The
bacterial wilt disease

            Bacterial
wilt disease is considered to be a major plant disease since its first report
from USA in potato at the end of 19th century (Burril, 1890). The
disease caused by R. solanacearum is
considered as one of the most destructive and wide spread bacterial disease of
solanaceous crop plants in the tropic, subtropics and warm temperate regions in
the world (Kelman, 1953; Hayward, 1991; Denny, 2006; Guidot et al., 2014). There is huge genetic
diversity in R. solanacearum across the globe and thus it is termed as R.
solanacearum species complex (RSSC) (Fegan and Prior, 2005). These marked
differences in geographical distribution suggest separate evolutionary
development. The variability in R. solanacearum species complex is
classified by different workers on the basis of different criteria like host
range, utilization of different carbon sources, phylogenetic relationship, etc.
It constitutes to be one of the largest known host ranges including plants of
economic importance such as potato (Solanum
tuberosum L.), tomato (Lycopersicon
esculentum L.), tobacco (Nicotiana
tabacum L.), chilli (Capsicum annum
L.), groundnut (Arachis hypogea L.),
Ginger (Zingiber officinale Rosc.),
pepper (Piper nigrum L.) and banana (Musa spp. Colla.) (Nayar and Mathew,
1982).

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            The genus Ralstonia belongs
to the ?-proteobacteria (Palleroni et al.,
1973). R. solanacearum is a gram-negative aerobic bacterium, which is
rod-shaped and has polar flagella, non-sporulating bacteria, produces colonies
which are fluid, smooth, white with red central whirling pattern on Triphenyl
tetrazolium chloride medium (Kelman, 1953; Holt et al., 1994). Ralstonia solanacearum has a wide host
range. Orignially known as Pseudomonas solanacearum (Yabuuchi et al., 1995), the pathogen is
considered as one of the most important plant pathogenic bacteria due to
the economic losses that occur globally resulting from bacterial wilt
disease caused by R. solanacearum (Hayward, 1991). The characteristic
symptoms of the disease are wilting, stunting, yellowing of foliage and finally
collapse of the whole plant. For confirmation of the pathogen ooze test is
done, where smoke like bacterial oozes comes out from cut ends (Singh, 2005 and
Mondal, 2011).

The
pathogen can survive for a long period of time in a nutrient depleted environment
(Grey and Steck, 2001). It has
the ability to survive in the soil in the absence of a host for extended
periods as well as in the protected niche of a weed’s rhizosphere (Hayward,
1991). High soil moisture in well-drained soils is conducive to R.
solanacearum survival, however, its survival in the soil is temperature
dependent. A high day temperature of 40oC maintained for more than
four hours has been shown to reduce bacterial populations. Although an increase
in ambient temperature between 30-35oC has been correlated with an increase
in disease incidence and rate of onset of bacterial wilt on hosts such as
tomato (Hayward, 1991). A host may often be regarded as healthy since disease
symptoms are not visible however the pathogen can be present in the plant at
high inoculum levels. The pathogen over-winters in diseased plants or plant
debris, in vegetative propagative organs such as potato tubers or banana
rhizomes, on the seeds of some crops like capsicum and tomato, and in the rhizosphere
of weed hosts e.g. Solanum dulcamara, Solanum carolinense and Solanum
cinereum (Hayward, 1991; Elsas et
al., 2000). This results in latent infection as the host is further
cultivated (Denny et al., 2001).

Reports
on severe wilt in crops, planted for the first time in virgin soils in
Indonesia, Central America and Florida indicated that the pathogen has survived
in widely separated area (Budenhagen and Kelman, 1964). The loss in yield due
to bacterial wilt ranges from about 10.8 to 90.6 per cent depending on the
environmental conditions and stage of the crops during infection. The maximum
yield loss was reported to occur during summer season when the crop was
infected within the age of 60 days from planting (Kishun, 1989). In India
bacterial wilt of brinjal (Solanum
melongena L.) was first reported by Das and Chattopadhyay in 1955 from West
Bengal and the annual yield loss was estimated to be about 54.6 to 62.5 per cent
and also reported that the pathogen persisted for about 16 months in soil being
soil borne and soil inhabitant in nature.

            Insect dissemination of R. solanacearum in banana (Moko Disease)
has been uniquely important for spreading of the disease. Reddy (2013) reported
that in crops like chilli, disease spread through infected plants, weeds,
irrigation water, soil and implements. The pathogen has an extremely wide and
diverse host range including both monocots and dicots. Many weeds also served
as a host of R. solanacearum in
Bangladesh and Brazil (Dey et al.,
2001).

