Diagnostics PhytosanitationDiagnostics, Phytosanitation, Phytosanitary Regulations and Germplasm Transfer

An overview

Lava KumarIITA, PMB 5320, Ibadan, Nigeria

L k @ iL.kumar@cgiar.org

Some terms

Pest = Pathogens, insects, mites, nematodes, etc.Pest   Pathogens, insects, mites, nematodes, etc.Any species, strain, biotype injurious to plants and plant products 

Virus negative: plants tested negative to a particular virusCBSD free: Free of all virus species presently known to cause CBSDCBSD‐free: Free of all virus species presently known to cause CBSDVirus clean or clean germplasm: Seldom used in official documents 

Diagnosis: Identification of the cause of the problemDiagnosis: Identification of the cause of the problem 

Diagnostic tools: To ascertain presence, absence or quantify pests 

Germplasm(Gene Bank / Breeders)

Collection missions Distribution missions

C ti / d ti Utilization/marketingConservation/production Utilization/marketing

Indexing for Germplasm HealthGlobalization of Pathogens

Germplasm exchange: risks

•Movement of germplasm involves a risk of accidentally introducing quarantine pests.

•Germplasm sometimes are contaminated ith h f l t

quarantine pests.

with harmful pests.

•Pathogens harmful to the same species (e.g. cassava to cassava)

•Can also carry pathogens that do not harm the carrier, but harmful to other species

(e.g. cassava carrying spores of maize pathogen)(spores, insect eggs, etc)p gg

Clean germplasm is prerequisite for safety of conserved germplasm andClean germplasm is prerequisite for safety of conserved germplasm and for the safe distribution of germplasm

I i f i di USInterception of non‐indigenous pests at US ports

• 725,000 pest (non‐indigenous insects, mites, molluscs, nematodes, plant pathogens and weeds) interceptions recorded in US Port‐interception network from 1984 to 2000.

• 62% of intercepted pests were associated with baggage, p p gg g

• 30% were associated with cargo and 

• 7% were associated with plant propagative material• 7% were associated with plant propagative material. 

• 50,750 in 17 years (ca. 3,000 interceptions per year)

Source: McCullough et al., 2006, Biological invasions 8: 611‐630.

•Measures to remove pests infested/infected plants •Elimination of pests from infected/infested tissues (therapy)

Phytosanitatoin

Elimination of pests from infected/infested tissues (therapy)

Phytosanitaory Germplasm y yRegulations

pTransfer

•Policy framework and  exing

international standards for risk assessment, risk reduction, phytosanitation, 

Inde

Diagnostics, p y ,

safe exchange of germplasm and otherwww iipc int •Tools for detection and identification 

of pests in the germplasm

www.iipc.int

International regulations and technical guidelinestechnical guidelines

International Standards for Phytosanitary Measures (ISPMs) of FAO IPPCInternational Standards for Phytosanitary Measures (ISPMs) of FAO‐IPPC

ISPM 1 to 36www.ippc.int

Challenges for cassava conservation 

•Pest matters, irrespective of disease causing potential

Pests

Endophytes

Regulated Not regulated

EndophytesCommon pests of insignificant consequence

Quarantine pestsRegulated non-

quarantine pests

consequence

quarantine pests

Endemic pests, know to cause debilitating impact on host: eg. 

Endemic pests, know to cause debilitating impact on host (eg. 

b k) Cassava mosaic virusescassava brown streak)

NPPO provides the list of quarantine pests and non‐qurantine pests

Major cassava pests in Africa( l d i )(regulated non‐quarantine pests)

Indigenous pestsIndigenous pests• Cassava mosaic viruses• Whiteflies, fungal diseases, root scales etc

Pests of regional importance• Cassava brown streak virus

, g ,

• Cassava brown streak virus• East African cassava mosaic virus – Uganda (EACMV‐UG)

Exotic pests• Green spider mite (Mononychellus tanajoa) 

