Transcript Aissata Thera`s PowerPoint Presentation Master`s Thesis Research

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)
4/13/2015
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Mali
Introduction
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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
4/13/2015
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An overview of the
Bacterial Wilt problem
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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
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Generality on Ralstonia
solanacearum
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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
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Disease Symptoms
Vascular discoloration
Diagnostic
bacterial streaming
in water
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Wilting often without
leaf death
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Ralstonia solanacearum is
a species complex
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Traditionally divided into 5 races based
on host range and six biovars based on
biochemical properties
2 geographic divisions
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Division 1- biovars 3,4,5 primarily from
Asia
Division 2. biovars 1,2,N2-primarily from
Americas
Taxonomy is in transition
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Race and Biovar
classification
Table 1. Races and Biovars of Ralstonia solanacearum
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Table 2: Proposed New Classification
Taxonomic level
Taxonomic
Equivalent
Nomenclature
Species
Species
Ralstonia
solanacearum
complex
Phylotype
subspecies
Sequevar
Intraspecific
groups
Phylotypes
I, II, III, IV
Sequevars 1-23
Clone
Clonal lines
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Ralstonia solanacearum
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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 freezingonce imported to Montana on geranium
cuttings from Kenya
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Host range: very broad depending on
Race/Biovar
Important aquatic weed hosts (Solanum
dulcamara -bittersweet nightshade)
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Potato: importance and potential
of the crop in Mali and in West
Africa .
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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
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competitive
In West Africa
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CFA-Free Trade Area
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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.
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The obstacles to improved Malian potato
production and profitability are:
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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
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Current Seed Potatoes Situation
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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.
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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
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Lack of efficient pest
management
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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.
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The disease have been
reported in the main
production areas of
potato and tomato in
Sikasso, Koulikoro, Kati,
and Baguinda
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The current Ralstonia
situation on potato in Mali
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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
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Seed production in
Mali?
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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.
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What was missed in this first effort to establish
effective production of seed potato?
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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.
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How to improve?
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Improving the technical capability to
detect potato diseases.
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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.
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No reported presence of
bacterial wilt is reported
in the dry sahelian
northern part of Mali
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Impact of the bacterial wilt on seed
production in Mali
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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
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heavily infested with Rs
Managing the Ralstonia solanacearum
disease for a best production of certified
seed potato in Mali.
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Race identification
Pathogen distribution
Pathogen survival and spread
Identification of resistant or tolerant
varieties
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1.- Race identification
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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:
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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
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1.- Race identification
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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.
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Result
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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
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Number of dead plants 40 days after inoculation
Sikasso Isolates
Frozen isolates
Non frozen isolates
Tomato
Tobacco
Peanut
Tomato
Tobacco
Peanut
Potato from
Sikasso
5/5
5/5
0/5
5/5
5/5
2/5
Potato :Clausta
from Farako
5/5
5/5
0/5
5/5
5/5
2/5
Tomato from
Sikasso1
5/5
5/5
1/5
5/5
5/5
2/5
Potato :
Mondial from
Sikasso1
5/5
5/5
2/5
5/5
5/5
1/5
Potato:
Mondial from
Sikasso2
5/5
5/5
1/5
5/5
5/5
2/5
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Potato:
Mondial from
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5/5
5/5
0/5
5/5
5/5
4/5
Number of dead plants 40 days after inoculation
Koulikoro
Isolates
Frozen isolates
Non frozen isolates
Tomato
Tobacco
Peanut
Tomato
Tobacco
Peanut
Potato from
Sotuba1
5/5
5/5
2/5
5/5
5/5
2/5
Potato from
Sonityeni
5/5
5/5
3/5
5/5
5/5
3/5
Potato from
Sotuba 2
5/5
5/5
1/5
5/5
5/5
3/5
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Interpretation
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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?
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2.- Disease distribution
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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.
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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
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Interpretation
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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.
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3.- Pathogen survival
and spread
Hand dug wells
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Irrigation canals
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.
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Bacteria streaming from a cut infected
stem test is always done first before any
over test.
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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
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Immunostrip Results
Sample
Resul
t
Sikasso I
Tomato
+
Locality
Sample
Result
Bguineda
Tomato
+
Sikasso
II
Tomato
+
Sonityeni
( Kati )
Claustar
(P.T)
+
Farako
Claustar
(P.T)
+
Farako
Appoline
(P.T)
+
Sikasso 1
Modial
(P.T)
+
Sikasso 2
Modial
(P.T)
+
Sikasso 3
Modial
(P.T)
+
Sonityeni
Sotuba
Kogniba
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Locality
Tabacco
Potato
Tomato
+
+
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AgDia Immunostrip Tests
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Table 7: Elisa test for water and soil samples of
different regions in Mali
Type
of sample
and origin
Soil/Sotuba
Water from
Canal in
Baguineda
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Result of
Elisa
+
+
Type
of sample
and origin
Result
of Elisa
Soil in
Samogossoni
-
Water from
wells in
Samogossoni
+
Soil/Farako
Water from
creek in
Farako
+
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4.- Identification of resistant or
tolerant Varieties
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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.
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Table 8: Results of potato varietal test in
Sikasso.
Varieties
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Percentage
established
Plants
Percentage
infected
with Rs*1
Spunta
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0.8
Daifla
35
0.8
Claustar
38
0.8
Liseta
31
0.0
Appoline
37
0.5
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*1Mondial
Determinate by visual analysis
2.0
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Interpretation
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Seed dormancy and establishment
were probably the most limiting
factors.
Level of bacterial wilt was quite
low.
Lowest infection with varieties
Liseta and Appoline.
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Table 9: Results of potato varietal test in
Sotuba.
Varieties
Percentage
established
plants
Percentage
plants
infected
with Rs*1
Spunta
10
1.4
Appoline
32
0
Liseta
31
10.0
Daifla
22
4.0
Claustar
24
3.0
Mondial
31
3.0
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*1 Determined by visual and bacterial streaming test
Interpretation
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Appoline and Spunta lowest
infection, while Liseta was the
highest.
Seed dormancy problem on all
varieties.
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Table 10: Results of potato varietal test in
Soniteyi.
Percentage
established
Percentage
dead plants
from Rs
Claustar
37
100
Spunta
77
60
Mondial
67
83
Appoline
80
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Liseta
72
90
Daifla
87
78
Varieties
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*1 Determined by visual and bacterial streaming test
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Interpretation
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Appoline had the lowest infection
with Rs in all 3 field tests
Soniteyi had a better plant
establishment than Sikasso and
Sotuba
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Impact of these results
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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
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Required Actions needed
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Img_0032.jpg
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
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Study genomics of Mali races / biovars
Conclusion
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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.
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Conclusion
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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:
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Pathogen free seed
Use crop rotations
Use sanitation with shallow wells.
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Next Steps
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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
Img_0034.jpg
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.
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Thank You to:
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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
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Montana Potato Lab group
Thank you for your attention
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