In this article we will discuss about the bacterial wilt disease of potato caused by ralstonia solanacearum.
Potato (Solarium tuberosum L.) a poor man’s food, is an important solanaceous vegetable crop. Potato ranks fourth in production, after rice, wheat and maize and provides wholesome food. Potato is grown almost worldwide over 37 countries. Annually about 300 million tones of potatoes are produced and consumed by over one billion people world over.
Potato production has increased most rapidly in the far Eastern countries such as China, India and Indonesia. Potato is a native of Andes in South America. According to Scott (1976), the Inca Indians first cultivated potato in 200 B.C., which they called “papa”.
In 1537, Spanish conquistadors introduced potato in Spain, from where it spread into Italy and then into Central Europe, especially in Germany, Russia and Ireland.
Potato can contribute substantially towards both food and nutritional security in the years to come. It holds a great potential as food for the ever increasing world population per unit time and area as well as its versatility to adapt to a wide range of climate. Potato is a starch rich food (14-16%). which also contains protein (2.8%) and minerals (0.9%).
Potato constituted 18-20 per cent of the total production of the agricultural commodities for utilization as human and animal feed in addition to industrial use. The biological value of potato protein is very high and it contains sufficient quantity of vitamin C and considerable quantities of vitamins of B group.
Potato was introduced from Europe into India by British missionaries in late seventeenth century. The average potato yield in India in 2002-03 was 19.4 t/ha as compared to world average yield of 16.4 t/ha. The total area under potato in 2002- 03 was 1.4 million ha, which accounted for yield of 25.0 million tonnes.
India is the third largest potato producer in the world. According to the International Food Policy Research Institute (IFPRI) and International Potato Centre (CIP), India is likely to have the highest growth rates in production and productivity of potatoes during 1993-2020. India’s potato export has not been exploited to potential (<0.5 % of world export).
In spite of the suitable conditions for the cultivation, of potato, many factors are responsible for its low yield. Fungi, bacteria, viruses, phytoplasma, viroid and nematode are reported to attack and parasitize potato in field during transportation and storage. In India, important diseases of’, potato are reported to cause nearly 30- 40 per cent reduction in yield and this is about 6-10 million tonnes of potato every year.
In addition to foliage blight pathogens, bacteria are among the most potential pathogens, which have been found to cause six diseases in potato crop viz., bacterial wilt or brown rot (Ralstonia solanacearum), soft rot of stem or tubers (Erwinia carolovora, Bacillus sp., Pseudomonas sp.), ring rot (Corynebacierium sepedonicum), common scab (Streptomyces sp.), pink eye (Pseudomonas sp) and leaf spot (Xanthomonas vesicatoria). The ring rot and pink eye do not occur in India.
Bacterial wilt caused by Ralstonia solanacearum (Smith) Yabuuchi is one of the most important and destructive bacterial disease of plants, widely distributed in tropical, subtropical land and some warm temperate regions of the world. It is first bacterial disease recorded in India from Pune, Maharashtra and also first record of its occurrence on potato.
Now the disease has gradually become a problem of increasing importance and the damage caused by it in certain areas seems to be considerable. In India, losses caused by bacterial wilt vary from 20-100 per cent.
The disease may damage the crop due to premature wilting which results in causing yield losses and rotting of tubers both in field and stores. Several non-host crops and weeds can harbour the pathogen in their root systems. The pathogen could also survive in sheltered sites left over in the soil.
Since the pathogen is mainly transmitted through seed tubers, as latent infection in vascular tissues of progeny tubers. There is need to detect latent infection in tubers for disease free seed (tuber) production. Imrnunodiagnostid techniques have been standardized for proper detection of Ralstonia solanacearum in infected plants and soil.
Keeping in view the economic importance of potato crop, detection and diagnosis ofRalstonia solanacearum causing bacterial wilt pathogen by serodiagnosis is discussed here.