C l b (Ph ih ti)• Cassava mealy bug, (Phenacoccus manihoti) • Cassava Bacterial Blight (Xanthomonas axonopodis)• Indian cassava mosaic virusIndian cassava mosaic virus

Quarantine pests of cassava in Africa

Pests not present in the continentPests not present in the continent1. Cassava common mosaic virus (??)2 Cassava green mosaic virus2. Cassava green mosaic virus 3. Cassava vein virus 4 Cassava X virus4. Cassava X virus5. Frog Skin Disease (complex etiology)6 Cassava antholysis (phytoplasma like)6. Cassava antholysis (phytoplasma‐like)7. Cassava witches broom (Phytoplasma)8 Super Elongation (Sphaceloma manihoticola)8. Super Elongation (Sphaceloma manihoticola) 

Risk of pest spread through planting materials

Pest Stems Sterileti lt

seed Steriletifi d titissue culture certified tissue 

culture plants*Bacteria  + ‐ ‐ ‐Fungi + ‐ ‐ ‐Virus  + + ‐ (?) ‐Insects & other + ‐ ‐ ‐

Stems: carriers of all pests

Ti lt l t O l i ( d h ti b t i )Tissue culture plants: Only viruses (some endophytic bacteria)

Virus elimination requires combination of  tissue culture and therapy q pytechniques cryotherapy, etc) and selection of plants following virus indexing. 

The viruses of cassava in Africa

African cassava mosaic virus (ACMV)Indian cassava mosaic virus

CMGCMGEast African cassava mosaic virus (EACMV)South African cassava mosaic virus (SACMV) East African cassava mosaic Cameroon virus

CMGsCMGs

East African cassava mosaic Cameroon virus East African cassava mosaic Malawi virusEast African cassava mosaic Zanzibar virus East African cassava mosaic Kenya virusEast African cassava mosaic Kenya virusEACMV-Uganda (recombinant virus)Cassava mosaic Madagascar virus

Cassava brown streak virus (Ipomovirus)Cassava brown streak Uganda virus

Cassa a I o ian bacillifo m i s*

CBSVCBSV

Cassava Ivorian bacilliform virus* Cassava Kumi virus*Cassava ‘Q’ virus*Cassava common mosaic virus* (Potexvirus)Cassava common mosaic virus (Potexvirus)

Challenges to cassava conservation

•Cassava is seriously affected by Wh t i t accumulation of pathogens in the 

propagation material. What goes in stays

• Gene banksGene banks conserves cassava

• Cassava conserves•High risk of pathogen spread through stems

Cassava conserves pests, particularly viruses

•Constraint to field gene banks High risk of infection & re‐infection; cause germplasm decline and genetic erosion

•Restrictions on exchanging stems

•International exchanges require in vitro plants  

Challenges to cassava conservation:Vi h i iVirus threat to micropropagation

Tissue culture cleanses

•In vitro conservation is emerging as mainstay for conserving clonal crops.

Tissue culture cleanses pests and pathogens, but not viruses.

•Effectiveness of in vitro therapy procedures depends on the genotype, host p g yp ,susceptibility, infection status, virus strains, type of therapy procedure and practice

•Procedures capable of eradicating cassava viruses are time consuming. 

•Requires expensive capacity and facilities, and round the clock access to water andand round the clock access to water and power. 

Challenges to cassava conservation:Vi di iVirus diagnostics

•ELISA, and PCR‐based tests (conventional and RT‐PCR) are used for the detection of cassava viruses. R tl iRNA b d th d

High diversity is a bane to accurate diagnosis of

Recently even siRNA based methods are used for diagnosis.

accurate diagnosis of viruses •High diversity poses challenges to 

the virus detection.

•Generic (broad‐specific) diagnostics b tt f i d t ti fare better for virus detection for 

quarantine monitoring, and virus indexing.indexing.  

