Asterix (4,5,6). Kennebec (7). Innovator (8). Home Guard (3). Atlantic (4). Lugovska (6). Meduza (8). Irys (3). Irene (5,7). Maxilla (7). Robijn (8,9). Kathadin (2,4,6).
Evaluation of resistance to Phytophthora infestans: A survey 1
Ewa Zimnoch-Guzowska and Bogdan Flis
1
This paper presents the results of a survey on the evaluation of resistance to late blight in potato. The methods applied for resistance evaluation are major factors influencing the progress in the breeding of late blight (LB) resistant cultivars. A questionnaire was prepared with 17 points, starting from general questions concerning the importance of LB in the potato breeding program and ending with specific details on the screening methods used. The questionnaire was sent to GILB members, who indicated an interest in breeding, and to other breeding centers known to the authors. In total, 39 centers participated in the survey, from which 32 are engaged directly in commercial breeding (breeding programs), five are involved in breeding research (research programs) and two are specialized in screening for LB resistance (screening programs). The centers are localized in 25 countries — the majority of them are in Europe (17countries), four are in South America, two in North America and two in Asia (Table 1). Table 1. Countries of the participating centers. The number in parenthesis is the number of centers if more than 1. Asia
Hungary Ireland Italy Latvia Netherlands (6) Poland (3) Russia (2) Slovakia Spain Ukraine
India Vietnam Europe Austria Belarus Czech Republic Denmark France Germany (3)
United Kingdom North America Mexico (2) USA (4) South America Argentina Colombia Ecuador Peru
The results of the survey are summarized below, independently for the first to the seventeenth question. First question: Estimate the importance of resistance to late blight in your program. The importance of LB resistance in the breeding program was assessed in 1–5 grades, where 5 is the highest. About 38 percent of respondents scored resistance to late blight as the most important trait (score 5), 26 percent estimated the importance of LB as grade 4, about 23 percent as grade 3, and 13 percent considered LB resistance of minor importance (scores 1 and 2). 11
Number of programs
12 9
10
8
8
Breeding programs
6 4
Research programs
3 2
1
1
2
0
2
0 0
0 0
2
Screening programs 0
0 1
2
3
4
5
Importance rating using a scale of 1 - 5 where 5 is the highest
Figure 1. Declared importance of resistance to late blight in the breeding program. 1
Plant Breeding and Acclimatization Institute (IHAR), Mlochow Research Center, 05-832 Mlochow, 19 Platanowa Street, Poland
37
Second question: Estimate the part of your breeding program devoted to LB resistance
15
Number of programs
Breeding programs
10
5
Screening programs
Research programs
0 0-20
20-40
40-60
60-80 80-100 20-40
40-60
60-80 80-100 80-100
P e rce n ta g e
Figure 2. Percentage of effort in breeding, research and screening only dedicated to the development of LB resistance. Third question: Estimate the size of your breeding program in thousands of first year seedlings grown. In the surveyed breeding programs the overall sum of first year seedlings grown each year is 1.4 million, with approximately one third under selection for LB resistance. The average size of the breeding programs is 38,000 seedlings (ranging from 5,000 to 170,000 seedlings). The average size of the LB breeding materials is 14,000 seedlings (ranging from 0 to 56,000 seedlings). Fourth question: Indicate the sources of resistance used including cultivars, wild species and transgenes. Figure 3 provides data on the number of programs that use cultivars, wild species, or cultivars and wild species as sources for LB resistance, and those that also use transgenes. Table 2 lists the names of these sources.
23
Number of programs
25 20
15 10
6 4 2
5
2
0
1
0 Breeding program s Cultivars
Wild species
Research program s Cultivars & w ild species
Also transgenes
Figure 3. Sources of resistance to LB used by the breeding and research programs.
