Postulation of virulence groups and resistance factors inthe quinoa/downy mildew pathosystem using material fromEcuador

J. Ochoaa, H. D. Frinkingb and Th. Jacobsc*aInstituto Nacional Autonomo de Investigaciones Agropecuarios (INIAP), Estacion Experimental Santa Catalina, Casilla postal 17–01–340,Quito, Ecuador; bDepartment of Phytopathology, Wageningen Agricultural University, Wageningen, the Netherlands; cDepartment ofPlant Breeding, Wageningen Agricultural University, PO Box 386, 6708 AJ Wageningen, the Netherlands

Twenty downy mildew isolates were collected in south, central and north Ecuador and inoculated on seedlings of 60quinoa lines. A scale for scoring quinoa downy mildew infections was developed. Four virulence groups and threeresistance factors were identified. In southern Ecuador only isolates of virulence group 2 were collected, while in thecentral region only virulence group 4 was found. In the north of Ecuador isolates of all four virulence groups werecollected. Resistance factor R3 was found most frequently in seedlings of 13 high-yielding Ecuadorian quinoa lines.No effective resistance against group 4, the most virulent group, was observed. Lines ECU-288 (susceptible), ECU-291(R1), ECU-470 (R2) and ECU-379 (R3) could be used as a preliminary quinoa differential set for identifyingdowny mildew virulence groups.

Keywords: quinoa downy mildew, resistance factors, virulence factors

Introduction

Quinoa (Chenopodium quinoa) originates from thehighlands of the Andean regions of Peru and Bolivia(Gandarillas, 1979). Before the discovery of America byEuropeans, quinoa was extensively cultivated by Incasand Araucanians from southern Chile to northernColombia. Today the crop is cultivated on a smallscale within the boundaries of the old Inca Empire(National Research Council, 1989). Owing to the highnutritional value, drought resistance and high adap-tation capacity, this species has become a promising cropwithin and outside the Andes.

Quinoa is a traditional crop in Ecuador and is grownin three different regions. The northern and centralregions are traditional areas of production and quinoa isnow being promoted in the south. Quinoa breeding inEcuador started in the early eighties and four cultivarshave been released so far: Cochasquı and Imbaya (Nietoet al., 1986); and Tunkahuan and Ingapirca (Nieto et al.,1992).

Downy mildew (Peronospora farinosa f.sp. chenopo-dii) is the most important disease of quinoa and developswherever quinoa is cultivated (Alandia et al., 1979).

Under conditions favourable for the disease, crop lossescan be 100% (Salas & Otazu, 1976). Little is knownabout resistance against the disease, although differencesin incidence have been reported under field conditions(Ochoa, 1987; Vasques, 1976; Vega, 1984).

The nature and types of resistance to downy mildew inquinoa have not been studied. Seedling tests were carriedout using a set of quinoa lines and downy mildewisolates to study plant resistance and variation invirulence of the pathogen.

Materials and methods

Twenty isolates of downy mildew were tested onseedlings of 60 quinoa lines from INIAP, Ecuador; 30of these lines had been scored resistant and 30susceptible during field evaluations in Ecuador by Vega(1984) and Ochoa (1987) (Table 1).

The isolates were collected from different regions inEcuador during a survey in June 1994 (Table 2). For eachlocation, leaves with one sporulating or nearly sporulat-ing lesion were placed in a Petri dish with moist paper.To avoid contamination only one leaf was used per dish.The dishes were sealed with parafilm, stored at 58C andtransported to Wageningen. Leaves with lesions wereimmersed in de-mineralized water and carefully shaken.The fluid was poured onto cheese-cloth and the waterwith spores collected underneath. The isolates were

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*Present address: ORIGIN, Service Line Baan, PO Box 546,3770 AM Barneveld, the Netherlands.

Accepted 20 January 1999.

