RAPIDSEED- A MODIFIED SCHEME FOR RAPID SEED-TUBER POTATO PRODUCTION Developed by The Virology Management Team (VMT) and contributions by Paul Demo and Peter Gildemacher, SSA Region, Nairobi, Kenya CIP Dec. 2004 (The authors thank Dr. Sylvie Priou from CIP and Eng. Hebert Torres from SENASA, Peru, who kindly provided some illustrations of potato diseases)
INTRODUCTION • The method proposed (Rapidseed) is based in two “old” technologies: positive selection and plant index. • This alternative is proposed to overcome the difficulties that programs in some countries encounter to operate efficiently a tissue culture lab as a way of producing virus-free stocks (high cost of operation, technical difficulties including development of mutations, lack or shortage of electrical power, lack of trained personnel, difficulties in adapting plants to the greenhouse or field, etc.) • For this method to be effective it is required that users have a good knowledge of potato diseases and that they can apply simple but advanced pathogen detection technologies, specially for viruses and bacteria. • Further multiplication of seed after the second generation will depend on the quality of the seed determined by standard procedures used in seed production.
SUMMARY OF BASIC STEPS IN RAPIDSEED A. Positive selection B. Plant – index system for the production of first generation tubers C. Second multiplication (Bulking)
A. POSITIVE SELECTION Positive selection is a process aimed at selecting healthy plants from the fields and relies in the experience of seed producer to identify those plants that have large and better chances of being free of dangerous pathogens. For more detailed information on positive selection click symbol Plants selected must be free of any of the symptoms or defects described in the following links: For viruses and similar agents click symbol For bacteria click symbol For fungi click symbol Pests click symbol
B. PLANT – INDEX SYSTEM FOR THE PRODUCTION OF FIRST GENERATION TUBERS The following steps are required: 1. Examining tubers for disease symptoms 2. Testing for virus and bacteria infection by ELISA 3. Storing the healthy tubers 4. Cutting tubers 5. Planting in the field and evaluation
Examining tubers for disease or pest symptoms • Plant foliage may not show symptoms or signs of somediseases or pests that affect the tubers. Therefore, it is advisable to examine the tubers of positively selected plants at harvest and discard the tuber progeny if symptoms and signs are evident or above acceptable levels. All tubers from plants having signs or symptoms of systemic diseases (e.g. bacterial wilt, soft rot, viruses,etc.) or organisms that can transmit systemic diseases (fungi such as Spongospora subterranea, or virus-transmitting nematodes) should be discarded. Some common symptoms are the following: Click symbol to see some examples
2. Testing for virus and bacteria infection by DAS-ELISA At harvest three small tubers from each plant should be tested (individually or as a composite sample) for the most important viruses. It is advisable to test for PLRV, PVY, PVM, PVA, PVX (or the potexvirus group) and PVS (or the carlavirus group). Also, latent infection by Ralstonia solanacearum must be tested by NCM-ELISA. If other viruses are known to be limiting factors for potato production in the region/country a test for them must be included. For instance, for the Andean region detection of PMTV (by NCM-ELISA) and APMoV should be included. Samples should be taken from the region where the stolon attaches to the tuber. Composite testing commonly used to reduce the cost of testing by ELISA should be evaluated before use at this stage. Tubers tested should be discarded.
Detection of viruses and R. solanacearum in tubers (See Manuals on the techniques developed by CIP) Recommended sample site in tuber for testing by ELISA click symbol for detailson techniques Stolon end Extract tissue with a disinfected knife Discard tuber after test
3. Storing tubers Tubers from each plant selected and tested for most important pathogens should be stored in paper bags or any other container that allow each plant progeny to be individualized and identified. Diffuse light stores (DLS) are an excellent alternative if cold stores are not available. It is recommended that tubers be disinfected before storing by treating them with 1% Sodium hypochlorite, dry the tubers and treat them with a powder fungicide.
