Sampling protocol for mln and introduction to immunostrips for mln pathogen detection suresh l m adama oct 2019
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This document provides information about Maize Lethal Necrosis Disease (MLN), including its symptoms, causative viruses, geographic distribution, screening and management. MLN is caused by a combined infection of Maize Chlorotic Mottle Virus and maize-infecting potyviruses. It was first reported in Africa in 2011 and has since spread across several countries. Symptoms include yellowing leaves, stunted growth, and sterile cobs. Two strains of the Maize Chlorotic Mottle Virus have been identified. The document outlines protocols for sampling and testing maize plants using immunostrips to detect MLN pathogens, as well as establishing quarantine facilities to screen and distribute disease-
![Cover the leaf tissue and place it in perforated
paper bag:
Fold the tissue paper so that it covers the leaf sample. Place
the single collected leaf sample into a paper bag perforated
with air holes (Fig 3) [NB: Only 1 leaf sample should be
placed into each leaf sample bag]. Caution must be used not
to touch the interior of the sample bag with fingers,
implements or any other leaves.
a b 3. Placing the samples
Between the layers of
Tissue paper (a) and
Inserting the samples
In the perforated paper
bag (b).](https://image.slidesharecdn.com/samplingprotocolformlnandintroductiontoimmunostripsformlnpathogendetectionsureshlmadamaoct2019-191113164519/85/Sampling-protocol-for-mln-and-introduction-to-immunostrips-for-mln-pathogen-detection-suresh-l-m-adama-oct-2019-32-320.jpg)
![ Sending Samples
1. If MLN symptomatic plants (or suspected MLN symptomatic plants)
are observed on the survey (i.e., any bulk sample that test positive
with the immunostrip test), the samples should be sent to a
recommended laboratory for ELISA testing for confirmation as soon
as possible (within 48 hours).
2. If possible, mail the positive / suspected samples to a recommended
laboratory in the country [NB: NO samples should be mailed to
another country]. Place the bulk sample bag(s) inside a small
cardboard box. Please mention on the box “Plant sample“ “RUSH
IMMEDIATELY” and mail using either a courier service or express mail
to the laboratory.
3. If the samples cannot be mailed immediately, keep them in a
refrigerated condition or in a cool dark place. Samples must be
refrigerated at 4oC and kept for no longer than 48 hours. After that
time, leaf samples can deteriorate and the results will not be reliable.](https://image.slidesharecdn.com/samplingprotocolformlnandintroductiontoimmunostripsformlnpathogendetectionsureshlmadamaoct2019-191113164519/85/Sampling-protocol-for-mln-and-introduction-to-immunostrips-for-mln-pathogen-detection-suresh-l-m-adama-oct-2019-43-320.jpg)
Sampling protocol for mln and introduction to immunostrips for mln pathogen detection suresh l m adama oct 2019
- 1. MLN disease symptoms Sampling protocol for MLN and Introduction to Immunostrips for MLN pathogen detection Dr. SURESH, L.M. Maize Pathology Lead – Sub Saharan Africa Global Maize Program (GMP) CIMMYT, Nairobi, Kenya Training on MLN rapid diagnosis Kits and MLN management, Dates 22nd – 23rd October Adama, Ethiopia
- 2. MLN is a viral disease caused by combined infection of maize with Maize Chlorotic Mottle Virus (MCMV) and any of the Potyviruses infecting cereals, especially Sugarcane Mosaic Virus (SCMV) The disease was first reported in Africa, particularly in Kenya in Sept 2011, and since then reported in Uganda, Tanzania, Rwanda, D.R. Congo, and Ethiopia.
- 3. Disease Symptoms • Dying leaves, leading to premature plant death • Failure to tassel and sterility in male plants • Malformed or no ears • Rotting cob
- 4. Series of reports on MLN in the last 4-5 years… 2016
- 5. Global Occurrence and distribution of MLN/MCMV Pic Source : Suresh,L.M.
