Ralstonia solanacearum management: A prerequisite for a potato seed certification program in Mali By Aissata T. Thera Plant Pathologist : Fruits and Vegetables Diseases- IER (Rural Institute of the Economy) 4/11/2017

Mali Introduction Problem: How can we establish a certified seed potato production program in Mali where Bacterial Wilt (Ralstonia solanacearum) is an important disease on Solanaceous plants? Objective: Develop practical approaches for control of Bacterial Wilt for potato growers and develop a Certified Pathogen-Free Seed Potato Program in Mali-cooperative project IER /IPR and MSU 4/11/2017

An overview of the Bacterial Wilt problem The causal agent: Ralstonia solanacearum Potato: importance and potential of the crop in Mali and in West Africa. The current Ralstonia situation on potato in Mali Impact of the disease on a tentative seed production in Mali 4/11/2017

Generality on Ralstonia solanacearum Strict aerobic,Gram negative, non spore forming, non capsulated and rod shaped bacterium affecting a wide range of crops plant world wide. In Wet tropics, sub tropics and some warm temperate regions and recently in cool temperate regions of northern and southern hemisphere. Important plant hosts: potato, tomato, geranium, banana, tobacco, peanut, ginger, pepper. More severe where Root Knot (Meloidogyne) nematodes occur 4/11/2017

Disease Symptoms Vascular discoloration Diagnostic bacterial streaming in water Wilting often without leaf death 4/11/2017

Ralstonia solanacearum is a species complex Traditionally divided into 5 races based on host range and six biovars based on biochemical properties 2 geographic divisions Division 1- biovars 3,4,5 primarily from Asia Division 2. biovars 1,2,N2-primarily from Americas Taxonomy is in transition 4/11/2017

Race and Biovar classification Table 1. Races and Biovars of Ralstonia solanacearum 4/11/2017

Table 2: Proposed New Classification Taxonomic level Taxonomic Equivalent Nomenclature Species Ralstonia solanacearum complex Phylotype subspecies Phylotypes I, II, III, IV Sequevar Intraspecific groups Sequevars 1-23 Clone Clonal lines 4/11/2017

Ralstonia solanacearum Soil inhabitant- up to 7 years, but can use dry fallow and 4 year rotations with non-host crops (cowpea, soybean, corn) Race 1 will not survive freezing Race 3 biovar 2-will survive freezing-once imported to Montana on geranium cuttings from Kenya Host range: very broad depending on Race/Biovar Important aquatic weed hosts (Solanum dulcamara -bittersweet nightshade) 4/11/2017

Potato: importance and potential of the crop in Mali and in West Africa . Potato was introduced to Mali in 1938 Crop have been well integrated in Mali’s food culture and field production system Mali is the biggest producer in the region with 60 000-70 000 mt/year and average 20-24 mt/ha of yield Market is growing through both domestic consumption and exportation to Ivory Coast, Ghana, Burkina Faso, Togo and Benin (CFA free-trade) following the devaluation of the CFA. Land and water resource are available to increase potato production Increased export to CFA area and other countries if production is improved and competitive 4/11/2017

In West Africa Countries such as Ivory Coast are not able to produce potatoes because of wet conditions. Others countries such as Burkina Faso and Ghana need more potato than they can produce. Prior to CFA Free Trade it was most common to import potato from Europe, but now those countries are opening their market to The Malian potato which is fresh and less expensive. Export to EU and other African countries has been difficult. CFA-Free Trade Area 4/11/2017

The obstacles to improved Malian potato production and profitability are: Farmers to access to credit Access to disease-free, non dormant seed potatoes of adapted varieties. Lack of efficient pest management. Lack of storage for seed and commercial potatoes 4/11/2017

Current Seed Potatoes Situation High price ( $40-$60/25 kg-$72-94/100lbs), farmers can’t buy the amount they really need so they cut one tuber into 10 or more seed pieces each with one eye. Excessive cutting results in lower yield and increased pathogen transmission 1500 metric ton of seed potato is imported from EU every year Seed cost is 50% of production cost 96% of seed is imported from EU without any competition Seed often arrives from Europe to late for optimal planting and limits crop to one/year No stability in price, amount and varieties imported Europeans imported varieties are long day not well adapted to our tropical conditions –need day neutral cultivars Seed often arrives for planting in dormant condition 4/11/2017