4. Cutting tubers When tubers sprout proceed to cut large tubers in pieces that contain at least 2 sprouts each. For cutting tubers a number of precautions should be followed such as the use of cutting knives (several) dipped in 10% Calcium hypochlorite for several minutes and flamed before use. Suberization of cut pieces should proceed for a few days (7-10) under shade before planting. Treating cut surfaces with a powder mixture of a fungicide and calcium hydroxide may help to prevent infections with fungi or bacteria. The value of this latter procedure should be evaluated for every condition. click symbol to see disinfections and cutting of tubers
5. Planting in the field (first generation) and evaluation The progeny of each selected plant should be planted in the field one after the other but maintaining their identification (click symbol for details ). The field should be located in areas away from commercial plantings and its management should follow the recommended procedures for seed production including rouging of infected and atypical plants. If symptoms of a systemic disease (such a virus disease) develops in SOME plants the whole plant progeny should be discarded. If the disease develops in only ONE plant it will be advisable to check the remaining plants in the unit with a sensitive and efficient method. Discard infected plants. At harvest the progeny of all plant units that are disease-free can be bulked (to form a seed lot stock) and properly disinfected and stored until they are ready for planting.
C. SECOND MULTIPLICATION (BULKING) The seed lot should be multiplied as a second generation seed lot following standard procedures for seed production including rouging of infected and atypical plants. At harvest a sample of 250 to 400 tubers should be tested for virus infection in each seed lot. If seed from this second field multiplication has low incidence of diseases and defects (according to tolerances set by the country) it can be used for a further cycle of multiplication (third generation).
RAPIDSEED-AN IMPROVED “OLD”SCHEME FOR SEED POTATO PRODUCTION (Summary of steps and example of seed produced)
Cut tubers according to size >200 g >150 g >100 g <50 g Tuber size Cut tubers dusted with a powder mixture fungicide-calcium hydroxide (return)
Mosaic or Mottle (next) (return) Pale green or chlorotic areas in the leaves. Sometimes these predominate, leaving potions of normal tissue as “islands” or blotches. In mottle borders of pale green areas are better defined than in mosaics. These symptoms are the result of the decreased production of chlorophyll or destruction of chloroplast.
Chlorosis (next) (return) The normal green color lacks uniformity in part or all of the foliage. The color change usually starts at the top of the plant in leaves that develop following infection. Sometimes only restricted to the interveinal tissue. This symptom occurs commonly with PLRV in some cultivars.
Calico (next) (return) Large, bright yellow areas with irregular borders are usually dispersed throughout the foliage.
Aucuba (next) (return) Small , usually round spots or flecks of bright yellow occur irregularly in leaves. They may coalesce, but can still be individualized.
Yellow patterns (next) (return) These include yellow lines forming defined shapes such as rings, incomplete rings or arcs, and sinuous lines. This is a typical symptom caused by PMTV in some regions
Vein yellowing (next) (return) A bright yellow coloration on the veins contrasts greatly with the green color of the leaf lamina. This symptom has been found only with PYVV in South America
Anthocyanescense (next) (return) Because of the abnormal production and accumulation of anthocyanin, leaves may show a range of purple, red, or blue colors. These variations are usually associated with changes in leaf shape. Somewhat similar symptoms can be caused by Rhizoctonia under some conditions.
Little Leaf (next) (return) Leaves are small compared with those of healthy plants. This is usually associated with some color changes on the leaf borders. Easily confused with abnormal nutrients supply
Leafroll (next) (return) This involves severe upward rolling of leaflets having the central vein as an axis. It is usually more severe toward the tip of the leaflet. Texture is also affected in rolled leaves (leathery leaves). Leaflets crack easily when crushed between the fingers. This is caused by accumulation of starch in the leaf cells from impaired translocation of carbohydrates. It is the typical symptom caused by PLRV-secondarily infected plants.
