MLN Artificial Inoculation Protocol

Janet Kimunye, Biswanath Das, Anne Wangai

Training Workshop on MLN Diagnosis and ManagementMLN Screening Facility Naivasha -17th March 2015

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

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.

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)

Symptoms in artificially inoculated maize plants in screen house

MCMV SCMV

Inoculum amplification

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

Inoculum preparation

Harvest infected leaves SCMV and MCMV separately

Chop and weigh the leaf tissue

Blend separately in cold 0.1M potasiumphosphate buffer at PH 7.0

Sieve to remove plant debris and package

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§ Mix the extracted MCMV and SCMV inoculum in a large mixing tank, add carborandum rate 1g/Liter

Keep stirring and maintain the inoculum cool

Artificial field inoculation

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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.

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

• Disease severity: Scale of 1-5 to assess of the intensity of the disease symptoms

1 = no MLN symptoms 2 = fine chlorotic streaks on leaves3 = chlorotic mottling throughout plant4 = 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

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

Rating of 1

Scale description • 1 = no MLN symptoms

Rating 2

Scale Description. • 2 = fine chlorotic streaks on

new / emerging leaves

Rating 3

Scale Description.• 3 = severe chlorotic mottling

throughout plant

Rating 4

Scale description. • 4 = excessive chlorotic

mottling and leaf necrosis. In some cases, have the dead heart symptoms

Rating 5

Scale Description. • 5 = complete plant necrosis