- 6. MCMV – diversity studies - SSA Source : Luke Braidwood et.al.,, Scientific reports | (2018) 8:1173 | DOI:10.1038/s41598-018-19607-4
- 7. MCMV • MCMV first reported to infect maize in Peru (Herbert and Castillo, 1973) – yield loss of 10-15% in some cultivars (Nault et al, 1978) • Subsequently reported in Argentina, Brazil, Peru, Mexico, USA, Thailand and China (Nyvall, 1999) • Major problem for temperate seed production in Hawaii in the 1990s (Nelson et al, 2011) • From Kenya (Wangai et al., 2012) Two geographically and genetically different strains of MCMV have been reported (Nyvall,1999): • MCMV (K) – Kansas • MCMV (P) – Peru • There may be others…
- 8. Surveyed Areas in the Region-2017, 2018, 2019
- 9. MLN status in Ethiopia as on date
- 10. Disease Symptoms
- 11. Symptoms of the disease • Symptoms observed vary widely depending on; -Germplasm -Time of infection -Prevailing environmental conditions -Ratios of the viruses infecting the plant • The symptoms can easily be confused with drought , micro- nutrient deficiency or stalk borer infestation
- 12. SCMV and MCMV symptoms • Chlorotic spots/mosaics on lower leaves developing into streaks along leaf veins. • As plants approach maturity, leaves become yellow • Slight stunting (higher incidence of ear and stalk rot if early disease infection) • Seed set is affected.
- 13. MCMV symptoms Mottling Chlorotic stripes Inter-veinal necrosis / severe chlorosis Coalition of chlorotic spots forms chlorotic stripes Mild chlorotic stripes Mosaic symptoms with chlorosis Shorter intermodal distance
- 14. MLN Symptoms Mild mosaic and mottling Mild mosaic and mottling Chlorosis and Mottling Diffuse mottling and chlorosis
- 15. MLN Symptoms • Mottling symptoms on leaves, usually starting from base of young leaves in the whorl and extending outwards • Stunting and shortened internodes • Dead heart and necrosis • Sterility, poor seed set, shrivelled seeds Tassel Sterility/Blast Dead Heart
- 16. Shortened Internodes Early leaf necrosis Poor seed set and shrivelled ears
- 17. Why is the MLN devastating in EA? • MCMV is new to the region • Potentially new strains of SCMV/MDMV • Conducive environment – continuous maize cropping in certain areas leading to continuous build-up of virus inoculum • Seed contamination by MLN-causing viruses, especially MCMV, besides local spread through insect vectors • Widespread cultivation of susceptible germplasm that has never been screened for MCMV • A very large proportion of commercial maize varieties in eastern Africa as well as other regions in sub-Saharan Africa are highly vulnerable to MLN.
- 18. Symptoms of the disease • Symptoms observed vary widely depending on; -Germplasm -Time of infection -Prevailing environmental conditions -Ratios of the viruses infecting the plant • The symptoms can easily be confused with drought , micro- nutrient deficiency or stalk borer infestation
- 19. Tackling the MLN challenge
- 20. MLN screening facility – Naivasha
- 21. Key Phytosanitation steps in MLN Screening facility Environment and water Vehicle Equipment and Tools Plant material People Objective : Deliver and implement phytosanitation guidelines that are specific to the Naivasha Maize MLN Research Site to ensure the desired level of insect and pathogen control that is essential for successful research. • Training • Avoid movements with maize field • Restricted movements. • No food and drink, tobacco • Use PPE’s • Only receive maize seeds • Stop moving out all plant and plant materials from site. • After screening - incinerate • Wash and Disinfect the tools and implements. • Restricted use. • Use them only in site. • Restricted Access • Wash and disinfect • Safe and clean water use • Drain the used water in soak pit • Use safe insecticides and chemicals
- 22. MLN Phenotyping facility – Center of Excellence Inoculation Protocol Facilities Process Phytosanitation
- 23. MLN Quarantine Facility at Mazowe, Harare Established with support from USAID, DR&SS- Zimbabwe, and CRP MAIZE. Enables CIMMYT maize germplasm flow from eastern Africa to southern Africa after evaluation under quarantine conditions
- 24. MLN Quarantine and Screening facilities in SSA MLN Quarantine facility Harare, Zimbabwe • Surveillance and diagnosis for MLN • Seed production, selfing and Crossing. • Destroy the maize crop in the farm and 50 KM vicinity, if found MCMV positive. • Distribute Maize germplasm, after confirming negative to MCMV MLN Screening and Quarantine facility Naivasha, Kenya • Germplasm Screening for MLN, MCMV & SCMV • No seed production, selfing and Crossing. • No return of seeds after screening, only provide the data.