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Table 1 Line, origin, name, field scores, seedling scores and postulated resistance factors of 60 quinoa lines from the Ecuadorian nationalcollection (INIAP)

Field SeedlingLines Country, location Name of cultivar scorea reactionb

PART A , lines with low field scoresECU-222 Ecuador, Pichincha Chaucha de Rayuquı 2 SECU-233 Ecuador, Pichincha Chaucha de Cotopaxi 1 R1ECU-238 Ecuador, Pichincha PA 1-32/80 1 MECU-239 Ecuador, Pichincha PGC 2-33/80 1 MECU-240 Ecuador, Pichincha Violacea 38/80 2 SECU-254 Ecuador, Pichincha Kancolla Blanca 46/80 1 MECU-245 Ecuador, Pichincha Land race 2 MECU-255 Ecuador, Pichincha Land race 2 SECU-272 Ecuador, Pichincha Enana 1-5E-119/80 2 MECU-288 Ecuador, Pichincha Especial 1E-203/80 2 SECU-290 Ecuador, Pichincha Chaucha de Gonzales 1E-209/80 1 MECU-291 Ecuador, Pichincha Chaucha de Gonzales 1E-211/80 1 R1ECU-292 Ecuador, Pichincha PGC 9-1E-215/80 2 MECU-304 Ecuador, Pichincha Quinua Roja 245/80 2 R1ECU-325 Ecuador, unknown Quinua Blanca 2 MECU-345 Ecuador, Pichincha Land race 2 SECU-348 Ecuador, Pichincha Land race 2 MECU-377 Ecuador, Tungurahua Chauca Blanca 2 SECU-379 Ecuador, Chimborazo Land race 2* R3ECU-415 Peru, Lima La Molina 28 2* MECU-423 Peru, Puno 03-02-00554 1* MECU-424 Ecuador, Cotopaxi Land race 1* R1ECU-439 Ecuador, Chimborazo Land race 2* MECU-494 Ecuador, Pichincha Quinua Blanca 2* SECU-497 Ecuador, Pichincha Quinua Morada 0* R1ECU-500 Peru, unknown Kello Jiura 1* MECU-509 Peru, unknown 03-08-00140-1C-1E 1* SECU-521 Peru, unknown 03-08-0545-2e-1E 2* SECU-542 Peru, Huancayo H-20B 1* R1ECU-547 Ecuador, Imbabura Quinua Morada 0* MPART B , lines with high field scoresECU-218 Ecuador, Pichincha Glomerulada 6/80 7 SECU-219 Ecuador, Pichincha Glomerulada 2-7/80 7 MECU-223 Ecuador, Pichincha Glomerulada 3-11/80 6 SECU-279 Ecuador, Pichincha Sajama de Cayambe 2E-183/80 6 MECU-321 Ecuador, Imbabura Rosada de San Rafael 6 MECU-332 Peru, unknown 03-7-0596 8 SECU-336 Peru, unknown 03-07-0656 6 MECU-393 Peru, Puno 03-08-00065 7 SECU-398 Peru, Puno Anco Jiura 03-08-0072 6 MECU-436 Ecuador, Tungurahua Land race 6* MECU-455 Ecuador, Pichincha Land race 7* SECU-384 Peru, Puno 03-08-00022 7 MECU-394 Peru, Puno 03-08-00066. Real 7 MECU-397 Peru, unknown 03-08-00071 7 MECU-465 Peru, Puno Sajama Juliaca 8* SECU-470 Bolivia, La Paz 217 7* R2ECU-475 Peru, Puno 04-02-0505 8* MECU-499 Peru, unknown Coitojiura 3-8-44-4E 1e 1E 8* SECU-528 Ecuador, Cotopaxi Pujilı 3-2e-1E 7* MECU-531 Ecuador, Pichincha Pifo 1-2e-1E 6* R3ECU-538 Bolivia, Omasuyos Sajama 7* SECU-541 Ecuador, Chimborazo Land race 7* SECU-550 Bolivia, Omasuyos 539-6 (1)X3-80 7* MECU-552 Bolivia, Omasuyos 642-7(1)X2-30 6* MECU-554 Bolivia, Omasuyos 545-7(2)X2-55 7* SECU-556 Bolivia, Omasuyos 612-5(1)X2-20 7* SECU-575 Bolivia, Omasuyos 03-08-0738 6* SECU-562 Bolivia, Omasuyos 2-31 7* SECU-565 Bolivia, Omasuyos 3-49 7* MECU-578 Bolivia, Omasuyos Jiwaki 6* M

aDowny mildew scores under field conditions on a scale 0–9: 0, no disease symptoms; 9, plants showing abundant sporulation. Data from Vega,1984. *Data from Ochoa (1987) are marked with an asterisk. bDisease score and postulated resistance factors from seedling data (this study), S ¼

susceptible, M ¼ mixed reaction, R1, R2, R3 are postulated resistance factors.

