1

1

INTEGRATED CONTROL OFBACTERIAL WILT OF POTATO

Authors: S. Priou, P. Aley, E. Chujoy,B. Lemaga and E. R. French

Ralstonia solanacearum is thecausal agent of the disease knownas potato brown rot or bacterial wilt(BW). The bacterium affects morethan 30 plant species, the mostsusceptible crops being potato,tomato, eggplant, pepper, bananaand groundnut. Spread of thepathogen worldwide has beenassociated with its disseminationin latently infected planting mate-rial. Bacterial wilt is the secondmost important constraint to potatoproduction in tropical and sub-tropical regions of the world.Quarantine measures necessaryto avoid spread of the disease toBW-free areas often restrict theproduction of seed potatoes andlimit the commercialization of warepotatoes between countries andbetween infected and non-infectedregions within a country. Limitedavailability of high-quality seed,and lack of farmer knowledge onproper agronomic practices,threaten the sustainability of thepotato crop in the developingworld. BW incidence can bereduced only if various controlcomponents are combined. Thisinvolves mainly the planting of

INTEGRATED CONTROL OFBACTERIAL WILT OF POTATO

S. Priou, P. Aley, E. Chujoy, B. Lemaga and E. French

2

3

4

healthy seed in clean soil, plantingtolerant varieties, rotation withnon-susceptible crops, and, theapplication of various sanitationand cultivation practices. Anintegrated disease managementapproach can lead to significantreduction, or even eradication ofBW.

WORLDWIDE DISTRIBUTION OFR. solanacearum ON POTATOBacterial wilt has spread to mostpotato producing countries. Itsoccurrence in Australia and in thesoutheastern United States hasresulted in concentration of scien-tific research on BW in theseareas. In Latin America, thedisease has been reported from allpotato producing countries exceptEcuador. BW occurs throughoutcentral and southern Africa, and isa serious production constraint inUganda, Ethiopia, Kenya, Mada-gascar, Rwanda, Burundi, Nigeriaand Cameroon. Although not aserious disease in Egypt, latentinfection of tubers has resulted ina strong decline of potato exportsto Europe. In South Asia, BWoccurs in the mid-hills of theHimalayas in India, Pakistan,Nepal and Bhutan; also in the mid-hills and plains of India, where it iseffectively controlled. In East andSoutheast Asia, bacterial wilt is

important in Indonesia, the Philip-pines, southern Vietnam, Laos,Japan and southern China. In theearly 1990s, BW became a seri-ous threat to potato production inEuropean countries includingBelgium, England, France, TheNetherlands, Spain, Italy andPortugal. It has also been reportedin Russia. Earlier incidence inSweden was followed by eradica-tion.

SURVEY AND DIAGNOSISInspection of potato fields is ofprime importance in seed certifica-tion schemes. This is the way toidentify diseased areas wherenational crop protection servicesmay apply quarantine and controlmeasures. Likewise, early diseasediagnosis allows potato growers todefine an appropriate manage-ment strategy. Field diagnosis ofbacterial wilt disease can be doneby identifying the foliage and tubersymptoms during the growingseason or at harvest.

Foliage symptoms

2 3

Foliage symptoms

WORLDWIDE DISTRIBUTION OFRalstonia solanacearum ON POTATO

SURVEY AND DIAGNOSIS

5

6

Total and unilateral wilting ofpotatoAbove ground symptoms of BWinclude wilting, stunting and yel-lowing of the foliage. The browningof vascular bundles may be seenexternally, or when the cortex ispeeled. Characteristic too, is theinitial wilting of only part of thestems of a plant, or even one sideof a leaf or stem. If disease devel-opment is rapid, the entire plantwilts quickly, without yellowing.Alternatively, the diseased stemcan wilt completely and dry up,while the remainder of the plantappears healthy.

Tuber symptoms

Bacterial exudate in tuber eyesExternal symptoms on the tuberare visible at harvest when infec-tion is severe. Bacterial oozecollects at tuber eyes or on theend of the stolon, causing soil toadhere to the secretions.

7

Bacterial exudate from vascularringTuber symptom is often describedas brown rot. Cut tubers showbrownish discoloration of thevascular ring, and slight squeezingforces a pus-like slime out of thering, or it may exude naturally.

Necrosis involving secondaryinfectionThe vascular ring, or the wholetuber, may disintegrate completelyat more advanced stages ofnecrosis development. This ofteninvolves secondary infection withsaprophytic bacteria (Erwinia spp.,Clostridium sp.) or fungi (Fusariumsp., Pythium sp.).

