1 1 INTEGRATED CONTROL of BACTERIAL WILT of POTATO Ralstonia Solanacearum Is the Causal Agent of the Disease Known As Potato

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1 INTEGRATED CONTROL OF BACTERIAL WILT OF POTATO

INTEGRATED CONTROL OF Authors: S. Priou, P. Aley, E. Chujoy, BACTERIAL WILT OF POTATO B. Lemaga and E. R. French

S. Priou, P. Aley, E. Chujoy, B. Lemaga and E. French Ralstonia solanacearum is the causal agent of the disease known as potato brown rot or bacterial wilt (BW). The bacterium affects more than 30 plant species, the most susceptible crops being potato, tomato, eggplant, pepper, banana and groundnut. Spread of the pathogen worldwide has been associated with its dissemination in latently infected planting material. Bacterial wilt is the second most important constraint to potato production in tropical and sub- tropical regions of the world. Quarantine measures necessary to avoid spread of the disease to BW-free areas often restrict the production of seed potatoes and limit the commercialization of ware potatoes between countries and between infected and non-infected regions within a country. Limited availability of high-quality seed, and lack of farmer knowledge on proper agronomic practices, threaten the sustainability of the potato crop in the developing world. BW incidence can be reduced only if various control components are combined. This involves mainly the planting of

1 healthy seed in clean soil, planting important in Indonesia, the Philip- tolerant varieties, rotation with pines, southern Vietnam, Laos, non-susceptible crops, and, the Japan and southern China. In the application of various sanitation early 1990s, BW became a seri- and cultivation practices. An ous threat to potato production in integrated disease management European countries including approach can lead to significant Belgium, England, France, The reduction, or even eradication of Netherlands, Spain, Italy and BW. Portugal. It has also been reported 2 in Russia. Earlier incidence in WORLDWIDE DISTRIBUTION OF Sweden was followed by eradica- WORLDWIDE DISTRIBUTION OF Ralstonia solanacearum ON POTATO R. solanacearum ON POTATO tion. Bacterial wilt has spread to most 3 potato producing countries. Its SURVEY AND DIAGNOSIS occurrence in Australia and in the Inspection of potato fields is of southeastern United States has prime importance in seed certifica- resulted in concentration of scien- tion schemes. This is the way to tific research on BW in these identify diseased areas where areas. In Latin America, the national crop protection services disease has been reported from all may apply quarantine and control

potato producing countries except SURVEY AND DIAGNOSIS measures. Likewise, early disease Ecuador. BW occurs throughout diagnosis allows potato growers to central and southern Africa, and is define an appropriate manage- a serious production constraint in ment strategy. Field diagnosis of Uganda, Ethiopia, Kenya, Mada- bacterial wilt disease can be done gascar, Rwanda, Burundi, Nigeria by identifying the foliage and tuber and Cameroon. Although not a symptoms during the growing serious disease in Egypt, latent season or at harvest. infection of tubers has resulted in 4 a strong decline of potato exports Foliage symptoms to Europe. In South Asia, BW occurs in the mid-hills of the Himalayas in India, Pakistan, Nepal and Bhutan; also in the mid- Foliage symptoms hills and plains of India, where it is effectively controlled. In East and Southeast Asia, bacterial wilt is

2 3 5 Total and unilateral wilting of 8 Bacterial exudate from vascular potato ring Above ground symptoms of BW Tuber symptom is often described include wilting, stunting and yel- as brown rot. Cut tubers show Total wilt lowing of the foliage. The browning brownish discoloration of the of vascular bundles may be seen vascular ring, and slight squeezing Unilateral wilt externally, or when the cortex is forces a pus-like slime out of the peeled. Characteristic too, is the ring, or it may exude naturally. initial wilting of only part of the Bacterial exudate from vascular ring stems of a plant, or even one side of a leaf or stem. If disease devel- 9 Necrosis involving secondary opment is rapid, the entire plant infection wilts quickly, without yellowing. The vascular ring, or the whole Alternatively, the diseased stem tuber, may disintegrate completely can wilt completely and dry up, at more advanced stages of while the remainder of the plant necrosis development. This often appears healthy. involves secondary infection with 6 saprophytic bacteria (Erwinia spp., Tuber symptoms Necrosis involving secundary infection Clostridium sp.) or fungi (Fusarium sp., Pythium sp.). 10 Field diagnosis Tuber symptoms