Geographical Distribution of Ralstonia Solanacearum:
Bacterial wilt of potato caused by Ralstonia (Pseudomonas) solanacearum (Smith) Yabuuchi is widely distributed in tropical, subtropical and some warm temperate regions of the world. Table 1 shows that Ralstonia solanacearum Race 3, Bio var II has a much greater distribution into both higher latitudes of the globe and greater altitudes in the tropics.
Bacterial wilt is the first bacterial disease recorded in India from Pune, Maharashtra by Cappel (1882) and the first record of occurrence on potato was made by Butler (1903).
Now the disease has gradually become a problem of increasing importance and the damage caused by it in certain areas seems to be considerable. In India, the disease is epidemic in West coast from Trivendrum in Kerala to Khera in Gujarat, in Central Karnataka, Western Maharashtra and Madhya Pradesh, in Eastern plains of Assam, Orrisa, Chhota Nagpur Plateau and Andaman Nicobar Islands (Table 26.2).
Economic Importance:
The major crops affected by Ralstonia solanacearum, belongs to solanaceous vegetables such as summer grown tomato, brinjal in the plains and tomato and potato in hills. Serious incidence of wilt on chillies has also been reported. In India losses caused by bacterial wilt vary from 20-100 per cent.
Verma and Shekhawat (1990) observed the endemic occurrence of bacterial wilt in Mukteswar region of Uttarakhand where potato seed production was taken. They reported that nearly 37 per cent potato produce was lost every year due to brown rot.
In potato, losses reported are upto 13.8- 55 per cent in Kumaon hills, 0.33-40.00 per cent in Maharashtra. 20-25 per cent at Hyderabad and over 75 per cent in some locations of Karnataka. According to Olonya (2002) the potato production and yield losses due to bacterial wilt as high as 100 per cent have been reported in parts of tropical Africa.
Pathogen:
Ralstonia solanacearum has been associated with bacterial wilt of potato since it was reported in 1903. With the introduction of molecular technology the generic nominal of the wilt pathogen underwent rapid change from Pseudomonas to Burkhalderia to Ralstonia. Yabuuchi (1992) proposed new genus Burkholderia to accommodate RNA homology group II including Pseudomonas solanacearum with P. cepacia as type species.
Later work based on 16S rRNA genes and polyphasic taxonomy showed dichotomy in genus Burkhoderia and the new genus Ralstonia was proposed with R. picketti as type species.
Ralstonia solanacearum is a gram negative rod measuring approximately 0.5-0.7 x 1.5-2.5 pm. Virulent isolates are mainly non-flagellated, non-motile and are surrounded by extracellular slime. Avirulent isolates are devoid of any extracelluar slime, usually they bear 1-4 polar flagella and are highly motile. Polar fimbrae present with which twitching motility and spreading growth on solid media are associated.
Cells contain inclusion of poly-beta-hydroxybutyrate which are sudanophilic and retractile under phase microscope and commonly show bipolar staining. It is a chemo-organotroph with aerobic respiratory metabolism; catalase and kovac’s oxidase positive; optimum, temperature for growth varies from 27-37°C depending on the strain.
Diversity in Pathogen:
Intraspecific diversity in Ralstonia solanacearum was evident since early years of 20th century. Smith (1914) described isolates from Sumatra affecting tobacco as Ralstonia solanacearum var. asiaticum on the basis of acidification of litmus milk containing cream. The strains of Ralstonia solanacearum differ in host range, geographical distribution, pathogenicity, epidemiological relationships, and physiological properties.
It is therefore important to have a classification of strains that is sufficient in information content of’ epidemiology and control of bacterial wilt. For almost the past three decades, a binary system has been in use reflecting two different approaches to differentiation, one placing emphasis on host affinity and establishment of races (Table 4).
The other making use of selected biochemical properties as the basis for separation into biovars.