Field diagnosis

The vascular flow testA diagnostic test is necessarybecause plant wilting caused bythe bacterium Ralstoniasolanacearum can be confusedwith symptoms induced by otherpathogens such as Fusarium

8

10

9

11

4 5

Tuber symptoms

Field diagnosis

Total wilt

Unilateral wilt

Bacterial exudate in tuber eyes

Bacterial exudate from vascular ring

Necrosis involving secundary infection

Vascular flow test

eumartii, Verticillium sp., Erwiniachrysanthemi, or by insect ormechanical damage at the stembase. Diagnosis in the field can beeasily accomplished by cutting apiece of stem 2-3 cm long from thebase, and suspending it in clearwater in a glass container. The cutstem can be held with an openedpaper clip to maintain a verticalposition. Within a few minutes, thesmoke-like milky threads streamdownward from the cut stem. Thismilky slime exuding from the stemis characteristic of R.solanacearum present within thevascular system.

The KOH testThe brown rot symptom on tuberscan be confused with ring rotcaused by Clavibactermichiganensis subsp. sepedonicus(previously called Corynebacteriumsepedonicum). A quick differentialtest can be performed directly onthe bacterial exudate on the tuberto differentiate between the twobacteria. Two drops of 3%potassium hydroxide (KOH) areplaced on the ooze and mixed witha laboratory loop or a woodentoothpick for 10 seconds. Theformation of a milky thread uponlifting the toothpick indicates thepresence of R. solanacearum (aGram-negative bacterium),whereas with C. michiganensissubsp. sepedonicus (a Gram-

12

13

14

positive bacterium) the thread isnot produced.

Laboratory identification

Isolation of R. solanacearum onmodified Kelman’s mediumThe BW agent can be isolatedfrom a diseased tuber or stem byplating the bacterial tuber exudateor two drops of the suspensionobtained in the vascular flow teston modified Kelman’s medium(TZC containing only 2.5 gdextrose). After 48 h incubation at30°C, the typically fluid, slightlyred-tinted colonies of virulent R.solanacearum are easily distin-guished from other saprophyticbacteria (round shaped, uniformlydark red-colored colonies).

Pathogenicity testThe purified culture of R.solanacearum on Kelman’s me-dium can be inoculated to tomatoor potato seedlings to confirm itspathogenicity. Young tomato orpotato transplants (third to fifthtrue leaf) are inoculated by injecting

15

6 7

Laboratory identification

KOH testIsolation of Ralstonia solanacearum

on modified Kelmans medium

Pathogenicity testPotato

Tomato

16

17

the stems with 20 µl (e.g., a drop)of a bacterial suspension of R.solanacearum at 108 cells/ml witha 1 ml-propylene syringe and ahypodermic needle, just above thecotyledons. Another inoculationmethod is to insert (at the level ofthe third leaf axil) a toothpickcarrying the bacteria from aKelman’s agar plate. Greenhouseconditions that favor diseasedevelopment are 28 ± 4º C, 80-90% R.H., and natural daylight.Plants are irrigated normally,except one day before inoculation.If the isolate is a pathogenic strainof R. solanacearum, the wilting oftomato or potato seedlings maybegin in less than a week, but willcertainly appear within 4 weeks.After symptoms appear, thevascular flow test can be per-formed, and the pathogen can bere-isolated from the vascular flowas described previously.

Differentiation ofR. solanacearum into biovarsTwo classification systems areused for R. solanacearum: therace and the biovar systems. Thebiovar system consists of bio-chemical tests based on the abilityof the bacterium to utilize threedisaccharides and/or oxidize threehexose alcohols. Biovar classifica-tion is based on the following:Bv 1 = utilization of disaccharides

negative, oxidation of alcoholsnegative;Bv 2 = utilization of disaccharidespositive, oxidation of alcoholsnegative;Bv 3 = utilization of disaccharidespositive, oxidation of alcoholspositive;Bv 4 = utilization of disaccharidesnegative, oxidation of alcoholspositive;Bv 5 = utilization of disaccharidespositive, oxidation of only mannitol.

Biovar determinationThese biochemical tests can beperformed together in microtitreplates. The utilization of the disac-charide and oxidation of thealcohol result in the acidification ofthe medium, expressed by thechange in the medium color fromgreen (neutral pH) to yellow (acidicpH).