Field diagnosis

7 Bacterial exudate in tuber eyes External symptoms on the tuber are visible at harvest when infec- 11 tion is severe. Bacterial ooze The vascular flow test collects at tuber eyes or on the Vascular flow test A diagnostic test is necessary end of the stolon, causing soil to because plant wilting caused by adhere to the secretions. the bacterium Ralstonia

Bacterial exudate in tuber eyes solanacearum can be confused with symptoms induced by other pathogens such as Fusarium

4 5 eumartii, Verticillium sp., Erwinia positive bacterium) the thread is chrysanthemi, or by insect or not produced. mechanical damage at the stem 13 base. Diagnosis in the field can be Laboratory identification easily accomplished by cutting a piece of stem 2-3 cm long from the base, and suspending it in clear water in a glass container. The cut Laboratory identification stem can be held with an opened paper clip to maintain a vertical position. Within a few minutes, the smoke-like milky threads stream downward from the cut stem. This milky slime exuding from the stem 14 Isolation of R. solanacearum on is characteristic of R. modified Kelman’s medium solanacearum present within the The BW agent can be isolated vascular system. from a diseased tuber or stem by 12 plating the bacterial tuber exudate The KOH test or two drops of the suspension The brown rot symptom on tubers obtained in the vascular flow test can be confused with ring rot on modified Kelman’s medium KOH test caused by Clavibacter Isolation of Ralstonia solanacearum (TZC containing only 2.5 g on modified Kelmans medium michiganensis subsp. sepedonicus dextrose). After 48 h incubation at (previously called Corynebacterium 30°C, the typically fluid, slightly sepedonicum). A quick differential red-tinted colonies of virulent R. test can be performed directly on solanacearum are easily distin- the bacterial exudate on the tuber guished from other saprophytic to differentiate between the two bacteria (round shaped, uniformly bacteria. Two drops of 3% dark red-colored colonies). potassium hydroxide (KOH) are 15 placed on the ooze and mixed with Pathogenicity test a laboratory loop or a wooden Potato The purified culture of R. Pathogenicity test toothpick for 10 seconds. The solanacearum on Kelman’s me- formation of a milky thread upon Tomato dium can be inoculated to tomato lifting the toothpick indicates the or potato seedlings to confirm its presence of R. solanacearum (a pathogenicity. Young tomato or Gram-negative bacterium), potato transplants (third to fifth whereas with C. michiganensis true leaf) are inoculated by injecting subsp. sepedonicus (a Gram-

6 7 the stems with 20 µl (e.g., a drop) negative, oxidation of alcohols of a bacterial suspension of R. negative; solanacearum at 108 cells/ml with Bv 2 = utilization of disaccharides a 1 ml-propylene syringe and a positive, oxidation of alcohols hypodermic needle, just above the negative; cotyledons. Another inoculation Bv 3 = utilization of disaccharides method is to insert (at the level of positive, oxidation of alcohols the third leaf axil) a toothpick positive; carrying the bacteria from a Bv 4 = utilization of disaccharides Kelman’s agar plate. Greenhouse negative, oxidation of alcohols conditions that favor disease positive; development are 28 ± 4ºC, 80- Bv 5 = utilization of disaccharides 90% R.H., and natural daylight. positive, oxidation of only mannitol. Plants are irrigated normally, except one day before inoculation. 17 If the isolate is a pathogenic strain Biovar determination of R. solanacearum, the wilting of These biochemical tests can be tomato or potato seedlings may performed together in microtitre begin in less than a week, but will plates. The utilization of the disac- certainly appear within 4 weeks. charide and oxidation of the After symptoms appear, the alcohol result in the acidification of vascular flow test can be per- the medium, expressed by the formed, and the pathogen can be Biovar determination change in the medium color from re-isolated from the vascular flow green (neutral pH) to yellow (acidic as described previously. pH). 16 Differentiation of 18 Differentiation of R. solanacearum R. solanacearum into biovars Equivalence between biovar and into biovars EquivalenceR. between solanacearum biovar race in R. solanacearum BIOCHEMICAL BIOVAR Two classification systems are and race in TESTS 1 2 3 4 5 used for R. solanacearum: the Race BiovarsHosts Location The race system is based on host : Solanaceous Utilization of Lowland tropics 1 1, 3, 4 + many m ore • lactose, maltose -++-+ race and the biovar systems. The range under field conditions. Four and cellobiose Musaceae American and 1, 3 2 = Moko disease Asian tropics Oxidation of : biovar system consists of bio- races can be distinguished: Potato 3 2A Cool climates • mannitol --+++ Tomato • Race 1 affects a wide range of • sorbitol, dulcitol chemical tests based on the ability --++- 4 5 Mulberry China