multiplied on seedlings of the susceptible cultivarBordeaux grown in separate pots. After inoculation theisolates were kept separately from each other. Leaveswith each sporulating isolate were stored at – 208C. Thespores remained viable for over three months.

Mildew inoculation for scale development

Tests were carried out under controlled conditions at theDepartment of Phytopathology, Wageningen AgriculturalUniversity, the Netherlands. The susceptible and resis-tant lines were sown in separate trays. Three seeds ofeach line were sown together in the same hole and theholes were 4 cm apart. Trays were placed in a growthchamber with a day/night regime of 12 h light (20 000lux) and 12 h darkness at 258C. Inoculation was carriedout 18 days after sowing, when the seedlings had twolayers of expanded leaves, each layer with two leaves.

Spores of each isolate were collected as describedabove. The concentration of spores was determined andadjusted as required and 10 mL of inoculum of about105 spores per ml of water were applied with a Vilbisssprayer on each tray of seedlings. After inoculation, theseedlings were incubated in plastic bags for 24 h in agrowth chamber held at 158C with a day/night regime of16 h light (10 000 lux) and 8 h darkness. After incuba-tion, the trays were transferred to a growth chamber at208C with the same day/night regime. Six days afterinoculation, when symptoms were clearly visible, thetrays were placed in plastic cages for one day with high

humidity to create a suitable environment for sporula-tion. After sporulation started, the disease reactionswere described and classified into a scale for scoringdisease resistance (see below).

Virulence analysis

After the scale had been developed, the virulence of20 downy mildew isolates was characterized using 39quinoa lines inoculated as described. Readings of indivi-dual leaves were averaged to obtain one value per line.

Postulation of resistance factors

Resistance factors were postulated for 13 quinoa linesfrom the Ecuadorian breeding program, and for twolines from the Center for Genetic Resources, Wagenin-gen, the Netherlands. The 13 lines were inoculated with11 isolates representing the four virulence groupsidentified in previous experiments. Inoculation methods,experimental conditions and disease scoring weresimilar to those described. As no genetic basis has beendetermined the term ‘resistance factor’ is preferredrather than ‘resistance gene’.

Results

Development of the scale for disease scoring

Six reaction types were described based on the amountsof necrosis/chlorosis and sporulation (Table 3). Reactiontypes 0, 1 and 2 were classified as resistant, involving noor some necrosis and little sporulation and 3, 4 and 5 assusceptible, showing chlorotic lesions and some orabundant sporulation. The scale describes the reactionsof individual seedling leaves.

Reaction type 0 is characterized by the absence ofperceptible necrosis, although sometimes a few necroticspots were observed. Reaction type 1 involves visiblenecrosis through which the truncated development ofmycelium can be seen, suggesting a delayed response ofthe plant to the presence of the pathogen. Reaction type1 was always observed in the second (upper) layer ofleaves, mostly accompanied by some chlorosis andsporulation in the first (lower) layer. The average forthis reaction was scored as 1 þ 0. Reaction type 1 þ ismost likely an incomplete race-specific resistance asreported by Crute & Norwood (1978) for lettuce downymildew, a similar disease.