Equivalence between biovar andrace in R. solanacearumThe race system is based on hostrange under field conditions. Fourraces can be distinguished:• Race 1 affects a wide range of

solanaceous crops includingpotato and many weeds.Some strains can affectgroundnut, ginger and diploidbanana. It is prevalent at lower

18

8 9

Differentiation of Differentiation of R. R. solanacearumsolanacearuminto into biovarsbiovars

- - + + +- - + + -

Oxidation of :

• mannitol

• sorbitol, dulcitol

1 2 3 4 5

BIOVAR

- + + - +

BIOCHEMICALTESTS

Utilization of :

• lactose, maltose and cellobiose

Equivalence between Equivalence between biovarbiovarand race in and race in R.R. solanacearum solanacearum

Biovars Location

11 1, 3, 41, 3, 4So lan ac eo usSo lan ac eo us

+ m a ny m ore+ m a ny m oreLo w lan d t rop icsLo w lan d t rop ics

2 1, 3M usa ce ae

= M ok o d iseas eA m e ric an an dA sian t rop ics

33 2A2A Po ta toPo ta toTo m atoTo m atoC ool c l im a tesC ool c l im a tes

N otkn ow n

5

Pr im arily S ou th A m e ric an lo w la nd s

4

2T N um ero u s

C hin a

HostsRace

M ulb erry

Biovar determination

elevations in the tropics andsubtropics.

• Race 2 affects plants of themusaceae family, such astriploid banana, plantain andHeliconia spp. in the tropics.

• Race 3 affects mainly potatoand occasionally tomato andother solanaceous crops andweeds. It is common in higherelevations or latitudes (coolclimates).

• Race 4 affects mulberry andoccurs only in China.

There is some equivalence be-tween race and biovar: race 3coincides with biovar 2A; race 1strains are of biovar 1, 3 or 4,biovar 1 being the most commonon potato. No race has beenestablished for biovar 2T, primarilyfound in the lowlands of the Ama-zon basin. It has numerous strainswith a wide host range as is thecase with race 1; however, biovar2T strains are less aggressive.

DISEASE CYCLEThe source of inoculum can beinfected potatoes (seed tubers,harvest leftover and infectedplants) or infested soil, or both.The pathogen can survive in soil(mostly on plant debris) and in therooting system and rhizosphere ofmany hosts (weeds, other

19

20

21

Infestedsoil

Soil

Harvestleftovers

Irrigationwater

Seed tubers

Wilt ofBacterial

potato

DISEASE CYCLE OF BACTERIAL WILTDISEASE CYCLE OF BACTERIAL WILTDISEASE CYCLE OF BACTERIAL WILT

Soil in tools,hooves and shoes

Nematodes andinsects in soil

Host cropsand weeds

Volunteers

Diseased potatotubers and plants

host crops, potato volunteers). R.solanacearum is mainly spread bymovement of infected seed tubers.Contaminated surface water usedfor irrigation and infested soiladhered to farmers’ shoes andtools also contribute to diseasedissemination. Bacterial entranceinto potato roots is facilitated bywounds made by tools during post-emergence cultivation, and bynematodes and insects in the soil.

CONTROL

Integrated management ofbacterial wiltTo control and eradicate bacterialwilt, the main components are theuse of healthy seed and planting inclean soils. However, many addi-tional factors influence the inci-dence of the bacterium, such asenvironmental conditions (tem-perature and soil moisture), rota-tion with non host plants, the useof less susceptible varieties andcultural practices (crop sanitationand nematode control).

10 11

CONTROL

Healthy seedHealthy seed

•• Plant resistance Plant resistance

Crop sanitationCrop sanitation

•• Rotation Rotation•• Nematode control Nematode control

CONTROLCONTROL

COMPONENTSCOMPONENTS

Seed infectionSeed infection Soil infestationSoil infestation

Integrated management of bacterial wiltIntegrated management of bacterial wilt

22

Thus only an integrated controlstrategy can succeed in reducingBW incidence or eradicating it.This strategy is site-specific. Fromthe existing and available controlfactors, those that are feasible,suitable and effective should beselected for a given location (seeslide #47). Moreover, social andeconomic factors that influencefarmers’ decision-making shouldbe factored into the managementscheme.

Use of healthy seedInfected seed tubers are the mainmeans of dissemination of R.solanacearum (particularly for race3 strains). In cool conditions, suchas tropical elevations above 2500m, infected but symptomlessplants may harbor the bacteriumand transmit it to progeny tubersas latent infection, leading tosevere disease outbreaks whengrown at warmer locations. Inseed certification schemes, nobacterial wilt must be toleratedduring the growing season. Forseed production, only BW-freeseed tubers originating fromdisease-free areas must be used.The original planting material usedin clonal (pre-basic and basic seedtubers) or positive selection multi-plication scheme must be testedfor latent infection with R.solanacearum. In endemic areas

23

where healthy seed tubers arehardly available, an alternative toplanting seed tubers is the use oftrue potato seeds (TPS) or cleancuttings obtained under controlledgrowing conditions frommicropropagation.