Lack of efficient pest management Diseases like black leg, Fusarium tuber rot in storage, viruses and early blight are present but have not been a big concern since farmers are able to get good yields. Viruses very uncommon Bacterial wilt is currently a big problem . Farmers have ceased to grow potato in a lot of villages in Sikasso, the main production area of potato. Farmers are growing potato in the same place year after year. Potato is rotated with alternative hosts to R. solanacearum and most growers do not use the sanitary measures to avoid disease spread. Growers do not have enough information about how to control the diseases of potato. The country does not have a good program of quarantine and certification. 4/11/2017

The disease have been reported in the main production areas of potato and tomato in Sikasso, Koulikoro, Kati, and Baguinda 4/11/2017

The current Ralstonia situation on potato in Mali The disease have been reported to be the most important disease on potato and other Solanceous crop (tobacco, peanut, tomato, pepper, eggplant) production by farmers. Most of the Europeans varieties are susceptible to the disease Lost of 100% of the potato production during successive years in some villages of Sikasso made farmers stop growing potato; the same thing happens on tomato at Baguinda (tropical humid area) Tobacco, tomato, eggplant, peanut and pepper are produced in rotation with potato. Potatoes are grown under irrigation-surface water or hand dug wells 4/11/2017

Seed production in Mali? Mali has a laboratory with a fully developed capability for in vitro multiplication of potato (Biotechnology laboratory in IPR/IFRA in Koulikoro). Soc International NGO in collaboration with IER (varietals Test) and IPR (tuber multiplication) introduced the seed production in Sikasso region. In spite of difficult cultural conditions in Mali, the average yield varies from 6 to 12 t/ha for a microtuber plantation and from 8 to 14 t/ha for minitubers. The profitability of the local seed production scheme seems interesting. The cost price of the second generation is already below the price of imported seed potato. I0 years of presence of farmer’s pilot unit, able to multiply prebase material Poor quality of seed Yield 10-12 t/ha compared to 20-24t/ha for normal production. 4/11/2017

What was missed in this first effort to establish effective production of seed potato? The capability to ensure quality control and project management throughout the production chain. There is not enough knowledge on how to scale up production. No national certification and standards were established relative to varietal purity or freedom from pathogens. Rapid diagnostics tests for virus and bacteria were not as widely available or adaptable to Malian conditions. Identification of seed production areas that are separate from table stock production and free of bacterial wilt. 4/11/2017

How to improve? Improving the technical capability to detect potato diseases. To develop the capacity to do immunological and/or DNA based assays for a relevant range of diseases. Production of nuclear minitubers. Identify the diseases, their epidemiology and the infested areas. Identification of areas free of soilborne pathogens for nuclear and G-1 production. The setting up of field and bin inspections (quality control process) for field production of certified seed. Storage facilities that will maintain high quality. 4/11/2017

No reported presence of bacterial wilt is reported in the dry sahelian northern part of Mali 4/11/2017

Impact of the bacterial wilt on seed production in Mali Seed produced in infested fields will spread the disease Seed quality is reduced With low quality seed there is no possibility to compete with EU imported seed in West Africa Potato production will decrease, farmers poverty levels increase Seeds production areas have to be selected through efficient techniques of rapid detection of pathogens in soil and water The first tentative of seed production with micro and minituber from the IPR lab did not work at Sikasso-soil and water heavily infested with Rs 4/11/2017

Pathogen distribution Pathogen survival and spread Managing the Ralstonia solanacearum disease for a best production of certified seed potato in Mali. Race identification Pathogen distribution Pathogen survival and spread Identification of resistant or tolerant varieties 4/11/2017

1.- Race identification Identifying the race will help to set up a plan for rotation by knowing the specific host range of the race. 10 isolates collected from throughout main production areas in Mali Method: hypersensitivity test on tobacco: 10 plants of tobacco were inoculated by tiny syringe in the mesophyle of the leaf. Race1 brown necrosis after 60h; Race 2 white necrosis in interveinal area after 60h and Race 3 chlorosis after 2-8 days 4/11/2017