Virulence analysis

Of the 60 lines studied, 22 showed susceptibility to allisolates and 29 showed mixed reactions. Mixed in thisrespect means that the reaction types of the leaves of thethree seedlings of one line differed markedly from oneanother. As seed impurity or mixture of different seedscould not be excluded, these results were discarded.Nine lines showed clear compatible or incompatible

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Table 2 Geographical origin and virulence groups of quinoa downymildew isolates collected in Ecuador in June 1994

GeographyIsolate Virulencenumbera Region City group

1 South Saraguro V23a South Biblian V24 South Canar V25 South Canar V26 South Canar V27 South Canar V210a Central Guamote V412 Central Colta V413 Central Colta V414 Central Riobamba V415 North Pedro Moncayo V416 North Pedro Moncayo V417 North Pedro Moncayo V418 North Otavalo V119 North Tulcan V220 North Tulcan V321 North Montufar V422a North El Angel V223 North Latacunga V224a North Mejıa V4

All isolates were collected from quinoa landraces except 3, 10, 22 and24, which were collected from the cultivar Tunkahuan.

interactions (Tables 1 and 4). None of the lines wasresistant to all the isolates.

The results with the 9 lines that showed clearcompatible–incompatible reactions were used to iden-tify virulence and resistance factors (Table 4). Fourvirulence factors and three resistance factors weredetected. Line ECU-288 was the most susceptible, so itwas assumed not to carry resistance factors expressed inseedlings and was used as a susceptible check for allisolates. Lines with the postulated resistance factor R1were resistant to isolate 18 (virulence group 1). LineECU-470 carries resistance factor R2, which gaveresistance to isolates from virulence group 2. ECU-379and ECU-531 carry R3 for resistance to virulence group3. None of the lines showed resistance to isolates ofvirulence group 4, including the 29 lines with mixedreactions, indicating the presence of an additional andpossibly new type of virulence in the quinoa downymildew pathogen. Resistance factors R1 and R3originated from Ecuadorian lines and R2 from aBolivian cultivar. All resistance factors are present inlandraces and breeding material (Table 2). There was norelationship between the incidence on adult plants in the

field, as reported by Vega (1984) and Ochoa (1987), andthe reactions of seedlings as described in the presentstudy (Table 1).

The interaction of plants carrying resistance factorsR2 and R3 with isolate 18, the least virulent isolate, wasdenoted reaction type 0, indicating that both leaf layersshowed no perceptible necrosis (Table 4). All otherresistant reactions were scored as reaction type 1 þ,indicating chlorosis and some sporulation in the first leaflayer and a hypersensitive reaction with small necroticlesions in the second (upper) leaf layer.

Isolates collected in the south of Ecuador all belongedto virulence group 2, but in the central region onlyisolates of virulence group 4 were found (Table 2). Inthe northern regions isolates of all virulence groupswere collected. Isolates collected from landraces belongedto all the virulence groups. On the cultivar Tunkahuanonly isolates of virulence groups 2 and 4 were found.

Postulation of resistance factors in breeding lines

From the test with 11 isolates representing the fourvirulence groups, the resistance factors in seedlings of

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Table 3 Scale for scoring disease reactionsof downy mildew (Peronospora farinosa f.sp.chenopodii) in primary leaves of quinoa(Chenopodium quinoa)

Reactiontype Classification Symptoms

0 Resistant No evidence of symptoms, no perceptible necrosis1 Resistant Small chlorotic–necrotic lesions (2–5 mm) with

truncated mycelium in the mesophyl of the leaf2 Resistant Small chlorotic lesions (4–8 mm) with a little sporulation3 Susceptible Medium-sized and confined chlorotic lesions with

sporulation, sporulation mainly on the lower leaf surface4 Susceptible Large, not clearly confined chlorotic lesions with

sporulation, sporulation mainly on the lower leaf surface5 Susceptible Mild chlorosis with abundant sporulation on both upper

and lower leaf surfaces

Table 4 Downy mildew reaction types of seedlings of 10 quinoa lines inoculated with 20 downy mildew isolates