Not Prim arily South solanaceous crops including of the bacterium to utilize three 2T Numerous known A merican low land s disaccharides and/or oxidize three potato and many weeds. hexose alcohols. Biovar classifica- Some strains can affect tion is based on the following: groundnut, ginger and diploid Bv 1 = utilization of disaccharides banana. It is prevalent at lower

8 9 elevations in the tropics and host crops, potato volunteers). R. subtropics. solanacearum is mainly spread by • Race 2 affects plants of the movement of infected seed tubers. musaceae family, such as Contaminated surface water used triploid banana, plantain and for irrigation and infested soil Heliconia spp. in the tropics. adhered to farmers’ shoes and • Race 3 affects mainly potato tools also contribute to disease and occasionally tomato and dissemination. Bacterial entrance other solanaceous crops and into potato roots is facilitated by weeds. It is common in higher wounds made by tools during post- elevations or latitudes (cool emergence cultivation, and by climates). nematodes and insects in the soil. • Race 4 affects mulberry and occurs only in China. 20 CONTROL There is some equivalence between race and biovar: race 3 coincides with biovar 2A; race 1 strains are of biovar 1, 3 or 4, CONTROL biovar 1 being the most common on potato. No race has been established for biovar 2T, primarily found in the lowlands of the Ama- zon basin. It has numerous strains 21 with a wide host range as is the Integrated management of case with race 1; however, biovar Integrated management of bacterial wilt bacterial wilt 2T strains are less aggressive. To control and eradicate bacterial wilt, the main components are the Seed infection Soil infestation 19 CONTROL use of healthy seed and planting in DISEASE CYCLE COMPONENTS clean soils. However, many addi- Crop sanitation Healthy seed DISEASE CYCLE OF BACTERIAL WILT The source of inoculum can be • Rotation tional factors influence the inci- • Plant resistance • Nematode control Irrigation Host crops water and weeds infected potatoes (seed tubers, dence of the bacterium, such as Seed tubers Volunteers harvest leftover and infected environmental conditions (tem- Harvest Soil in tools, leftovers hooves and shoes Bacterial Wilt of plants) or infested soil, or both. perature and soil moisture), rota- Soil Nematodes and potato insects in soil The pathogen can survive in soil tion with non host plants, the use

Infested (mostly on plant debris) and in the of less susceptible varieties and Diseased potato soil tubers and plants rooting system and rhizosphere of cultural practices (crop sanitation many hosts (weeds, other and nematode control).

10 11 Thus only an integrated control where healthy seed tubers are strategy can succeed in reducing hardly available, an alternative to BW incidence or eradicating it. planting seed tubers is the use of This strategy is site-specific. From true potato seeds (TPS) or clean the existing and available control cuttings obtained under controlled factors, those that are feasible, growing conditions from suitable and effective should be micropropagation. selected for a given location (see 23 slide #47). Moreover, social and Plant in R. solanacearum-free economic factors that influence soil farmers’ decision-making should After a bacterial wilt infected crop, be factored into the management potato and other hosts must not be scheme. Plant in R. solanacearum-free soil planted for at least two years. 22 Rotation with cereals or grami- Use of healthy seed neous pastures can be imple- Infected seed tubers are the main mented to eliminate soil inoculum. means of dissemination of R. The duration of the rotation neces- solanacearum (particularly for race sary to eliminate soil inoculum is 3 strains). In cool conditions, such variable because R. solanacearum as tropical elevations above 2500 survivability in soil varies accor- Use at healthy seed m, infected but symptomless ding to environmental conditions plants may harbor the bacterium (temperature, moisture) and soil and transmit it to progeny tubers characteristics (biotic and abiotic as latent infection, leading to factors). Some soils are suppres- severe disease outbreaks when sive, e.g., the bacterium does not grown at warmer locations. In survive over a long period of time, seed certification schemes, no however, the mechanisms respon- bacterial wilt must be tolerated sible for this suppressiveness are during the growing season. For unknown. seed production, only BW-free 24 seed tubers originating from Plant Resistance disease-free areas must be used. Plant resistance Plant resistance is one of the most The original planting material used effective means of controlling BW. in clonal (pre-basic and basic seed However, although many potato tubers) or positive selection multi- Resistant varieties have been found to have plication scheme must be tested a degree of resistance to BW, they for latent infection with R. still transmit latent infection to their

