T. Ralstonia solanacearum strains in Phylotype IV have also been found in Australia and Japan.
This phylotype also contains the two close relatives of Ralstonia solanacearum, P. syzygii and the BDB. The phylotype to which a strain belongs can be rapidly identified using a multiplex PCR based upon sequence information from the ITS region.
This phylotype-specific multiplex-PCR employs four forward primers one specific for each phylotype and a single reverse primer which is specific for the species (Table 27.2, Fig. 27.1) and also includes the 759/760 primer pair described by Opina (1997).
All Ralstonia solanacearum, BDB and P. syzygii strains generate the 280 bp Ralstonia solanacearum species complex-specific fragment produced by amplification of template DNA by the 759/ 760 primer pair.
All Ralstonia solanacearum, BDB and P. syzygii strains tested with the multiplex PCR produce a phylotype specific amplicon with the exception of strain ACH0732. Strain ACH0732 is the only Ralstonia solanacearum strain that varies in its phylogenetic position depending on the genomic region sequenced.
Therefore using the phylotype specific multiplex PCR ACH0732 only produces the 280 bp Ralstonia solanacearum species complex-specific fragment. Each phylotype is composed of a number of sequevars. A sequevar or sequence variant is defined as a group of strains with a highly conserved sequence within the area sequenced.
Only if two or more strains sequenced have similar sequences has a sequevar been defined. Therefore single sequence clusters have not been given sequevar status (for example CIP10 in Fig. 27.2).
Sequevars are primarily defined upon partial endoglucanase gene sequences as a large number of strains have been sequenced in this region. In the future it is hoped that sequence information from more strains from other areas of the genome, such as the hrpB gene, will be generated to confirm these sequevars.
The endoglucanase gene of > 140 Ralstonia solanacearum isolates has been sequenced and over 20 sequevars have been identified (Fig. 27.8).
Each sequevar may be composed of a number of clonal lines which may be identified using genomic fingerprinting methods such as PFGE, AFLFs or rep-PCR. In our experience rep-PCR is a fast and reproducible method for identification of clonal lineages within a sequevar. The phylotyping scheme is highly discriminatory, flexible and additive allowing identification of further sequevars or even phylotypes.
This phylotyping scheme is based upon genetic variation that accumulates relatively slowly in the genome of organisms at the level of the phylotypes and sequevars thus giving a long term global epidemiological perspective. However, the scheme also incorporates the finer resolving power of the genomic fingerprinting techniques to identify clonal lines below the level of the sequevar.
Because PCR facilities are not yet routinely applicable worldwide, an attempt has also been made to identify phenotypes associated with phylotypes or sequevars. This will allow phenotypic identification of these genetic groups. This approach is fundamentally different from the biovar scheme as it is attempting to identify a phenotype associated with an already identified genetic cluster of isolates.
This work is ongoing but it is hoped that we will be able to use biochemical tests to identify the major genetic groups and thus allow laboratories that do not have ready access to sequencing technologies to accurately identify and place unknown isolates into this new typing scheme. The biotyping scheme described here is based upon the work of Harris (1972) and Hayward (1964).
Our results have confirmed the great degree of phenotypic diversity within biovar 1 isolates identified by Harris (1972). This diversity is not surprising as this biovar is based on negative criteria for both disaccharides and hexose alcohols.
The biotype scheme generates a unique metabolic profile for many sequevars based on a set of six substrates, namely maltose, manitol, malonate, trehalose inositol and hippurate (Table 27.3).
Of the 56 strains tested in our collection, only the strain UW170 did not provide a biotyping profile consistent with the sequevar to which the strain belongs. Genetically this strain falls in phylotype II/sequevar 4 and it was expected that it would be of biotype 4. However, UW170 produced a biotype 3 profile which is characteristic of phylotype II/sequevar 3.
The biotyping system needs to be fully validated on many other strains. Nevertheless, the scheme has already been useful in typing unknown strains of Ralstonia solanacearum isolated from pothos and from anthurium. The biotype of the isolates from these hosts was used to predict the phylotype and sequevar to which they belonged. The predicted phylogenetic positions were confirmed later by sequence analysis.
Comparison of the Phylotyping Classification Scheme to Previous Schemes:
In comparison to race and biovar classification schemes we feel the phylotyping scheme discussed here more accurately reflects the diversity that we now know to be present in the Ralstonia solanacearum species complex. Race 1, defined as strains “affecting tobacco, tomato, many Solanaceous crops and other weeds and certain diploid bananas” is a very broad definition.
Strains belonging to race 1 are found in phylotypes I and II and probably in phylotypes III and IV if host of origin can be used as a guide to which race a strain belongs. In contrast to Race 1, Races 2 and 3 have narrow host ranges and this is reflected in the narrow genetic diversity included within races. Race 3 strains belong to phylotype II, sequevars 1 and 2.
Strains belonging to race 2 belong to phylotype II, sequevars 3, 4 and 6. Strains representing biovars 1 and 2T are present in three of the four phylotypes and it is clear that simply identifying a strain as biovar 1 or 2T does not tell you much about the strain. The large degree of variation within strains of biovar 1 has previously been recognised phenotypically and this is mirrored in the genetic variation found in biovar 1 strains.
Most strains belonging to biovar 2 are equivalent to race 3 and therefore belong to phylotype II sequevars 1 and 2. However, some biovar 2 strains do not belong to race 3 and are found in phylotype IV sequevars 8 and 9.
This new scheme largely confirms the RFLP typing scheme Cook (1989). Phylotypes I and II are equivalent to the divisions 1 and 2 defined by Cook (1994).