Plant in R. solanacearum-freesoilAfter a bacterial wilt infected crop,potato and other hosts must not beplanted for at least two years.Rotation with cereals or grami-neous pastures can be imple-mented to eliminate soil inoculum.The duration of the rotation neces-sary to eliminate soil inoculum isvariable because R. solanacearumsurvivability in soil varies accor-ding to environmental conditions(temperature, moisture) and soilcharacteristics (biotic and abioticfactors). Some soils are suppres-sive, e.g., the bacterium does notsurvive over a long period of time,however, the mechanisms respon-sible for this suppressiveness areunknown.

Plant ResistancePlant resistance is one of the mosteffective means of controlling BW.However, although many potatovarieties have been found to havea degree of resistance to BW, theystill transmit latent infection to theirprogeny tubers. The use of

24

12 13

Plant in R. solanacearum-free soil

Use at healthy seed

Resistant

Susceptible

Plant resistance

25

27moderately resistant varieties mustbe thus coupled with a seedprogram that provides BW-freeseed tubers. Resistance is strain-specific and is overcome when thelevels of factors that favor BW areincreased: high temperature,excessive soil moisture, woundingof roots and stolons, etc. Anessential step in the developmentof resistant varieties is localscreening. Locally grown varietiesshould be tested for tolerance toBW since some not bred for BWresistance have been found tohave lower susceptibility (var.Achat in Brazil, Cruza 148 in EastAfrica and Peru).

Sanitation and cultivationpracticesCrop sanitation and cultivationmeasures aim to avoid or limitpathogen survival and dissemina-tion. These measures are com-monly used to manage otherpotato diseases and pests.

Removal of potato haulmsAfter harvest of a BW-infestedcrop, potato haulms must beremoved from the field and burieddeep, down-slope and far fromirrigation canals. Alternatively, theycan be burned.

26

Removal of rotted tubersAfter harvest, sorted-out andleftover diseased tubers must alsobe removed from the field anddestroyed using the same proce-dure as for haulms.

WeedingR. solanacearum survives inweeds, so weeding must be donebefore planting potato and anyother crop used in the rotation.

Roguing volunteer potato plantsVolunteer potato plants are anothermeans of survival of R.solanacearum and must be re-moved in the subsequent crop(potato or any other crop) soonafter their emergence.

Roguing wilted potato plantsIf the incidence of BW is low in afield, wilted potato plants must beremoved as soon as they areobserved to avoid contamination ofhealthy neighboring plants. Theymust be destroyed carefully as

28

29

30

14 15

Sanitation and cultivation practices

Removal of potato haulms

... and of rotted tubers

Weeding

Roguing volunteer potato plants

Roguing wiltedpotato plants

31

33

34

3532

36

described previously for potatohaulms and sorted-out tubers.Tubers from neighboring diseasedplants should not be used forseed.

Tool decontaminationTo prevent movement of soil froman infested to a disease-free field,all tools must be decontaminatedby washing with water and calciumhypochlorite (or other availablebactericide) or sterilized by flame.

Tractor decontaminationMachinery, vehicles, hooves ofanimals used for traction, andshoes of the personnel comingfrom an infested field must bewashed at least with water beforeentering another field.

Uncontaminated waterThe flow of water from an infestedfield to adjacent fields must beavoided. In infested areas, irriga-tion by well water is preferred oversurface water from rivers or irriga-tion canals.

Other cultivation practices

• Minimum post-emergencecultivation: hilling only atplanting time to avoid wound-ing roots with tools. Subse-quent hand weeding is prefer-able for the same reason.

• Flooding of paddy rice: inAsian countries this signifi-cantly decreases soil popula-tions of R. solanacearum.

• In very warm areas, exposing plowed soil to summer heat also decreases soil infestation.

RotationRotation with non-host plants is aneffective means of decreasing thelevel of R. solanacearum popula-tions in soil, provided volunteerpotato plants are continuallyremoved by uprooting the entireplants from the field as theyemerge.

Rotate with cereal cropsRotation with cereals andgramineous pastures rapidlydecreases soil inoculum potential.However, the time necessary foreradication has to be determinedin each location.

16 17

Other cultural practices

. Minimum post-emergence cultivation

. Flooding of paddy rice

. Exposing plowed soil to summer heat

Rotation

Rotate withcereal crops

Wheat

Maize

Uncontaminated water

Tractor decontamination

Tool decontamination

37

38

41

40

Rotate with liliaceous andbrassicaceousAllium sp. (onion, garlic, leak) andbrassicaceous (cabbage, cauli-flower) can be planted after a BW-infected potato crop. Furthermore,these crops promote the elimina-tion of weeds and volunteers thatmaintain the pathogen.