solanacearum. In endemic areas Susceptible progeny tubers. The use of

12 13 moderately resistant varieties must 27 Removal of rotted tubers be thus coupled with a seed After harvest, sorted-out and program that provides BW-free leftover diseased tubers must also seed tubers. Resistance is strain- be removed from the field and specific and is overcome when the destroyed using the same proce- levels of factors that favor BW are dure as for haulms. increased: high temperature, excessive soil moisture, wounding of roots and stolons, etc. An ... and of rotted tubers essential step in the development of resistant varieties is local 28 screening. Locally grown varieties Weeding should be tested for tolerance to R. solanacearum survives in BW since some not bred for BW weeds, so weeding must be done resistance have been found to before planting potato and any have lower susceptibility (var. other crop used in the rotation. Achat in Brazil, Cruza 148 in East Africa and Peru). Weeding 25 Sanitation and cultivation 29 practices Roguing volunteer potato plants Crop sanitation and cultivation Roguing volunteer potato plants Volunteer potato plants are another measures aim to avoid or limit means of survival of R. Sanitation and cultivation practices pathogen survival and dissemina- solanacearum and must be re- tion. These measures are com- moved in the subsequent crop monly used to manage other (potato or any other crop) soon potato diseases and pests. after their emergence.

26 Removal of potato haulms 30 After harvest of a BW-infested Roguing wilted potato plants Removal of potato haulms crop, potato haulms must be Roguing wilted If the incidence of BW is low in a potato plants removed from the field and buried field, wilted potato plants must be deep, down-slope and far from removed as soon as they are irrigation canals. Alternatively, they observed to avoid contamination of can be burned. healthy neighboring plants. They must be destroyed carefully as

14 15 described previously for potato 34 Other cultivation practices haulms and sorted-out tubers. Tubers from neighboring diseased • Minimum post-emergence plants should not be used for cultivation: hilling only at seed. Other cultural practices planting time to avoid wounding roots with tools. Subse- . Minimum post-emergence cultivation . Flooding of paddy rice quent hand weeding is prefer- . Exposing plowed soil to summer heat able for the same reason. 31 Tool decontamination • Flooding of paddy rice: in To prevent movement of soil from Asian countries this signifi- an infested to a disease-free field, cantly decreases soil popula- all tools must be decontaminated tions of R. solanacearum. by washing with water and calcium • In very warm areas, exposing hypochlorite (or other available plowed soil to summer heat bactericide) or sterilized by flame. also decreases soil infestation. Tool decontamination

32 35 Tractor decontamination Rotation Machinery, vehicles, hooves of Rotation with non-host plants is an animals used for traction, and effective means of decreasing the shoes of the personnel coming level of R. solanacearum popula- from an infested field must be Rotation tions in soil, provided volunteer washed at least with water before potato plants are continually entering another field. removed by uprooting the entire Tractor decontamination plants from the field as they emerge. 33 Uncontaminated water 36 The flow of water from an infested Rotate with cereal crops field to adjacent fields must be Rotation with cereals and Rotate with avoided. In infested areas, irriga- cereal crops gramineous pastures rapidly tion by well water is preferred over decreases soil inoculum potential. surface water from rivers or irriga- However, the time necessary for

tion canals. Wheat eradication has to be determined Uncontaminated water in each location.

Maize

16 17 37 Rotate with liliaceous and 41 Avoid other solanaceous crops brassicaceous Eggplant, pepper and tobacco Avoid other Allium sp. (onion, garlic, leak) and solanaceous crops must never be planted after a BW brassicaceous (cabbage, cauli- infected potato crop Rotate with flower) can be planted after a BW- liliaceous and infected potato crop. Furthermore, brassicaceous these crops promote the elimina- tion of weeds and volunteers that Pepper maintain the pathogen. Eggplant

