The procedure for evaluation of advanced clones for resistance to BW involves trials over 3 seasons according to the following scheme:
1. First season: Plant 5 tubers per plot with 4 replications.
2. Second season: Plant 5 tubers per plot with 5 replications, to re-evaluate clones presenting mean wilt scores of between 1 and 2 in the first season and no plant with a maximum wilt score greater than 2 (see below).
3. Third season: Plant 5 tubers per plot with 6 replications, to re-evaluate clones presenting average scores of between 1 and 2 in the second season and no plant with a maximum wilt score greater than 2 (see below).
1. Field Preparation
Each test uses a Randomized Complete Block Design. The distance between plants is 0.40 m and between rows, 0.90 m. Cultural operations are as recommended for commercial crops:
- N-P-K is applied at 200-180-180 U/ha or according to local recommendations. Half of the nitrogen is applied at planting and the other half at first hilling.
- Water is supplied by furrow irrigation according to requirement of plants.
- Pesticide applications are carried out against insects (for example Furadan, Ambush) and late blight (Dithane M-45 and Ridomil MZ 72) as needed.
1.1. With enhanced inoculum
One month after plant emergence, soil inoculum is enhanced by burying one eighth of a 9-cm diameter culture plate containing a 48 h-agar culture of R. solanacearum (local isolate) on Kelman's medium without tetrazolium chloride (each culture piece contained approximately 3 x 109 colony forming units) at the level of the roots. Sanitation precautions are taken to avoid pathogen spread such as (i) shoes and tools of workers are washed and disinfected with 1% sodium hypochlorite when leaving the field, (ii) the plot is surrounded by maize crops, (iii) a well (2-m deep) is located in the lowest corner of the field considering both slopes to collect run-off waters and is regularly disinfected with approximately 1% sodium hypochlorite, (iv) harvested tubers that are not taken to the laboratory for evaluation are used exclusively for consumption or burned.
1.2. Without inoculation (In the region)
Screening potato for resistance requires a field with reasonably uniform and moderate level of infestation (between 30 % to 50% wilt incidence in the previous potato crop). Previous to the evaluation trial, a susceptible potato variety should be planted in efforts to homogenize and enhance soil inoculum levels. Rotted left-over tubers from this planting can be buried randomly in the plot. The plot design should be chosen according to observations on uniformity of the inoculum spread in the field. The biovar / race of the strains present in the field should be identified (both races 1 and 3 can be present).
2. Disease Assessment
2.1 Plant wilt
For each plant, the degree of wilt (disease severity) is evaluated at approximately 15 day intervals for two months starting 45 days after planting (two weeks after inoculation) and continuing until 15 days before harvest using the CIP scale of 1 to 5 (1 = 0%, 2 = 1-25%, 3 = 26-50%, 4 = 51-75% and 5 = 76-100% of the plant is wilted). For each plot, the average score (i.e. the sum of wilt scores for all plants divided by the number of plants scored) and the maximum score are recorded at each evaluation date. Only clones with mean wilt score for the plot less than or equal to 2 and with maximum wilt score no exceeding 2 at the last reading are selected for confirmation of resistance in the next season. Local susceptible and moderately resistant varieties are used as controls. The test is qualified by verifying that the moderately resistant control (e.g. Cruza 148) has not shown an average wilt score higher than 1.5 and the susceptible varieties showed wilt score greater than 3. At harvest, the yield of the selected clones is recorded as follows: the weight of asymptomatic tubers and of tubers exhibiting symptoms of bacterial wilt (visible oozing at the eyes or vascular oozing or rot upon slicing out of tubers at harvest) is recorded separately, and the percent of weight of symptomatic tubers is calculated. It is not necessary to determine the yield of the susceptible clones except for the moderately resistant and susceptible control varieties for comparison purposes.
2.2 Tuber latent infection
During each cropping season, clones that have shown no plant wilt (mean wilt score 1) and with no visible tuber symptoms at harvest are tested for latent infection. Tubers of the moderately resistant control varieties (e.g. Cruza 148) are also processed in order to compare with the clones being tested. For this purpose, 30 tubers (of any size above 30 mm) for each clone are selected at random in the experimental plots or two tubers per plant for 15 plants can also be taken at random. Tubers are taken to the laboratory and processed individually in order to evaluate frequency of latent infection by R. solanacearum using CIP post-enrichment NCM-ELISA kit according to CIP protocol indicated in the manual of instructions for use. The number (n) of infected tubers (positive ELISA) is converted to a percentage per clone (n/30 x 100).
French, E.R. (1986) Field-screening CIP clones developed for resistance to bacterial wilt. Technology Evaluation Series No. 1982-6, 9 p. International Potato Center, Lima (Peru).
Priou, S., L. Gutarra and P. Aley. 1999. Highly sensitive detection of Ralstonia solanacearum in latently infected potato tubers by post-enrichment ELISA on nitrocellulose membrane. Bulletin EPPO/OEPP Bulletin 29: 117-125.
Priou, S. and L. Gutarra. 2001 (updated edition). Manual of instructions for use of the CIP NCM-ELISA kit for the detection of Ralstonia solanacearum in potato. 26 pp. (English and Spanish versions), International Potato Center, Lima, Peru.
Priou, S., C. Salas, F. de Mendiburu, L. Gutarra and P. Aley. 2001. Assessment of latent infection frequency in progeny tubers of advanced potato clones resistant to plant wilt: a new selection criterion for breeding for bacterial wilt resistance. CIP Program Report 1999-2000, in press. International Potato Center, Lima, Peru.