- 25. Safe seed movement with effective quarantine measures Kiboko,Kenya Scouting - Scout for MLN suspect samples - Diagnosis - Destroy plants with MLN +ve - Arrange seed for diagnosis SHL-Nairobi Diagnosis •- Seed Diagnosis •- Destroy +ve sample •- Comply check list •- Arrange –ve sample to KEPHIS KEPHIS,Kenya Certification - Field inspection - Seed Diagnosis - Destroy +ve sample - Arrange Phyto certificate - Seed Shipment from CIMMYT QF-Harare,Zimbabwe Surveillance - Seed Diagnosis - Grow out in quarantine facility - Scouting - Diagnosis - Destroy maize crop if any suspect or positive - Multiply and distribute seed Year Field inspection Seed testing Seed Shipment entries from Kenya lots Samples tested for causing MLN tested Lines planted and released from facility Incidence of MLN in pre- released seed 2016 854 906 3249 Established Quarantine facility in Mazoe, Harare, 2016 2017 503 1481 1338 >35000 >6800 0 2018 649 995 8415 Current MLN Status As on Dec 2018 Progress made in Kenya Progress made in Zimbawe Tanya
- 26. Overview Field sampling Sample processing Sample testing Documentation Sample shipping for ELISA testing.
- 27. How to collect and ship Fresh samples for the VIRUS diagnosis
- 28. •The sampling procedure must ensure that all parts of the field are adequately and proportionately represented in the plants inspected with in the various usual microclimates of the field. •The pattern of field inspection can vary as far as the above condition is met (CDFA Phytosanitary Certification Manual, 1985) FIELD INSPECTION and LEAF SAMPLING:
- 29. (CDFA Phytosanitary Certification Manual, 1985)
- 30. Procedure for leaf sampling Precautions : • Choose the right plant and leaf. • Avoid the plants with abiotic symptoms such as (mechanical ) wilt, nutrition deficiency, pesticide injury, due to drought. • Try to avoid samples with multiple symptoms such as leaf blight, yellowing along with mosaic. • If you are in doubt, please look for fresh plant sample. • It is important that the samples should be collected before pesticide application.
- 31. Selection of plant and collection of leaf tissue • The sample should be taken from the youngest leaf of the plant. To carry out the sampling procedure, the diagnostician should wear laboratory gloves. • Leaf samples should be collected as follows: – Select approximately a leaf of newly developed symptomatic tissue. Using a clean sheet of fresh tissue paper, enclose and hold the leaf to be sampled. Use bleach-cleaned scissors to cut off a 5-6 cm leaf segment (Fig 2). Carefully avoid cross-contamination of samples with exudate from leaves onto hands or implements. Scissors or any other implements must be cleaned thoroughly with bleach solution between each sample ( between farm). a b 2. Samples with no symptoms (a) and symptoms (b)
- 32. Cover the leaf tissue and place it in perforated paper bag: Fold the tissue paper so that it covers the leaf sample. Place the single collected leaf sample into a paper bag perforated with air holes (Fig 3) [NB: Only 1 leaf sample should be placed into each leaf sample bag]. Caution must be used not to touch the interior of the sample bag with fingers, implements or any other leaves. a b 3. Placing the samples Between the layers of Tissue paper (a) and Inserting the samples In the perforated paper bag (b).
