Details on the this workshop on maize lethal necrosis (MLN) disease at: http://bit.ly/1GZ6KoX

1. MLN Artificial Inoculation Protocol
Janet Kimunye, Biswanath Das, Anne Wangai
Training Workshop on MLN Diagnosis and Management
MLN Screening Facility Naivasha -17th March 2015

2. Why artificial inoculation?
• Natural infection is sporadic
• Disease pressure under natural infection is highly variable
• Attain uniform infection across germplasm
• Helps in timing of infection time

4. Collection and Purification of
MLN Viruses
• SCMV and MCMV identified as causal agents of MLN
in Kenya
• Collect symptomatic leaf samples from MLN hotspots
• Conduct diagnostic assay ELISA, or PCR to identify
samples only infected with either SCMV or MCMV
• Grind individual leaves mortar & pestle /blender in cold
(0.1M Potassium phosphate at buffer -pH7(ratio 1:10)
• Add carborandum and inoculate susceptible plants (3-4
leaf stage) in the greenhouse by rubbing sap onto
leaves with fingers.

5. Current Protocol
• When symptoms appear, conduct ELISA to
confirm purity and transfer to new seedlings
• Maintain pure cultures of both viruses on
susceptible variety in the screen house
• Amplify inoculum of each virus for field
inoculation in greenhouses(ready after 6 weeks)

6. Symptoms in artificially inoculated maize plants
in screen house
MCMV SCMV

7. Inoculum amplification

8. Inoculum preparation steps(1ha)
• Require 120L inoculum
• Virus mixing ratios1:4 i.e. MCMV:SCMV
• Dilution ratio 1:20 i.e. Leaf material: Buffer(based on
weight)
Harvest infected leaves from the greenhouse
• Weighing and chopping
– 4.8 kg of MCMV
– 1.2 kg of SCMV
• Blending
• Sieving
• Mixing and carborandum
• inoculation

9. Inoculum preparation
Harvest infected leaves
SCMV and MCMV
separately
Chop and weigh the
leaf tissue
Blend separately in
cold 0.1M potasium
phosphate buffer at
PH 7.0
Sieve to remove
plant debris and
package

10. 10
§ Mix the extracted MCMV and SCMV inoculum
in a large mixing tank, add carborandum rate
1g/Liter
Keep stirring and maintain the inoculum cool

11. Artificial field inoculation
11
Inoculate field trials at 4-6 leaf stage
using a motorized mist blower (Solo
423 MistBlower, 11 liter capacity).
Symptoms should appear after 10
days.
A repeat inoculation is done after1
week.

12. Planting trials for MLN screening
• Hybrids and inbreds planted separately
• Include both hybrid and inbred tolerant and susceptible
checks as borders
• Plant according to collaborator instruction on 3m
rows(single or double)
• Plant two seeds/hill thinned to 1 plant ( Total 13 plants)
• Inoculate seedling (3 weeks old) in the field using
motorized backpack sprayers
• Proper tagging and coding of partner germplasm
• All required agronomic management applied

13. • Disease severity: Scale of 1-5 to assess of the intensity of
the disease symptoms
1 = no MLN symptoms
2 = fine chlorotic streaks on leaves
3 = chlorotic mottling throughout plant
4 = excessive chlorotic mottling , necrosis on leaves and in some cases
dead heart
5 = complete plant necrosis
• Plant stand
• Agronomic traits – Plant height, Ear height, Flowering &
silking dates grain yield (hybrids)
Data recorded

14. Disease rating system
• Rating system is visual
• Starts 2 weeks after second inoculation
• Conducted every 7 days for inbred lines and 14 days for
hybrids
• The disease score is given on row basis
• Collect a minimum of 3 ratings to allow calculation of the
area under disease progress curve

15. Rating of 1
Scale description
• 1 = no MLN symptoms

16. Rating 2
Scale Description.
• 2 = fine chlorotic streaks on
new / emerging leaves

17. Rating 3
Scale Description.
• 3 = severe chlorotic mottling
throughout plant

18. Rating 4
Scale description.
• 4 = excessive chlorotic
mottling and leaf necrosis.
In some cases, have the
dead heart symptoms

19. Rating 5
Scale Description.
• 5 = complete plant necrosis