Virulence groups and isolates factor

Vb V2 V3 V4

Linea Resistance 18 1 3 4 5 6 7 19 22 23 20 21 10 12 13 14 15 16 17 24

ECU-288 – 4c 5 5 4 5 5 5 5 4 5 5 5 5 5 4 5 5 5 5 5ECU-233 R1 1þd 5 5 5 5 5 5 5 4 4 5 5 5 5 5 5 5 5 4 5ECU-304 R1 1þ 5 5 5 4 5 4 4 4 4 5 4 4 5 4 4 5 5 4 5ECU-424 R1 1þ 5 5 4 5 5 4 5 3 4 5 4 3 5 3 4 5 4 4 5ECU-497 R1 1þ 5 5 4 4 5 5 5 4 4 4 4 4 5 4 4 5 4 4 5ECU-542 R1 1þ 5 5 5 5 5 5 5 3 4 5 5 5 5 4 4 5 3 3 5ECU-291 R1 1þ 5 5 5 4 5 5 5 4 4 5 3 5 5 4 4 5 5 4 5ECU-470 R2 0 1 1 1þ 1þ 1þ 1þ 1þ 1þ 1þ 3 3 4 5 5 5 5 5 4 5ECU-379 R3 0 1 1 1þ 1þ 1þ 1þ 1þ 1þ 1þ 1þ 1þ 5 5 5 4 5 5 4 5ECU-531 R3 0 1 1 1þ 1þ 1þ 1þ 1þ 1þ 1þ 1þ 1þ 5 4 5 4 5 4 4 5

aIdentification number in the Ecuadorian national collection.bVirulence group identification: V1, V2, V3 and V4.cDisease reaction type at seedling stage (18 days) on a scale of 0–5. Types 0–2: resistant; types 3–5: susceptible.dScore 1 þ indicates that some sporulation was observed on the first developed leaves.

13 high-yielding lines from the Ecuadorian quinoabreeding program were postulated (Table 5). Noresistance factor was found for virulence group 4. Thelines Faro, Bordeaux, Otavalo 4 and the newly releasedcultivar Tunkahuan lacked seedling resistance factorsand were susceptible to isolates belonging to all fourvirulence groups. Lines ECU-621 and ECU-262 carry R1and were resistant to virulence group 1. CultivarIngapirca, originally introduced from Peru, carried R2,and was resistant to virulence groups 1 and 2. The linesInagrofa, Masal 3-89-7E, Colorado, ECU-223 1e1E,Amarilla de Olmedo and Masal 3-89-5e1E carried R3,and were resistant to isolates of virulence groups 1, 2and 3. No resistance factors could be determined for thecultivar Imbaya and line ECU-362, which showed largedifferences in seedling reactions and were discarded.

Discussion

A scale was developed in which six different reactiontypes could be distinguished, ranking from fully resistantto completely susceptible. The scale describes the diseasereaction of individual leaves. Sometimes small differencescould be observed among the four leaves of the two layersand these values were averaged. Use of the scale allowedrapid classification of large numbers of seedlings.

Reaction type 0 was observed in interactions betweenweakly virulent groups (group V1) and resistance factorsR2 and R3. Reaction type 1 was observed in interactionsbetween isolates of group V1 with resistance factor R1and between highly virulent isolates (V2 and V3) withresistance factor R2 and R3. This suggests that the effectof resistance factor R1 on growth of the pathogen isgreater than that of R2 and R3.

Isolates lacking virulence were able to develop some

infection in incompatible interactions when resistancefactor R1 was present, but no infection in plants carryingR2 and R3. Virulent isolates gave some infection even inincompatible interactions indicating that isolates lackingvirulence were less aggressive than isolates carryingvirulence. This suggested that the presence of virulencecontributed to the fitness of the parasite. This couldexplain why complex isolates (V4) have been collectedfrom cultivars such as Tunkahuan, which lacks inresistance factors (Table 5). However, this could alsobe caused by the presence of resistance factors in quinoalandraces grown nearby, or because cultivars withresistance factors were been grown previously.