Rotate with legumesLegumes, e. g. fabaceous (pea,bean), can be used as rotationcrops for potatoes. Moreover, theyhave the additional benefit ofincreasing soil fertility by fixingatmospheric nitrogen. A bean/maize rotation has reportedlyreduced soil inoculum potential.

Rotate with cucurbitaceouscropsCucurbitaceous crops (pumpkin,cucumber, zucchini) are not hostsfor R. solanacearum and can thusbe used in the rotation.

Avoid tomato cropTomato crop should be avoidedsince it is highly susceptible tobacterial wilt caused by either race1 or 3 strains of R. solanacearum.

39

Avoid other solanaceous cropsEggplant, pepper and tobaccomust never be planted after a BWinfected potato crop

Avoid ginger and groundnutGinger and groundnut must neverbe planted after a BW-infectedpotato crop in infected areaswhere race 1 strains of R.solanacearum are prevalent (low-elevation tropics).

Nematode controlNematodes open a path for thebacterium to enter the plantthrough root injuries. Therefore,they must be controlled to avoidtheir interaction with R.solanacearum. Major nematodecontrol components are soil fumi-gation, rotation with cereals,application of high quantities oforganic amendments (free of R.solanacearum), and plantingnematode resistant varieties.

42

43

18 19

Nematode control

Avoid othersolanaceous crops

Avoid

Groundnut

Ginger

PepperEggplant

Rotate withliliaceous andbrassicaceous

Rotate with legumes

Rotate with cucurbitaceous crops

Avoid tomato crop

45

46

48

Symptoms caused by the root-knot nematodeThe major nematode interactingwith BW is the root-knot nematode(Meloidogyne incognita), occurringmainly in warm climates and sandysoil.

QuarantineOnce BW is introduced to an area,quarantine regulations have to beapplied to avoid spreading of BWto non-infested areas. Measuresrestrict the production of seedpotatoes and impede the commer-cialization of ware potatoes to BW-free countries or regions, affectingthe economy of the quarantinedregions.

No transport of infected seedtubers to non-infected areasAvoid transport of ware or seedpotatoes from an infected area toa BW-free to avoid spreading thedisease. While it is often difficult tofully control all potato trading, thisis one of the best preventativemeasures available.

DEFINING A CONTROLSTRATEGYThe most relevant components forthe control of bacterial wilt havebeen presented. Based on thatinformation, the following table

47

lists the main factors to be consid-ered in developing a strategy forcontrol of bacterial wilt that iscaused by either race 3 or 1. Eachfactor has been rated on a scale of1 to 7. The higher the number, themore the factor is judged to controlthe disease. The rates allocated toeach factor may change accordingto the importance of a factor in agiven location. A sum of at least14 would usually be adequate forgood control or potential eradica-tion.

Factors to be rated to formulate a control strategy Race 3 Race 1Healthy seedPotato resistance or toleranceR. solanacearum-free soilSuppressive soilRotation with non-hostsRoguing wilted potato plantsRoguing volunteersWeed controlRemoval of potato haulms and/or harvest leftoversControl of spread in waterMinimal post-emergence cultivationNematode controlTool decontamination, washing of hooves and shoesExposure of plowed soil to summer heatFlooding of paddy rice

DETECTION METHODS OFR. solanacearum

20 21

44

7 74 27 72 25 34 34 22 23 32 14 43 32 11 31 3

Quarantine

DETECTION METHODS

DEFINING A CONTROL STRATEGYDEFINING A CONTROL STRATEGY

Factors to be rated to formulate a control strategy Race 3 Race 1

Healthy seedPotato resistance or toleranceR. solanacearum-free soilSuppressive soilRotation with non-hostsRoguing wilted potato plantsRoguing volunteersWeed controlRemoval of potato haulms and/or harvest leftoversControl of spread in waterMinimal post-emergence cultivationNematode controlTool decontamination, washing of hooves and shoesDry / heat soilFlooding of paddy rice

747254423243211

727233223143133

Root-Knot Nematode (Meloidogine incognita)

No transport ofinfected seed tubersto non-infected areas

DEFINING A CONTROL STRATEGY

49

50

51

52

Detection of latent infection inseed tubersThe classical method of detectingtuber infection consists of incubat-ing tubers for 3 to 4 weeks at 30°Cand observing oozing from theeyes or stolon ends, or cutting thetubers to observe oozing from thevascular ring. However, thismethod is time and space con-suming and may not reveal lowinfection rates. Because of this,CIP developed a simple, sensitive,quick and low-cost technique todetect latent infection in tubersthat is suitable for seed testing:post-enrichment NCM-ELISA(Enzyme linked immunosorbentassay on nitrocellulose mem-brane). The kit is being distributedto seed programs worldwide.