            Sarkar and Chaudhuri (2016) reported
that bacterial wilt disease is now endemic in west coast of Thiruvanthapuram in
Kerala to Gujarat, Karnataka, Maharashtra and Madhya Pradesh, the eastern
plains of Assam, Orissa, West Bengal and the Andaman & Nicobar islands. It
is also endemic in eastern hills of West Bengal, Meghalaya, Manipur, Mizoram,
Nagaland, Tripura and Arunachal Pradesh.

Characterization of R. solanacearum

            The race, biovar and phylotype
classification is considered to be an important key for subdividing R.
solanacearum species complex. R.
solanacearum are grouped based on utilization of disaccharides and hexose
alcohols into biovars and based on host range into races (Hayward, 1964; Kumar et al., 1993). A PCR-RFLP method has
been used to assess the genetic diversity of worldwide collection of R. solanacearum (Poussier et al., 1999).

Strains of R. solanacearum are diverse in host
range, pathogenicity, biochemical and physiological properties, geographical
distribution and epidemiological relationships (Poussier et al., 1999; Horita and Tsuchiya, 2001). The species was
subdivided into five races according to host range (Buddenhagen and Kelman,
1964; Pegg and Moffett, 1971) and into five biovars (Hayward, 1964; Hayward et al., 1990) based on carbon source
utilization. There are numerous subtypes within the biovars that may be
associated with particular geographical locations (Buddenhagen and Kelman,
1964). The classification of R.
solanacearum into races and biovars is superficial and donot give any
phylogenetic information. Although
the biovar and race systems are widely accepted for the classification of R.
solanacearum, there is no definite correlation between biovar and race.
The only positive correlation between
the biovar and race systems exists for biovar 2 and race 3 (Patrice, 2008). Few
years ago, based on sequence analysis of the internal transcribed spacer
region, Fegan and Prior (2005) proposed a new classification scheme and based
on which R. solanacearum was
subdivided into four phylotypes.

R. solanacearum is divided into races based on host
range. Race 1 is known to affect wide range of species including potato,
tomato, eggplant, tobacco, chillies and several weed species families (French,
1994; Denny, 2006). Race 1 is more frequent in warm areas and lower elevations
of the tropics. It has a high optimum temperature requirement of 35-37°C as
races 2, 4, and 5 (Martin and French, 1985). Race 2 of the R. solanacearum is
indigenous to Central and South America, and attacks members of the Musaceae
family such as plantain, triploid bananas, and Heliconia (French, 1994). It causes moko disease on bananas and Heliconia in Central and South America,
and bugtok disease on plantains in the Philippines (Martin and French, 1985).
Race 3 occurs at higher altitudes (in the tropics) and higher latitudes than
race1. It mainly attacks potato, tomato (especially when planted after infected
potato), geranium, occasionally Pelargonium zonale, eggplants, Capsicum,
and some solanaceous weeds like Solanum nigrum and S. dulcamara
(Martin and French, 1985; Janse, 1991; French, 1994). Race 3 also infects a
number of non-solanaceous weed asymptomatically (Wenneker et al., 1999;
Pradhanang et al., 2000). This race has a long association with potatoes
and has an optimum temperature range of 27 – 28°C (French, 1994). Race 4 has
been reported to affect ginger in Asia and Hawaii, while race 5 affects
mulberry in China (EPPO, 2004).

 R. solanacearum species is further
subdivided into biovars based on utilization of the disaccharide cellobiose,
lactose and maltose and oxidation of the hexose alcohols dulcitol, mannitol and
sorbitol (Oslon, 2005). Martin and French, (1985) added that five biotypes I to
V can be distinguished depending on biochemical tests. Biotype II coincides
with race 3, biotype V with race 4 and Biotype I, III and IV are in race I.

            Four
phylotypes of R. solanacearum are
grouped based on molecular technique which are determined after phylogenetic analysis of sequences of
particular genes which corresponds to four broad genetic groups, each of them is
related to a geographic origin. Phylotype I contains all strains belonging to
biovars 3, 4 and 5, isolated primarily from Asia. Phylotype II includes biovar
1 and 2 strains, and 2T (a subgroup of biovar 2 for tropical areas) isolated
from America, all race 3 strains pathogenic to potato and the race 2 banana
pathogen. Phylotype III comprises strains belonging to biovars 1 and 2T from
Africa and surrounding islands. Phylotype IV is more heterogeneous, with biovar
1, 2 and 2T strains from Indonesia, strains isolated in Australia and Japan.