1.- Race identification Inoculation of tobacco with frozen and non frozen bacteria to evaluate the bacteria pathogenicity after freezing Race 3 biovar 2, and race 3 can survive freezing. Race 1 should not survive the freezing. 4/11/2017

Result After 3 days tobacco hypersensitivity tests were negative-Race 1-brown necrosis on leaves. After 2 weeks 3 plants began to show wilt symptom-Race 1 A month after those 3 plants were died The wilt of tobacco plant allows us to confirm that the race in Mali is the race 1, which is the only one able to wilt tobacco 4/11/2017

Number of dead plants 40 days after inoculation Sikasso Isolates Number of dead plants 40 days after inoculation Frozen isolates Non frozen isolates Tomato Tobacco Peanut Potato from Sikasso 5/5 0/5 2/5 Potato :Clausta from Farako Tomato from Sikasso1 1/5 Potato : Mondial from Sikasso1 Potato: Mondial from Sikasso2 Potato: Mondial from Sikasso3 4/5 4/11/2017

Number of dead plants 40 days after inoculation Koulikoro Isolates Number of dead plants 40 days after inoculation Frozen isolates Non frozen isolates Tomato Tobacco Peanut Potato from Sotuba1 5/5 2/5 Sonityeni 3/5 Sotuba 2 1/5 4/11/2017

Interpretation Infection from frozen bacteria does occur on the 3 crops, only peanut did show a increased infection from the unfrozen bacteria Infection of tobacco and peanut indicates Race 1 of Rs. But why infection with frozen bacteria? Not frozen in soil-used agar cultures Not frozen long enough? 4/11/2017

2.- Disease distribution It is important to know where the disease is really distributed to set up a quarantine and seed production program. Method: soils from main potato and tomato production areas were brought to Sotuba, then planted with tobacco. 4/11/2017

Places Number of plants dead from Rs % dead plants Baguineda 6/54 11 Kati 40/60 67 Koulikoro 44/60 76 Sikasso 58/72 98 4/11/2017

Interpretation The soils from Sikasso and Koulikoro are more infested than Kati and Baguinda. The number of dead plant could be related to the pathogen concentration in soil or pathogenicity of resident Rs. 4/11/2017

3.- Pathogen survival and spread Hand dug wells Where is Rs while potato is not growing Method: sampling of potato, tomato, weeds, soil, and irrigation water (from wells, creek, canal), in the main infested areas of potato production in Mali. Bacteria streaming from a cut infected stem test, Immunostrip test, and ELISA test were used. 4/11/2017 Irrigation canals

Bacteria streaming from a cut infected stem test is always done first before any over test. This test is done automatically to any sample with bacterial wilt All the samples tested positives in immunostrip test were first positives to bacteria streaming test Only the tobacco from Sonityeni was negative to bacteria streaming test, and also to immunostrip test 4/11/2017

Immunostrip Results Locality Sample Result Sikasso I Tomato + Sikasso II Farako Claustar (P.T) Appoline Sikasso 1 Modial Sikasso 2 Sikasso 3 Locality Sample Result Bguineda Tomato + Sonityeni ( Kati ) Claustar (P.T) Tabacco - Sotuba Potato Kogniba 4/11/2017

AgDia Immunostrip Tests 4/11/2017

4/11/2017

Table 7: Elisa test for water and soil samples of different regions in Mali Type of sample and origin Result of Elisa Soil/Sotuba + Water from Canal in Baguineda Type of sample and origin Result of Elisa Soil in Samogossoni - Water from wells in + Soil/Farako creek in Farako 4/11/2017

4.- Identification of resistant or tolerant Varieties 6 varieties of potato were compared in station at Sikasso and Sotuba and in farmer fields at Sonityeni for their resistance to Rs. 40 plants of each variety. Data: Visual incidence, Number of plants established, and bacteria streaming from a cut infected stem test were used. 4/11/2017