38
0
Table 2. Cited sources for late blight resistance indicating how many programs list it as a source when greater than 1. Varieties Stirling (5) Jacqueline Lee (3) Kuras (3) Lugovska (3) Zarevo (3) Bionta (2) Cara (2) Innovator (2) Torridon (2) Algodona Atlanta Atzimba Awiza Belosowski Brodick Charodey Cramond Cruza 148 Effect Ireri Juanita Monserrate Nikulinsky Norteña Nevsky Pampanea Pana Blanca Panda Pokra Polesskyi Rozovyi Pushkiniec Pimpernel Qompis Reserv Robijn Skoroplodny Sjamero Suzorye Teina Udacha White Lady
Clones AWN 86514-2 (3) A90586-11 (3) BO718-3 (2) INTA clones ABPT sources (Netherlands) Advanced breeding populations (CIP) CIP 3753331 CIP 573268 CIP 575010 CIP 720091 CIP 676008 CIP 391013 CIP 391137 Mexican clones Black's differential R9 J-Lines (S. bulbocastanum) J103-K7 B0767-2 G6582-3 EGAO from IHAR, Poland LB resistant selections (A. Rivera INIFAP) F87084 (Agriculture Canada) VIR 90-7-2 VIR 90-7-7 VIR 97-152-6
Wild species S. demissum (16) S. bulbocastanum (13) S. microdontum (9) S. stoloniferum (8) S. andigena (6) S. berthaulti (6) S. hougasii (6) S. phureja (6) S. verrucosum (6) S. chacoense (5) S. pinnatisectum (4) S. vernei (4) S. iopetalum (3) S. acaule (2) S. circaeifolium (2) S. fendlerii (2) S. papita (2) S. polytrichon (2) S. stenotomum (2) S. sucrense (2) S. tarijense (2) S. arnezii x hondelmahii S. avilesii S. brachycarpum S. bukasovii S. commersonii S. guerreroense S. hjertingii S. multiinterruptum S. neoantipovichii S. okadae S. schenckii S. simplicifolium S. yungasense
39
Fifth question: Indicate stages of breeding cycle when the screening for LB is done. The selection of resistance to LB can start from the first year of breeding cycle with the screening of the first year seedlings, and it continues for several years. More than 10 breeding centers select until the sixth clonal generation. Usually, breeding materials are tested from the second to the fifth year. The most frequent situation is screening for LB for three or four seasons, as shown in Figure 4. 14
Number of programs
12 breeding programs research programs
10 8 6 4 2 0 1
2
3
4
5
6
7
8
9
10
11
12
Stage in breeding cycle (year)
Figure 4. The years in the breeding cycle when selection for LB resistance takes place in the breeding and research programs. Sixth question: Indicate difficulties met in breeding cultivars highly resistant to LB. To obtain a ranking of the difficulties in breeding for late blight resistance, the participants were asked to choose up to five factors that hamper progress in breeding for LB resistance. Number values were assigned to each factor, 5 for the most important difficulty, 4 for the next, etc. After the thematic grouping and summing up of the points, the following factors were found to be the most important (in descending order): •
late blight resistance with agronomic traits
•
combination of earliness with LB resistance (maturity)
•
pathogen diversity and resistance evaluation (screening methods)
•
identification and utilization of new sources of resistance
•
inheritance of resistance
•
cost of the selection
•
combination of foliage and tuber resistance
•
lack of MAS (marker assisted selection) applicable to selection for LB resistance
When the weighted means were calculated, the inheritance of resistance to LB and the negative correlation between resistance and maturity were considered most important by the breeding programs, followed by the cost of the evaluation (selection), pathogen diversity and resistance evaluation (Figure 5). For research programs, the most important problems were: the resistance sources and their utilization, lack of MAS, the negative correlation between resistance and maturity, difficulty in combining of foliage and tuber resistance and inheritance of resistance. The costs of resistance evaluation were the most important for screening programs. 40
inheritance of resistance earliness & resistance (maturity) cost of selection pathogen diversity & resistance evaluation combination of foliage & tuber resistance resistance & agronomic traits lack of marker assisted selection (MAS) identification & utilization of sources
0
1
2
3
4
5
1 - 5 scale, where 5 = most important
Figure 5. Ponderated ranking of difficulties in breeding for LB resistance. Seventh question: Indicate methods applied for testing resistance The different methods for evaluating resistance and the number of breeding, research and screening programs that use them are listed in Table 3. Table 3. Number of programs and methods applied for testing resistance. Evaluation Method
Breeding
Field Laboratory or Greenhouse Field and Laboratory or Greenhouse Field and Laboratory or Greenhouse & others
a
Research
Screening
13
1
1
–
–
–
13
3
1
4
1
–
None
2
–
–
Total
32
5
2
a
indirect methods, including molecular markers.