Pest detection: requirements 

•Symptomatic stage: Confirmation of pathogen cs 

•Asymptomatic stage: Confirmation of infection & pathogen

gnostic

•Undetectable stage Germplasm (dormant)

Diag

Low‐titers of pathogens 

Knowledge on pathogens and their distribution

Availability of diagnostic tools

Fund

s

Capacity (human skills and infrastructure)

Choice of methods depends on the need

Identify known pest( i i i d i

Identify new pest ( f(repetitive; e.g. indexing, 

surveillance)(one time; e.g. cause of 

new disease)

Specific test(target pest of interest)

Non‐specific (generic) test(no prior knowledge) 

ELISA, PCR, NASH( p g )Morphometrics, bioassay, EM, 

ELISA, PCR, NASH, sRNAs

•PCR‐based assays are commonly used for cassava indexing

Source: A Adkins

•Small RNA‐based approach is gaining popularity as a useful broad‐specific tools

Guidelines for use of diagnostics

International Plant Protection Convention (IPPC) WTO‐SPS guidelinesWTO SPS guidelines

Regional and Country‐specific guidelines

Product quality For monitoring infectious diseasescertification

Pl ti t i l

For monitoring infectious diseases

Pests ‐Planting material

‐Product quality

Pests

Regulated Not regulated Product quality(inputs as well as plant products) 

Quarantine pests Regulated non-quarantine pests

Plenty of options for cassava pest diagnostics 

•Diagnostics is a very well established field, but not saturated

•Tremendous progress over last 3 years

•Low‐tech to very high‐tech tools

•Simple cost‐effective tools to most expensive tools

•Availability is not an issue application and practice matters•Availability is not an issue, application and practice matters. 

Sustainability of diagnostics

Diagnostic labs 2. Establishment‐Infrastructure ‐Equipment‐Supplies and reagentsMaintenance

1. ScopeD fi h f h l b ‐Maintenance 

‐Human skills‐Water and power

Define the scope of the lab•Pests, pathogens, pesticides, food‐borne toxins etc

3 Sustainability

Water and power‐Maintenance strategy

food‐borne toxins, etc.•Specific or generic lab•Indexing & certification,  3. Sustainability

‐Regular use‐Funding supportFunding support

Cassava Phytosanitation

Total plants tested

Number that tested negative to CBSV

Varietytested to CBSV

1 MM06/0011 20TC 15

2 MM06/0024 20TC 10

3 MM06/0138 20TC 3

4 MM06/0131 20TC 3

5 MM06/0019 20TC 9

6 MM06/0079 20TC 116 MM06/0079 20TC 11

7 MM06/0013 20TC 1

8 MM06/0045 20TC 20

9 MM06/0012 20TC 20

10 MM06/0023B

20TC

8

11 MM06/0139

20TC

2

12 MM06/0124

20TC

4

13 MM06/0112

20TC

3

14 MM06/0076

20TC

6

IPCC guidelines for cassava germplasm exchangeg g p g

V t ti (i it ) t i lVegetative (in vitro) material: • Import permit and phytosanitary certificate 

• Declaration certifying the absence of virus, bacteria, and nematodes (particular those quarantine/regulated pests (based on country pest lists)

• Compulsory treatment of consignment before shipment into Africa.

• Exchange only virus tested virus‐free materials

• Cassava seed: Seed sanitation and in vitro plants derived from meristems of t d l t

• Exchange only virus tested, virus‐free materials.

regenerated plants

Country specific guidelines can differ from IPPC and IAPSC guidelines

IITA Germplasm Health Unit 

•Adjunct to NPPO•Prevent the spread of pests and pathogens through germplasm exchangePrevent the spread of pests and pathogens through germplasm exchange•Produce healthy planting material & safe conservation and exchange procedures•Ensure compliance with national and international regulations 

Indexing for insect pests, fungi and bacteria: Visual inspection, blotter 

test, and  sedimentation assays (Optional seed treatment / fumigation)

Health Testing(For pathogens under

Release of pathogen free‐G l

seed treatment / fumigation)

Import / Export (For pathogens under containment facilities)

pathogen freematerial

Germplasm

Indexing for viruses: Grow‐out tests, evaluation for target 

virus by ELISA,  PCR or EM

Requirements for germplasm export

Material owner(GRC, Breeding Units, etc.)