38 42 Rotate with legumes Avoid ginger and groundnut Legumes, e. g. fabaceous (pea, Avoid Ginger and groundnut must never bean), can be used as rotation be planted after a BW-infected crops for potatoes. Moreover, they potato crop in infected areas have the additional benefit of where race 1 strains of R. increasing soil fertility by fixing solanacearum are prevalent (low- Groundnut atmospheric nitrogen. A bean/ elevation tropics). Rotate with legumes maize rotation has reportedly Ginger reduced soil inoculum potential. 39 43 Rotate with cucurbitaceous Nematode control crops Nematodes open a path for the Cucurbitaceous crops (pumpkin, bacterium to enter the plant cucumber, zucchini) are not hosts through root injuries. Therefore, Nematode control for R. solanacearum and can thus they must be controlled to avoid be used in the rotation. their interaction with R. solanacearum. Major nematode Rotate with cucurbitaceous crops control components are soil fumi- gation, rotation with cereals, 40 application of high quantities of Avoid tomato crop organic amendments (free of R. Avoid tomato crop Tomato crop should be avoided solanacearum), and planting since it is highly susceptible to nematode resistant varieties. bacterial wilt caused by either race 1 or 3 strains of R. solanacearum.

18 19 44 Symptoms caused by the root- lists the main factors to be consid-

Root-Knot Nematode (Meloidogine incognita) knot nematode ered in developing a strategy for The major nematode interacting control of bacterial wilt that is with BW is the root-knot nematode caused by either race 3 or 1. Each (Meloidogyne incognita), occurring factor has been rated on a scale of mainly in warm climates and sandy 1 to 7. The higher the number, the soil. more the factor is judged to control the disease. The rates allocated to each factor may change according 45 to the importance of a factor in a Quarantine given location. A sum of at least Once BW is introduced to an area, 14 would usually be adequate for quarantine regulations have to be good control or potential eradica- applied to avoid spreading of BW tion. Quarantine to non-infested areas. Measures Factors to be rated to formulate a control strategy Race 3 Race 1 restrict the production of seed Healthy seed 7 7 potatoes and impede the commer- Potato resistance or tolerance 4 2 cialization of ware potatoes to BW- R. solanacearum-free soil 7 7 free countries or regions, affecting Suppressive soil 2 2 the economy of the quarantined Rotation with non-hosts 5 3 regions. Roguing wilted potato plants 4 3 Roguing volunteers 4 2 46 No transport of infected seed Weed control 2 2 Removal of potato haulms and/or harvest leftovers 3 3 tubers to non-infected areas Control of spread in water 2 1 Avoid transport of ware or seed Minimal post-emergence cultivation 4 4 potatoes from an infected area to Nematode control 3 3 a BW-free to avoid spreading the Tool decontamination, washing of hooves and shoes 2 1 No transport of disease. While it is often difficult to Exposure of plowed soil to summer heat 1 3 infected seed tubers fully control all potato trading, this Flooding of paddy rice 1 3 to non-infected areas is one of the best preventative measures available. 48 47 DETECTION METHODS OF R. solanacearum DEFINING A CONTROL STRATEGY DEFINING A CONTROL DEFINING A CONTROL STRATEGY Factors to be rated to formulate a control strategy Race 3 Race 1 STRATEGY Healthy seed 7 7 Potato resistance or tolerance 4 2 R. solanacearum-free soil 7 7 The most relevant components for Suppressive soil 2 2 Rotation with non-hosts 5 3 DETECTION METHODS Roguing wilted potato plants 4 3 the control of bacterial wilt have Roguing volunteers 4 2 Weed control 2 2 Removal of potato haulms and/or harvest leftovers 3 3 been presented. Based on that Control of spread in water 2 1 Minimal post-emergence cultivation 4 4 Nematode control 3 3 Tool decontamination, washing of hooves and shoes 2 1 information, the following table Dry / heat soil 1 3 Flooding of paddy rice 1 3

20 21 49 Detection of latent infection in 51 Evaluation of latent infection in seed tubers potato seed lots Sample size = 250 tubers per ha Detection of latent infection in seed PROTOCOL FOR THE DETECTION OF ⊇ 10 sub-samples of 25 tubers tubers using CIP NCM-ELISA kit The classical method of detecting LATENT INFECTION IN POTATO SEED LOTS The minimal sample size required Remove pieces of the vascular ring of each tuber (< 0.5 g per tuber) Combine the pieces removed from 25 tubers in a separate bag, weigh and add 2 ml of extraction tuber infection consists of incubat- buffer per gram of tuber tissue is 250 tubers per hectare (sampled ...... ing tubers for 3 to 4 weeks at 30°C 12 910 at harvest or in storage) for analy- Crush tissue Crush tissue