Eighth question: Indicate the method of field testing Ninety-four percent of the programs use field testing. In Table 4 are shown the data on infection sources used and in Table 5 the field plot designs utilized. Table 4. Infection source used for field testing by different programs a.
Program
Natural infection without infectors
Natural infection with infectors
Artificial infection with infectors
Breeding
13
3
7
Research Screening
1 2
1
1
a Number
Combination of infection sources 7 2
of programs/infection source 41
Table 5. Conditions of field testing used by the different programs. Conditions
Breeding
Research
Screening 1 – 20
Number of plants per plot
5 – 20
1–5
Prevalent number of replications
2–3
1–3
1–5
Number of locations
2–5
1–3
1
b
a
a One b 13
research program uses >20 plants breeders screen in more than one location.
Ninth question: Briefly describe the conditions used for artificial field inoculations. The conditions for the use of isolates (single or mixed combinations) and their inoculum concentrations for artificial field inoculations used by the breeding and research programs and the number of programs that use them are shown in Table 6. The screening programs in the survey do not use artificial inoculations in the field. Table 6. Conditions for artificial field inoculations used in by the breeding and research programs. Conditions Programs using single isolate (A) Inoculum concentration a Programs using mixture of isolates (B) Inoculum concentration a Programs using (A) and (B) Inoculum concentration a
Breeding
Research
7
1
104 – 105
–
5
1
4.3 x 102 – 2.3 x 105
1.1 x 105
1
1
104
104
a sporangia/ml
AUDPC (area under the disease progress curve) is used most frequently in breeding, research and screening programs for the evaluation of infection. The participants listed 50 cultivars used as standards, several of which have variable scored foliage resistance, depending on the cited source. The preferred standard potato cultivars are highlighted by bold letters (Table 7). Table 7. Standards used in field screening with their known resistance scores a given in brackets on a scale of 1 – 9, where 1 is the most susceptible. Bold lettering indicates most frequently used standards.
1–3 Bintje (3) Erstling (2) Bildstar (2,3,4) Duke of York (3,4) Erntestolz (3,6,7) Home Guard (3) Irys (3) Kathadin (2,4,6) Paloma Pimpernel (2,7,8) Rus. Burbank (3,4,7) White Rose (3,4) Wilja (2,4,8)
a Resistance
42
Foliale resistance in 1 – 9 scale 4–5 6–7 Desiree (4,6) Cara (7) Agria (4,6) Astarte (7) Adretta (4,6) Danva (7, 9) Arinda (4) Escort (7) Asterix (4,5,6) Kennebec (7) Atlantic (4) Lugovska (6) Irene (5,7) Maxilla (7) Izora (5,7) Milva (7) Karlena (5) Remarka (6,7) Maris Peer (4,7) Signal (7) Maris Piper (4) Storm. Enterprice (6) Monalisa (4,5,7) Tomensa (6) Oleva (5) Pentland Javelin (4,8) Raja (4) Sante (5,6)
scores from participants or from the European Cultivated Potato Database (www.europotato.org)
8–9 Kuras (8) Arka (8) Aziza (8) Bzura (8) Innovator (8) Meduza (8) Robijn (8,9) Sava (8) Stirling (8)
Tenth question: Briefly describe the conditions for seedling resistance testing. The conditions of use of isolates (single or mixed combinations) and their inoculum concentrations for seedling resistance testing used by the breeding and research programs and the number of programs that use them are shown in Table 8. The screening programs in the survey do not use seedling testing. Table 8. Conditions for seedling testing used by the different programs. Conditions Programs using single isolate (A) Inoculum concentrations a Programs using mixture of isolates (B) Inoculum concentrations a Programs using (A) and (B) Inoculum concentrations a
Breeding
Research
6
–
0.5 x 102 – 1.5 x 105
–
5
–
1.5 x 103 – 4 x 105
–
–
1
–
7.5 x 104 – 2.3 x 105
a sporangia/ml