- 33. Labelling and packing • Stick completed sample labels and a unique QR code on each sample bag and record the unique sample code. Place each labelled sample bag inside a zip lock plastic bag, this will protect the label from damage). Fig 4. A. Individual leaf sample bag lableled with QR code and sample label. B. Labeled leaf sample bag inside ziplock bag a b
- 34. Sample bulking • Place 6 individually labelled leaf sample bags from the same plot inside one large plastic bag (Bulk sample bag). This will keep the 6 leaf samples from the plot to be used for one bulk immunostrip assay together (Fig. 5). It is essential to stick another unique QR label onto this bulk sample bag and record the unique bulk sample code. Always use separate bags for each set of 6 leaf samples i.e., if 12 leaf samples in total are collected from a plot there should be 2 uniquely labeled bulk sample bags for the plot. Fig. 5. Labelled bulk sample bag containing 6 individual labelled leaf sample bags
- 35. Storing the samples in the container Place the labeled bulk sample bags inside a cool box, and ensure that ice packets are placed around the samples to keep them cool during sample processing and subsequent transportation. Fig. 6. Keeping the samples inside the ice box using ice pack Keep samples in a cool place until shipping. (Do not place in the refrigerator). When ready to ship, place the double bagged box containing in a styrofoam container with a coolpack. Place the styrofoam container in a cardboard box and ship as one unit. Label the outside of the box (Styrofoam) with sample ID, date sample was collected, grower and field where sample was collected. ( place a sample log details inside the box) Fill shipping container with packing material to avoid movement of the sample in the container
- 36. Y Both tests are based on the unique nature of the way in which the antigen (example: a plant virus) and an antibody molecule fit together Y Immunostrips and ELISA E FIELD AND LABORATORY DETECTION of MCMV: FIELD AND LABORATORY DETECTION of MCMV:
- 37. • The technology is based on a series of capillary beds, such as pieces of porous paper, that has the capacity to transport fluid (e.g., plant sap) spontaneously. • The porous paper acts as a sponge, once soaked, the plant sap migrates to the second section of the porous paper in which conjugate is stored. • The analyte (plant sap containing the virus) binds to the particles while migrating further through the third capillary bed. • This material has one or more areas where a third molecule has been immobilized by the manufacturer. By the time the sample-conjugate mix reaches these strips, analyte has been bound on the particle and the third 'capture' molecule binds the complex. • After a while, when more and more fluid has passed the stripes, particles accumulate and the stripe-area changes color. Typically there are at least two stripes: • one (the control) that captures any particle and thereby shows that reaction conditions and technology worked fine, • the second contains a specific capture molecule and only captures those particles onto which an analyte molecule has been immobilized. Fast detection with immunostrips: Principle:
- 38. The benefits of immunochromatographic, also called lateral flow tests or simply strip tests, include: 1. User-friendly format. 2. Very short time to get test result. 3. Long-term stability over a wide range of climates. 4. Relatively inexpensive to make.
- 39. Immunostrip test steps • Selection of test place • Keep at most hygiene as possible • Carefully handle the leaf sample using glouce • Keep all the items sanitized • Keep all the material check list ready • Ensure the test documentation is done before you leave the place • If needed or in doubt please contact the nearest contact or send mail to : l.m.suresh@cigar.org or whats up +254392664 • Avoid cross contamination between the bulk samples • Avoid taking food, using phone, smoking etc.. • After the test is done, leave the sample in the same farm
- 40. 1. For the correct execution of the test the ratio between leaf tissue and extraction buffer must be as close as possible to 1:40 w/v (or the one indicated in the instructions of the kit) 2. To maintain this proportion the total amount of tissue to be processed in the test must not exceed the size of a coin 6. Wearing gloves, open the first collection bag and detach a small portion of the tissue, introduce it in the plastic extraction bag 7. Close the collection bag where the samples was preserved and store the bag again in the cool box 8. Change gloves between samples 9. Wear a new pair of gloves and proceed as in point 6 until you obtain the 6 leaf fragments to test in the extraction bag 10. Add the buffer as indicated on the instructions included with in the kit 11. Homogenize the sample with …..on a flat surface; for this purpose a plastic box or a plastic tray can be used as working bench. This action should not be longer than 2-5 seconds 12. Transfer a total of 4 drops of the extract into the disposable cuvette 13. Insert 1 strip as indicated on the instructions included with the kit 14. Leave the strip for at least 10-15 minuets before reading the results 15. Interpretation of the results: Preparation of the sample:
- 41. Immunostrip test steps Sample collection Sample Preparation Immunostrp test Documentation 5 min x6 5 min 1 drop 3 drop sap buffer 15 min 5 min
- 42. MCMV immunostrip documentation MCMV Diagnostic Testing of Maize Plant Samples Location : Harare Farmer's Name : Jan De Vries Trial Name or Number (if applicable): Plot Number (if applicable): Virus symptoms present: Yes / No Yes Date of sample : 3rd March 2014 Sample location : Harare Name of the person who sampled: Kevin Conn Bulk Sample ID Immunostrip (paste the strip here) Reaction start time (in min and sec) Reaction confirmation time (in min and sec) Remarks ZWEweodfrtdds12 2.13 4.13 Positive Signature : Name : Kevin Conn Institution : ZARI Location : Harare Farmer's Name : Jan De Vries Trial Name or Number (if applicable): Plot Number (if applicable): Virus symptoms present: Yes / No Yes Date of sample : 3rd March 2014 Sample location : Harare Name of the person who sampled: Kevin Conn Bulk Sample ID Immunostrip (paste the strip here) Reaction start time (in min and sec) Reaction confirmation time (in min and sec) Remarks ZWEweodfrtdds12 2.13 4.13 Positive Signature : Name : Kevin Conn Institution : ZARI Disclaimer: CIMMYT does not specifically endorse any specific commercial product / brand / company mentioned in this experiment.