Phylotypes III and IV were not recognised by Cook (1989) as they did not study strains belonging to these two phylotypes. It would be expected that if strains belonging to these two phylotypes had been analysed using RFLP’s then these two groups would have been identified earlier.
Below the level of the phylotype at the sequevar level the RFLP and phylotyping schemes are also congruent with strains which belong to different MLG’s also belonging to distinct sequevars. For example strains belonging to MLG’s 24, 25 and 28, which contain moko disease causing strains of Ralstonia solanacearum, are equivalent to sequevars 3, 4 and 6 respectively.
How useful is the Phylotyping Scheme:
The DNA-DNA hybridisation values of certain strains of Ralstonia solanacearum are lower than would be expected of organisms belonging to the same species. Further, they used strains representing biovars 1-4; the puzzling result from their analysis was the low DNA-DNA hybridisation values between strains representing biovars 1.
The biovar 1 strains used by these authors originated from the USA, South America, Zimbabwe and Reunion Island. When the same strains were analysed using the phylotyping scheme the strains from the USA and South America were found to belong to Phylotype II whereas the strains from Zimbabwe and Reunion Island belonged to Phylotype III.
This probably explains the low hybridisation values. However, it does raise the question as to the taxonomic relationship of strains belonging to each phylotype.
The phylotyping scheme has also been used to help elucidate the unexplained results of Marin and El-Nashaar (1993). The isolated biovar 1 strains from potato in Peru that apart from the biochemical tests used to differentiate the biovars were phenotypically and in pathogenicity the same as biovar 2/race 3 strains.
When these strains were placed in the phylotyping scheme they were found to belong to Phylotpe II, sequevar 1 which contains only Ralstonia solanacearum biovar 2/race 3 pathogens of potato. When the strains were compared to other strains belonging to sequevar 1 using rep-PCR they produced a fingerprint the same as other strains within sequevar 1.
7. Horizontal Gene Transfers in Ralstonia Solanacearum:
Bacterial genomes are extremely dynamic and mosaic in nature. A substantial amount of genetic information is inserted into or deleted from such genomes through the process of horizontal transfer.
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Through the introduction of novel physiological traits from distantly related organisms, horizontal gene transfer often causes drastic changes in the ecolagical and pathogenic character of bacterial species and thereby promotes microbial diversification and speciation.
Different works suggested the importance of horizontal gene transfers (HGT) in genome plasticity of Ralstonia solanacearum. Sequencing and annotation of the genome of strain GMI1000 established that this bacterium harbours a 2.1 Mb megaplasmid in addition to a 3.7 Mb chromosome.
It also demonstrated that these two replicons have a mosaic structure where fragments having the expected base composition and codon usage of Ralstonia solanacearum alternate with ACURs (alternate codon usage regions) characterized by a biased GC% and often flanked with mobile genetic elements suggesting that they have been acquired by HGT.
The fundamental role of HGT in Ralstonia solanacearum genome evolution has been supported by the findings of several studies. Bertolla (1997, 1999) demonstrated that this bacterium is able to naturally develop the physiological state of competence required to exchange genetic material by transformation under in vitro as well as in planta conditions.
Recently, Coupat (2008) demonstrated that transformability is ubiquitously shared among the four phylotypes of Ralstonia solanacearum and that large DNA fragments ranging from 30 to 90 kb can be exchanged.
In silico analysis of the complete genome sequence of the Ralstonia solanacearum GMI1000 strain revealed the mosaic structure of both the 3.7 Mb chromosome and the 2.1 Mb megaplasmid that constitute the bacterium’s genome.
More than 7% of the genome contains regions with a GpC-biased composition and alternative codon usage regions (ACURs), frequently surrounded by IS and phage elements. The authors assumed that these genomic regions were acquired from other species by HGT.
Complementary analysis based on phylogenetic reconstruction of prokaryote homologous gene families or on nucleotide composition detected that 13-15% of the Ralstonia solanacearum GMI1000 genes originated from HGT events.
Recently, an analysis of gene distribution among Ralstonia solanacearum strains by comparative genomic hybridization (CGH) on the microarray identified a list of 2338 variable genes (40% of the genome) within the species. These variable genes often cluster within genomic islands, suggesting that they might originate from HGT.
8. Conclusions, Predictions and Speculations of Ralstonia Solanacearum:
The Ralstonia solanacearum species complex is composed of at least 4 genetic groups or phylotypes. Within these phylotypes there are subgroupings, sequevars, which correspond to clusters of isolates with similar pathogenicity or isolates of common geographic origin.
The additive nature of the scheme gives it groat flexibility and allows addition of more genotypes as they are discovered. It is hoped that this scheme will be of use to plant breeders, plant pathologists and quarantine officials as it is able to distinguish epidemiological and ecological groupings of Ralstonia solanacearum strains and will thus help predict the biological properties of unknown strains.
As more strains are isolated from environmental sources and other natural hosts of Ralstonia solanacearum it is expected that greater genetic diversity will be uncovered. Although, it is possible that this new genetic diversity may lead to the description of new phylotypes in our opinion.
However, it is almost certain that as more strains are sequenced more sequevars will be described. Collecting and cataloguing, strains of Ralstonia solanacearum although very important, are a relatively simple endeavour.
However, it is more difficult to gather information on the biological, ecological and epidemiological properties of strains. Without both pieces of the puzzle it is impossible to use any taxonomic scheme to predict pathogenicity of strains or to aid in control of the disease.