The infection rate for seedlings is evaluated as percentage or grades. In this type of test the standards are potato cultivars with resistance levels that can be differentiated, or seedling progenies from selfing of susceptible and/or resistant materials (Table 9). Table 9. Standards used for seedling testing in the greenhouse or growth chamber. Cultivars Susceptible Pampeana Binje Arinda Paloma Maris Piper Kerrs Pink Erstling
Selfing progenies
Resistant Innovator Raja Pimpernel Stirling
selfing Yungay selfing Adretta selfing of a susceptible clone and resistant cv. Stirling seedlings of S. demissium & S. chacoense
Eleventh question: Briefly describe the conditions of tests on whole plants inoculated in the greenhouse or in the growth chamber. The conditions for the use of isolates (single or mixed combinations), their inoculum concentration and the methods used for the infection assessment for the testing of whole plants that is applied by the breeding, research and screening programs, and the number of programs that use them are shown in Table 10. Table 10. Conditions for whole plant testing used by the different programs. Conditions
Breeding
Research
Screening
Programs using single isolate
5
1
1
5 x 102 – 104
104 – 2 x 104
104
1
–
–
1.5 x 103
–
–
Grades, %
IE, LP, LGR, SPOR2
%
Inoculum concentrations1 Programs using mixture of isolates Inoculum concentrations1 Infections assessment 1sporangia/ml 2 IE
= Infection Efficiency, LP = Latency Period (LP), LGR = Lesion Growth Rate, SPOR = Sporulation density. 43
Twelfth question: Briefly describe the conditions of the leaflet test. The use of leaflets or leaf discs, the conditions for the use of isolates (single or mixed combinations), their inoculum concentrations, and the methods of infection assessment for testing employed by the breeding, research and screening programs, and the number of programs that use them are shown in Table 11. The standard cultivars utilized are listed in Table 12. Table 11. Conditions for the leaflet test used by the different programs. Conditions Programs using leaflet test
Breeding
Research
Screening
12
3
1
Programs using leaf disc
1
Programs using single isolate Inoculum concentration a
6
4
1
104 – 5 x 104
104 – 105
104
5
1
–
7 x 102– 3.5 x 105
7 x 103– 3.5 x 105
–
Grades
Grades, IE, LP, LGR, SPOR b
ni b
Programs using mixture of isolates Inoculum concentration a Infections assessment a sporangia/ml b IE
= Infection Efficiency, LP = Latency Period (LP), LGR = Lesion Growth Rate, SPOR = Sporulation density; ni – not indicated
Table 12. Standards used for the leaflet test by the different programs. Cultivars Adretta Agria Aksamit Arinda Bildstar Bzura Cara Desiree
Gambria Innovator Irys Kuras Meduza Maxilla Paloma Pampeana
Pimpernel Raja Rebecca Tomensa Ute Yavar White Rose White Lady
Others Black’s differentials – with and without R genes Diploid clones DG94-14 and DG94-668
Thirteenth question: Briefly describe the conditions of stem assays. None of the participants reported using this test.
Fourteenth question: Briefly describe the conditions for tuber assays. Forty-nine percent of the participants use this assay. The conditions employed by the breeding, research and screening programs, and the number of programs that use them are given in Table 13, and the potato cultivars used as standards are listed in Table 14.
44
Table 13. Conditions for the tuber assay used by the different programs. Conditions
Breeding
Research
Screening
Programs using tuber slices
2
–
–
Programs using whole tuber
9
–
1
Programs using tuber slices and whole tuber
4
3
Inoculum concentration for whole tubers a
1.5 x 103 – 7 x 104
104 – 2.5 x 105
–
Inoculum concentration for tuber slices a
1.5 x 102– 5 x 104
5 x 104
–
Mainly grades
Grades, IE, IA b
%
Infections assessment a sporangia/ml b
IE = Infection efficiency; IA = invasive ability
Table 14. Standards used in tuber screening with their known resistance scores a given in brackets (on a scale of 1 – 9, where 1 is the most susceptible). Bold lettering indicates most frequently used standards.