*Requirements:-Accession details-Geographic origin of the material (site of nt

atio

n

Submit request to GHU with necessary details*

g p g (production) -Year of propagation/production-Previous indexing history (if any)-Import permit from the recipient-Any other instructions D

ocum

e

-Any other instructions

Indexing at GHUfor fungal and bacterial virus, as

Dh

per the standard procedures, as well as importing country

requirements.

disp

atch

Pest-free material selected for dispatch

catio

n &

NAQS certificate and GHU health statement on health status

Pack and dispatch

certi

fic

Pack and dispatch pest-free material

Procedure for importing 

l ti t i l

Importer at IITA (GRC, breeders, etc.)

Routed throughGermplasm Health Unit

atio

n

planting material

Importer to sign MTA as applicable;

NAQS may put specific conditions depending on the crop species/ material and country of origin.

Submit request to NAQS for import permitPermit issued

ocum

ent

Consignment arrives in Nigeria

Exporter to comply with the importer’s phytosanitary needs. Communicate any difficulties to requester. A solution will be worked out to get the material.

Submit to exporter Do

e

Process germplasm (seed/ in vitro material / vegetative

propagules) as per the standard procedure

Material that meets importing country standards

Material that do not satisfy importing country standards

Mandator health testing rele

asestandard procedure

Certified consignments Uncertified consignments

y

Mandatory health testing •Material held in post-entry quarantine facility•Visual inspection•Biochemical analysis for quarantine pathogens

ectio

n &

Depends on the

Grow-out in post-entry quarantine isolation

Weekly inspections Inse

pe

Depends on the NAQS advise

Release ‘clean’ material to requisitioner

Depends on the NAQS advise

Distribution of imported material

Certified Clones that meets the importer NPPO guidelines are released

International distribution In‐country distribution

Follow established regulations. 

Depends on NPPO regulations:

•This could include certification of clones by authorized labs/agencies present in‐country

Or Depends on NPPO regulations:•Material can be supplied to end‐users for further propagation and distribution.

or

•Distribute directly

•Plant in ‘quarantine plots’ for certification by local agencies and release to end‐users.

Germplasm exchange with a t ith l ticountry with regulations

Regulator:• Create awareness about the policy and standards• Create awareness about the policy and standards• Assess the producer constantans and find solutions (policy/technical) 

Producer / client•Strictly comply with the regulatory guidelinesStrictly comply with the regulatory guidelines•Discuss any constrains with the regulators  

Germplasm exchange with a t ith t l ticountry without regulations

Regulator:g•Follow the IPPC standards •Identify gaps and establish a feasible model

Producer/client• Adopt international standards• Adopt international standards • Work with regulators to establish guidelines 

Other issues

• Phytosanitary permits are issued based on the NPPO analysis of risk priority end use etcof risk, priority, end use, etc. 

• Permit to import prohibited (quarantine) plant material for research purposeresearch purpose 

• Permit to import plant material for research purpose 

• Permit to import plant material for processing and industrial use (not meant for propagation)

• Most stringent regulations apply for the material imported or exported for ‘propagation purpose’.

• Retrospective indexing 

Risk of transferring material

• Within Africa, biggest risk is CBSD, which is already present in TanzaniaWithin Africa, biggest risk is CBSD, which is already present in Tanzania• Requires a perfect phytosanitary system for re‐distribution 

Endnote 

• Risk assessment, analysis and phytosanitary system should be kept inRisk assessment, analysis and phytosanitary system should be kept in place prior to commissioning collection missions for conservation of germplasm in a central facility (perform in consultation with NPPO)

• Create awareness about the advantages (benefits) of the process, facility and phytosanitary procedures in place, to the participatory y p y y p p , p p ycountries.  

• Establish a virus indexing certification and documentation system• Establish a virus indexing, certification and documentation system (traceability), and obtain accreditation from TPPC.

• Establish a safe measures to harden virus‐free plants for exchange with countries with inadequate capacity to handle TC plants.