            A number of different
phenotypic and genotypic methods are presently being employed for the
identification and classification of bacteria, including Ralstonia. Each
of these methods permits a certain level of phylogenetic classification from
the genus, species, subspecies, biovar to the strain level. Modern phylogenetic
classification is based on 16S rRNA sequence analysis (Cook et al., 1989;
Gillings et al., 1993; Seal et al., 1992; Seal et al., 1993;
Poussier et al., 2000; Popoola et al., 2015).

            There
is a lot of controversy regarding the prevalence of strains in the various
parts of the world. In India, however, very limited information is available
about the prevalence of biovars, races and strains in various parts of the
country. There is also no enough information on incidence and distribution of
bacterial wilt in North-east India. Ability of the pathogen to stay long in
soils with its wide host range and farmers practice of saving their own seed
for planting the following season also increase the probability of the disease
occurrence.   Understanding local
pathogen genetic diversity is the first step in a successful breeding and
integrated disease management programme. A study was therefore needed to fill
the current knowledge gap on occurrence, incidence and distribution of the
disease and variability of the disease causing organism throughout North-east
India.

Identification
of races

There is no
biochemical test for race identification of bacterial wilt pathogen R. solanacearum.
The races of R. solanacearum are identified by pathogenicity
tests in wide host range such as brinjal, tomato and chilli etc. Denny and
Hayward (2001) identified race of R. solanacearum by host range.
Aragaki and Quinon (1965) reported that race 4 infected ginger in the Philippines.
He et al. (1983) reported race 5 from
mulberry in China. Five races have been described so far, but they differ in
host range, geographical distribution and ability to survive under different
environmental conditions (French, 1986). Patrice (2008) reported that R.
solanacearum was initially subdivided into races and biovars based on
variability in host range and five races have been identified within the
species. Strains of R. solanacearum have also been divided into
five host-specific races by Pradhanang et al. (2000).

            Dhital
et al. (2000) characterized strains
of R. solanacearum, causal agent of
potato wilt disease based on pathogenicity, biochemical/physiological and
serological tests.

            Kubota
et al. (2008) reported rapid and
sensitive detection of R. solanacearum
pathogen in soil and water using an isothermal method for copying DNA known as
loop-mediated amplification (LAMP).

            Kumar,
R. (2014) reported phylotype I strain of R.
solanacearum species complex in chilli plant collected from a field nearby
to Tezpur university. 

Sakthivel
et al., (2016) reported the presence
of race 1 biovar 3 and race 1 biovar 4 from Andaman islands in India isolated
from tomato, chilli, eggplant.

Gaitonde
and Ramesh (2014) characterized 50 isolates, of which 33 were isolated from
brinjal, tomato and chilli. Results reveald that all the isolates belong to
phylotype I and biovar 3, except one which is grouped under biovar 6. Host
range studies showed that 39 isolates were pathogenic on brinjal and tomato
while 11 were pathogenic on chilli.

Sikirou
et al. (2017) isolated 75 strains of R. solanacearum from tomato, brinjal and
peeper in Benin and reported that the disease was more severe in ferralitic
soil, in valleys and lowlands and in highlands. Strains identified as R. solanacearum were more widely distributed
in the south than the center and the north of Benin. Based on biochemical
characteristics, Beninese R. solanacearum
strains were grouped into biovar I/race1 and biovar III/race 1.

Rahman et al. (2010) reported occurrence of
biovar III and race I of R. solnacearum
in Bngladesh isolated from wilted brinjal plant.

Popoola et al. (2015) reported race 1/biovar 3
and race 3/biovar 2 of R. solanacearum,
causal agent of bacterial wilt of tomato

Gutarra et al. (2017) reported presence of phylotype
I, II & III from Peru.  Gallal et al. (2003) studied the race and
biovar of R. solanacerum by adapting
three detection methods, viz.,
Immuno-fluorescent staining test (IF), semi selective culture medium and
indicator plant (tomato) or BOX-PCR, and found that BOX-PCR could detect race
and biovar of R. solanacearum by
using a reference isolates.

Janse et al. (2004) reported bacterial wilt in
Pelargonium nurseries in Belgium and characterized them as R. solanacearum biovar 2, race 3 on the basis of host range,
morphology, serology, PCR and fatty acid pattern. Stefani et al. (2005) reported the presence of biovar 2 and race 3 of R. solanacearum isolated from wilted
tomato crop.