1–2 Bintje (2,3,4,5,8) Rus. Burbank (1,3,4) Pentl. Javelin (2,5,7,8) King Edward (1,2) Ranger Rus. (2,3) White Lady (1,3,4)
a
Tuber resistance in 1 – 9 scale 3–4 5–6 Maris Piper (3) Pimpernel (5,7) Altantic (3,7) Adretta (5,6) Bzura (4, 5) Agria (6,7,8) Cara (4,5,7,8,9) Dianella (5) Desiree (4,5,6,7,8) Dunja (6) Estima (4,5) Erstling (5) Granola (3,5) Fecuva (5,7) Home Guard (3) Hansa (5) Irys (3) Izora (5) Kerrs Pink (3) Karnico (5,6) Maris Peer (4) Lvovjanka (5) Marabel (3) Maxilla (5,7) Provost (3) Meduza (5,7) Spunta (4,5,6,7) Monalisa (5,6,7) Redskin (3) Satina (6) Ukama (3,5,6,7) Stor. Enterprise (5,8) Ute (3) Wilja (3,5,6,7)
7–8 Oleva (7) Stirling (8)
see footnote to Table 7.
No standard cultivars were listed for a score of 9 by any of the programs. The tuber resistance scores given to the standards by the different evaluators vary significantly and may well limit the reliability of tuber resistance comparisons among various programs. Fifteenth question: How important is tuber resistance in relation to foliage resistance? Twenty-four percent of the surveyed programs considered tuber resistance more important than foliage resistance, 38 % considered foliage resistance more important, and 38% considered both resistances of equal importance.
Sixteenth question: Do you check the virulence and aggressiveness of isolate(s) used in screening? The virulence and aggressiveness of isolates used in screening for LB resistance is checked in 19 out of 24 breeding programs responding to this question. Six of them check virulence once a year, two twice a year and some check the virulence at each evaluation. Five breeding programs do not do the virulence check 45
themselves. The four research programs check virulence at each evaluation. One screening center does virulence checking twice a year. This information is summarized in Figure 6.
25
5 20
yes
no
15
19
10
1
5
4 1
0 Screening
Research
Breeding
Figure 6. Breeding, research and screening programs, which do or do not check isolates used in screening for virulence and aggressiveness. Seventeenth question: Which type of LB resistance is preferable in your breeding programs? Among the 34 responses to this question 70% preferred resistance in which R-genes and polygenes are operating together, 26 % preferred polygenic resistance alone and one respondent preferred resistance determined by R-genes. This is summarized in Figure 7.
19
R- and polygenes
5 8
Polygenes
Breeding
1
Research 0 R genes
1 0
5
10
15
Number of programs
Figure 7. Type of LB resistance preferred by the different programs.
46
20
Conclusion This survey presents a picture of a global breeding potential directed to the improvement of LB resistance in newly bred cultivars. Assuming that each new cultivar is selected out of approximately 100,000 seedlings, the 450,000 seedlings cited in this survey as selected for LB resistance guarantee four to five cultivars released each year with outstanding resistance to LB. If this survey represents about 20 % of global breeding activities, these figures might be higher. The breeding centers surveyed are exploring various sources of resistance to P.infestans from both cultivars and wild species (most of which are carrying R-genes.). Non-conventional sources of resistance are not widely used. The majority of participants of the survey listed the combination of R–genes and polygenes as the most preferable type of resistance in breeding materials. According to the survey the most important problem indicated in breeding resistant cultivars is insufficient agronomic value of resistance sources, followed by the negative correlation between earliness and resistance. The most popular method applied in selection for resistance to LB is field testing followed by tuber testing. The conditions under which the different tests are carried out vary substantially between programs. Also, there is significant variation in the evaluation of resistance level for often used standards. The creation of agreed sets of standards for each type of tests on a global or regional scale seems highly recommendable. The Authors wish to express grateful thanks to all participants of the survey.
47