Table 8: Results of potato varietal test in Sikasso. Varieties Percentage established Plants infected with Rs*1 Spunta 28 0.8 Daifla 35 Claustar 38 Liseta 31 0.0 Appoline 37 0.5 Mondial 25 2.0 4/11/2017 *1 Determinate by visual analysis

Interpretation Seed dormancy and establishment were probably the most limiting factors. Level of bacterial wilt was quite low. Lowest infection with varieties Liseta and Appoline. 4/11/2017

Table 9: Results of potato varietal test in Sotuba. Varieties Percentage established plants infected with Rs*1 Spunta 10 1.4 Appoline 32 Liseta 31 10.0 Daifla 22 4.0 Claustar 24 3.0 Mondial 4/11/2017 *1 Determined by visual and bacterial streaming test

Interpretation Appoline and Spunta lowest infection, while Liseta was the highest. Seed dormancy problem on all varieties. 4/11/2017

Table 10: Results of potato varietal test in Soniteyi. Varieties Percentage established dead plants from Rs Claustar 37 100 Spunta 77 60 Mondial 67 83 Appoline 80 46 Liseta 72 90 Daifla 87 78 4/11/2017 *1 Determined by visual and bacterial streaming test

Interpretation Appoline had the lowest infection with Rs in all 3 field tests Soniteyi had a better plant establishment than Sikasso and Sotuba 4/11/2017

Impact of these results n RS Race 1 seems to be predominant race We know we can not grow seed in food potato production areas which are mostly tropical humid due to soil and water contamination with Rs. Mopti region has potential as foundation seed production region (Free of Rs, few Rs host crops, deep water wells). Surface water and shallow water wells are contaminated with Rs-weed hosts? Appoline show some resistance in Sikasso, Sotuba and Sonyteni We are studying resistance of 11 tropical day neutral varieties from CIP. Research on rotation and soil treatments underway Rotations: Cowpea, soybean, rice and corn Neem-Root Knot nematode control; compost 4/11/2017

Required Actions needed Required Actions Needed in Mali   Know more pathogen biology / epidemiology Introduce tropical adapted varieties from CIP resistant to bacterium Exactly identify seed potato production areas Establish seed certification standards / test protocols Improve tissue culture/ minituber nuclear production capacity / quality control Improve Mali quarantines Define sanitary norms for each steps of certified seed production Train seed potato / table potato producers in certification process Establish legislation supporting certification Study genomics of Mali races / biovars 4/11/2017

Conclusion The Rs race 1 is present in all the most important areas of potato and tomato production. Considering that race 1 is present will be necessary to establish a rotation program without hosts of this race. Disease is transmitted through soil, equipment, farmer's movement and irrigation water. It is necessary to develop source of disease free seed without dormancy problems. Mali has infrastructures, trained personal, and pathogen free area for developing a certified potato seed production program. 4/11/2017

Conclusion Certified seed potato can not be produced in Sikasso, Baguineda, Koulikoro and Kati Some varieties have been found that are less susceptible (Appoline) Growers can establish a good integrated management programs using: Pathogen free seed Use crop rotations Use sanitation with shallow wells. 4/11/2017

Next Steps Identify the pathogen races/ biovar (s) in Mali Design primers for efficient identification of the pathogen races / biovars Assessment of hypersensitivity test on tobacco and pathogenicity test on tobacco, pepper, tomato, peanut and eggplant using various inoculation techniques at different stages at MSU Varietal test of CIP varieties in Mali-MSU for productivity and resistance to Rs Seed multiplication in field –Mopti area To distribute pathogen free seed to research stations for farmer field trials-performance of European and CIP varieties. 4/11/2017

Thank You to: USAID-Washington who provided my training funding through HED in the grant Linking Biotechnology / Bioengineering with Mali-based Agribusiness-Florence Dunkel grant coordinator IER, MSU, IPR CIP PEO Barry Jacobsen, Oliver Neher, Eva Grimme, Nina Zidack, Ernesto Moya, John Ansley Montana Potato Lab group 4/11/2017

Thank you for your attention 4/11/2017