Nath et al. (2015) characetrized tomato
isolate of R. solanacearum in Assam
based on 16S rDNA and reorted maximum homology using ntBLAST (NCBI).

Shekhawat et al., (1978) recorded strains causing
brown rot of potato belonged to race 1 and biovar 3 & 4 was encountered
among the isolates from eastern parts of India. Race 3 and biovar 2 were
obtained only from a few places in the central plains and Deccan plateau.
Bhattacharya et al. (2003) reported
prevalence of race 1 and biotype 3 infecting potato, tomato, chilli, jute and
banana from West Bengal.

Sreidhar et al. (2015) screened 28 isolates of R. solanacearum from infected chilli,
tomato, brinjal and potato for race detection based on host range and
hypersensitivity testy on capsicum leaves and confirmed that all isolates
collected from different agro-climatic regions of Karnataka and Kerela belong
to race 1 and biovar 3.

Chandrasekhara et al. (2012) screened 54 isolates as
race 1, biovar 3 and 3 isolates were confirmed as race 1, biovar 3B by morphological,
physiological, biochemical and pathogenicity studies. Two sets of primers were
used to authenticate the organism and further confirmed by using a serological
diagnostic kit and a single chain variable fragment antibody specific to R. solanacearum.

In Assam various
starins of R. solanacearum belonging
to race 1 have been reported to cause serious wilt disease in different
solanaceous crops ( Addy et al.,
1980). Nath et al. (1996) reported
the prevalence of  3 races of pathogen in
Assam, while Devnath (2000) reported the prevalence of race 4 of the pathogen
affecting ginger crop.

Biovars
determination

For biovar test
modified minerals media of Ayers et al.
(1919) will be followed.

Test for biovar determination in R. solanacearum

Utilization
of

Biovar
1

Biovar
2

Biovar
3

Biovar
4

Biovar
5

Maltose

+

+

+

Lactose

+

+

+

Cellobiose

+

+

+

Mannitol

+

+

+

Sorbitol

+

+

Dulcitol

+

+

Pathogenicity
of R. solanacearum

            The pathogen
enters the host via root wounds, which may be caused by insects, nematodes,
cultural practices or sites of secondary root emergence (Kelman and Sequeira,
1965). The bacteria move towards the xylem vessels where they multiply and
spread (Salanoubat et al., 2002). The
root cortex and vascular parenchyma are colonised and cell walls are disrupted.
This facilitates the spread of the pathogen through the vascular system. The
bacteria accumulate in pockets filled with slimy masses and cellular debris
(Hayward, 1991; Vasse et al., 1995; Genin and Boucher, 2002). The colonising R.
solanacearum bacteria cause rot and tissue disintegration as a result of
secreted extracellular products. These include an acidic, high molecular mass,
extracellular polysaccharide (EPS1) and several plant cell wall degrading
enzymes: endo-polygalacturonase (PehA), two exo-polygalacturonases (PehB and
PehC), endoglucanase (Egl) and a pectinmethylesterase (Pme). Recently a new
cell wall degrading enzyme has been identified following sequencing of R.
solanacearum: an exoglucanase 1,4 ?-cellobiosidase (Salanoubat et al., 2002). Together the endopolygalacturonases and
exo-polygalacturonases are thought to contribute substantially to the virulence
of R. solanacearum (Genin and Boucher, 2002).

            The
accumulation of the bacteria in pockets in the vascular bundle, pith and the
cortex, effectively destroys the plant’s vascular system. Stems, roots and
tubers discolour through necrosis. These tissues will also exude
whitish-coloured exudates under conditions of severe infection. The plants wilt
completely, with younger plants wilting more rapidly than the older plants,
followed by rotting and disintegration of the roots.

            For
pathogenicity studies different methods have been evolved by different workers.
Out of which root inoculation and stem inoculation techniques are most widely
followed. Kelman and Johnson (1951) noticed that the most effective method for
inoculation for patrhogenicity test of bacteria was stem inoculation technique.
Winstead and Kelman (1952) developed the root inoculation technique by cutting
lateral roots and by pouring the bacterial suspension over the roots which
later become popular and is being adopted by several workers./ Kelman (1953)
reported stem inoculation technique as most effective technique for testing
pathogenicity of R. solanacearum.
Artar et al. (2012) evaluated three
inoculation methods to screen solanaceous vegetable crops for bacterial wilt
resistance, viz., soil drenching,
leaf clipping and axil puncturing method and he recorded highest wilt incidence
in soil drenching method.

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