General scheme of seed testingThe main steps of post-enrichmentNCM-ELISA to detect R.solanacearum in latently infectedtubers are:1) Preparation of tuber extracts

from the tuber vascular ring;2) Incubation of tuber extracts for

48 h at 30°C in a semi-selective broth (= enrich ment procedure); and3) Loading (Dot-blotting) of the enriched samples on nitrocellu lose membrane (NCM), and serological test (ELISA).

Evaluation of latent infection inpotato seed lotsThe minimal sample size requiredis 250 tubers per hectare (sampledat harvest or in storage) for analy-sis in ten sub-samples of 25tubers. The tissue of the vascularring removed from each of the 25tubers is crushed in one individualbag after adding the volume ofextraction buffer corresponding todouble the tissue weight. For eachsub-sample, 0.5 ml (500 µl) of thetuber extract is mixed with thesame volume of SMSA broth in anEppendorf tube and incubated for48 h at 30°C with agitation twotimes a day. Two dots of eachincubated extract are put on themembrane.

The five steps of NCM-ELISANCM-ELISA is an immuno-enzy-matic assay. It uses a nitrocellu-lose membrane instead of amicrotitre plate as support for thesamples and reagents. The testconsists of the following steps:1) Loading of a very small

amount of the tuber extract (20µl e.g. one drop) on a nitrocel-lulose membrane (= Dot-blotting);

2) Blocking of the area of themembrane that is free ofextract with milk casein(1 h incubation);

3) Binding of the samples with

Crushing Crushing of tissueof tissue

Tuber extractTuber extractWashings Washings

andand disinfection disinfection Removal of Removal of pieces of pieces of

vascular ringvascular ring

ENRICHMENT=Incubation for 48 h at 30°C

in SMSA broth

DOT - BLOTTINGon nitrocellulose membrane (NCM)

SEROLOGICAL TEST:

NCM-ELISA

SAMPLE EXTRACTION

Scheme for the detection of Scheme for the detection of R. R. solanacearumsolanacearum in latently in latently infected potato tubers using CIP NCM-ELISA kitinfected potato tubers using CIP NCM-ELISA kit

22 23

Detection of latent infection in seedtubers using CIP NCM-ELISA kit

NCM-ELISANCM-ELISABlocking

Polyclonal rabbit antibody

Color development

Goat anti-rabbit conjugate

Washings

Washings

Dot-blotting

Antigen

Polyclonal rabbitantibody

Goat anti-rabbit conjugate

Blocking protein

Enzyme substrate

Incubation for 48 h at 30°C Dot - Blotting (two dots per sub-sample )

Remove pieces of the vascular rin gof each tuber (< 0.5 g per tuber)

Sample size = 250 tubers per ha⊇ 10 sub-samples of 25 tubers

. . . . . .

Crush tissue

500 µlextract

Agitate

500 µlSMSA

1 2

500 µlextract

500 µlSMSA

Crush tissue

500 µlextract

Agitate

500 µlSMSA

9 10

500 µlextract

500 µlSMSA

Combine the pieces removed from 25 tubersin a separate ba g, weigh and add 2 ml of extractionbuffer per gram of tuber tissue

PROTOCOL FOR THE DETECTION OFPROTOCOL FOR THE DETECTION OFLATENT INFECTION IN POTATO SEED LOTSLATENT INFECTION IN POTATO SEED LOTS

NCM-ELISA

53

54

R. solanacearum-specificrabbit antibodies (2 h incuba-tion);

4) Binding of the R.solanacearum-antibodiescomplex with enzyme-labeled(alkaline phosphatase) goatanti-rabbit antibodies (1 hincubation); and

5) Revealing of the bound en- zyme by adding the substrate leading to a coloration reaction (5 to 20 min).

Sensitivity of NCM-ELISA withand without enrichmentThe presence of R. solanacearumin the tuber extract leads to thedevelopment of a purple colora-tion. All races and biovars of R.solanacearum can be detectedwith the polyclonal antiboides.Theintensity of the coloration is pro-portional to the bacterial concen-tration. After enrichment (e.g.,multiplication of the bacterialpopulation in the extract), as fewas 10 bacteria per ml of tuberextract (cells/ml) can be detected,whereas 107 cells/ml or more arenecessary if enrichment is notpracticed before conducting theimmunoassay. Thus, enrichmentincreases sensitivity of the sero-logical test by a million-fold,allowing the detection of R.solanacearum in potato tubers (orstems) that are latently infected,e.g., with very low infection levelsthat produce no visible symptoms.