Crinkle This symptom is evident toward the edge of leaves that show a wavy appearance, but the leaf lamina is sometimes also affected. Crinkle is commonly associated with mosaic or mottle. (return) (next)
Paracrinkle (next) (return) This is a type of crinkle. Leaves are bent upwards having the central vein as an axis. It resembles the leafroll symptom.
Leaf deformation (next) (return) Leaves lose their normal shape by elongation, reduction or widening of the leaf lamina. In most cases, this is associated with thickening or another deformation in the main leaflet vein or necrosis. It is usually caused by a virus complex.
Rugosity (next) (return) The leaf surface is uneven, as illustrated (arrows) by the presence of blisters or by different growth rates of veins and leaf lamina. This symptom may occur only in some stems of the infected plant but more commonly throughout the foliage.
Erectness (next) (return) Leaves form acute angles of insertion relative to the stem (in most cultivars less than 60º). Commonly associated to other symptoms such as color or shape deviation in the leaves. In addition to PLRV in some cultivars this symptoms is typical of PSTVd infection
Apical necrosis (next) (return) This is also called acronecrosis. Though necrosis starts at the top of the plant and the branches, it may descend to lower leaves and, sometimes, affects the whole plant.
Systemic necrosis (next) (return) These are necrotic streaks, solid spots, or rings distributed in part or all of the foliage without any definite distribution pattern.
Vein necrosis (next) (return) Systemic necrosis in the veins, observed particularly on the underside of leaves, this may occur in the entire length of the veins, partially, or as necrotic streaks. Leaves usually bend downwards. It is a typical infection of some strains of PVY
Marginal flavescence (next) (return) Entire leaf borders usually pre-necrotic or necrotic. It differs from little leaf because the symptom is found in the entire plant
Dwarfing (next) (return) Plants showing this symptom usually emerge late and stay smaller than healthy plants. Some plants may start developing normally, but their growth stops suddenly. Some deformation of leaves or stems occurs.
Stunting (next) (return) Reduction in plant size is a common symptom of several virus infections. This symptom is usually confused with dwarfing, but stunted plants do not show deformation.
Bunchiness (next) (return) Leaves are small and so severely crinkled that they grow tightly together along the stem. This symptom is sometimes called bouquet or rosette. It is in a way a severe type of crinkle with stunting
Witches’-broom (next) (return) This is a proliferation of axillary branches on the main stems at the mother tuber level. It is associated with chlorosis, leaf reduction, stunting.
Purple top (return) The upper part of the stems turn purple. This symptom is related to anthocyanescense.
Typical bacterial wilt symptom. Somewhat similar symptoms are produced by Erwinia sp. Plants selected should be away (as far as possible) from those showing symptoms. Return to basic presentation
Spindle tuber There is usually a gradual decrease in the diameter of tubers, and it is more severe at the stolon (stem) end. Return to basic presentation Next
Aerial tubers (next) (Return) Tubers are produced in the leaf axillae. They may or may not have stolon-like connections.
Overgrowths (next) (Return) These represent a bulking of tuber eyes (buds) or growth of tuberlets on the main tuber. Tuber deformation is usually associated to this symptom.
Fine cracking (next) (return) Tubers may show superficial or deep cracks. Sometimes this symptom is also called “russeting”
Deep cracks and deformation (Next) (Return) This type of symptom that resembles infrequent and/or inappropriate water management can be caused by some strains of PVY. Even symptomless tubers may carry the virus. It is advisable to check for PVY before collecting the tubers or discard the tuber progeny
Necrotic patterns (next) (return) These are lines, arcs, or rings on the surface or deep in the tuber flesh. Some of these symptoms are commonly called “spraing”.
Necrotic ringspots (next) (return) Lesions are usually circular and dry. Most times are restricted to the surface or few millimeters inside the tuber flesh. Sizes can vary.
Net necrosis (next) (return) These are necrotic spots or blotches, circular or irregular in shape. Usually coalesce to form net patterns or large necrotic blotches
Plants having tubers showing vascular rots or bacteria oozing from the vascular tissue must be discarded (next) (return)