Isolates collected from different areas of India belonged to Race-1 and 3 and biotype- II, III and IV. The Isolates from cool humid hilly areas belonged to race-3 and biotype- II. However, prevalence of Race-1 in Panchagani hills of Western Ghat, Palni-hills of Tamil Nadu, Kumaon hills of North India have been recorded.
A large number of Ralstonia solanacearum isolates from Indian plains and plateau belong to Race-1 and Biovar-III. Biovar-IV has been recorded only from two locations in Eastern plains, two locations in plateau and in mid-hills of Kumaon. Sunaina (1997) reported an odd type of strain oxidizing only maltose and mannitol from North- Eastern hills.
In contrast to normal white-coloured colonies, this strain produced cream- coloured fluidal colonies on tetrazolium chloride medium. It did not cause disease symptoms in aubergines, Datura streamonium or Capsicum annum but was slightly pathogenic on tomatoes and potatoes respectively, although it did not cause vascular discolouration.
Five potato isolates infecting potato cv. Kufri Jyoti and two isolates infecting tomato cv. Arka Sourabh and groundnut cv. TMV 2 we’re collected from different agro-climatic zones of Karnataka. All these isolates belonged to race-1 and biotype-III on the basis of pathogenicity to various solanaceous and non-solanaceous plants and utilization of carbohydrates.
Detection of Plant Diseases:
Diagnosis of plant diseases is an art as well as a science. The ability > f Ralstonia solanacearum to infect and colonize the potato plant as well as other crops and weed spp., without causing symptoms, has resulted in its widespread dispersal and subsequent establishment in different environments worldwide.
One of the main constraints to developing strategies to efficient management of bacterial wilt has been the lock of rapid and accurate methods to detect the pathogen in large numbers of plant, soil, and water samples on a routine basis.
The authors reviewed diagnostic methods for Ralstonia solanacearum in potatoes under few heads: Simple techniques including syptomatology, Indicator plants, ooze test, culture techniques, biochemical tests, serological techniques and methods involving detection of pathogen specific DNA.
Symptomatology:
The pathogen affects both above and underground parts of the crop. The first symptom in the potato plant is partial wilting of the upper leaves. These become pale green and even yellow. A slight yellowing may be observed on the lower leaves.
Plant affected by brown rot pathogen frequently show wilting of only one branch with a leaf wilt, and later a progressive wilting, stunting and death of the whole plant.
Smith (1997) reported that initially, leaves wilt during the day but recover during the night time. Leaves may develop a bronze cast and petioles may develop epinasty. In advanced stage, the lower stem will have a streaked brown appearance.
According to Hingorani (1956) the characteristic symptoms of the disease include wilting, stunting and yellowing of the foliage followed by collapse of the affected plants (Fig. 26.1).
Diseased plants show dark narrow stripes beneath the epidermis of the stem, which correspond to the affected vascular bundles. In cross section of a stem, these bundles show brown colouring and in advanced stage white ooze forms on the cut surface within a few minutes.
The infected plant roots at advanced stage turn dark and decay. Symptoms on tubers are very important and diagnostic for this disease. Infected tuber eyes show grayish brown discolouration at advanced stages.
Tubers from the diseased plants show a brown discolouration (ring) in vascular tissue (Fig. 26.2) and blacking of eye buds in a severely decayed potato. In Simla, Meghalaya and Darjeling hills, lenticel infection of potato tubers was also observed occasionally.
It has been reported that less virulent forms of some Indian isolates cause no wilting in potato plants but when inoculated to tomato plants produce swelling around inoculated site giving bottle-shaped appearance. Petioles of such plants become swollen and show epinasty and roots remain stunted. Sometimes cracks are noticed around inoculated portions. Some variants may also cause scorching/burning in the leaves.
The yellowing of lower leaves and stunting of plants are not observed in India. Although vascular browning has been reported to be a characteristic symptoms of the disease but strains of Ralstonia solanacearum that do not produce vascular browning in potato tubers have also been recorded in. Portugal, Kenya and Australia.