500 µl 500 µl 500 µl 500 µl sis in ten sub-samples of 25 and observing oozing from the extract extract extract extract tubers. The tissue of the vascular eyes or stolon ends, or cutting the 500 µl 500 µl 500 µl 500 µl SMSA SMSA SMSA SMSA Agitate Agitate ring removed from each of the 25 tubers to observe oozing from the Dot - Blotting Incubation for 48 h at 30°C (two dots per sub-sample vascular ring. However, this ) tubers is crushed in one individual method is time and space con- bag after adding the volume of suming and may not reveal low extraction buffer corresponding to infection rates. Because of this, double the tissue weight. For each CIP developed a simple, sensitive, sub-sample, 0.5 ml (500 µl) of the quick and low-cost technique to tuber extract is mixed with the detect latent infection in tubers same volume of SMSA broth in an that is suitable for seed testing: Eppendorf tube and incubated for post-enrichment NCM-ELISA 48 h at 30°C with agitation two (Enzyme linked immunosorbent times a day. Two dots of each assay on nitrocellulose mem- incubated extract are put on the brane). The kit is being distributed membrane. to seed programs worldwide. 52 50 The five steps of NCM-ELISA General scheme of seed testing Dot-blotting NCM-ELISA is an immuno-enzy- Blocking SAMPLE EXTRACTION NCM-ELISANCM-ELISA The main steps of post-enrichment matic assay. It uses a nitrocellu- Tuber extract Polyclonal rabbit antibody Washings Removal of and disinfection Removal of Crushing NCM-ELISA to detect R. Washings lose membrane instead of a pieces of of tissue vascular ring solanacearum in latently infected microtitre plate as support for the ENRICHMENT= Antigen Incubation for 48 h at 30°C Goat anti-rabbit conjugate SEROLOGICAL TEST: Polyclonal rabbit in SMSA broth tubers are: antibody Washings samples and reagents. The test NCM-ELISA Goat anti-rabbit conjugate 1) Preparation of tuber extracts Blocking protein consists of the following steps: DOT - BLOTTING Enzyme substrate on nitrocellulose membrane (NCM) from the tuber vascular ring; Color development 1) Loading of a very small Scheme for the detection of R. solanacearum in latently infected potato tubers using CIP NCM-ELISA kit 2) Incubation of tuber extracts for amount of the tuber extract (20 48 h at 30°C in a µl e.g. one drop) on a nitrocel- semi-selective broth (= enrich lulose membrane (= Dot- ment procedure); and blotting); 3) Loading (Dot-blotting) of the 2) Blocking of the area of the enriched samples on nitrocellu membrane that is free of lose membrane (NCM), and extract with milk casein serological test (ELISA). (1 h incubation); 3) Binding of the samples with

22 23 R. solanacearum-specific Methods to detect soil inoculum rabbit antibodies (2 h incuba- 54 tion); Tomato bioassay 4) Binding of the R. Detection of soil inoculum Since tomato is highly susceptible solanacearum-antibodies to bacterial wilt at warm tempera- complex with enzyme-labeled tures, tomato bioassay is the (alkaline phosphatase) goat simplest way to evaluate soil anti-rabbit antibodies (1 h infestation with R. solanacearum. incubation); and However, it requires greenhouse 5) Revealing of the bound en- Tomato bioassay space and is time consuming zyme by adding the substrate since it can take up to six weeks to leading to a coloration reaction observe symptom development. It (5 to 20 min). can also be done in the field by 53 planting rows of tomato and Sensitivity of NCM-ELISA with observing wilt development. For Sensitivity of NCM-ELISA without (-E) and with (+E) 48 h enrichment and without enrichment Sensitivity of NCM-ELISA- E + withoutE (-E) greenhouse tests, 2 kg of soil are Cells / ml Cells / ml and with (+E)8 48 h enrichment 10 106 The presence of R. solanacearum placed in a plastic tray and 50 two- 10 7 105 10 6 104 in the tuber extract leads to the 3 10 5 10 week old tomato seedlings (about 102 10 development of a purple colora- 10 cm high) are transplanted. For 1 0.1 tion. All races and biovars of R. each soil sample, 100 seedlings Negative control: TE (+E) solanacearum can be detected R. solanacearum Inoculated tuber are transplanted in two trays, and in citrate buffer extract (TE) with the polyclonal antiboides.The irrigated normally. From 7 days intensity of the coloration is pro- until 45 days, the percentage of portional to the bacterial concen- wilted tomato plants is recorded tration. After enrichment (e.g., two times a week. The test tube multiplication of the bacterial assay and the KOH test with the population in the extract), as few bacteria oozing from the tomato as 10 bacteria per ml of tuber stems can be used to confirm that extract (cells/ml) can be detected, wilt results from R. solanacearum whereas 107 cells/ml or more are and not from other soil pathogens necessary if enrichment is not such as Pythium sp., Verticillium practiced before conducting the sp. and Clavibacter michiganensis immunoassay. Thus, enrichment subsp. sepedonicus. increases sensitivity of the serological test by a million-fold, allowing the detection of R. solanacearum in potato tubers (or stems) that are latently infected, e.g., with very low infection levels that produce no visible symptoms.