In Ecuador, the distribution of virulence groupsappeared to be restricted to the different quinoa growingregions. In the south, where quinoa is not extensivelycultivated, the isolates collected belonged only tovirulence group 2, which is not highly virulent. Theisolates seemed to be adapted to the landraces grownand the small number of recently introduced cultivars. Inthe central region, where both landraces and newlyreleased cultivars are grown, only isolates of virulencegroup 4 were collected. The north is the traditionalquinoa growing region, where in many locations oldlandraces have been cultivated for many years. In otherplaces new cultivars have been introduced. Here, isolatesof all four virulence groups were collected. The virulencegroups are probably restricted to those areas in whichsuitable plant genotypes are present. For instance, theleast virulent isolate (18) was collected in the mosttraditional quinoa growing area of Ecuador, around thetown of Otavalo, where mainly old landraces are grown.It is possible that isolates belonging to virulence group 1,being the least complex, were predominant in thetraditional quinoa growing areas until recently, when

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Table 5 Reaction type and postulated resis-tance factors for 15 quinoa lines against isolatesof 4 virulence groups of downy mildew

Reaction type to isolates of virulence groupsd Postulatedresistance

Line V1 V2 V3 V4 factor

Tunkahuana 5 5 5 4 –Otavalo 4 4 5 5 5 –ECU-621 2 5 5 5 R1ECU-262 1þ 4 5 5 R1ECU-362 ? 4 5 5 ?Ingapircaa 0 1þ 5 5 R2Inagrofa 0 1þ 2 4 R3Masal 3-89-7E 0 1þ 2 5 R3Colorado 0 1þ 2 4 R3ECU-223 1e1E 0 2 1þ 5 R3Amarilla de Olmedo 0 1þ 2 5 R3Masal 3-89-5e1E 0 1þ 2 4 R3Imbayab ? 1þ ? 5 ?Faroc 5 5 5 5 –Bordeauxc 4 5 5 5 –

aCultivar released in Ecuador in 1992.bCultivar released in Ecuador in 1986.cTwo lines from the quinoa collection from CPRO-DLO, the Netherlands.dScores on a scale 0–5. Isolates used V1¼ 18; V2¼ 1, 4, 22, 23; V3¼ 20, 21; V4¼ 10, 12, 14, 2.

new cultivars with other resistance factors were intro-duced. The range of virulence present in the northernregion suggests that the fungus is able to adapt to newresistance factors.

The lack of a relationship between the results reportedby Vega (1984) and Ochoa (1987) and the findings ofthis study (Table 1) may be attributed to differences inexpression of resistance factors in the seedling and adultplant stages. These differences can, in part, also becaused by differences in the virulence composition of thefungal populations used for this study and those presentin the field.

Three resistance factors are postulated for the 13quinoa lines of the Ecuadorian breeding program(Table 5). Lines ECU-621 and ECU-262 carry resistancefactor R1, which was probably effective at the beginningof the breeding programme, when isolates of virulencegroup 1 were dominant. R2 is present only in the cultivarIngapirca, which was originally introduced as a linefrom Peru, and this resistance factor was previouslyidentified in a Bolivian cultivar and probably originatedfrom the highlands of Peru and Bolivia. The presence ofR3 in many of the lines indicates that new resistancefactors are being used in the quinoa breeding pro-gramme. Resistance to downy mildew is one of the goalsof the quinoa breeding program (Yanez et al., 1994).Unfortunately, no source of resistance to virulence group4 was found.

A differential set of quinoa lines is proposed: ECU-288 (universally susceptible), ECU-291 (R1), ECU-270(R2) and ECU-379 (R3). This enables classification ofall isolates into the presently postulated virulencegroups. In an additional and separate experiment, thedifferential set was tested against isolates 18 (virulencegroup 1), 1, 4, 22 and 23 (virulence group 2), 20 and 21(virulence group 3) and 10, 12, 14 and 24 (virulencegroup 4). Results were identical to those reported inTable 4. Inclusion of a line resistant to virulence group 4is anticipated.

The present breeding strategy to control the disease isto find sources of resistance to isolates from virulencegroup 4, to develop alternatives to the unstablehypersensitive resistance, and to include durable typesof resistance when identified.

Acknowledgements

The Directorate General for International Cooperation

of the Netherlands Ministry of International Relations isgratefully acknowledged for financial support. The firstauthor wishes to thank INIAP-Ecuador for allowinghim to study and work at Wageningen AgriculturalUniversity.

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