Indicator Plants:
Since the use of the hypersensitive reaction (dark brown necrotic lesions) technique in tobacco leaves, there has been a number of attempts made to detect Ralstonia solanacearum.
Potato shoots cut from healthy tubers or surface-sterilized tomato seeds and tomato seedlings of susceptible cultivars also have been used as planting materials for indicator plants which showed symptoms within 4 weeks under controlled conditions when inoculated or planted in the soil sick with pathogen. Ravnika (1999) used tomato as well as brinjal as indicator plants.
Ooze Test:
Ooze test from wilt suspected stem/root or tuber in clear water from the vascular ring is a simple field test to confirm whether a diseased sample has bacterial wilt or not (Fig. 26.3). Shiny white exudates in the eyes of severely infected tubers can be observed with naked eye.
Cut surface of, infected tubers, when slightly pressed exudes milky white droplets from the vascular bundles. Latently infected tubers stored or grown in pots of sterilized soil above 25°C for 4-6 weeks can encourage bacterial wilt symptom development from infected tubers.
Culturing Technique:
Kelman (1954) developed a tetrazolium medium which allows to identify colonies of Ralstonia solanacearum among those of other bacteria by their typical fluidal, smooth, white appearance with red internal whirling patterns.
Several recipes for selective media have been developed that rely on the specific resistance of Ralstonia solanacearum to different antibiotics.
Biochemical Test:
Suslow 1982 suggested the gram reaction can be predicted by determining solubility of the bacterial ooze in 3 per cent aqueous KOH solution (KOH test) and a milky thread upon lifting the toothpick indicates the presence of the gram-negative pathogen.
Kovac (1956) determined oxidase test by smearing young bacterial culture on strip of filter paper soaked with 1% solution of tetramethyl-p-phenyldiamine resulting purple colour. This test can be performed directly on diseased tubers.
Shekhawat (2000) reported change of media colour containing urea to violet when incubated for an hour at 35-37°C with bacteria (Urease test).
Staining:
Lelliot and Stead (1987) and Venkatesh (2000) observed fluorescent bright orange colour under fluorescent microscope when poly-3-hydrobutyrate (PHV) granules, an intracellular inclusions produced by Ralstonia solanacearum has been stained with 1% aqueous solution of Nile blue A.
Venkatesh (2000) observed pink discoloration at the vascular region in the potato slices having latent infection when dipped in 1 per cent tetrazolium chloride (TZC) solution (Fig. 26.4).
Cultural and Physiological Test:
He (1983); Schaad (2001) and Williamson (2002) observed the cultural and physiological test of Ralstonia solanacearum on different media and reported that bacteria was gram negative, oxidase and catalase positive, non-fluorescent on King’s B medium and produced cream coloured colonies on yeast dextrose calcium carbonate medium (YDC).
Serodiagnosis:
The correct diagnosis is a prerequisite of effective disease management. The more rapidly and accurately the causal organism is identified, the sooner proper control can be instituted.
Biochemical and physiological tests which are routinely used to identity plant pathogenic bacteria are not entirely satisfactory. Serological techniques now offer sensitive and easily used alternative methods for detecting Ralstonia solanacearum in plant samples (Fig. 26.5).
Perez (1962) developed antisera against polysaccharide-containing bacterial extracts were used successfully to identify P. solanacearum isolates but did not differentiate strains from different host. Morton (1966) prepared race specific antisera against Races 1, 2 and 3 of Ralstonia solanacearum and reported that Race 2 and 3 were more closely related.
Digat and Cambra (1976) described cell surface antigens in four categories: exopolysaccharide (EPS), cell wall structural (somatic) components (“O” antigens), extra cellular glycoprotein, and flagella components (“H” antigens). Glycoproteins tend to be strain specific whereas EPS and somatic antigens are common to all strains of Ralstonia solanacearum.