24 25 55 Methods to detect soil inoculum 3) Loading of the R. Post-enrichment DAS-ELISA solanacearum-specific rabbit DetectionDetection of soil of soil inoculum inoculum Post-enrichment DAS-ELISA A kit has been set up at CIP to IgG conjugated to alkaline Rs in soil extract Post-enrichment RsDAS-ELISA in citrate buffer - E + E 108 108 detect low population levels of R. phosphatase (3 h incubation); 107 107 106 106 105 105 solanacearum in soil. The method 4) Development of the coloration 104 Sensitivity of DAS-ELISA 103 reaction by adding the enzyme without (-E) and 102 is based on the simple preparation with (+E) 48 h enrichment 20 of soil extracts by mixing 10 g of substrate (1 h). Negative controls: Citrate - E + E buffer Non-infested soil with 90 ml of PBS buffer. After 57 soil extract 30 min. constant agitation, the METHODS TO ASSESS POTATO solution is allowed to stand for one METHODS TO ASSESS RESISTANCE TO BW minute and 2 ml of the supernatant POTATO RESISTANCE TO BW Field evaluation is removed, mixed in a sterile flask Field evaluation Screening potato for resistance to to 38 ml of SMSA broth comple- BW requires a field with a reason- mented with potato broth and ably uniform and moderate level of ° incubated for 48 h at 30 C with Greenhouse inoculation infestation (between 30% to 50% agitation (enrichment procedure). wilt incidence in the previous The enriched extracts are ana- potato crop). The biovar/race of lyzed in double-antibody sandwich the strains present in the field ELISA (DAS-ELISA) in microtitre should be identified (both races 1 plates. As few as 20 bacteria per and 3 can be present). The plot gram of soil can be detected. For design should be chosen accord- semi-quantification of the popula- ing to the number of tubers per tions, the soil extracts can be clone to be tested and the unifor- diluted previous to their enrich- mity of the inoculum spread in the ment. field, but at least 20 tubers should 56 be used (4 replications of 5 tu- The four steps of DAS-ELISA bers). During the growing season Double-antibody sandwich ELISA the number of wilted plants is DAS - ELISA Coating with the rabbit IgG DAS - ELISA Washings (DAS-ELISA) is an immunoassay recorded once a week from 40

Sample loading conducted in microtitre plates, with days to 80 days after emergence. Microtitre plate Washings

Antigen (sample) the following four steps: At harvest, visible infection of Rabbit immunoglobulin (IgG) specific to the antigen Loading of the tubers (and latent infection if no conjugated rabbit IgG 1) Coating of the microtitre plate Rabbit immunoglobulin (IgG), specific to the Washings antigen, conjugated to alkaline phosphatase wells with the R. wilting occurred during the growing Enzyme substrate

Color development solanacearum-specific rabbit season), and yield of marketable immunoglobulins (IgG) sized tubers are also recorded. (3 h incubation); 2) Loading of the samples in the wells (overnight incubation);