- 43. Sending Samples 1. If MLN symptomatic plants (or suspected MLN symptomatic plants) are observed on the survey (i.e., any bulk sample that test positive with the immunostrip test), the samples should be sent to a recommended laboratory for ELISA testing for confirmation as soon as possible (within 48 hours). 2. If possible, mail the positive / suspected samples to a recommended laboratory in the country [NB: NO samples should be mailed to another country]. Place the bulk sample bag(s) inside a small cardboard box. Please mention on the box “Plant sample“ “RUSH IMMEDIATELY” and mail using either a courier service or express mail to the laboratory. 3. If the samples cannot be mailed immediately, keep them in a refrigerated condition or in a cool dark place. Samples must be refrigerated at 4oC and kept for no longer than 48 hours. After that time, leaf samples can deteriorate and the results will not be reliable.
- 44. Packing and shipping samples : • After placing the bulk sample bags into the cool box take the samples to a suitable site / laboratory in the plot to undertake the ELISA – After completing the immunostrip tests and recording all sample information, place the Styrofoam / cool box with all the collected plot samples inside a cardboard box, to provide a good and sturdy transport arrangement (Fig. 9). • Note: Inside each box, please keep a list of samples that has all the information collected during the sampling . Fig. 9. Sample box ready for transportation
- 45. MLN Management • As with diagnosis, management of disease depends on thorough knowledge of the three major components of a disease: susceptible host plant, virulent pathogen, and favorable environment. For a disease to develop, all the three factors must be present. Therefore, understanding these host- pathogen-environment dynamics is essential in devising disease management strategies. Such strategies target these three areas and manipulate them so that disease is not possible or its development is hampered.
- 51. HARMONIZED MLN MANAGEMENT CHECKLIST & SOPs No. TASK 1. Monitor crop disease history of seed production fields to enable adequate control plans. This includes an evaluation of Maize diseases recorded in the field/farm in the last three years and the documented management and yield loses if any. 1. Maintain adequate levels of soil fertility based on soil tests to ensure healthy crops. Consultation with relevant authorities for the appropriate fertilizer is important. 1. Timely planting to facilitate disease escape and eliminate disease incidence due to late planted crop 1. Use of disease free seed stocks in subsequent seed production. There is need to establish the origin of the seed and if it is certified as per the country seed certification regulations. 1. Clean farming equipment to remove contaminated soil debris and minimize spread of disease from one field to another. Use of recommended disinfectants can be used in cleaning the equipment and clothing. 1. Eliminate grasses and other weeds from fields and plot borders to remove vector hosting plants. Look out on neighboring farms for maize and sorghum that can harbor NLN vises is paramount. 1. Monitor and control insect vector population through tested spraying regimes. The target insect vectors are maize aphid and maize thrips (Franklinellia williamsi) 1. Scout for viral symptoms to ensure early detection and control. Use the MLN symptom catalogue to be sure of the occurrence of the disease. 1. Rogue symptomatic plants and burn/bury to minimize spread of disease. Be careful not to handle healthy plants during rouging for can infect them mechanically. 1. Sample suspect plants for diagnostic testing within the internal quality control to confirm disease presence. You can use the in MCMV immunostrips for on farm testing. You can take samples to recognized labs for confirmatory tests in there in need. 1. Post-harvest cob selection and seed testing for MLN causing viruses. Seed companies are encouraged to have mini labs for their self-regulation 1. Seed treatment using systemic insecticides to ensure early control of the disease. This s done mainly by the seed companies. 1. Crop rotations with non-cereal crops for 1- 3 seasons depending on disease history and pressure.