Methods to detect soil inoculum

Tomato bioassaySince tomato is highly susceptibleto bacterial wilt at warm tempera-tures, tomato bioassay is thesimplest way to evaluate soilinfestation with R. solanacearum.However, it requires greenhousespace and is time consumingsince it can take up to six weeks toobserve symptom development. Itcan also be done in the field byplanting rows of tomato andobserving wilt development. Forgreenhouse tests, 2 kg of soil areplaced in a plastic tray and 50 two-week old tomato seedlings (about10 cm high) are transplanted. Foreach soil sample, 100 seedlingsare transplanted in two trays, andirrigated normally. From 7 daysuntil 45 days, the percentage ofwilted tomato plants is recordedtwo times a week. The test tubeassay and the KOH test with thebacteria oozing from the tomatostems can be used to confirm thatwilt results from R. solanacearumand not from other soil pathogenssuch as Pythium sp., Verticilliumsp. and Clavibacter michiganensissubsp. sepedonicus.

24 25

Detection of soil inoculum

Tomato bioassay

Sensitivity of NCM-ELISA without (-E)and with (+E) 48 h enrichment

Sensitivity of NCM-ELISA without (-E)Sensitivity of NCM-ELISA without (-E) and with (+E) 48 h and with (+E) 48 h enrichmentenrichment

Inoculated tuber extract (TE)

R. solanacearum in citrate buffer

10 8

10 6

10 5

10 7

+ ECells / mlCells / ml

106

104

103

101

105

0.1

102

- E

Negative control: TE (+E)

55

56

57

Methods to detect soil inoculumPost-enrichment DAS-ELISAA kit has been set up at CIP todetect low population levels of R.solanacearum in soil. The methodis based on the simple preparationof soil extracts by mixing 10 g ofsoil with 90 ml of PBS buffer. After30 min. constant agitation, thesolution is allowed to stand for oneminute and 2 ml of the supernatantis removed, mixed in a sterile flaskto 38 ml of SMSA broth comple-mented with potato broth andincubated for 48 h at 30°C withagitation (enrichment procedure).The enriched extracts are ana-lyzed in double-antibody sandwichELISA (DAS-ELISA) in microtitreplates. As few as 20 bacteria pergram of soil can be detected. Forsemi-quantification of the popula-tions, the soil extracts can bediluted previous to their enrich-ment.

The four steps of DAS-ELISADouble-antibody sandwich ELISA(DAS-ELISA) is an immunoassayconducted in microtitre plates, withthe following four steps:1) Coating of the microtitre plate

wells with the R.solanacearum-specific rabbitimmunoglobulins (IgG)(3 h incubation);

2) Loading of the samples in thewells (overnight incubation);

3) Loading of the R.solanacearum-specific rabbitIgG conjugated to alkalinephosphatase (3 h incubation);

4) Development of the colorationreaction by adding the enzymesubstrate (1 h).

METHODS TO ASSESS POTATORESISTANCE TO BWField evaluationScreening potato for resistance toBW requires a field with a reason-ably uniform and moderate level ofinfestation (between 30% to 50%wilt incidence in the previouspotato crop). The biovar/race ofthe strains present in the fieldshould be identified (both races 1and 3 can be present). The plotdesign should be chosen accord-ing to the number of tubers perclone to be tested and the unifor-mity of the inoculum spread in thefield, but at least 20 tubers shouldbe used (4 replications of 5 tu-bers). During the growing seasonthe number of wilted plants isrecorded once a week from 40days to 80 days after emergence.At harvest, visible infection oftubers (and latent infection if nowilting occurred during the growingseason), and yield of marketablesized tubers are also recorded.

26 27

Coatin g with the rabbit I gGDASDAS - ELISA - ELISA

Antigen (sample)

Rabbit immunoglobulin(IgG) specific to the antigen

Rabbit immunoglobulin(IgG), spec ific to theantigen, conjugated toalkaline phosphatase

Enzyme substrate

Microtitre plate

Antigen (sample)

Rabbit immunoglobulin(IgG) specific to the antigen

Enzyme substrate

Sample loadin g

Loadin g of theconju gated rabbit I gG

Washin gs

Washin gs

Washin gs

Color development

Detection of soil Detection of soil inoculuminoculum

Sensitivity of Sensitivity of DASDAS-ELISA -ELISA without (-E) and without (-E) and

with (+E) 48 hwith (+E) 48 h enrichmentenrichment

108

107

20

102

106

105

104

103

Rs in citratebuffer

Rs in soil extract

Non-infestedsoil extract

- E + E

108

107

106

105

Citrate buffer

Negative controls:

- E + E

Post-enrichmentPost-enrichment DAS DAS-ELISA-ELISA

DAS - ELISA

Detection of soil inoculumPost-enrichment DAS-ELISA

METHODS TO ASSESSPOTATO RESISTANCE TO BW

Field evaluation

Greenhouse inoculation

Greenhouse inoculationApical cuttings of potato plants orcuttings obtained from sproutedtubers are rooted in small potscontaining a soil substrate (amixture of soil, sand and peat2:1:1). However, the best resultshave been obtained using thePromix BX® substrate (PremiersBrands, INC, Stamford, Canada).A complete fertilizer (N-P-K 20-20-20, diluted at 0.5% in water) isapplied at the time of planting.Plants should be transferred to thegreenhouse at least one daybefore the inoculation. Green-house conditions that favor dis-ease development are 28±4º C,and 85-90% R.H. Plants areirrigated daily, except for one daybefore inoculation. Each potcontaining a one-stem 20-day-oldplant (about 15 cm tall) is inocu-lated with 25 ml of bacterial sus-pension of R. solanacearum at 108

cells/ml. Twenty plants per geno-type are inoculated. Plants areincubated for 30 days and thedisease indices are recordedweekly on the following scale:• 1 = no symptoms;• 2 = wilting of one leaf;• 3 = wilting of up to 50% of the leaves;• 4 = wilting of up to 75% of the leaves;• 5 = complete wilting of the plant.

Cultivars Cruza 148 (resistant tofoliage wilt), Molinera (= BR63.65,moderately susceptible),Revolucion (susceptible) andYungay (susceptible) are used ascontrols at CIP Lima. At a mini-mum, Cruza 148 should be usedas the less-susceptible control,together with a local susceptiblevariety.

28 29

REFERENCES

Aley, P., French, E. R. and Nydegger, U. (1994) Methods of greenhouse inoculation with Pseudomo-nas solanacearum to select resistant potato clones. Fitopatologia 29:20 (Abstract).

Anonymous. (1994) Bacterial wilt: the disease and its causative agent Pseudomonas solanacearum(eds. Hayward, A. C. and Hartman, G. L.). 259 p. CAB International, Wallingford (UK).

Anonymous. (1998) Bacterial wilt disease: molecular and ecological aspects (eds. Prior, Ph., Allen, C.and Elphinstone, J.) 447 p. INRA edn, Springer Verlag, Berlin (Germany).

Commonwealth Mycological Institute. (1977) Distribution maps of plant diseases: Pseudomonassolanacearum (E. F. Smith) E. F. Smith on potato (Solanum tuberosum), tobacco (Nicotianatabacum and banana (Musa) etc. Map No. 138 Edition 5.

Elphinstone, J. G., Hennessy, J., Wilson, J. K. and Stead, D. E. (1996) Sensitivity of differentmethods for the detection of Pseudomonas solanacearum (Smith) in potato tuber extracts. EPPO/OEPP Bulletin, 26 :663-678.

French, E.R. (1986) Field-screening CIP clones developed for resistance to bacterial wilt. TechnologyEvaluation Series No. 1982-6, 9 p. International Potato Center, Lima (Peru).

French, E.R. (1994) Strategies for integrated control of bacterial wilt of potatoes. In Bacterial wilt:the disease and its causative agent Pseudomonas solanacearum (eds. Hayward, A. C. andHartman, G. L.) p. 199-207, CAB International, Wallingford (UK).

French, E. R., Gutarra, L., Aley, P. and Elphinstone, J. (1995) Culture media for Ralstoniasolanacearum isolation, identification and maintenance. Fitopatologia 30 (3): 126-130.

Hayward, A. C. (1991) Biology and epidemiology of bacterial wilt caused by Pseudomonassolanacearum. Annual Review of Phytopathology 29, 65-87.

Hayward, A. C., El-Nashaar, H. M., de Lindo, L. and Nydegger, U. (1991) The use of microtiterplates in the phenotypic characterization of phytopathogenic Pseudomonads. Proc. 7th Int. Conf.Plant Pathog. Bact., Budapest, Hungary, pp 593-598.

Janse, J. D. (1996) Potato brown rot in Western Europe –history, present occurrence and someremarks on possible origin, epidemiology and control strategies. Bulletin OEPP/EPPO Bulletin26:679-695.

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

Martin, C. and French, E.R. (1985) Bacterial wilt of potato: Pseudomonas solanacearum. TechnicalInformation 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 detectionof 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 inlatently infected potato tubers by post-enrichment ELISA on nitrocellulose membrane. EPPO/OEPPBulletin 29 (1), in press.

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

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

30