26 27 Greenhouse inoculation Cultivars Cruza 148 (resistant to Apical cuttings of potato plants or foliage wilt), Molinera (= BR63.65, cuttings obtained from sprouted moderately susceptible), tubers are rooted in small pots Revolucion (susceptible) and containing a soil substrate (a Yungay (susceptible) are used as mixture of soil, sand and peat controls at CIP Lima. At a mini- 2:1:1). However, the best results mum, Cruza 148 should be used have been obtained using the as the less-susceptible control, Promix BX® substrate (Premiers together with a local susceptible Brands, INC, Stamford, Canada). variety. A complete fertilizer (N-P-K 20-20- 20, diluted at 0.5% in water) is applied at the time of planting. Plants should be transferred to the REFERENCES greenhouse at least one day Aley, P., French, E. R. and Nydegger, U. (1994) Methods of greenhouse inoculation with Pseudomo- before the inoculation. Green- nas solanacearum to select resistant potato clones. Fitopatologia 29:20 (Abstract). house conditions that favor dis- Anonymous. (1994) Bacterial wilt: the disease and its causative agent Pseudomonas solanacearum ease development are 28±4ºC, (eds. Hayward, A. C. and Hartman, G. L.). 259 p. CAB International, Wallingford (UK). and 85-90% R.H. Plants are Anonymous. (1998) Bacterial wilt disease: molecular and ecological aspects (eds. Prior, Ph., Allen, C. irrigated daily, except for one day and Elphinstone, J.) 447 p. INRA edn, Springer Verlag, Berlin (Germany). before inoculation. Each pot Commonwealth Mycological Institute. (1977) Distribution maps of plant diseases: Pseudomonas containing a one-stem 20-day-old solanacearum (E. F. Smith) E. F. Smith on potato (Solanum tuberosum), tobacco (Nicotiana plant (about 15 cm tall) is inocu- tabacum and banana (Musa) etc. Map No. 138 Edition 5.

lated with 25 ml of bacterial sus- Elphinstone, J. G., Hennessy, J., Wilson, J. K. and Stead, D. E. (1996) Sensitivity of different pension of R. solanacearum at 108 methods for the detection of Pseudomonas solanacearum (Smith) in potato tuber extracts. EPPO/ cells/ml. Twenty plants per geno- OEPP Bulletin, 26 :663-678. type are inoculated. Plants are French, E.R. (1986) Field-screening CIP clones developed for resistance to bacterial wilt. Technology incubated for 30 days and the Evaluation Series No. 1982-6, 9 p. International Potato Center, Lima (Peru). disease indices are recorded French, E.R. (1994) Strategies for integrated control of bacterial wilt of potatoes. In Bacterial wilt: weekly on the following scale: the disease and its causative agent Pseudomonas solanacearum (eds. Hayward, A. C. and • 1 = no symptoms; Hartman, G. L.) p. 199-207, CAB International, Wallingford (UK). • 2 = wilting of one leaf; French, E. R., Gutarra, L., Aley, P. and Elphinstone, J. (1995) Culture media for Ralstonia • 3 = wilting of up to 50% of the solanacearum isolation, identification and maintenance. Fitopatologia 30 (3): 126-130. leaves; • Hayward, A. C. (1991) Biology and epidemiology of bacterial wilt caused by Pseudomonas 4 = wilting of up to 75% of the solanacearum. Annual Review of Phytopathology 29, 65-87. leaves; • 5 = complete wilting of the Hayward, A. C., El-Nashaar, H. M., de Lindo, L. and Nydegger, U. (1991) The use of microtiter plates in the phenotypic characterization of phytopathogenic Pseudomonads. Proc. 7th Int. Conf. plant. Plant Pathog. Bact., Budapest, Hungary, pp 593-598.

28 29 Janse, J. D. (1996) Potato brown rot in Western Europe –history, present occurrence and some remarks on possible origin, epidemiology and control strategies. Bulletin OEPP/EPPO Bulletin 26:679-695.

Kelman, A. (1954) The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathology 64:693-695.

Martin, C. and French, E.R. (1985) Bacterial wilt of potato: Pseudomonas solanacearum. Technical Information Bulletin 13, 8 p. International Potato Center, Lima (Peru).

Priou, S., Gutarra, L. (1998) Video demonstration on the use of CIP NCM-ELISA kit for the detection of Ralstonia solanacearum in latently infected tubers. English, Spanish and Chinese versions, 37 min, International Potato Center, Lima (Peru).

Priou, S., Gutarra, L. and Aley, P. (1999) Highly sensitive detection of Ralstonia solanacearum in latently infected potato tubers by post-enrichment ELISA on nitrocellulose membrane. EPPO/OEPP Bulletin 29 (1), in press.

Priou, S., Gutarra, L., Fernandez, H. and Aley, P. (1999) Sensitive detection of Ralstonia solanacearum in latently infected potato tubers and soil by postenrichment ELISA. CIP Program Report 1997-98, in press, International Potato Center, Lima, Peru.

Priou, S., Gutarra, L., Fernandez, H. and Aley, P. (1999) Sensitive detection of Ralstonia solanacearum (race 3) in soil by post-enrichment DAS-ELISA. ACIAR Bacterial Wilt Newsletter 16: 10-13.