Chakrabarti (1993) standardized a protocol of alkaline phosphatase based DAS- ELISA to detect Ralstonia solanacearum either from infected stems or tubers using antiserum produced against formalin killed bacteria (Race 1).
Further, Chakrabarti (1995) produced antisera against formalinised whole cell, glycoprotein and lipopolysaccharide (LPS) fractions of the bacterium. In slide agglutination test, no race specificity was observed. The antiserum against whole cells proves effective for ELISA of low concentration of pathogen from infected plants.
Robinson (1993) developed strains specific monoclonal antibody against Ralstonia solanacearum to detect it and reported that the indirect ELISA system can detect as few as 104 cfu/ml in pure or mixed bacterial suspensions or plant extracts.
Elphinstone and Standford (1998) detected the latent infection of the potato tubers using all methods including culture on semi-selective medium, ELISA, indirect immunofluorescent-antibody staining (IFAS) of fixed cells, immunofluorescent colony staining (IFAS), detection of specific DNA sequences following amplification by the PCR and bioassay in tomato seedlings.
ELISA and PCR were improved by pre-enrichment of samples in semi-selective broth prior to testing.
Griep (1998) developed recombinant single chain antibodies (ScFvs) against the lipopolysaccharide of Ralstonia solanacearum (Biovar 2, Race 3) were successfully selected, by phage display from a large combinatorial antibody library.
The selected antibodies had improved characteristics when compared with the polyclonal antiserum that was used for diagnosis of brown rot of potato in the Netherlands. The isolates monoclonal ScFvs reacts in broth ELISA immunofluorescence cell staining with all Race 3 strains tested but with only some strains belonging to other races.
Several methods were compared for the detection of Ralstonia solanacearum in tubers, including indirect ELISA and sensitivity of each method was determined in artificially inoculated potato extracts. Reliability of ELISA PCR was improved by incubating samples in semi-selective broth prior to testing.
Priou (1999) developed post-enrichment NCM-ELISA (enzyme linked immunosorbent assay on nitrocellulose membrane using enriched sample) was as sensitive as the double-antibody sandwich (DAS-ELISA), but was much easier and quicker.
Wolf (1999) described a comparative test of an immunofluorescence cell-staining (IF) and ELISA methods based on both polyclonal and recombinant monoclonal antibodies for the detection of Ralstonia soanacearum in potato tuber extracts.
No differences in detection levels were found in sensitivity of the assays based on polyclonal or monoclonal antibodies nor between water and potato tubers extracts as diluents.
Kumar (2002) have evaluated the suitability of NCM-ELISA kit, developed at CIP, Lima, Peru, for detecting bacterial wilt pathogen in ginger. The result indicated that the antibodies were sensitive enough to detect Ralstonia solanacearum from ginger, chilli, chromolaena and tomato.
The sensitivity of the kit was determined to be 42 cell/ml of ginger extract. Caruso (2002) developed a sensitive and specific routine detection of Ralstonia solanacearum in symptomless potato tubers was achieved by efficient enrichment followed by a reliable DAS-Indirect ELISA based as the specific monoclonal antibody 8B- IVIA.
This monoclonal antibody reacted with 168 typical Ralstonia solanacearum strains and did not recognize 174 other pathogenic or unidentified bacteria isolated from potato. Analysis of 233 commercial potato lots by this method provided results that coincided with the results of conventional methods.
In India, losses caused by bacterial wilt vary from 20-100 per cent. Since pathogen is mainly transmitted through seed tubers, as latent infection in vascular tissues of progeny tubers which may be detrimental factor for epidemic occurrence of bacterial wilt in disease free regions.
There are several tests including biochemical and physiological tests routinely used to detect bacterial infection which are not entirely satisfactory. Therefore to produce disease free breeder/certified seed tubers, it urgently needed to detect latent infection in seed tubers using antibody for serodiagnosis.